作　　者： ; ; ; ; ; ; ;

机构地区： 广东海洋大学

出　　处： 《中国兽医科学》 2007年第10期896-900,共5页

摘　　要： 采用巢式PCR方法克隆了牛18ku-bFGF基因完整的编码序列,并构建了原核表达载体pET-28a-bFGF,将其转化大肠杆菌BL21,在25℃低温条件下,用0.5 mmol/L IPTG诱导表达5 h,用Ni-NTA亲和纯化细胞裂解上清液,经Western-blotting检测,结果显示,在特定的诱导条件下,重组牛bFGF基因在大肠杆菌中获得了表达,并且主要以可溶性状态存在于细胞中。经检测,纯化后的重组蛋白能显著促进成纤维细胞的增殖(P<0.05),其活性与商品用重组人18ku-bFGF没有差异(P>0.05)。表明,所获得的可溶性重组牛18ku-bFGF蛋白具有较高的生物学活性,可用于后续研究工作。 A complete bovine 18ku-bFGF gene was cloned by nested-PCR and subcloned into expression vector pET-28a. The recombinant plasmid pET-28a-bFGF was transformed into Escherichia coli BL21. At 25 ℃, recombinant protein was induced by 0. 5 mmol/L IPTG for 5 hours. Supernatant of cell lysate was purified using Ni-NTA method and the products was submitted to detect the bioactivity by Western-blotting,which indicated that the fusion protein contained recombinant bovine bFGF. Results showed that recombinant bovine bFGF was produced and solubly existed in the supernatant. NIH-3T3 fibroblasts were used to detect the bioactivity of the fusion protein. The purified fusion protein was able to significantly stimulate proliferation of the cells（P〈0.05）. The bioactivity of the purified fusion protein was equivalent to that of the commercial recombinant human bFGF （P〉0. 05）. The recombinant bovine 18ku-bFGF with high expression, solubility and bioactivity,was harvested and can be used for further studies.

关 键 词： 牛碱性成纤维细胞生长因子 原核表达 活性检测

领　　域： [农业科学] [农业科学] [农业科学]