肺炎链球菌假想的溶菌酶样蛋白的活性分析和鉴定  

Analysis and Identification of the Activity of Hypothetical Lysozyme-like Protein in Streptococcus Pneumoniae

作　　者：杨晓亮[1] 孟江萍[2] 崔亚利[1] 黄健[1] 刘鑫[1] 胥文春[1] 

机构地区：[1]重庆医科大学临床检验诊断学教育部重点实验室,重庆市400016 [2]重庆医科大学附属第一医院,重庆市400016

出　　处：《医学分子生物学杂志》2010年第6期471-475,共5页Journal of Medical Molecular Biology

基　　金：国家自然科学基金（No.30700914）

摘　　要：目的 探索肺炎链球菌中一种假想的溶菌酶样蛋白的活性.方法 生物信息学分析该基因的功能结构域;利用长臂同源PCR对该基因进行敲除,观察D39野生菌和缺陷菌在生物学性状的改变;利用底物PNP-（GIcNAc）和溶壁微球菌,构建过表达载体,绘制生长曲线,对截断和全长蛋白的溶菌酶活性进行鉴定.结果 生物信息学分析结果显示该基因的编码产物为β-1,4-N-乙酰胞壁酸糖苷酶,属于糖基水解酶25家族;野生菌为长链生长,缺陷菌呈短链状;溶菌酶和假想的溶菌酶样蛋白均可使底物释放出游离的对硝基苯酚,A405nm吸光度值分别为1.166和0.792;同时也可使得溶壁微球菌发生溶解;含过表达质粒的肺炎链球菌较之野生菌,溶解较快.结论 假想的溶菌酶样蛋白具有溶菌酶活性,是一种新的溶菌酶.Objective To investigate the activity of a hypothetical lysozyme-like protein （Hllp） in Streptococcus pneumoniae. Methods The function domain of Hllp was analyzed by bioin- formatics. Hllp mutant strain was obtained by long flanking homology polymerase chain reaction. The biological changes of the mutant strain were compared to D39 wild strain. The activities of the short and full length of recombinant Hllp proteins were analyzed by use of the substrates PNP- （ Glc- NAc） 5 and Microcoecus lysodeikticus Fleming. Hllp expression vector was established to study Hllp＇s growth curve. Results Bioinformaties revea~[ed Hllp as a 13- 1, 4-N-acetylmuramidase be- longing to glycosyl hydrolase family 25 ; The long chain was representative for wild type Hllp, while the short chain for the mutant. Both lysozyme and the hypothetical lysozyme-like protein released p nitrophenol from the substrate PNP- （ GlcNAc）, showing 1. 166 and 0. 792 absorbance value at A405,m respectively and simultaneously leading to lysis of the Micrococcus lysodeiktieus fleming. Streptococcus pneumoniae containing over-expression vector were lysed significantly quick- ly. Conclusion The hypothetical lysozyme-like protein was proved to be a novel lysozyme.

关 键 词：肺炎链球菌 假想的溶菌酶样蛋白 溶菌酶 

分 类 号：R378.12[医药卫生—病原生物学] Q936[医药卫生—基础医学]