人角质化细胞生长因子2基因克隆、表达及其产物的纯化和鉴定

背景:人角质化细胞生长因子2具有广泛的生理功能,在胚胎发育、组织的修复、神经的再生、血管的生成和肿瘤发展中具有重要作用。目的:克隆人角质化细胞生长因子2基因,于大肠杆菌中表达,并检测其生物学活性,为进一步开发提供实验依据。设计:开放性实验。单位:中国疾病预防控制中心病毒病预防控制所。材料:实验于2000-09/2002-05在中国疾病预防控制中心病毒病预防控制所病毒基因工程国家重点实验室完成。pBV220温控表达载体为病毒基因工程国家重点实验室构建,EcoRⅠ、BamHⅠ、T4DNA连接酶(Promega公司);人角质化细胞生长因子2特异性聚合酶链反应引物(上海博亚生物技术有限公司合成);肝素-SepharoseCL-6B(Pharmacia公司);快速聚合酶链反应产物纯化试剂盒、总RNA提取Trizol试剂盒、反转录-聚合酶链反应试剂盒(GIBCO公司);质粒DNA快速提取试剂盒(博大公司);BL-21-codonpluscompentcells(Stratagene公司)。方法:用高表达菌株BL-21-codonpluscompententcells表达重组人角质化细胞生长因子2蛋白并初步纯化和检测其活性。通过反转录-聚合酶链反应从自然流产胎儿肺组织中钓取人角质化细胞生长因子2cDNA,将其克隆入pBV220载体质粒。在大肠杆菌BL-21-codonpluscompentcells中表达人角质化细胞生长因子2蛋白。采用亲和层析和离子交换层析分离纯化,以细胞增殖实验测定表达蛋白的生物活性。主要观察指标:人角质化细胞生长因子2cDNA的片段长度和序列,人角质化细胞生长因子2基因在大肠杆菌中的表达,纯化人角质化细胞生长因子2的活性。结果:人角质化细胞生长因子2cDNA片段大小约500bp,人角质化细胞生长因子2蛋白在BL-21中得到高效表达,于上清中可溶性表达,SDS-PAGE显示相对分子质量约20000;人角质化细胞生长因子2蛋白对NIH3T3细胞具有显著的促有丝分裂活性,1,5,10μg/L人角质化细胞生长因子2A值(490nm)高于空白对照组,差异有非常显著性意义(分别为0.174±0.022,0.220±0.029,0.306±0.050,0.066±0.004,P<0.001)。结论:成功克隆人角质化细胞生长因子2基因,并于大肠杆菌BL-21中得到高效表达;纯化的人角质化细胞生长因子2蛋白能刺激NIH3T3细胞的增殖,具有显著的促有丝分裂活性。

Cloning and expression of human keratinocyte growth factor-2 and the purification and identification of its products

BACKGROUND: Human keratinocyte growth factor-2 (hKGF-2) has extensive physiological functions, which plays an important role in embryonic development, tissue-repairing, nervous regeneration, vascularization and development of tumor.OBJECTIVE: To clone hKGF-2 gene, obtain the expression of hKGF-2 in Escherichia coli(E.coli) and determine its bioactivity, so as to provide experimental basis for further investigation.DESIGN: Open experiment.SETTING: Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention.MATERIALS: The experiment was conducted in State Key Laboratory of Viral Genetic Engineering, Institute for Viral Disease Control and Prevention of Chinese Center for Disease Control and Prevention. The temperature control expression vector pBV220 was constructed by State Key Laboratory of Viral Genetic Engineering; EcoR Ⅰ , BamH Ⅰ , T4 DNA ligase (Promega Co., Ltd.); The specific polymerase chain reaction (PCR) of hKGF-2 (Manufactured by Shanghai Boya Biotechnology Co., Ltd.); Heparin-Sepharose CL-6B (Pharmacia Company); PCR rapid purification kit,Trizol kits for total RNA extract, Kits for RT-PCR (GIBCO Co., Ltd.); Kits for rapid extraction of plasmid DNA (Boda Company); BL-21-codon plus compent cells (Stratagene Co., Ltd.).METHODS: High-expression strain BL-21 codon plus competent cells was used to express and purify initially recombinant hKGF-2 protein, and its activity was detected. RT-PCR was adopted to obtain hKGF-2 cDNA from lung tissues of naturally aborted fetus and clone it into pBV220 carri er plasmid. The hKGF-2 protein expressed in BL-21 codon plus competent cells of E.coli. Affinity chromatography and ion exchange chromatography were applied in isolation and purification, and the bioactivity of expression protein was determined in cell proliferation test.MAIN OUTCOME MEASURES: The length and sequence of cDNA segment in hKGF-2, the expression of hKGF-2 gene inE.coli and the purification of hKGF-2 activity.RESULTS: The segment of hKGF-2 cNDA was about 500 bp, and hKGF-2 protein highly expressed in BL-21, which had soluble expression in the supernatant. SDS-PAGE showed that the relative molecular mass was about 20000, and hKGF-2 protein could significantly promote the mitotic activity of NIH3T3 cells. The A value (490 nm) of hKGF-2 in the 1 μg/L, 5 μg/L and 10 μg/L groupswere higher than that in the blank control group, and the differences were significant (which were 0.174±0.022,0.220±0.029,0.306±0.050,0.066±0.004 respectively,P ＜ 0.001).CONCLUSION: hKGF-2 gene is successfully cloned, which highly expresses in BL-21 of the E.coli. Purified hKGF-2 protein can stimulate the proliferation of NIH3T3 cells and significantly promote its mitotic activity.