人基质蛋白酶2(MMP-2)血红素结合蛋白样结构域原核表达、复性及生物学活性验证

Expression of Hemopexin-like Domain of Human Matrix Metalloproteinase 2 in E.coli and It Bioactivity Analysis

用RT-PCR从人神经胶质瘤U87细胞扩增人基质金属蛋白酶-2(MMP-2)血红素结合蛋白样结构域(PEX)基因,克隆到pMD18-T载体然后测序;将PEX定向克隆到PET-25b(+)中构建原核表达载体PET-PEX,转化大肠杆菌BL21(DE3),诱导表达产生表达量近40%的包涵体蛋白,表达蛋白分子量大小约28.0KD,与预期大小相符。包涵体经优化洗涤,8 mol/L尿素变性,采用透析法进行复性。鸡胚绒毛尿囊膜(CAM)试验显示复性后的PEX蛋白能显著抑制鸡胚新生血管生成(P<0.05),并且其抑制作用表现出一定的剂量依赖性。

To express hemopexin-like domain of human matrix metalloproteinase 2 （PEX） in Escherichia coli and study its primary biological characteristics, the eDNA of PEX was amplified from human U87 glioma cells by RT-PCR. The amplified eDNA sequence was sequenced and its fragment was cloned into the prokaryotic expression vector PET25（a） to construct the recombinant plasmid of PET- PEX. The recombinant PET-PEX vector was transformed into E. coli BL21（DE3） and the PEX expression was induced by IPTG. The expressed PEX accounted for approximately 40 %of the total bacteria proteins,and existed mainly as inclusion bodies. The PEX proteins was refolded by dialysis after optimized-washed and denatured with 8mol/L urea. The refolded PEX was tested on chicken CAM model, and a large number of newly formed blood vessels was significantly regressed （P〈 0.05）, and the study results also shows the bacterial-expressed PEX effectively inhabited angiogenesis of the chicken embryo in a dose-depentment manner through CAM assay.