Construction and Expression of A Prokaryotic Expression Vector Containing Human Resistin Gene
HE Xiang-liang1,HE Dong-hua2,LI Feng3 ,YANG Chuan3,CHENG Hua3,FU Zu-zhi 3 (1Department of Emergency,The First Affiliated Hospital,2.Da An Gene Co mpany,3.Department of Endocrinology,The Second Affiliated Hospital,SUN Yat-sen University,Guangzhou 510080,China)
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.
【CateGory Index】： R346
【CateGory Index】： R346