粉尘螨Ⅰ类变应原基因的多态性分析及表达蛋白的特性鉴定

目的克隆表达我国深圳地区粉尘螨I类变应原Der f 1基因,并进行该基因的多态性分析和鉴定该纯化蛋白的变应原性,为研究具有我国区域特色的粉尘螨变应原奠定基础。方法从深圳地区挑取经形态鉴定的活粉尘螨,提取总RNA,RT-PCR扩增Der f 1基因,克隆到T载体测序确认。通过计算机软件进行该基因的多态性分析。将该目的基因克隆到pET-His表达载体上得到重组质粒pET-Der f 1。工程菌经IPTG诱导培养,表达Der f 1目的蛋白。重组Der f 1蛋白通过6 His-tag蛋白纯化系统进行分离、纯化,并进行Western-Blot检测纯化的Der f 1蛋白与粉尘螨过敏病人血清中IgE的反应性,以鉴定重组Der f 1的变应原性。结果以粉尘螨总RNA为模板,经RT-PCR从深圳地区克隆了4株Der f 1基因。该基因与GenBank公布的Der f 1(No.AB034946.1)比较发现核苷酸的同源性在99.48%-100%之间,理论推导的氨基酸序列同源性在99.69%-100%之间。工程菌经IPTG诱导后高效表达的Der f 1重组蛋白,并主要以包涵体形式存在。Western-Blot试验证实该4株Der f 1基因表达的重组蛋白都具有变应原性。结论本研究克隆了4株深圳地区粉尘螨I类过敏原基因并实现了原核表达,为进一步开展Der f 1蛋白研究和重组变应原免疫治疗疫苗研究奠定基础。

Polymorphic analysis of gene encoding Der f 1 allergen and identification on the bioactivity of its prokaryotic expression products

The live mites were collected from Shenzhen City,which was identified as Dermatophagoides farinae,cultured and picked out to extract the total RNA.Then the Der f 1 gene fragment was amplified by RT-PCR and sequenced.The Der f 1 gene was sub-cloned into the expression vector-pET-His and the recombinant plasmid pET-Der f 1.was induced to express Der f 1 coding protein by IPTG.The recombinant Der f 1 with 6 his-tag was purified by chelating resin and its allergic activity was identified by Western-blotting.In this way,4 strains of Der f 1 gene fragment with 969 bases was determined.Its sequence homology with the published one(GenBank No.AB034946.1) was from 99.48% to 100% at nucleotide level and from 99.69% to 100% at amino acid level respectively.They were sub-cloned into expressing vector pET-His and the four kinds of recombinant allergen Der f 1 were highly expressed as inclusion bodies under induction with IPTG,then purified by 6-His-tag purification system.Using Western-blotting method,the allergic activity of the purified recombinant allergens could be demonstrated.It is concluded that the results obtained in the present study may provide a basis for further study of Der f 1 protein and development of recombinant allergen vaccine for immunotherapy.