【摘要】：目的在大腸埃希菌中表達(dá)球形節(jié)桿菌尿酸酶(uricase,Uri),并進(jìn)行純化和檢測(cè)其活性。方法根據(jù)大腸埃希菌遺傳密碼子偏愛性,結(jié)合原核翻譯起始序列的局部二級(jí)結(jié)構(gòu)自由能最小化原則,優(yōu)化設(shè)計(jì)編碼Uri蛋白的核苷酸序列,經(jīng)PCR擴(kuò)增球形節(jié)桿菌uri基因,克隆至載體pET43.1a,構(gòu)建重組表達(dá)質(zhì)粒pET43.1a-uri,轉(zhuǎn)化感受態(tài)大腸埃希菌BL21-odonPlus(DE3)-RIPL,IPTG誘導(dǎo)表達(dá)。表達(dá)產(chǎn)物經(jīng)硫酸銨粗純及DEAE瓊脂糖凝膠層析純化后,經(jīng)SDS-PAGE分析純度;參考Uri產(chǎn)品說明書測(cè)定蛋白酶活性,并確定其最佳檢測(cè)溫度及pH值。結(jié)果重組表達(dá)質(zhì)粒pET43.1a-uri經(jīng)酶切及測(cè)序鑒定構(gòu)建正確;重組蛋白相對(duì)分子質(zhì)量約33 000,以可溶性形式存在,表達(dá)量約占菌體總蛋白的40%;純化后純度可達(dá)90%以上;酶活性達(dá)13.2 U/mg,酶活性檢測(cè)最佳反應(yīng)溫度為40℃,最佳反應(yīng)pH值為9.0。結(jié)論成功于大腸埃希菌中表達(dá)了uri基因,并獲得高純度的Uri蛋白,其酶學(xué)性質(zhì)與天然的球形節(jié)桿菌尿酸酶基本一致,為其大規(guī)模穩(wěn)定生產(chǎn)及尿酸酶法檢測(cè)試劑的配制研究奠定了基礎(chǔ)。
[Abstract]:Objective to express uricase (uricase,Uri) in Escherichia coli, purify and detect its activity. Methods according to the genetic codon preference of Escherichia coli and the principle of minimizing the local secondary structure free energy of prokaryotic translation initiation sequence, the nucleotides encoding Uri protein were optimized. The uri gene of Bacillus globularis was amplified by PCR and cloned into vector pET43.1a, to construct the recombinant expression plasmid pET43.1a-uri, to transform the receptive Escherichia coli BL21-odonPlus (DE3)-RIPL,IPTG to induce expression. After purification by ammonium sulfate and DEAE agarose gel chromatography, the purity of the expressed product was analyzed by SDS-PAGE, and the protease activity was determined with reference to Uri product specification, and the optimum detection temperature and pH value were determined. Results the recombinant expression plasmid pET43.1a-uri was constructed correctly by restriction enzyme digestion and sequencing, the relative molecular weight of the recombinant protein was about 33000, which existed in soluble form, accounting for 40% of the total bacterial protein, the purity was over 90%, the enzyme activity was 13.2 U 鈮，

本文編號(hào)：2508725