Cloning Of Envelope Protein Genes Vp26 And Vp28 Of Shrimp White Spot Syndrome Virus (WSSV) And The Expressions In Pichia Pastoris

White spot syndrome virus (WSSV) is one of the main shrimp pathogens causing serious losses in shrimp aquaculture. At present, the infection and pathogenesis of WSSV on molecular level have not been illuminated. Research on structure and function genes and pathogenic mechanism of the virus is in hotspot. WSSV is an enveloped double-strand DNA virus, VP26 and VP28 are two major envelope proteins. VP28 had been proved to play a key role in the initial steps of WSSV invasion, and VP26 may perform the similar functions as VP28. In this study, recombinant proteins (VP26 and VP28) were successfully expressed and secreted from host Pichia Pastoris. The results will provide important foundation for future studies on preparation of specific antiserum, effective detection of WSSV and development of subunit vaccine. Besides, the results will help improving the methods for expression foreign genes in eukaryotic host.1. Cloning of vp26 and vp28 genes: According to the complete genome sequences of White Spot Syndrome Virus (WSSV) published on GeneBank, two pairs of primers were designed for PCR amplification of genes vp26 and vp28. Both of the amplified products were 615bp. The amplified products were ligated into the pGEM-T-easy vectors and then transformed into competent cells of Escherichia coli DH5α. The positive recombinants were named pT-vp26 and pT-vp28 respectively. Sequencing of target fragments indicated that the sequences of vp26 and vp28 genes have more than 99% similarity with the published sequences in GenBank database, suggesting that the two genes are evolutionarily conserved.2. Expression and immunoactivity of recombinant proteins in Pichia Pastoris: The vp26 and vp28 genes were inserted into Pichia Pastoris secretory expression vectors pPIC9K. The recombinant plasmids pPIC9K-VP26 and pPIC9K-VP28 were linearized with BglⅡ, and then were transformed into Pichia Pastoris GS115 via electroporation. After G418 selection and PCR confirmation, the transformants were induced with methanol (0.5%) at 30℃for target proteins expression. The expression products were subjected for analysis by SDS-PAGE, ELISA and Western blot. The results showed that the vp26/28 genes were successfully expressed and secreted from host Pichia Pastoris and the expression products were about 30kDa. The Western blot and the ELISA analysis indicated that the expressed protein could react specifically with mouse polyclonal anti-serum.