中国免疫学杂志2011，Vol.27Issue(7)：642-647,6.DOI:10.3969/j.issn.1000-484X.2011.07.015
淡水小龙虾主要过敏原原肌球蛋白基因的克隆、表达及其免疫活性鉴定
Cloning and expression of Red swamp crayfish major allergen tropomyosin gene and identification of its immunoglogic activity
摘要
Abstract
Objective:To clone the major allergen tropom yosin gene from Red swamp crayfish and produce its recombinant protein and investigate its immunologic activity.Methods:Degenerate primers were designed according to the conserved sequence of trpomyosin.The RT-PCR was applied to clone the full-length tropom yosin allergen genes from Red swamp crayfish.The cDNA was sequenced and then subcloned into pFT28a expression vetor.The cloned tropom yosin cDNA gene was expressed in Escherichia coliRosetta(DE3)by IPTG induction.The re-combinant tropom yosin was purified by metal(N12+)chelating affinity chromatography.The immunologic activity of the recombinant tropomyosin was examined by Western blot mehtod.Results:Sequence analysis showed that this gene included an open reading frame coding for 284 am inoacids and shared high identities(＞80％) with tropomyosin allergens from shrimp,crab and lobster.The deduced protein was three-fore regarded as allergen and named as Proc1(EM BL/GenBank database entry No.FJ769183).The recombinant allergen tropomyosin was highly expressed in Escherichia coliRosetta(DE3).Western blot analysis showed that the recombinant allergen has IgE-reactivity.Conclusion:The tropomyosin of Red swamp crayfish was successfully expressed and showed good immunologic activity,which laid a foundation of the specific diagnosis and immunotherapy in shrimphypersensitivity disease.关键词
淡水小龙虾/过敏原/原肌球蛋白/免疫活性Key words
Red swamp crayfish(Procam barus clarkia)/Allergen/Tropom yosin/Immunologic activity分类
医药卫生引用本文复制引用
黄素文,杨文潮,朱海,王建峰,倪健波,吕志平,冼静雯,刘志刚..淡水小龙虾主要过敏原原肌球蛋白基因的克隆、表达及其免疫活性鉴定[J].中国免疫学杂志,2011,27(7):642-647,6.基金项目
本文为国家质检总局资助项目(2009IK173) （2009IK173）
