猪伪狂犬病病毒流行毒株gE/gI缺失突变株的构建及生物学特性研究

摘要

[目的]为研制针对猪伪狂犬病病毒流行毒株的疫苗提供候选毒株.[方法]构建针对猪伪狂犬病病毒流行毒株的gE/gI缺失重组转移质粒pMD-LA-RA及携带EGFP标记基因的重组转移质粒pMD-LA-EGFP-RA,将pMD-LA-EGFP-RA与伪狂犬病病毒流行毒株PRV AH进行同源重组,利用EGFP为筛选标记,获得携带EGFP基因的gE/gI基因缺失突变株PRV AHgE–/gI–/EGFP+,以此毒株与pMD-LA-RA进行第2次同源重组,筛选去除EGFP基因的gE/gI基因缺失突变株PRV AHgE–/gI–,并通过生长曲线、易感细胞连续传代和动物免疫评价其增殖能力、遗传稳定性及免疫原性.[结果]通过2次同源重组,结合荧光观察、空斑纯化和PCR检测,成功获得了PRV AH gE–/gI–,经PCR鉴定、荧光观察及测序鉴定,证实该毒株gE和gI基因被成功缺失,且不携带EGFP标记基因.生物学特性研究结果表明,该毒株增殖能力与亲本毒株相当,遗传稳定性及免疫原性良好.[结论]采用同源重组技术成功构建了免疫原性良好的猪伪狂犬病病毒流行毒株gE/gI基因缺失突变株,为研制针对流行毒株的基因缺失疫苗奠定了一定的基础.%[Objective]To obtain a candidate vaccine strain against epidemic porcine pseudorabies virus.[Method]A gE/gI-deleted transferring plasmid pMD-LA-RA and a recombinant plasmid carrying EGFP gene were constructed according to the sequence of epidemic porcine pseudorabies virus. The homologous recombination was operated between pMD-LA-EGFP-RA and PRV AH, and then the recombinant mutant virus PRV AHgE–/gI–/EGFP+ was selected using EGFP as screening marker. In order to obtain the gE/gI-deleted mutant strain PRV AHgE–/gI– without theEGFP gene, the second homologous recombination was carried out between p MD-LA-RA and PRV AH gE–/gI–/EGFP+. The proliferation ability, genetic stability and immunogenicity of PRV AH gE–/gI– were evaluated by its growth curve, continuous passage in susceptible cells and animal immunization.[Result]PRV AH gE–/gI– was obtained through two homologous recombinations with fluorescence observation, plaque purification and PCR. The deletion of gE,gI genes and EGFP gene marker in PRV AH gE–/gI– was identified by PCR, fluorescence observation and sequencing. PRV AH gE–/gI–had similar proliferation ability to the parental strain and had good genetic stability and immunogenicity.[Conclusion]A gE/gI-deleted mutant strain of epidemic porcine pseudorabies virus with good immunogenicity was constructed successfully, which provides a basis for the development of gene deleted vaccine targeting epidemic strains.