Construction and primary characterization of cDNA expression library of Humulus pollen

Abstract

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 spin-400 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 × 105 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. Show more