蠕形螨cDNA文库的构建及鉴定

Construction and identification of the cDNA library for Demodex

目的构建蠕形螨cDNA文库,以便进一步研究蠕形螨相关的基因。方法采用自然沉降法收集山羊蠕形螨,用TransZol Up试剂盒提取其总RNA,然后反转录合成第1链cDNA,通过LD-PCR获得第2链cDNA,将双链cDNA纯化去除小片段,收集大于500 bp的cDNA片段,与pGM-T载体连接,将重组质粒转入大肠杆菌感受态细胞TOP10建成蠕形螨cDNA文库,检验文库容量和重组效率,并采用PCR方法对插入cDNA片段大小进行分析。结果 cDNA文库的库容量为2.56×106cfu,重组效率达98%,平均插入片段长度大于1 000 bp。结论构建的蠕形螨cDNA文库质量良好,为进一步对蠕形螨基因探索奠定基础。

Objective To construct the cDNA library of Demodex for further studying its related genes.Methods Mites were collected by natural settlement in double distilled water and total RNA was extracted from the mites with the TransZol Up kit.The first-strand cDNA was synthesized by reverse transcription and the second-strand cDNA was synthesized and amplified by LD（long-distance） PCR.After size fractionation with a universal DNA purification kit,the cDNA fragments longer than 500 bp were ligated to the pGM-T vector.The ligation mixture was transformed into the competent cells of E.coli TOP10 to complete the construction of the cDNA library for the mites.The content and recombination rate of the cDNA library were tested and the length of inserted fragments was analyzed by PCR.Results The content of the cDNA library was estimated as 2.56×106 cfu with the recombination rate reaching 98%.The average length of the inserted cDNA fragment was longer than 1 000 bp.Conclusion The results showed that the quality of the cDNA library is good enough to set the basis for further exploring the genes of Demodex.