重组小鼠IFN-β原核表达载体的构建及包涵体溶解条件的筛选  

Construction of prokaryotic expression vector of recombinant mouse IFN-β and screening for solubilization conditions of inclusion body

作　　者：苏雅拉图[1] 张永胜[2] 陈宇杰[1] 胡小平[2] 王丽颖[2] 万敏[2] 吴柒柱[1] 

机构地区：[1]内蒙古民族大学生命科学学院,内蒙古通辽028000 [2]吉林大学白求恩医学院生物化学与分子生物学教研室,吉林长春130021

出　　处：《吉林大学学报（医学版）》2013年第4期841-846,共6页Journal of Jilin University：Medicine Edition

基　　金：国家自然科学基金资助课题(81060098);吉林省科技厅科研基金资助课题(20060416)

摘　　要：目的:通过重组小鼠干扰素β(IFN-β)原核表达载体的构建及对包涵体溶解条件的筛选,建立IFN-β原核表达模型,为筛选适合的包涵体溶解条件提供实验依据。方法:小鼠新鲜肝组织中提取DNA,传统方法构建小鼠IFN-β原核表达载体并命名为IFN-β′。SDS-PAGE检测以传统方法、pH环境、溶解液成分、干菌与溶解液比例、溶解温度和溶解时间的改变对包涵体溶解效果的影响。结果:IFN-β′成熟蛋白鉴定结果与预测结果吻合,诱导表达正常。改变pH值、在溶解液中加入DTT对包涵体溶解效果无影响,包涵体经洗涤液洗涤后亦不能被溶解液所溶解,温度对包涵体溶解几乎无影响,溶解液的比例由1g菌∶5mL溶解液增加为1g菌∶20mL溶解液时能溶解部分IFN-β′蛋白包涵体。结论:成功构建重组小鼠IFN-β原核表达载体,其蛋白以包涵体形式存在,且不易被溶解,在筛选的包涵体溶解条件中仅溶解液比例为影响包涵体蛋白溶解条件。Objective To establish the prokaryotic expression model of IFN-βby constructing the prokaryotic expression vector of recombinant mouse IFN-βand screening the conditions for its inclusion body solubilization,and to provide experimental basis for screening the proper solubilization conditions.Methods DNA was extracted from fresh liver tissue of mice.The prokaryotic expression vector of mouse IFN-βwas constructed by traditional methods and was named IFNβ ′.Based on the traditional method of solution,the inclusion body dissolving process conditions were improved,such as solution composition pH environment,bacteria and dissolved liquid ratio,and the effect of dissolved temperature and the dissolution time of inclusion body.Results The mature IFNβ ′protein was identical with the predicted results and the induced expression was normal.The changes in pH and DTT in dissolved liquid had no effects on inclusion body solubilization.The inclusion body could not be dissolved by washing liquid after liquid washing.Temperature had almost no effect on inclusion body solubilization.When the ratio of dry bacteria to solution was increased from 1g∶5mL to 1g∶20mL,the IFNβ ′protein inclusion body could be partially dissolved.Conclusion The recombinant mouse IFNβ prokaryotic expression vector is constructed successfully.The target protein is expressed with the form of inclusion body but not easy to be dissolved,and the dissolved liquid proportion is the only condition to affect the dissolution of in clusion body protein among the screened inclusion body solubilization conditions.

关 键 词：干扰素Β 原核表达 包涵体 

分 类 号：Q786[生物学—分子生物学]