新城疫病毒F、NP、M和HN基因在昆虫细胞内的共表达

Co-expression of F,NP,M and HN gene of NDV in insect cell

为构建杆状病毒转移载体,通过PCR的方法将F48E9株新城疫病毒F基因上的Stu I位点和NP基因上的Xba I位点进行突变,扩增出全长的F、NP以及F48E9株新城疫病毒M和HN基因片段,将其克隆到pMD18-T载体,再将F、NP、M和HN基因依次亚克隆到杆状病毒转移载体pAcAB_4上,F基因和M基因均在p10启动子的操控之下,NP基因和HN基因构建到两个polyhedrin启动子下游,构建重组杆状病毒转移载体pAcAB4_4-F-NP-M-HN。pAcAB_4-F-NP-M-HN与线性化的杆状病毒DNA共转染昆虫细胞Sf9,获得重组杆状病毒rBac-F-NP-M-HN。重组杆状病毒感染Sf9细胞,72h后收集细胞和培养上清。Western blot分析显示:M、NP、F和HN蛋白在培养上清中得到了共表达,大小与预期结果一致;感染细胞内只检测到了HN蛋白的表达。这表明M、NP、F和HN蛋白在昆虫细胞内共表达可以自我装配成病毒样颗粒,并且以出芽的方式释放到培养基中。该重组杆状病毒的获得为研究新城疫病毒各结构蛋白之间的相互作用和确定病毒粒子出芽的驱动力等方面奠定了基础。

To construct baculovirus transfer vector, Stu Ⅰ site in F gene and Xba Ⅰ site in NP gene of newcastle disease virus （NDV） F48E9 strain were mutated by polymerase chain reaction （PCR） methods. Four pairs of primers were designed to amplify F, NP, M and HN full-length genes, respectively. The PCR products were cloned into pMD18-T vector and sequenced. The F, NP, M and HN genes were then subcloned into baculovirus transfer vector pAcAB4 to construct recombinant baculovirus transfer vector pAcABg-F-NP-M-HN, in which the Fand M genes were under the control of the promoter p10 and NP HN genes were under the polyhedrin promoter. Insect cell Sf9 was co-transfected with pAcAB4-F-NP-M-HN and linearized baculovirus DNA. Recombinant baculovirus rBac-F-NP-M-HN was obtained and identified by PCR. Supernatant from Sf9 cells alone and from Sf9 cells infected with recombinant baculovirus rBac-F-NP-M-HN were collected at 72 h post infection. The expression of F, NP, M and HN protein in culture medium was confirmed by western blot. In contrast, only HN protein was detected in the infected cell lysate. The results suggested that virus-like particles can self-assemble under co-expression of M, NP, F and HN protein and bud into the culture supernatant.