人源性食管鳞癌cDNA文库的构建及鉴定  

Construction and identification of the cDNA expression library for human esophageal squamous carcinoma

作　　者：赵英芳[1] 赵素莲[1] 史天良[2] 

机构地区：[1]山西医科大学微生物免疫教研室,山西太原030001 [2]山西省肿瘤研究所生物技术部,山西太原030013

出　　处：《中华肿瘤防治杂志》2007年第18期1379-1382,共4页Chinese Journal of Cancer Prevention and Treatment

基　　金：山西省高校科技研究开发项目(200138)

摘　　要：目的:构建人源性食管鳞癌cDNA噬菌体表达文库。方法:从人食管鳞癌组织中提取总RNA并分离纯化mRNA,用RT-PCR合成cDNA单链,LD-PCR扩增双链cDNA,除去<500bp的小片段,与载体λTriplEx2噬菌体左右臂连接,经体外包装即构成全长cD-NA噬菌体表达文库。转化E.coliXL1-Blue感受态细胞,测定文库的滴度,确定文库的容量大小,用蓝白筛选测定文库的重组率,PCR鉴定cDNA插入片段的大小。结果:构建的人源性食管鳞癌cDNA噬菌体表达文库滴度为1.64×106pfu/mL,重组率为98.6%,cDNA插入片段的大小为0.7～2.5kb,平均长约1.5kb。结论:成功构建1个人源性食管鳞癌cDNA噬菌体表达文库,适合于大批量筛选cDNA克隆的食管鳞癌肿瘤抗原基因。OBJECTIVE： To construct a cDNA phage expression library for human esophageal squamous carcinoma. METHODS： After the total RNA was extracted from human esophageal squamous carcinoma tissues, mRNA was isolated and purified from total RNA. The single-strand and double-strand cDNA were synthesized through reverse transcriptase PCR and long-distance PCR, with the cDNA fragments smaller than 500 bp removed and the remaining cDNA combined with the right and left arms of λTriplEx2 phage vector. The recombinant phage were packaged in vitro and then small portion packaged phage was used to infect E. coli XL1-Blue for determing titration and the percentage of recombinant clones. PCR was used to identify the size of inserted cDNA. RESULTS： The constructed cDNA phage expression library for human esophageal squamous carcinoma was consisted of 1.64× 10^4 pfu/mL bacteriophages with a recombination rate of 98. 6%. The range of the fragment lenth of inserted cDNA was between 0.7 and 2.5 kb, with the average of 1.5 kb. CONCLUSION： The cDNA phage expression library of human esophageal squamous carcinoma is successfully constructed to meet the currently recognized standards, and can be well applicable in screening cDNA-cloned genes of human esophageal squamous carcinoma-associated antigens by immunoscreening.

关 键 词：食管肿瘤/遗传学 DNA 互补/遗传学 基因表达 

分 类 号：R735.1[医药卫生—肿瘤]