重组人角质细胞生长因子-2基因的克隆、表达及活性

Gene Clone,Expression and Its Biological Activity of Keratinocyte Growth Factor-2

从人胚肺成纤维细胞中提取总RNA,利用RT-PCR法获得人角质细胞生长因子-2(hKGF-2)cDNA,并通过PCR方法扩增,再与原核表达载体pET3c连接后转化至BL21(DE3)宿主细胞中获得表达菌株.通过流加补料方式和发酵条件的控制,进行高密度培养,并通过IPTG诱导获得可溶性表达的目的产物.结果表明:目的蛋白占菌体总蛋白的30.0%以上;经阳离子交换层析、肝素亲和层析两步柱层析方法获得的rhKGF-2纯度高于99.0%.建立了利用转受体FGFR2-Ⅲb的BaF3细胞株进行rhKGF-2促增殖作用的活性测定方法,活性测定结果呈典型的S型曲线,并在一定范围内具有剂量依赖性.

We extracted total RNA from human embryonic lung fibroblast cells and obtained cDNA by means of RT-PCR. Then the PCR products were inserted into pET3c that was transformed into BL21 （DE3） host cells, which was induced to develop solubility protein products. High-density culture was conducted in fed-batch mode under the control of fermentation conditions. Purification was carried out by cation exchange chromatography and heparin affinity chromatography. And the effect of rhKGF-2 on proliferative activity was tested via transferring receptor FGFR2-m b of the BaF3 cells. We constructed gene engineering bacteria of recombinant human keratinocyte growth factor-2 （rhKGF-2） , studied the high-density culture of recombinant engineering bacteria and purification process and established the method of activity determination. Totalprotein is more than 30.0% of the total bacterial protein over consecutive three batches of fermentation. The purity of rhKGF-2 was higher than 99.0% after two-step column chromatography. We built a new way to test its biological activity. The result shows that it seemed obviously curved letter S, which had some relation with the dose in some extent.