人类风湿性关节炎滑膜组织cDNA文库的构建  

Construction and Identification of a cDNA Library of Human Rheumatoid Arthritis Synovial Tissue

作　　者：闫永毅[1] 任蕾[1] 高飞[1] 卢秀敏[1] 刘彦虹[1] 

机构地区：[1]哈尔滨医科大学第二附属医院,黑龙江哈尔滨150086

出　　处：《现代生物医学进展》2012年第16期3045-3048,共4页Progress in Modern Biomedicine

摘　　要：目的:构建人类风湿关节炎(RA)滑膜组织cDNA文库。筛查与RA相关的特异基因,为探讨RA的发病机制及基因治疗奠定基础。方法:提取人类风湿关节炎滑膜组织RNA并使用Trizol法纯化mRNA;运用mRNA5'末端的模板转换方法以powerscriptTM逆转录酶进行转录,使用COS III/3'PCR引物合成第1链cDNAs;长距离聚合酶链反应(LD-PCR)合成双链cDNA;PCR产物经蛋白酶K水解并纯化后,经Sfi I酶切、柱层析洗脱,重组于TripIEx2载体并包装后,测定滴度、重组率、扩增文库,随机挑取40个噬菌斑,用载体克隆位点两端的通用引物进行PCR扩增,以检测所构建cDNA文库的质量。结果:未扩增文库的滴度为6.89×106pfu/mL;扩增后文库滴度为2.63×109pfu/mL,重组效率为93%;插入片段主要集中在300～1800bp。结论:成功构建了一个人类风湿关节炎(RA)滑膜组织cDNA文库,可以用探针抗体等做免疫学筛选,为进一步探寻与RA疾病相关的基因奠定坚实基础。Objective： To construct a cDNA library of human synovial tissue of RA and indentify the quality of the library.Methods： Total RNA was extracted and mRNA was purified.mRNA was reversed to first-strand cDNA which was amplified to double-strand cD-NA by long distance PCR.PCR products were digested by proteinase K and Sfi I,and were fractionated by CHROMA SPIN-400 column.The cDNA of length longer than 0.4kb were collected and ligated to TriplEx2 vector.The phage packaging reaction for the ligated DNA was performed to produce an unamplified library.Thereafter,the unamplified library was titered and the percentage of recombinant clones were detected.In the end,fourty plaques were randomly selected and amplified by PCR using universal primers from vector in or-der to test the quality of the obtained library.Results： The titers of unamplifed and amplified libraries were 6.89×106 pfu/mL and 2.63×109 pfu/mL respectively.The rate of recombinant was 93%.The insert size range from 300 to 1800 bp.Conclusions： A high quality cD-NA 1ibrary from human synovial tissue of RA was constructed successfully,and it lays solid foundation not only for screening and cloning new specia1 genes associated with the occurrence of RA,but also for gene therapy of it.

关 键 词：类风湿性关节炎 基因文库 CDNA 

分 类 号：R593.22[医药卫生—内科学]