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粉尘螨主要变应原基因Derf1和Derf3改组的研究 预览

Recombining the major allergens of Der f 1 and Der f 3 from Dermatophagoides farinae by DNA shuffling
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摘要 目的:探索粉尘螨变应原基因Der f 1和Der f 3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 f 3基因,用DnaseⅠ分别酶解Der f 1和Der f 3基因10、15、20、25、30 min;酶解相同时间的Der f 1和Der f 3基因片段两两混合,采用DNA shuffling技术对粉尘螨变应原Der f 1和Der f 3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 f 2 R为引物在酶切10、15、20、25、30 min均可扩增出清晰条带;以Der f 1 F和Der f 1 R、Der f 1 F和Der f 2 R、Der f 2 F和Der f 1 R为引物在酶切10、15、20、25、30 min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 f 1和Der f 3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. 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.
作者 姜玉新 郭伟 马玉成 刘志明 陈琪 李朝品 JIANG Yu-xin, GUO Wei, MA Yu-cheng, LIU Zhi-ming, CHEN Qi ,L1 Chao-pin Department of Medical Parasitology, Wannan Medical College, Wuhu 241002, China
出处 《皖南医学院学报》 CAS 2013年第2期87-91,共5页 Acta Academiae Medicinae Wannan
基金 基金项目:国家自然科学基金项目(30872367 81172790 81270091) 安徽省自然科学基金项目(070413088)
关键词 DNA改组 Derf1 Derf3 融合基因 DNA shuffling Der f1 Der f 3 chimeric gene
作者简介 作者简介:姜玉新(1971-),男,讲师,博士,(电话)13615535709,(电子信箱)jiangyuxin2000@hotmail.com; 李朝品,男,教授,博士生导师,(电子信箱)cpli001@126.com,通讯作者.
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  • 1石连,姜玉新,王海宁,李朝品.粉尘螨变应原Derf1基因的原核表达及多克隆抗体的制备[J].皖南医学院学报,2012,31(1):3-6. 被引量:11
  • 2SCHOU C,LIND P. The antigenicity of house dust mites [ J]. Al- lergy,1991,46(Suppl. 11 ) :10 - 13. 被引量:1
  • 3MUELLER GA, SMITH AM, CHAPMAN MD, et al. Hydrogen ex- change nuclear magnetic resonance spectroscopy mapping of anti- body epitopes on the house dust mite allergen Der p2 [ J ]. J Biol Chem,2001,276(12) :9359 -9365. 被引量:1
  • 4CRAMERI A, RAILLARD SA, BERMUDEZ E, et al. DNA shuff- ling of a family of genes from diverse species accelerates directed e- volution [ J ]. Nature, 1998,391 (6664) :288 - 291. 被引量:1
  • 5CRAMERI A, WHITEHOM EA, TATE E, et al. Improved green fluorescent protein by molecular evolution using DNA shuffling [ J ]. Nat Biotechnol, 1996,14 ( 3 ) :315 - 319. 被引量:1
  • 6CRAMERI A, CWIRLA S, STEMMER WPC. Construction and evo- lution of antibody - phage libraries by DNA shuffling [ J ]. Nat Med, 1996,2 ( 1 ) :100 - 102. 被引量:1
  • 7ZHAO H, GIVER L, SHAD Z, et al. Molecular evolution by stag- gered extension process ( StEP ) in vitrorecombination [ J ]. Nat Biotechnol, 1998,16 ( 3 ) :258 - 261. 被引量:1
  • 8CHANG CC, CHEN TT, COX BW, et al. Evolution of a cytokine u- sing DNA family shuffling [ J ]. Nat Biotechnol, 1999,17 ( 8 ) :793 - 797. 被引量:1
  • 9MILI:N E, D:AZ AM. Allergy to house dust mites and asthma [ J]. P R Health Sei J,2004,23 (1) :47 -57. 被引量:1
  • 10BOUSQUET J, LOCKEY R, MALLING HJ. Allergen immunothera- py:therapeutic vaccines for allergic diseases. A WHO position pa- per :J]. J Allergy Clin Immunnl,1998,102(4 Pt 1 ) :558 -562. 被引量:1

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