人T-bet基因的体外扩增及克隆和鉴定

Amplification,clone and identification of human T-bet gene in vitro

目的:克隆人T-bet基因,构建其真核表达载体pEGFP-C1-T-bet。方法:实验于2005-07/2006-07在北京大学深圳医院中心实验室完成。①根据Genebank的人T-bet基因的全长cDNA序列,设计合成一对附加BglⅡ和SalⅠ两个限制性内切酶酶切位点的特异性引物。②分离人外周血单个核细胞,提取RNA,用反转录-聚合酶链反应方法将T-bet的编码序列cDNA扩增,装入pMD18-T载体并送去测序。③BglⅡ+SalⅠ双酶切质粒pMD18-T-bet,经琼脂糖电泳切胶回收T-bet片段,将T-bet插入载体pEGFP-C1构建成重组真核表达载体pEGFP-C1-T-bet。④用双酶切和聚合酶链反应对插入片段进行分析和验证。结果:①反转录-聚合酶链反应产物经琼脂糖电泳结果显示在预期位置有相对分子质量为1608bp的特异性扩增带。②测序结果证实,T-bet的编码序列和Genbank中T-bet mRNA序列相同。③双酶切和聚合酶链反应结果证实插入片段序列正确。结论:实验成功扩增出人T-bet基因,构建了基因重组真核表达载体pEGFP-C1-T-bet,为探索T-bet基因对免疫细胞的调节作用和肿瘤基因治疗奠定了基础。

AIM： To clone human T-bet gene and construct a eukaryotic expression vector, pEGFP-C1-T-bet. METHODS： The experiment was conducted at the Central Laboratory, Shenzhen Hospital of Peking University from July 2005 to July 2006. ①A pair of specific primers with restriction enzyme sites of Bgl Ⅱ and SalⅠ were designed and synthesized according to the gene sequence of human T-bet cDNA of Genbank. ②The total RNA was isolated from human peripheral blood mononuclear cells （PBMC） and the T-bet cDNA was amplified from the mRNA by RT-PCR technique, and then the PCR products of T-bet was inserted into pMD18-T vector and sequencing. ③The pMD18-T-bet was double enzyme digested by Bgl Ⅱ and SalⅠ and the T-bet segment was extracted by agarose electrophoresis gel. Then the T-bet gene was inserted into pEGFP-C1 to construct the recombinant expression vector, pEGFP-C1-T-bet.④The inserted segment was analyzed and identified with double enzyme digestion and PCR. RESULTS：①Gel electrophoresis results of the product of RT-PCR showed that specific proliferative band with relative molecular mass of 1 608 bp appeared at the prospective site.②The coding sequence of T-bet was confirmed by sequencing that it was identical with the sequence of human T-bet mRNA in Genbank. ③The inserted fragment was confirmed by double enzyme digestion and PCR analysis. CONCLUSION： The recombinant eukaryotic expression vector pEGFP-C1-T-bet is successfully constructed after successful amplification of complete T-bet gene. It lays a foundation for the tumor immunity and cancer gene therapy.