本文關(guān)鍵詞：葡激酶聚乙二醇修飾及活性鑒定研究　出處：《重慶醫(yī)科大學學報》2017年07期 　論文類型：期刊論文

  更多相關(guān)文章： 蛋白質(zhì)改造 聚乙二醇修飾 定點突變 活性驗證

【摘要】：目的:通過對葡激酶(staphylokinase,SAK)基因序列進行定點突變,表達突變蛋白,并對其進行聚乙二醇(polyethylene glycol,PEG)修飾。通過分子篩分離得到較高純度修飾蛋白,在體內(nèi)外鑒定其溶栓活性。方法:根據(jù)SAK晶體結(jié)構(gòu)及抗原位點選擇突變位點,構(gòu)建突變質(zhì)粒,并在大腸桿菌中表達半胱氨酸(cysteine,Cys)突變蛋白S-cys;分離純化目的蛋白;對目的蛋白在不同條件下進行聚乙二醇修飾,篩選最優(yōu)修飾條件;分離修飾蛋白與未修飾蛋白,得到較高純度的修飾蛋白peg-S-cys,通過纖維蛋白平板溶圈實驗和動物栓塞模型的構(gòu)建初步對其生物活性進行驗證。結(jié)果:原核表達、純化后得到純度較高的S-cys突變體蛋白;優(yōu)化篩選S-cys蛋白的PEG修飾實驗參數(shù),得到最佳修飾條件;應(yīng)用分子篩分離純化,成功獲得純度較高的peg-S-cys蛋白(純度大于90%);初步驗證peg-S-cys具有溶栓活性。結(jié)論:通過對蛋白質(zhì)基因序列的定點突變,成功實現(xiàn)了蛋白質(zhì)的聚乙二醇定點修飾,在體外和體內(nèi)初步驗證其活性,為蛋白質(zhì)改造提供了一種新的方法與思路。
[Abstract]:Objective: to express the mutant protein by site-directed mutation of staphylokinase staphylokinase (staphylokinase) SAK gene sequence. The modified protein was modified by polyethylene glycol polyethylene (PEG) and purified by molecular sieve. Methods: according to the crystal structure and antigenic site of SAK, the mutant plasmid was constructed, and cysteine was expressed in Escherichia coli. Cys) mutant protein S-cys. Isolation and purification of the target protein; The target protein was modified with polyethylene glycol under different conditions. High purity modified protein peg-S-cys was obtained by isolating modified protein and unmodified protein. The biological activity was preliminarily verified by fibrinolysis assay and animal embolization model. Results: the purified S-cys mutant protein was purified and expressed in prokaryotic expression. The PEG modification parameters of S-cys protein were optimized and the optimum modification conditions were obtained. Peg-S-cys protein with high purity was successfully obtained by molecular sieve separation and purification. Conclusion: through the site-directed mutation of protein gene sequence, the protein was successfully modified with pegyleneglycol. Its activity was preliminarily verified in vitro and in vivo, which provided a new method and train of thought for protein modification.
【作者單位】： 重慶醫(yī)科大學附屬第一醫(yī)院心血管內(nèi)科;重慶醫(yī)科大學感染性疾病分子生物教育部重點實驗室;
【分類號】：R3411
【正文快照】： 蛋白質(zhì)改造即通過對蛋白質(zhì)化學、蛋白質(zhì)晶體學和蛋白質(zhì)動力學的研究,獲得有關(guān)蛋白質(zhì)理化特性和分子特性的信息,在此基礎(chǔ)上對編碼蛋白質(zhì)的基因進行有目的的設(shè)計和改造[1]。目前蛋白質(zhì)改造的手段主要有定點突變、融合蛋白、功能域表達、蛋白修飾等。研究表明,通過對蛋白質(zhì)進行聚

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