葎草花粉cDNA表达文库的构建和初步鉴定

Construction and primary characterization of cDNA expression library of Humulus pollen

目的构建葎草花粉cDNA表达文库,为进一步筛选葎草花粉的主要致敏蛋白组分、制备重组变应原奠定基础。方法采集新鲜葎草花粉,过筛后液氮保存,利用Trizol法抽提葎草花粉总RNA,纯化后PCR法反转录合成cDNA。经SfiⅠ酶切,过凝胶柱层析,去除小于400 bp的片断,收集大于400 bp的cDNA片段,加工后与λTripIEx2连接,文库体外包装,取出一小部分感染宿主菌XL1-Blue MRF,检测文库容量;PCR分析插入的cDNA片段的大小及多样性。结果成功构建一个含有5×105重组子的葎草花粉表达文库,重组率为90%,平均插入片段长度约为1.02 kb。结论所建文库容量及插入片段大小合适,适用于筛选克隆的目的cDNA。

Objective To construct a cDNA expression library of Humulus pollen and provide the basis for screening the major allergenic components and producing recombinant allergen of Humulus pollen. Methods Verdure Humulus pollen were collected and preserved in liquid nitrogen after being sift out. Total RNA was extracted from the Humulus pollen with trizol reagent, and cDNA was synthesized by RT-PCR with purified total RNA. Then the cDNA was digested by Sfi Ⅰ and the fragments smaller than 400 bp were removed by chroma spin400 column, and the fragments longer than 400 bp were ligated with λTripIEx2 Vector. The library was packaged in vitro and a small portion of packaged phage was used to infect E. coli XL1-Blue MRF＂ for titration. The diversity of the library and the length of the inserted fragments were analyzed by PCR. Results The cDNA expression library contained 5 × 10^5 recombinants and the percentage of recombination was 90%. The average length of inserted cDNA fragments was about 1.02 kb. Conclusion The constructed cDNA expression library contains appropriate contents and sizes of cDNA fragments and is qualified for screening target cDNA clone.