小鼠周脂素基因的原核表达及纯化

目的构建小鼠周脂素（prilipin,Plin）原核表达载体,表达周脂素蛋白并进行初步纯化,为研究周脂素的功能奠定基础。方法通过酶切从pMD18-Plin重组载体上酶切下周脂素目的基因,定向插入原核表达载体pET-32a（＋）中,转化大肠杆菌BL21（DE3）,IPTG诱导表达。表达产物经SDS-PAGE及Western blot鉴定后,进行纯化。结果构建的pET-32a-Plin载体能够表达周脂素重组蛋白,表达量约占菌体总蛋白的50%,经初步纯化后,纯度达95%以上。结论成功构建了周脂素原核表达载体,并在原核系统中表达了重组蛋白,纯化的蛋白纯度较高,可用于后续试验。

Prokaryotic Expression and Purification of Mouse Prilipin Gene

Objective To structure the prokaryotic expression vector of mouse prilipin gene,was expressed in prokaryotic cells and purify the expressed product.Methods By enzyme digestion from pMD18-Plin recombinant enzyme cut the prilipin gene.The target gene was recovered and cloned into expression vector pET-32a（＋）,and the constructed recombinant plasmid pET-32a-Plin was transformed to E.coli BL21（E3） for expression under induction of IPTG.The expressed product was identified by SDS-PAGE and Western blot,then purified by His label chromatography.Results The prokaryotic expression vector of mouse prilipin gene can express prilipin.The expressed recombinant prilipin,with a relative molecular mass of about 57 kDa,contained about 50% of total somatic protein.It showed good reactogenicity and reached a purity of more than 95% after purification.Conclusion The prokaryotic expression vector of mouse prilipin gene was successfully structured and expressed in prokaryotic cells,and the expressed product reached a high purity,which laid a foundation of study on the function of prilipin.