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目的获得粉尘螨(Dermatophagoides farinae)变应原Der f 8全长基因并构建其原核表达质粒。方法参考Gen Bank登录号为AY283295的Der f 8部分序列设计并合成引物。以粉尘螨总RNA为模板,RT-PCR扩增获得Der f 8部分片段。采用5′cDNA末端快速扩增技术(rapid amplification of cDNA ends,RACE)获得Der f 8全长序列,连接至pMD19-T载体,热转化至大肠埃希菌(Escherichia coli),挑取阳性菌落,抽提质粒后测序。根据Der f 8全长序列设计并合成引物,以粉尘螨总RNA为模板进行RT-PCR扩增,产物回收后连接pCold TF载体,热转化至E.coli,涂板过夜培养后,挑取阳性菌落,抽提质粒后测序。将pCold TF-Der f 8质粒转化至E.coli BL21,异丙基-β-D-硫代半乳糖苷诱导表达,采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析表达产物。采用生物信息学软件预测Der f 8的理化特性、结构和功能,并构建系统进化树。结果以Der f 8部分序列设计的引物行RT-PCR扩增获得Der f 8部分片段,长度为600 bp;5′RACE获得Der f 8剩余序列,长度为300 bp;以全长基因序列设计的引物进行RT-PCR扩增获得了Der f 8基因CDS区,长度为696 bp,测序均正确。SDSPAGE结果显示,目的蛋白为可溶性表达,相对分子质量(M_r)为81 000,与预期大小一致。序列经生物信息学分析结果显示,Der f 8全长序列与参考序列(Gen Bank登录号为AY283295)同源性为98.49%,预测其编码的疏水性蛋白具有谷胱甘肽S转移酶活性,二级结构包括α-螺旋(45.45%)、延伸主链(11.26%)和无规卷曲(43.29%)。系统进化树结果显示,粉尘螨与户尘螨(Dermatophagoides pteronyssinus)聚成一簇。结论获得了Der f 8全长基因及其原核表达质粒。
Objective To obtain the full length Der f Derm of Dermatophagoides farinae and to construct its prokaryotic expression plasmid. Methods The primers of Genbank Accession No. AY283295 Der f 8 partial sequence were designed and synthesized. The Der f 8 fragment was amplified by RT-PCR using the total RNA of dust mite as a template. The full-length sequence of Der f 8 was obtained by rapid amplification of cDNA ends (RACE), ligated into pMD19-T vector, and transformed into Escherichia coli by heat. Positive colonies were picked out, After plasmid extraction sequencing. Based on the full-length sequence of Der f 8, primers were designed and synthesized. The total RNA was used as a template for RT-PCR amplification. The product was recovered and ligated into pCold TF vector. The vector was transformed into E.coli after thermal incubation. After overnight incubation, Colonies, plasmid extraction after sequencing. The pCold TF-Der f 8 plasmid was transformed into E.coli BL21 and induced by isopropyl-β-D-thiogalactopyranoside. SDS-PAGE and SDS- The expression product was analyzed. Bioinformatics software was used to predict the physicochemical properties, structure and function of Der f 8, and phylogenetic tree was constructed. Results The fragment of Der f 8 was amplified by RT-PCR with primers designed by Der f 8, and the length was 600 bp. The remaining sequence of Der f 8 was 5’RACE and the length was 300 bp. The full-length gene sequence was designed Primer RT-PCR amplification Der f 8 gene CDS region, a length of 696 bp, sequencing correct. SDSPAGE results showed that the target protein was soluble, the relative molecular mass (M_r) was 81 000, consistent with the expected size. The results of bioinformatics analysis showed that the full-length sequence of Der f 8 was 98.49% homologous to the reference sequence (GenBank accession number AY283295), and the predicted hydrophobic protein encoded glutathione S-transferase activity. Secondary structures include a-helix (45.45%), extended backbone (11.26%) and random coil (43.29%). Phylogenetic tree analysis showed that dust mites clustered with Dermatophagoides pteronyssinus. Conclusion The full-length Der f 8 gene and its prokaryotic expression plasmid were obtained.