重组共表达载体pSNAV2.0-HSV1-tk-IRES-hIL-1Ra的构建及其在滑膜细胞中的表达  

Construction of co-expression vector pSNAV2.0-HSV1-tk-IRES-hIL-1Ra and its expression in synovial cells

作　　者：刘青春[1] 李伟[1] 张伟[1] 王来城[2] 张捷[2] 杨东[1] 吴帅[1] 

机构地区：[1]山东大学附属省立医院关节外科,济南250021 [2]山东大学附属省立医院科研中心,济南250021

出　　处：《山东大学学报（医学版）》2009年第9期81-85,共5页Journal of Shandong University:Health Sciences

基　　金：国家自然科学基金资助项目(30672115);山东省优秀中青年科学家科研基金资助项目(02BS082)

摘　　要：目的引入内部核糖体进入位点(IRES),构建HSV1-tk与hIL-1Ra双基因共表达AAV重组载体并检测其在滑膜细胞中的表达。方法PCR扩增HSV1-tkI、RES及hIL-1Ra基因片段,分别与pMD18-T simple载体连接,测序后,hIL-1Ra与HSV1-tk先后插入pMD-IRES,构建重组质粒pMD-HSV1-tk-IRES-hIL-1Ra,酶切出HSV1-tk-IRES-hIL-1Ra片段,克隆至AAV载体质粒pSNAV2.0上,构建成重组共表达质粒pSNAV2.0-HSV1-tk-IRES-hIL-1Ra,酶切鉴定后,用脂质体转染上述质粒至原代骨关节炎(OA)滑膜细胞,RT-PCR法鉴定共表达质粒在滑膜细胞中的表达。结果酶切鉴定证实重组共表达质粒构建正确;RT-PCR检测到转染后的OA滑膜细胞中表达HSV1-tk与hIL-1Ra。结论成功构建了重组共表达质粒pSNAV2.0-HSV1-tk-IRES-hIL-1Ra;重组质粒转染OA滑膜细胞后,能表达HSV1-tk与hIL-1Ra。这为OA的基因治疗研究提供了理论依据。Objective To construct an adeno-associated virus(AAV) vector co-expressing the herpes simplex virus 1 thymidine kinase(HSV1-tk) and human interleukin-1 receptor antagonist(hIL-1Ra) gene by utilizing an internal ribosome entry site(IRES) sequence to link the two cDNAs,and to detect its expression in synovial cells.Methods The HSV1-tk、 IRES and hIL-1Ra were amplified by the polymerase chain reaction(PCR) and inserted into pMD18-T simple vector,respectively.After being verified by sequencing,the hIL-1Ra and HS...

关 键 词：胸苷激酶 受体 白细胞介素 滑膜 骨关节炎 

分 类 号：R684[医药卫生—骨科学]