摘要
目的构建粉尘螨6类变应原(Dermatophagoides farinae,Derf6)基因的高效原核表达载体,并进行表达、纯化及生物学功能分析。方法根据Derf6基因已知序列,设计1对引物,通过对纯培养的粉尘螨提取总RNA,采用RT-PCR方法扩增出Derf6基因片段,PCR产物克隆入pMD18-T载体,转化大肠埃希菌(E.coliTop10),经PCR和酶切鉴定并测序。将上述所得阳性克隆菌株扩大培养,碱裂解法提取质粒,所得重组质粒pMD18-T-Derf6和空质粒pET-24a同时用限制性内切酶EcoRⅠ和XhoⅠ双酶切,经纯化后连接并转化至E.coliTop10。构建的重组质粒pET24a-Derf6经PCR、酶切和测序鉴定后,再转化至E.coliBL21(DE3),异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达。用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和蛋白质印迹法(Westernblotting)鉴定其表达效果,用Ni+离子亲和层析柱纯化重组质粒pET24a-Derf6表达产生的组氨酸重组蛋白。结果构建了重组质粒pMD18-T-Derf6和pET24a-Derf6。SDS-PAGE结果表明Derf6基因在E.coliBl21(DE3)中获得良好的表达,所得重组蛋白相对分子质量(Mr)为31000,与理论值一致,经亲和层析纯化后,SDS-PAGE结果显示单一条带。该蛋白以尘螨过敏患者血清进行Westernblotting,结果表明具有良好的IgE结合活性。结论克隆、表达并纯化了具有良好尘螨致敏患者IgE结合活性的Derf6。
Objective To construct, purify and characterize a recombinant expression plasmid containing Derf6 gene of Dermatophagoides farinae. Methods A pair of primers was designed according to the known sequence of Der f6 gene. The live mites identified and cultured locally were picked and the total RNA was extracted. The Der f6 gene fragment was amplified by RT-PCR, and cloned into pMD18-T vector, and then transferred into E.coli Top10. The target gene obtained from the recombinant plasmid by digestion with EcoR Ⅰ and Xho Ⅰ was connected to the prokaryotic expression vector pET-24a. The expressed recombinant plasmid containing Der f6 gene was constructed by cloning target gene into pET-24a and first transferred into E.coli Top10, then into E.coli B121 （DE3）. The expressed recombinant protein was analyzed by SDS-PAGE and Western blotting, and was purified by immobilized metal ion affinity chromatography （IMAC）. Results The two recombinant plasmids, pMD18-T-Derf6 and pET24a-Der f6, were constructed. SDS-PAGE showed a correct molecular weight of the recombinant Der f6 protein. After purification by affinity chromatography, the protein showed only one strip on SDS-PAGE gel and appropriate combination ability with IgE in sera of allergic patients. Conclusion The Der f6 gene has been cloned into plasmid pMD18-T vector and sub-cloned into the expression vector pET-24a, the recombinant plasmid pET24a-Der f6 has been expressed in E.coli BL21 （DE3）, purified by IMAC, and showed approprriate IgE-combined ability.
出处
《中国寄生虫学与寄生虫病杂志》
CAS
CSCD
北大核心
2006年第4期241-246,共6页
Chinese Journal of Parasitology and Parasitic Diseases
基金
国家863高技术研究发展计划(No.2002AA214011)
国家自然科学基金(No.30271226
30471505)~~
关键词
粉尘螨
Der
f6
基因表达
纯化
蛋白质印迹
Dermatophagoides farinae
Derf6
Expression
Purification
Western blotting
