杆状病毒AcMNPV p48基因在昆虫细胞中的表达

Expression of baculovirus AcMNPV p48 gene in insect cell

摘要:

摘要：【目的】p48（ac103）基因在昆虫杆状病毒中高度保守，暗示其具有重要的生物学功能。为了研究该基因的功能，我们首先对该基因的表达特征进行描述。【方法】以杆状病毒代表种——苜蓿银纹夜蛾核型多角体病毒（Autographa californica multiple nucleopolyhedrovirus, AcMNPV）的p48基因为研究对象，利用Bac-to-Bac杆状病毒表达载体系统分别构建了在P48蛋白N-端和C-端融合HA-标签，并且携带绿色荧光蛋白基因和多角体蛋白基因的重组Bacmid。将

Abstract:

Abstract: [Objective] p48 (ac103) gene is highly conserved in baculovirus, implying that p48 might play a fundamental role in the life cycle of baculovirus.[Methods] In this study, We studied the expression of p48 gene of Autographa californica multiple nucleopolyhedrovirus (AcMNPV)---the type baculovirus species. With Bac-to-Bac system, we constructed two p48 repair viruses, in which HA-tag was fused to C-terminus and N-terminus of P48, respectively. To examine the effect on occlusion body morphogenesis and to facilitate examination of virus infection, the green fluorescence protein (gfp) gene and polyhedrin (polh) gene were also inserted into the recombinant viruses. Sf9 cells were transfected with each bacmid constructed, and the supernatants containing the budded viruses (BVs) were used to infect Sf9 cells. At the indicated time points, cells were harvested and used for SDS-PAGE and Western blot analysis. The expression of fusion protein was detected with the monoclonal antibody to the HA-epitope. [Results] Western blot analysis indicated that a specific 43 kDa protein was detected at 12 hours postinfection (hpi) and remained detectable up to 96 hpi in cells infected with p48 repair virus with hemagglutinin (HA)-tag fused in C-terminus. Meanwhile, another protein of 26 kDa was also detected from 12 hpi to 96 hpi. However, no signals were detected in cells infected with p48 repair virus with HA-tag fused in N-terminus till 96 hpi. [Conclusion] The result indicated that p48 is a late gene and expressed in late infection. P48 might be cleaved in N-terminal when it is expressed in insect cells.