基因体外随机突变的两种方法的研究  

Studies on the Two Methods of Gene Random Mutation in vitro

作　　者：李弘剑[1] 张毅[2] 周天鸿[1] 李月琴[1] 

机构地区：[1]广东省暨南大学生命科技学院生物工程系,广州510632 [2]华南理工大学生物工程系,广州510641

出　　处：《遗传》2000年第2期96-100,共5页Hereditas（Beijing)

基　　金：:广东省自然科学基金资助项目

摘　　要：引导蛋白质功能进化常用的方法是模拟和加速蛋白质基因自然重组的进程 ,即在蛋白质的基因中引进随机突变。因此 ,蛋白质基因体外随机突变的方法影响着引导蛋白质功能进化的效果。本文描述两种简单而有效的基因体外随机突变发生方法。一种是化学诱变法 :将蛋白质基因用1.0mol/L硝酸钠在室温下处理1h,然后将突变基因插入质粒 ,导入大肠杆菌中表达 ;另一种方法是延伸诱变法 :将10个随机氨基酸短肽基因连接到蛋白质基因上 ,使蛋白质C末端连接随机短肽 ,通过增大蛋白质分子来达到延伸蛋白质序列空间的目的。来自嗜热脂肪芽孢杆菌的过氧化氢酶I基因用这两种方法进行了随机突变 ,获得了大量突变体酶基因。通过对突变体酶基因表达产物性质变化的测定 ,证明这两种基因诱变方法能够有效地诱发基因的随机突变。Two novel and simple methods were described in the paper for in vitro mutagenesis and recombinatin of polynucleotide sequence to mimic and accelerate nature's recombination strategy to direct the evolution of protein function.One is chemical mutagenesis: protein gene was inserted into M13mp18,and the single-stranded DNA was treated with 1 0mol/L sodium nitrite at room temperature for 1h for mutation and converted into duplex,then the mutated gene was ligated to plasmid for expression.Another is elongation mutagenesis: random peptide of 10 amino acid was connected at C terminal of protein to expand the sequence space by increasing protein's dimensions,then the elongation mutated gene expressed in E.coli.We have used these two methods to recombine the thermostabilized catalase I, and these two methods were found to be efficient to form a lot of catalase I mutates by identifying the properties of mutated enzyme.

关 键 词：定向进化 化学突变 延伸突变 蛋白质 随机突变 

分 类 号：TQ93[化学工程] Q754[生物学—分子生物学]