抗大鼠HSP70蛋白单克隆抗体的制备及特性研究

目的制备大鼠HSP70蛋白单克隆抗体并对其性质进行研究。方法利用PCR技术扩增大鼠全长hsp70,插入表达质粒pET-28a,pMAL-c2x,诱导表达,采用亲和层析纯化融合蛋白;用HIS-HSP70融合蛋白免疫BALB/c小鼠,杂交瘤技术制备大鼠HSP70单克隆抗体;采用Western印迹和免疫组化方法对抗体性质进行鉴定,ELISA方法确定抗体的表型。结果亲和层析纯化HIS-HSP70蛋白纯度在97.6%,获得6株可稳定分泌抗HSP70mAb的杂交瘤细胞系;抗体的亚类均是IgG1类κ链的。Western印迹结果显示6株单抗均能识别天然的大鼠HSP70蛋白;免疫组化结果显示3株抗体可以识别组织的HSP70蛋白。结论成功获得6株有活性HSP70单克隆抗体,可以识别HSP70蛋白不同表位,为进一步研究HSP70单克隆抗体在疾病中的作用奠定基础。

Preparation of monoclonal antibody against rat HSP70 protein and its characterization

Objective To prepare rat HSP70 protein monoclonal antibody（mAb）and explore its properties.Methods Full-length of hsp70 was amplified by PCR and inserted into the two expression plasmids pET-28a and pMAL-c2x.BALB/c mice were immunized with recombinant protein HIS-HSP70,and HSP70 mAb was prepared by hybridoma technique.The properties of mAb were identified by Western blot and immunohistochemical method while the phenotype of the antibody was analyzed by ELISA method.Results The purity of HIS-HSP70 recombinant protein was about 98%,and six hybridomas secreting specific mAb against HSP70 were established.The Ig subclass of these mAbs was IgG1 κ.The mAbs could be used in ELISA,Western blot,and immunohistochemistry.All the 6 mAb could recognize the natural HSP70 of rats,and 3 mAb could recognize tissue HSP70 protein.Conclusion Six hybridoma cell lines stably secreting specific mAb against HSP70 are established.The 6 active HSP70 mAb could recognize different epitopes of rat HSP70 protein.The specific mAb against HSP70 would be useful for the studies of its role in diseases.