人源溶菌酶基因LYZL4在毕赤酵母中的重组表达及活性测定  

Recombinant Human Lysozyme LYZL4: Expression in Pichia pastoris and Its Antibacterial Activity

作　　者：李新新[1] 陶建军[1] 余龙[1] 

机构地区：[1]复旦大学遗传工程国家重点实验室遗传学研究所,上海200433

出　　处：《中国生物工程杂志》2014年第1期79-85,共7页China Biotechnology

基　　金：国家重大新药创制科技重大专项项目资助(2011ZX09102-007-01)

摘　　要：LYZL4是本实验室克隆的一种c型人溶菌酶基因,根据毕赤酵母密码子偏爱性对LYZL4基因进行优化设计,优化后的基因克隆至具有乙醇氧化酶启动子(AOX1)的pPIC9K载体中。通过电转化方法整合到甲醇营养型毕赤酵母表达菌株GS115基因组上,再利用不同浓度梯度的G418抗性YPD固体培养基筛选高拷贝转化子,经摇瓶发酵后,其上清液在SDS-PAGE胶上14.4 kDa处出现明显的蛋白条带,该蛋白条带经质谱鉴定证实是人LYZL4蛋白。以人溶菌酶(164 071U/mg)作为标准品,使用艳红微球菌作为底物,通过比色法测定重组LYZL4溶菌酶蛋白的活性,结果显示重组人溶菌酶LYZL4蛋白具有明显的溶菌活性,其酶活力可达38893U/ml。LYZIA is a kind of c type human lysozyme gene and cloned by our laboratory. It was optimized according to the yeast eodon preference. The code sequences optimized LYZL4 gene was cloned under the control of the alcohol oxidase 1 promoter （AOX1） in the vector pPICgK and integrated into the genome of the methylotrophic yeast Pichia pastoris （P. pastoris ） strain GS115 by electroporation. The screening of multiple copies of transformants was operated on YPD medium adding different concentration of G418 geneficin. We could get the multiple copies after screening. After methanol inducing by the flasks, the supernatant was analyzed by SDS-PAGE electrophoresis. The protein bands showed on 14.4 kDa. With human lysozyme （164 071U/mg） as the standard and the red micrococcus as the substrate, The activity of the human lysozyme LYZL4 was detected. The recombinant protein of LYZIA showed bactericidal activity to micrococeus and its activity was up to 38893U/ ml.

关 键 词：重组人溶菌酶LYZL4 毕赤酵母 溶菌活性 

分 类 号：Q78[生物学—分子生物学]