重组共表达载体pSNAV2.0 HSV1tkIREShIL1Ra的构建及其在滑膜细胞中的表达

山东大学学报(医学版) ›› 2009, Vol. 47 ›› Issue (9): 81-85.

重组共表达载体pSNAV2.0 HSV1tkIREShIL1Ra的构建及其在滑膜细胞中的表达

刘青春¹，李伟¹，张伟¹，王来城²，张捷^2，杨东¹，吴帅¹

山东大学附属省立医院 1. 关节外科； 2. 科研中心，济南 250021

收稿日期:2008-12-29 发布日期:2009-09-16
通讯作者: 李伟(1969- )，男，博士后，副主任医师，主要从事关节外科的基础与临床研究。Email:greatli2000@yahoo.com.cn
作者简介:刘青春(1980- )，男，医师，硕士研究生，主要从事关节外科的基础与临床研究。
基金资助:
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）。

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

LIU Qingchun¹, LI Wei¹, ZHANG Wei¹, WANG Laicheng², ZHANG Jie², YANG Dong¹, WU Shuai¹

1. Department of Joint Surgery; 2. Department of Scientific Research , Shandong Provincial Hospital Affiliated to Shandong University, Jinan 250021, China

Received:2008-12-29 Published:2009-09-16

摘要/Abstract

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tk、IRES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。

关键词: 胸苷激酶, 受体，白细胞介素, 滑膜, 骨关节炎

Abstract:

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. MethodsThe 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 HSV1tk were cloned into pMDIRES to construct pMDHSV1tkIREShIL1Ra. The HSV1tkIREShIL1Ra fragment was then cloned into pSNAV2.0. The recombinant plasmid pSNAV2.0HSV1tkIREShIL1Ra was constructed and identified by restriction enzyme. The recombinant plasmid was transfected into synovial cells with the Lipofectamine 2000 method. The mRNA level change of HSV1tkIREShIL1Ra was identified by reverse transcription polymerase chain reaction(RTPCR). ResultsThe recombinant plasmid was confirmed by restriction endonucleases digestion. The RTPCR results indicated that HSV1tk and hIL1Ra were effectually expressed in the transfected synovial cells. ConclusionThe recombinant plasmid pSNAV2.0HSV1tkIREShIL1Ra was successfully constructed and effectually expressed in the transfected synovial cells, which provided a sound base for gene therapy of osteoarthritis.

Key words: Thymidine kinase; Receptors, interleukin1; Synovial m embrane; Osteoarthritis

. 重组共表达载体pSNAV2.0 HSV1tkIREShIL1Ra的构建及其在滑膜细胞中的表达[J]. 山东大学学报(医学版), 2009, 47(9): 81-85.

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