导向性IFN-α2a-α-MSH基因的克隆、表达、纯化及鉴定

Cloning,expression,purification and identification of IFN-α2a-α-MSH fusion gene

目的:构建导向性IFN-α2a-α-MSH融合基因的原核表达载体,并建立重组蛋白的原核高效表达体系.方法:将本实验室已构建的pET-22b(+)IFN-α2a-NGR用BamH I和SalI限制性内切酶进行双酶切,将得到的大片段与设计的α-MSH多肽片段退火后的产物进行连接,构建原核表达载体pET-22b(+)IFN-α2a-α-MSH,将序列鉴定正确的重组质粒转化入Rosetta-gamiTM2(DE3)大肠杆菌,IPTG诱导表达目的蛋白.对表达产物进行SDS-PAGE及Western Blot鉴定.结果:在大肠杆菌中成功实现了IFN-α2a-α-MSH的稳定高效表达,表达产物以包涵体形式存在,采用超声裂菌的方法,用疏水作用层析技术获得较高纯度的重组蛋白.结论:成功克隆、表达和纯化了IFN-α2a-α-MSH蛋白.

AIM ： To construct interferons-α2a （ IFN-α2a）-α melanocyte-stimulating hormone （MSH） fusion gene and a higt prokaryotic expression system of IFN-α2a-α-MSH. METHODS： Plasmid pET-22b （ ＋ ） IFN-α2a-NGR in our laboratory, was digested with the BamH I and Sal I restriction enzymes, and then connected to the annealed α-MSH peptide sequences to construc prokaryotic expression vector pET-22b（ ＋ ） IFN-α2a-α-MSH . The correct sequence that had been identified was transfected int（ Rosetta-gamiTM2（ DE3 ） cell. IPTG induced fusion protein IFN-α2a-α-MSH expression. The expression of IFN-α2a-α-MSH was detected by SDS-PAGE and Western Blot. RESULTS： The IFN-α2a-α-MSH fusion protein in form of inclusion body,was successfully and stably expressed in E. coli. After the E. coli was lysed by ultrasonic wave, Phenyl Sepharose 6 Fast Flow （ high sub） was used to get the purified recombinant protein. CONCLUSION： The fusion protein of IFN-α2a-α-MSH has been successfully expressed and purified.