吸烟者肺癌组织T7噬菌体cDNA文库构建

Construction of a T7 Phage Display cDNA Library from Lung Cancer of Smokers

摘要: 目的：为筛选吸烟人群中早期肺癌肿瘤标志物，构建吸烟者肺癌组织T7噬菌体展示cDNA文库。方法：用RNeasy Mini Kit提取吸烟者肺癌组织总RNA，分离纯化mRNA，逆转录合成双链cDNA，经末端修饰、定向EcoRI/HindIII接头连接、接头消化，使其两端分别带有EcoRI和HindIII粘性末端。用Mini Column分离cDNA片段，收集300 bp以上的双链cDNA，与T7Select 10-3b载体连接，体外包装后，以BLT5615为受体菌得到吸烟者肺癌组织T7噬菌体展示cDNA文库。结果：经测定，库容为1.35 × 106 pfu，扩增后文库滴度为4.4 × 1010 pfu/ml。PCR鉴定从原始文库中随机挑取的100个噬菌斑，重组率为97%，重组体中91%的插入片段长度>300 bp。结论：成功构建了吸烟者肺癌组织T7噬菌体cDNA文库，为下一步筛选吸烟人群中早期肺癌肿瘤标志物奠定基础。

Abstract: Objective: To screen tumor markers of smokers, the T7 phage display cDNA library from the lung cancer of smokers was constructed. Methods: The mRNA was isolated from total RNA extracted from the lung cancer of smokers by RNeasy Mini Kit, and was used to synthesize double strain (ds) cDNA by the reverse transcription. Then the directional EcoRI/HindIII linkers were liquated into the ends of ds cDNA and the ds cDNA was further digested with EcoRI and HindIII, which resulted in ds cDNA with EcoRI and HindIII ends. The digested ds cDNA fragments longer than 300 bp in length were fractionated by Mini Column, and then liquated into the T7 Select 10-3b vertor with EcoRI and HindIII ends. After packaging in vitro, the T7 Select 10-3b vertor was tansformed into BLT5615 to construct the T7 phage display cDNA library. Results: Analysis showed that the library contained 1.35 × 106 clones and the titer of the applied library was 4.4 × 1010 pfu/ml. The PCR identification results of 100 clones picked at random showed that 97% clones were recombinant and 91% of recombinant clones contained cDNA fragments longer than 300 bp in length. Conclusion: A T7 phage display cDNA library from the lung cancer of smokers is successfully constructed.