Fuctional Analysis Of AcMNPV ORF9 And HaSNPV ORF2

Baculovirus is a kind of double-stranded circle large DNA virus, which has been developed as an environment sound pesticide and a powerful vector of foreign protein expression as well as a potential vector of gene therapy. Revealing the molecular mechanism of viral replication, package, and transportation will lead the better understanding of baculovirus itself and enhance its application. Sequence analysis found that Heliocoverpa armigera single nucleocapsid nucleopolyhedrovirus (HaSNPV) containing a baculoviruses conserved gene Ha orf2, the homologue of AcMNPV P78/83, which shares the common motifs with WASP proteins indicating that Ha2 and P78/83 might involve in baculovirus transportation. This thesis reports the results of studies on functional analysis of HaSNPV ORF2 and it's homologous gene AcMNPV ORF9. It encompasses three chapters.In Chapter one we present a brief introduction of baclovirus, including the classification status, replication, characteristics of the baculovirus genome. We also summary the identified genes in some detail especially the baculovirus conserved genes, finally we give the aims of this study.Chapter two describes the materials and methods used.The major results are presented in the Chapter three which contains three parts: part one: results of studies on Ac ORF9; part B: results of studies on Ha ORF2 and part C: comparison studies on Ac ORF9 and Ha ORF2.Trying to understand P78/83 function, we first constructed recombinant virusvAc-orf9-gfp with Bac-to-Bac system, within which AcORF9 was fused with EGFP. Sf21 cells were infected with the recombinant virus and Laser Confocal Microscopy was used to determine the subcellular location of P78/83. Green fluorescences are major distribution of in cell plasms after 12h infection, and transferred into neucleus at 24h pi. This means that P78/83 is located at neucleus. Infection assay in vitro showed that overexpression of P78/83 hadn't any evident effect on the virus growth and viral assemble. It is notable that the fusion protein can be assembled to virions. Via actin dissemination in vAc-orf9-gfp infected cells, P78/83-EGFP might colocalize with actin.In Part two, amino acid sequence of HaORF2 was analysed, and conserved domains of WASP protein were found in HaORF2, which suggests HaORF2 functions similarly to WASP family proteins. Furtherly expression phase analysis showed that ORF2 appeared in different form during infection. To reveal the function of HaORF2, vHa-orf2-gfp recombinant virus which tagged with EGFP-ORF2 fusion protein was generated. HaORF2 protein was found to localize in cell nucleus with this recombinant virus, and the fusion protein appeared to assemble into virions normally. According to the study on the interaction between HaORF2 protein and actin, HaORF2 seemed to colocalize with actin during infection.Part three aimed to answer the question whether HaORF2 can replace AvORF9. Two recombinant viruses: vAc-Ha2-gfp and delAc9-Ac-Ha2-gfp was expected to give the answer. The former one was generated successfully, and subcellular location of HaORF2 protein in Sf21 cells showed a different situation compared with in HzAMI cells, but there is no distinct difference between vAc-Ha2-gfp and AcMNPV-gfp when observed with electron microscopy. The deleted recombinant aiming to answer whether Ha2 can replace Ac9 hasn't been got yet.