摘要
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。
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.
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
2007年第2期156-160,共5页
Chinese Journal of Zoonoses
基金
国家863计划(No.2002AA214011)
国家自然科学基金(No.30471505
30271226)
广东省科技重大专项(2003A3080502)
粤港关键领域重点突破项目(No.20054982207)
深圳市科技计划资助项目
关键词
粉尘螨
Derf1
基因多态性
原核表达
Dermatophagoides farinae
Der f 1
gene polymorphism
prokaryotic expression
