Construction and primary characterization of cDNA expression library of psilgramma menephorn / 西安交通大学学报(医学版)

ABSTRACT

Objective To construct a cDNA expression library of psilgramma menephorn and provide the basis for screening the major allergen and producing recombination allergen of psilgramma menephorn. Methods Total RNA was extracted from the whole body of psilgramma menephorn with TRIZOL mRNA purified with Oligo (dT) Spin-Column. ds cDNA was synthesized through reverse transcription. EcoRI adapters were ligated to the blunt ends and the ends were phosphorylated. The XhoI digestion released EcoRI adapter. The fragments smaller than 400 bp were removed by GHROMA SPIN-400 column and the fragments longer than 400 bp were ligated to the Uni-ZAP XR vector. The lambda library was packaged in vitro and is plated on the E.coli cell line XL1-Blue MRF. The content and recombination rate of cDNA library were tested. The length of the inserted fragments was analyzed by PCR. Results The cDNA expression library contained 5?10 5 recombinants and the recombination rate was 67%. The average length of inserted cDNA fragments was about 1.49 kb. Conclusion The constructed cDNA expression library contains appropriate contents and size of cDNA fragments for screening target cDNA to clone.