人瘦素蛋白在大肠杆菌中的融合表达和纯化

Fusion expression and purity of human leptin in E. coli

目的构建人瘦素因子(Leptin)的原核表达质粒pGEX-4T-3-LEP,并诱导该基因在原核细胞中大量表达。方法采用逆转录．聚合酶链反应(RT-PCR)和DNA重组技术,从人皮肤成纤维细胞内将编码人Leptin的cDNA序列克隆至原核表达载体pGEX-4T-3上,选取阳性克隆扩增后,提取DNA进行酶切鉴定和测序。同时应用SDS．PAGE和蛋白质印迹方法对其表达产物进行特异性鉴定。结果克隆出的Leptin基因的cDNA由469bp组成,包括翻译起始密码子、编码序列和终止密码子等部分,含有Letinp基因的cDNA的表达质粒pGEX-4T-3在DH5a细菌体内能够表达,并且随着诱导时间的延长,Leptin基因的表达量增加,重组Leptin蛋白主要存在于包涵体中。结论Lepfin基因可以在原核细胞中获得表达,为深入研究该蛋白在创伤愈合中的具体作用奠定了基础。

Objective To construct the plasmid of pGEX-4T-3-LEP for Leptin with prokaryotic expression vector pGEX-4T-3 and induce the Leptin protein expression in E. coli DH5α. Methods RTPCR technology in vitro was used to clone cDNA （complementary deoxyribonucleic acid） sequence coding human leptin. DNA recombination method was adopted to insert the sequence into the multiple cloning sites of pGEX-4T-3. Results The cDNA sequence encoding leptin was cloned into the prokaryotic expression vector pGEX-4T-3 to construct pGEX-4T-3-LEP. The sequence of cDNA was identical to coding framework of leptin gene, which was composed of translating original codon, coding sequence and terrninating-codon. Conclusion The prokaryotic expression plasmid pGEX-4T-3-LEP was successfully constructed. From the basis of the work, further research of wound heeling with leptin could be carried out.