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

作　　者：田海山[1,2] 唐禄[2] 王晓杰[2] 王艳芳[1] 官丽莉[1] 李校堃[1] 

机构地区：[1]吉林农业大学生物反应器与药物开发教育部工程研究中心,长春130118 [2]温州医学院药学院,浙江温州325035

出　　处：《吉林大学学报（理学版）》2011年第6期1150-1156,共7页Journal of Jilin University:Science Edition

基　　金：吉林省教育厅"十一五"科技发展规划项目(批准号:吉教科合字2009第65号);长春市应用技术计划研究项目(批准号:08YZ45);2009年科技型中小企业技术创新基金(批准号:09C26212203306)

摘　　要：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.

关 键 词：重组角质细胞生长因子-2(rhKGF.2) 高密度培养 柱层析 转受体FGFR2-Ⅲb BaF3细胞株 生物活性 

分 类 号：Q78[生物学—分子生物学]