粉尘螨Der f5克隆表达、纯化和免疫原性鉴定  

Cloning,Expression,Purification and Immunogenicity of House Dust Mite Allergen Der f5

作　　者：袁小惠[1] 高安健[1] 邬玉兰[1] 刘志刚[1] 

机构地区：[1]深圳大学医学院过敏反应与免疫学研究所,广东深圳518060

出　　处：《南昌大学学报（医学版）》2013年第2期1-5,共5页Journal of Nanchang University：Medical Sciences

基　　金：国家863计划(2006AA02A231);国家自然科学基金(81071388);广东省自然科学基金(04554);广东省高等学校国际暨港澳台科技合作创新平台项目(2012gjhz0009);深圳市重点实验室组建项目(SW201110010);深圳市科技计划基础研究重点项目(JCYJ20120613100657482);深圳市南山区一般研发资助项目(KC2012JSYB0003A)

摘　　要：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HindⅢ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。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 ac- cording to the known sequences of Der f5 gene. Total RNA 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 Topl0. 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 re- combinant 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 fS, were constructed successfully. SDS-PAGE showed a correct molecular weight of the recombinant Der f5 protein. After purification by affinity chromatography,the pro- tein showed only one strip on SDS-PAGE gel and appropriate ability to bind to IgE in sera of al- lergic patients. Conclusion The Der f5 gene has been cloned, expressed and purified and the re- combinant Der f5 protein shows appropriate IgE-combined ability. The study provides a basis for specific diagnosis and treatment of dust mite-induced allergic disease.

关 键 词：粉尘螨 Der F5 蛋白表达 纯化 免疫印迹 

分 类 号：R392.11[医药卫生—免疫学]