人resistin基因原核表达载体构建和表达  

Construction and Expression of A Prokaryotic Expression Vector Containing Human Resistin Gene

作　　者：何祥梁[1] 何东华[2] 黎锋[3] 杨川[3] 程桦[3] 傅祖植[3] 

机构地区：[1]中山大学附属第一医院急诊科,广东广州510080 [2]中山大学达安基因公司,广东广州510080 [3]中山大学附属第二医院内分泌科,广东广州510080

出　　处：《中山大学学报（医学科学版）》2004年第5期413-416,共4页Journal of Sun Yat-Sen University:Medical Sciences

基　　金：中国博士后科研启动基金资助项目(第31期)

摘　　要：【目的】构建人resistin基因原核表达载体并观察其表达,为开展对resistin的研究打下实验基础。【方法】酚氯仿一步法从一位2型糖尿病合并胆管癌患者腹壁皮下脂肪组织抽取总RNA,RT-PCR法扩增出re-sistin基因cDNA,经测序后,PCR产物亚克隆入T载体,用EcoRI和KpnI分别酶切重组T质粒和原核表达载体pGENKS,然后resistin基因片段和表达载体通过T4DNA连接酶进行定向连接,用核酸内切酶EcoRI、KpnI酶切和测序方法鉴定出重组表达质粒。重组表达质粒转化大肠杆菌JM109,经1mmol/LIPTG在32℃诱导表达2h,裂解细菌,进行PAGE凝胶电泳鉴定融合蛋白的表达。【结果】RT-PCR扩增产物分子大小为327bp,序列分析表明为人resistin基因完整编码区。PCR产物与T载体连接、转化大肠杆菌后,从细菌质粒中酶切回收了目的片段。重组表达质粒经EcoRI和KpnI双酶切后能产生出327bp、4980bp大小的两个片段,序列分析表明重组表达质粒包含人resistin基因完整编码区,基因编码无移位,PAGE凝胶电泳表明在细菌裂解上清有分子质量为39.5kD的融合蛋白。【结论】成功构建了人resistin基因原核表达载体,且构建的载体能表达出含目的蛋白的融合蛋白,为下一步研究打下了实验基础。To construct a prokaryotic expression vector containing human resistin gene and observe its expression,to lay a f oundation for further study on resis tin.The total RNA was extracted from abdominal subcut aneous tissue of type 2diabetic pati ents using routine method.The resistin cDNA was amplified by reverse transcription polyme rase chain reaction(RT-PCR)and sequenced,and subcloned into T v ector.The recombinant T plasmid was identified by PCR and digestion analysis of restriction endonuclease EcoR I and kpn I.Then,the gene fragment of resistin was obtained by the digestion of recombined T plasmid with endon uclease EcoR I and Kpn I.The prokaryotic expression vector pGENKS was digested with same endonucleases.The gene fragment of resistin was orientationly linked into vector pGENKS by T4DNA ligase,and the recombined expression plasmid was identified b y digestion analysis of endonuclease EcoR I and Kpn I and sequence analysis.The recombined e xpression plasmid was transduced in to JM109by heatshock,and the expression of resistin protein was induced by 1mmol /L IPTG at 32℃for 2hours,and the fusion protein of GST /resistin was analyze d by SDS-PAGE after lysis of bacteria.The molecular size of RT-PCR products was about 327bp by gel electrophoresis analysis.Sequence analysis of RT-PCR products demonstrated that i t contained the complete code region of human resistin gene.The recombined expression plasmid pGEN KS-re was digested into two fragment s of about 327bp and 4980bp by endonuclease EcoR I and Kpn I,and sequence analysis of the recom bined expression plasmid demonstrated that it contained a com plete code region of human resistin g ene.A GST /resistin fusion protein molecule of 39.5kD was found in supernatant of lysis of bacteria b y SDS-PAGE electrophoresis.[Conclusion]A prokaryotic expression plasmid containing human resistin gene is successfully constructed,and it can express out objective protein,which has laid a concrete fundation for future study on resistin.

关 键 词：原核表达载体 重组表达质粒 融合蛋白 细菌 编码区 腹壁 核酸内切酶 酶切 T载体 PCR产物 

分 类 号：R378.99[医药卫生—病原生物学] Q78[医药卫生—基础医学]