粉尘螨第十六类变应原的克隆表达、纯化及免疫原性鉴定  

Cloning,Expression,Purification and Immunogenic Identification of Dust Mite Allergen Der f16

作　　者：李维中[1,2] 王月明[3] 吴莹莹[1] 邬玉兰[1] 刘志刚[1,2] 

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 [2]南昌大学医学院免疫教研室,南昌330006 [3]蚌埠医学院研究生院,2011级安徽蚌埠233000

出　　处：《南昌大学学报（医学版）》2013年第12期7-10,15,共5页Journal of Nanchang University：Medical Sciences

基　　金：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)

摘　　要：目的获得大量具有良好IgE结合活性的粉尘螨第十六类变应原(Der f16)的重组变应原,以促进粉尘螨变态反应性疾病的特异性诊断及治疗的研究。方法挑取经纯培养的粉尘螨,提取总RNA,根据已知Der f16基因序列设计引物,经RT-PCR扩增Der f16基因片段,产物连入pMD32-T载体中。扩增后,利用限制性内切酶EcoRⅠ和XhoⅠ双酶切将目的基因片段连接到pET32a表达载体上,转化到大肠杆菌(E.coli BL21)中经IPTG诱导表达。表达载体经亲和层析纯化,SDS-PAGE检测蛋白纯度,Western bolt检测变应原免疫学活性。结果以粉尘螨总RNA为模板成功克隆出Der f16基因,与数据库中Der f16基因同源性为100%;经IPTG诱导后,大肠杆菌大量表达Der f16蛋白,所获得的重组蛋白分子质量为73ku,上清及沉淀物均有蛋白表达,且上清表达量高于沉淀物。重组Der f16能够与螨过敏患者血清中的IgE反应,而不与健康者血清中的IgE反应。结论成功构建了Der f16的原核表达载体,并高效表达和纯化出具有免疫原性的Der f16重组蛋白。Objective To obtain the recombinant dust mite allergen Der f16 with high IgE binding activity, and to promote the specific diagnosis and treatment of dust mite induced allergic disease. Methods Total RNA was extracted from cultured dust mites. The primers were designed according to Der f16 sequences and Der f16 gene fragment was amplified by RT-PCR. The PCR product was cloned into pMD32-T vector, and then subcloned into pET32a vector by restriction endonuclease EcoR I and Xho E. coli BL21 and was induced wi I. The recombinant plasmid pET-Der f16 was transformed into th IPTG. The expressed Der f16 protein was purified by immobi lized metal ion affinity chromatography and detected by SDS-PAGE. The allergenic activity of Der f16 protein was detected by Western blot. Results Der f16 gene was successfully cloned using to-tal RNA for template and the cloned gene has a homology of 100 %to the published sequence. Af- ter induction with IPTG, Der f16 protein was largely expressed in E. coli BL21. The molecular weight of the recombinant Der f16 protein was 73 ku. In addition, Der f16 protein was also ex- pressed in the supernatant and sediment, and the Der f16 expression in supernatant was higher than that in sediment. The fusion protein showed high IgE binding activity with serum from dust mite-allergic patients,but did not bind to serum IgE in healthy subjects. Conclusion Der f16 ex- pression vector was successfully constructed and the recombinant Der f16 protein with immunoge- nicity was largely expressed and purified.

关 键 词：粉尘螨 Derf16 克隆 表达 纯化 

分 类 号：R392.8[医药卫生—免疫学]