人睾丸基因TDRG1重组真核载体的构建及其表达  

Construction of TDRG 1 eukaryotic expression recombinant vector and its expression in 293 cell lines

作　　者：汤育新[1] 蒋先镇[1] 文甲明[1] 陈厚仰[1] 阳建福[1] 肖小旺[1] 戴英波[1] 

机构地区：[1]中南大学湘雅三医院中美男性病研究中心中南大学湘雅三医院泌尿外科,长沙410013

出　　处：《中国男科学杂志》2010年第8期6-9,共4页Chinese Journal of Andrology

基　　金：国家自然科学基金资助课题(30672090)

摘　　要：目的构建人睾丸基因TDRGl的真核表达质粒,并研究其表达。方法取人新鲜正常睾丸组织,提取总RNA,采用逆转录-聚合酶联链反应(RT-PCR)扩增TDRG1基因编码序列;将该基因克隆到真核表达载体pYD5中,构建真核细胞表达载体pYD5-TDRG1,用限制性内切酶酶切分析,DNA序列分析鉴定重组质粒;将测序正确的重组质粒pYD5-TDRG1在脂质体介导下转染293细胞,间接免疫荧光法和Western Blot法鉴定目的蛋白质的表达。结果 RT-PCR扩增出TDRG1基因编码序列,目的插入片段长约303bp;产物行限制性内切酶酶切后连接到真核表达载体pYD5,重组质粒pYD5-TDRG1经酶切及DNA测序鉴定构建成功;该质粒转染293细胞48h后在荧光显微镜下可观察到绿色荧光,阳性细胞率96.42%;行Western blot分析检测到约39.8kD的目的蛋白表达。结论成功构建了人类睾丸基因TDRG1的真核表达载体pYD5-TDRG1,TDRG1基因在293细胞内成功表达。Objective To construct TDRG1 eukaryotic expression recombinant vector and identify its expression in 293 cell lines. Methods Total mRNA was extracted from normal human testis tissue, and the coding sequence of TDRG1 was obtained by RT-PCR. The TDRG1 gene was cloned into pYD5 vector and the recombinant vector of pYD5-TDRG1 was constructed and transfected into 293 cell lines. The expression of target protein TDRG1 was deteced by immunofluorescence analysis and Western blot. Results A fragment of 5 000bp and inserted fragment of 303bp were identified by positive recombinant plasmid of pYD5-TDRG1 digested with dual endonuclease BamHI / HindⅢ, Automatic DNA sequence analysis demonstrated that sequence of the recombinant plasmid pYD5-TDRG1 was totally the same as the sequence of TDRG1. The expression of green fluorescence protein was detected by fluorescence microscope and a protein band with 39.8 kD was detected by Western Blot. Conclusion The eukaryotic expression recombinant vector of pYD5-TDRG1 was constructed and expressed successfully in 293 cell lines.

关 键 词：TDRG1基因 克隆 真核载体 基因转染 

分 类 号：R392-33[医药卫生—免疫学]