斑马鱼原肌球蛋白基因克隆表达及免疫学鉴定  

Cloning and Expression of Tropomyosin Gene and Identification of Allergic Activity in Zebra Fish(Danio rerio)

作　　者：李荔[1] 陈樱麟[1] 闫浩[1] 刘志刚[1] 

机构地区：[1]深圳大学过敏反应与免疫学研究所,广东深圳518060

出　　处：《水产科学》2013年第1期26-30,共5页Fisheries Science

基　　金：国家"863"计划项目(No.2006AA100308);深圳市过敏反应与免疫学重点实验室组建项目(No.SW201110010);深圳大学校创新科研团队基金资助项目(No.200904)

摘　　要：克隆斑马鱼原肌球蛋白的基因并表达纯化出重组蛋白,对其免疫学特性进行鉴定。提取斑马鱼总RNA,采用RT-PCR克隆斑马鱼原肌球蛋白的全长基因,根据序列设计带有酶切位点的特异性引物,扩增斑马鱼原肌球蛋白基因的完整开放阅读框,连入载体pET-28a,在大肠杆菌BL21(DE3)菌株中通过IPTG诱导得到重组斑马鱼原肌球蛋白。重组蛋白经Ni 2+亲和层析柱纯化后进行Western-blot检测。获得斑马鱼原肌球蛋白基因,Western-blot结果显示重组蛋白具有良好的免疫活性。本研究成功克隆并表达了斑马鱼原肌球蛋白基因,经免疫学鉴定发现其具有过敏原性。The tropomyosin gene was cloned and expressed in zebra fish （Danio rerio） and the immunological activity of the tropomyosin was identified. The tropomyosin ORF sequence containing 855 bp was amplified through RT-PCR by specificity primer from total RNA. The recombinant tropomyosin was inserted in expression vector PET-28a and expressed in Escherchia coli BL21 （DE3）. Then the recombinant protein was purified by affinity chromatography with Ni2＋ coupled to sepharose. The cloning of tropomyosin gene and western-blot analysis revealed that the recombinant tropomyosin showed good IgE-binding capacity. The tropomyosin gene from zebra fish was successfully cloned and expressed, which was identified as an allergen of zebra fish.

关 键 词：斑马鱼 原肌球蛋白 过敏原 

分 类 号：Q786[生物学—分子生物学]