摘要
目的:实现重组人角质细胞生长因子-2(rhKGF-2)的原核可溶性高效表达,并获得活性良好的重组蛋白。方法:将编码人成熟KGF-2的cDNA构建至pET/21 a表达载体中,利用大肠杆菌BL21进行表达,对表达条件(如诱导温度和诱导时间等)进行优化,并对目的蛋白的表达情况进行电泳分析;利用阳离子交换柱对重组蛋白进行纯化,对纯化的rhKGF-2利用MTT法检测其促IEC-6及NIH/3T3细胞的增殖活性。结果:通过对表达条件的优化,rhKGF-2的表达量可占到全菌总蛋白的25%,并且基本上全部为可溶性形式表达;利用阳离子交换柱对rhKGF-2进行纯化,薄层扫描分析其纯度高于90%。利用MTT法对rhKGF-2的活性进行研究,结果表明,低浓度的rhKGF-2就能够促进IEC-6细胞增殖,但对于NIH/3T3细胞需高浓度的rhKGF-2才能促进其增殖。结论:本研究实现了对rhKGF-2的可溶性高效表达,纯化后的rhKGF-2具有较好的活性,这为进一步的功能研究以及基因工程重组药物的开发奠定了基础。
Objective： To realize the high effective soluble expression of recombinant human keratinocyte growth factor- 2（rhKGF-2） in Escherichia coli and obtain the purified rhKGF-2 with better activity. Methods： The KGF-2 cDNA was inserted into pET/21 a plasmid and expressed in E. coli BL21. The expression conditions, such as induced concentration, induced temperature, induced time were optimized, and the recombinant protein was analyzed by 15% SDS-PAGE. The recombinant protein was subsequently purified by ion exchange column chromatography, and its effects on IEC-6 and NIH/3T3 cells were identified by MTT, respectively. Results：The optimized expression level of rhKGF-2 was about 25% of the total cell protein and almost completely expressed as a soluble form. rhKGF-2 could stimulate the proliferation of IEC-6 at a concentration as low as 78 ng/ml, while at the same concentration it couldn＇t exert any effects on NIH/3T3 until the concentration reached 5 000 ng/ml. Conclusion： This study proved that rhKGF-2 with better activity can be effectively expressed as a soluble form in E. coli, which sheds light on further functional studies and biological technical R＆D of rhKGF-2.
出处
《军事医学科学院院刊》
CSCD
北大核心
2008年第1期34-38,共5页
Bulletin of the Academy of Military Medical Sciences
