小麦蛋白质二硫键异构酶基因的克

发布时间：2018-08-15 12:07

【摘要】：目的:克隆小麦蛋白质二硫键异构酶(wheat protein disulfide isomerase,wPDI)基因,实现其在大肠杆菌中的表达并探究其酶学性质。方法:以小麦种子总RNA为模板,逆转录并扩增得到wpdi,并以pET-30b为表达载体、大肠杆菌BL21(DE3)为宿主菌进行原核表达,表达产物经金属螯合层析纯化后进行了酶学性质研究。结果:克隆的基因全长1 548 bp,与"Wyuna"品种小麦wpdi基因相似性达99%。构建了pET-30b-wpdi表达载体,获得w PDI的最佳表达条件为:诱导温度22℃,诱导时间6 h,诱导剂浓度0.5 mmol/L。该酶含4个硫氧还蛋白结构域,分子质量约为66.2 kD,具有二硫键的还原酶和异构酶活性以及分子伴侣活性。结论:对重组wPDI的表达和酶学性质研究,为wPDI在面制品加工及其他方面的应用提供参考依据。
[Abstract]:Aim: to clone wheat protein disulfide isomerase (wheat protein disulfide isomerasewPDI gene and express it in Escherichia coli and investigate its enzymatic properties. Methods: using total RNA of wheat seed as template, wpdil was obtained by reverse transcription and amplification. PET-30b was used as expression vector and Escherichia coli BL21 (DE3) was used as host strain for prokaryotic expression. The expression product was purified by metal chelate chromatography and its enzymatic properties were studied. Results: the total length of the cloned gene was 1 548 BP, which was similar to the wpdi gene of "Wyuna" wheat. The pET-30b-wpdi expression vector was constructed and the optimal expression conditions of w PDI were obtained as follows: induction temperature 22 鈩，

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