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牛碱性成纤维细胞生长因子基因在大肠杆菌中的表达
及其表达产物的生物学活性
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刘华忠1,2，杨兴元2，李晴2，安晓荣2，陈永福2，钟杰平1，李琨1
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（1.广东海洋大学 现代生物化学中心，广东 湛江524088；
2.中国农业大学 农业生物技术国家重点实验室，北京100094）
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摘要：
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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0.5mmol/L IPTG诱导表达5h，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。 |
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关键词：
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牛碱性成纤维细胞生长因子；原核表达；活性检测 |
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中图分类号：S 852.2 文献标识码：
A 文章编号：1673-4696(2007)10-0896-05 |
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Expression of bovine basic fibroblast growth factor gene in
Escherichia coli and bioactivity of the expressed product |
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LIU Hua-zhong1,2,YANG Xing-yuan2,LI
Qing2，AN Xiao-rong2,CHEN Yong-fu2,
ZHONG Jie-ping1,LI Kun1 |
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(1.Center for Modern Biochemistry,Guangdong Ocean University,Zhanjiang 524088,China;
2.State Key Laboratory for Agrobiotechnology/China Agricultural University,Beijing 100094,China) |
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Abstract: 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 0.5mmol/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.
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Key words: bovine basic fibroblast growth factor;prokaryotic expression;activity analysis |
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