The Prokaryotic Expressions And Establishments Of Indirect ELISA Of GB Gene Of Porcine Pseudorabies Virus

As we all known in recent years pseudorabies(PR) explode throughout the country from2011. At present, PR is already become one of the major viral disease which affects the economic interests and development of farm. ELISA method not only has a high specificity and easy to operate, but also economic and time saving, it can meet the demand of large sample testing and very suitable for monitoring the antibody levels. gB protein is the most conservative protein of pseudorabies virus’glycoprotein. And gB is an important component and the main immunogen of the PRV envelope protein. There is an important significance to build an indirect-ELISA to detect gB antibody for monitoring immunization status.The study has a gene sequence analysis of PRV gB gene, and according to the main antigen domain of gB, we divide it into four parts clone to prokaryotic expression vector, pET-28a(+), such as gB1(98aa-369aa)、gB2(370aa-578aa)、gB3(541aa-743aa)、 gB4(820aa-916aa). Then, this four recombinant plasmids expressed in E.coli bacterias, Rosetta(DE3)pLysS. And purify the four protein by Ni+affinity chromatography, verify the immunogenicity of the four protein by western-blot.Using the purified gBl、gB2、gB3、gB4proteins as antigens to coating plates, determine the best condition to build an indirect-ELISA method and choose a best protein to optimize. The results show that gB3protein is the best one to be the antigen for building the indirect-ELISA method. As the optimized gB3-ELISA and Biochek-gB kit have the sensitivity of95.12%, the specificity of89.13%, the coincidengce rate of92.97%, this indirect-ELISA method has a high senseitivity and specificity, it should be have a great application prospect.