MMP-9-PEX原核表达、纯化及其对结肠癌细胞侵袭能力的影响  

Expression,Purification of the MMP-9-PEX in E.coil and Its Effect on Invasive Capacity of Colonic Cancer Cells

作　　者：严春霞[1] 何俊琳[1] 刘学庆[1] 丁裕斌[1] 陈雪梅[1] 王应雄[1] 

机构地区：[1]重庆医科大学公共卫生学院遗传优生教研室,重庆400016

出　　处：《细胞生物学杂志》2006年第5期717-720,共4页Chinese Journal of Cell Biology

基　　金：重庆市科委攻关项目资助(No.7668)~~

摘　　要：用RT-PCR法扩增出基质金属蛋白酶-9(MMP-9)C末端血红素结合蛋白样结构域(PEX),克隆至表达载体pET32a中并转化至E.coilBL21(DE3),经IPTG诱导后在约为42kDa处发现有外源基因的表达,密度扫描显示表达蛋白含量占菌体总蛋白的50%左右。重组蛋白质pET32a/PEX主要以包涵体形式表达,其在上清液中的含量极微。含8mol/L尿素和10mmol/LDTT的裂解缓冲液溶解的包涵体采用金属整合层析有效分离出目标蛋白,纯化后pET32a/PEX蛋白质的纯度大于90%。在Transwell实验中发现复性纯化后的重组蛋白质pET32a/PEX能抑制结肠癌细胞的侵袭,且呈剂量依赖性。Matrix metalloproteinase 9 hemopexin domain （PEX）, which locates the C-terminal, was amplified by RT-PCR and was inserted into expression vector pET32a. The recombinant plasmid was induced by IPTG for 4 h and a 42 kDa recombinant protein was produced. Amount of the fusion protein pET32a/PEX expression was 50% of total bacterial protein, mostly in form of inclusion, few in form of supernatant. Inclusion was dissolved in 8 mol/L urea and 10 mmol/L DTT, carried out affinity purification under denaturing condition. The expressed 42 kDa fusion protein is confirmed by SDS-PAGE. The pET32a/PEX expression vector was successfully constructed and highly expressed in E.coli. The purified pET32a/PEX protein can inhibitor the invasion of the colonic cancer cell in Transwell experiments and show a dose-dependent manner.

关 键 词：基质金属蛋白酶-9 血红素结合蛋白样结构域 基因表达 细胞侵袭 

分 类 号：R735.35[医药卫生—肿瘤]