人T细胞免疫球蛋白黏蛋白-4 cDNA全长真核表达载体的构建及其在16HBE细胞中的表达  

Construction of eukaryotic expression vector for human T cell immunoglobulin mucin protein-4 and its expression in 16HBE cells

作　　者：吴小慧[1] 李一荣[1] 陈凤花[1] 王琳[1] 胡丽华[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院检验科,武汉430022

出　　处：《免疫学杂志》2010年第4期304-308,共5页Immunological Journal

基　　金：国家自然科学基金资助项目(30672008)

摘　　要：目的克隆人T细胞免疫球蛋白黏蛋白-4(TIM-4)基因cDNA,构建其真核表达载体pEGFP-C2-TIM-4,并转染16HBE细胞。方法根据GeneBank中人TIM-4 cDNA序列(编号:NM-138379.2),设计出带有EcoRI和BamHI的上下游引物,应用RT-PCR技术,从人骨髓中扩增出TIM-4基因编码区序列,克隆到pMD18-T载体,形成pMD18-T-TIM-4重组质粒,经菌落PCR,双酶切,测序鉴定获得人TIM-4基因cDNA片段,然后将其亚克隆入pEGFP-C2绿色荧光载体中,并通过菌落PCR,双酶切,测序鉴定其重组体。将测序正确的pEGFP-C2-TIM-4质粒转染人气道上皮细胞(16HBE),用实时定量PCR检测目的基因的表达。结果成功扩增出人TIM-4 cDNA全长1134 bp,经T-A克隆构建的pMD18-T-TIM-4重组质粒经菌落PCR,双酶切,测序证实载体中含有正确的TIM-4编码区片段。由此构建的真核表达载体pEGFP-C2-TIM-4,经菌落PCR扩增出1134 bp左右的片段,经双酶切后产生4.7 kb和1134 bp左右的2条带,DNA测序显示与GeneBank中人TIM-4 cDNA序列一致。重组质粒转染16HBE细胞在荧光显微镜下可见绿色荧光,经实时定量PCR分析,转染细胞TIM-4基因的表达显著增加。结论首次从人骨髓中扩增出TIM-4基因cDNA全长,构建了其真核表达载体pEGFP-C2-TIM-4,并在16HBE细胞内成功高表达,为进一步研究TIM-4的生物学功能奠定了基础。In this study,we construct a eukaryotic expression vector of the entirely coding human T cells immunoglobulin mucin protein -4（TIM-4） and transfect it into 16HBE cell line,for benefiting the further study on biological function of TIM-4.According to TIM-4 cDNA sequence in GeneBank（NO：NM-138379.2）,we designed two primers which have sites of EcoRI and BamHI,respectively.The TIM-4 cDNA was amplified by RT-PCR from human bone marrow and then cloned into pMD18-T vector to obtain recombinant vector pMD18-T-TIM-4,which was verified by colony PCR,double enzyme digestion,and sequencing.Then the identified TIM-4 cDNA fragment was subcloned into pEGFP-C2 plasmid.The recombination vector pEGFP-C2-TIM-4 was identified by colony PCR,double enzyme digestion and sequencing.After confirmation with sequence analysis,the expression vector pEGFP-C2-TIM-4 was transfected into the 16HBE cells,and then the expression of TIM-4 in transfected cells was detected by Real-Time PCR.The entirely coding human TIM-4 gene fragment was about 1 134 bp.The recombinant pEGFP-C2-TIM-4 was identified by colony PCR,and the amplification fragment was about 1 134 bp.By double enzyme digestion,the recombinant was digested into about 1134 bp and 4.7 kb fragments.The DNA sequencing revealed the inserted fragment was identical with human TIM-4 cDNA in GeneBank.Green fluorescence could be observed in the pEGFP-C2-TIM-4-transfected cells;the expression of TIM-4 was significantly increased in the transfected cells.In our studies,the human TIM-4 cDNA was amplified from human bone marrow for the first time;furthermore its eukaryotic expression vector pEGFP-C2-TIM-4 was successfully constructed and highly expressed in 16HBE cells,which will provide a foundation for the further research.

关 键 词：T细胞免疫球蛋白黏蛋白-4(TIM-4) 基因克隆 真核表达载体 聚合酶链反应(PCR) 转染 

分 类 号：R392.11[医药卫生—免疫学]