人脂联素基因的原核表达、纯化及活性检测

Prokaryoti Expression,Purification and Activity Analysis of Human Adiponectin Gene

目的:在大肠杆菌中表达具有活性的重组人脂联素蛋白。方法:利用PCR技术扩增人脂联素基因完全编码序列,选用Gateway原核表达体系,经过BP反应、LR反应两步反应,将PCR产物克隆入pET-DEST42质粒,构建融合表达载体pET-DEST42/attB-adiponectin,转化大肠杆菌BL21(DE3),以异丙基硫代-β-D-半乳糖苷(IPTG)诱导表达脂联素融合蛋白,经镍柱亲和层析纯化,NDSB201高效复性后,使重组蛋白作用于体外培养的肝细胞,RT-PCR法检测重组脂联素对肝细胞葡萄糖-6-磷酸酶转录水平的影响。结果:成功构建了表达载体pET-DEST42/attB-adiponectin,DNA序列测定结果与预期一致。IPTG诱导表达的融合蛋白经纯化后Western Blot鉴定具有人脂联素抗原性,并可使培养的肝细胞中葡萄糖-6-磷酸酶转录水平下降。结论:获得具有生物学活性的重组人脂联素为进一步研究脂联素的生理机制奠定了基础。

Objective： To express recombinant human adiponectin with biological activity in E coli. Methods： The cDNA coding fragment of human adiponectin gene was amplified by PCR. Gateway prokaryoti expression system was used. PCR products were cloned into pET-DEST42 plasmid to construct fusion expression vector pET-DEST42/attB-adiponectin after BP reaction and LR reaction, pET-DEST42/attB-adiponectin was transformed into E coli BL21 and the bacteria were induced by IPTG expressed adiponectin,fusion protein was purified by Ni·NTA affinity chromatography. The effect of purified recombinatant adiponectin on glucose-6-phosphatase was detected by RT-PCR. Results： The DNA sequencing showed that expression vector pET-DEST42/attB-adiponectin was constructed successfully. Purified adiponectin showed antigenicity and reduced glucose-6-phosphatase transcriptional level. Conclusion： We have obtained the recombinant human adiponectin with biological activity, which is significant for further study.