Expression Of Helicovapa Armigera Nucleopolyhedravirus (HearNPV) HA2 In Insect Cell

Baculoviruses from lepidopteran insects contain the homologous gene of theHelicovapa armigera nucleopolyhedravirus (HearNPV) open reading frame 2 (ha2),which belongs to WASP family existing in eukaryotic lives. Sequence analysisindicates that ha2 is 1242 bp long and potential codes 414 amino acids (aa) withmolecular weight of 46.1kDa. HearNPV HA2 shares WASP converted domains suchas proline-rich domain,WH2 domain,acidic domain and Cofilin domain.WASP, Wiscott-Aldrich syndrome protein, is a component of signaling pathwaysthat control reorganization of the actin cytoskeleton. It can activate the Arp2/3complex, increase the affinity of Arp2/3 complex and actin, leading to rapid actinpolymerization.In Autographa californica multiple NPV (AcMNPV), P78/83, the homologue ofHA2,locates at the basment of viral nuclearcapsid, recent experment indciated thatP78/83 performs the similar function to WASP to activate Arp2/3 complex leading torapid actin polymerization. The polymerized F-actin migth promote nuclearcapsidtransportation to nucleus. This suggested that HA2 might have the same function ofP78/83 which involves in baculovirus transportation.Recent studies have further clarified that the phosphorylation of WASP plays animportant role in WASP activation process. Two phosphorylation sites in the VCAdomain of WASP at serines 483 and 484 are substrates for casein kinase 2.Phosphorylation of these residues increases the affinity of the VCA domain for theArp2/3 complex and leading to rapid actin polymerization indirectly. Thus it shouldplay a very important role in function of HA2 that HA2 is modified post-translation ininsect cells. In this thesis, to reveal the structure and the function of HA2 which can clarify the role of HA2 in HearNPV replication more clearly, we choosed thebaculovirus expression system to express 4 truncated HA2 proteins for functionalanalysis in next step. This thesis contains three chapters.In Chapter one, we present a brief introduction about baclovirus, including theclassification status, replication, structures and interaction between baculovirus andactin. We also introduce the baculovirus expression system. In the last of this chapter,the purpose of experiment is described.In Chapter two, some tools and software have been used to predict possiblemodification sites of HA2 protein, and to analyze functional domains.In Chapter three, four truncated proteins of HA2 have been expressed in Sf9insect cells using baculovirus expression system. These four proteins are named asHA2-VCA,HA2-385,HA2-580,HA2-785, which contain different functionaldomains. In chapter 3, we will introduce the expression of 4 proteins in detail.