摘要
目的对人内皮抑素(hES)基因进行定点突变,提高其在大肠杆菌中的可溶性表达量。方法根据Internet网站提供的关于hES的结构信息及其结构与功能方面的研究文献,设计突变位点;利用生物信息学的相关网站,对hES突变体进行结构预测,验证突变位点设计的合理性;采用重叠延伸PCR方法进行hES基因的定点突变,并将突变基因和未突变基因分别亚克隆至表达载体pGEX-4T-3,在大肠杆菌中进行融合表达,比较突变前后目的蛋白的可溶性表达量。结果三级结构预测结果表明突变位点设计合理,序列分析结果表明实现了hES基因的定点突变。突变基因的表达产物中,可溶性部分约占总表达量的40%,而未突变基因的表达产物几乎全部为包涵体。结论已成功对hES基因进行了定点突变,突变后的hES可溶性表达量得到了提高。
Objective To increase the soluble expression level of human endiostatin （hES） gene in E. coli by site-directed mutagenesis. Methods The mutation sites were designed according to the information on structure as well as relationship between structure and function of hES from internet sites. The rationality of the designed mutation sites was confirmed by predicting the three-dimensional structure of hES mutant in relevant internet sites of bioinformatics. The site-directed mutagenesis of bES was achieved by overlapping extension PCR, and the mutated and original hES genes were subcloned into vector pGEX-4T-3 respectively for fusion expression in E. coli. The soluble expression levels of target protein before and after mutation were compared. Results The prediction of three-dimensional structure of hES showed rational design of mutation sites. Sequencing result proved the site-directed mutation of hES gene. Of the expressed product of hES gene mutant, 40% existed in a soluble form. However, almost all the expressed product of original hES gene were in forms of inclusion bodies. Conclusion The site-directed mutagenesis of hES gene was successfully achieved, which increased the soluble expression level of target protein.
出处
《中国生物制品学杂志》
CAS
CSCD
2009年第5期438-442,共5页
Chinese Journal of Biologicals
基金
福建省自然科学基金资助项目(C0740012)
集美大学中青年创新团队专项基金资助项目(2006A002)
关键词
人内皮抑素
定点突变
可溶性表达
重叠延伸PCR
Human endostatin （hES）
Site-directed mutagenesis
Soluble expression
Overlapping extension PCR
