【摘要】 目的克隆和表达粉尘螨第7类变应原基因,并鉴定重组蛋白的免疫原性。方法提取粉尘螨总RNA,根据GenBank提供的Der f 7编码区(CDS)(登录号为AY 283292)序列设计特异性引物,逆转录PCR(RTPCR)克隆Der f 7基因。将测序正确的目的片段克隆至pET-32a表达载体,得到的重组质粒pET-32a-Der f 7在大肠埃希菌(E.coli)BL21(DE3)中用异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,通过镍离子亲和层析纯化目的蛋白。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)检测目的蛋白表达和纯化结果,蛋白质印迹(Western Blotting)分析重组蛋白的免疫原性。结果RT-PCR结果显示,Der f 7基因片段大小约为650 bp。测序结果表明,Der f 7基因片段与已发表的粉尘螨Der f 7基因(登录号为FJ436108)同源性为99%。SDS-PAGE结果显示,重组质粒pET32a-Der f 7在BL21(DE3)中高效表达,重组蛋白相对分子质量(M r)约为23 000。Western Blotting分析结果表明,Der f 7重组蛋白可被尘螨过敏患者血清识别。结论成功构建了粉尘螨第7类变应原的原核表达载体,并获得具有免疫原性的Der f 7重组蛋白。更多还原

【Abstract】 Objective To clone and express Der f 7 gene of Dermatophagoides farinae, and identify its immunogenicity. Methods Total RNA was extracted from D. farinae mites. A reference sequence(Accession No. AY283292) was used to design specific primers. The Der f 7 gene fragment was amplified by RT-PCR, and cloned into pET-32a vector. The recombinant plasmid was transformed into E. coli BL21(DE3) and induced with IPTG for protein expression. The recombinant protein was purified by Ni2 +chelating affinity chromatography and analyzed by SDS-PAGE and Western blotting.Results The Der f 7 gene fragment was about 650 bp, and shared 99% homology with the published one(Accession No.FJ436108). SDS-PAGE result showed its relative molecular weight(Mr) of 23 000. The recombinant protein showed appropriate combination ability with IgE in sera of mite allergic patients. Conclusion Der f 7 gene has been expressed in prokaryotic expression system and shows allergenicity.更多还原