犬瘟热病毒核蛋白基因的克隆及原核及真核表达.pdf

犬瘟热病毒核蛋白基因的克隆与原核及真核表达研究生:宗卫峰导师:李厚达教授季明春副教授中文摘要犬瘟热是严重威胁我国养犬业发展的主要传染病之一,免疫接种是预防此病的重要措施;为克服常规疫苗的不足,我们构建了犬瘟热核蛋白洲)基因的重组质粒,这为研制新一代安全高效、使用方便的核酸疫苗打下基础。以犬瘟热病毒OP株感染Vero细胞收获病毒提取的RNA为模扳,根据已发表的犬瘟热病毒OP株序列设计两对引物,RT—PCR扩增出N基因的822bp、897bp片段。(GST)基因的下游,再将重组质粒转化进宿主菌BL2l中,在37℃ IPIG的诱导下,核蛋白基因分段获得了良好的表达,表达产物经聚丙烯酰胺凝胶电泳鉴定,确定其表达融合蛋白质的大小分别52kDa、54kDa。将表达产物回收后混合免疫小鼠,间接免疫荧光试验(IFA)显示免疫小鼠的血清能与病毒感染的细胞呈现特异反应,表明体外表达核蛋白基因保留了天然蛋白部分的抗原性。设计含EcoR I和BamH I酶切位点的一对引物,采用RT—PCR法从犬瘟热OP 株感染Vero细胞收获的病毒中扩增出CDV N全段,定向克隆到真核表达载体 (一)中,转化感受态大肠杆菌DHs。,经氨苄抗性和限制性内切酶酶切等鉴定,证实构建成功;,:并与PEI混合肌注小鼠,ELISA检测证明获得了较高的抗体滴度,为进一步研究CDV核蛋白基因的功效及制备核酸疫苗奠定物质基础。关键词:犬瘟热病毒、核蛋白基因、克隆、表达、转染、间接免疫荧光 Cloning and Expression ofCanine Distemper Virus Nucleoc'apsid Protein Gene — :ZongWeifeng Advisor: Houda ABSTRACT Canine distemper is ahighly infectious,frequently lethaldiseaseindogs and other order todevelop DNA ines which are safe,convenient for use for canine distemper,we designed aseriesofexperimentS toreachthegoal. The templates were produced fromthe reverse transcripfion reaction thatRNA WaS isolated from OP—CDV infected Vero pairs of primers had been designed andsynthesized based on t11eNucteocapsidm、protein gene oftheonderstepoort strainofcanine distemper virus(CDV).822bp and 897bp fragments of N gene were amplified bypolymerase chainreaction(PeR) products were cloned intotheexpressing plasmid vector pGEX-6P— binant was verified by restricfion endonuclease analysis and two binants were transformed intO thehoststrainBL2l and expressed by IPTG indueed at37℃. n埭expression products were identifiedby SDS—PAGE and found tobe52kDa and 54kDa band insize astwo fusionproteins withglutathione Stransferase(GST)proteiu. nlespecific bands ofexpression wereexcised from thegel mixed andinjeeted intothe mice aweek andlast specifi