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作 者:高宇[1] 赵红蕾[1] 冯新[1] 刁昱文[1] 刘珊珊[1] 李林溪[1] 顾敬敏[1] 韩文瑜[1] 雷连成[1]
出 处:《动物医学进展》2011年第8期32-36,共5页Progress In Veterinary Medicine
基 金:吉林省科技平台计划项目(20070210)
摘 要:能分泌表达重组人溶菌酶-鲎素融合蛋白的巴斯德毕赤酵母(Pichia pastoris)工程菌(X33/pPICZαA-human lysozyme-TachyplesinⅠ构建的)。除了基因本身内在特性外,表达条件对外源基因的表达量也有着极显著的影响。本文在摇瓶水平上通过发酵液起始pH、甲醇诱导浓度、诱导时间这3个因素进行表达条件优化。通过生长曲线,Bradford检测以及平板扩散试验分析,获得摇瓶发酵表达人溶菌酶-鲎素融合蛋白的最佳条件为发酵液初始pH 5.0,甲醇诱导浓度为2.0%,诱导时间为72 h;Bradford检测法分析显示表达量最高,平板扩散试验显示融合蛋白对金黄色葡萄球菌(ATCC25923)抑菌能力最明显,为进一步大规模发酵表达人溶菌酶-鲎素融合蛋白奠定了基础。The engineering strain of Pichia pastoris, X33/pPICZaA-human lysozyme-Tachyplesin I , which was constructed by our lab, can express secretory recombinant human lysozyme - Tachyplesln I fusion protein, gecides their internal properties of foreign gene, the culture and inducing conditions also has a sig- nificant impact to the expression . In this paper, the expression conditions of recombinant HLYZ-Tachyplesin I were optimized of three factors such as initial medium pH, methanol concentration and the induction time. Growth curve, Bradford and the plate diffusion test had been used to analysis and we found that the optimum induction conditions for recombinant HLYZ-Tachyplesin I expression were :an initial pH of 5.0, a final methanol concentration of 2.0 % ,and 72 hours for induction Under these conditions ,the Bradford analysis showed the high level expression and the agar diffusion tests showed that the fusion protein antimicrobial activity for Staphylococcus aureus(ATCC25923). Thus sets up an optimized expression system for hLYZ-Tachyplesin I fusion protein, which will play an important role in its further large-scale fermentation and industrial production.
关 键 词:毕赤酵母 人溶菌酶-鲎素融合蛋白 表达条件 优化
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