摘要
目的克隆表达粉尘螨(Dermatophagoides farinae)α-烯醇化酶(alpha-enolase,ENOA)基因,初步研究其N端B细胞表位与自身免疫性疾病的关系。方法 1设计特异性引物,从文库中克隆ENOA cDNA;2通过BamH I/EcoR I双酶切位点,将ENOA基因克隆至pET-His原核表达载体,表达纯化,获得了重组ENOA蛋白;3测定重组ENOA酶活性;4通过Swiss-Model软件模拟ENOA蛋白3D结构,并分析其潜在的N端B细胞表位;合成ENOA的N端表位肽,测定其与类风湿性关节炎患者血清IgG的反应性。结果 1克隆了ENOA全长cDNA(GenBank登录号KJ421213.1);2成功构建了pETHis-ENOA表达载体,经纯化获得了重组ENOA蛋白。3重组蛋白具有烯醇化酶活性;4瓜氨酸修饰的合成表位肽与类风湿性关节患者血清IgG-ELISA呈阳性反应,与健康对照组血清均不呈反应(P<0.05)。结论本课题首次克隆表达的重组ENOA蛋白具有烯醇化酶活性;其N端B细胞表位与类风湿性关节炎相关。本研究为尘螨与自身免疫性疾病的相关性研究提供了新的思路。
We aimed to clone and express cDNA of alpha-enolase from the dust mite Derrnatophagoides farina (Der f), and study the relationship between its N-terminal B-cell epitope and autoimmune disease. The cDNA of α-enolase was amplified from Der f cDNA library and sub-cloned into a pET-His vector to express in E. coli BL21(DE3)plysS. Recombinant α-enolase was purified and its enzyme activity was determined. The 3D structure was constructed by Swiss-ModeL The N-terminal epitope peptide was synthesized and subjected to IgG-ELISA with the patients' serum of rheumatoid arthritis vs healthy con- trois. Results showed that the gene encoding α-enolase was cloned from Der f (GenBank accession No. KJ421213.1). The pET-ENOA expression vector was constructed. High pure recombinant a-enolase was obtained with enzyme activity. ELISA demonstrated IgG binding to the citrullinated peptide from 11 of 64 rheumatoid arthritis (RA) patients' serum samples, but was not bound by IgG antibodies from 26 healthy controls (P〈0.05). The present study cloned and expressed α-enolase cDNA from the dust mite. The findings have revealed the dust mite α-enolase might be associated with human rheumatoid arthritis.
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
2015年第7期649-654,共6页
Chinese Journal of Zoonoses
基金
国家自然科学基金(No.31328014
No.31400786)
广东省自然科学基金(No.2014A030313563)
深圳市科技计划(No.JCYJ20120613113021045
JCYJ20130326112225593)~~
关键词
粉尘螨
α-烯醇化酶
类风湿性关节
表位
Dermatophagoides farina
alpha-enolase
rheumatoid arthritis
epitope