Bioscience, Biotechnology, and Biochemistry
ISSN: 0916-8451 (Print) 1347-6947 (Online) Journal homepage: http://www.tandfonline.com/loi/tbbb20
Molecular Cloning of Feline Interferon cDNA by Direct Expression Naoko Nakamura, Tetsuo Sudo, Susumu Matsuda & Akira Yanai To cite this article: Naoko Nakamura, Tetsuo Sudo, Susumu Matsuda & Akira Yanai (1992) Molecular Cloning of Feline Interferon cDNA by Direct Expression, Bioscience, Biotechnology, and Biochemistry, 56:2, 211-214, DOI: 10.1271/bbb.56.211 To link to this article: http://dx.doi.org/10.1271/bbb.56.211
Published online: 12 Jun 2014.
Submit your article to this journal
Article views: 22
View related articles
Citing articles: 16 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=tbbb20 Download by: [205.217.235.162]
Date: 05 November 2015, At: 16:20
Biosci. Biotech. Biochem., 56 (2), 211-214, 1992
Molecular Cloning of Feline Interferon cDNA by Direct Expression' Naoko NAKAMURA, Tetsuo SUDO, Susumu MATSUDA, and Akira Y ANAl Basic Research Laboratories, TORAY Industries, Inc., 1111 Tebiro, Kamakura 248, Japan Received June 26, 1991
Downloaded by [205.217.235.162] at 16:20 05 November 2015
A cDNA sequence coding for feline interferon has been cloned for the first time by screening a cDNA library constructed using Okayama-Berg vector and mRNA derived from the feline cells (LSA-I) induced by TPA (12-o-tetradecanoylphorbor 13-acetate) for the ability of transient expression to produce feline interferon in COSI monkey cells. The amino acid sequence, deduced from the nucleotide sequence by comparing it with the sequences of other mammalian IFNs, consists of 171 amino acids with 6 cysteins and an N-glycosylation site at the amino acid position 79, and has about 60% homology to human IFN alpha 1. The interferon was partially purified through Blue Sepharose, and its molecular weight was estimated to be 2.4 x 104 • The antiviral activity was acid stable, and glycosylation was suggested.
Interferon is a physiologically active substance the main ingredient of which is a protein showing antiviral activity and which is abbreviated IFN. By the progress of cellular and molecular biology, many mammalian IFNs have been reported, some of which have been mass-produced for anticancer and/or antiviral drugs. We tried to clone the cDNA of feline IFN (FeIFN) for the purpose of mass-production by genetic engineering aiming at an antiviral drug for cats that are suffering from viral diseases such as feline herpes virus, feline calici virus, feline leukaemia virus, feline infectious peritonitis virus, or feline immunodeficiency virus. When we started the work, no FeIFN had been mass-produced, and nor was there nucleotide or amino acid sequence information available for the synthesis of specific DNA probes, though a few preliminary reports had been published on FeIFN. 1 - 5) Therefore we took advantage of the characteristics of highly antiviral specific-activity of IFN molecule and used an expression vector of mammalian cells for the screening of cDNA by the direct expression of composite plasmids followed by assaying the transfected cell supernatants for antiviral activity. The obtained cDNA in th pFeIFNI will lead to a tool for mass production of FeIFN.
Materials and Methods Cell lines and cultivation. LSA-I is a cloned T-cell line derived from a thymic tumor of a feline leukaemia virus-infected cat by Yamamoto et al.,3) 1986, and has weak productivity of FeIFN. Fc9 (Felis catus 9) is a kind gift from Dr. J. K. Yamamoto. Both were maintained and grown in 50% MEM (Nissui Seiyaku Co., Japan) and 50% LIS medium (Gibco Lab) (MEM/LIS) containing 10% fetal bovine serum (FBS) (MEM/LI5/ FBS). COSI was maintained in RPMII640 (Gibco Lab) medium supplementded with 10% fetal bovine serum (RPMII640/FBS). Biological assay offeline interferon. The antiviral activity was measured by the cytopathic effect inhibition method 6 ) using Fc9 and Vesicular Stomatitis virus. Isolation of mRNA and construction of cDNA library. One of recloned cells named LSA-D6 was proliferated by spinner culture in 200 ml of MEM/LI5/FBS. At the cell concentration 5 x lOs/ml, TPA (12-0tetradecanoyl phorbol-13-acetate (Sigma Chemical Co.)) was added to 5 ng/ml for induction of mRNA production. After 20 hr of cultivation, cells were harvested by centrifugation. Total cellular RNA was extracted by using the guanidinium thiocyanate method,7) and the poly(A) + RNA was selected by oligo(dT)-cellulose chromatography. Before the con-
struction of the eDNA library, the RNA was confirmed for translational ability to produce FeIFN activity. Samples of the eluted RNA were injected into 20 individual Xenopus laevis oocytes (50 to 60ngjoocyte) and the supernatant from smashed oocytes were collected after 20 hr incubation,8) and assayed for antiviral activity. The eDNA library was constructed using an Okayama-Berg cDNA Expression Vector Strain Kit (Pharmacia LKB Biotechnology) consisting of the pcDVI oligo(dT)-tailed plasmid-primer, the pLI oligo(dG)-tailed linker and E. coli according to the attached procedure manual. 9 ) Screening of FeIFN-producing plasmids. The FeIFN-producing plasmids were screened by their transient-expression ability of IFN activity. COSI monkey cells were grown to confluency in RPMII640/FBS in 90-mm plates, and transfections were done with 20 J.lg of plasmid DNA in 4 ml of RPMII640 medium containing 50mM Tris-HCI (pH 7.4) and 400 J.lg of DEAE-Dextran per m1. 10 ) After 4 hr of incubation at 37°C, cells were washed and then incubated with RPMII640 medium containing ISO J.lM chloroquine for 3 hr. This was then replaced with 10 ml of RPMII 640/FBS, and the medium was collected 72 hr later, then assayed for FeIFN activity. DNA sequence analysis. A eDNA-containing fragment for nucleotide sequence analysis was prepared from a composite plasmid consisting of the sequencing plasmid pUCl8 and an inserted DNA fragment, BamHI-BamHI or two kinds of BamHI-Bg/II containing eDNA from pFeIFNl. Sequence analysis was done by a combination of the dideoxy method 11) and the primer extension method 12 ) by Shozo Kanai of the Toray Research Center Inc. Partial purification of FeIFN. Culure medium containing FeIFN activity was put on a 50-ml fast flow type Blue Sepharose column that had been equilibrated with 2.5 mM KCI-1.5 mM phosphate buffer (pH 7.0). After washing the column with 500 ml of 50 mM phosphate buffer (pH 7.0) containing 0.5 MNaC!, the adsorbed FeIFN was eluted with 50 ml of 50 mM phosphate buffer (pH 7.0) containing 1M NaCI followed by 500 ml of 50mM phosphate buffer (pH 7.0) containing 1 M NaCI and 20% ethylene glycol.
Results and Discussion Induction of FeIFN synthesis and evaluation of mRNA LSA-I cell was known to produce FeIFN activity and release it into supernatant medium. 3) The highest subclones, LSA-D4-C and LSA-D4-KI7, were obtained by repeated recloning, and showed 500 U /ml productivity at most. However, these highest productivities easily decreased through serial passages. The decrease was observed to be faster in RPMI1640/FBS than in the MEM/LI5/FBS which was used for cell propagation for mRNA preparation. To increase the quality of the cDNA library, a greater abundance of FeIFN mRNA is required in poly(A) + RNA
NII-Electronic Library Service
212
N. NAKAMURA
preparation. To make the production yield of the RNA of IFN higher, inductive ability of known IFN inducers were investigated on LSA-I subclonal cells by assaying IFN activity found in the supernatant of the cultures. As is shown in Table I, TPA was shown to have highly inductive activity. The optimal TP A concentration and optimal induction stage were examined, using LSA-D6 cells which are a subclone of LSA-D4-KI7, as shown in Table II and Table III. On the other hand, the FeIFN produced by transient expression in supernatant of COSI cell culture showed stable in the medium for 3 days at 37°C. The high amount of FeIFN in medium at 30 hr and later in Table III should be due to accumulation of FeIFN and the time of maximum mRNA content in the cells was expected before that. So the extraction of mRNA was done from cells harvested at 20 hr of cultivation in MEM/Ll5/FBS after TPA induction. The yield of poly(A)+ RNA was 301 p,g from about 5.8mg of total RNA from 7 x 108 cells. To confirm that the extracted
Downloaded by [205.217.235.162] at 16:20 05 November 2015
Table I.
et al.
poly(A) + RNA fraction had translational activity ofFeIFN, both poly(A)+ RNA fractions from LSA-D6 cells, induced or uninduced, was injected into X. laevis oocytes, and the supernatants from the oocytes were assayed for FeIFN activity. mRNA preparation from induced and uninduced cells showed FeIFN productivity of 13 and < 2 U Iml respectively. Screening of the cDNA library by transient expession
The cDNA library was constructed with 6 x 10 5 clones. Before screening of the cDNA library, the size of inserted cDNA was estimated by electrophoresis. Seven among 12 clones of composite plasmids were shown to have inserted BamHI DNA fragments longer than 0.5 kb. The cDNA Table IV. DNA Transfection Assay for FeIFN Activity from Pools of Plasmid DNA DNA
Production of FeIFN by LSA-I Induced by Inducers
1st screen
Supernatant activity (unitjml)
MEMjL15 None
PHAa TPA b ConAc a b C
LSA-D4-KI7
LSA-D4-C
Inducers
14> 12> 776 12>
RPMI1640 14 12 1,725 12
MEMjL15 261 320 7,890 182
38 46 6,215 34
PHA: 10mgjml of phytohemagglutinin. TPA: 5 ngjml. ConA: 5 jlgjml of Concanavalin A. Cells grown in 24-well plates to 4 x 105jml in MEMjL15 and RPMI1640, both of which were supplemented with 10%FBS, were harvested and resuspended to 5 x 105 jml in fresh media with 2%FBS and inducers. FeIFN activity was assayed after 14 hr of incubation.
Table II.
2nd screena
RPMI1640
Inductive Ability by TPA TPA (ngjml)
FeIFN activity (Ujml)
10 25 50
15,850 590
ISSN: 0916-8451 (Print) 1347-6947 (Online) Journal homepage: http://www.tandfonline.com/loi/tbbb20
Molecular Cloning of Feline Interferon cDNA by Direct Expression Naoko Nakamura, Tetsuo Sudo, Susumu Matsuda & Akira Yanai To cite this article: Naoko Nakamura, Tetsuo Sudo, Susumu Matsuda & Akira Yanai (1992) Molecular Cloning of Feline Interferon cDNA by Direct Expression, Bioscience, Biotechnology, and Biochemistry, 56:2, 211-214, DOI: 10.1271/bbb.56.211 To link to this article: http://dx.doi.org/10.1271/bbb.56.211
Published online: 12 Jun 2014.
Submit your article to this journal
Article views: 22
View related articles
Citing articles: 16 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=tbbb20 Download by: [205.217.235.162]
Date: 05 November 2015, At: 16:20
Biosci. Biotech. Biochem., 56 (2), 211-214, 1992
Molecular Cloning of Feline Interferon cDNA by Direct Expression' Naoko NAKAMURA, Tetsuo SUDO, Susumu MATSUDA, and Akira Y ANAl Basic Research Laboratories, TORAY Industries, Inc., 1111 Tebiro, Kamakura 248, Japan Received June 26, 1991
Downloaded by [205.217.235.162] at 16:20 05 November 2015
A cDNA sequence coding for feline interferon has been cloned for the first time by screening a cDNA library constructed using Okayama-Berg vector and mRNA derived from the feline cells (LSA-I) induced by TPA (12-o-tetradecanoylphorbor 13-acetate) for the ability of transient expression to produce feline interferon in COSI monkey cells. The amino acid sequence, deduced from the nucleotide sequence by comparing it with the sequences of other mammalian IFNs, consists of 171 amino acids with 6 cysteins and an N-glycosylation site at the amino acid position 79, and has about 60% homology to human IFN alpha 1. The interferon was partially purified through Blue Sepharose, and its molecular weight was estimated to be 2.4 x 104 • The antiviral activity was acid stable, and glycosylation was suggested.
Interferon is a physiologically active substance the main ingredient of which is a protein showing antiviral activity and which is abbreviated IFN. By the progress of cellular and molecular biology, many mammalian IFNs have been reported, some of which have been mass-produced for anticancer and/or antiviral drugs. We tried to clone the cDNA of feline IFN (FeIFN) for the purpose of mass-production by genetic engineering aiming at an antiviral drug for cats that are suffering from viral diseases such as feline herpes virus, feline calici virus, feline leukaemia virus, feline infectious peritonitis virus, or feline immunodeficiency virus. When we started the work, no FeIFN had been mass-produced, and nor was there nucleotide or amino acid sequence information available for the synthesis of specific DNA probes, though a few preliminary reports had been published on FeIFN. 1 - 5) Therefore we took advantage of the characteristics of highly antiviral specific-activity of IFN molecule and used an expression vector of mammalian cells for the screening of cDNA by the direct expression of composite plasmids followed by assaying the transfected cell supernatants for antiviral activity. The obtained cDNA in th pFeIFNI will lead to a tool for mass production of FeIFN.
Materials and Methods Cell lines and cultivation. LSA-I is a cloned T-cell line derived from a thymic tumor of a feline leukaemia virus-infected cat by Yamamoto et al.,3) 1986, and has weak productivity of FeIFN. Fc9 (Felis catus 9) is a kind gift from Dr. J. K. Yamamoto. Both were maintained and grown in 50% MEM (Nissui Seiyaku Co., Japan) and 50% LIS medium (Gibco Lab) (MEM/LIS) containing 10% fetal bovine serum (FBS) (MEM/LI5/ FBS). COSI was maintained in RPMII640 (Gibco Lab) medium supplementded with 10% fetal bovine serum (RPMII640/FBS). Biological assay offeline interferon. The antiviral activity was measured by the cytopathic effect inhibition method 6 ) using Fc9 and Vesicular Stomatitis virus. Isolation of mRNA and construction of cDNA library. One of recloned cells named LSA-D6 was proliferated by spinner culture in 200 ml of MEM/LI5/FBS. At the cell concentration 5 x lOs/ml, TPA (12-0tetradecanoyl phorbol-13-acetate (Sigma Chemical Co.)) was added to 5 ng/ml for induction of mRNA production. After 20 hr of cultivation, cells were harvested by centrifugation. Total cellular RNA was extracted by using the guanidinium thiocyanate method,7) and the poly(A) + RNA was selected by oligo(dT)-cellulose chromatography. Before the con-
struction of the eDNA library, the RNA was confirmed for translational ability to produce FeIFN activity. Samples of the eluted RNA were injected into 20 individual Xenopus laevis oocytes (50 to 60ngjoocyte) and the supernatant from smashed oocytes were collected after 20 hr incubation,8) and assayed for antiviral activity. The eDNA library was constructed using an Okayama-Berg cDNA Expression Vector Strain Kit (Pharmacia LKB Biotechnology) consisting of the pcDVI oligo(dT)-tailed plasmid-primer, the pLI oligo(dG)-tailed linker and E. coli according to the attached procedure manual. 9 ) Screening of FeIFN-producing plasmids. The FeIFN-producing plasmids were screened by their transient-expression ability of IFN activity. COSI monkey cells were grown to confluency in RPMII640/FBS in 90-mm plates, and transfections were done with 20 J.lg of plasmid DNA in 4 ml of RPMII640 medium containing 50mM Tris-HCI (pH 7.4) and 400 J.lg of DEAE-Dextran per m1. 10 ) After 4 hr of incubation at 37°C, cells were washed and then incubated with RPMII640 medium containing ISO J.lM chloroquine for 3 hr. This was then replaced with 10 ml of RPMII 640/FBS, and the medium was collected 72 hr later, then assayed for FeIFN activity. DNA sequence analysis. A eDNA-containing fragment for nucleotide sequence analysis was prepared from a composite plasmid consisting of the sequencing plasmid pUCl8 and an inserted DNA fragment, BamHI-BamHI or two kinds of BamHI-Bg/II containing eDNA from pFeIFNl. Sequence analysis was done by a combination of the dideoxy method 11) and the primer extension method 12 ) by Shozo Kanai of the Toray Research Center Inc. Partial purification of FeIFN. Culure medium containing FeIFN activity was put on a 50-ml fast flow type Blue Sepharose column that had been equilibrated with 2.5 mM KCI-1.5 mM phosphate buffer (pH 7.0). After washing the column with 500 ml of 50 mM phosphate buffer (pH 7.0) containing 0.5 MNaC!, the adsorbed FeIFN was eluted with 50 ml of 50 mM phosphate buffer (pH 7.0) containing 1M NaCI followed by 500 ml of 50mM phosphate buffer (pH 7.0) containing 1 M NaCI and 20% ethylene glycol.
Results and Discussion Induction of FeIFN synthesis and evaluation of mRNA LSA-I cell was known to produce FeIFN activity and release it into supernatant medium. 3) The highest subclones, LSA-D4-C and LSA-D4-KI7, were obtained by repeated recloning, and showed 500 U /ml productivity at most. However, these highest productivities easily decreased through serial passages. The decrease was observed to be faster in RPMI1640/FBS than in the MEM/LI5/FBS which was used for cell propagation for mRNA preparation. To increase the quality of the cDNA library, a greater abundance of FeIFN mRNA is required in poly(A) + RNA
NII-Electronic Library Service
212
N. NAKAMURA
preparation. To make the production yield of the RNA of IFN higher, inductive ability of known IFN inducers were investigated on LSA-I subclonal cells by assaying IFN activity found in the supernatant of the cultures. As is shown in Table I, TPA was shown to have highly inductive activity. The optimal TP A concentration and optimal induction stage were examined, using LSA-D6 cells which are a subclone of LSA-D4-KI7, as shown in Table II and Table III. On the other hand, the FeIFN produced by transient expression in supernatant of COSI cell culture showed stable in the medium for 3 days at 37°C. The high amount of FeIFN in medium at 30 hr and later in Table III should be due to accumulation of FeIFN and the time of maximum mRNA content in the cells was expected before that. So the extraction of mRNA was done from cells harvested at 20 hr of cultivation in MEM/Ll5/FBS after TPA induction. The yield of poly(A)+ RNA was 301 p,g from about 5.8mg of total RNA from 7 x 108 cells. To confirm that the extracted
Downloaded by [205.217.235.162] at 16:20 05 November 2015
Table I.
et al.
poly(A) + RNA fraction had translational activity ofFeIFN, both poly(A)+ RNA fractions from LSA-D6 cells, induced or uninduced, was injected into X. laevis oocytes, and the supernatants from the oocytes were assayed for FeIFN activity. mRNA preparation from induced and uninduced cells showed FeIFN productivity of 13 and < 2 U Iml respectively. Screening of the cDNA library by transient expession
The cDNA library was constructed with 6 x 10 5 clones. Before screening of the cDNA library, the size of inserted cDNA was estimated by electrophoresis. Seven among 12 clones of composite plasmids were shown to have inserted BamHI DNA fragments longer than 0.5 kb. The cDNA Table IV. DNA Transfection Assay for FeIFN Activity from Pools of Plasmid DNA DNA
Production of FeIFN by LSA-I Induced by Inducers
1st screen
Supernatant activity (unitjml)
MEMjL15 None
PHAa TPA b ConAc a b C
LSA-D4-KI7
LSA-D4-C
Inducers
14> 12> 776 12>
RPMI1640 14 12 1,725 12
MEMjL15 261 320 7,890 182
38 46 6,215 34
PHA: 10mgjml of phytohemagglutinin. TPA: 5 ngjml. ConA: 5 jlgjml of Concanavalin A. Cells grown in 24-well plates to 4 x 105jml in MEMjL15 and RPMI1640, both of which were supplemented with 10%FBS, were harvested and resuspended to 5 x 105 jml in fresh media with 2%FBS and inducers. FeIFN activity was assayed after 14 hr of incubation.
Table II.
2nd screena
RPMI1640
Inductive Ability by TPA TPA (ngjml)
FeIFN activity (Ujml)
10 25 50
15,850 590
