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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6.89×106 pfu/mL；扩增后文库滴度为2.63×109 pfu/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。

英文摘要:

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 cDNA 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 toλ TriplEx2 vector. The λ 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 order 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 cDNA 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.

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