肉鸡HSP70的基因克隆、原核表达及单克隆抗体制备

 【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。

Gene Cloning and Expression of Broiler Chicken HSP70 in E.coil and Preparation of HSP70 Monoclonal Antibody

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.