表达PEDV S1蛋白重组PRV的构建与鉴定  

Construction and characterization of recombinant PRV expressing S1 protein of PEDV

作　　者：侯晓璇 穆永 王彪 王同燕 颜世君 王孟月 谭菲菲 田克恭 HOU Xiao-xuan;MU Yong;WANG Biao;WANG Tong-yan;YAN Shi-jun;WANG Meng-yue;TAN Fei-fei;TIAN Ke-gong(College of Veterinary Medicine,Henan Agricultural University,Zhengzhou 450046,China;National Research Center for Veterinary Medicine,Luoyang 471000,China;PULIKE Biological Engineering,INC.,Luoyang 471000,China)

机构地区：[1]河南农业大学动物医学院,河南郑州450046 [2]国家兽用药品工程技术研究中心,河南洛阳471000 [3]普莱柯生物工程股份有限公司,河南洛阳471000

出　　处：《中国兽医科学》2023年第8期1003-1009,共7页Chinese Veterinary Science

基　　金：郑洛新自创区创新引领型产业集群专项(201200211200)。

摘　　要：为了有效预防PED的感染,本研究将PEDV流行毒株的S1基因克隆至pcDNA3.1(+)载体中构建真核表达质粒,经鉴定S1蛋白表达后,获得S1基因表达盒,再将S1基因表达盒克隆至含有gG基因上下游同源臂以及抗性筛选基因的转移质粒PUC-ABK中,构建供体质粒PUC-ABK-CMV-S1,获得线性同源重组片段A-CMV-S1-K-B。利用Red同源重组技术将线性同源重组片段电转化至含有pPRVBac-GFP-TK--gE--gI--US9--2-的感受态中获得重组pPRVBac-S1,最后拯救获得重组病毒rPRV-S1。PCR及IFA鉴定结果显示S1基因成功转录且表达,为研制PEDV活载体疫苗提供了实验依据。In order to effectively prevent PED infection,S1 gene of PEDV epidemic strain was cloned into pcDNA3.1(+)vector in this study to construct eukaryotic expression plasmid,and S1 gene expression box was obtained after S1 protein expression was identified.Then the S1 gene expression box was cloned into the transfer plasmid PUC-ABK containing the upper and downstream homologous arms of gG gene and the resistance screening gene.The donor plasmid PUC-ABK-CMV-S1 was constructed,and the linear homologous recombinant fragment A-CMV-S1-K-B was obtained.Red homologous recombination technique was used to electrically transform the linear homologous recombination fragment into the receptive state containing pPRVBac-GFP-TK--gE--gI--US9--2-to obtain recombinant pPRVBac-S1,and finally to rescue and obtain recombinant virus rPRV-S1.PCR and IFA identification results showed that S1 gene was successfully transcribed and expressed,providing experimental basis for the development of PEDV live vector vaccine.

关 键 词：PEDV S1蛋白 RED同源重组 重组PRV 

分 类 号：S852.659.6[农业科学—基础兽医学]