粉尘螨3类变应原基因的克隆、表达、纯化与变应原性鉴定

Cloning,Expression and Purification of Dust Mite Allergen Der f 3 and Identification of its Allergic Activity

目的克隆表达粉尘螨3类变应原(Der f 3)基因,并鉴定纯化蛋白的变应原性。方法取华南地区采集经实验室培养的粉尘螨(约500只)提取总RNA,经RT-PCR扩增Der f 3基因。将目的基因克隆到pET-His表达载体上得到重组质粒pET-Der f 3。大肠埃希菌(E.coli)BL21(DE3)经异丙基-β-D-硫代半乳糖苷(IPTG)诱导培养,表达Der f 3目的蛋白。重组rDer f 3蛋白经6His-tag蛋白纯化系统进行分离、纯化,蛋白质印迹法(Western blotting)分析该纯化Der f 3蛋白与粉尘螨过敏患者血清IgE反应性,以鉴定重组Der f 3的变应原性。结果以粉尘螨总RNA为模板克隆出Der f3基因,该基因与GenBank (D63858)公布的Der f3比较有4个核苷酸差异,但同源性为99.5%。E.coliBL21(DE3)经IPTG诱导后高效表达Der f 3重组蛋白,主要以包涵体形式存在。经Western blotting分析该重组蛋白Der f 3能与粉尘螨过敏患者血清的IgE反应,而不与健康者血清IgE反应。结论构建了华南地区粉尘螨3类变应原的原核表达载体,并高效表达和纯化出具变应原性的Der f3重组蛋白。

Objective To clone, express and identify Der f3 gene. Methods Live mites were collected from southern China region, identified as Dermatophagoides farinae, and cultured. The total RNA was extracted. The Der f 3 gene fragment was amplified by RT-PCR and sequenced. The Der f 3 gene fragment encoding a serine protease mature peptide was sub-cloned into the expression vector pET-His. The recombinant pET-Der f 3 plasmid was inserted into E.coli BL21 and induced to express Der f 3 coding protein by IPTG. The recombinant Der f 3 with 6 his-tag was then purified by chelating resin and its allergic activity was identified by Western blotting. Results The Der f3 gene fragment with 840 bases was determined. Its sequence homology with the published one （GenBank No.D63858） was 99.5% at nucleotide level. It was sub-cloned into expressing vector pET-His and the recombinant allergen rDer f 3 was highly expressed in E.coli BL21 （DE3） under induction of IPTG, and purified by 6-His-tag purification system. Using Western blotting method, the allergic activity of the purified recombinant allergen was identified as its affinity to IgE antibodies from the mite-allergic patient sera. Conclusion Der f 3 gene has been successfully cloned and its prokaryotic expression vector is constructed.