Preliminary Identification Of Envelope Protein VP32 And Collagen-like Protein CLP Of White Spot Syndrome Virus

White Spot Syndrome Virus(WSSV) is a large,rod-shaped,enveloped double-stranded DNA virus.The WSSV has been classified to be the sole species of Whispovirus in genus of Nimaviridae,and the main pathogen harming shrimp industry.At present,our research mainly focuses on the main proteins of WSSV.These proteins play important roles in the formation construction,invasion,proliferation, etc.This paper has two parts as below:(1) Identification by mass spectrum indicated that VP32 is the production of the ORF wsv198.We constructed expression vector pGEX-2Th-vp32 and pMAL-vp32, and expressed and purified GST-VP32 and MBP-VP32 in E.coli BL21(DE3) repectively,and then anti GST-VP32 serum was prepared.By using western-blot,we confirmed that VP32 is viral envelop protein and immune-gold microscope(IEM) further approved it.When treated with the detergent 0.1%Tween-20 mildly,we found VP32 apparently located in the inner of viral envelope.The tag of GST/MBP might exert influence on VP32's folding to correct native state,which made against further research of VP32 function(mainly protein interaction aspect).(2)The collagen-like proteins(CLP) protein coded by WSSV ORF wsv 001 is a really unique envelop protein of WSSV which is consisted of 1,684 amino acids and the predicted molecular weight is 168.2 KDa.Research showed that protein has a particular collagen-like sequence(Gly-X-Y,161—1327 aa) which is rare in the virus.We chose N-terminal,middle,C-terminal sequence and expressed them in E.coli BL-21(DE3) respectively,then the corresponding antibodies were prepared. We detected the distribution of CLP in some tissues of infected shrimps and a certain difference was disclosed.The weak signal of the western blotting in C-terminus indicated the degeneration might occured.All the above laid the foundation for further research on this protein's function.