人Leptin基因的克隆及其在大肠杆菌中的表达

目的克隆Leptin基因,构建其重组表达菌株,并对表达产物进行免疫学鉴定。方法利用RT-PCR方法从脂肪细胞RNA中扩增出人瘦素前体(pre-Leptin)的cDNA片段和去信号肽序列的Leptin成熟基因片段,并插入pET32a中,转化大肠杆菌BL21(DE3),获得表达重组人瘦素(rh-Leptin)的菌株。结果DNA序列分析显示获得的pre-Leptin和成熟基因片段的序列与预计序列一致,菌株诱导后经SDS-PAGE检测,rh-Leptin表达量达菌体总蛋白的40%以上。Western blot分析显示重组Lep-tin具有免疫学活性。结论已获得高效表达Leptin的重组菌株。

Cloning of Human Leptin Gene and Its Expression in E. coli

Objective To clone Leptin gene,construct a recombinant expression bacterial strain and identify the expressed product.Methods Amplify the cDNA fragment of human pre-Leptin and the mature gene fragment of Leptin,with the signal peptide sequence deleted,from the RNA of fat cells by RT-PCR.Insert the mature gene fragment into vector pET32a,then transform the recombinant plasmid to E.coli BL21(DE3) to construct a recombinant bacterial strain for the expression of human Leptin.Results The DNA sequences of the amplified pre-Leptin and mature Leptin gene fragments were consistent with those expected.SDS-PAGE proved that the expressed product contained more than 40% of total somatic protein.Western blot showed immunological activity of recombinant Leptin.Conclusion A recombinant E.coli strain for high expression of Leptin was constructed.