粉尘螨主要变应原基因Der f2和Der f3基因改组的优化

摘要

Objective To explore optimized recombinant conditions of major allergens Der f2 and Der f3 genes from Dermatophagoides farinae under DNA shuffling technology, and obtain fused mutation genes. Methods Der f2 and Der f3 genes were amplified by RT- PCR and digested using Dnase I for 10, 15, 20, 25, 30 min, respectively. The digested fragments of Der f2 and Der f3 genes at the same time point were mixed and amplified using non- primer and different primers by DNA shuffling technology, and then recombinant mutants were detected using electrophoresis. Results The target fragments were appeared using primer pairs of Der f1 F, Der f1 R and Der f2 F, Der f2 R, respectively in the templates digested 10min, 15min, 20min, 25min; and the same results were also shown using primer pairs of Der f2 F and Der f3 R in the templates digested 10min, 15min and 30min. Conclusion Multiple recombinant mutants of Der f2 and Der f3 were obtained using various optimized conditions, it was laid the foundation for preparation of efficient and low-cost asthma therapeutic vaccine in large scale.%目的：优化粉尘螨变应原Der f2和Der f3基因改组条件，以期获得粉尘螨变应原融合突变基因。方法 RT- PCR方法扩增粉尘螨变应原Der f2和Der f3基因，用DnaseⅠ分别酶解Der f2和Der f3基因10、15、20、25、30min；酶解相同时间的Der f2和Der f3基因两两混合，采用DNA shuffling技术对粉尘螨变应原Der f2和Der f3基因进行重组，电泳检测重组子。结果以Der f1 F和Der f1 R、Der f2 F和Der f2 R为引物分别在酶切10min、15min、20min、25min均可扩增出清晰条带；以Der f2 F和Der f3 R为引物在酶切10min、15min和30min的模板中亦出现PCR片段。结论通过多种条件的组合，可获得多个粉尘螨变应原Der f2和Der f3基因间的融合基因，为大规模制备高效，低价的哮喘疫苗奠定基础。