肠道病毒71型真核双基因表达载体的构建  

Construction and identification of the eukaryotic coexpression plasmid p-IRES-P1-3CD

作　　者：姚相杰[1] 何雅青[1] 蔡春林[2] 杨洪[1] 冼慧霞[1] 张海龙[1] 罗敏[1] 张仁利[1] 

机构地区：[1]广东省深圳市疾病预防控制中心微生物检验科,518055 [2]广东省深圳市罗湖区疾病预防控制中心微生物检验科,518020

出　　处：《检验医学与临床》2012年第23期2916-2918,共3页Laboratory Medicine and Clinic

基　　金：广东省医学科研基金(B2010287);广东省深圳市罗湖区软科学计划(2010069)

摘　　要：目的通过克隆肠道病毒71型结构前体蛋白P1和非结构蛋白3CD,构建共表达P1前体蛋白和非结构蛋白3CD的真核双基因表达载体。方法设计合成脑心肌炎病毒(EMCV)IRES序列的特异性引物,克隆至真核表达载体pcDNA3.0上,命名为pc-IRES。从手足口病重症患者分离EV71病毒株,经逆转录聚合酶链式反应(RT-PCR)技术分别扩增出EV71病毒的P1前体蛋白区与3CD区的片段,并将其定向克隆至真核表达载体pc-IRES载体中IRES序列的上游和下游,随后转化到大肠埃希菌DH5α中,最后经PCR和双酶切鉴定转化菌落。结果通过重组质粒p-IRES-P1-3CD进行酶切和DNA序列分析,重组真核双基因表达载体p-IRES-P1-3CD构建成功。结论成功构建共表达EV71病毒P1前体蛋白和非结构蛋白3CD真核双基因表达载体,这将为下一步制备基于DNA载体的EV71病毒的类病毒颗粒疫苗打下基础。Objective To amplify P1 and 3CD gene from patient infected with EV71 and construct the eukaryotic co-expression plasmid encoding P1 and 3CD. Methods The internal ribosomal entry site（IRES） from encephalomyocarditis virus（EMCV） was amplified and inserted into eukaryotic vector pcDNA3.0, named pc-IRES. Then the cDNA encoding P1 and 3CD were obtained by RT-PCR amplification from the fecal of patient infected with EVT1 and cloned into pc-IRES vector. The constructed plasmid p-IRES-P1-3CD was confirmed by restriction enzymolysis and PCR. Results A 2.5 kb and 1.9 kb fragment were obtained from amplification,the sequences were confirmed to be EV71 P1 and 3CD gene by sequence analysis. PCR and double endonucleases results confirmed that the eukaryotic coexpression plasmid p-IRES-P1-3CD was successfully constructed. Conclusion The successful construction of eukaryotic expression plasmid p-IRES-P1-3CD establishes an experimental basis for virus like particle vaccine of EV71.

关 键 词：肠道病毒71型 类病毒颗粒 疫苗 

分 类 号：R392.1[医药卫生—免疫学]