粉尘螨全长cDNA文库的构建及初步鉴定

目的构建粉尘螨全长cDNA文库,为粉尘螨相关基因研究奠定基础。方法用RNAiso Reagent试剂盒提取粉尘螨总RNA,然后以SMART IVTM Oligo-nucleotide和CDSIII/3’PCR Primer为引物,在反转录酶作用下合成cDNA第1链,利用LD-PCR方法合成cDNA第2链。合成的双链cDNA经蛋白酶K消化及SfiⅠ酶切,得到的cDNA克隆入λTripIEx2载体中,构建粉尘螨全长cDNA文库,测定分析文库滴度及插入片段大小。结果成功构建粉尘螨全长cDNA文库。检测cDNA文库未扩增时滴度为7.3×106pfu/ml,文库扩增后滴度为5.0×109pfu/ml,插入片段为0.5~2.5kb,平均1.2kb左右。结论成功构建了高质量的粉尘螨全长cDNA表达文库,所建文库容量及插入片段大小适用于对粉尘螨相关基因的进一步研究。

Construction and identification of a full-length cDNA library for Dermatophagoides farinae

Objective s To construct a full-length eDNA library of Dermatophagoides farinae and to provide a basis to study its related genes encoding allergens. Methods Total RNA was isolated from D. farinae using an RNA isolator （TaKaRa） in accordance with the manufacturer＇s instructions. A modified CDSIII/3＇ PCR Primer and a SMART IVTM Oligo-nucleotide primer were used to prime the first-strand synthesis reaction and second-strand eDNA was synthesized and amplified using LD-PCR. After proteinase K digestion and Sfi I digestion, the double-strand eDNA fragments were ligated to a XTripIEx2 vector to construct a full length eDNA library of Dermatophagoides farinae. The library titer and length of insert fragments were determined. Results A full-length eDNA library of D. farinae has been constructed. The unamplified library was estimated to have a titer of 7.3 ×10^6 pfu/ml while the amplified library was estimated to have a titer of 5×10^9 pfu/ml. PCR results indicated that the length of inserted fragments varied from 0.5--2.5 kb, with an average size larger than 1.2 kb. Conclusion A high-quality full-length eDNA library has been constructed for D. farinae. This eDNA library is appropriate for further study of related genes in D. farinae.