人正常胃黏膜细胞全长cDNA文库的构建及鉴定  

Construction and identification of a cDNA library of human gastric mucosa

作　　者：何震[1] 谢玉波[2] 肖强[1] 

机构地区：[1]广西医科大学第一附属医院胃肠腺体外科,广西南宁530021 [2]广西医科大学第一附属医院麻醉科,广西南宁530021

出　　处：《成都医学院学报》2010年第1期8-11,共4页Journal of Chengdu Medical College

基　　金：国家自然科学基金资助项目(30860273)

摘　　要：目的构建人正常胃黏膜细胞的cDNA文库并鉴定文库质量。方法运用mRNA5’末端的模板转换方法以powerscript逆转录酶进行转录，在mRNA的5’末端添加一段5’oligo做为延伸后的模板，从而富集全长cDNA．扩增后的cDNAs经XhoI酶切、柱层析洗脱，重组于pGADT7载体并包装后，测定滴度、重组率，扩增文库。随机挑取16个克隆行PCR反应扩增插入片段。结果构建的cDNA文库滴度为4．00×10^6pfu／ml，重组率〉95％，扩增后滴度达9．50×10^9pfu／ml，16个插入片段长度为0．5～2．0kb．结论构建的人正常胃黏膜细胞cDNA文库为高效全长的酵母双杂交cDNA文库，符合cDNA文库的要求，可进一步用酵母双杂交的方法探寻与胃疾病相关的基因。Objective To construct a full-length cDNA library of human gastric mucosa and identify the quality of the library. Methods The library was constructed by using the template-switching technique at 51 end of mRNA. A powerscript reverse transcriptase was used to transcribe,and a 5＇ oligo fragment as an extended template was added t o 5＇end of mRNA to enrich full-length cDNAs. After amplification, the cDNAs digested by XhoI and size-fractionated by columns were recombined into pGADT7 vectors. After package, the titer of recombinant vectors and the recombinant rate（blue/white）were determined, then the library was amplified. We identified the library using PCR reaction to determine the size of the inserts. Results The titer of cDNA library was 4. 00×10^6 pfu/ml, the rate of recombinant was above 95%,and the titer of amplified library was 9. 50×10^9 pfu/ml. The insert size ranged from 0. 5 to 2. 0 kb. Conclusion The yeast two-hybrid eDNA library of human gastric mucosa is successfully constructed and can be used for screening by Yeast Two-HyBrid to find the genes related to gastric diseases.

关 键 词：胃黏膜细胞 CDNA文库 全长 

分 类 号：R318[医药卫生—生物医学工程] R573[医药卫生—基础医学]