表达非洲猪瘟病毒外膜蛋白CD2v的重组伪狂犬毒株的构建  

Construction of recombinant Pseudorabies virus strain expressing the out membrane CD2v protein of African swine fever virus

作　　者：李鸿鑫[1] 邵洋洋 蔺文成[1] 陈伟国 张祥斌[1] 谢青梅[1] LI Hongxin;SHAO Yangyang;LIN Wencheng;CHEN Weiguo;ZHANG Xiangbin;XIE Qingmei(College of Animal Science,South China Agricultural University,Guangzhou 510642,China)

机构地区：[1]华南农业大学动物科学学院,广州510642

出　　处：《黑龙江畜牧兽医》2021年第15期93-97,共5页Heilongjiang Animal Science And veterinary Medicine

基　　金：国家重点研发计划项目(2017YFD0502303-5);广东省重点领域研发计划项目(2019B020211004)。

摘　　要：为了构建表达非洲猪瘟病毒(African swine fever virus, ASFV)外膜蛋白CD2v的重组伪狂犬毒株并研究其生物学特性,试验利用RED/ET同源重组技术和ccdB反向筛选技术将EP402R pUC57-CMV-Flag-EP402R-BGH质粒(EP402R序列来源Georgia 2007/1株,GenBank登录号为FR682468)的EP402R基因片段替换掉pBeloBAC11-Bartha-K61载体中的TK基因片段,构建pBeloBAC11-Bartha-K61-(TK)CMV-Flag-EP402R-BGH重组质粒;再将该重组质粒转染至Vero细胞中进行病毒拯救,通过观察细胞病变、PCR方法和Western-blot鉴定得到重组伪狂犬毒株rBartha-K61-EP402R,并对其稳定性和增殖特性进行研究。结果表明:构建的重组伪狂犬毒株连续传代20次后仍能检测到EP402R基因和Flag标签蛋白;重组伪狂犬毒株与亲本毒株的增殖曲线保持一致,且病毒效价峰值可达到1×10^(5.6)TCID_(50)/mL。说明试验成功构建了表达ASFV外膜蛋白CD2v的重组伪狂犬毒株,且该毒株具有良好遗传稳定性和增殖性能。In order to construct the recombinant Pseudorabies virus strain expressing the out membrane CD2 v protein(encoded by EP402 R gene) of African swine fever virus(ASFV) and study its biological characteristics, this experiment used homologous recombination and ccdB counter-selection technique to replace TK gene of pBeloBAC11-Bartha-K61 vector with EP402 R gene of PUC57-CMV-Flag-EP402 R-BGH plasmid(the EP402 R belonged to Georgia 2007/1 strain, GenBank accession number FR682468) and construct pBeloBAC11-Bartha-K61-(TK)CMV-Flag-EP402 R-BGH recombinant plasmid. Then, the recombinant plasmid was transfected into Vero cells for virus rescue, and the recombinant Pseudorabies virus strain rBartha-K61-EP402 R was obtained through cytopathic observation, PCR and Western-blot identification;its stability and proliferation characteristics were studied. The results showed that the EP402 R gene and Flag tag protein of the constructed recombinant Pseudorabies virus strain after 20 consecutive passages could be still detected. The proliferation curve of the recombinant virus was consistent with that of the parent strain, and the peak value of the virus titer could reach 1×10^(5.6)TCID_(50)/mL. The results suggested that the experiment successfully constructed a recombinant Pseudorabies virus strain expressing the outer membrane protein CD2 v of ASFV,and the strain had good genetic stability and proliferation performance.

关 键 词：非洲猪瘟病毒 伪狂犬病毒 外膜蛋白CD2v TK基因 同源重组 载体疫苗 

分 类 号：S852.659.1[农业科学—基础兽医学]