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目的:制备针对人Fkbp19的多克隆抗体,为进一步研究Fkbp19基因的功能奠定基础。方法:利用鉴定正确的重组原核表达载体pET21a-Fkbp19,在大肠杆菌BL21-DE3中诱导表达,应用Ni-NTA亲和层析法获得纯度较高的原核表达蛋白,并免疫家兔制备多克隆抗体,Western blot及免疫荧光的方法检测抗体对乳腺癌细胞中内源性Fkbp19蛋白的识别能力。结果:成功地在大肠杆菌中实现了His-Fkbp19融合蛋白的表达,经纯化后免疫家兔得到了高滴度的多克隆抗体,该抗体可以用Western blot的方法检测内源性Fkbp19蛋白。结论:所制备的多克隆抗体可以用于Fkbp19的Western blot检测。
OBJECTIVE: To prepare polyclonal antibody against human Fkbp19 and lay the foundation for further study on the function of Fkbp19 gene. Methods: The recombinant prokaryotic expression vector pET21a-Fkbp19 was used to induce the expression in E. coli BL21-DE3. The purified prokaryotic expression protein was obtained by Ni-NTA affinity chromatography and the rabbit was immunized with polyclonal antibody Western blot and immunofluorescence were used to detect the ability of the antibody to recognize the endogenous Fkbp19 protein in breast cancer cells. Results: His-Fkbp19 fusion protein was successfully expressed in E. coli. After being purified, the rabbit was immunized with a high titer polyclonal antibody. The antibody can be used to detect the endogenous Fkbp19 protein by Western blot. Conclusion: The prepared polyclonal antibody can be used for Western blot detection of Fkbp19.