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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 major causative agent of severe viral diarrhea in infants and young children. VP6 is one of the major structural proteins of rotavirus. The natural VP6 has strong antigenicity and immunogenicity. A group of rotavirus T114 Chinese isolate VP6 gene was cloned into the transfer vector pBacPAK8 by gene recombination technique and the recombinant plasmid pBacPAK8-VP6 and the linearized silkworm baculovirus Bm-BacPAK6DNA were co-transfected into BmN cells to obtain a recombinant virus BmNPV-VP6. BmN cells infected with BmNPV-VP6 and 5th instar larvae of silkworm were detected by Western blotting. The specific bands of 120 kD were detected in the hemolymph of BmN cells and 5th instar larvae of Bombyx mori, indicating that the recombinant VP6 protein was expressed in silkworms , Consistent with the size of VP6 trisomy. The results of ELISA showed that the recombinant VP6 protein reached the highest level on the 4th and 6th day after infection in the hemolymph of BmN cells and 5th instar larvae of Bombyx mori. The successful expression of VP6 in the silkworm baculovirus expression system can provide a new way for the development of new rotavirus vaccine.