携带小鼠HVEM的重组腺相关病毒载体的构建及鉴定  

Construction and Identification of Recombinant Mouse HVEM Adeno-associated Virus

作　　者：马衣努尔.买提托合提 韩凌斐[1] 廖术杰[1] 周志刚[1] 董红[1] 马丁[1] 王世宣[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院妇产科,武汉430030

出　　处：《华中科技大学学报（医学版）》2010年第2期179-183,共5页Acta Medicinae Universitatis Scientiae et Technologiae Huazhong

基　　金：国家重点基础研究发展计划资助项目(No.2009CB521800);国家自然科学青年基金资助项目(No.30901586)

摘　　要：目的克隆小鼠疱疹病毒侵入介体(HVEM)基因,构建其重组腺相关病毒载体,鉴定目的基因在真核细胞中表达。方法用RT-PCR法从小鼠脾脏淋巴细胞克隆出全长HVEM基因,构建携带HVEM基因的穿梭质粒pAAV-IRES-HVEM-hrGFP和辅助质粒pAAV-RC及pAAV-Helper,利用磷酸钙共沉淀法将穿梭质粒共转染入AAV-293细胞中,采用细胞内质粒DNA同源重组法构建重组腺相关病毒rAAV-HVEM。用氯仿-PEG8000法纯化病毒,实时荧光定量PCR测病毒滴度。RT-PCR检测rAAV-HVEM在中国仓鼠卵巢(CHO)细胞中的表达情况。结果成功构建组装重组小鼠HVEM腺相关病毒,纯化后病毒滴度为5×1010v.g/mL,且在CHO转导细胞中能有效表达目的基因。结论构建的重组腺相关病毒载体rAAV-HVEM为组织工程相关细胞转基因构建及临床应用提供先进的载体系统,为今后进一步研究HVEM的功能及其在部分疾病基因免疫治疗中的应用奠定基础。Objective To clone mouse herpes virus entry mediator（HVEM） gene into adeno-associated virus（AAV） vector pAAV-IRES-hrGFP and obtain high titer and purify recombinant mouse HVEM-AAV,and identify the expression of targeting gene in CHO cells.Methods Framework plasmid of recombinant HVEM-AAV was constructed by gene recombination.The recombinant plasmids pAAV-IRES-HVEM-hrGFP,pAAV-RC and pAAV-Helper were co-transfected into AAV-293 cells according to the calcium phosphate based protocol and produced recombinant virus rAAV-HVEM was purified by PEG/chloroform.The purity of recombinant rAAV-HVEM was verified by SDS-PAGE.The titer of the virus particles was detected by quantitative real-time PCR.HVEM expression in CHO cells which were infected by recombinant rAAV-HVEM was detected by RT-PCR and FACS analysis.Results Recombinant mouse rAAV-HVEM was successfully constructed with high titer and high purity.After transfection recombinant AAV-HVEM mediated a stable expression of HVEM mRNA in CHO cells.Conclusion Recombinant mouse rAAV-HVEM is successfully constructed,which will benefit for further study on the function of HVEM and its applications.

关 键 词：疱疹病毒侵入介体 腺相关病毒 基因治疗 

分 类 号：R349.6[医药卫生—基础医学]