深圳地区粉尘螨Ⅱ类变应原基因的多态性分析及其表达蛋白的变应原性鉴定  

Polymorphism of Der fⅡ gene in Shenzhen City and the allergic activity of its prokaryotic expression products

作　　者：白羽[1] 吉坤美[1] 刘志刚[1] 蔡成郁[1] 

机构地区：[1]深圳大学生命科学学院,广东深圳518060

出　　处：《昆虫学报》2007年第6期560-566,共7页Acta Entomologica Sinica

基　　金：国家"863"计划(2002AA214011);国家自然科学基金项目(30471505;30271226);广东省科技重大专项(2003A3080502);粤港关键领域重点突破项目(20054982207);深圳市科技计划资助项目

摘　　要：粉尘螨Ⅱ类变应原（DerfⅡ）是粉尘螨Dermatophagoides farinae主要变应原之一,可引发Ⅰ型变态反应。从深圳地区挑取活粉尘螨,经形态鉴定后纯培养,提取其总RNA,RT-PCR扩增出DerfⅡ基因,克隆到pMD18-T载体后测序。通过计算机软件分析该基因的多态性。将该目的基因克隆到pET-His表达载体上得到重组质粒pET-DerfⅡ。工程菌经IPTG诱导培养,表达DerfⅡ目的蛋白,该蛋白主要以包涵体形式存在。重组DerfⅡ蛋白通过6His-tag蛋白纯化系统进行分离、纯化,并进行Western blot检测该重组蛋白与对粉尘螨过敏患者血清中IgE的反应性。结果表明,克隆的5株深圳地区的DerfⅡ基因与GenBank公布的DerfⅡ（AB195580.1）核苷酸序列同源性为96.8%～99.3%,推导的氨基酸序列同源性为93.8%～98.6%。表达纯化的5种重组DerfⅡ蛋白经Western blot检测,表明都具有良好的变应原性。Abstract： Der fⅡ is a major allergen of Dermatophagoides farinae, which can induce the type Ⅰ allergy. In this study, live mites were collected from Shenzhen City, and these identified as D. farinae were picked out and bred in the laboratory. The total RNA was extracted from the bred mites. The Der fⅡ gene fragment was amplified by RT-PCR and sequenced. The Der fⅡ gene was sub-cloned into the expression vector pET-His. The recombinant pET-Der fⅡ plasmid was induced to express Der fⅡ coding protein by IPTG. The recombinant Der fⅡ with 6His-tag was then purified by chelating resin, and its allergic activity was identified by Western blotting. As results, five strains of Der fⅡ gene fragment with 411 bases were determined. Comparison showed that the Der fⅡ gene sequences of the five strains from Shenzhen had an identity from 96.8 % to 99.3 % at the nucleotide level and 93.8 % to 98.6 % at the amino acid level with the homologous gene sequence deposited in GenBank （GenBank No. AB195580.1）. Then they were sub-cloned into expressing vector pET-His, and five kinds of recombinant allergen Der fⅡ were highly expressed as inclusion bodies, which were then purified by 6-His-tag purification system. Using Western-blotting method, the allergic activities of purified recombinant allergens were positively identified as their affinity to IgE antibodies from the mite-allergic sera of patients.

关 键 词：粉尘螨 DerfⅡ 变应原 基因多态性 原核表达 

分 类 号：Q966[生物学—昆虫学]