人源性大肠癌cDNA噬菌体库的构建及PCR鉴定  

CONSTRUCTION OF HUMAN COLORECTAL CARCINOMA cDNA PHAGE EXPRESSION LIBRARY AND ITS IDENTIFICATION BY PCR

作　　者：刘宇虎[1] 张振书[1] 武金宝[1] 钟东[1] 崔海宏[1] 王亚东[1] 

机构地区：[1]第一军医大学南方医院,广州510515

出　　处：《解放军医学杂志》2003年第7期608-610,共3页Medical Journal of Chinese People's Liberation Army

基　　金：国家自然科学基金资助课题 (编号 30 1 71 0 53)

摘　　要：为构建人源性大肠癌cDNA噬菌体表达文库 ,从人大肠癌组织中提取总RNA ,纯化mRNA ,用RT PCR反转录合成cDNA第一链 ,用LD PCR合成cDNA第二链 ,除去小于 5 0 0bp的小片段 ,与λTriplEx2噬菌体连接 ,体外包装。转化E .coliXL1 blue大肠杆菌 ,滴定文库的滴度 ,用IPTG和X gal测定重组率。用PCR鉴定插入cDNA片段大小。构建成含 2 .0 7× 10 6 pfu/ml重组子的人大肠癌cDNA噬菌体表达文库 ,重组率为 94.5 % ,插入外源cDNA片段大小从 60 0bp～ 4kb ,平均约 1.4kb。文库合符要求 。The aim of this experiment was to construct a human colorectal carcinoma cDNA phage expression library. Total RNA was extracted from the cancer tissue of human colorectal carcinoma, and the mRNA was purified. The single-strand and double-strand of cDNA were synthesized through reverse transcription-PCR and LD-PCR. cDNA fragments, after removal of those smaller than 500bp, were combined with λTriplEx2 phage vector. The recombinant cDNA were packaged in vitro with MaxPlax TM Packaging Extract, then a small portion of packaged phage was used to infect E.coli XL1-blue for titration and determination of the percentage of recombinant clones. PCR method was used to identify the size of inserted cDNA. A human colorectal carcinoma cDNA phage library consisting of 2.07×10 6 pfu/ml recombinant bacteriophages was successfully constructed, the recombinant percentage was 94.5%, and the range of the fragment length of exogenous inserted cDNA was between 600bp～4kb, with an average of about 1.4kb. It met the universally accepted standards, and it could be useful in screening cDNA clones to find out the human colorectal carcinoma associated antigen genes.

关 键 词：结肠直肠肿瘤 噬菌体 CDNA文库 

分 类 号：R735[医药卫生—肿瘤]