大肠杆菌中高效表达rhBMP-4的探讨及初步活性测定

目的 在大肠杆菌中表达具有生物活性的rhBMP-4。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BMP-4成熟肽基因全长进行定点突变，将之重组入pET-3c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0.348 kb的BMP-4 DNA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rhBMP-4有良好的诱骨生成活性。结论 该方案能够实现rhBMP-4在大肠杆菌中的高效表达。

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

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.