人溶菌酶基因的克隆和生物信息学分析  

Cloning and Bioinformatic Analysis of Human Lysozyme Gene

作　　者：卢雪梅[1] 金小宝[1] 朱家勇[1] 沈娟[1] 梅寒芳[1] 马艳[1] 肖明珠[1] 褚夫江[1] 

机构地区：[1]广东药学院药用生物活性物质研究所广东省生物活性药物研究重点实验室,广东广州510006

出　　处：《生物技术》2010年第6期26-29,共4页Biotechnology

基　　金：国家自然科学基金项目(30671832)资助

摘　　要：目的:克隆人溶菌酶基因并进行生物信息学分析及构建其原核表达载体。方法:采用RT-PCR法扩增人溶菌酶基因,运用相关生物信息学软件对其理化性质、疏/亲水性、功能结构域及蛋白质二级结构等进行分析。将该基因克隆入载体pET32a,PCR、双酶切鉴定和核苷酸序列测定。结果:获得约400 bp的人溶菌酶基因,其序列与GenBank中公布序列完全一致。生物信息学分析显示人溶菌酶基因编码130个氨基酸,分子量为14.7 kDa,理论等电点为9.28,含有一个功能结构域,二级结构主要由α-螺旋、无规则卷曲、延伸链和β-转角组成。经PCR、双酶切鉴定和核苷酸序列测定,表明重组表达质粒构建成功。结论:该基因的克隆、生物信息学分析及原核表达质粒的构建为进一步研究其功能奠定了基础。Objective：To clone the human lysozyme（Hly） gene,analyze it by bioinformatics analysis software and construct the prokaryotic expression plasmid of Hly/pET-32a.Method：The human lysozyme gene was amplified by reverse-transcription polymerase chain reaction（RT-PCR） using specific primers.The physical-chemical properties,the conserved domains,hydrophilicity or hydrophobicity and second structure of Hly were analysised by bioinformatics analysis software.The Hly gene fragment was subcloned into expression vector of pET-32a.The resulting plasmids were transformed into E.coli BL21 cells to verify by PCR screening,restriction endonuclease analysis and DNA sequencing.Result： The sequence of Hly was identifying to the sequence reported in Genbank.Hly is cationic molecules contains 130 amino acids,with molecular weight of 14.7 kDa,theoretical PI of 9.28,containing a conserved domain of lysozyme superfamily.The second structures of Hly contain α-helix,random coil,extended strand and β-turn.The prokaryotic expression plasmid Hly/pET-32a was constructed successfully.Conclusion：The cloning,bioinformatic analysis and construction of prokaryotic expression plasmid of human lysozyme gene could provide foundation for further study of its biological activity.

关 键 词：人溶菌酶 克隆 生物信息学分析 原核表达载体 

分 类 号：Q786[生物学—分子生物学]