A Study On Optimization And Anti-tuberculosis Of TAT-Ag85B Vaccine

ObjectivesIn order to obtain a large number of proteins,we optimized the conditions of pET28a-TAT-Ag85B expressed in E.coli BL21(DE3).And through the immune function of experimental observation expression protein in vivo and in vitro, provide the basis for the prevention and control of diseases such as tuberculosis (TB).Methods1. Transformed the plasmid pET28a-TAT-Ag85B into E.coli BL21(DE3) to express TAT-Ag85B by the induction of (Isopropyl glucosinolates galactose glucoside) IPTG.Useing polyacrylamide gel electrophoresis (SDS-PAGE) analysis the expressed product.2.Respectively optimized the conditions of protein expression from induced temperature and concentration of inducer, induction time.3.Using Ni-IDA His-band resin separated and purified the expressed product.the expression of fusion protein TAT-Ag85B were detected by SDS-PAGE and Western blotting.3.BALB/c mice were immunized by three times of subcutaneous injection with TAT-Ag85B.Two weeks after the end of the immunization,the anti-Ag85B antibody titres in sera were tested by ELISA.Meanwhile,taking out of mice spleen cells and macrophages,ane then co-culture,tested the concentrations of cytokines in culture supernatant by the method of ELIS A.Results1. Double digestion with restriction endonucleases proved that the plasmid pET28a-TAT-Ag85B into successed. Confirmed that the induction protein is right by sds-page.2.Through contrast test,using1tendency/L IPTG in28℃under the condition of the induction8h is one of the best fusion protein expression conditions, and obtain higher expression.3.The protein obtained is TAT-Ag85B,which is our needed.4.ELISA results showed that IgG titer was significantly increased in TAT-Ag85B group. The levels of IL-2in culture supernatant detected by ELISA,and found that TAT-Ag85B proteins stimulated cells which increased the ability of antigen presenting. ConclusionsThe experiments making a series of optimization for the expression of the fusion protein TAT-Ag85B,getting a large number of protein. Its immune effect laid a foundation for further study.Through animal experiments found that TAT-Ag85B has the ability of antigen presenting by improving the secretion of IL-2. As a result, the protein has certain curative effect for autoimmune diseases such as tuberculosis (TB),provides basis for further study in tuberculosis and other autoimmune disease drug.