Identification And Functional Research Of White Spot Syndrome Virus(WSSV) Envelope Protein VP52B And Screening For Immediate-early Gene Promoters

White spot syndrome virus (WSSV) is the main pathogen of shrimp. This thesis focuses on viral envelope proteins. Also, we concern about the screening and identification of WSSV immediate-early genes. The work mainly includes three parts below:(1)We characterized and investigated the functionality of WSSV envelope protein VP52B (ORF wsv256). The ORF wsv256 was cloned and expressed in E. coli BL-21 with GST-tag and polyclonal antibody of VP52B was prepared afterwards. GST pull-down suggests that VP52B interacts with VP26, a major viral envelope protein which had been reported as a crucial linking element. For the purpose of finding out the specific interacting regions of VP26 with VP52B, six mutations of VP26 were constructed and expressed. The 135～170Aa region of VP26 found out to play an important role on the interaction of VP26 and VP52B.(2)WSSV genome was randomly fragmented to 400bp～900bp using ultrasonic process and then inserted to vector PIZΔIE/V5-EGFP-His to build a library. After transfecting Hi5 insect cells with this library, a part of the cells proved to express enhanced green fluorescent protein, suggesting DNA fragments in these cells might function as immediate-early gene promoters. Therefore our study would be helpful for the screening and identification of WSSV immediate-early genes and immediate-early gene promoter studies.(3) Twenty WSSV ORFs, including zinc finger structure or other transcript regulating factor were selected. Their upstream sequences with a size of 400-500bp were cloned and inserted into promoter-activity-detecting vector. The result of transfecting Hi5 insect cells suggests that two ORFs (ORF240,ORF403) concern with WSSV immediate-early gene promoter activity. Both of them include Ring-H2 finger motif, suggesting a relationship between the motif and promoter activity.