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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6.89×106pfu/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 synovial tissue of human rheumatoid arthritis (RA). Screening for specific genes associated with RA, to lay the foundation for the pathogenesis of RA and gene therapy. Methods: RNA was extracted from human rheumatoid arthritis synovial tissue and mRNA was purified by Trizol method. The cDNA was transcribed by powerscript TM reverse transcriptase using the template transformation method of mRNA at the 5 ’end. The first strand cDNAs were synthesized using COS III / 3’ Double-stranded cDNA was synthesized by polymerase chain reaction (LD-PCR). After proteolytic cleavage and purification, the PCR product was digested by Sfi I and eluted by column chromatography. After the recombinant was placed on TripIEx2 vector and packaged, the titer and recombination rate , Amplifying the library and randomly selecting 40 plaques. PCR amplification was performed by using universal primers at both ends of the vector cloning site to detect the quality of the constructed cDNA library. Results: The titer of the untreated library was 6.89 × 106pfu / mL. The amplified library titer was 2.63 × 109pfu / mL, and the recombination efficiency was 93%. The inserted fragments mainly concentrated in the range of 300 ~ 1800bp. CONCLUSION: A cDNA library of synovial tissue of human rheumatoid arthritis (RA) has been successfully constructed and can be immunologically screened with probe antibodies to lay a solid foundation for further exploration of RA-related genes.