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目的制备抗Ras相关C3肉毒毒素底物1(Rac1)兔多克隆抗体并进行初步应用。方法以人宫颈癌细胞系He La细胞c DNA为模板,构建重组表达质粒p ET-32a-Rac1。His-Rac1融合蛋白在大肠杆菌中成功表达,将纯化后的融合蛋白作为免疫原免疫家兔制备兔多克隆抗体。分别采用ELISA、Western blot法和免疫组织化学技术对抗Rac1抗血清的效价及特异性进行鉴定。结果 His-Rac1融合蛋白成功构建并高效表达。制备的抗Rac1兔多克隆抗体可以检测到He La细胞、MCF-7细胞的Rac1蛋白,并能识别人肾组织中的Rac1蛋白。结论成功制备了兔抗人Rac1多克隆抗体,该抗体可以识别多种天然样本中的Rac1蛋白。
Objective To prepare polyclonal antibody against ras-related C3 botulinum toxin substrate 1 (Rac1) and to conduct preliminary application. Methods The recombinant plasmid p ET-32a-Rac1 was constructed by using c DNA of He La cell as a template. His-Rac1 fusion protein was successfully expressed in E. coli, and the purified fusion protein was used as immunogen to immunize rabbit to prepare rabbit polyclonal antibody. The titer and specificity of anti-Rac1 antiserum were identified by ELISA, Western blot and immunohistochemistry respectively. Results His-Rac1 fusion protein was successfully constructed and highly expressed. The prepared anti-Rac1 rabbit polyclonal antibody can detect Hela cells, Rac1 protein of MCF-7 cells and recognize Rac1 protein in human renal tissues. Conclusion The rabbit anti-human Rac1 polyclonal antibody was successfully prepared, which can recognize Rac1 protein in many natural samples.