【摘要】 为克隆原核表达并纯化出粉尘螨过敏原脂肪酶蛋白,鉴定其免疫学活性,并分析其分子特征.通过重组合成粉尘螨新过敏原脂肪酶基因,与p ET-28a载体连接后转化大肠埃希菌E.coli Top10,用异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达重组新过敏原脂肪酶蛋白;经镍柱亲和层析纯化出粉尘螨重组脂肪酶蛋白,用Western Blot、ELISA方法检测其免疫原性.用生物信息学软件预测其理化性质、二级结构,并构建分子进化树.成功表达纯化出高纯度的粉尘螨重组脂肪酶蛋白,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)结果显示表达重组产物分子质量约为40 k Da,与理论值一致,纯化后的表达产物经Western blot印迹检测有明显条带显示.信息学分析显示蛋白二级结构由α螺旋(16.38%)、延伸主链(18.93%)、无规则卷曲(64.69%)组成.成功原核表达出粉尘螨过敏原脂肪酶蛋白,并纯化获得较高纯度及较强免疫学活性的重组脂肪酶蛋白,为尘螨过敏性疾病的特异性诊断和免疫治疗奠定理论基础.更多还原

【Abstract】 To clone,express and purify of dust mite( lipase),test its immunogenicity,and analysis of its structure and function. Synthesize the lipase gene of dust mite,link it to the p ET-28 a vector,and the recombinant plasmid was transfected into E. coli Top10 and IPTG induces the expression of lipase protein. The protein was purified by nickelaffinity chromatography and its immunological identification was analyzed by Western blot and ELISA. Bioinformatics software were used to predict physicochemical properties,secondary structure and to construct its molecular phylogenetic tree. High purify recombinant lipase protein of dust mite was successful expressed purified. SDS-PAGE results showed that the expression of recombinant allergen was about 40 k Da. After chromatography,the recombinant allergen could band with the serum Ig E from patients with asthma in Western Blot. Its secondary structure contained an alpha helix( 16. 38%),extended strand( 18. 93%) and random coil( 64. 69%). The success of prokaryotic expression and of dust mite allergen( lipase),and purified with high purity and strong immunological activity of the recombinant lipase protein. It can lay the foundation for the special diagnosis of allergic diseases.更多还原