Prokaryotic Expression And Preliminary Functional Study Of Vp36a Of White Spot Syndrome Virus (wssv)

White Spot Syndrome Virus (WSSV) is the major pathogen of the worldwide epidemic disease of cultured shrimp since 1992, until now, it still has being brought serious economic loss to the shrimp culture industry of china. In the aims of developing relevant blocking vaccines, the studies of WSSV presently mainly focused on searching its attachment proteins, by which the virals are able to attach to the host cells. Previous research showed that VP36A(named by apparent molecular weight) is a minor envelope protein of WSSV containing the RGD motif, the phage displayed VP36A has binding ability with shrimp gill cell membrane and competitively bind to shrimp gill cell membrane against WSSV, which indicated that VP36A may be a attachment protein of WSSV. On this basis, this paper studies the function of VP36A using prokaryotic expressed VP36A by ELISA^ cell culture,immunoblotting and so on.Primers were designed bases on the vp36a gene of the Chinese WSSV isolate, containing NheⅠand HindⅢrestriction sites. After amplification by PCR, the VP36A DNA fragment was directional cloned into prokaryotic expression plasmid pBADgⅢB, the recombinant prokaryotic expression vector was successfully constructed and identified by double digestion PCR and DNA sequencing. Then the recombinant vector pBADgIIIB-VP36A was transformed into E.coli and induced with gradient concentrations of L-arabinose and induced for gradient hours respectively to optimize culture conditions.Results of SDS-PAGE and Western-blot indicated that recombinant VP36A was about 40KDa and over expressed as inclusion body after induced with 0.2%L-arabinose for 4h. Finally the fusion protein was purified by Metal Chelating Affinity Chromatography.To prepare polycolonal antibodies against VP36A, two peptide segments of the VP36A with high antigenicity were synthesized and immunized new zealand white rabbits separately. The ELISA titers of affinity purified antibodies against two peptides of VP36A were all above 1:10000. Western blot analysis showed that the two kinds of antibodies could bind to expressed fusion protein specifically, but failed to bind the naive VP36A of WSSV at 36KD, the reason needs to be further studied.The binding ability of VP36A to shrimp gill cell membrane and haemocytes were vertified with ELISA and fluorescence microscopic method separately. Furthermore Far Western Blot was used to fish the VP36A binding proteins from WSSV structure proteins and shrimp gill cell membrane proteins. The results indicated that there is one structure protein of WSSV about 22KDa and one protein about 32 KDa of shrimp gill cell membrane may bind to VP36A.The research found that one protein of WSSV and one protein of shrimp gill cell membrane could bind with VP36A obviously, the binding activity needs to be vertified,which will help to enrich the basic research on the molecular virology of WSSV。...