Construction Of S2 Cell Lines Expressing Rabbit Hemorrhagic Disease Virus VP60 Protein And Development Of Indirect ELISA Assay Based VP60 Protein

Rabbit hemorrhagic disease virus(RHDV) genome is a single-stranded RNA, which contains two open reading frame (ORF). The capsid protein VP60, encoded by the open reading frame (ORF) located in the 5’ terminus, which is the dominant protective antigen of RHDV.Currently, the serological diagnosis method of RHDV is the traditional human "O" erythrocytes hemagglutination inhibition test. As the RHDV cannot be culture in vitro at present, we still use the outdated tissue inactivated vaccine to prevent disease.The hemagglutination inhibition test has obvious defects, such as strong subjectivity, low repetitiveness and inconvenience of preparing human erythrocytes. Meanwhile the tissue inactivated vaccine possess low immunization efficiency and high risk of bio-safety. These disadvantages severely limit the healthy development of rabbit breed, while the reality requires us to develop new RHDV detection method and genetic engineering vaccine.This study consists of two parts:1) establish a rapid and fidelity ELISA method to detect the specific antibody of RHDV; 2) select a recombinant insect cell lines,which can able to express the VP60 protein stably. Given this, first we cloned the VP60 gene and obtaining the recombinant expression plasmid pET32a/VP60.Then pET32a/VP60 were transformed in to BL21, and recombinant protein can be highly expressed in the inclusion bodies. We gained highly purified protein and immunized with rabbits, at last obtained the specific polyclonal antibody. Simultaneously, two dominant antigen regions genes of VP60 capsid protein:A (31-250 AA) and B (470-579 AA), amplified by overlapping PCR. Then the genes were inserted into pET-30a, obtaining the recombinant expression vector pET30a/AB.The fusion protein AB can be detected in inclusion bodies after transfected into BL21. The molecular weight of the recombinant protein is 36 kD. Western blot assay was carried out to analysis the immunogenicity of the purified protein, and the results showed that the dominant antigen regions were successfully expressed in Escherichia coli. Then we use the purified protein AB as the diagnostic antigen, through multiple optimizing the Antigen-antibody reaction conditions, the indirect ELISA assay was established.In order to get high yield, low cost and better immunogenicity antigen, we choose S2 cell to express VP60 protein. First,we cloned VP60 gene into the eukaryotic expression vector pMT/BIP-V5-HisA, and co-transfected the recombinant plasmid BIP/VP60 with helper plasmid pCoBlast into Drosophila S2 cells. By using the blasticidin and limiting dilution assay to screen monoclonal positive cells, we successfully established a stable transfected S2 cell lines.In conclusion,in this research we developed a indirect ELISA assay to detect RHDV antibody and provided a good preparation for the produce cheap, efficient and safe RHDV genetic engineering vaccine.