A组轮状病毒结构蛋白VP6在家蚕杆状病毒表达系统中的表达

Expression of Structural Protein VP6 of Group A Rotavirus in Bombyx mori Baculovirus Expression System

A组轮状病毒(rotavirus,RV)是婴幼儿严重病毒性腹泻的主要病原,VP6为轮状病毒的主要结构蛋白之一,天然VP6具有很强的抗原性和免疫原性。通过基因重组技术将A组轮状病毒T114中国地方株VP6基因克隆入转移载体质粒pBacPAK8中,并将重组质粒pBacPAK8-VP6与线性化家蚕杆状病毒Bm-BacPAK6DNA共转染BmN细胞,获得重组病毒BmNPV-VP6。用BmNPV-VP6感染BmN细胞和家蚕5龄幼虫,Western blotting检测表明在BmN细胞和家蚕5龄幼虫的血淋巴中均可检测到120 kD的特异性条带,提示重组VP6蛋白在家蚕中获得表达,与VP6三体形式的大小相符合。ELISA检测结果显示,重组VP6蛋白在BmN细胞和家蚕5龄幼虫血淋巴中的表达分别在感染后第4天和第6天达到最高水平。VP6在家蚕杆状病毒表达系统中的成功表达可为新型轮状病毒疫苗的研制提供新的途径。

Group A rotavirus（RV） is the main pathogen causing diarrhea in children.As one of the main rotavirus structural proteins,natural VP6 has very high antigenicity and immunogenicity.In this paper,VP6 gene from Chinese local strain T114 of group A rotavirus was cloned into the transfer vector pBacPAK8 by employing gene recombination technique,then the recombinant plasmid pBacPAK8-VP6 was co-transfected into silkworm BmN cells together with DNA of linearized Bombyx mori baculovirus Bm-BacPAK6 to construct the recombinant virus BmNPV-VP6.After BmNPV-VP6 was used to infect BmN cells and the 5th instar silkworm silkworm larvae,Western blotting detected a specific band of 120 kD molecular weight both in BmN cells and in hemolymph of the 5th instar silkworm larvae,indicating that the recombinant protein VP6 was expressed in silkworm and the expressed protein was consistent with the size of trimeric form of VP6.ELISA assay indicated that the expression of recombinant protein VP6 reached maximal level in BmN cells on the 4th day post-infection and in hemolymph of the 5th instar larvae on the 6th day post-infection.The successful expression of VP6 in Bombyx mori baculovirus expression system may provide a new way to develop novel vaccines against rotavirus.