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粉尘螨α-烯醇化酶基因的克隆表达、纯化及其N端B细胞表位的初步鉴定 被引量:1

Cloning and expression of alpha-enolase gene from Dermatophagoides farinae and identification of its N-terminal B-cell epitope
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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
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  • 1Subramanian A,Miller D M.Structural analysis of alpha-enolase.Mapping the functional domains involved in down-regulation of the c-myc protooncogene[J].J Biol Chem,2000,275 (8):5958-5965. 被引量:1
  • 2Piast M,Kustrzeba-Wojcicka I,Matusiewicz M.Molecular evolution of enolase[J].Acta Biochim Pol,2005,52(2):507-513. 被引量:1
  • 3Fougerousse F,Edom-Vovard F,Merkulova T,et al.The musclespecific enolase is an early marker of human myogenesis[J].J Muscle Res Cell Motil,2001,22 (6):534-544. 被引量:1
  • 4Keller A,Rouzeau J D,Farhadian F,et al.Differential expression of α-and β-enolase genes during rat heart development and hypertrophy[J].Am J Physiol,1995,269 (6Pt2):H1843-H1851. 被引量:1
  • 5Pancholi V.Multifunctional α-enolase:its role in disease[J].Cell Mol Life Sci,2001,58(7):902-920. 被引量:1
  • 6Reed G H,Poyner R R,Larsen T M,et al.Structure and mechanistic studies of enolase[J].Curr Opin Struct Biol,1996,6 (6):736-743. 被引量:1
  • 7Brewer J M.Specificity and mechanism of action of metal ions in yeast enolase[J].FEBS Lett,1985,182 (1):8-14. 被引量:1
  • 8Mcalister L,Holland M J.Targeted deletion of a yeast enolase structural gene.Identification and isolation of yeast enolase isozymes[J].J Biol Chem,1982,257(12):7181-7188. 被引量:1
  • 9Sousa L P,Brasil B S,Silva Bde M,et al.Characterization of alpha-enolase as an interferon-alpha 2 alpha 1 regulated gene[J].Front Biosci,2005,10(5):2534-2547. 被引量:1
  • 10Ghosh A K,Steele R,Ray R B.C-myc promoter-binding protein 1 (MBP-1) regulates prostate cancer cell growth by inhibiting MAPK pathway[J].J Biol Chem,2005,280 (14):14325-14330. 被引量:1

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