Structural And Functional Analysis Of ORF44(Ha44)of Helicoverpa Armigera Nucleopolyherovirus (HearNPV)

In this thesis, the structure and function of ORF44 (Ha44) of Helicoverpaarmigera nucleopolyhedrovirus (HearNPV) were studied.Chapter 1 is a general introduction of baculovirus, including the overview ofbiology, molecular biology and structure protein of baculovirus. And the aim ofthe thesis was presented.In chapter 2, the HA44 was expressed in E. coli with His-tag. And the His-HA44protein was purified and used to generate antiserum against HearNPV HA44. Westernblot analysis indicated that the obtained anti-HA44 antiserum was able to identify theHA44 in HearNPV infected cells.In chapter 3, the expression time course of HA44 was analysis. Western blotanalysis showed that HA44 was expressed from 18h to 96h post infection. Todetermine whether Ha44 is essential for viral replication, a Ha44-deleted bacmid anda Ha44-repair bacmid were constructed. The results of tranfection and infection assaydemonstrated that the Ha44 is essential for viral replication.In chapter 4, the functional domains of HA44 were identified by fusing enhancedgreen fluorescence protein (EGFP) tag. The fluorescent images of transfected cellsindicated that EGFP-HA44 was localized in nucleus with aggregation dot. A series ofHA44 truncations with EGFP fused in frame to the N-terminus were constructed andanalyzed by transient transfection into HzAM1 cells. The results revealed that thenuclear localization signal (NLS) was resided within the N-terminal 130 residues ofHA44 and the domain required for aggregation was resided within 187-255 residues of HA44. And the experiment of site-directed mutagenesis of HA44 showed that R²⁵was a key amino acid for the nuclear localization of HA44.In chapter 5, a series of HA44-rescued bacmids were constructed. Tranfection andinfection assay showed that HA44_1-130, HA44_187-378 and HA44_187-255could notrescue the function of HA44, but HA44L²⁵L²⁶ could rescue the function of HA44.Growth curve indicated that the HA44L²⁵L²⁶ repaired virus had a lower replicationefficiency than wild-type HA44 repaired virus.