人脂联素球状结构域(gAd)基因的克隆、融合表达和纯化

目的建立人脂联素球状结构域(gAd)融合组氨酸标签的原核高效表达体系,并分离纯化gAd。方法从淋巴细胞中提取人基因组DNA,PCR扩增出含有脂联素编码序列的片段,经T-A克隆入pMD18-T载体中,然后设计适当的引物,引入起始密码、C端连续6个组氨酸编码序列、终止密码以及相应的酶切位点,把脂联素球状结构域(gAd)基因亚克隆到表达载体pBV220,将序列鉴定正确的重组质粒转化入大肠杆菌DH5α中,用42℃温控诱导表达目的蛋白。结果在大肠杆菌中成功实现了gAd稳定高效表达,表达产物以包涵体形式存在,表达量约占全菌蛋白的19%,用超声破菌,经金属离子(Ni2+)配体亲和层析一步纯化得到了均一蛋白,经聚丙烯酰胺凝胶电泳鉴定得到了较高纯度的gAd。结论已成功表达了gAd融合蛋白,为进一步研究其生物学功能和作用机制奠定了基础。

Cloning and Fusion Expression of Globular Domain of Human Adiponection(gAd) in E. coli and Purification of Expressed Product

Objective To construct a high prokaryotic expression system of globular domain of human adiponection (gAd) and purify the expressed product.Methods Extract human genomic DNA from lymphocytes and amplify the gene fragment containing adiponectin-encoding sequence.Clone the amplified gene fragment into pMD18-T vector by T-A cloning,then introduce start codon,His 6-encoding sequence at C-terminus,end codon and the corresponding restriction site by designing appropriate primers,and subclone gAd gene into expression vector pBV220.Identify the recombinant by gene sequencing and transform to E.coli DH5α for expression of fusion protein at 42℃.Results The gAd fusion protein containing about 19% of total somatic protein,in form of inclusion body,was successfully and stably expressed in E.coli.After ultrasonication and purification by immobilized metal chelation affinity chromatography,homogenous protein was obtained and reached a high purity as proved by SDS-PAGE.Conclusion The globular domain of human adiponectin was successfully expressed.It laid a foundation of further study on its biological function and action mechanism.