中国农业科学 ›› 2008, Vol. 41 ›› Issue (7): 2154-2161 .doi: 10.3864/j.issn.0578-1752.2008.07.038

• 兽医 • 上一篇    下一篇

肉鸡HSP70的基因克隆、原核表达及单克隆抗体制备

孙培明,吴晓东,赵永刚,刘雨田,王志亮,鲍恩东   

  1. 南京农业大学动物医学院
  • 收稿日期:2006-06-05 修回日期:2007-08-15 出版日期:2008-07-10 发布日期:2008-07-10
  • 通讯作者: 鲍恩东

Clone and expression of broiler HSP70 gene in E. coli and preparation of HSP70 monoclonal antibody

  

  1. 南京农业大学动物医学院
  • Received:2006-06-05 Revised:2007-08-15 Online:2008-07-10 Published:2008-07-10

摘要: 【目的】制备高特异性的肉鸡热休克蛋白70(heat shock protein 70,HSP70)单克隆抗体,研究肉鸡HSP70与热应激损伤的关系。 【方法】 利用RT-PCR方法扩增肉鸡HSP70 mRNA,将其扩增产物克隆到PGEM-T Easy 载体和原核表达载体pET-28a(+)上,进一步转化至大肠杆菌BL-21中,用IPTG诱导表达重组肉鸡HSP70。以纯化的重组肉鸡HSP70制备多克隆抗体,免疫印迹分析说明重组肉鸡HSP70具有很好的免疫原性。以纯化的重组肉鸡HSP70免疫Balb/c小鼠,通过淋巴细胞杂交瘤技术制备单克隆抗体。【结果】获得2株分泌肉鸡HSP70单克隆抗体的杂交瘤细胞株,进一步通过免疫印迹分析筛选出1株高特异性的肉鸡HSP70单克隆抗体,命名为BH70-1。 【结论】免疫组织化学结果显示,单克隆抗体BH70-1是一种理想的抗体。

关键词: 热休克蛋白70, 原核表达, 单克隆抗体制备, 肉鸡

Abstract: The mRNA of broiler HSP70 gene was amplified by RT-PCR and the production was cloned into the PGEM-T Easy vector and the expression vector pET-28a(+). The recombinant expression vector was transformed into E. coli BL-21 and induced by IPTG to express. The recombinant broiler HSP70 was purified to prepare the polyclonal antibody and the immunel activity of recombinant broiler HSP70 was proved by western-blot. The recombinant broiler HSP70 was used to immunize BALB/ c mice and two hyridoma cell lines secreting monoclonal antibody of broiler HSP70 were established by hybridoma technique. One of the high-specific monoclonal antibody for broiler HSP70 was selected by western-blot and named as BH70-1. The immunohistochemistry results showed that the monoclonal antibody BH70-1 has high affinity with broiler tissues. The results suggest that the prepared monoclonal antibody BH70-1 is effective.

Key words: Heat shock protein 70, Expression in E.coli, Monoclonal antibody, Western-blot, Immunohistochemistry, Broiler