屋尘螨变应原Derp 5在大肠埃希菌中的表达及纯化  

Expression and purification of house hust mite allergen Derp 5 in E.coli

作　　者：王罗[1] 贺鹏飞[2] 窦侠[3] 张杰[3] 张伟[1] 魏准[1] 

机构地区：[1]深圳北京大学香港科技大学医学中心生物医学研究所,广东深圳518036 [2]中国食品药品检定研究院,北京100050 [3]北京大学深圳医院皮肤科,广东深圳518036

出　　处：《中国生物制品学杂志》2017年第12期1264-1268,1273,共6页Chinese Journal of Biologicals

摘　　要：目的克隆表达屋尘螨(Dermatophagoides pteronyssinus,Derp)第5组变应原Derp 5基因,在E.coli中表达、纯化重组Derp 5蛋白,并分析其血清Ig E反应原性。方法以合成的Derp 5核酸序列为模板进行PCR扩增,产物经双酶切后与载体pCold-TFM连接,构建重组表达质粒pCold-TFM-Derp 5,转化E.coli BL21(DE3),IPTG诱导表达。用镍离子亲和层析柱和凝胶过滤层析柱纯化目的蛋白,尘螨过敏患者血清鉴定其IgE反应原性。结果重组表达质粒pCold-TFM-Derp 5经双酶切、PCR及测序鉴定证明构建正确;在E.coli BL21(DE3)中实现了目的蛋白的可溶性高表达,毎升细菌表达产物经纯化可得到纯度95%以上的Derp 5蛋白约6 mg。纯化的重组Derp 5蛋白与尘螨过敏患者血清IgE有结合活性。结论成功构建了Derp 5原核表达载体,高效表达并纯化了目的蛋白,纯化的重组Derp 5蛋白具有良好的IgE反应原性,为屋尘螨变态反应性疾病的特异性诊断和治疗以及致敏蛋白的进一步研究奠定了基础。Objective To clone the gene encoding group 5 allergen of Dermatophagoides pteronyssinus （Der p 5）,express in E.coli,purify the expressed protein and determine its IgE reactogenicity.Methods The DNA sequence of Der p 5 was synthesized and used as a template for amplification by PCR.The PCR product was cloned into pCold-TFM vector,and the constructed recombinant plasmid pCold-TFM-Der p 5 was transformed to E.coli BL21 （DE3） for expression under induction of IPTG.The recombinant proteins were purified by nickel ion affinity column and size-exclusion column chromatography,and identified for IgE reactivity with serum of patients allergic to house dust mite （HDM） via indirect ELISA.Results Recombinant plasmid pCold-TFM-Der p 5 was constructed correctly as proved by restriction analysis,PCR and sequencing.The target protein was highly expressed in solube form in E.coli BL21 （DE3）.About 6 mg of expressed product with a purity of more than 95% was obtaiend from each liter of bacteria.The purified Der p 5 showed binding activity to serum IgE of patients allergic to HDM.Conclusion The prokaryotic expression vector for Der p 5 was constructed successfully,and target protein was highly expressed and purified.The purified Der p 5 showed good IgE reactogenicity,which laid a foundation of specific diagnosis and treatment of HDM associated allergic diseases and further study on allergic protein.

关 键 词：屋尘螨 第5组变应原 原核细胞 基因表达 特异性免疫治疗 反应原性 

分 类 号：R392.8[医药卫生—免疫学] Q786[医药卫生—基础医学]