Construction Of Staphylococcus Aureus Enterotoxin B Mutants(N23S, H32N And K172E) And Study Its Effect On The Broiler As Adjuvant

Staphylococcus aureus enterotoxin B(SEB) is a kind of exotoxin that secreted by Staphylococcus aureus,it is also a kind of superantigen. SEB can activate immune cells nonspecifically,especially T cells. It can cause the body to produce numerous cytokines and improve the immunogenicity of specific antigen. Therefore SEB has the potential to become immune adjuvant. However, since SEB have greater side effects to humans and mammals, its adjuvant activity of application is limited in the field of animal husbandry and veterinary.In order to reduce or eliminate the toxic effects of SEB, we used PCR site-directed mutagenesis techniques to build SEB mutants(N23S, H32 N and K172E). Then,we screened a mutant which has better superantigen activity. Both SEB and its mutant are also studied their safety and adjuvant activity in vivo in broiler. Our purpose is to lay the foundation for the promotion of SEB as an immune adjuvant used in the veterinary field. The test consists of three parts:1.The mutant protein obtainedWe choosed three key antigenic sites including SEB N23 S, H32 N and K172 E and used PCR site-directed mutagenesis techniques, constructed prokaryotic expression vector p ET-28a-SEB,p ET-28a-SEBM23(N23S),p ET-28a-SEBM32(H32N),p ET-28a-SEB M172(K172E),p ET-28a-SEBM23/172(N23SK172E),p ET-28a-SEBM32/172(H32NK172E), p ET-28a-SEBM23/32/172(N23SH32NK172E). Induced expression in E.coli and purified to obtain seven kinds of recombinant proteins.2. Study SEB and its mutant SEBM32/172 adjuvant effect of the AIV-H9/NDV combined vaccines.In this experiment, broilers were immunized with AIV-H9/NDV combined vaccines and recombinant SEB or its mutant SEBM32/172, respectively. Detection of H9 avian influenza, Newcastle disease antibody titers and lymphocyte activity by HI test and MTT experimental to evaluate the adjuvant effect of SEB and its mutant SEBM32/172 on inactivated vaccine. The results showed: 14-28 days after intramuscular injection of 5μg recombinant protein SEBM32/172, can increase the antibody levels of AIV-H9 inactivated vaccine in different degree, but there is no effect on NDV. Adjuvant effects of 1μg or 10μg SEBM32 / 172 and SEB for inactivated vaccines is instability. Lymphocyte activity test results show, 7 days after immunization 1μg SEB and 5μg SEBM32 / 172, experimental group was significantly higher than the control vaccinegroup. There are also some differences in other times.3. Evaluation the safety of SEB and its mutants in vivo on broilers.In order to study the site-directed mutagenesis SEBM32 / 172 is able to reduce the toxic effects of SEB on the body. This test, we intramuscular injection 10-fold dose adjuvant of recombinant proteins were SEB and SEBM32 / 172 to the broiler,7、14、21 days after immunization, anatomy broilers of experimental group and the control group, produced tissue slices and observed pathological damage. During the experiment,both the experimental group and the control group had no obvious symptoms. However, under the microscope, the experimental group of broilers presence of renal pathological damage in varying degrees, such as deformation of tubular necrosis, renal enlargement, glomerulonephritis, pathological damage increases with time and aggravation. The pathological damage SEBM32 / 172 group was lower than pathological damage SEB group. The results showed that double mutant SEBH32NK172 E can reduce the toxic effects of SEB for broilers.