Xu Chuang, Chen Yuanyuan, Chang Qiaocheng, Xia Cheng, Yang Wei, Zhang Hongyou
College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University , Daqing, P.R. China .
Monoclon Antib Immunodiagn Immunother. 2015 Aug;34(4):263-9. doi: 10.1089/mab.2014.0099.
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.
