基础医学与临床 ›› 2012, Vol. 32 ›› Issue (6): 634-638.

• 研究论文 • 上一篇    下一篇

尘螨变应原Der f2和Der p2基因的DNA改组及生物信息学分析

马玉成1,李朝品2,姜玉新1,朱涛1   

  1. 1. 皖南医学院
    2. 安徽省芜湖市皖南医学院
  • 收稿日期:2011-10-19 修回日期:2012-03-01 出版日期:2012-06-05 发布日期:2012-05-25
  • 通讯作者: 李朝品 E-mail:cpli001@126.com
  • 基金资助:
    国家自然科学基金;安徽省自然科学基金

DNA shuffling and bioinformatics analysis of the dust mites group 2 allergen genes, Der f2 and Der p2

  • Received:2011-10-19 Revised:2012-03-01 Online:2012-06-05 Published:2012-05-25

摘要: 目的 构建尘螨变应原Der f2和Der p2嵌合基因文库,并进行生物信息学分析。方法 PCR分别扩增尘螨Ⅱ类变应原Der f2和Der p2基因,扩增产物等量混合后DNaseⅠ随机酶解;胶回收50bp-100bp之间的片段,连续进行3轮无引物PCR扩增,随后以Der f2基因的特异性引物进行有引物PCR扩增,扩增片段连接于pUCm-T载体,转化E. coli DH-5α后,随机挑取60个单菌落测序,应用DNAMAN软件进行序列比对分析和NetMHCII 2.2 Server软件进行辅助性T细胞(helper T cell)抗原表位的预测。结果 获得了10个基因片段发生明显交换的重组子,且生物信息学分析结果显示其编码蛋白不仅具有Der f2和Der p2所共有的Th细胞表位同时也出现了新的Th细胞表位。结论 成功应用DNA改组技术获得了Der f2和Der p2嵌合基因疫苗库,为后期筛选和制备高免疫原性和低变应原性的高效尘螨哮喘疫苗奠定了基础。

关键词: 变应原, 嵌合基因, DNA改组

Abstract: Objective A chimeric gene library of the dust mites group 2 allergen, Der f2 and Der p2, was constructed by using DNA shuffling method and analyzed by bioinformatics. Method Der f2 and Der p2 genes were amplified by polymerase chain reaction (PCR), respectively; the PCR products were mixed equally (v/v) and digested with DNase I. DNA fragments of 50bp-100bp were purified. DNA shuffling was performed through three rounds of primerless PCR and additional PCR with Der f2 specific primers. A single product of correct size was inserted into pUCm-T vector and transformed into E. coli DH-5α. Sixty clones were picked out randomly, sequenced and analyzed by multiple sequence alignment using DNAMAN software. T helper cell epitopes were predicted by NetMHCII 2.2 Server. Results Ten recombinants were obtained. The amino acid sequences deduced from these recombinants were predicted to not only share the common Th cell epitopes with Der f2 and Der p2 but also possess new epitopes. Conclusion A chimeric gene library of Der f2 and Der p2 was successfully established, which could provide the basis for selection of vaccine with high immunogenicity and low allergenicity for asthma treatment in future.

Key words: allergens, chimeric gene, DNA shuffling