大肠杆菌中高效表达rhBMP-4的探讨及初步活性测定  

Strong Expression of Recombinant Human Morphogenetic Protein-4 in Escherichia coli and its Bioassay in vivo

作　　者：高思红[1] 汪炬[1] 董群伟[2] 刘侃[1] 刘雪婷[1] 洪岸[1] 谢秋玲[1] 孙奋勇[1] 

机构地区：[1]暨南大学生物工程研究所,广州510632 [2]广东药学院附属第一医院骨科,广州510632

出　　处：《中国生物工程杂志》2006年第3期51-56,共6页China Biotechnology

基　　金：广东省科技厅资助项目(2004B34001009);广州市科技局科技攻关计划资助项目(2004Z3-E4041)

摘　　要：目的:在大肠杆菌中表达具有生物活性的rhBMPBMP4。方法:在不改变氨基酸序列的前提下,以全基因合成的方式对人BMP4成熟肽基因全长进行定点突变,将之重组入pET3c表达载体并转化至大肠杆菌BL21(DE)plysS。IPTG诱导和包涵体复性后,利用C2C12细胞横向成骨细胞分化实验以及小鼠肌袋异位骨形成实验检测其活性。结果:获得0.348kb的BMP4DNA序列,表达的目的蛋白主要以包涵体的形式存在。经纯化及复性后,体内与体外的活性检测表明rhBMP4有良好的诱骨生成活性。结论:该方案能够实现rhBMP4在大肠杆菌中的高效表达。Objective：To produce rhBMP-4 with bioactivity in E. coli. Methods： The full-length human BMP-4 gene was mutated by PCR without changes in amino acid sequence, then the synthesized gene was cloned into plasmid pET-3c, transducted into BL21 （DE）plysS, and induced by adding IPTG to a final concentration of 1.0 mmol/L. The protein product was purified using ion-exchange chromatography method and then renaturated, bioactivity was checked by C2C12 differentiation in vitro and mouse ectopic bone formation in vivo. Results： A 438 bp gene fragment encoding mature peptide of hBMP-4 was cloned , the protein product was mostly in the form of inclusion body, after renaturation, the engineering protein shows better bioactivity. Conclusion： The mutant strategy can enhance the expression of bioactive rhBMP-4 in E. coli expression system.

关 键 词：rhBMP-4 定点突变 包涵 体活性 

分 类 号：Q78[生物学—分子生物学] Q781