Cloning And Expression Gp90 Gene Of Reticuloendotheliosis Virus (REV) And Development Of Indirect-ELISA For Detection Of Antibody To REV

Abstract:Avian reticuloendothelial hyperplasia is a division of the avian retrovirus reticuloendothelial hyperplasia virus (reticuloendotheliosis virus, REV), which could led to chickens, ducks, geese and other poultry pathological syndrome occurring, REV infection can cause tumor growth small syndrome, glandular gastritis, more importantly, thymus and bursa of infection often cause tissue and organ atrophy, reduced immunity of poultry and even lead to immune suppression, a great following for the viruses and bacteria provides a convenient infections RE reported trends in China have a tendency to expand, its rate of infection in poultry in China has reached 20% to 30%, prevention of significant detection of REV. By cloning, expression REV immune dominant protein, this study was to establish a rapid, accurate ELISA detection featuresBased on the nucleotide sequence of avian reticuloendotheliosis virus (REV) SNV strain, Sichuan isolates REV-SC1 used as a template, a pair of primers was designed to amplify the 121-1023 bp of gp90 gene fragment encoding high antigenic and hydrophilic domain of C-terminal protein. The gp90 gene was inserted into pMD-19T vector and sequenced, using the online software of NCBI blast to analysis the sequencing result. The gp90 gene was inserted to expression plasmid pET-32a(+), and the recombinant plasmid was transformed into E. coli BL21(DE3), then using IPTG to induce expression of protein, at last, an indirect ELISA method was established for detection the antibody to REV. The results showed that:1) Compared with other REV strain nucleotide sequences, the maximum matching rate was 99%, and there was a mutation separately at the 231 and 870 sites.2) SDS-PAGE analysis showed that the expected protein was got, and the Western-blot result indicated good reactogenicity of the target protein.3) An indirect enzyme-linked immunosorbent assay (ELISA) was established by using the recombinant protein. The optimal antigen coating concentration was set as 1.5 mg·mL-1, and the best anti-serum dilute degree was at 1:320, and the cut-off value (OD450nm) was 0.126. The detection results revealed that the method had good diagnostic sensitivity and specificity.