Abstract:

Objective To explore optimized recombinant conditions of major allergens Der f2 and Derf3 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 lowcost asthma therapeutic vaccine in large scale.

Key words: DNA shuffling, Der f2, Der f3, Recombinant gene, Optimization