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罗氏沼虾原肌球蛋白基因的克隆表达及变应原性鉴定
引用本文:罗伟芝,符春荣,邬玉兰,陈献雄,刘志刚,黄海珍*. 罗氏沼虾原肌球蛋白基因的克隆表达及变应原性鉴定[J]. 江西师范大学学报(自然科学版), 2012, 0(4): 425-430
作者姓名:罗伟芝  符春荣  邬玉兰  陈献雄  刘志刚  黄海珍*
作者单位:深圳大学过敏反应与免疫学研究所, 广东 深圳 518000
基金项目:国家自然科学基金(31101280,81271950);深圳市深港创新圈课题(CX Q2008026);深圳市重点实验室组建项目(SW201110010);深圳大学基础研究课题(201101)资助项目
摘    要:通过NCBI查找到罗氏沼虾原肌球蛋白的mRNA,根据其CDS区域设计特异引物,通过反转录聚合酶链反应(RT-PCR)克隆出目的基因片段,测序后将该片段克隆到原核表达载体 pET-28a 上,转化到 E. coli BL21(DE3)和E. coli Rosatta后,经异丙基-B-D-硫代乳糖苷(IPTG)诱导表达,用Ni2+亲和层析柱对重组变应原进行纯化.采用免疫印迹检测其与对虾过敏患者血清的 IgE 结合活性.对罗氏沼虾变应原进行了表达、鉴定及纯化,成功地获得了具有变应原活性的重组罗氏沼虾原肌球蛋白,为罗氏沼虾过敏性疾病的诊断、治疗奠定了基础.

关 键 词:罗氏沼虾  原肌球蛋白  变应原  克隆和表达

The Cloning, Expression and Purification of Tropomyosin from Haliotis Discus and Identification of Its Allergic Activity
LUO Wei-zhi,FU Chun-rong,WU Yu-lan,CHEN Xian-xiong,LIU Zhi-gang,HUANG Hai-zhen. The Cloning, Expression and Purification of Tropomyosin from Haliotis Discus and Identification of Its Allergic Activity[J]. Journal of Jiangxi Normal University (Natural Sciences Edition), 2012, 0(4): 425-430
Authors:LUO Wei-zhi  FU Chun-rong  WU Yu-lan  CHEN Xian-xiong  LIU Zhi-gang  HUANG Hai-zhen
Affiliation:(Allergy Reaction and Immunology Institute,Shenzhen University,Shenzhen Guangdong 518060,China)
Abstract:Bioinformatic method was used for the comparative analysis of numerous homologous animal food tropomyosin sequences.Conservative domains among the sequences were determined for degenerate primer de-signing.The RT-PCR was applied to clone the full length allergen genes from Macrobrachium rosenbergii and the sequences were analyzed.The specific primers were designed.The complete coding cDNA sequence including the start and the stop codons of tropomyosin of Macrobrachium rosenbergii was subcloned into the expression vector pET 28a.Expression of the recombinant Macrobrachium rosenbergii tropomyosin was carried out in Escherichia coli BL21(DE3) and the purification of the recombin ant protein was performed viaaffinity chromatography with Ni2 + coupled to sepharose.Protein from E.coli lysate and purified recombinant tropomyosin were analyzed by SDS-PAGE.IgE reactivity of recombinant Macrobrachium rosenbergii tropomyosin was investigated by Western-blotting.The recombinant Macrobrachium rosenbergii tropomyosin with good allergenicity in this study was obtained,which would be used as a base for further study on Macrobrachium rosenbergii related allergy.
Keywords:macrobrachium rosenbergii  allergen  tropomyosin  cloning and expression
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