伪狂犬病病毒流行株gE/gI双基因缺失弱毒株的制备  

Construction of gE/gI double gene deletion attenuated strain of Pseudorabies virus

作　　者：于泽坤 只勇 张伦 段笑笑 李思菲 马广斌 单学强 YU Zekun;ZHI Yong;ZHANG Lun;DUAN Xiaoxiao;LI Sifei;MA Guangbin;SHAN Xueqiang(Shandong Sinder Technology Co.,Ltd.,Qingdao 266000,China;Qingdao Animal Disease Prevention and Control Center,Qingdao 266000,China)

机构地区：[1]山东信得科技股份有限公司,山东青岛266000 [2]青岛市动物疫病预防控制中心,山东青岛266000

出　　处：《黑龙江畜牧兽医》2021年第13期75-80,147,148,共8页Heilongjiang Animal Science And veterinary Medicine

基　　金：山东新旧动能转换重大工程重大课题攻关项目“基于大规模细胞悬浮培养技术的畜禽病毒基因工程疫苗的研究与创制”。

摘　　要：为了降低伪狂犬病病毒(Pseudorabies virus,PRV)QD株毒力,获得候选疫苗株,试验通过同源重组方法同时缺失非必需蛋白gE和gI,构建含同源臂及EGFP表达盒片段的重组质粒pB-arm、pB-EGFP-arm,将重组质粒pB-EGFP-arm与母本毒株PRV QD基因组共转染至PK-15细胞后通过蚀斑纯化得到PRV QD-gE^(-)/gI^(-)/EGFP+株,再将重组质粒pB-arm与PRV QD-gE^(-)/gI^(-)/EGFP+株基因组共转染至PK-15细胞,通过蚀斑纯化删除EGFP基因得到PRV QD-gE^(-)/gI^(-)株;采用PCR扩增和Western-blot分别测定PRV QD-gE^(-)/gI^(-)株gE、gI基因mRNA转录和gE蛋白表达情况;绘制缺失毒株的增殖曲线并测定缺失基因的传代稳定性,比较缺失毒株与母本毒株的小鼠半数致死量(LD50)差异。结果表明:PRV QD-gE^(-)/gI^(-)株无gE和gI基因mRNA转录,gE蛋白未表达。缺失毒株对PK-15细胞的半数感染量(TCID50)为1×10^(-8.65)/0.1 mL,与母本毒株比较差异不显著(P>0.05),连续传至15代时缺失基因未改变。缺失毒株对小鼠的LD50为1×10^(-1.84)/0.2 mL,低于母本毒株的1×10^(-4.16)/0.2 mL。说明PRV QD株通过缺失gE和gI基因后毒力减弱,可作为制备PRV疫苗的候选毒株。In order to reduce the virulence of Pseudorabies virus(PRV) QD strain to obtain a candidate vaccine strain, the experiment was conducted to delete the non-essential proteins gE and gI simultaneously through homologous recombination. Recombination plasmids pB-arm and pB-EGFP-arm containing the homologous arms on both sides of the deleted gene and the EGFP reporter gene were constructed, and the recombinant plasmid pB-EGFP-arm and the maternal strain PRV QD genome were co-transfected into PK-15 cells and then purified by plaque to obtain PRV QD-gE^(-)/gI^(-)/EGFP+ strain;then the recombinant plasmid pB-arm and the PRV QD-gE^(-)/gI^(-)/EGFP+ strain genome were co-transfected into PK-15 cells, and the EGFP gene was deleted through plaque purification to obtain PRV QD-gE^(-)/gI^(-)strain. PCR amplification and Western-blot identification were used to determine the mRNA transcription of gE and gI genes and the expression of gE protein of PRV QD-gE^(-)/gI^(-)strain. The proliferation curve of the deleted strain was drawn, and the passage stability of the deleted gene was determined, as well as the difference in LD50 between the deleted strain and the maternal strain. The results showed that PRV QD-gE^(-)/gI^(-)strain had no gE and gI gene mRNA transcription, and gE protein was not expressed. The half-infectious dose(TCID50) of the deleted strain on PK-15 cells was 1×10^(-8.65)/0.1 mL,which was not significantly different from the maternal strain(P>0.05);the deletion gene remained unchanged until the 15 th generation. The LD50 of the deletion strain to mice was 1×10^(-1.84)/0.2 mL,which was lower than the maternal strain(1×10^(-4.16)/0.2 mL). The results suggested that the virulence of PRV QD strain weakened after the deletion of gE and gI genes, and it can be used as a candidate strain for preparing PRV vaccine.

关 键 词：伪狂犬病病毒 非必需蛋白 糖蛋白E 糖蛋白I 基因缺失 致弱毒株 

分 类 号：S852.655[农业科学—基础兽医学]