联合利用嵌套缺失和酚筛选法构建和鉴定目的基因的缺失突变体  

Constructing and venfication of deletion mutants of target gene by using nested deletion method and phenyl hydrate screening

作　　者：程璘令[1] 李冰[2] 涂洪斌[2] 刘启才[2] 冉丕鑫[1] 

机构地区：[1]广州医学院第一附属医院广州呼吸疾病研究所,510120 [2]广州医学院实验医学研究中心

出　　处：《现代临床医学生物工程学杂志》2006年第3期288-290,共3页Journal of Modern Clinical Medical Bioengineering

基　　金：广东省自然科学基金(06301136)

摘　　要：目的介绍一种快捷、无突变的目的基因缺失体构建和鉴定方法。方法用MluI和SacI酶将GCLC/pGL3在GCLC基因与pGL3连接处切开,形成以GCLC端为3’凹端而pGL3载体端为3’凸端的线型载体。利用外切核酸酶Ⅲ从GCLC基因端(3’凹端)单向逐一切除GCLC基因上的核苷酸,并在不同的时间终止其反应以获得一系列的GCLC基因缺失体。将GCLC基因缺失体与pGL3载体自身环化构建成GCLC基因序列缺失体的pGL3报道载体。最后用酚抽提和凝胶电泳法粗略挑选出不同长度的GCLC/pGL3缺失突变体。结果利用嵌套缺失法构建出各种长度的GCLC基因的缺失突变体,且不存在基因突变的现象。利用酚筛选法快速鉴定出29种不同长度的缺失突变体。结论联合利用嵌套缺失和酚筛选法可以高保真而快速地构建和鉴定目的基因的缺失突变体。Objective To introduce a quick and no mutational method for constructing deletion mutants of target gene and the construction of deletion mutants of GCLC promoter is used as an example. Methods Mlu Ⅰ and Sac Ⅰ were used to cut the GCLC/pGL3 gene at the conjunction of GCLC gene and pGL3, and the linearized gene which had one susceptible site at one end and one resistant site at the other was obtained. Exonuelease Ⅲ was used to cut the 5＇- flanking region of linearized GCLC gene. The serial deletion mutants were obtained by terminating the deleting reaction at different time. Each deletion mutant was reeircularized to get different mutants of GCLC-Luc. All mutants of GCLC-Luc were screened by phenyl hydrate exact and gel electrophoresis. Results The nested deletion method for constructing deletion mutants of GCLC promoter was advanced, with no gene mutation. Twenty-nine mutants of GCLC-Luc were screened by phenyl hydrate exact. Conclusion The deletion mutants of target gene can be obtained quiekly and with no mutation by using nested deletion method and phenyl hydrate screening.

关 键 词：嵌套缺失法 酚筛选法 缺失突变体 

分 类 号：R346[医药卫生—基础医学]