Cloning and screening of cDNA of Psilgramma menephorn allergen / 药物分析学报

Objective To construct a cDNA expression library of Psilgramma menephorn to screen its major allergen so as to provide the basis for producing recombinant allergen vaccine of Psilgramma menephorn. Methods Total RNA was extracted from the whole body of Psilgramma menephorn with Trizol and mRNA was purified with Oligo (dT) Spin-Column. And dscDNA was synthesized through reverse transcription. After blunting, the cDNA fragments were ligated with EcoRⅠ adapters. Then the cDNAs were digested by XhoⅠ, and the fragments less than 400 bp were removed by using GHROMA SPIN-400 column. The remaining fragments longer than 400 bp were ligated with Uni-ZAP XR vector. The recombinants were packaged in vitro and a small portion of the packaged phage was used to infect E.coli XL1-Blue MRF′ for titration. The recombinants were examined by color selection. The size of cDNA inserts and the diversity of library were analyzed by PCR. The library was screened using SPT positive sera from patients with Psilgramma menephorn allergy repeatedly. Results The cDNA expression library consisting of a 5×105 recombinant bacteriophages was constructed with the recombinant ratio of 67%. The average length of recombinant exogenous inserts was about 1.49 kb. Five positive cDNA clones were obtained. Conclusion The constructed cDNA expression library shows appropriate contents and size of cDNA fragments and the related genes of Psilgramma menephorn major allergens were harbored successfully, which lays the foundation for the positive clone identification and further analysis.

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

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.