屋尘螨cDNA表达文库的构建及初步鉴定

Construction and identification of a cDNA library for Dermatophagoides pteronyssinus

目的:构建屋尘螨cDNA表达文库.方法:用RNAisoReagent试剂盒提取屋尘螨Total RNA,用Poly AT tract mRNA分离试剂盒提取mRNA,用Clontech公司SMARTTM PCR cD-NA library kit反转录合成第1链cDNA,用LD-PCR合成第2链cDNA并扩增,PCR产物与MaxPlaxTM试剂盒体外连接包装,建成未扩增的cDNA文库,计算其滴度和重组效率后进行文库扩增并测定扩增文库的滴度.结果:cDNA文库未扩增时滴度为9.148×106,重组效率达93.88%以上,文库扩增后滴度为7.628×109,插入片段平均1.63kb.结论:成功地构建了高质量的屋尘螨cDNA表达文库.

AIM： To construct a cDNA library for Dermatophagoides pteronyssinus. METHODS： Total RNA was isolated from the mites using RNA isolator （TaKaRa） according to the manufacturer＇s instructions, from which mRNA was separated with Poly（A） Tract mRNA Isolation System Ⅱ （Promega） and cDNA was synthesized by SMARTTM cDNA protocol. Products were ligated and packed with MaxPlaxTM reagent, and the h phage packaging reaction for the ligations was performed to produce an unamplified library. After the titer of unamplified library and the recombination efficiency were respectively worked out, the library amplification was executed and the titer of the amplified one was counted. RESULTS： The titers of the unamplified and the amplified libraries were 9. 148×10^6 and 7.628×10^9 pfu/mL, respectively. The recombination efficiency was above 93.88%. The average length of the inserted cDNA fragment was 1.63 kb. CONCLUSION： A qualified cDNA library has been constructed for Dermatophagoides pteronyssinus.