人角质细胞生长因子2基因的克隆及其在大肠杆菌中的表达

Cloning and Expression of Human Keratinocyte Growth Factor 2

目的:构建人角质细胞生长因子2(hKGF2)基因的高效原核表达载体pET-30a(+)-hKGF2,并在大肠杆菌BL21(DE3)中获得表达。方法:从培养的人胚胎肺成纤维细胞中提取总RNA,采用RT-PCR技术扩增出去除了信号肽部分的hKGF2基因,克隆到pMD18-T载体,经DNA序列分析后与pET-30a(+)表达载体连接,在大肠杆菌BL21(DE3)诱导表达6×His-hKGF2,用SDS-PAGE及Western印迹鉴定表达蛋白。结果:构建了表达载体pET-30a(+)-hKGF2,经IPTG诱导后,表达的重组蛋白理论相对分子质量约为23000,约占菌体总蛋白的20%。结论:6×His-hKGF2蛋白在大肠杆菌BL21(DE3)中为可溶性高效表达,为获得高纯度、高活性的产物,以及进一步的大规模生产和应用研究奠定了基础。

Objective： To construct pET-30a（＋） expression vector containing human keratinocyte growth factor 2（hKGF2） gene, and to obtain the recombinant hKGF2 protein in E.coli BL21（DE3）. Methods： The total RNA was extracted from the cultured human embryonic lung fibroblast. The hKGF2 gene absent signal peptide was amplified by using RT-PCR. Then the hKGF2 gene was cloned into pMD18-T vector. After DNA sequencing, the hKGF2 gene was ligated with pET- 30a （＋） and the 6xHis-hKGF2 was over expressed in E.coli BL21 （DE3）. The expressed protein was detected by SDSPAGE and Western blot. Results： The hKGF2 protein was successfully expressed. After induced by IPTG, the target protein amounted to 20% of total bacteria proteins and the molecular weight is approximately 23kD. Conclusion： The 6xHishKGF2 protein was over expressed and soluable. It lays a good foundation for obtaining large scale production and further exploratory development.