穿膜肽KGF-2的构建及其对HaCaT增殖作用的影响

Construction of TAT-KGF-2 and Its Proliferation Effect on Human Keratinocyte Cell Line HaCaT

目的:构建穿膜肽KGF-2(TAT-KGF-2),研究其对人角质形成细胞HaCaT的增殖作用。方法:采用普通PCR技术和特殊引物从人胚胎肺成纤维细胞cDNA中扩增出目的基因TAT-KGF-2,并将其插入pET-28a(+)载体中构建pET28a-TAT-KGF-2重组质粒;将该质粒转化大肠杆菌Rosetta(DE3),IPTG诱导表达重组蛋白TAT-KGF-2;通过Ni-NTA柱纯化获得目标重组蛋白并利用Western blot进行鉴定;MTT法研究TAT-KGF-2对HaCa的增殖作用。结果:成功构建pET28a-TAT-KGF-2重组质粒,经序列比对,与GenBank上公布的人KGF-2基因序列同源性达到100%;获得穿膜肽KGF-2蛋白,分子量约为19 kDa,纯度达到95%以上;TAT-KGF-2对HaCaT在12.5ng/ml时具有增殖作用,并呈浓度依赖性,而且与不具有穿膜功能的KGF-2相比有更强的增殖效果。结论:成功纯化出TAT-KGF-2蛋白,并验证其对HaCaT具有增殖作用,为进一步研究该蛋白的结构和功能奠定了基础。

Objective： To construct TAT - KGF - 2 and research its proliferation effect on human keratinocyte ceil line HaCaT. Method： TAT - KGF - 2 gene was amplified from human embryonic lung fibroblast cell eDNA by two - step PCR and then was cloned into pET28a （ ＋ ） plasmid to construct the pET28a - TAT - KGF - 2 recombinant plasmid. The recombinant plasmid was transformed into Escherichia coli bacteria Rosetta（ DE3 ）. After being induced by IPTG,the recombinant protein was expressed and purified by using Ni -NTA affinity chromatography and then the purified protein was identified by western blot; The MTT proliferation test was used to revealed that if TAT - KGF - 2 has promoting activity for HaCaT. Result： The TAT - KGF - 2 recombinant plasmid was successfully constructed, sequence align- ment result showed that the sequence shares 100% of sequence homology with the Homo KGF -2 reported in GenBank; the TAT -KGF - 2 protein were obtained with the molecular weight which was about 19kDa and the purity was more than 95% ; the TAT- KGF- 2 ,com- pared to the positive control which didn＇ t have the cell penetrating peptides, had a stronger promoting activity for HaCaT at 12. 5ng/ml in a concentration - dependent manner. Conclusion： The TAT - KGF - 2 protein was successfully purified and was confirmed that it has a stronger promoting activity for HaCaT. These results laid foundation for the further studies on the structure and function of KGF -2.