【摘要】 为构建大片吸虫成虫cDNA表达文库,用Trizol试剂提取大片吸虫成虫总RNA,经反转录合成cDNA第一链,应用LD-PCR扩增方法,合成双链cDNA。用SfiⅠ内切酶修饰此双链cDNA,使形成两端分别带有SfiⅠA和SfiⅠB的黏性末端。经CHROMA SPIN-400柱纯化,收集400 bp以上的双链cDNA片段,将其连接于带有SfiⅠA和SfiⅠB末端的λTriplEx2噬菌体载体,经体外包装后,以XL1-Blue为受体菌构建cDNA表达文库。经测定,库容量为1.08×106PFU/mL,重组率为96.6%。扩增后的文库滴度为2.41×109PFU/mL,插入片段平均大小约为1 000 bp。这些结果表明已成功构建大片吸虫成虫cDNA表达文库,适合进一步筛选大片吸虫新基因。更多还原

【Abstract】 To construct a cDNA library from adult Fasciola gigantica.Total RNA was extracted from Fasciola gigantica by Trizol regent.The first strand cDNA was synthesized and the second strand was amplified by Long-Distance PCR.The Chroma Spin-400 was applied to purify cDNA fragments longer than 400bp.The purified fragments were ligated with λTriplEx2,then were packaged in vitro,at last the cDNA phage expression library was constructed by infecting XL₁-Blue.The unamplified library capacity was 1.08×10⁶ PFU/mL,the library recombination rate was 96.6%.The titer of the amplied library was 2.41×10⁹PFU/mL.The cDNA library of adult Fasciola gigantica has been constructed using SMART LD-PCR technology for the first time.更多还原