Construction Of Whole Genome Bacterial Two-Hybrid System Library And Primary Research Of Structural Proteins VP664 And VP41A Of White Spot Syndrome Virus

White spot syndrome virus (WSSV),an enveloped, non-occluded, bacilliform and circular dsDNA virus, is one of the major pathogens of shrimp diseases, causing considerable mortality in penaeid shrimp. Bacterial two-hybrid system is a newly developed method for studying protein-protein interaction. We used BacterioMatch two-hybrid system (Stratagene) to study the interaction of WSSV protein. First, the random segments (about 1 kb) of WSSV genome were inserted into the plasmids pBT and pTRG to construct recombinant plasmids pBT-wssv and pTRG-wssv respectively. Then these two plasmids were co-transformed into the competent bacteria XL1-Blue MRF'. And a reporter gene had been embedded into this strain. Therefore, we used LB-TCK agar plate to screen the WSSV protein in order to find the interaction of them. In current research we had constructed the WSSV gene group bacterial two-hybrid system, which had been used to screen the WSSV genome to find the protein interaction. And we also built a good foundation for this viral functional genome research.We had previously reported a simple and effective method to extract and purificate the intact viral particles from the tissues of experimentally infected crayfish (Procambarus clarkii) with high yield, which provided a good foundation to identificate the structure protein. White spot syndromes virus nucleocapsid protein VP664 is a hitherto biggest structural protein.The genome is the presence of a giant open reading frame (ORF) of 18,234 nucleotids that encodes a long polypeptide of 6,077 amino acids. Two 600bp were chosen from two terminals of gene that encoded VP664 ,and respectively each was given the name vp664n and vp664c. They were separate cloned and inserted into pET-his vector, The gene was expressed in E. coli BL21 (DE3) and purified by Ni²⁺-NTA agarose. Then two polyclonal antibodies related to these proteins were obtained. These antibodies did only react with the proteins of WSSV nucleocapsids and the intact virions in western blot assay, indicating these proteins should be categorized to viral nucleocapsids.