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Expression of bovine viral diarrhoea virus envelope glycoprotein E2 in yeast Pichia pastoris and its application to an ELISA for detection of BVDV neutralizing antibodies in cattle a
a
a
a
Sthita Pragnya Behera , Niranjan Mishra , Ram Kumar Nema , Pooja Dubey Pandey , a
a
b
Semmannan Kalaiyarasu , Katherukamem Rajukumar & Anil Prakash a
National Institute of High Security Animal Diseases (formerly High Security Animal Disease Laboratory, Indian Veterinary Research Institute), Indian Council of Agricultural Research, Anand Nagar, Bhopal, Madhya Pradesh, India-462022. b
Department of Microbiology, Barkatullah University, Bhopal, Madhya Pradesh, India Accepted author version posted online: 02 Apr 2015.
Click for updates To cite this article: Sthita Pragnya Behera, Niranjan Mishra, Ram Kumar Nema, Pooja Dubey Pandey, Semmannan Kalaiyarasu, Katherukamem Rajukumar & Anil Prakash (2015): Expression of bovine viral diarrhoea virus envelope glycoprotein E2 in yeast Pichia pastoris and its application to an ELISA for detection of BVDV neutralizing antibodies in cattle, Journal of Immunoassay and Immunochemistry, DOI: 10.1080/15321819.2015.1032305 To link to this article: http://dx.doi.org/10.1080/15321819.2015.1032305
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Title: Expression of bovine viral diarrhoea virus envelope glycoprotein E2 in yeast Pichia pastoris and its application to an ELISA for detection of BVDV neutralizing antibodies in cattle Running title: BVDV E2 ELISA using yeast P. pastoris expressed antigen
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Sthita Pragnya Behera(1), Niranjan Mishra(1)*, Ram Kumar Nema(1), Pooja Dubey Pandey(1), Semmannan. Kalaiyarasu(1), Katherukamem Rajukumar(1), and Anil Prakash(2) Affiliations and addresses of the authors (1)
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National Institute of High Security Animal Diseases (formerly High Security Animal Disease Laboratory, Indian Veterinary Research Institute), Indian Council of Agricultural Research, Anand Nagar, Bhopal, Madhya Pradesh, India-462022. Department of Microbiology, Barkatullah University, Bhopal, Madhya Pradesh, India
*Corresponding author.
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Tel.: +91 755 2750647; fax: +91 755 2758842 E-mail address: [email protected]
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Abstract
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Postal address: Principal Scientist, National Institute of High Security Animal Diseases, Indian Council of Agricultural Research, Anand Nagar, Bhopal, Madhya Pradesh, India-462022.
The aim of this study was to express envelope glycoprotein E2 of bovine viral diarrhoea virus
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Authors:
(BVDV) in yeast Pichia pastoris and its utility as a diagnostic antigen in ELISA. The BVDV E2 gene was cloned into the pPICZαA vector followed by integration into the Pichia pastoris strain
X-33 genome for methanol induced expression. SDS-PAGE and Western blot results showed that the recombinant BVDV E2 protein (72 kDa) was expressed and secreted into the medium at
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a concentration of 40mg/L of culture under optimized conditions. An indirect ELISA was then developed by using the yeast-expressed E2 protein. Preliminary testing of 300 field cattle serum samples showed that the E2 ELISA showed a sensitivity of 91.07% and a specificity of 92.02%
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compared to the reference virus neutralization test. The concordance between the E2 ELISA and
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VNT was 91.67%. This study demonstrates feasibility of BVDV E2 protein expression in yeast
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neutralizing antibodies in cattle.
Keywords: Bovine viral diarrhoea virus; E2 glycoprotein; ELISA; Pichia pastoris; Virus
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neutralization test; Yeast
Introduction
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Bovine viral diarrhoea (BVD) causes significant economic losses in cattle farming and is prevalent worldwide. The causative agents of BVD, Bovine viral diarrhoea virus 1 (BVDV-1)
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and Bovine viral diarrhoea virus 2 (BVDV-2) together with border disease virus (BDV) and [1]
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classical swine fever virus (CSFV) belong to the genus Pestivirus in the family Flaviviridae.
The BVDV genome is approximately 12.3 kb long single stranded positive sense RNA containing a single ORF encoding about 4000 amino acids flanked by un-translated regions
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Pichia pastoris for the first time and its efficacy as an antigen in ELISA for detecting BVDV
(UTR) at 5’ and 3’ ends.
[2]
The viral polyprotein (NH2-Npro-C-Erns-E1-E2-p7-NS2-3-NS4A-
NS4B-NS5A-NS5B-COOH) is processed by viral and cellular proteases to generate structural and non-structural proteins. The structural proteins are represented by capsid protein C and three envelope glycoproteins, namely, Erns, E1 and E2. The E2 envelope glycoprotein contains the
2
major antigenic determinants of BVDV with most of the neutralizing epitopes are conformational and are located within the N-terminal half of the protein.
[3]
Following natural
infection, the majority of the immune response is directed at viral proteins E2 and NS2-3 and a
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much weaker response is elicited at viral proteins Erns and E1.
of BVDV infection in a herd due to varied clinical features and complexity of BVDV
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pathogenesis. The virus neutralization test (VNT) is the gold standard test for the serological diagnosis of BVD and it is also used as the reference potency test for commercial vaccines.
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However, the requirement of cell culture, live virus and a dedicated facility with constant monitoring of serum and cells to prevent viral contamination makes the test expensive and difficult to deploy. Alternatively, commercially available NS3 ELISA is more frequently used due to their ability to screen large number of serum samples at short notice. However, often the
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results of NS3 ELISA do not correlate well with the results of VNT, since neutralizing antibodies
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are elicited only against BVDV E2 protein. These constraints support the development of a
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simple and rapid BVDV E2 ELISA as an alternative to VNT. Recently, Pichia pastoris yeast system has been used successfully for high level production of several recombinant viral proteins, such as human immunodeficiency virus type 1 (HIV-1) Gag
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Serological test for detection of BVDV antibodies is a valuable tool for diagnosis and monitoring
protein,
[4]
domain III
CSFV Erns protein, [7]
[5]
CSFV E2 protein,
[6]
dengue virus type 2 envelope protein
and reticuloendotheliosis virus gp90 protein
[8]
due to its advantages of both
prokaryotic and eukaryotic expression systems. BVDV E2 protein has previously been expressed in mammalian expression system,
[9, 10]
baculovirus expression system,
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[9, 11]
Drosophila
melanogaster system,
[12]
Saccharomyces cerevisiae yeast system
[13]
and alfalfa plants
[14]
. In
this study, BVDV E2 protein was expressed in Pichia pastoris yeast for the first time and its potential use as a candidate antigen was evaluated in ELISA for detection of BVDV neutralizing
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Amplification and cloning of BVDV E2 gene
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An Indian BVDV-2 cattle isolate Ind 141353 [15] was used for amplification of BVDV E2 gene. Following RNA extraction by QIAamp viral RNA mini kit (Qiagen, Hilden, Germany), the cDNA was synthesized in 20 µl volume using random hexamer primers and Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA, USA) and was used as a template in PCR to
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amplify a 1160 bp fragment (position in BVDV-1 strain SD1: nt 2256-3415) covering the Cterminal part of E1 gene (206 bp) and almost the entire E2 gene from N-terminus (954 bp of
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1116 bp, encoding amino acid residues 1-318 of E2 glycoprotein). The PCR reaction was carried
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out in 25μl volume by using 2μl of cDNA, 2.5μl of 10x PCR buffer (Invitrogen, Carlsbad, CA, USA), 2μl Mgcl 2 (25mM), 0.5μl dNTP (10mM), 1μl (10 pmol) of each forward (P2256F) and reverse (P3422R) primers [16] and 0.2μl Taq (1U/μl) DNA polymerase (Invitrogen, Carlsbad, CA,
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MATERIALS AND METHODS
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antibodies in cattle.
USA). The amplified BVDV-2 E2 DNA fragment was purified from agarose gel using QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany). The purified DNA was then cloned into pCR 2.1TOPO vector (Invitrogen, Carlsbad, CA, USA) by T/A cloning strategy to generate recombinant plasmid TOPO-BVDV-2-E2 following the protocol supplied by the manufacturer. All the
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cloning steps were performed in E. Coli TOP 10F’ (Invitrogen). The plasmid TOPO-BVDV-2E2 was sequenced using an automatic DNA Sequencer (ABI 3130, Applied Biosystems, Foster
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Construction of recombinant expression plasmid
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City, CA, USA) for ascertaining the correct orientation of the insert in the vector.
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study, contains the methanol-inducible AOX1 promoter, a multiple cloning site and the AOX1 transcription termination sequence. It also contains the zeocin-resistance marker to allow for the
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selection of multi-copy transformants and plasmid Ori sequences for propagation in E. coli. The P. pastoris host strain X33 was used as the expression host. Both the yeast host and the plasmid vector were purchased from Invitrogen (Carlsbad, CA, USA). TOPO-BVDV-2-E2 plasmid was digested with Kpn1/Not1 restriction enzymes and the purified product was ligated into the
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Kpn1/Not1digested pPICZαA vector in frame with the α-factor secretion signal sequence to construct the expression plasmid pPICZαA/E2 and cloned using E. Coli TOP 10F’. The clones
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were selected in low salt LB agar (1% tryptone, 0.5% NaCl, 0.5% yeast extract, 1.5% agar, pH
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7.5) containing 25µg/ml Zeocin and were screened for the presence of insert by BVDV-2 E2 gene specific primers in PCR. The constructed recombinant expression plasmid was sequenced using 3’AOX1 and 5’AOX1 primers to confirm the orientation of E2 gene in yeast vector.
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The P. Pastoris integrative plasmid pPICZαA (3.6 kb), used for heterologous expression in this
Transformation and selection in yeast Transformation of P. pastoris strain X33 was carried out by chemical transformation method using Pichia Easy Comp transformation kit (Invitrogen, Carlsbad, CA, USA) following the
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manufacturer’s protocol. Briefly, P. pastoris strain X33 was made competent by using the protocol and reagents provided in the kit, and competent X33 strain was transformed with 10 µg of PmeI linearized expression plasmid pPICZαA/E2. The cells were then plated onto yeast
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extract peptone dextrose (YPD, 1% yeast extract, 2% peptone, 2% dextrose) agar (2%),
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containing 100 µg/ml of Zeocin (Invitrogen, Carlsbad, CA, USA) and was incubated for 5 days
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colonies were plated onto YPD agar plates containing higher amount of Zeocin (1mg/ml). The presence of BVDV E2 gene insert in the clones was analyzed by direct colony PCR using gene
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specific primers. The strain X33 containing empty vector pPICZαA was selected as negative control.
Expression of BVDV E2 in yeast
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A single recombinant yeast colony was inoculated in 10 ml of YPD medium in a 100 ml baffled flask and was grown over night at 30°C in a shaker incubator with shaking at 250 rpm until the
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culture reached the log phase of growth (OD 600 = 2-6). The cells were pelleted at 2000 rpm for 5
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min at room temperature and then resuspended in 25 ml of fresh YPD medium containing 0.5% (v/v) methanol in a 250 ml baffled flask with two layers of sterile gauze and was incubated for 5 days in a shaker incubator at 30°C. Methanol at a final concentration of 0.5% was added to the
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in dark at 30°C until single colonies were formed. To obtain multi-copy recombinants, individual
medium after every 24 h to maintain the induction. The strain X33 containing empty vector pPICZαA was treated similarly in parallel as negative control. To analyze the level of expression
and to determine the optimal time following induction, 1ml of culture was collected periodically
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(0 , 24, 48, 72 and 96 h) and centrifuged at 13000 rpm for 5 min and both the pellet and supernatant fractions were stored at -80°C.
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SDS-PAGE and Western blotting Culture supernatants and pellets were mixed with equal volumes of 2 x SDS-PAGE sample
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bands were visualized by staining with Coomassie brilliant blue R250. For Western blotting, following SDS-PAGE the proteins were transferred by electroblotting onto nitrocellulose (NC)
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membrane using semidry buffer and blotting apparatus (Hoefer, Holliston, MA, USA) according to the manufacturer’s manual. The NC membrane was then blocked in 5% skimmed milk diluted in phosphate buffered saline (pH 7.4) for 2 h at 37oC. Following washing, the membrane was treated initially with 1:200 diluted anti-BVDV goat polyclonal serum (VMRD, Pullman, WA,
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USA), 1:200 diluted anti-BVDV-2 cattle polyclonal serum (Animal Health & Veterinary Laboratory Agency, Weybridge, U.K.), 1: 100 diluted anti-BVDV-1 cattle polyclonal serum
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(NIHSAD, Bhopal, India), 1:100 diluted BVDV common E2 specific
monoclonal antibody
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(MAb) 348 (VMRD, Pullman, WA, USA ) or 1:100 diluted BVDV-2 E2 specific MAb BA2 (VMRD, Pullman, WA, USA) and then with anti goat IgG donkey antibody (1:2000 dilution), anti bovine IgG rabbit antibody (1:4000 dilution) or 1: 3000 diluted anti mouse IgG goat
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loading buffer, boiled for 10 min and the proteins were separated by 12% SDS-PAGE. Protein
antibody conjugated with horse radish peroxidise (Sigma-Aldrich, St. Louis, MO, USA). Finally the membrane was treated with substrate 3,3’-diaminobenzidine tetrahydrochloride (DAB, Sigma-Aldrich, St. Louis, MO, USA) for colour development.
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Standardization of BVDV E2 ELISA The secreted protein in the culture supernatant was concentrated by precipitation with 60%
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ammonium sulphate followed by dialysis against PBS and the protein concentration was
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determined (Qubit, Invitrogen, Carlsbad, CA, USA). The optimal dilutions of antigen, antibody
negative cattle sera. The ELISA plate (Nalgene Nunc, Penfield, NY, USA) was coated with 50 µl
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of partially purified P. pastoris expressed recombinant BVDV E2 antigen in carbonate-
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bicarbonate buffer (pH 9.6, Sigma-Aldrich, St. Louis, MO, USA) ranging from 500 ng to 1500 ng per well and the plate was incubated overnight at 4oC. Following blocking and washing, reference positive and negative sera diluted 2-fold from 1:50 to 1:400 were added to the wells in duplicate and the plate was incubated at 37oC for 1 h to determine the optimum serum dilution.
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The working dilution of anti-bovine-IgG HRPO conjugate (Sigma), TMB substrate (Sigma) reaction temperature and time were also optimized. The dilutions that provided maximum
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difference in optical density (OD) values at 405 nm between positive and negative cattle serum
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(P/N) were selected for testing the serum samples in test proper.
Dose-response curve
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and conjugate were determined by checker board titration using BVDV antibody positive and
The dose-response curve was prepared between the antigen concentrations (500-1500 ng/well) and different dilutions of positive control serum (1/50-1/400) after subtracting the OD value of the negative control serum in the corresponding dilution.
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BVDV E2 ELISA test procedure Following optimization, 50 µl of 1000 ng/ml BVDV E2 protein diluted in carbonate-
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bicarbonate buffer was coated onto wells of the 96-well ELISA plate. After washing the wells
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thrice with PBST (PBS pH7.4, 0.05% Tween-20), all the wells were blocked with 100 µl of
washed and 50 µl of positive, negative and test serum samples diluted 1:200 in blocking buffer
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was added in duplicate and then incubated at 37oC for 1h. After washing, 50 µl of 1:40000
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diluted anti-bovine-IgG HRPO conjugate (Sigma-Aldrich, St. Louis, MO, USA) was added to each well and incubated at 37oC for 1 h. Then the wells were washed thrice and 50 µl of TMB substrate solution (Sigma-Aldrich, St. Louis, MO, USA) was added to each well and incubated for 10 min in dark at room temperature. Reactions were stopped by adding 100 µl of stop
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solution (0.2M sulphuric acid) to each well. The OD of each well was measured at 450 nm by an ELISA multiwell plate reader (Tecan, Mannedorf, Switzerland).
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Virus neutralization test
A total of 390 field cattle sera were tested for presence of virus neutralizing antibodies
against both BVDV-1 and BVDV-2 by VNT. Briefly, the sera were heat inactivated at 56°C for
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blocking buffer (3% non fat milk powder diluted in PBST) for 90 min at 37oC. The wells were
30 min, diluted 1:5 and were tested in triplicate by microplate indirect-immuno peroxidase
monolayer assay (IPMA) as described in our previous report
[17]
using 200 tissue culture
infectious dose 50 (TCID 50 ) of BVDV-1 cattle isolate Ind S-1449 [18], BVDV-2 cattle isolate Ind 141353 [15] and MDBK cells. Immunostaining was carried out using anti-BVDV goat polyclonal
9
antibody (VMRD, Pullman, WA, USA), anti goat IgG-HRPO conjugate (Sigma-Aldrich, St. Louis, MO, USA), substrate H 2 O 2 with chromogen 3-amino-9-ethyl carbazole (Sigma-Aldrich, St. Louis, MO, USA) and the results were noted after examination under microscope.
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Appropriate positive, negative, cell and virus controls were included in each plate. Serum sample
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was considered positive for BVDV-1 and/or BVDV-2 neutralizing antibodies when cells mixed
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otherwise it was considered as negative.
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ELISA cut-off value
A total of 90 negative sera samples as determined by virus neutralization test were tested by BVDV-E2 ELISA to determine the cut-off values. Each serum was tested twice in duplicate
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wells. The cut off value was set based on OD value of the mean of all negative sera
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and standard deviation (S.D.) between them.
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Sensitivity and specificity of BVDV-E2 ELISA For validation of E2 ELISA, 300 cattle sera samples available from the field were tested
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with serum at an initial dilution of 1:5 did not show any pestivirus specific cytoplasmic staining,
both by BVDV-E2 ELISA and VNT. The relative sensitivity and specificity of BVDV-E2 ELISA was determined as compared to the gold standard virus neutralization test using the
following formulae. Sensitivity % = [True positive / (True positive + False negative)] × 100
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Specificity % = [True Negative / (True negative + False positive)] × 100 Agreement % = [(True positive + True Negative) / Total number of samples] × 100
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Kappa value = [(Observed agreement-Expected agreement)/ (1- Expected agreement)]
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A total of 10 serum samples were selected to evaluate the reproducibility of the E2-ELISA. The coefficients of variation (CV) were calculated between plates (inter-assay variation) and within
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the same plate (intra-assay variation) for each sample. Each sample was tested in five different plates on different occasions to determine the inter-assay CV and five replicates within each plate were used to calculate the intra-assay CV.
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Robustness of E2 ELISA
The robustness of the assay was determined by evaluating 84 coded serum samples by two
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compared.
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different operators in other divisional laboratories within the Institute and the results were
RESULTS
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Reproducibility of the E2 ELISA
Creation of the recombinant P. pastoris strain In order to create a recombinant P. pastoris strain capable of expressing BVDV E2 protein, at first, the 1160 bp E2 gene amplified product (Fig. 1, lane 3) was purified and cloned to generate
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the plasmid TOPO-BVDV-E2. The recombinant plasmid was confirmed by restriction endonuclease digestion and DNA sequencing (data not shown). DNA sequencing of recombinant expression plasmid pPICZαA-BVDV-E2 confirmed integration of 1160 bp BVDV E2 gene in
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proper frame with initiation codon. This plasmid was then transformed into the genome of P.
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pastoris host strain X-33 and the recombinant yeast colonies appeared 3-4 days after incubation
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specific primers amplified a specific 1160 bp DNA product (Fig. 1, lane 2) confirming integration of BVDV E2 gene into the P. pastoris host genome. In contrast, an X-33 clone
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transformed with the empty pPICZαA plasmid did not produce any amplification in PCR (Fig. 1, lane 1).
Expression of BVDV E2 protein in P. pastoris
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To identify the best expressing clone, three PCR-positive Pichia transformants were grown and both the supernatant and pellet fractions of clones collected at 24, 48, 72 and 96 h after induction
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were assayed for BVDV E2 protein by SDS-PAGE and western blot. Expression of BVDV-E2
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protein with a molecular weight of 72 kDa was visualized both in the pellet and supernatant fractions of all the selected P. pastoris integrants at 72 h following induction (Fig. 2). As shown in Fig. 2 (lanes 4, 6 and 8), the recombinant protein, almost in a purified form was expressed
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at 28°C in dark. PCR analysis of genomic DNA of zeocin-resistant X-33 clones using gene
mostly in the supernatant fraction, whereas it was expressed at a minor amount along with other yeast proteins in the pellet fraction suggesting predominant extracellular expression of BVDVE2 protein in P. pastoris. The observed size of the expressed BVDV E2 protein (72 kDa) under reducing conditions was higher than the predicted size of the polypeptide backbone. No similar
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band was observed in negative control strain X-33 containing empty pPICZαA plasmid (Fig. 2, lanes 1 and 2). One of the clones was selected for optimization of induction conditions to achieve maximal BVDV E2 protein expression levels. The results showed that the concentrations of
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recombinant protein increased with increase in induction time and maximum level of BVDV E2
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protein expression occurred at 96 h post induction (Fig. 3, lane 5), which amounted to be 40 mg
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Characterization of yeast expressed BVDV E2 protein
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The antigenicity of BVDV E2 protein was characterized by western blot using anti-BVDV polyclonal serum and BVDV E2 specific monoclonal antibodies (MAB). The results revealed that the BVDV E2 protein (72 kDa) could be recognized not only by BVDV specific polyclonal antibodies (Fig. 4, lane 2), but also by BVDV-1 specific (Fig. 4, lane 5) or BVDV-2 specific
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(Fig. 4, lane 8) polyclonal antibodies indicating its utility as a candidate E2 antigen. The identity of yeast expressed BVDV E2 protein was further confirmed by its reactivity with BVDV E2
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specific MAB 348 and BA2 (Fig. 5, lanes 2 and 4).
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Standardization of BVDV E2 ELISA The BVDV E2 protein expressed in P. pastoris was tested for its utility as a diagnostic antigen in
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/L of supernatant.
indirect ELISA. The checker board titration tests showed that there was maximum difference in OD values between positive and negative serum (P/N ratio) when the concentration of coating antigen was 1000 ng/ well and serum dilution was 1:200 (Fig. 6). The dose response curve (Fig.
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7) showed that the slopes were linear and maximum correlation coefficient was found for 1000 ng/well of antigen. To determine the cut-off value, a total of 90 BVDV antibody negative cattle serum samples as
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determined by VNT were tested by the optimized BVDV E2 ELISA, which revealed a mean OD
(mean+1S.D.). The serum samples with an OD value greater than or equal to 0.200 was scored as
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BVDV antibody positive, otherwise it was scored to be BVDV antibody negative.
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Sensitivity and specificity of BVDV E2 ELISA
The relative sensitivity and specificity of the E2 ELISA was determined by comparison with reference virus neutralization test. Out of 300 serum samples tested, 102 samples were found
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positive by E2 ELISA whereas 112 samples were found positive by virus neutralization test (Table 1). The E2 ELISA showed a sensitivity of 91.07% and a specificity of 92.02% and the
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concordance between E2 ELISA and VNT was 91.67%.
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Reproducibility of the E2 ELISA The inter assay coefficient of variation was in the range of 1.25-4.36%, while the intra assay
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value of 0.12 with standard deviation (S.D.) of 0.08. Hence, the cut off value was set as 0.20 OD
coefficient of variation was in the range of 1.14-3.26% (Table 2) indicating good reproducibility of the ELISA.
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Robustness of E2 ELISA The E2 ELISA was found to be robust, since similar results were found by the different operators
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(Results not shown).
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BVDV is prevalent in cattle populations worldwide. The E2 envelope protein of BVDV is the most immunogenic protein of this virus with high titres of neutralizing antibodies against E2 [3]
Hence, this protein is important for development of
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being found in the host after infection.
diagnostic tools and vaccines. VNT has traditionally been used as a reference test for serological diagnosis of BVD and evaluation and efficacy of BVDV vaccines. But the test is laborious, time consuming, resource demanding and require cell culture facilities and runs the risk of live virus
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handling. In contrast, ELISA is rapid, sensitive, inexpensive and useful for screening of large number of samples. Different forms and sources of antigen have been used in different ELISAs:
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whole viral particles, BVDV infected cell cultures extracts, viral antigens immobilized with
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monoclonal antibodies or recombinant proteins produced in bacteria.
[19 - 22]
Although several
NS3 based ELISAs are now commercially available, they fail to detect BVDV neutralizing antibodies that are important for protection and there is a need to develop a test alternative to
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DISCUSSION
VNT. Hence, in this study, BVDV E2 protein was expressed in Pichia pastoris and its potential use as diagnostic antigen in ELISA was evaluated.
15
We selected yeast Pichia pastoris expression system, since it is a highly successful system that combines advantages of both prokaryotic and eukaryotic systems for the expression of heterologous genes in native form due to adequate glycosylation of expressed proteins, low cost,
[23, 24]
Moreover, it
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facilitates its culturing at high cell densities relative to fermentative yeasts.
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easy to use and the strong preference for respiratory growth, a key physiological trait that greatly
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expression when compared with baculovirus and mammalian systems. [25]
The coding region of almost entire BVDV E2 gene was engineered successfully into the genome
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of the yeast P. pastoris by the use of secreted expression vector pPICZαA, and upon methanol induction, a 72 kDa protein was expressed and secreted into the medium almost in a pure form. The expression level of E2 protein in culture medium was estimated at 40 mg/L which is a reasonable yield normally by a secreted expression. It has been shown earlier that since P.
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pastoris secretes only low level of endogenous proteins and its medium contains no added
28]
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proteins, the secreted heterologous protein comprises most of the total protein in the medium. [26 The expressed BVDV E2 protein was detected by SDS-PAGE and western blot which
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revealed that the size of the expressed protein (72 kDa) was higher than expected (60 kDa), which may be due to hyperglycosylation of the E2 protein. Hyperglycosylation of some of the expressed proteins such as Toxoplasma gondii recombinant surface antigen 2 in yeast P. pastoris
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has higher yields, either intracellularly or extracellularly, and is more suitable for a large-scale
has been reported earlier.
[29]
However, increase in size of the E2 protein had little effect on its
immunogenic reactivity, since the expressed 72 kDa protein could be recognized efficiently by polyclonal antibodies against both BVDV-1 and BVDV-2 and BVDV E2 specific monoclonal antibodies. It is well known that the major neutralizing epitopes of BVDV are located within the
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N-terminal half of the E2 protein and BVDV-1 and BVDV-2 strains possess an immunodominant neutralizing epitope mapped to amino acid positions 71-74. [30, 31] The BVDV E2 ELISA developed here was capable of differentiating between BVDV
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neutralizing and non-neutralizing antibodies in control sera and was able to detect antibodies
ELISA and results were correlated with VNT, a sensitivity of 91.07% and a specificity of
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92.02% were achieved, with a concordance of 91.67%. The diagnostic sensitivity of P. pastoris expressed E2 ELISA obtained in this study was better than that reported earlier with a drosophila
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expressed E2 indirect ELISA which yielded 82% correlation with the virus neutralization test results and a Baculovirus expressed E2 ELISA that showed a sensitivity of 88.3%.
[11, 12]
But it
was lower than an avidity blocking ELISA using drosophila expressed E2 antigen that showed a sensitivity of 98.10%. [32] This may be explained by the reason that most ELISAs probably detect
ed
preferentially IgG isotypes, while VNT detects both IgM and IgG isotypes.
[33]
A slightly lower
pt
specificity of the developed E2 ELISA was observed in our study when compared to the VNT. This may be explained by the reasons that the yeast expressed E2 antigen used for the
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development of ELISA contains 318 of the total 372 amino acids of the E2 protein and possibility of the presence of minor amount of yeast proteins in the antigen preparation.
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against both BVDV-1 and BVDV-2 in field sera. When field cattle sera were evaluated by this
Although BVDV E2 protein has been expressed in several heterologous expression systems, it has been evaluated mostly as candidate antigens for development of vaccines and its evaluation as a diagnostic antigen is limited.
cerevisiae
[13]
[9 - 14]
and in yeast K. lactis,
BVDV E2 protein was recently expressed in yeast S.
[34]
but no information is available for its utility as a
17
diagnostic antigen for detection of BVDV antibodies. Hence, this is first time that BVDV E2 protein was expressed in yeast P. pastoris and its utility as antigen in ELISA was demonstrated.
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CONCLUSIONS
antigenic reactivity. The recombinant protein was used as the coating antigen to develop an
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indirect ELISA. The BVDV E2-ELISA showed a good level of sensitivity and specificity and shows advantages over virus neutralization test in terms of rapidity and requirement of cell
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culture and infectious virus usage. The test has the potential for use in detecting BVDV neutralizing antibodies during sero-surveillance of BVDV infection in cattle. ACKNOWLEDGEMENT
ed
N. Mishra was supported by a project grant (BT/PR4415/AAQ/01/165/2003) from Department
pt
of Biotechnology, Govt. of India.
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Standardization of enzyme-linked immunosorbent assays (ELISAs) for quantitative estimation of antibodies specific for infectious bovine rhinotracheitis virus, respiratory syncytial virus, parainfluenza-3 virus, and bovine viral diarrhea virus. J. Vet. Diagn. Invest. 1997, 9, 24–31.
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34. Krijger, J. J.; Baumann, J.; Wagner, M.; Schulze, K.; Reinsch, C.; Klose, T.; Onuma, O. F.; Simon, C.; Behrens, S. E.; Breunig, K. D. A novel, lactase-based selection and strain
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ce
Fact. 2012, 11, 112.
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blocking ELISA as an alternative to the bovine viral diarrhea virus neutralization test. J. Virol.
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Table 1: Relative sensitivity and specificity of BVDV E2 ELISA with VNT
102
10
Total
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Positive
Negative
112
___________________________________________________________________________ 15
173
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Negative
188
___________________________________________________________________________ 117
183
ed
Total
Relative sensitivity (a/a+c): 91.07% (95% CI-84.34, 95.08)
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Relative specificity (d/b+d): 92.02% (95% CI-87.25, 95.11)
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Concordance: 91.67% (95% CI-87.99, 94.29) Kappa value: 0.823
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Positive
t
E2 ELISA
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Virus neutralization test
24
300
Table 2: Reproducibility of the BVDV E2-ELISA
2
3
4
5
6
1.14
1.23
1.41
2.26
1.35
3.26
assay 1.25
2.12
2.25
3.35
1.36
8
ed
CV%(Mean)
ce
pt
CV: Coefficient of variation
25
1.68
9
1.25
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Inter
4.36
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CV%(Mean)
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Samples
Intra-assay
7
10
t
1
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Serum
2.35
1.45
3.16
1.25
4.24
2.34
Fig. 1. PCR analysis of P. pastoris transformants in X33 strain. Lane 1: negative transformant carrying empty vector pPICZαA; Lane 2: positive transformant carrying BVDV E2 gene; Lane 3: Amplification of BVDV E2 gene before its integration into P. pastoris genome; Lane M: 1kb
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DNA molecular marker.
26
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Fig. 2. Analysis of P. pastoris yeast expressed BVDV-E2 protein by SDS-PAGE. Lane 1: pellet fraction of negative control (strain X-33 containing empty pPICZαA plasmid); Lane 2: supernatant fraction of negative control; Lanes 3, 5 and 7: pellet fractions of strain X-33 containing pPICZαA-BVDV-E2 recombinants; Lanes 4, 6 and 8: supernatant fractions of strain X-33 containing pPICZαA-BVDV-E2 recombinants Lane M: protein molecular weight markers in kDa.
27
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Fig. 3. Time course study of BVDV E2 protein expression in P. pastoris strain X-33. The X33 culture supernatants were harvested at 24 h (lane2), 48h (lane3), 72 h (lane4) and 96 h (lane5) and the proteins were analyzed by 12% SDS-PAGE followed by western blot using anti-BVDV polyclonal antibodies. The supernatant fraction of strain X-33 containing empty pPICZαA plasmid harvested at 96 h (lane 1) was used as negative control. Lane M: protein molecular weight marker in kDa.
28
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Fig. 4. Characterization of yeast P. pastoris expressed BVDV E2 protein. The supernatant and pellet fractions of X-33 cells expressing BVDV- E2 recombinant protein were analyzed by SDS-PAGE followed by western blot with anti-BVDV polyclonal antibodies (lanes 1-3), antiBVDV-2 polyclonal antibodies (lanes 4-6) and anti-BVDV-1 polyclonal antibodies (lanes 7-9). Lanes 1, 4 and 7: pellet fractions expressing BVDV E2 protein; Lanes 2, 5 and 8: supernatant fractions expressing BVDV E2 protein; Lanes 3, 6 and 9: negative control (supernatant fractions of X-33 carrying PPICZαA empty vector); Lane M: protein molecular weight marker in kDa.
29
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Fig. 5. Characterization of P. pastoris expressed BVDV E2 protein using monoclonal antibodies. The supernatant fractions of X-33 cells expressing BVDV E2 recombinant protein were analyzed by SDS-PAGE followed by western blot with anti-BVDV mAb 348 (lanes 1 and 2) and mAb BA2 (lanes 3 and 4). Lanes 2 and 4: yeast expressed E2 protein; Lanes 1 and 3: negative control. Lane M: protein molecular weight marker in kDa.
30
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Fig. 6. Optimum antigen concentration and serum dilution for the development of BVDV E2 ELISA. X axis: concentration of protein in ng and serum dilutions; Y axis: P/N ratio
31
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Figure 7. Effect of antigen and serum dilutions on absorbance values in control positive serum.
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Journal of Immunoassay and Immunochemistry Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ljii20
Expression of bovine viral diarrhoea virus envelope glycoprotein E2 in yeast Pichia pastoris and its application to an ELISA for detection of BVDV neutralizing antibodies in cattle a
a
a
a
Sthita Pragnya Behera , Niranjan Mishra , Ram Kumar Nema , Pooja Dubey Pandey , a
a
b
Semmannan Kalaiyarasu , Katherukamem Rajukumar & Anil Prakash a
National Institute of High Security Animal Diseases (formerly High Security Animal Disease Laboratory, Indian Veterinary Research Institute), Indian Council of Agricultural Research, Anand Nagar, Bhopal, Madhya Pradesh, India-462022. b
Department of Microbiology, Barkatullah University, Bhopal, Madhya Pradesh, India Accepted author version posted online: 02 Apr 2015.
Click for updates To cite this article: Sthita Pragnya Behera, Niranjan Mishra, Ram Kumar Nema, Pooja Dubey Pandey, Semmannan Kalaiyarasu, Katherukamem Rajukumar & Anil Prakash (2015): Expression of bovine viral diarrhoea virus envelope glycoprotein E2 in yeast Pichia pastoris and its application to an ELISA for detection of BVDV neutralizing antibodies in cattle, Journal of Immunoassay and Immunochemistry, DOI: 10.1080/15321819.2015.1032305 To link to this article: http://dx.doi.org/10.1080/15321819.2015.1032305
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Title: Expression of bovine viral diarrhoea virus envelope glycoprotein E2 in yeast Pichia pastoris and its application to an ELISA for detection of BVDV neutralizing antibodies in cattle Running title: BVDV E2 ELISA using yeast P. pastoris expressed antigen
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Sthita Pragnya Behera(1), Niranjan Mishra(1)*, Ram Kumar Nema(1), Pooja Dubey Pandey(1), Semmannan. Kalaiyarasu(1), Katherukamem Rajukumar(1), and Anil Prakash(2) Affiliations and addresses of the authors (1)
2
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National Institute of High Security Animal Diseases (formerly High Security Animal Disease Laboratory, Indian Veterinary Research Institute), Indian Council of Agricultural Research, Anand Nagar, Bhopal, Madhya Pradesh, India-462022. Department of Microbiology, Barkatullah University, Bhopal, Madhya Pradesh, India
*Corresponding author.
ed
Tel.: +91 755 2750647; fax: +91 755 2758842 E-mail address: [email protected]
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Abstract
pt
Postal address: Principal Scientist, National Institute of High Security Animal Diseases, Indian Council of Agricultural Research, Anand Nagar, Bhopal, Madhya Pradesh, India-462022.
The aim of this study was to express envelope glycoprotein E2 of bovine viral diarrhoea virus
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Authors:
(BVDV) in yeast Pichia pastoris and its utility as a diagnostic antigen in ELISA. The BVDV E2 gene was cloned into the pPICZαA vector followed by integration into the Pichia pastoris strain
X-33 genome for methanol induced expression. SDS-PAGE and Western blot results showed that the recombinant BVDV E2 protein (72 kDa) was expressed and secreted into the medium at
1
a concentration of 40mg/L of culture under optimized conditions. An indirect ELISA was then developed by using the yeast-expressed E2 protein. Preliminary testing of 300 field cattle serum samples showed that the E2 ELISA showed a sensitivity of 91.07% and a specificity of 92.02%
t
compared to the reference virus neutralization test. The concordance between the E2 ELISA and
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VNT was 91.67%. This study demonstrates feasibility of BVDV E2 protein expression in yeast
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neutralizing antibodies in cattle.
Keywords: Bovine viral diarrhoea virus; E2 glycoprotein; ELISA; Pichia pastoris; Virus
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neutralization test; Yeast
Introduction
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Bovine viral diarrhoea (BVD) causes significant economic losses in cattle farming and is prevalent worldwide. The causative agents of BVD, Bovine viral diarrhoea virus 1 (BVDV-1)
pt
and Bovine viral diarrhoea virus 2 (BVDV-2) together with border disease virus (BDV) and [1]
ce
classical swine fever virus (CSFV) belong to the genus Pestivirus in the family Flaviviridae.
The BVDV genome is approximately 12.3 kb long single stranded positive sense RNA containing a single ORF encoding about 4000 amino acids flanked by un-translated regions
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Pichia pastoris for the first time and its efficacy as an antigen in ELISA for detecting BVDV
(UTR) at 5’ and 3’ ends.
[2]
The viral polyprotein (NH2-Npro-C-Erns-E1-E2-p7-NS2-3-NS4A-
NS4B-NS5A-NS5B-COOH) is processed by viral and cellular proteases to generate structural and non-structural proteins. The structural proteins are represented by capsid protein C and three envelope glycoproteins, namely, Erns, E1 and E2. The E2 envelope glycoprotein contains the
2
major antigenic determinants of BVDV with most of the neutralizing epitopes are conformational and are located within the N-terminal half of the protein.
[3]
Following natural
infection, the majority of the immune response is directed at viral proteins E2 and NS2-3 and a
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much weaker response is elicited at viral proteins Erns and E1.
of BVDV infection in a herd due to varied clinical features and complexity of BVDV
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pathogenesis. The virus neutralization test (VNT) is the gold standard test for the serological diagnosis of BVD and it is also used as the reference potency test for commercial vaccines.
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However, the requirement of cell culture, live virus and a dedicated facility with constant monitoring of serum and cells to prevent viral contamination makes the test expensive and difficult to deploy. Alternatively, commercially available NS3 ELISA is more frequently used due to their ability to screen large number of serum samples at short notice. However, often the
ed
results of NS3 ELISA do not correlate well with the results of VNT, since neutralizing antibodies
pt
are elicited only against BVDV E2 protein. These constraints support the development of a
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simple and rapid BVDV E2 ELISA as an alternative to VNT. Recently, Pichia pastoris yeast system has been used successfully for high level production of several recombinant viral proteins, such as human immunodeficiency virus type 1 (HIV-1) Gag
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Serological test for detection of BVDV antibodies is a valuable tool for diagnosis and monitoring
protein,
[4]
domain III
CSFV Erns protein, [7]
[5]
CSFV E2 protein,
[6]
dengue virus type 2 envelope protein
and reticuloendotheliosis virus gp90 protein
[8]
due to its advantages of both
prokaryotic and eukaryotic expression systems. BVDV E2 protein has previously been expressed in mammalian expression system,
[9, 10]
baculovirus expression system,
3
[9, 11]
Drosophila
melanogaster system,
[12]
Saccharomyces cerevisiae yeast system
[13]
and alfalfa plants
[14]
. In
this study, BVDV E2 protein was expressed in Pichia pastoris yeast for the first time and its potential use as a candidate antigen was evaluated in ELISA for detection of BVDV neutralizing
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Amplification and cloning of BVDV E2 gene
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An Indian BVDV-2 cattle isolate Ind 141353 [15] was used for amplification of BVDV E2 gene. Following RNA extraction by QIAamp viral RNA mini kit (Qiagen, Hilden, Germany), the cDNA was synthesized in 20 µl volume using random hexamer primers and Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA, USA) and was used as a template in PCR to
ed
amplify a 1160 bp fragment (position in BVDV-1 strain SD1: nt 2256-3415) covering the Cterminal part of E1 gene (206 bp) and almost the entire E2 gene from N-terminus (954 bp of
pt
1116 bp, encoding amino acid residues 1-318 of E2 glycoprotein). The PCR reaction was carried
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out in 25μl volume by using 2μl of cDNA, 2.5μl of 10x PCR buffer (Invitrogen, Carlsbad, CA, USA), 2μl Mgcl 2 (25mM), 0.5μl dNTP (10mM), 1μl (10 pmol) of each forward (P2256F) and reverse (P3422R) primers [16] and 0.2μl Taq (1U/μl) DNA polymerase (Invitrogen, Carlsbad, CA,
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MATERIALS AND METHODS
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antibodies in cattle.
USA). The amplified BVDV-2 E2 DNA fragment was purified from agarose gel using QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany). The purified DNA was then cloned into pCR 2.1TOPO vector (Invitrogen, Carlsbad, CA, USA) by T/A cloning strategy to generate recombinant plasmid TOPO-BVDV-2-E2 following the protocol supplied by the manufacturer. All the
4
cloning steps were performed in E. Coli TOP 10F’ (Invitrogen). The plasmid TOPO-BVDV-2E2 was sequenced using an automatic DNA Sequencer (ABI 3130, Applied Biosystems, Foster
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Construction of recombinant expression plasmid
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City, CA, USA) for ascertaining the correct orientation of the insert in the vector.
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study, contains the methanol-inducible AOX1 promoter, a multiple cloning site and the AOX1 transcription termination sequence. It also contains the zeocin-resistance marker to allow for the
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selection of multi-copy transformants and plasmid Ori sequences for propagation in E. coli. The P. pastoris host strain X33 was used as the expression host. Both the yeast host and the plasmid vector were purchased from Invitrogen (Carlsbad, CA, USA). TOPO-BVDV-2-E2 plasmid was digested with Kpn1/Not1 restriction enzymes and the purified product was ligated into the
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Kpn1/Not1digested pPICZαA vector in frame with the α-factor secretion signal sequence to construct the expression plasmid pPICZαA/E2 and cloned using E. Coli TOP 10F’. The clones
pt
were selected in low salt LB agar (1% tryptone, 0.5% NaCl, 0.5% yeast extract, 1.5% agar, pH
ce
7.5) containing 25µg/ml Zeocin and were screened for the presence of insert by BVDV-2 E2 gene specific primers in PCR. The constructed recombinant expression plasmid was sequenced using 3’AOX1 and 5’AOX1 primers to confirm the orientation of E2 gene in yeast vector.
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The P. Pastoris integrative plasmid pPICZαA (3.6 kb), used for heterologous expression in this
Transformation and selection in yeast Transformation of P. pastoris strain X33 was carried out by chemical transformation method using Pichia Easy Comp transformation kit (Invitrogen, Carlsbad, CA, USA) following the
5
manufacturer’s protocol. Briefly, P. pastoris strain X33 was made competent by using the protocol and reagents provided in the kit, and competent X33 strain was transformed with 10 µg of PmeI linearized expression plasmid pPICZαA/E2. The cells were then plated onto yeast
t
extract peptone dextrose (YPD, 1% yeast extract, 2% peptone, 2% dextrose) agar (2%),
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containing 100 µg/ml of Zeocin (Invitrogen, Carlsbad, CA, USA) and was incubated for 5 days
us
colonies were plated onto YPD agar plates containing higher amount of Zeocin (1mg/ml). The presence of BVDV E2 gene insert in the clones was analyzed by direct colony PCR using gene
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specific primers. The strain X33 containing empty vector pPICZαA was selected as negative control.
Expression of BVDV E2 in yeast
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A single recombinant yeast colony was inoculated in 10 ml of YPD medium in a 100 ml baffled flask and was grown over night at 30°C in a shaker incubator with shaking at 250 rpm until the
pt
culture reached the log phase of growth (OD 600 = 2-6). The cells were pelleted at 2000 rpm for 5
ce
min at room temperature and then resuspended in 25 ml of fresh YPD medium containing 0.5% (v/v) methanol in a 250 ml baffled flask with two layers of sterile gauze and was incubated for 5 days in a shaker incubator at 30°C. Methanol at a final concentration of 0.5% was added to the
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in dark at 30°C until single colonies were formed. To obtain multi-copy recombinants, individual
medium after every 24 h to maintain the induction. The strain X33 containing empty vector pPICZαA was treated similarly in parallel as negative control. To analyze the level of expression
and to determine the optimal time following induction, 1ml of culture was collected periodically
6
(0 , 24, 48, 72 and 96 h) and centrifuged at 13000 rpm for 5 min and both the pellet and supernatant fractions were stored at -80°C.
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t
SDS-PAGE and Western blotting Culture supernatants and pellets were mixed with equal volumes of 2 x SDS-PAGE sample
us
bands were visualized by staining with Coomassie brilliant blue R250. For Western blotting, following SDS-PAGE the proteins were transferred by electroblotting onto nitrocellulose (NC)
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membrane using semidry buffer and blotting apparatus (Hoefer, Holliston, MA, USA) according to the manufacturer’s manual. The NC membrane was then blocked in 5% skimmed milk diluted in phosphate buffered saline (pH 7.4) for 2 h at 37oC. Following washing, the membrane was treated initially with 1:200 diluted anti-BVDV goat polyclonal serum (VMRD, Pullman, WA,
ed
USA), 1:200 diluted anti-BVDV-2 cattle polyclonal serum (Animal Health & Veterinary Laboratory Agency, Weybridge, U.K.), 1: 100 diluted anti-BVDV-1 cattle polyclonal serum
pt
(NIHSAD, Bhopal, India), 1:100 diluted BVDV common E2 specific
monoclonal antibody
ce
(MAb) 348 (VMRD, Pullman, WA, USA ) or 1:100 diluted BVDV-2 E2 specific MAb BA2 (VMRD, Pullman, WA, USA) and then with anti goat IgG donkey antibody (1:2000 dilution), anti bovine IgG rabbit antibody (1:4000 dilution) or 1: 3000 diluted anti mouse IgG goat
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loading buffer, boiled for 10 min and the proteins were separated by 12% SDS-PAGE. Protein
antibody conjugated with horse radish peroxidise (Sigma-Aldrich, St. Louis, MO, USA). Finally the membrane was treated with substrate 3,3’-diaminobenzidine tetrahydrochloride (DAB, Sigma-Aldrich, St. Louis, MO, USA) for colour development.
7
Standardization of BVDV E2 ELISA The secreted protein in the culture supernatant was concentrated by precipitation with 60%
t
ammonium sulphate followed by dialysis against PBS and the protein concentration was
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determined (Qubit, Invitrogen, Carlsbad, CA, USA). The optimal dilutions of antigen, antibody
negative cattle sera. The ELISA plate (Nalgene Nunc, Penfield, NY, USA) was coated with 50 µl
us
of partially purified P. pastoris expressed recombinant BVDV E2 antigen in carbonate-
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bicarbonate buffer (pH 9.6, Sigma-Aldrich, St. Louis, MO, USA) ranging from 500 ng to 1500 ng per well and the plate was incubated overnight at 4oC. Following blocking and washing, reference positive and negative sera diluted 2-fold from 1:50 to 1:400 were added to the wells in duplicate and the plate was incubated at 37oC for 1 h to determine the optimum serum dilution.
ed
The working dilution of anti-bovine-IgG HRPO conjugate (Sigma), TMB substrate (Sigma) reaction temperature and time were also optimized. The dilutions that provided maximum
pt
difference in optical density (OD) values at 405 nm between positive and negative cattle serum
ce
(P/N) were selected for testing the serum samples in test proper.
Dose-response curve
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and conjugate were determined by checker board titration using BVDV antibody positive and
The dose-response curve was prepared between the antigen concentrations (500-1500 ng/well) and different dilutions of positive control serum (1/50-1/400) after subtracting the OD value of the negative control serum in the corresponding dilution.
8
BVDV E2 ELISA test procedure Following optimization, 50 µl of 1000 ng/ml BVDV E2 protein diluted in carbonate-
t
bicarbonate buffer was coated onto wells of the 96-well ELISA plate. After washing the wells
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thrice with PBST (PBS pH7.4, 0.05% Tween-20), all the wells were blocked with 100 µl of
washed and 50 µl of positive, negative and test serum samples diluted 1:200 in blocking buffer
us
was added in duplicate and then incubated at 37oC for 1h. After washing, 50 µl of 1:40000
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diluted anti-bovine-IgG HRPO conjugate (Sigma-Aldrich, St. Louis, MO, USA) was added to each well and incubated at 37oC for 1 h. Then the wells were washed thrice and 50 µl of TMB substrate solution (Sigma-Aldrich, St. Louis, MO, USA) was added to each well and incubated for 10 min in dark at room temperature. Reactions were stopped by adding 100 µl of stop
ed
solution (0.2M sulphuric acid) to each well. The OD of each well was measured at 450 nm by an ELISA multiwell plate reader (Tecan, Mannedorf, Switzerland).
ce
pt
Virus neutralization test
A total of 390 field cattle sera were tested for presence of virus neutralizing antibodies
against both BVDV-1 and BVDV-2 by VNT. Briefly, the sera were heat inactivated at 56°C for
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blocking buffer (3% non fat milk powder diluted in PBST) for 90 min at 37oC. The wells were
30 min, diluted 1:5 and were tested in triplicate by microplate indirect-immuno peroxidase
monolayer assay (IPMA) as described in our previous report
[17]
using 200 tissue culture
infectious dose 50 (TCID 50 ) of BVDV-1 cattle isolate Ind S-1449 [18], BVDV-2 cattle isolate Ind 141353 [15] and MDBK cells. Immunostaining was carried out using anti-BVDV goat polyclonal
9
antibody (VMRD, Pullman, WA, USA), anti goat IgG-HRPO conjugate (Sigma-Aldrich, St. Louis, MO, USA), substrate H 2 O 2 with chromogen 3-amino-9-ethyl carbazole (Sigma-Aldrich, St. Louis, MO, USA) and the results were noted after examination under microscope.
t
Appropriate positive, negative, cell and virus controls were included in each plate. Serum sample
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was considered positive for BVDV-1 and/or BVDV-2 neutralizing antibodies when cells mixed
us
otherwise it was considered as negative.
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ELISA cut-off value
A total of 90 negative sera samples as determined by virus neutralization test were tested by BVDV-E2 ELISA to determine the cut-off values. Each serum was tested twice in duplicate
ed
wells. The cut off value was set based on OD value of the mean of all negative sera
pt
and standard deviation (S.D.) between them.
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Sensitivity and specificity of BVDV-E2 ELISA For validation of E2 ELISA, 300 cattle sera samples available from the field were tested
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with serum at an initial dilution of 1:5 did not show any pestivirus specific cytoplasmic staining,
both by BVDV-E2 ELISA and VNT. The relative sensitivity and specificity of BVDV-E2 ELISA was determined as compared to the gold standard virus neutralization test using the
following formulae. Sensitivity % = [True positive / (True positive + False negative)] × 100
10
Specificity % = [True Negative / (True negative + False positive)] × 100 Agreement % = [(True positive + True Negative) / Total number of samples] × 100
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t
Kappa value = [(Observed agreement-Expected agreement)/ (1- Expected agreement)]
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A total of 10 serum samples were selected to evaluate the reproducibility of the E2-ELISA. The coefficients of variation (CV) were calculated between plates (inter-assay variation) and within
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the same plate (intra-assay variation) for each sample. Each sample was tested in five different plates on different occasions to determine the inter-assay CV and five replicates within each plate were used to calculate the intra-assay CV.
ed
Robustness of E2 ELISA
The robustness of the assay was determined by evaluating 84 coded serum samples by two
ce
compared.
pt
different operators in other divisional laboratories within the Institute and the results were
RESULTS
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Reproducibility of the E2 ELISA
Creation of the recombinant P. pastoris strain In order to create a recombinant P. pastoris strain capable of expressing BVDV E2 protein, at first, the 1160 bp E2 gene amplified product (Fig. 1, lane 3) was purified and cloned to generate
11
the plasmid TOPO-BVDV-E2. The recombinant plasmid was confirmed by restriction endonuclease digestion and DNA sequencing (data not shown). DNA sequencing of recombinant expression plasmid pPICZαA-BVDV-E2 confirmed integration of 1160 bp BVDV E2 gene in
t
proper frame with initiation codon. This plasmid was then transformed into the genome of P.
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pastoris host strain X-33 and the recombinant yeast colonies appeared 3-4 days after incubation
us
specific primers amplified a specific 1160 bp DNA product (Fig. 1, lane 2) confirming integration of BVDV E2 gene into the P. pastoris host genome. In contrast, an X-33 clone
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transformed with the empty pPICZαA plasmid did not produce any amplification in PCR (Fig. 1, lane 1).
Expression of BVDV E2 protein in P. pastoris
ed
To identify the best expressing clone, three PCR-positive Pichia transformants were grown and both the supernatant and pellet fractions of clones collected at 24, 48, 72 and 96 h after induction
pt
were assayed for BVDV E2 protein by SDS-PAGE and western blot. Expression of BVDV-E2
ce
protein with a molecular weight of 72 kDa was visualized both in the pellet and supernatant fractions of all the selected P. pastoris integrants at 72 h following induction (Fig. 2). As shown in Fig. 2 (lanes 4, 6 and 8), the recombinant protein, almost in a purified form was expressed
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at 28°C in dark. PCR analysis of genomic DNA of zeocin-resistant X-33 clones using gene
mostly in the supernatant fraction, whereas it was expressed at a minor amount along with other yeast proteins in the pellet fraction suggesting predominant extracellular expression of BVDVE2 protein in P. pastoris. The observed size of the expressed BVDV E2 protein (72 kDa) under reducing conditions was higher than the predicted size of the polypeptide backbone. No similar
12
band was observed in negative control strain X-33 containing empty pPICZαA plasmid (Fig. 2, lanes 1 and 2). One of the clones was selected for optimization of induction conditions to achieve maximal BVDV E2 protein expression levels. The results showed that the concentrations of
t
recombinant protein increased with increase in induction time and maximum level of BVDV E2
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protein expression occurred at 96 h post induction (Fig. 3, lane 5), which amounted to be 40 mg
us
Characterization of yeast expressed BVDV E2 protein
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The antigenicity of BVDV E2 protein was characterized by western blot using anti-BVDV polyclonal serum and BVDV E2 specific monoclonal antibodies (MAB). The results revealed that the BVDV E2 protein (72 kDa) could be recognized not only by BVDV specific polyclonal antibodies (Fig. 4, lane 2), but also by BVDV-1 specific (Fig. 4, lane 5) or BVDV-2 specific
ed
(Fig. 4, lane 8) polyclonal antibodies indicating its utility as a candidate E2 antigen. The identity of yeast expressed BVDV E2 protein was further confirmed by its reactivity with BVDV E2
pt
specific MAB 348 and BA2 (Fig. 5, lanes 2 and 4).
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Standardization of BVDV E2 ELISA The BVDV E2 protein expressed in P. pastoris was tested for its utility as a diagnostic antigen in
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/L of supernatant.
indirect ELISA. The checker board titration tests showed that there was maximum difference in OD values between positive and negative serum (P/N ratio) when the concentration of coating antigen was 1000 ng/ well and serum dilution was 1:200 (Fig. 6). The dose response curve (Fig.
13
7) showed that the slopes were linear and maximum correlation coefficient was found for 1000 ng/well of antigen. To determine the cut-off value, a total of 90 BVDV antibody negative cattle serum samples as
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t
determined by VNT were tested by the optimized BVDV E2 ELISA, which revealed a mean OD
(mean+1S.D.). The serum samples with an OD value greater than or equal to 0.200 was scored as
us
BVDV antibody positive, otherwise it was scored to be BVDV antibody negative.
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Sensitivity and specificity of BVDV E2 ELISA
The relative sensitivity and specificity of the E2 ELISA was determined by comparison with reference virus neutralization test. Out of 300 serum samples tested, 102 samples were found
ed
positive by E2 ELISA whereas 112 samples were found positive by virus neutralization test (Table 1). The E2 ELISA showed a sensitivity of 91.07% and a specificity of 92.02% and the
pt
concordance between E2 ELISA and VNT was 91.67%.
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Reproducibility of the E2 ELISA The inter assay coefficient of variation was in the range of 1.25-4.36%, while the intra assay
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value of 0.12 with standard deviation (S.D.) of 0.08. Hence, the cut off value was set as 0.20 OD
coefficient of variation was in the range of 1.14-3.26% (Table 2) indicating good reproducibility of the ELISA.
14
Robustness of E2 ELISA The E2 ELISA was found to be robust, since similar results were found by the different operators
cr ip
t
(Results not shown).
us
BVDV is prevalent in cattle populations worldwide. The E2 envelope protein of BVDV is the most immunogenic protein of this virus with high titres of neutralizing antibodies against E2 [3]
Hence, this protein is important for development of
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being found in the host after infection.
diagnostic tools and vaccines. VNT has traditionally been used as a reference test for serological diagnosis of BVD and evaluation and efficacy of BVDV vaccines. But the test is laborious, time consuming, resource demanding and require cell culture facilities and runs the risk of live virus
ed
handling. In contrast, ELISA is rapid, sensitive, inexpensive and useful for screening of large number of samples. Different forms and sources of antigen have been used in different ELISAs:
pt
whole viral particles, BVDV infected cell cultures extracts, viral antigens immobilized with
ce
monoclonal antibodies or recombinant proteins produced in bacteria.
[19 - 22]
Although several
NS3 based ELISAs are now commercially available, they fail to detect BVDV neutralizing antibodies that are important for protection and there is a need to develop a test alternative to
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DISCUSSION
VNT. Hence, in this study, BVDV E2 protein was expressed in Pichia pastoris and its potential use as diagnostic antigen in ELISA was evaluated.
15
We selected yeast Pichia pastoris expression system, since it is a highly successful system that combines advantages of both prokaryotic and eukaryotic systems for the expression of heterologous genes in native form due to adequate glycosylation of expressed proteins, low cost,
[23, 24]
Moreover, it
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facilitates its culturing at high cell densities relative to fermentative yeasts.
t
easy to use and the strong preference for respiratory growth, a key physiological trait that greatly
us
expression when compared with baculovirus and mammalian systems. [25]
The coding region of almost entire BVDV E2 gene was engineered successfully into the genome
M an
of the yeast P. pastoris by the use of secreted expression vector pPICZαA, and upon methanol induction, a 72 kDa protein was expressed and secreted into the medium almost in a pure form. The expression level of E2 protein in culture medium was estimated at 40 mg/L which is a reasonable yield normally by a secreted expression. It has been shown earlier that since P.
ed
pastoris secretes only low level of endogenous proteins and its medium contains no added
28]
pt
proteins, the secreted heterologous protein comprises most of the total protein in the medium. [26 The expressed BVDV E2 protein was detected by SDS-PAGE and western blot which
ce
revealed that the size of the expressed protein (72 kDa) was higher than expected (60 kDa), which may be due to hyperglycosylation of the E2 protein. Hyperglycosylation of some of the expressed proteins such as Toxoplasma gondii recombinant surface antigen 2 in yeast P. pastoris
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has higher yields, either intracellularly or extracellularly, and is more suitable for a large-scale
has been reported earlier.
[29]
However, increase in size of the E2 protein had little effect on its
immunogenic reactivity, since the expressed 72 kDa protein could be recognized efficiently by polyclonal antibodies against both BVDV-1 and BVDV-2 and BVDV E2 specific monoclonal antibodies. It is well known that the major neutralizing epitopes of BVDV are located within the
16
N-terminal half of the E2 protein and BVDV-1 and BVDV-2 strains possess an immunodominant neutralizing epitope mapped to amino acid positions 71-74. [30, 31] The BVDV E2 ELISA developed here was capable of differentiating between BVDV
cr ip
t
neutralizing and non-neutralizing antibodies in control sera and was able to detect antibodies
ELISA and results were correlated with VNT, a sensitivity of 91.07% and a specificity of
us
92.02% were achieved, with a concordance of 91.67%. The diagnostic sensitivity of P. pastoris expressed E2 ELISA obtained in this study was better than that reported earlier with a drosophila
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expressed E2 indirect ELISA which yielded 82% correlation with the virus neutralization test results and a Baculovirus expressed E2 ELISA that showed a sensitivity of 88.3%.
[11, 12]
But it
was lower than an avidity blocking ELISA using drosophila expressed E2 antigen that showed a sensitivity of 98.10%. [32] This may be explained by the reason that most ELISAs probably detect
ed
preferentially IgG isotypes, while VNT detects both IgM and IgG isotypes.
[33]
A slightly lower
pt
specificity of the developed E2 ELISA was observed in our study when compared to the VNT. This may be explained by the reasons that the yeast expressed E2 antigen used for the
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development of ELISA contains 318 of the total 372 amino acids of the E2 protein and possibility of the presence of minor amount of yeast proteins in the antigen preparation.
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against both BVDV-1 and BVDV-2 in field sera. When field cattle sera were evaluated by this
Although BVDV E2 protein has been expressed in several heterologous expression systems, it has been evaluated mostly as candidate antigens for development of vaccines and its evaluation as a diagnostic antigen is limited.
cerevisiae
[13]
[9 - 14]
and in yeast K. lactis,
BVDV E2 protein was recently expressed in yeast S.
[34]
but no information is available for its utility as a
17
diagnostic antigen for detection of BVDV antibodies. Hence, this is first time that BVDV E2 protein was expressed in yeast P. pastoris and its utility as antigen in ELISA was demonstrated.
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t
CONCLUSIONS
antigenic reactivity. The recombinant protein was used as the coating antigen to develop an
us
indirect ELISA. The BVDV E2-ELISA showed a good level of sensitivity and specificity and shows advantages over virus neutralization test in terms of rapidity and requirement of cell
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culture and infectious virus usage. The test has the potential for use in detecting BVDV neutralizing antibodies during sero-surveillance of BVDV infection in cattle. ACKNOWLEDGEMENT
ed
N. Mishra was supported by a project grant (BT/PR4415/AAQ/01/165/2003) from Department
pt
of Biotechnology, Govt. of India.
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immunogenicity of recombinant surface antigen 2 (SAG2) expressed in the yeast Pichia pastoris. Exp. Parasitol. 2008, 119, 373-378. 30. Paton, D. J.; Lowings, J. P.; Barrett, A. D. Epitope mapping of the gp53 envelope protein of
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bovine viral diarrhea virus. Virology. 1992, 190, 763–772.
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31. Deregt, D., Bolin, S. R., Van den Hurk, J.; Ridpath, J. F.; Gilbert, S. A. Mapping of a type 1specific and a type-common epitope on the E2 (gp53) protein of bovine viral diarrhoea virus with neutralization escape mutants. Virus Res. 1998, 53, 81-90.
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32. Franco Mahecha, O. L.; Ogas Castells, M. L.; Combessies, G.; Lavoria, M. A.; Wilda, M.; Mansilla, F. C.; Seki, C.; Grigera, P. R.; Capozzo, A. V. Single dilution avidity-
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Methods. 2011, 175, 228-235.
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Standardization of enzyme-linked immunosorbent assays (ELISAs) for quantitative estimation of antibodies specific for infectious bovine rhinotracheitis virus, respiratory syncytial virus, parainfluenza-3 virus, and bovine viral diarrhea virus. J. Vet. Diagn. Invest. 1997, 9, 24–31.
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34. Krijger, J. J.; Baumann, J.; Wagner, M.; Schulze, K.; Reinsch, C.; Klose, T.; Onuma, O. F.; Simon, C.; Behrens, S. E.; Breunig, K. D. A novel, lactase-based selection and strain
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improvement strategy for recombinant protein expression in Kluyveromyces lactis. Microb. Cell
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Fact. 2012, 11, 112.
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blocking ELISA as an alternative to the bovine viral diarrhea virus neutralization test. J. Virol.
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Table 1: Relative sensitivity and specificity of BVDV E2 ELISA with VNT
102
10
Total
us
Positive
Negative
112
___________________________________________________________________________ 15
173
M an
Negative
188
___________________________________________________________________________ 117
183
ed
Total
Relative sensitivity (a/a+c): 91.07% (95% CI-84.34, 95.08)
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Relative specificity (d/b+d): 92.02% (95% CI-87.25, 95.11)
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Concordance: 91.67% (95% CI-87.99, 94.29) Kappa value: 0.823
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Positive
t
E2 ELISA
cr ip
Virus neutralization test
24
300
Table 2: Reproducibility of the BVDV E2-ELISA
2
3
4
5
6
1.14
1.23
1.41
2.26
1.35
3.26
assay 1.25
2.12
2.25
3.35
1.36
8
ed
CV%(Mean)
ce
pt
CV: Coefficient of variation
25
1.68
9
1.25
us
Inter
4.36
M an
CV%(Mean)
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Samples
Intra-assay
7
10
t
1
cr ip
Serum
2.35
1.45
3.16
1.25
4.24
2.34
Fig. 1. PCR analysis of P. pastoris transformants in X33 strain. Lane 1: negative transformant carrying empty vector pPICZαA; Lane 2: positive transformant carrying BVDV E2 gene; Lane 3: Amplification of BVDV E2 gene before its integration into P. pastoris genome; Lane M: 1kb
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DNA molecular marker.
26
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Fig. 2. Analysis of P. pastoris yeast expressed BVDV-E2 protein by SDS-PAGE. Lane 1: pellet fraction of negative control (strain X-33 containing empty pPICZαA plasmid); Lane 2: supernatant fraction of negative control; Lanes 3, 5 and 7: pellet fractions of strain X-33 containing pPICZαA-BVDV-E2 recombinants; Lanes 4, 6 and 8: supernatant fractions of strain X-33 containing pPICZαA-BVDV-E2 recombinants Lane M: protein molecular weight markers in kDa.
27
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Fig. 3. Time course study of BVDV E2 protein expression in P. pastoris strain X-33. The X33 culture supernatants were harvested at 24 h (lane2), 48h (lane3), 72 h (lane4) and 96 h (lane5) and the proteins were analyzed by 12% SDS-PAGE followed by western blot using anti-BVDV polyclonal antibodies. The supernatant fraction of strain X-33 containing empty pPICZαA plasmid harvested at 96 h (lane 1) was used as negative control. Lane M: protein molecular weight marker in kDa.
28
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Fig. 4. Characterization of yeast P. pastoris expressed BVDV E2 protein. The supernatant and pellet fractions of X-33 cells expressing BVDV- E2 recombinant protein were analyzed by SDS-PAGE followed by western blot with anti-BVDV polyclonal antibodies (lanes 1-3), antiBVDV-2 polyclonal antibodies (lanes 4-6) and anti-BVDV-1 polyclonal antibodies (lanes 7-9). Lanes 1, 4 and 7: pellet fractions expressing BVDV E2 protein; Lanes 2, 5 and 8: supernatant fractions expressing BVDV E2 protein; Lanes 3, 6 and 9: negative control (supernatant fractions of X-33 carrying PPICZαA empty vector); Lane M: protein molecular weight marker in kDa.
29
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Fig. 5. Characterization of P. pastoris expressed BVDV E2 protein using monoclonal antibodies. The supernatant fractions of X-33 cells expressing BVDV E2 recombinant protein were analyzed by SDS-PAGE followed by western blot with anti-BVDV mAb 348 (lanes 1 and 2) and mAb BA2 (lanes 3 and 4). Lanes 2 and 4: yeast expressed E2 protein; Lanes 1 and 3: negative control. Lane M: protein molecular weight marker in kDa.
30
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Fig. 6. Optimum antigen concentration and serum dilution for the development of BVDV E2 ELISA. X axis: concentration of protein in ng and serum dilutions; Y axis: P/N ratio
31
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Figure 7. Effect of antigen and serum dilutions on absorbance values in control positive serum.
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