【摘要】：目的 PEX是基質(zhì)金屬蛋白酶(matrix metalloproteinase, MMPs)C端的血紅素樣結(jié)構(gòu)域(hemopexin domain),它主要通過抑制MMPs的活性來抑制血管發(fā)生、細(xì)胞增殖和侵襲。本實(shí)驗(yàn)通過構(gòu)建MMP9-PEX原核表達(dá)質(zhì)粒,對(duì)人基質(zhì)蛋白酶9(matrix metalloproteinase, MMP9)C末端血紅素結(jié)合蛋白樣結(jié)構(gòu)域的原核表達(dá)、透析復(fù)性條件進(jìn)行優(yōu)化,以獲得高表達(dá)的活性蛋白。 方法 ①Trizol裂解法自人肝組織中提取總RNA, RT-PCR法擴(kuò)增MMP9-PEX基因片段;②利用基因重組技術(shù)將MMP9-PEX基因片段插入PET-his質(zhì)粒,構(gòu)建原核表達(dá)質(zhì)粒;③將構(gòu)建好的原核表達(dá)質(zhì)粒PET-his-MMP9-PEX轉(zhuǎn)化大腸埃希菌菌株BL21(DE3),異丙基β-D硫代半乳糖苷( IPTG)誘導(dǎo)后產(chǎn)生包涵體蛋白,探討不同IPTG濃度、不同誘導(dǎo)時(shí)間、不同菌液密度值對(duì)目的蛋白表達(dá)產(chǎn)量的影響;④鹽酸胍裂解包涵體,用鎳瓊脂糖凝膠純化重組蛋白,蛋白印跡(western blot)方法檢測(cè)MMP-9-PEX表達(dá);⑤對(duì)純化蛋白的透析復(fù)性條件進(jìn)行優(yōu)化,明膠酶譜方法檢測(cè)MMP9-PEX活性。 結(jié)果 酶切和基因測(cè)序結(jié)果表明,成功構(gòu)建了重組質(zhì)粒PET-his-MMP9-PEX;重組質(zhì)粒轉(zhuǎn)化至大腸桿菌中,經(jīng)IPTG誘導(dǎo)發(fā)現(xiàn)30kDa處蛋白質(zhì)條帶明顯增強(qiáng);包涵體蛋白經(jīng)洗滌、裂解、純化后純度在95%以上,蛋白經(jīng)復(fù)性條件優(yōu)化后回收率為36.86%;明膠酶譜方法檢測(cè)表明,膠酶降解作用由于加入MMP-9-PEX而減弱,說明復(fù)性得到的蛋白具有生物學(xué)活性。 結(jié)論 成功構(gòu)建了重組質(zhì)粒PET-his-MMP9-PEX,優(yōu)化了MMP9-PEX原核表達(dá)、透析復(fù)性條件,獲得了有活性的MMP9-PEX蛋白,為進(jìn)一步研究MMP9-PEX的功能提供了實(shí)驗(yàn)基礎(chǔ)。
[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.
【學(xué)位授予單位】：遼寧醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R346

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