摘要
目的 建立人肝细胞生长因子α链 (rhHGFα)高效原核表达体系。方法 以pRC/CMV hHGF为模板 ,扩增hHGFαcDNA ,继以XhoI酶切为 386和 95 4bp 2个片段 ,亚克隆入pBSKS,DNA测序后 ,将上述两个片段与pBV2 2 0连接 ,构建重组质粒。转化E .coliJM10 9、DH5α和BL2 1(DE3) ,筛选高效表达菌株。分离包涵体 ,8mol/L尿素溶解 ,梯度复性后利用反相和凝胶过滤层析纯化重组蛋白 ,经MTT法检测活性。结果 所克隆的hHGFα基因序列正确 ,筛选出高效表达菌株BY ,经SDS-PAGE显示在相对分子质量 5 2 0 0 0处出现特异的目的蛋白表达带 ,表达量约占全菌蛋白的 2 5 %。表达产物以不溶性的包涵体 (IBs)形式存在 ,复性后具有刺激原代大鼠肝细胞生长的作用。结论 已成功表达了rhHGFα蛋白 ,为研究rhHGFα结构与功能及中试生产奠定了基础。
Objective To construct a prokaryotic system for high expression of human hepatocyte growth factor α(rhHGFα).Methods hHGFα cDNA was amplified using pRC/CMV-hHGF as a template, digested with Xho I into two fragments at lengths of 386 and 954 bp respectively, and subcloned into plasmid pBS KS.After DNA sequencing, insert the two fragments into plasmid pBV220.Transform the recombinant plasmid into E.coli and screen the bacterial strain in which rhHGFα is highly expressed.Separate the expressed protein existing in a form of inclusion body, dissolve with 8 mol/L urea, re-naturalize and purify by reverse and gel filtration chromatography and detect for activity by MTT method.Results The cloned hHGFα gene showed a correct sequence, and an E.coli strain BY for high expression of rhHGFα was screened.SDS-PAGE showed a specific protein band with a relative molecular weight of 52 000.The expressed product existed in a form of insoluble inclusion body and contained about 25% of total somatic protein.After renaturation, the expressed protein showed stimulating effect on the growth of primary rat hepatocytes.Conclusion rhHGFα protein was successfully expressed in E.coli.It laid a foundation of study on the structure and function as well as pilot production of hHGFα.
出处
《中国生物制品学杂志》
CAS
CSCD
2005年第1期19-22,共4页
Chinese Journal of Biologicals
基金
山西省高等学校科技研究开发项目 (2 0 0 413 16)
