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目的:纯化CVN重组蛋白,检测其抗病毒活性,制备CVN多克隆抗体,并对其进行纯化和修饰。方法:利用两步Ni-NTA亲和层析和一步SUMO酶切制备CVN抗原,CPE法观察其抗病毒活性,活性抗原免疫新西兰白兔制备多克隆抗体,ELISA及Western blot鉴定其效价和特异性,抗血清经硫酸铵初步沉淀和DEAE离子交换层析获得纯化抗体,纯化抗体通过辣根过氧化物酶(HRP)标记获得修饰抗体。结果:纯化获得纯度达95%以上且具有明显抗流感病毒作用的非融合CVN,利用所得CVN抗原成功制备CVN多克隆抗体,酶联免疫分析显示其效价达到1∶16 000以上,Western blot分析表明其具明显特异性。抗血清经盐析和DEAE离子柱层析后获得效价为1∶6 400高纯度纯化抗体,经HRP修饰后获得具有较高生物活性的标记抗体。结论:获得高纯度,高效价兔抗CVN多克隆抗体,以及HRP标记的兔抗CVN多克隆抗体,为后续研究奠定了基础。
OBJECTIVE: Purification of CVN recombinant protein, detection of its antiviral activity, preparation of polyclonal antibody to CVN and its purification and modification. Methods: CVN antigen was prepared by two-step Ni-NTA affinity chromatography and one-step SUMO digestion. The anti-viral activity was observed by CPE method. New Zealand white rabbits were immunized with the active antigen to prepare polyclonal antibody. The titer and specificity The purified antibody was obtained by primary precipitation of ammonium sulfate and DEAE ion exchange chromatography. The purified antibody was modified by horseradish peroxidase (HRP). Results: The non-fusion CVN with the purity of more than 95% and significant anti-influenza virus activity was purified and the polyclonal antibody against CVN was successfully prepared using the obtained CVN antigen. The titer of the polyclonal antibody against CVN was over 1:16 000 by Western blot analysis Show that it has obvious specificity. Antiserum was purified by salting-out and DEAE ion-exchange column chromatography. The titer of purified antiserum was 1:6 400. After purification by HRP, a labeled antibody with high biological activity was obtained. CONCLUSION: The high purity and high titer rabbit anti-CVN polyclonal antibody and the HRP-labeled rabbit anti-CVN polyclonal antibody were obtained, which laid the foundation for further study.