title: Preparation and Preliminary Application of MAdCAM-1 Polyclonal Antibody in Dairy Cows with Subclinical Mastitis.
abstract: 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-28a-MAdCAM-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 SDS-PAGE. 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.
authors: College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University , Daqing, P.R. China .
authors: Chuang
authors: C
authors: Xu
authors: College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University , Daqing, P.R. China .
authors: Yuanyuan
authors: Y
authors: Chen
authors: College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University , Daqing, P.R. China .
authors: Qiaocheng
authors: Q
authors: Chang
authors: College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University , Daqing, P.R. China .
authors: Cheng
authors: C
authors: Xia
authors: College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University , Daqing, P.R. China .
authors: Wei
authors: W
authors: Yang
authors: College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University , Daqing, P.R. China .
authors: Hongyou
authors: H
authors: Zhang
mesh_terms: D000818:Animals; D000906:Antibodies / Q000235:genetics / Q000276:immunology*; D000911:Antibodies, Monoclonal / Q000276:immunology; D058345:Asymptomatic Infections; D002417:Cattle; D003001:Cloning, Molecular / Q000379:methods; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D005822:Genetic Vectors / Q000235:genetics; D007114:Immunization / Q000379:methods; D008414:Mastitis, Bovine / Q000276:immunology*; D051379:Mice; D009088:Mucoproteins / Q000276:immunology*; D010957:Plasmids / Q000235:genetics; D051381:Rats; D017208:Rats, Wistar; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology
chemical_list: D000906:Antibodies; D000911:Antibodies, Monoclonal; D009088:Mucoproteins; D011993:Recombinant Fusion Proteins