KSHV K15P慢病毒载体的构建及其滴度测定  

Construction and the titer determination of lentivirus vector containing KSHV K15P gene

作　　者：许常青[1,2] 陈伟[1] 方圆[1] 汪小五[1] 王林定[1] Xu Changqing Chen Wei Fang Yuan et al(Dept of Microbiology, Anhui Medical University, Hefei 230032 Dept of Clinical Laboratory, The Third People＇ s Hospital of Hefei, Hefei 230000)

机构地区：[1]安徽医科大学微生物学教研室,合肥230032 [2]合肥市第三人民医院检验科,合肥230000

出　　处：《安徽医科大学学报》2016年第10期1417-1420,共4页Acta Universitatis Medicinalis Anhui

基　　金：国家自然科学基金(编号:81271837);安徽省高校省级科学研究项目(编号:KJ2012A161);安徽医科大学博士科研经费资助项目(编号:XJ200914)

摘　　要：目的构建含卡波肉瘤相关疱疹病毒(KSHV)ORF K15P基因的慢病毒载体,并对其在293T细胞内的表达进行研究。方法 PCR法扩增获得KSHV K15P基因,限制性内切酶Nhe I酶切和无缝克隆法构建重组慢病毒载体p CDHCMV-K15P-GFP,通过脂质体转染试剂将重组慢病毒载体与3种辅助质粒p MDL-PRRE、pRSV-Rev和p MD2.g共转染HEK 293T细胞包装产生慢病毒,Western blot法检测K15P蛋白的表达。结果经酶切及测序鉴定,成功构建慢病毒载体,重组慢病毒质粒与辅助质粒共转染HEK 293T细胞可见有荧光表达,说明K15P基因在细胞内有表达并且96 h后产生4×106TU/ml滴度为慢病毒。结论成功构建了KSHV K15P慢病毒表达载体p CDH-CMV-K15P-GFP,获得高效表达K15P基因的慢病毒颗粒,为后续其在细胞内的功能研究奠定实验基础。Objective To construct the lentivirus vector containing Kaposi sarcoma-associated herpesvirus（KSHV） ORF K15P and study its expression and functions in 293T cells. Methods The gene region of K15P, which was amplified by ordinary PCR, was cloned into pCDH-CMV-GFP vector with Nhe I restriction enzyme. The recombi- nant plasmid was then transiently tansfeeted into HEK 293T cell. The pCDH-CMV-K15P-GFP plasmid was co- transfected with three assisted plasmids of pMDL-PRRE, pRSV-Rev,pMD2, g into HEK 293T cell to produce lenti- virus particles after 96 h and Western blot was performed to detect the expression of K15P protein. Results The lentivirus vector was successfully constructed and the titer of lentivirus was 2 ×106 transducing units/ml at the time of 96 hours after eo-transfected. Conclusion K15P expression lentiviral vector is successfully constructed. It buihs an experimental basis for the K15P functional study in 293T cells.

关 键 词：卡波肉瘤相关疱疹病毒 K15P基因 慢病毒 

分 类 号：R394.3[医药卫生—医学遗传学] R373.9[医药卫生—基础医学]