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1、华南农业大学学报 2018, 39(2): 9-15http://xuebao.scau.edu.cnJournal of South China Agricultural Universitydoi:10.7671/j.issn.1001-411X.2018.02.002潘慧, 李艳华, 向柯宇, 等. 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[J]. 华南农业大学学报, 2018, 39(2): 9-15.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潘 慧†，李艳华†，向柯宇，唐 栋，程珍珠，罗永文，琚春
2、梅(华南农业大学兽医学院/广东省动物源性人兽共患病预防与控制重点实验室，广东广州 510642)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E
3、GFP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 AHgE–/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
4、的基础。关键词:猪伪狂犬病病毒；流行毒株；基因缺失；同源重组；生物学特性中图分类号:S852.651 文献标识码:A文章编号:1001-411X(2018)02-0009-07ConstructionofagE/gI-deletedmutantstrainofepidemicporcinepseudorabiesvirusanditsbiologicalcharacteristicsPAN Hui†, LI Yanhua†, XIANG Keyu, TANG Dong, CHENG Zhenzhu, LUO Yongwen, JU Chunmei(Co
5、llege of Veterinary Medicine, South China Agricultural University /Key Laboratory of Zoonosis Prevention andControl of Guangdong Province, Guangzhou 510642, China)Abstract:【Objective】To obtain a candidate vaccine strain against epidemic porcine pseudorabies virus.【Method】AgE/gI-
6、deleted transferring plasmid pMD-LA-RA and a recombinant plasmid carryingEGFPgenewere constructed according to the sequence of epidemic porcine pseudorabies virus. The homologousrecombination was operated between pMD-LA-EGFP-RA and PRV AH, and then the recombinant mutant virusPR
7、V AHgE–/gI–/EGFP+was selected using EGFP as screening marker. In order to obtain thegE/gI-deletedmutant strain PRV AHgE–/gI–without theEGFPgene, the second homologous recombination was carried outbetween pMD-LA-RA and PRV AHgE–/gI–/EGFP+. The proliferation ability, genetic stabi
8、lity andimmunogenicity of PRV AHgE–/gI–were eva
