【摘要】 目的克隆人脂联素(hAPM)基因及构建真核表达重组体PGEX-4T-1/hAPMcDNA,诱导表达谷胱甘肽巯基转移酶-hAPM(GST-hAPM)融合蛋白。方法应用RT-PCR法从中国汉族人大网膜脂肪垫总RNA中扩增出APMcDNA全长基因,并在基因上下游分别克隆上EcoR1、Sal1两个酶切位点,将其定向导入PGEX-4T-1多克隆位点,构建PGEX-4T-1/hAPM融合表达载体,转化大肠杆菌BL21(DE3)后进行诱导表达。结果从脂肪组织总RNA中扩增得到760bp片段APM基因,经双酶切鉴定及测序分析,证明hAPMcD-NA已克隆到PGEX-4T-1载体中。免疫印迹证实,诱导表达的融合蛋白可被多克隆抗体Acrp30(N-20)-R识别。结论成功诱导表达出融合蛋白GST-hAPM,为进一步获得大量可供实验研究和临床应用的重组hAPM创造条件。更多还原

【Abstract】 Objective To clone hAPM cDNA in the PGEX-4T-1 vector and construct PGEX-4T-1/hAPM cDNA to express GST-hAPM fusion protein. Methods APM mRNA from greater omentum fat pat of Chinese was amplified by RT-PCR. Then complete hAPM cDNA was orientally inserted and its fusion expression vector was obtained. The recombinant vector was transformed into E.coli BL21(DE3) and induced by IPTG. Results About 760 bp of hAPM fragment was amplified from total RNA of Chinese omentum fat pat. The recombinant vector was digested with EcoR1 and Sal1 and hAPM gene was testified by DNA sequencing. These quenching results for amplified target gene showed that the sequence of hAPM from omentum fat pat of Chinese was identical to that of hAPM in Genbank. The results demonstrated that the hAPM was successfully inserted into the PGEX-4T-1 vector. Western blot analysis of fusion protein confirmed that it could be specially recognized by the polyclonal antibody of Acrp30(N-20)-R. Conclusion Expression of GST-hAPM fusion protein provides a foundation for obtaining a larger quantity of recombinant hAPM for experimental and clinic studies.更多还原