摘要
目的制备和鉴定抗Hsp83蛋白的多克隆抗体。方法利用PCR技术从果蝇cDNA中获得hsp83基因片段,构建重组质粒;将其转化到BL21(DE3)菌株中诱导蛋白表达,利用Ni-NTA亲和法纯化重组蛋白;再将纯化的蛋白免疫BALB/C小鼠制备多克隆抗体;利用免疫印迹法(Western blot)和免疫荧光染色法检测多克隆抗体的特异性。结果构建的pET28ahsp83质粒在大肠杆菌中成功表达了Hsp83融合蛋白,蛋白纯化后作为抗原免疫小鼠,获得了抗Hsp83的多克隆抗体。免疫印迹法和免疫荧光染色法检测显示,抗果蝇Hsp83多克隆抗体具有较高的特异性,并能检测出内源性Hsp83蛋白。果蝇卵巢免疫荧光染色显示,Hsp83蛋白定位在卵巢细胞的细胞质中。结论成功制备了小鼠抗Hsp83蛋白的特异性抗体,此工作为深入研究Hsp83蛋白的功能奠定了基础。
Objective To prepare and identify the polyclonal antibody against Drosophila Hsp83 protein.Methods The fragment of gene ksp83 was cloned from Drosophila cDNA library by PCR and then inserted into the expression vector pET28 a to construct recombinant plasmids.Hsp83 fusion protein was expressed in BL21(DE3),purified by Ni-NTA affinity chromatography and then used to immunize BALB/C mice.The polyclonal antibody against Drosophila Hsp83 was gained from the mice serum.The specificity of the antibody was analyzed by Western blotting and immunofluorescent staining.Results The Hsp83 fusion protein was successfully expressed in BL21(DE3) from the pET28a-hsp83 plasmids constructed.The purified fusion protein as antigen immunized BALB/C mice and stimulated the generation of polyclonal antibody against Hsp83.The anti-Hsp83 antibody showed relatively high specificity in the western blot and immunofluorescent staining,and was able to detect endogenous Drosophila Hsp83 protein.In addition,Hsp83 was localized in the cytoplasm of ovarian cells by immunofluorescent staining.Conclusion The polyclonal antibody against Hsp83 with high specificity was successfully prepared,and this lays the foundation for further study of the functions of Hsp83 protein.
出处
《中国组织化学与细胞化学杂志》
CAS
CSCD
2016年第5期454-459,共6页
Chinese Journal of Histochemistry and Cytochemistry
基金
国家自然科学基金面上项目(31071266)
安徽省高校自然科学基金重点项目(KJ2015A082)
安师大研究生科研创新与实践项目(2015cxsj172)
安徽高校科研平台创新团队项目
