三个猪戊型肝炎病毒ORF2基因连续片段核酸疫苗的构建  

Construction of three nucleic acid vaccines of swine hepatitis E virus ORF2 gene continuous fragments

作　　者：李斌[1,2] 苏乾莲[1,2] 赵武[1,2] 秦毅斌[1,2] 梁家幸[1,2] 肖爱欢[1,2,3] 卢冰霞[1,2,3] 李莹莹[1,2,3] 何颖[1,2] 段群棚[1,2] 姜佳佳[1,2,3] 梁保忠[1,2] 

机构地区：[1]广西兽医研究所,广西南宁530001 [2]广西畜禽疫苗新技术重点实验室,广西南宁530001 [3]广西大学动物科学技术学院,广西南宁530005

出　　处：《中国兽医科学》2013年第10期1059-1066,共8页Chinese Veterinary Science

基　　金：广西基本科研业务费专项(桂科专项10-1;桂科专项11-1);广西科技攻关项目(桂科攻10100014-4);广西水产畜牧兽医局科技计划项目(桂渔牧科1204931);广西畜禽疫苗新技术重点实验室系统性研究课题(12-071-28-A-5)

摘　　要：为了研制猪戊型肝炎(HE)基因工程疫苗,设计引物扩增猪戊型肝炎病毒(HEV)的基因片段LB1、LB2、LB3,分别与真核表达载体pEASY-M1Expression Vector直接进行T-A连接,构建得到了重组真核表达质粒pEASY-LB1、pEASY-LB2、pEASY-LB3。将这3个重组真核表达质粒分别转染293T细胞,采用实时荧光定量RT-PCR检测目的基因,并进行SDS-PAGE电泳分析和Western-blot鉴定。结果显示,重组真核表达质粒pEASY-LB1、pEASY-LB2、pEASY-LB3在转染的293T细胞中均可表达一约20.5ku的目的蛋白,且具有良好的HEV抗原性。本试验获得的3个HEV ORF2连续片段的候选核酸疫苗为今后进一步研制HEV核酸疫苗奠定了基础。In order to develop swine hepatitis E（HE） genetically engineering vaccines,specific primers of genes LB1, LB2, LB3 of swine hepatitis E virus were designed and used for amplification, DNA amplicons generated by PCR assays,were directly cloned into T-A plasmid and expressed using pEASY-M1 expression vector. Three recombinant eukaryotic expression plasmids of pEASY-LB1, pEASY-LB2 and pEASY-LB3 were constructed. The eukaryotic expression plasmids of pEASY-LB1, pEASY-LB2,pEASY-LB3 were transfected into 293T cells,and three target genes were detected by real-time fluorescent quantitative RT-PCR. The results confirmed that three eukaryotic expression plasmids were transfected into 293T cells and target protein were expressed. Analysis by SDS-PAGE electrophoresis and Western-blot analysis indicated that three target proteins were expressed 293T cells transfected by eUkaryotic expression plasmids of pEASY-LBI,pEASY-LB2 and pEASY-LB3. Antigenicity studies indicated good HEV responses. Therefore,three recombinant DNA of HEV ORF2 nucleic acid vaccine candidates were obtained, which might lay the foundation for further studies in the future.

关 键 词：猪 戊型肝炎病毒 ORF2 真核表达质粒 核酸疫苗 

分 类 号：S852.659.6[农业科学—基础兽医学]