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

【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。将重组Bacmid转染Sf9细胞,收集含病毒的上清去感染Sf9细胞,在感染后不同时间点收集细胞进行SDS-PAGE电泳,利用商业化的HA抗体进行Western blot分析以检测融合蛋白在昆虫细胞中的表达情况。【结果】用C-端融合HA-标签的重组病毒感染细胞后12h即可检测到一条43kDa左右、能与HA抗体发生特异性结合的蛋白条带,该特异性蛋白的表达一直持续到病毒感染后96h。从感染后48h起一直到96h,均能检测到另外一条约26kDa的蛋白条带也能与HA抗体发生特异性结合。在N-端融合HA-标签的重组病毒感染的细胞中没有检测到与HA抗体特异结合的蛋白。【结论】结果表明,p48基因是个晚期基因,在病毒感染的晚期表达,并且该蛋白在昆虫细胞中表达时N-端可能被剪切。

Objective p48（ac103） gene is highly conserved in baculovirus,implying that p48 might play a fundamental role in the life cycle of baculovirus.We studied the expression of p48 gene of Autographa californica multiple nucleopolyhedrovirus（AcMNPV）—the type baculovirus species.MethodsWith Bac-to-Bac system,we constructed two p48 repair viruses,in which hemagglutinin（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.ResultsWestern 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 HA-tag fused in C-terminus.Meanwhile,another protein of 26 kDa was also detected from 48 hpi to 96 hpi.However,no signals were detected in cells infected with p48 repair virus with HA-tag fused in N-terminus even at 96 hpi.Conclusionp48 is a late gene and expressed in late infection.P48 might be cleaved in N-terminal when it is expressed in insect cells.