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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 Demodex mite cDNA library for further study on Demodex related genes. METHODS: Demodex mites were collected by natural sedimentation method. The total RNA was extracted by TransZol Up kit. The first strand cDNA was reverse transcribed. The second strand cDNA was obtained by LD-PCR. The double-stranded cDNA was purified to remove small fragments. The cDNA fragments larger than 500 bp were collected and ligated with pGM-T vector. The recombinant plasmids were transformed into E.coli competent cells TOP10 to construct Demodex mite cDNA library. The library capacity and recombination efficiency were tested. The size of inserted cDNA fragments analysis. Results The cDNA library had a capacity of 2.56 × 106 cfu and the efficiency of recombination was 98%. The average insert length was more than 1 000 bp. Conclusion The constructed Demodex mite cDNA library is of good quality, which lays the foundation for the further exploration of the gene of Demodex mite.