Recombinant Expression Of Brucella VirB12 And It's Used For Immunoassay Of Labeled Vaccine Cells

BACKGROUND: Brucellosis is a zoonotic disease that seriously affects animal husbandry.Animal brucellosis is mainly prevented and controlled through vaccine immunization.At present,there are three main vaccine strains in China,namely S2(mainly used for Brucella suis epidemic prevention),A19(used for Brucella abortus epidemic prevention),and M5/M5-90(used for Brucella.ovis epidemic prevention).These vaccines play an important role in the prevention and control of Brucella transmission in animals.There are some problems in the practical application of these vaccines.Serological surveillance is the most important technical means of animal brucellosis diagnosis.There are commonly used methods such as Rose-Bengal Plate Agglutination Test,Standard Tube Agglutination Test and ELISA,etc.But these methods cannot distinguish natural infection antibodies from vaccine immune antibodies,which bring great difficulties to the development of epidemic prevention.Gene marker vaccine is considered to be a new type of vaccine to solve the problem of differential diagnosis between brucellosis vaccine immunization and natural infection.The A19-?Vir B12 gene marker vaccine developed by Tiankang Biological Co.,Ltd.has lower virulence than the maternal strain and good protective effect.It also has the function of distinguishing wild strain infection from vaccine immunization.It is of great significance for the prevention and control of ranch brucellosis.In this study,we recombined and expressed Vir B12 protein in vitro to analyze the cellular immune characteristics of Vir B12 protein and explored the feasibility of identification of vaccine immunity and natural infection.METHODS: In this study,we first constructed a prokaryotic expression strain of Brucella abortus vir B12 protein and detected the protein expression and immunogenicity.A pair of primers were designed and synthesized by using the molecular biology software prime premier 5.0 based on the vir B12 gene sequence of Brucella A19 strain registered on NCBI.vir B12 gene was amplified by PCR and cloned into p ET-28 a vector.Recombinant plasmids were transformed into E.coli BL21(DE3)and induced by IPTG.The expressed products were purified by nickel column.The purified products were identified by Western blot analysis.Brucella A19-?Vir B12 strain lacks vir B12 gene compared with its parent strain A19 and virulent strain 2308.A19-?Vir B12 strain does not express the corresponding Vir B12 protein during the growth process.Animals cannot produce Vir B12 antibody and corresponding immune memory after immunization.In this experiment,peripheral blood lymphocytes of experimental animals were immunized with strain A19 and strain A19-?Vir B12 in vitro.Lymphocytes were stimulated with Vir B12 protein as antigen after culture.Then IFN-gamma was detected and strain A19 and strain A19-Vir B12 were identified after immunization.Then IFN-gamma was detected and the strains A19 and A19-?Vir B12 were identified after immunization.RESULTS: The results showed that the prokaryotic expression system of vir B12 gene of Brucella A19 strain was successfully constructed.The purified Vir B12 protein showed high purity.Western blot test showed that the Vir B12 protein had good reactivity and had a reaction with Brucella wild virus positive serum while no reaction with A19-?Vir B12 strain immune serum.IFN-gamma detection in vitro using purified Vir B12 protein showed that there was a significant difference(P < 0.001)in IFN-gamma produced by peripheral blood lymphocytes(PBMC)stimulated by Vir B12 between A19 and A19-?Vir B12 immune groups.The results showed that the immunity of strain A19 and strain A19-?Vir B12 could be successfully distinguished by the PBMC extraction and Vir B12 protein stimulation in vitro.CONCLUSIONS: The prokaryotic expression strain of Vir B12 protein was successfully constructed in this study.The purified Vir B12 protein can be used as a stimulus antigen to differentiate A19 strain immunity from A19-Vir B12 strain at IFN-gamma level in vitro,which provides novel methods and ideas for the prevention,control and eradication of brucellosis.