粉尘螨变应原DerfMag29的克隆、表达及生物信息学分析  

Cloning,Expression and Analysis of Der f Mag 29 Allergen of Dermatophagoides farinae

作　　者：孙金霞[1] 俞黎黎[1] 周鹰[1] 卞勇华[1] 杨李[1] 王楠[1] 崔玉宝[1] 

机构地区：[1]盐城卫生职业技术学院医学检验学院,盐城224006

出　　处：《中国寄生虫学与寄生虫病杂志》2013年第6期480-482,共3页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：国家自然科学基金(No.31272369;81001330;30660166)~~

摘　　要：提取粉尘螨总RNA,根据GenBank中的序列(登录号为AY283281.1)设计并合成引物,RT-PCR扩增Mag 29全长基因,克隆至pCold TF DNA载体,转化至大肠埃希菌(E.coli)JM109,取阳性克隆测序鉴定。将pCold TF-Mag 29质粒转化至BL21,异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)验证表达产物,采用生物信息学软件预测Mag 29的结构、理化特性和生物进化关系。RT-PCR产物电泳结果显示,在429 bp处有一条带,与Mag 29基因大小一致,测序鉴定表明质粒构建成功。SDS-PAGE结果显示,表达产物的全细胞、上清和沉淀物中均有相对分子质量(M r)为63 000的蛋白表达,与预测值一致,其中上清中蛋白表达量高于沉淀。生物信息学分析显示,该蛋白是由142个氨基酸组成的M r15 100的分子,二级结构由α螺旋(55.63%)、延伸主链(3.52%)和无规则卷曲(40.85%)组成,是亲水性细胞质蛋白,与热休克蛋白70家族具有较高同源性。The full-length Mag 29 gene of Dermatophagoidesfarinae was amplified by RT-PCR with a pair of specific primers. The PCR product was cloned into pCold TF DNA vector. The constructed plasmid pCold TF-Mag 29 was transformed into E. coli BL21 and followed by expression of the protein induced by IPTG. The recombinant protein was analyzed by SDS-PAGE. The full-length Mag 29 gene was 429 bp. A specific band （Mr 63 000） were detected in the whole cells, the supernatant, and the precipitate. Bioinformatics analysis revealed that Mag 29 protein was composed with 142 amino acid residues with a calculated molecular weight of Mr 15 100, and its secondary structure was composed of alpha helix （55.63%）, extended strand （3.52%）, and random coil （40.85%）. The Mag 29 allergen was a hydrophilic and cytoplasmic protein, and shared a high degree homology with the heat shock protein 70 family.

关 键 词：粉尘螨 变应原 生物信息学 

分 类 号：R384.4[医药卫生—医学寄生虫学]