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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 eukaryotic expression plasmid of human testis gene TDRG1 and study its expression. Methods Fresh normal testis tissue was obtained and total RNA was extracted. The coding sequence of TDRG1 gene was amplified by RT-PCR. The gene was cloned into eukaryotic expression vector pYD5 to construct eukaryotic expression vector The vector pYD5-TDRG1 was digested with restriction endonuclease and identified by DNA sequence analysis. The recombinant plasmid pYD5-TDRG1 was transfected into 293 cells by indirect immunofluorescence and Western Blot Method to identify the expression of the target protein. Results The coding sequence of TDRG1 gene was amplified by RT-PCR and inserted into the eukaryotic expression vector pYD5 by restriction endonuclease digestion. The recombinant plasmid pYD5-TDRG1 was identified by restriction enzyme digestion and DNA sequencing The results showed that green fluorescent was observed under fluorescence microscope 48 hours after transfection of 293 cells and the positive rate was 96.42%. The expression of target protein of about 39.8 kD was detected by Western blot. Conclusion The eukaryotic expression vector pYD5-TDRG1 of human testis gene TDRG1 was successfully constructed and the TDRG1 gene was successfully expressed in 293 cells.