利用PCR筛选噬菌体文库人免疫球蛋白重链Cγ基因的一种新方法  

A new method used to screen the human immunoglobulin heavy chain Cγ gene by means of PCR-screening phage library

作　　者：孙剑华[1,2] 王宏 钱旻[1] 成国祥 

机构地区：[1]华东师范大学生命科学院,上海200062 [2]上海市转基因研究中心,上海201203 [3]同济大学生命科学与技术学院,上海200092

出　　处：《现代免疫学》2009年第1期30-34,共5页Current Immunology

基　　金：上海市科技发展基金资助项目(024319105)

摘　　要：扩增噬菌体人基因组文库以形成多个亚文库并富集稀有目的克隆。利用人免疫球蛋白Cγ基因启动子、编码区设计的多对探针引物,PCR筛选已扩增的亚文库,获得共阳性亚文库;利用多条同位素标记的探针筛选上述共阳性亚文库,经相应探针引物PCR鉴定,获得共阳性多克隆噬菌斑;2000倍稀释共阳性多克隆噬菌斑稀释液,PCR筛选出阳性单克隆噬菌斑。基因转换到pBSK(+)载体测序,筛到长12 kb的人免疫球蛋白重链Cγ基因,含有启动子、编码外显子、穿膜外显子及polyA序列。与传统的单条探针筛库相比,新策略利用多条探针一次同位素标记,就筛到完整的稀有Cγ基因;利用PCR在亚文库、多克隆噬菌斑稀释液、单克隆噬菌斑稀释液三个层面上筛选,逐步缩减的筛选范围是一次同位素标记筛到完整稀有基因的前提。新策略不仅提高一倍工作效率,降低几倍的耗材,同时为需要长片段基因的功能基因组领域提供筛选捷径。A new method used to screen the human immunoglobulin heavy chain Cγ gene by PCR-screening phage library was established,in which the phage human genomic library was amplified to form many sublibraries and to concentrate the rare target clones,and the amplified sublibraries were screened by PCR with multiple pairs of probe primers designed according to the promoter and the coding region of human immunoglobulin Cγ gene to obtain the co-positive sublibrary.This co-positive library was screened by probe labeled...

关 键 词：噬菌体基因组文库 PCR higCγ基因 筛选 同位素 

分 类 号：R392[医药卫生—免疫学]