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摘要:目的:为了更为准确地确定在肠激酶整个生产过程中是否一直含有目的物质，我们要进行检验分析，而且对分析方法进行不断优化。实验:重组大肠杆菌表达肠激酶轻链融合蛋白主要是以包涵体形式存在，待控温发酵后冰冻可以得到湿菌体，按1：8质量体积比加入到TE缓冲液中混匀。其次对混合液进行破碎、离心，收集沉淀即为粗包涵体并称重,然后将包涵体溶解，依次加入一定量的尿素、EDTA（0.5 mol)和DTT(20 mmol),变性处理20 h。将变性夜进行超滤除杂后，取样（200 ul）测蛋白浓度并确定复性缓冲液体积之后加入甘氨酸、谷胱甘肽（还原型和氧化型）配置成复性溶液进行复性处理20 h。然后将复性后溶液进行浓缩、酶切即可得到粗酶夜，对酶切后溶液进行聚丙烯酰胺凝胶电泳（SDS-PAGE）。结果:最后上柱进行层析分离得到较纯的肠激酶，由称得的1270 g大肠杆菌经以上一系列步骤得到肠激酶53.6 mg。38406
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毕业论文关键词：电泳;酶切;蛋白浓度测定;变性;复性
Quality Control of Recombinant Escherichia coli Expressing Enterokinase
In order to determine whether the whole production process of intestinal kinase has always been containing the target substance, we must carry out the inspection and analysis and optimize the analysis method.Enterokinase recombinant Escherichia coli eklc fusion protein mainly existed in the form of inclusion body，after temperature controlled fermentation and freezing we can get wet cell，then we will qualitify and by 1:8 quality volume than joining to TE buffer liquid mixing.Secondly on the mixture of crushing, centrifuging and collecting the precipitate is rough inclusion and weighed，and inclusion body dissolving in water stirring，followed by adding a certain amount of Urea and EDTA (0.5 mol) and DTT (20 mmol) for 20 h.Ultra filter impurities to sampling (200 ul )measure the protein concentration to determine the renaturation buffer volume then and added the glycine, glutathione (reduced and oxidized) configured to the refolding for 20 h.Then refolding solution concentration, enzyme digestion to obtain crude enzyme SDS-PAGE .Finally, the isolated intestinal kinase was obtained by chromatography,the intestinal kinase 53.6 mg was obtained from the 1270 g E.coli after a series of steps.
Keywords: electrophoresis；enzymeprotein concentration determination；denaturation；renaturation
目录
引言 3
1 材料与方法 5
1.1材料 5
1.1.1供试材料 5
1.1.2试剂 5
1.1.3仪器 7
1.2 实验方法 7
1.2.1大肠杆菌的发酵、破碎、离心 7
1.2.2包涵体的变复性 8
1.2.3浓缩液的酶切 8
1.2.4上柱层析 8
1.2.5电泳 8
1.2.6 蛋白质浓度测定（单点测定和曲线测定） 10
2 实验结果与分析 11
2.1大肠杆菌发酵后SDS-PAGE结果分析 11
2.2包涵体的变复性结果分析 11
2.3上柱层析后SDS-PAGE结果分析 12
2.4蛋白浓度测定结果分析 13
2.5酶活性分析 14
3 讨论 15
4 致谢 15
5 参考文献 15
引言
肠激酶是（Enterokinase,EK）是一种存在于高等动物的十二指肠粘膜中，它重要作用是能够将胰蛋白酶原水解成具有活性的胰蛋白酶。EK是一种蛋白内切酶，能够高度专一性识别蛋白质Asp-Asp-Asp-Asp-Lys序列，从而肠激酶被广泛应用于基因工程产品的开发研究当中，比如用于重组融洽蛋白的特异性断裂来进一步进行生物制药。