小鼠B、T淋巴细胞衰减因子真核表达载体的构建及在HEK293细胞中的表达  

Construction of eukaryotic expression vector of murine B and T lymphocyte attenuator and its expression in HEK293 cells

作　　者：夏丽坤[1] 张胜男[1] 陈琳琳[2] 周晶[2] 

机构地区：[1]中国医科大学附属盛京医院眼科,沈阳110004 [2]沈阳市第四医院眼科,110031

出　　处：《眼科研究》2010年第4期295-299,共5页Chinese Ophthalmic Research

基　　金：国家自然科学基金(30772394);辽宁省自然科学基金(20072091);辽宁省科技攻关计划项目(2007225018);沈阳市科学技术计划项目(1091171-1-02)资助

摘　　要：目的构建小鼠B、T淋巴细胞衰减因子(BTLA)的真核表达载体,并分析其在人胚肾HEK293细胞中的表达,为单纯疱疹性角膜基质炎的免疫治疗奠定基础。方法应用PCR法获得小鼠BTLA胞外功能区的cDNA片段,将其插入真核表达载体pcDNA3.1中,获得重组表达质粒pBTLA,经酶切鉴定和测序正确后转染HEK293细胞,并利用间接免疫荧光法、实时定量PCR(real-time PCR)、Western blot技术检测BTLA在细胞中的表达。结果重组质粒pBTLA经酶切鉴定和测序,确认插入序列正确,与基因文库中提供的基因序列同源性达98%～100%。Real-time PCR检测可见转染了重组质粒pBTLA的HEK293细胞内BTLA mRNA表达增强(P=0.00),3个组间HEK293细胞内BTLA mRNA表达差异有统计学意义(F=167.83,P=0.00);Western blot结果显示,转染重组质粒pBTLA的HEK293细胞内BTLA蛋白表达明显增强,转染后24～48 h表达最强。间接免疫荧光结果显示BTLA蛋白主要表达在HEK293细胞的细胞膜和细胞质中。结论成功构建了BTLA的真核表达载体,该重组质粒在HEK293细胞中能够表达BTLA,为后续研究BTLA在抗病毒免疫中的作用奠定基础。Background The combination of glycoprotein of herpes simplex virus type 1（HSV-1） with its ligant,herpes virus entry mediator（HVEM） to destroy the cellular membrane is a key step of invasion of virus into host and causing immunoreaction.And the association of B and T lymphocyte attenuator（BTLA） with HVEM can downregulate the immune response.ObjectiveThe aim of this study is to construct the eukaryotic expression vector of murine BTLA and investigate the expression of BTLA in the transfected HEK293 cells for the further study on the immunotherapy of herpetic stromal keratitis.MethodsThe cDNA fragment expressing murine BTLA was obtained by PCR and then inserted into the eukaryotic plasmid pcDNA3.1 to get recombinant plasmid pBTLA.The recombinant plasmid pBTLA was transfected into the HEK293 cell line to express the target protein BTLA after identified by restriction enzyme digestion and direct sequencing.The expression of BTLA gene,BTLA protein in transfected cells were detected by real-time PCR,indirect immunofluorescence and Western blot assay respectively.ResultsRestriction enzyme digestion and sequencing showed that BTLA cDNA fragment was successfully detected in the vector with the homology of 98%-100% with the data from GenBank.Transfection of recombinant plasmid pBTLA into HEK293 cells induced effective expression of BTLA,presenting the green fluorescence on cell membrane and cytoplasm under the fluorescence microscope.BTLA mRNA from transfected pBTLA cells was obviously increased by real-time PCR （F=167.83,P=0.00）,and Western blot assay revealed that BTLA protein showed the enhanced expression of BTLA protein in HEK293 cells in transfected pBTLA group with the strongest expression from 24 through 48 hours after transfected.ConclusionRecombinant eukaryotic expression vector harboring murine BTLA cDNA is successfully obtained and can be effectively expressed in HEK293 cells.The construction might offer a foundation for further researches on the immunotherapy of BTLA for herpetic stromal keratit

关 键 词：B、T淋巴细胞衰减因子 真核表达载体 基因转染 

分 类 号：R392.12[医药卫生—免疫学] R772.21[医药卫生—基础医学]