猪瘟流行毒株E2基因的克隆及其杆状病毒表达载体的构建

用RT PCR方法扩增了陕西省近期猪瘟流行野毒株E2基因 ,并将其克隆到 pMD 18 T载体 ;经转化、筛选、鉴定后 ,测定了核苷酸序列 ,根据C株、Brescia株和Alfort株确定起始氨基酸三联体的正确位置后进行 gp5 5氨基酸序列推导 ,并进行同源性比较。结果表明 ,该猪瘟流行毒株的E2核苷酸序列与C株、Brescia株和Alfort株的E2核苷酸序列同源性分别为 81.9%、83.4 %和83.5 % ;相应地 ,gp5 5氨基酸同源性分别为 94 .8%、95 .6 %和 95 .3%。将此E2基因亚克隆到杆状病毒转移载体 pFastBacHTb后获得了重组质粒 pFBHT E2 ,进而转化入含穿梭载体Bacmid的感受态细胞DH10Bac中 ,发生转座作用 ;经抗性及蓝白斑筛选得到了含E2基因的重组DNA ,将其命名为rBacmid E2。此研究为进一步在昆虫细胞中表达E2基因、开发研制猪瘟基因工程疫苗奠定了基础。

Cloning of E2 gene of classical swine fever virus and construction of baculovirus expression vector

E2 gene of prevalent classical swine fever virus(CSFV) virulent strain from Shaanxi Province of China was amplified by RT-PCR. The E2 fragment was cloned into pMD 18-T vector. The cDNA fragments of the E2 gene were sequenced,and on the basis of the amino acid sequences of China strain, Brescia strain and Alfort strain,the amino acid sequence encoded by the E2 gene was deduced. The nucleotide sequence homologies of the prevalent virulent strain with China strain, with Brescia strain and with Alfort strain were 81.9%, 83.4% and 83.5% respectively. The E2 gene cDNA was subcloned into baculovirus transfer vector and the recombinant baculovirus vector pFBHT-E2 was constructed successfully. Then it was transferred into E.coli DH10Bac and cultured in LB plate. The white bacterial colonies were positive recombinant bacmid named as rBacmid-E2. Thus the result mentioned-above laid down the theoretical and practical foundations for the expression of E2 gene in insect cells.