Objective To clone and express the antigen Der f5 from the dust mite dermatopha-goides farinae ,and to identify its immunogenicity. Methods A pair of primers were designed according to the known sequences of Der f 5 gene .Total RN A was extracted from dust mites and Der f5 gene fragments were amplified by RT-PCR .The PCR product was cloned into pMD18-T vector and transferred into E .coli Top10 .The clones were screened and identified by PCR and restriction enzyme digestion .The target gene obtained from the recombinant plasmid by digestion with Bam H Ⅰ and Hind Ⅲ was connected to the prokaryotic expression vector pET-32a.The expressed recombinant plasmid containing Der f5 gene was constructed by cloning target gene into pET-32a and transferred into E .coli Top10 .The recombinant plasmid was transfected into E .coli strain BL21 (DE3 ) and the transfected cells were grown in the presence of inducer IPTG .The expressed recombinant protein was analyzed by SDS-PAGE and Western blotting and was purified by immo-bilized metal ion affinity chromatography. Results The two recombinant plasmids ,pMD18-T-Der f5 and pET32a-Der f5,were constructed successfully .SDS-PAGE showed a correct molecular weight of the recombinant Der f 5 protein .After purification by affinity chromatography ,the protein showed only one strip on SDS-PAGE gel and appropriate ability to bind to IgE in sera of allergic patients .Conclusion The Der f5 gene has been cloned,expressed and purified and the recombinant Der f5 protein shows appropriate IgE-combined ability . The study provides a basis for specific diagnosis and treatment of dust mite-induced allergic disease.%目的 克隆表达粉尘螨第5组变应原(dermatophagoides farinae,Der f5)基因,并鉴定纯化蛋白免疫原性.方法 根据Der f5 基因已知序列,设计出相应的引物,提取粉尘螨总RNA,采用RT-PCR 方法扩增出Der f5 基因片段,PCR 产物克隆入pMD18-T 载体,转化大肠埃希菌 Top10,经PCR和酶切鉴定并测序.将上述所得阳性克隆菌株扩大培养,碱裂解法提取质粒,所得重组质粒pMD18-T-Der f5 和空质粒pET-32a 同时用限制性内切酶Bam HⅠ与Hind Ⅲ双酶切,经纯化后连接并转化至大肠埃希菌 Top10.构建的重组质粒pET32a-Der f5,经PCR、酶切和测序鉴定后,再转化至大肠埃希菌 BL21(DE3),异丙基-β-D-硫代半乳糖苷(IPTG) 诱导表达.用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE) 和蛋白质印迹法(Western blotting) 鉴定其表达效果,用Ni+离子亲和层析柱纯化重组质粒pET32a-Der f5 表达产生的组氨酸重组蛋白.结果 构建了重组质粒pMD18-T-Der f5 和pET32a-Der f5.SDS-PAGE 结果表明Der f5 基因在大肠埃希菌 BL21(DE3)中获得良好的表达,经亲和层析纯化后,SDS-PAGE 结果显示单一条带.该蛋白以尘螨过敏患者血清进行Western blotting,结果表明具有良好的IgE 结合活性.结论 克隆、表达并纯化了具有良好尘螨致敏患者IgE 结合活性的Der f5,为粉尘螨变态反应性疾病的特异性诊断和治疗以及进一步的实验研究奠定了基础.
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