TFPI-2/KD1的克隆表达及其生物学性质研究  

Cloning,expression and biological characterization of the first kunitz-type domain of human tissue factor pathway inhibitor-2

作　　者：时辉宁[1] 丁日高[1] 钟玉绪[1] 刘红岩[1] 徐琪寿[2] 马凤中 廖明阳[1] 

机构地区：[1]军事医学科学院毒物药物研究所,北京100850 [2]军事医学科学院放射医学研究所,北京100850 [3]汇泽生医药科技有限公司,北京100850

出　　处：《中国医药生物技术》2008年第6期445-450,共6页Chinese Medicinal Biotechnology

摘　　要：目的克隆表达人组织因子途径抑制物-2(hTFPI-2)Kunitz结构域1(KD1)基因。方法提取健康女性正常分娩胎盘中总RNA,采用RT-PCR法合成hTFPI-2全长cDNA后行PCR扩增,以扩增得到的hTFPI-2/KD1基因片段与原核表达载体pET22b进行连接并转化E.coliDH5α,获得重组质粒pET22b-hTFPI-2/KD1。将重组质粒pET22b-hTFPI-2/KD1转化至E.coliBL21,获得重组菌株E.coliBL21[pET22b-hTFPI-2/KD1]。以异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,对表达产物进行SDS-PAGE和蛋白质印迹分析,并利用Ni2+-NTA柱进行亲和层析纯化,采用发色底物法和明胶酶谱法分别检测hTFPI-2/KD1抑制胰蛋白酶和基质金属蛋白酶(MMP)的活性。结果核酸序列测定显示克隆的hTFPI-2/KD1基因序列与理论序列相符。SDS-PAGE和蛋白质印迹分析显示E.coliBL21[pET22b-hTFPI-2/KD1]诱导表达产物相对分子质量与预期相符,经纯化后获得了完整的hTFPI-2/KD1蛋白,发色底物法和明胶酶谱法检测显示其具有抑制胰蛋白酶、MMP-2和MMP-9的活性。结论通过原核表达系统成功获得高效表达并具有生物活性的hTFPI-2/KD1,为进一步研究其功能奠定了基础。Objective To clone the first Kunitz-type domain of human tissue factor pathway inhibitor-2 （hTFPI-2/KD1） in E.coli. Methods The hTFPI-2/KD1, obtained by PCR from a human placenta cDNA, was directly inserted into plasmid pET22b. The E.coli BL21 was transformed with the recombinant plasmids and induced to express the target proteins. Chromogenic and gelatin zymography methods were used to evaluate the inhibiting effects of purified KD1 on trypsin and MMPs individually. Results The coding fragment of hTFPI-2/KD1 was cloned successfully and the protein expressed in resoluble high production. The protein could inhibit the activities of trypsin and MME Conclusions The activated hTFPI-2/KD1 is obtained by using prokaryotic expressed system effectively. This formed a basis for further studying the characteristics of hTFPI-2/KD 1.

关 键 词：丝氨酸蛋白酶抑制剂 蛋白质结构 三级 蛋白质工程 克隆 分子 

分 类 号：R346[医药卫生—基础医学]