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

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.