MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY Volume 34, Number 4, 2015 ª Mary Ann Liebert, Inc. DOI: 10.1089/mab.2014.0099
Preparation and Preliminary Application of MAdCAM-1 Polyclonal Antibody in Dairy Cows with Subclinical Mastitis Chuang Xu,* Yuanyuan Chen,* Qiaocheng Chang, Cheng Xia, Wei Yang, and Hongyou Zhang
MAdCAM-1 plays an important role in mediating immune response and inflammation. This study aimed to express and purify a fusion protein of MAdCAM-1 in prokaryotic cells and to prepare rat anti-bovine MAdCAM-1 polyclonal antibodies. Prokaryotic expression vector pGEX-4T-1-MAdCAM-1 and pET-28aMAdCAM-1 were constructed, respectively. The above plasmids were transformed into BL21 Escherichia coli strain. These recombinant strains were induced by IPTG and identified by Western blot analysis and SDSPAGE. Wistar rats were immunized with recombinant protein (pET-28a-MAdCAM-1) emulsified with Freund’s adjuvant, and antibody titers were measured by indirect ELISA. Antibody titers reached the highest value (1:128,000) after the third immunization. Western blot showed that rat anti-bovine MAdCAM-1 polyclonal antibody can not only recognize recombinant MAdCAM-1 protein expressed in E. coli but also recognizes natural MAdCAM-1 protein extracted from bovine tissues. However, commercial anti-mouse MAdCAM-1 monoclonal antibodies did not recognize the recombinant MAdCAM-1 protein or natural protein, which indicated no cross-reactivity between bovine MAdCAM-1 and mouse MAdCAM-1. Real-time fluorescence quantitative polymerase chain reaction and Western blot analysis showed that MAdCAM-1 expression was limited in mammary lymphoid nodes of subclinical mastitis in dairy cows. We speculate that MAdCAM-1 expression is inconsistent in different periods of the dairy cows. The successful preparation of rat anti-bovine MAdCAM-1 polyclonal antibody and its preliminary application in dairy cows provide the foundation for further study of the mechanism of anti-inflammation of MAdCAM-1 in dairy cows with subclinical mastitis.
Introduction
D
airy cow mastitis could greatly impact animal husbandry and cause huge economic loss.(1,2) Subclinical mastitis especially is difficult to diagnose, and can cause up to 70% of total economic losses.(3) Antibiotic therapy is the first-line treatment for subclinical mastitis over a long period, but antibiotic residue and drug-resistant strains are becoming increasingly prominent. This makes treatment and prevention complicated,(4) but the antibiotic residue is also seriously harmful to human health. Therefore, it has become imperative to develop an effective gene drug or vaccine to prevent and cure subclinical mastitis.(5,6) MAdCAM-1, a specific mucous membrane tissue marker, can be recognized by circulating lymphocytes.(7) MAdCAM-1 is involved in mediating lymphocyte recirculation and homing, and is related to pathological and physiological processes.(8–10) MAdCAM-1 plays an important role in lymphocytes migrating to mucosal lymphoid tissues; its expression is markedly increased during chronic inflammation and other illnesses, such as
cholangitis, enteritis, and cirrhosis.(11,12) Thus, MAdCAM-1 plays an important role in certain diseases. Blocking MAdCAM1 is a viable therapeutic approach to enterocolitis.(13) Previous studies demonstrated that MAdCAM-1 is expressed in mouse mammary glands, especially in venous vascular endotheliocytes that surround the mammary gland lobules.(14) Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that MAdCAM-1 expression has stage specificity. MAdCAM-1 expression amounts are low in baby rat mammary glands, but MAdCAM-1 expression increases during pregnancy, peaks during lactation, and declines throughout the lactation period. Studies on MAdCAM-1 in rodent animals and humans have made meaningful progress. However, MAdCAM1 may have different roles in rodents and large animals, such as bovines. Therefore, research using large animals can help identify the mechanisms and roles of MAdCAM-1 in large animals, especially with regard to anti-inflammation mechanisms. MAdCAM-1 is an endothelial adhesion molecule belonging to the immunoglobulin superfamily and is constitutively expressed by endothelial venous cells in Peyer’s
College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, Daqing, P.R. China. *These authors contributed equally to this work.
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patches and mesenteric lymph nodes, and on flattened venous endothelial cells in the intestinal lamina propria.(15,16) MAdCAM-1 activity was first reported nearly 30 years ago. To date, seven MAdCAM-1 molecules have been identified in mammals, and the functional profiles of each family member have been characterized. Bovine MAdCAM-1 was first reported in 2006 by Wang and co-workers, who cloned its cDNA and confirmed that homology between bovine and mouse MAdCAM-1 is 36%. Because of the lack of a commercial antibody against bovine MAdCAM-1, studies on the mechanism of anti-inflammation of MAdCAM-1 in subclinical mastitis in dairy cows are limited. In this study, the cloning and expression of bovine MAdCAM-1 gene in E. coli, purification of recombinant proteins, and generation of polyclonal antibody against MAdCAM-1 are described. The prepared antibody can be useful for the study of expression and distribution of MAdCAM-1 in various tissues at the protein level and to elucidate its biofunctions and regulation mechanisms in inflammation of dairy cows with subclinical mastitis. Realtime fluorescence quantitative PCR was used to detect gene transcription levels of MAdCAM-1 in mammary lymphoid nodes from dairy cows with subclinical mastitis and healthy dairy cows. Results showed that MAdCAM-1 expression is low in these tissues. Western blot analysis to detect the expression of MAdCAM-1 protein in mammary lymphoid nodes from subclinical mastitis and healthy mammary glands used rat anti-bovine MAdCAM-1 polyclonal antibody as the primary antibody with alkaline phosphataselabeled anti-IgG antibody as the secondary antibody. Results showed that there was low MAdCAM-1 expression in dairy cows with subclinical mastitis. We speculate that little MAdCAM-1 expression is related to the dry period of the dairy cows. Materials and Methods Ethical statement
Animals were housed in accordance with the ethical principles and experimental procedures for animals and were approved by the Animal Care and Use Committee of the Faculty of Veterinary Medicine, Heilongjian Bayi Agricultural University Daqing. Experimental animals and tissues
The lymphoid nodes of mammary gland were obtained from clinically healthy dairy cows and dairy cows with subclinical mastitis. Peyer’s patches were from clinically healthy dairy cows. All tissues were maintained at -80C until use. Three Wistar rats (180–220 g) were purchased from the Experimental Animal Center of the Basic Medical College at Jilin University. RNA extraction and synthesis of first-strand cDNA
Total RNA was extracted from bovine Peyer’s patches using RNAiso reagent (Takara, Dalian, China). After extraction, total RNA was treated with RNase-free DNase I (Takara, Tokyo, Japan) to destroy potentially contaminating genomic DNA. The concentration of total RNA was measured using a protein and nucleic acid analyzer (Beckman Coulter, Miami, FL). The first-strand cDNA was synthesized
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using an ALV RT Kit (Takara), according to the manufacturer’s instructions. PCR amplification of MAdCAM-1
MAdCAM-1 full cDNA (GenBank accession DQ288286.1) was amplified using the Expand High Fidelity PCR system with a pair of gene specific primers (forward: 5¢-CTC GAG TTA TGA CAT CGT CGG GGA GC-3¢; reverse: 5¢-CTC GAG TTA TGA CAT CGT CGG GGA GC-3¢) containing the EcoRI and XhoI restriction sites (Invitrogen, Beijing, China), respectively. The reaction was carried out with the following parameters using a GeneAmp PCR System (Biometra, Go¨ttingen, Germany): initial denaturation at 94C for 5 min followed by 30 consecutive cycles of denaturation at 94C for 1 min, annealing for 30 s at 56C, extension at 72C for 1 min, and final extension at 72C for 10 min. Full-length cDNA cloning and sequencing
All the PCR products were purified using the Takara Agarose Gel DNA Purification Kit (Takara), ligated to pMD18-T vectors (Takara), transformed into E. coli and sequenced (Invitrogen Biotechnology). Construction of expression vectors
The amplified MAdCAM-1 gene was gel-purified by the Gel Extraction Kit (Axygen, Hangzhou, China). After digestion with EcoRI and XhoI, the purified product was inserted into the corresponding region of pGEX-4T-1 and pET-28a expression vector (Takara), respectively, then confirmed by restriction analysis and sequencing (Invitrogen Biotechnology). The correct recombinant prokaryotic expression vectors were named pGEX-4T-M and pET-28a-M, respectively. Expression and purification of bovine His/MAdCAM-1 and GST/MAdCAM-1 fusion protein
The pGEX-4T-M and pET-28a-M plasmid were transformed into E. coli BL21 (Takara). E. coli BL21strains harboring pGEX-4T-M and pET-28a-M plasmids were induced by treatment with 1 mM isopropyl-b-D-thiogalactopyranoside (IPTG) at 37C for 4 h. E. coli BL21 strains harboring the pGEX-4T-M and pET-28a-M plasmids were uninduced as negative control. E. coli BL21 pellets were dissolved in loading buffer and the recombinant proteins were identified on 12% sodium dodecylsulfate polyacrylamide gel electrophoresis. The expression of the recombinant pGEX-4T-M and pET-28a-M was detected by Western blot analysis. Protein concentrations were determined by using a bicinchoninic acid (BCA) protein assay kit (Bio-Rad, Hercules, CA). The recombinant fusion protein MAdCAM-1 containing His-tag was purified using His GraviTrap (GE Healthcare, Waukesha, WI) according to the manufacturer’s instructions. The recombinant fusion protein MAdCAM-1 containing GST-tag was purified by Gluthathione-Sepharose 4B (GE Healthcare) according to the manufacturer’s instructions. The purified His-MAdCAM-1 and GST-MAdCAM-1 concentration were measured by BCA method, respectively. The purified His-MAdCAM-1 fusion protein and GST-MAdCAM-1 fusion protein were identified by 12% SDS-PAGE followed by staining with Coomassie blue, respectively.
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Polyclonal antibody production
To produce polyclonal antibodies against bovine MAdCAM-1, three Wistar rats were immunized with 50 mg purified His-MAdCAM-1 fusion protein emulsified in complete Freund’s adjuvant (Sigma, St. Louis, MO) on day 1. Subsequent booster immunizations of 50 mg of the purified His-MAdCAM-1 in incomplete Freund’s adjuvant (Sigma) were administered to each rat on days 14, 28, and 42. The rats were bled 7 days after the final immunization, and the antiMAdCAM-1 serum was stored at -20C until use. Purification of immunoglobulin from rat serum
To obtain purified polyclonal immunoglobulin (IgG), the rat antiserum was purified with Protein G (GE Healthcare), according to the manufacturer’s instructions.
FIG. 2. Enzyme digestion profile of pGEX-4T-1MAdCAM-1. M, DL2000 marker; lane 1, identification of pGEX-4T-1-M with EcoRI and XhoI digestion.
SDS-PAGE and Western blot analysis
After 12% SDS-PAGE, the gel was immersed in transfer buffer, and the recombinant protein GST/MAdCAM-1 was transferred onto a nitrocellulose membrane (Millipore, Temecula, CA). The membrane was incubated for 1 h with blocking buffer (5% bovine serum albumin BSA] in TBS) at room temperature (RT). After being washed four times (5 min each) with TBS-Tween buffer, the membrane was incubated with anti-His-mAb (1:20,000; Sigma) for 2 h at RT. The membrane was then incubated for 1 h with horseradish peroxidase-conjugated goat anti-rat IgG (1:20,000; Sigma) at RT after thorough washing. The membrane was washed as described above and then analyzed using the enhanced chemiluminescence detection system (Amersham Pharmacia Biotech, Baie d’Urfe, Canada) and exposed to Kodak BioMax X-ray film (Eastman Kodak, Rochester, NY) for 2–5 min.
labeled, goat anti-rat IgG at 37C. The reaction was initiated by adding disodium 4-nitrophenyl phosphate (PNPP) substrate (Sigma), and the absorbance at 405 nm was determined using bichromatic microplate reader (Bio-Tek, Winooski, VT). Detection of recombinant protein MAdCAM-1 by Western blot
Membrane protein of Peyer’s patches was extracted according to the manufacturer’s instructions (BestBio, Shanghai, China). After SDS-PAGE, the gel was transferred onto a nitrocellulose membrane (Millipore). The membrane was incubated with the anti-bovine MAdCAM-1-IgG (1:500; Sigma) or anti-mouse MAdCAM-1-mAb (1:20,000; Sigma) for 2 h at RT. The other experimental procedures were performed as described above.
ELISA
Detection of natural protein MAdCAM-1 by Western blot
A microtiter plate was coated with 2 mg/mL of the purified His-MAdCAM-1 in carbonate-buffered saline and incubated for 16 h at 4C. The plate was then washed with phosphatebuffered saline (PBS), and excess binding sites were blocked with PBS containing 3% BSA (Sigma). After incubation, 50 mL aliquots of antiserum at different dilutions were added to the appropriate wells and incubated for 1 h at 37C. The plate was washed and incubated for 1 h with phosphatase-
Membrane protein of Peyer’s patches and membrane protein of mammary gland lymphoid node were extracted according to the manufacturer’s instructions (Bestbio). After SDS-PAGE, the gel was transferred onto a nitrocellulose membrane (Millipore). The membrane was incubated with the anti-bovine MAdCAM-1-IgG (1:20,000; Sigma) or for 2 h at RT. The other experimental procedures were performed as described above.
FIG. 1. PCR product of the MAdCAM-1 gene. M, DL2000 marker; lane 1, MAdCAM-1 gene.
FIG. 3. Enzyme digestion profile of pET-28a-M. M, DL2000 marker; lane 1, identification of pET-28a-M with EcoRI and XhoI digestion.
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FIG. 4. Expression of GST-M analyzed by 12% SDSPAGE. Lanes 2–5, whole lysate of cells harboring pGEX-4T1-MAdCAM-1 after IPTG induction; lane 1, whole lysate of cells harboring pGEX-4T-1-MAdCAM-1 without IPTG induction.
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FIG. 6. Western blot analysis of recombinant GSTMAdCAM-1 protein. M, pre-stained protein marker; lane 2, anti-GST antibody as the primary antibody. using Excel software. In all statistical tests, p value < 0.05 was considered significant.
Detection of MAdCAM-1 by fluorescence quantitative PCR
Total RNA from mammary gland lymphoid tissues by TRIzol reagent (Takara) were reverse transcribed using a Reverse transcription RNA PCR Kit (Takara) according to the manufacturer’s instructions. Quantitative RT-PCR was performed using GeneAmp-PCR system 7500 (ABI, Vernon, CA). MAdCAM-1 cDNA (GenBank accession DQ288286.1) was amplified using a pair of gene-specific primers (forward: 5¢-CAT GGC CAA CCT GTA TGT GG-3¢; reverse: 5¢-AGG AAC ACC AGG AGA AGG AGC-3¢), and b-actin cDNA (GenBank accession AY141970) was amplified using a pair of primers (forward: 5¢-GTG GGC CGC CCT AGG CAC CA-3¢; reverse: 5¢-GGG CCT CGG TCA GCA GCA C-3¢) on fluorescence quantitative PCR instrument (ABI 7500). Statistical analysis
Statistical analysis and student t-test results were analyzed by SPSS10.0 software (Chicago, IL), and images formed
Results Full-length cDNA for MAdCAM-1 cloning and construction of expression vector
The complete cDNA of MAdCAM-1 was amplified by RT-PCR from the Peyer’s patches of bovine. Gene-specific primers were designed according to the published sequence of bovine MAdCAM-1. One percent agarose gel electrophoresis demonstrated a clear band above 1100 bp consistent with the expected value (Fig. 1). The PCR product was ligated to pGEX-4T-1 and pET-28a. The recombinant plasmids pGEX-4T-M and pET-28a-M were identified by restriction analysis (Figs. 2 and 3) and then confirmed by sequencing. Results showed a clear band above 4900 bp and 1100 bp (Fig. 2). A band was also observed above 5300 bp and 1100 bp (Fig. 3). Production of polyclonal antibody against bovine MAdCAM-1 protein
Induction of recombinant plasmid pGEX-4T-M and pET28a-M using IPTG resulted in a high level of expression of a 45 kDa recombinant protein of GST-MAdCAM-1 (Fig. 4) and
FIG. 5. Expression of His-MAdCAM-1 analyzed by 12% SDS-PAGE. Lanes 2–5, whole lysate of cells harboring pET-28a-MAdCAM-1 after IPTG induction; lane 1, whole lysate of cells harboring pET-28a-MAdCAM-1 without IPTG induction.
FIG. 7. Western blot analysis of recombinant HisMAdCAM-1 protein. M, pre-stained protein marker; lane 2, anti-His antibody as the primary antibody.
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FIG. 8.
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Antibody titer dynamic changes of different dilutions in soluble supernatant after third immunization.
FIG. 9.
Changes in antibody levels.
a 45 kDa recombinant protein of His-MAdCAM-1(Fig. 5), respectively. The expressed product was purified by Ni2 + affinity chromatography using Ni-NTA beads. Peak A280 fractions eluted from the column were pooled and showed a predominant single His-MAdCAM-1 band (data not shown). Identification of recombinant protein
The recombinant protein was identified by Western blot analysis using an anti-GST monoclonal antibody as the primary antibody and an anti-His monoclonal antibody as the primary antibody (Figs. 6 and 7). Results showed a clear band above 66 kDa and 45 kDa, respectively. Detection of polyclonal antibody titers
Rats were immunized with recombinant protein M-His to produce polyclonal antibodies. The antibody titers reached their highest value (1:128,000) when M-GST was used as a test antigen and could maintain these levels for several weeks after the third immunization (Figs. 8 and 9).
FIG. 10. Western blot analysis of His-MAdCAM-1 polyclonal antibody to identify recombinant protein. M, prestained protein marker; lane 1, GST; lane 2, M-GST.
Identification of recombinant protein with rat anti-bovine polyclonal antibody by Western blot
The recombinant proteins M-GST were identified by Western blotting using the rat anti-MAdCAM-1 polyclonal antibody as the primary antibody and demonstrated a clear band above 66 kDa (Fig. 10). Thus, the prepared polyclonal antibody can recognize the recombinant protein MAdCAM1-GST specifically. Identification of natural protein with rat anti-bovine polyclonal antibody by Western blot
The membrane protein extracted from Peyer’s patches was identified by Western blot using rat anti-bovine MAdCAM-1 polyclonal antibody as the primary antibody, showing a clear band above 50 kDa (Fig. 11). Results showed that the prepared polyclonal antibody recognized the natural MAdCAM-1 protein specifically.
FIG. 11. Western blot analysis of His-MAdCAM-1 polyclonal antibody identifies primitive M protein. M, prestained protein marker; lane 1, membrane protein.
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scription levels in dairy cows with subclinical mastitis, but no MAdCAM-1 gene expression in healthy cows (Fig. 13). Discussion
FIG. 12. Detection of MAdCAM-1gene transcription differences in lymphoid tissues from sub-mastitis and healthy dairy cows by real-time PCR. Cross-reactivity
The commercial anti-mouse monoclonal antibody did not recognize recombinant bovine MAdCAM-1 or natural protein. Results showed that there was no cross-reactivity between bovine MAdCAM-1 and mouse MAdCAM-1 (data not shown). MAdCAM-1 gene transcription levels in lymphoid tissues from sub-mastitis and healthy bovines by real-time PCR
MAdCAM-1 gene transcription differences were investigated in lymphoid tissues from sub-mastitis and healthy dairy cows by real-time PCR. Peyer’s patches were used as a positive control and b-actin as an internal standard (Fig. 12). The results showed that low MAdCAM-1 gene transcription leveling in dairy cows with subclinical mastitis, but no MAdCAM-1 gene express in healthy cows. Detection of MAdCAM-1 protein expression in lymphoid tissues from sub-mastitis and healthy dairy cows by Western blot
To detect MAdCAM-1 protein expression, differences were investigated in lymphoid tissues from sub-mastitis and healthy dairy cows by Western blotting; Peyer’s patches were used as positive control and b-actin as an internal standard (Fig. 13). The results showed low MAdCAM-1 gene tran-
MAdCAM-1 is preferentially expressed on high endothelial venules of gut-associated lymphoid organs and on lamina propria venules, helping lymphocytes traffic to mucosal organs.(17) The interaction between MAdCAM-1 and integrin a4b7 is a key step in lymphocyte homing to the gut and plays vital roles in both gut mucosal immune homeostasis and intestinal inflammation.(18,19) The roles of antagonism against a4b7(20) b7(21) and MAdCAM-1(22) have been demonstrated in animal models. MAdCAM-1 expression is dramatically amplified during inflammation. To perform research on the mechanisms of MAdCAM-1 in dairy cows with subclinical mastitis, a highquality protein and highly efficient and specific anti-bovine MAdCAM-1 antibody is essential. However, no commercial anti-bovine MAdCAM-1 antibody exists; therefore, we investigated the use of a commercial mouse MAdCAM-1 antibody. However, it did not cross-react with bovine MAdCAM1 and therefore could not be used to detect bovine MAdCAM-1. We purified bovine MAdCAM-1 protein and prepared a rat anti-bovine MAdCAM-1 polyclonal antibody. In this study, His-tag recombinant MAdCAM-1 was used as the immunizing antigen, and a GST-tag recombinant MAdCAM-1 protein was used as the detection antigen because of the low immunogenicity of the His-tag in immunized mice. Immunodetection by recombinant proteins with different labels will avoid falsepositive antibodies against the recombinant protein tag. As an independent test to analyze the specificity of the antibody, we extracted natural MAdCAM-1 protein from bovine Peyer’s patches. Western blot analysis revealed that the anti-bovine MAdCAM-1 antibody specifically reacted with the natural MAdCAM-1 protein. Although MAdCAM-1 expression was low when assessed by fluorescent quantitative PCR and Western blotting, this might be caused by different levels of MAdCAM-1 expression during different periods in dairy cows. In this study, the full-length MAdCAM-1 gene was amplified by PCR, and pET-28a/MAdCAM-1 and pGEX-4T-1/MAdCAM-1
FIG. 13. Detection of MAdCAM-1 gene expression differences in lymphoid tissues from sub-mastitis and healthy dairy cows by Western blotting.
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plasmids were constructed. Bovine MAdCAM-1 was expressed at high levels in E. coli. Specific polyclonal antibodies against bovine MAdCAM-1 were successfully obtained, and the rat anti-bovine MAdCAM-1 polyclonal antibody identifies both the recombinant protein and the natural protein. Therefore, the MAdCAM-1 antibodies can be used to identify and analyze MAdCAM-1 expression in different periods of dairy cows. This experimental research has laid a foundation for further studies on the inflammation mechanisms of MAdCAM-1 during mastitis in dairy cows. Acknowledgments
This work was supported, in part, by the Fund for Imported Talents in Heilongjiang Bayi Agricultural University (XYB 2013-10), the National Nature Science Foundation of China (31372625), and the National Key Technology Support Program (2013BAD21B01). Author Disclosure Statement
The authors have no financial interests to disclose. References
1. Heald CW, Kim T, Sischo WM, Cooper JB, and Wolfgang DR: A computerized mastitis decision aid using farm-based records: an artificial neural network approach. J Dairy Sci 2000;83(4):711–720. 2. Seegers H, Fourichon C, and Beaudeau F: Production effects related to mastitis economics in dairy cattle herds. Vet Res 2003;34(5):475–491. 3. De Mol RM, and Ouweltjes W: Detection model for mastitis in cows milked in an automatic milking system. Prev Vet Med 2001;49(1-2):71–82. 4. Keefe G: Update on control of Staphylococcus aureus and Streptococcus agalactiae for management of mastitis. Vet Clin Food Anim 2012;28(2):203–216. 5. Talbot BG, and Lacasse P: Progress in development of mastitis vaccines. Livestock Prod Sci 2005;98(1-2):101–113. 6. Zecconi A, Piccinini R, and Fox LK: Epidemiologic study of intramammary infections with Staphylococcus aureus during a control program in nine commercial dairy herds. J Am Vet Med Assoc 2003;223(5):684–688. 7. Parikh A, Leach T, Wyant T, Scholz C, Sankoh S, Mould DR, Ponich T, Fox I, and Feagan BG: Vedolizumab for the treatment of active ulcerative colitis: a randomized controlled phase 2 dose-ranging study. Inflamm Bowel Dis 2012;18(8):1470–1479. 8. Butcher EC, Williams M, Youngman K, Rott L, and Briskin M: Lymphocyte trafficking and regional immunity. Adv Immunol 1999;72:209–253. 9. Finke D, Acha-Orbea H, Mattis A, Lipp M, and Kraehenbuhl J: CD4 + CD3- cells induce Peyer’s patch development: role of alpha4beta1 integrin activation by CXCR5. Immunity 2002;17(3):363–373. 10. Binion DG, West GA, Volk EE, Drazba JA, Ziats NP, Petras RE, and Fiocchi C: Acquired increase in leucocyte binding by intestinal microvascular endothelium in inflammatory bowel disease. Lancet 1998;352(9142):1742–1746. 11. Connor EM, Eppihimer MJ, Morise Z, Granger DN, and Grisham MB: Expression of mucosal addressin cell adhesion molecule-1(MAdCAM-1) in acute and chronic inflammation. J Leukoc Biol 1999;65(3):349–355.
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12. Hanninen A, Taylor C, Streeter PR, Stark LS, Sarte JM, Shizuru JA, Simell O, and Michie SA: Vascular addressins are induced onislet vessels during insulitis in nonobese diabetic mice and are involved in lymphoid cell binding to islet endothelium. J Clin Invest 1993;92(5):2509–2515. 13. Fong S, Jones S, Renz ME, Chiu HH, Ryan AM, Presta LG, and Jackson D: Mucosal addressin cell adhesion molecule-1 (MAdCAM-1). Its binding motif for alpha 4 beta 7 and role in experimental colitis. Immunol Res 1997;16(3):299–311. 14. Nishimura T, Koike R, and Miyasaka M: Mammary glands of Aly mice: developmental changes and lactation-related expression of specific proteins, a-casein, GLyCAM-1 and MAdCAM-1. Am J Reprod Immunol 2000;43(6):351–358. 15. Briskin MJ, McEvoy LM, and Butcher EC: MAdCAM-1 has homology to immunoglobulin and mucin like adhesion receptors and to IgA1. Nature 1993;363(6428):461–464. 16. Berlin C, Berg EL, Briskin MJ, Andrew DP, Kilshaw PJ, Holzmann B, Weissman IL, Hamann A, and Butcher EC: Alpha 4 beta 7 integrin mediates lymphocyte binding to the mucosal vascular addressin MAdCAM-1. Cell 1993;74(1): 185–195. 17. Clahsen T, Pabst O, Tenbrock K, Schippers A, and Wagner N: Localization of dendritic cells in the gut epithelium requires MAdCAM-1. Clin Immunol 2014;156(1):74–84. 18. Springer TA: Traffic signals on endothelium for lymphocyte recirculation and leukocyte emigration. Annu Rev Physiol 1995;57:827–872. 19. Pan WJ, Hsu H, Rees WA, Lear SP, Lee F, Foltz IN, Rathanaswami P, Manchulenko K, Chan BM, Zhang M, Xia XZ, Patel SK, Prince PJ, Doherty DR, Sheckler CM, Reynhardt KO, Krill CD, Harder BJ, Wisler JA, Brandvig JL, Lynch JL, Anderson AA, Wienkers LC, and Borie DC: Pharmacology of AMG 181, a human anti-a4 b7 antibody that specifically alters trafficking of gut-homing T cells. Br J Pharmacol 2013;169(1):51–68. 20. Fedyk ER, Wyant T, Yang LL, Csizmadia V, Burke K, Yang H, and Kadambi VJ: Exclusive antagonism of the a4 b7 integrin by vedolizumab confirms the gut-selectivity of this pathway in primates. Inflamm Bowel Dis 2012;18(11):2107–2119. 21. Stefanich EG, Danilenko DM, Wang H, O’Byrne S, Erickson R, Gelzleichter T, Hiraragi H, Chiu H, Ivelja S, Jeet S, Gadkari S, Hwang O, Fuh F, Looney C, Howell K, Albert V, Balazs M, Refino C, Fong S, Iyer S, and Williams M: A humanized monoclonal antibody targeting the beta7 integrin selectively blocks intestinal homing of T lymphocytes. Br J Pharmacol 2011;162(8):1855–1870. 22. Pullen N, Molloy E, Carter D, Syntin P, Clemo F, FincoKent D, Reagan W, Zhao S, Kawabata T, and Sreckovic S: Pharmacological characterization of PF-00547659, an antihuman MAdCAM monoclonal antibody. Br J Pharmacol 2009;157(2):281–293.
Address correspondence to: Yuanyuan Chen Department of Veterinary Clinical Medicine College of Animal Science and Veterinary Medicine Heilongjiang Bayi Agricultural University Daqing High-tech Industrial Development Zone Daqing 163319 P.R. China E-mail: [email protected] Received: December 9, 2014 Accepted: April 13, 2015
Preparation and Preliminary Application of MAdCAM-1 Polyclonal Antibody in Dairy Cows with Subclinical Mastitis Chuang Xu,* Yuanyuan Chen,* Qiaocheng Chang, Cheng Xia, Wei Yang, and Hongyou Zhang
MAdCAM-1 plays an important role in mediating immune response and inflammation. This study aimed to express and purify a fusion protein of MAdCAM-1 in prokaryotic cells and to prepare rat anti-bovine MAdCAM-1 polyclonal antibodies. Prokaryotic expression vector pGEX-4T-1-MAdCAM-1 and pET-28aMAdCAM-1 were constructed, respectively. The above plasmids were transformed into BL21 Escherichia coli strain. These recombinant strains were induced by IPTG and identified by Western blot analysis and SDSPAGE. Wistar rats were immunized with recombinant protein (pET-28a-MAdCAM-1) emulsified with Freund’s adjuvant, and antibody titers were measured by indirect ELISA. Antibody titers reached the highest value (1:128,000) after the third immunization. Western blot showed that rat anti-bovine MAdCAM-1 polyclonal antibody can not only recognize recombinant MAdCAM-1 protein expressed in E. coli but also recognizes natural MAdCAM-1 protein extracted from bovine tissues. However, commercial anti-mouse MAdCAM-1 monoclonal antibodies did not recognize the recombinant MAdCAM-1 protein or natural protein, which indicated no cross-reactivity between bovine MAdCAM-1 and mouse MAdCAM-1. Real-time fluorescence quantitative polymerase chain reaction and Western blot analysis showed that MAdCAM-1 expression was limited in mammary lymphoid nodes of subclinical mastitis in dairy cows. We speculate that MAdCAM-1 expression is inconsistent in different periods of the dairy cows. The successful preparation of rat anti-bovine MAdCAM-1 polyclonal antibody and its preliminary application in dairy cows provide the foundation for further study of the mechanism of anti-inflammation of MAdCAM-1 in dairy cows with subclinical mastitis.
Introduction
D
airy cow mastitis could greatly impact animal husbandry and cause huge economic loss.(1,2) Subclinical mastitis especially is difficult to diagnose, and can cause up to 70% of total economic losses.(3) Antibiotic therapy is the first-line treatment for subclinical mastitis over a long period, but antibiotic residue and drug-resistant strains are becoming increasingly prominent. This makes treatment and prevention complicated,(4) but the antibiotic residue is also seriously harmful to human health. Therefore, it has become imperative to develop an effective gene drug or vaccine to prevent and cure subclinical mastitis.(5,6) MAdCAM-1, a specific mucous membrane tissue marker, can be recognized by circulating lymphocytes.(7) MAdCAM-1 is involved in mediating lymphocyte recirculation and homing, and is related to pathological and physiological processes.(8–10) MAdCAM-1 plays an important role in lymphocytes migrating to mucosal lymphoid tissues; its expression is markedly increased during chronic inflammation and other illnesses, such as
cholangitis, enteritis, and cirrhosis.(11,12) Thus, MAdCAM-1 plays an important role in certain diseases. Blocking MAdCAM1 is a viable therapeutic approach to enterocolitis.(13) Previous studies demonstrated that MAdCAM-1 is expressed in mouse mammary glands, especially in venous vascular endotheliocytes that surround the mammary gland lobules.(14) Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that MAdCAM-1 expression has stage specificity. MAdCAM-1 expression amounts are low in baby rat mammary glands, but MAdCAM-1 expression increases during pregnancy, peaks during lactation, and declines throughout the lactation period. Studies on MAdCAM-1 in rodent animals and humans have made meaningful progress. However, MAdCAM1 may have different roles in rodents and large animals, such as bovines. Therefore, research using large animals can help identify the mechanisms and roles of MAdCAM-1 in large animals, especially with regard to anti-inflammation mechanisms. MAdCAM-1 is an endothelial adhesion molecule belonging to the immunoglobulin superfamily and is constitutively expressed by endothelial venous cells in Peyer’s
College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, Daqing, P.R. China. *These authors contributed equally to this work.
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patches and mesenteric lymph nodes, and on flattened venous endothelial cells in the intestinal lamina propria.(15,16) MAdCAM-1 activity was first reported nearly 30 years ago. To date, seven MAdCAM-1 molecules have been identified in mammals, and the functional profiles of each family member have been characterized. Bovine MAdCAM-1 was first reported in 2006 by Wang and co-workers, who cloned its cDNA and confirmed that homology between bovine and mouse MAdCAM-1 is 36%. Because of the lack of a commercial antibody against bovine MAdCAM-1, studies on the mechanism of anti-inflammation of MAdCAM-1 in subclinical mastitis in dairy cows are limited. In this study, the cloning and expression of bovine MAdCAM-1 gene in E. coli, purification of recombinant proteins, and generation of polyclonal antibody against MAdCAM-1 are described. The prepared antibody can be useful for the study of expression and distribution of MAdCAM-1 in various tissues at the protein level and to elucidate its biofunctions and regulation mechanisms in inflammation of dairy cows with subclinical mastitis. Realtime fluorescence quantitative PCR was used to detect gene transcription levels of MAdCAM-1 in mammary lymphoid nodes from dairy cows with subclinical mastitis and healthy dairy cows. Results showed that MAdCAM-1 expression is low in these tissues. Western blot analysis to detect the expression of MAdCAM-1 protein in mammary lymphoid nodes from subclinical mastitis and healthy mammary glands used rat anti-bovine MAdCAM-1 polyclonal antibody as the primary antibody with alkaline phosphataselabeled anti-IgG antibody as the secondary antibody. Results showed that there was low MAdCAM-1 expression in dairy cows with subclinical mastitis. We speculate that little MAdCAM-1 expression is related to the dry period of the dairy cows. Materials and Methods Ethical statement
Animals were housed in accordance with the ethical principles and experimental procedures for animals and were approved by the Animal Care and Use Committee of the Faculty of Veterinary Medicine, Heilongjian Bayi Agricultural University Daqing. Experimental animals and tissues
The lymphoid nodes of mammary gland were obtained from clinically healthy dairy cows and dairy cows with subclinical mastitis. Peyer’s patches were from clinically healthy dairy cows. All tissues were maintained at -80C until use. Three Wistar rats (180–220 g) were purchased from the Experimental Animal Center of the Basic Medical College at Jilin University. RNA extraction and synthesis of first-strand cDNA
Total RNA was extracted from bovine Peyer’s patches using RNAiso reagent (Takara, Dalian, China). After extraction, total RNA was treated with RNase-free DNase I (Takara, Tokyo, Japan) to destroy potentially contaminating genomic DNA. The concentration of total RNA was measured using a protein and nucleic acid analyzer (Beckman Coulter, Miami, FL). The first-strand cDNA was synthesized
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using an ALV RT Kit (Takara), according to the manufacturer’s instructions. PCR amplification of MAdCAM-1
MAdCAM-1 full cDNA (GenBank accession DQ288286.1) was amplified using the Expand High Fidelity PCR system with a pair of gene specific primers (forward: 5¢-CTC GAG TTA TGA CAT CGT CGG GGA GC-3¢; reverse: 5¢-CTC GAG TTA TGA CAT CGT CGG GGA GC-3¢) containing the EcoRI and XhoI restriction sites (Invitrogen, Beijing, China), respectively. The reaction was carried out with the following parameters using a GeneAmp PCR System (Biometra, Go¨ttingen, Germany): initial denaturation at 94C for 5 min followed by 30 consecutive cycles of denaturation at 94C for 1 min, annealing for 30 s at 56C, extension at 72C for 1 min, and final extension at 72C for 10 min. Full-length cDNA cloning and sequencing
All the PCR products were purified using the Takara Agarose Gel DNA Purification Kit (Takara), ligated to pMD18-T vectors (Takara), transformed into E. coli and sequenced (Invitrogen Biotechnology). Construction of expression vectors
The amplified MAdCAM-1 gene was gel-purified by the Gel Extraction Kit (Axygen, Hangzhou, China). After digestion with EcoRI and XhoI, the purified product was inserted into the corresponding region of pGEX-4T-1 and pET-28a expression vector (Takara), respectively, then confirmed by restriction analysis and sequencing (Invitrogen Biotechnology). The correct recombinant prokaryotic expression vectors were named pGEX-4T-M and pET-28a-M, respectively. Expression and purification of bovine His/MAdCAM-1 and GST/MAdCAM-1 fusion protein
The pGEX-4T-M and pET-28a-M plasmid were transformed into E. coli BL21 (Takara). E. coli BL21strains harboring pGEX-4T-M and pET-28a-M plasmids were induced by treatment with 1 mM isopropyl-b-D-thiogalactopyranoside (IPTG) at 37C for 4 h. E. coli BL21 strains harboring the pGEX-4T-M and pET-28a-M plasmids were uninduced as negative control. E. coli BL21 pellets were dissolved in loading buffer and the recombinant proteins were identified on 12% sodium dodecylsulfate polyacrylamide gel electrophoresis. The expression of the recombinant pGEX-4T-M and pET-28a-M was detected by Western blot analysis. Protein concentrations were determined by using a bicinchoninic acid (BCA) protein assay kit (Bio-Rad, Hercules, CA). The recombinant fusion protein MAdCAM-1 containing His-tag was purified using His GraviTrap (GE Healthcare, Waukesha, WI) according to the manufacturer’s instructions. The recombinant fusion protein MAdCAM-1 containing GST-tag was purified by Gluthathione-Sepharose 4B (GE Healthcare) according to the manufacturer’s instructions. The purified His-MAdCAM-1 and GST-MAdCAM-1 concentration were measured by BCA method, respectively. The purified His-MAdCAM-1 fusion protein and GST-MAdCAM-1 fusion protein were identified by 12% SDS-PAGE followed by staining with Coomassie blue, respectively.
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Polyclonal antibody production
To produce polyclonal antibodies against bovine MAdCAM-1, three Wistar rats were immunized with 50 mg purified His-MAdCAM-1 fusion protein emulsified in complete Freund’s adjuvant (Sigma, St. Louis, MO) on day 1. Subsequent booster immunizations of 50 mg of the purified His-MAdCAM-1 in incomplete Freund’s adjuvant (Sigma) were administered to each rat on days 14, 28, and 42. The rats were bled 7 days after the final immunization, and the antiMAdCAM-1 serum was stored at -20C until use. Purification of immunoglobulin from rat serum
To obtain purified polyclonal immunoglobulin (IgG), the rat antiserum was purified with Protein G (GE Healthcare), according to the manufacturer’s instructions.
FIG. 2. Enzyme digestion profile of pGEX-4T-1MAdCAM-1. M, DL2000 marker; lane 1, identification of pGEX-4T-1-M with EcoRI and XhoI digestion.
SDS-PAGE and Western blot analysis
After 12% SDS-PAGE, the gel was immersed in transfer buffer, and the recombinant protein GST/MAdCAM-1 was transferred onto a nitrocellulose membrane (Millipore, Temecula, CA). The membrane was incubated for 1 h with blocking buffer (5% bovine serum albumin BSA] in TBS) at room temperature (RT). After being washed four times (5 min each) with TBS-Tween buffer, the membrane was incubated with anti-His-mAb (1:20,000; Sigma) for 2 h at RT. The membrane was then incubated for 1 h with horseradish peroxidase-conjugated goat anti-rat IgG (1:20,000; Sigma) at RT after thorough washing. The membrane was washed as described above and then analyzed using the enhanced chemiluminescence detection system (Amersham Pharmacia Biotech, Baie d’Urfe, Canada) and exposed to Kodak BioMax X-ray film (Eastman Kodak, Rochester, NY) for 2–5 min.
labeled, goat anti-rat IgG at 37C. The reaction was initiated by adding disodium 4-nitrophenyl phosphate (PNPP) substrate (Sigma), and the absorbance at 405 nm was determined using bichromatic microplate reader (Bio-Tek, Winooski, VT). Detection of recombinant protein MAdCAM-1 by Western blot
Membrane protein of Peyer’s patches was extracted according to the manufacturer’s instructions (BestBio, Shanghai, China). After SDS-PAGE, the gel was transferred onto a nitrocellulose membrane (Millipore). The membrane was incubated with the anti-bovine MAdCAM-1-IgG (1:500; Sigma) or anti-mouse MAdCAM-1-mAb (1:20,000; Sigma) for 2 h at RT. The other experimental procedures were performed as described above.
ELISA
Detection of natural protein MAdCAM-1 by Western blot
A microtiter plate was coated with 2 mg/mL of the purified His-MAdCAM-1 in carbonate-buffered saline and incubated for 16 h at 4C. The plate was then washed with phosphatebuffered saline (PBS), and excess binding sites were blocked with PBS containing 3% BSA (Sigma). After incubation, 50 mL aliquots of antiserum at different dilutions were added to the appropriate wells and incubated for 1 h at 37C. The plate was washed and incubated for 1 h with phosphatase-
Membrane protein of Peyer’s patches and membrane protein of mammary gland lymphoid node were extracted according to the manufacturer’s instructions (Bestbio). After SDS-PAGE, the gel was transferred onto a nitrocellulose membrane (Millipore). The membrane was incubated with the anti-bovine MAdCAM-1-IgG (1:20,000; Sigma) or for 2 h at RT. The other experimental procedures were performed as described above.
FIG. 1. PCR product of the MAdCAM-1 gene. M, DL2000 marker; lane 1, MAdCAM-1 gene.
FIG. 3. Enzyme digestion profile of pET-28a-M. M, DL2000 marker; lane 1, identification of pET-28a-M with EcoRI and XhoI digestion.
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FIG. 4. Expression of GST-M analyzed by 12% SDSPAGE. Lanes 2–5, whole lysate of cells harboring pGEX-4T1-MAdCAM-1 after IPTG induction; lane 1, whole lysate of cells harboring pGEX-4T-1-MAdCAM-1 without IPTG induction.
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FIG. 6. Western blot analysis of recombinant GSTMAdCAM-1 protein. M, pre-stained protein marker; lane 2, anti-GST antibody as the primary antibody. using Excel software. In all statistical tests, p value < 0.05 was considered significant.
Detection of MAdCAM-1 by fluorescence quantitative PCR
Total RNA from mammary gland lymphoid tissues by TRIzol reagent (Takara) were reverse transcribed using a Reverse transcription RNA PCR Kit (Takara) according to the manufacturer’s instructions. Quantitative RT-PCR was performed using GeneAmp-PCR system 7500 (ABI, Vernon, CA). MAdCAM-1 cDNA (GenBank accession DQ288286.1) was amplified using a pair of gene-specific primers (forward: 5¢-CAT GGC CAA CCT GTA TGT GG-3¢; reverse: 5¢-AGG AAC ACC AGG AGA AGG AGC-3¢), and b-actin cDNA (GenBank accession AY141970) was amplified using a pair of primers (forward: 5¢-GTG GGC CGC CCT AGG CAC CA-3¢; reverse: 5¢-GGG CCT CGG TCA GCA GCA C-3¢) on fluorescence quantitative PCR instrument (ABI 7500). Statistical analysis
Statistical analysis and student t-test results were analyzed by SPSS10.0 software (Chicago, IL), and images formed
Results Full-length cDNA for MAdCAM-1 cloning and construction of expression vector
The complete cDNA of MAdCAM-1 was amplified by RT-PCR from the Peyer’s patches of bovine. Gene-specific primers were designed according to the published sequence of bovine MAdCAM-1. One percent agarose gel electrophoresis demonstrated a clear band above 1100 bp consistent with the expected value (Fig. 1). The PCR product was ligated to pGEX-4T-1 and pET-28a. The recombinant plasmids pGEX-4T-M and pET-28a-M were identified by restriction analysis (Figs. 2 and 3) and then confirmed by sequencing. Results showed a clear band above 4900 bp and 1100 bp (Fig. 2). A band was also observed above 5300 bp and 1100 bp (Fig. 3). Production of polyclonal antibody against bovine MAdCAM-1 protein
Induction of recombinant plasmid pGEX-4T-M and pET28a-M using IPTG resulted in a high level of expression of a 45 kDa recombinant protein of GST-MAdCAM-1 (Fig. 4) and
FIG. 5. Expression of His-MAdCAM-1 analyzed by 12% SDS-PAGE. Lanes 2–5, whole lysate of cells harboring pET-28a-MAdCAM-1 after IPTG induction; lane 1, whole lysate of cells harboring pET-28a-MAdCAM-1 without IPTG induction.
FIG. 7. Western blot analysis of recombinant HisMAdCAM-1 protein. M, pre-stained protein marker; lane 2, anti-His antibody as the primary antibody.
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FIG. 8.
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Antibody titer dynamic changes of different dilutions in soluble supernatant after third immunization.
FIG. 9.
Changes in antibody levels.
a 45 kDa recombinant protein of His-MAdCAM-1(Fig. 5), respectively. The expressed product was purified by Ni2 + affinity chromatography using Ni-NTA beads. Peak A280 fractions eluted from the column were pooled and showed a predominant single His-MAdCAM-1 band (data not shown). Identification of recombinant protein
The recombinant protein was identified by Western blot analysis using an anti-GST monoclonal antibody as the primary antibody and an anti-His monoclonal antibody as the primary antibody (Figs. 6 and 7). Results showed a clear band above 66 kDa and 45 kDa, respectively. Detection of polyclonal antibody titers
Rats were immunized with recombinant protein M-His to produce polyclonal antibodies. The antibody titers reached their highest value (1:128,000) when M-GST was used as a test antigen and could maintain these levels for several weeks after the third immunization (Figs. 8 and 9).
FIG. 10. Western blot analysis of His-MAdCAM-1 polyclonal antibody to identify recombinant protein. M, prestained protein marker; lane 1, GST; lane 2, M-GST.
Identification of recombinant protein with rat anti-bovine polyclonal antibody by Western blot
The recombinant proteins M-GST were identified by Western blotting using the rat anti-MAdCAM-1 polyclonal antibody as the primary antibody and demonstrated a clear band above 66 kDa (Fig. 10). Thus, the prepared polyclonal antibody can recognize the recombinant protein MAdCAM1-GST specifically. Identification of natural protein with rat anti-bovine polyclonal antibody by Western blot
The membrane protein extracted from Peyer’s patches was identified by Western blot using rat anti-bovine MAdCAM-1 polyclonal antibody as the primary antibody, showing a clear band above 50 kDa (Fig. 11). Results showed that the prepared polyclonal antibody recognized the natural MAdCAM-1 protein specifically.
FIG. 11. Western blot analysis of His-MAdCAM-1 polyclonal antibody identifies primitive M protein. M, prestained protein marker; lane 1, membrane protein.
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scription levels in dairy cows with subclinical mastitis, but no MAdCAM-1 gene expression in healthy cows (Fig. 13). Discussion
FIG. 12. Detection of MAdCAM-1gene transcription differences in lymphoid tissues from sub-mastitis and healthy dairy cows by real-time PCR. Cross-reactivity
The commercial anti-mouse monoclonal antibody did not recognize recombinant bovine MAdCAM-1 or natural protein. Results showed that there was no cross-reactivity between bovine MAdCAM-1 and mouse MAdCAM-1 (data not shown). MAdCAM-1 gene transcription levels in lymphoid tissues from sub-mastitis and healthy bovines by real-time PCR
MAdCAM-1 gene transcription differences were investigated in lymphoid tissues from sub-mastitis and healthy dairy cows by real-time PCR. Peyer’s patches were used as a positive control and b-actin as an internal standard (Fig. 12). The results showed that low MAdCAM-1 gene transcription leveling in dairy cows with subclinical mastitis, but no MAdCAM-1 gene express in healthy cows. Detection of MAdCAM-1 protein expression in lymphoid tissues from sub-mastitis and healthy dairy cows by Western blot
To detect MAdCAM-1 protein expression, differences were investigated in lymphoid tissues from sub-mastitis and healthy dairy cows by Western blotting; Peyer’s patches were used as positive control and b-actin as an internal standard (Fig. 13). The results showed low MAdCAM-1 gene tran-
MAdCAM-1 is preferentially expressed on high endothelial venules of gut-associated lymphoid organs and on lamina propria venules, helping lymphocytes traffic to mucosal organs.(17) The interaction between MAdCAM-1 and integrin a4b7 is a key step in lymphocyte homing to the gut and plays vital roles in both gut mucosal immune homeostasis and intestinal inflammation.(18,19) The roles of antagonism against a4b7(20) b7(21) and MAdCAM-1(22) have been demonstrated in animal models. MAdCAM-1 expression is dramatically amplified during inflammation. To perform research on the mechanisms of MAdCAM-1 in dairy cows with subclinical mastitis, a highquality protein and highly efficient and specific anti-bovine MAdCAM-1 antibody is essential. However, no commercial anti-bovine MAdCAM-1 antibody exists; therefore, we investigated the use of a commercial mouse MAdCAM-1 antibody. However, it did not cross-react with bovine MAdCAM1 and therefore could not be used to detect bovine MAdCAM-1. We purified bovine MAdCAM-1 protein and prepared a rat anti-bovine MAdCAM-1 polyclonal antibody. In this study, His-tag recombinant MAdCAM-1 was used as the immunizing antigen, and a GST-tag recombinant MAdCAM-1 protein was used as the detection antigen because of the low immunogenicity of the His-tag in immunized mice. Immunodetection by recombinant proteins with different labels will avoid falsepositive antibodies against the recombinant protein tag. As an independent test to analyze the specificity of the antibody, we extracted natural MAdCAM-1 protein from bovine Peyer’s patches. Western blot analysis revealed that the anti-bovine MAdCAM-1 antibody specifically reacted with the natural MAdCAM-1 protein. Although MAdCAM-1 expression was low when assessed by fluorescent quantitative PCR and Western blotting, this might be caused by different levels of MAdCAM-1 expression during different periods in dairy cows. In this study, the full-length MAdCAM-1 gene was amplified by PCR, and pET-28a/MAdCAM-1 and pGEX-4T-1/MAdCAM-1
FIG. 13. Detection of MAdCAM-1 gene expression differences in lymphoid tissues from sub-mastitis and healthy dairy cows by Western blotting.
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plasmids were constructed. Bovine MAdCAM-1 was expressed at high levels in E. coli. Specific polyclonal antibodies against bovine MAdCAM-1 were successfully obtained, and the rat anti-bovine MAdCAM-1 polyclonal antibody identifies both the recombinant protein and the natural protein. Therefore, the MAdCAM-1 antibodies can be used to identify and analyze MAdCAM-1 expression in different periods of dairy cows. This experimental research has laid a foundation for further studies on the inflammation mechanisms of MAdCAM-1 during mastitis in dairy cows. Acknowledgments
This work was supported, in part, by the Fund for Imported Talents in Heilongjiang Bayi Agricultural University (XYB 2013-10), the National Nature Science Foundation of China (31372625), and the National Key Technology Support Program (2013BAD21B01). Author Disclosure Statement
The authors have no financial interests to disclose. References
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Address correspondence to: Yuanyuan Chen Department of Veterinary Clinical Medicine College of Animal Science and Veterinary Medicine Heilongjiang Bayi Agricultural University Daqing High-tech Industrial Development Zone Daqing 163319 P.R. China E-mail: [email protected] Received: December 9, 2014 Accepted: April 13, 2015
