MMP9-PEX原核表达、纯化与复性研究

发布时间：2019-01-06 18:58

【摘要】：目的 PEX是基质金属蛋白酶(matrix metalloproteinase, MMPs)C端的血红素样结构域(hemopexin domain),它主要通过抑制MMPs的活性来抑制血管发生、细胞增殖和侵袭。本实验通过构建MMP9-PEX原核表达质粒,对人基质蛋白酶9(matrix metalloproteinase, MMP9)C末端血红素结合蛋白样结构域的原核表达、透析复性条件进行优化,以获得高表达的活性蛋白。方法 ①Trizol裂解法自人肝组织中提取总RNA, RT-PCR法扩增MMP9-PEX基因片段;②利用基因重组技术将MMP9-PEX基因片段插入PET-his质粒,构建原核表达质粒;③将构建好的原核表达质粒PET-his-MMP9-PEX转化大肠埃希菌菌株BL21(DE3),异丙基β-D硫代半乳糖苷( IPTG)诱导后产生包涵体蛋白,探讨不同IPTG浓度、不同诱导时间、不同菌液密度值对目的蛋白表达产量的影响;④盐酸胍裂解包涵体,用镍琼脂糖凝胶纯化重组蛋白,蛋白印迹(western blot)方法检测MMP-9-PEX表达;⑤对纯化蛋白的透析复性条件进行优化,明胶酶谱方法检测MMP9-PEX活性。结果酶切和基因测序结果表明,成功构建了重组质粒PET-his-MMP9-PEX;重组质粒转化至大肠杆菌中,经IPTG诱导发现30kDa处蛋白质条带明显增强;包涵体蛋白经洗涤、裂解、纯化后纯度在95%以上,蛋白经复性条件优化后回收率为36.86%;明胶酶谱方法检测表明,胶酶降解作用由于加入MMP-9-PEX而减弱,说明复性得到的蛋白具有生物学活性。结论成功构建了重组质粒PET-his-MMP9-PEX,优化了MMP9-PEX原核表达、透析复性条件,获得了有活性的MMP9-PEX蛋白,为进一步研究MMP9-PEX的功能提供了实验基础。
[Abstract]:Objective PEX is a heme-like domain (hemopexin domain), in the (matrix metalloproteinase, MMPs) C terminal of matrix metalloproteinase, which inhibits angiogenesis, cell proliferation and invasion by inhibiting the activity of MMPs. In this study, MMP9-PEX prokaryotic expression plasmid was constructed to express the heme-binding protein-like domain at the C-terminal of human matrix protease 9 (matrix metalloproteinase, MMP9, and the renaturation conditions were optimized to obtain the highly expressed active protein. Methods the MMP9-PEX gene fragment was amplified by total RNA, RT-PCR extraction from human liver tissue by 1Trizol cleavage method, and the MMP9-PEX gene fragment was inserted into PET-his plasmid by gene recombination technique, and the prokaryotic expression plasmid was constructed. 3 the constructed prokaryotic expression plasmid PET-his-MMP9-PEX was transformed into Escherichia coli strain BL21 (DE3) and induced by isopropyl 尾 -D-galactoside (IPTG) to produce inclusion body proteins. The effect of different liquid density on the expression yield of target protein; The recombinant protein was purified by nickel agarose gel and the expression of MMP-9-PEX was detected by Western blot (western blot). (5) the dialysis renaturation conditions of purified protein were optimized and the activity of MMP9-PEX was detected by gelatinase spectrum. Results the results of enzyme digestion and gene sequencing showed that the recombinant plasmid PET-his-MMP9-PEX; was successfully constructed and transformed into Escherichia coli, and the protein bands at the 30kDa site were significantly enhanced by IPTG induction. The purity of inclusion body protein was above 95% after washing, cleavage, and the recovery rate was 36.86 after renaturation. The results of gelatinase analysis showed that the degradation of gelatinase was weakened by adding MMP-9-PEX, which indicated that the refolding protein had biological activity. Conclusion the recombinant plasmid PET-his-MMP9-PEX, was successfully constructed to optimize the prokaryotic expression of MMP9-PEX, to obtain the active MMP9-PEX protein under dialysis renaturation conditions, and to provide an experimental basis for further study on the function of MMP9-PEX.
【学位授予单位】：辽宁医学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R346

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