粉尘螨变应原第2组分基因的原核表达及生物信息学分析

Construction of prokaryotic expression system for the dust mite allergen Der f 2 in full-length and analysis of its structure and function

目的:克隆并原核表达粉尘螨变应原第2组分重组蛋白,鉴定其变应原性,分析其分子特征,为制备用于临床诊治的变应原奠定基础。方法:提取粉尘螨Total RNA,根据国际基因库(AB195580)设计并合成引物,经RT-PCR合成Derf 2全长基因,与pMD19-T载体连接后,热转化至E.coliJM109;将测序正确的Der f 2基因与pET28a(+)Vector连接,转化BL21(DE3),IPTG诱导表达;经镍琼脂糖凝胶FF亲和柱层析,收集各阶段洗脱峰用梯度透析法进行复性,Western blot鉴定重组蛋白的变应原性。用生物信息学软件预测其理化性质、空间结构,并构建分子进化树。结果:RT-PCR成功扩增获得目的基因Der f2,全长441 bp,与参考序列同源性99.3%。将构建的pET28a(+)-Der f 2质粒转化宿主菌E.coliBL21诱导表达,SDS-PAGE电泳显示全细胞和沉淀物中均有蛋白表达。亲和柱层析后获得约3.86 mg重组蛋白纯品,Western blot分析表明其可与哮喘患儿血清IgE发生特异性结合。去除信号肽序列(1～17氨基酸)后,重组蛋白由129个氨基酸组成,相对分子质量为14.076,二级结构由延伸主链(30.82%),无规则卷曲(49.32%),α螺旋(19.86%)组成。该变应原可能位于线粒体,属ML域家族。序列比对显示,粉尘螨和屋尘螨、微角尘螨、梅氏嗜霉螨的相似度依次为88%、96%、83%,此四种螨在上述蜱螨变应原第2组分构建的分子进化树中聚成一簇,支持率达100%。结论:粉尘螨变应原第2组分原核表达获得成功,纯化后获得具有变应原性的重组蛋白,为进一步规模生产该变应原并用于临床诊治奠定基础;生物信息学分析初步获得了该变应原的分子特征,为进一步实验室验证指明方向。

Objective：To obtain the recombinant product of the group 2 allergen of Dermatophagoidesfarinae （Der f 2）, and its molecular characteristics. Methods：The total RNA of Dermatophagoides farinae was isolated for amplification of Der f 2 in full-length by RT-PCT with nucleic acid primers designed according to GenBank AB195580, whcih was then linked into pMD19-T simple vector and transformed into E. coli competent cells JM109. The fragment was then sequenced, sub-cloned into the plasmid pET28a （ ＋ ）, expressed in E. coli BL21 at the aid of the inducer IPI＇G. The protein was purified by nickel-affinity chromatography, and the elution peak at each stage was collected and re-natured with gradient dialysis. In the end, the allergenicity of the recombinant product was identified by Western blot. The pbysicecbemical properties, spatial structure of the allergen were analyzed with bioinformatics software, as well as molecular phylogenetic tree. Results： The target gene of Der f 2 was synthesized by RT-PCR successfully, which was 441 in full-length and 99.3 % identical with the reference sequence. The constructed plasmid pET28a（ ＋ ）-Der f 2 was trmrsformed into E. coli BL21 for gene expression, and SDS-PAGE showed a specific brand about 14 kD in the whole cell lysate and the pellet E. coli BI21 cells containing pET28a（ ＋ ）-Der f 2. After chromatography, the recombinant product about 3.86 mg were obtained, which could band with the serum IgE from asthma children. After elimination of the sequence for signal peptide （ from 1 to 17 amino acid）, the recombinant protein consisted with 129 amino acid with molecular weight 14. 076 kD, and its secondary structure contained an alpha helix （ 19.86 % ）, extended strand （ 30.82 % ）, and random coil （49.32 % ）. The subcellular localization for this allergen was predicted to be mitochondrial, further, its function was demonstrated to be associated with MD-2-related lipid-recognition （ML） domain. The similarity of the group 2 allergen between Dermatophacoide~ farinae and D . pteronyssinus , D . sibonev , Euroglyphus maynei were 88% ,96%,83 %, respectively, which was clustered with bootstrap 100 % in the molecular phylogenetic tree constructed with the group 2 allergens for different mite-species. Conclusion： Der f 2 has been expressed in E. coli successfully, the purified recombinant protein has good allergenicity, which provides a basis for production of the allergen in large scale for clinical diagnosis and treatment of allergic disorders. Through bioinformatics software, its molecular characteristics are obtained which point out the direction for laboratory study.