重组人脂联素基因构建、表达、纯化及活性鉴定

Gene Construction,Expression and Activity Analysis of Recombinant Human Adiponectin

根据脂联素cDNA序列设计并合成9条寡聚核苷酸片段,经重叠PCR技术获得重组人脂联素基因.构建pET-22b(+)/ADPN表达载体,转化大肠杆菌BL21(DE3),IPTG诱导表达.目的蛋白以包涵体的形式表达,表达量占菌体总蛋白的30%以上.表达产物通过Ni-NTA柱纯化,SDS-PAGE分析显示,其分子量约为30 ku,与预期结果一致.内皮细胞生长抑制实验证实,重组人脂联素生理浓度下具有抑制人内皮细胞生长的活性并呈时间依赖性.以上工作为进一步研究脂联素的生物学功能奠定了基础.

According to the eDNA sequence encoding the human Adiponectin（ADPN）, nine oligonucleotides were designed and synthesized. The ADPN gene was obtained by overlapping PCR technique. The expression vector pET22b（＋）/ADPN was constructed and transformed into E. coli BL21 （DE3）. The ADPN protein was expressed as inclusion bodies with the yield of more than 30 % of total bacterial proteins under IPTG induction. The expression products were purified by Ni-NTA affinity column,SDS-PAGE analysis showed that the relative molecular mass of ADPN is 30 ku which was consistent with the theoretically predicted value. The result of inhibition of endothelial cell growth confirmed that ADPN can inhibit the endothelial cell proliferation at subphysiological concentrations, which appears time-dependent. All these results would contribute to the further study of its biological functions.