粉尘螨主要变应原基因Der f1和Der f3改组的研究

目的:探索粉尘螨变应原基因Der f1和Der f3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 f 1和Der f3基因,用DnaseⅠ分别酶解Der f 1和Der f3基因10、15、20、25、30 min;酶解相同时间的Der f1和Der f3基因片段两两混合,采用DNAshuffling技术对粉尘螨变应原Der f1和Der f3基因进行重组,琼脂糖凝胶电泳检测重组子。结果:以Der f 2 F和Der f2 R为引物在酶切10、15、20、25、30min均可扩增出清晰条带;以Der f1 F和Der f1 R、Der f1 F和Der f2 R、Der f2 F和Der f1 R为引物在酶切10、15、20、25、30min的模板中均无条带出现;而其他引物组合则在不同酶切时间的模板中均出现条带。结论:通过多种条件的组合,可获得多个粉尘螨变应原基因Der f1和Der f3间的融合基因,为大规模制备高效、低价的哮喘疫苗奠定了基础。

Recombining the major allergens of Der f 1 and Der f 3 from Dermatophagoides farinae by DNA shuffling

Objective： To explore the optimized recombinant conditions for major allergens of Derf 1 and Derf3 gene from Dermatophagoides far- inae using DNA shuffling technology to obtain the fused mutants. Methods ：Derf 1 and Derf3 gene were amplified by RT-PCR and diges- ted using Dnase I for 10,15,20,25,30 rain, respectively. The digested fragments of Derf 1 and Derf3 gene at the equivalent time point were mixed and amplified using non-primer and different primers by DNA shuf- fling technology followed by detecting the recombinant mutants with elec- trophoresis. Results ： The target fragments appeared （ Derf2 F and Derf2 R as primer pairs, respectively） in the templates digested at 10,15,20,25 and 30 min, respectively, but were absent as expected at the corresponding time point when using respective Der f 1F and Der f 1 R,Der f 1 F and Derf2 R,Derf2 F and DerfiR as primer pairs. Still,more or less frag- ments were shown in the previous templates through different primer com- bination. Conclusion：Multiple recombinant mutants of Derf 1 and Derf3 were obtained under various optimized conditions, and this work has laid the foundation for preparation of efficient and low-cost therapeutic vaccine against allergic asthma in large scale.