表达猪源流感病毒HA基因重组伪狂犬病毒的构建

目的获得共表达H1N1和H3N2亚型猪源流感病毒(SIV)HA基因的重组伪狂犬病毒(PRV)。方法克隆H1N1以及H3N2亚型SIV的HA基因,将HA基因与PUC-TK转移载体进行酶切、连接来构建pUC-P-H1A-H3A重组表达质粒,用脂质体将其转染已感染PRV亲本病毒的Vero细胞,使其在Vero细胞内与PRV基因组发生同源重组。运用RT-PCR、间接免疫荧光等方法检测外源基因重组情况及其在Vero细胞内的表达,并通过Western-blot对表达产物进行免疫原性鉴定。结果实验结果显示,外源基因HA已重组入PRV基因组,并在Vero细胞中有效表达,且所表达蛋白具有免疫原性。结论HA基因重组PRV的构建成功为SIVHA基因重组PRV活载体疫苗的研究奠定了初步基础。

Construction of the recombinant pseudorabies virus vector expressing the HA genes of swine influenza virus

The HA genes of H1N1 and H3N2 swine influenza viruses were cloned,digested and ligated to the transfer vector pUC-TK to construct the recombinant plasmid pUC-P-H1A-H3A.This recombinant plasmid was used to transfer Vero cells infected with pseudorabies virus(PRV).RT-PCR and indirect immunofluorescence assay(IFA)were performed to examine the expression of HA genes.The immunogenicity of protein was identified by Western blot assay.The experimental results showed that the exogenous HA genes could recombine with PRV successfully and the immunogenic protein was expressed in vero cells.It is apparent that the successful construction of the recombinant pseudorabies virus expressing HA genes of swine influenza virus paves a way for the vaccine development of swine influenza.