Preliminary Study Of α4β7 Express Disciplinarian On Mastitis Dairy Cow

Mastitis is the most devastating disease in the development of the dairy industry. It can bring a serious economic loss to producers. The mammary gland is a complex organ. The mammary gland requires lymphocytes not only for immune protection from invading pathogens but also to generate antibodies that provide crucial passive immunity to the neonate. Trafficking of lymphocytes is not a random process and regional specificity is regulated by specific sets of cell-surface adhesion molecules on lymphocytes and complementary receptors expressed on the vascular endothelium of target tissues.The mastitis research from domestic and foreign scholars is mainly focused on pathogens. This study chooses lymphocyte homing receptorα4β7 as the breakthrough point for research. Theα4β7 express disciplinarian of lymphocytes was preliminary detected on mastitis dairy cow. It has laid a experiment foundation for further study on mammary gland immunity.In this study, the fragment of bovine integrin a4 was synthetized, which was 753 bp. It was cloned into pET-28a(+) to construct the recombinant plasmid, which was then transformed into BL21. After being induced by IPTG, the recombination protein with relative molecular mass of 30 kDa was expressed successfully. The expression product was mainly in the form of inclusion body. After purification, the target protein was renaturated. The polyclonal antibody titer of rat anti-bovine a4 was 1:16 000 which was detected by indirect ELISA. Western Blot tests showed that the polyclonal antibody could identify a4 subunit of bovine lymphocyte surface. It indicates that the antibody has good Immunogenicity and immune responsiveness.Meanwhile, the fragment ofβ7 subunit was synthetized. pET28a-β7 was constructed by using DNA recombination technology. After being induced by IPTG, the recombination protein was expressed successfully in E.coli. Then, the polyclonal antibody was prepared by inoculating rabbits withβ7 purified product. The ELISA and Western Blot tests showed that the rabbit anti-bovineβ7 polyclonal antibody has good Immunogenicity and immune responsiveness. It could identifyβ7 subunit of bovine lymphocyte surface.Blood and milk samples were collected from healthy and clinical mastitis dairy cow. Self-made polyclonal antibody of rat anti-bovineα4 and rabbit anti-bovineβ7 were used in flow cytometry tests, and then used fluorescently-labeled anti-IgG as secondary antibody. Result showed that positive rate of lymphocytes that expressα4β7 in milk and blood from clinical mastitis dairy cow was significantly higher than healthy group (p<0.05). Through this experimental research, it has laid a foundation for further study.