蓝藻抗病毒蛋白-N的制备、生物活性鉴定及其多克隆抗体的制备和修饰

Purification of Cyanovirin-N with antiviral activity and preparation as well as modification of its polyclonal antibody

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。

AIM： To purify the recombinant Cyanovirin-N（CVN） and determine its anti-influenza virus A（H1N1） activity,and to prepare the polyclonal antibody of CVN,purify it and label it with an enzyme for future application.METHODS： The recombinant CVN were rapidly purified by two rounds of Ni-NTA chelating chromatography intervened with a SUMO protease cleavage step.Anti-H1N1 activity of CVN was determined using cytopathogenic effect assay（CPE）.The rabbits were immunized with the purified CVN and the antibody was identified by ELISA and Western blot.IgG was purified by ammonium sulfate precipitation followed by DEAE chromatography and the purified IgG was labeled by HRP.RESULTS： The purity of the obtained CVN protein which showed obvious Anti-H1N1 activity in vitro was higher than 95%.The polyclonal antibody of CVN was successfully produced in the rabbits and the results of ELISA and Western blot showed that the antiserum had high titer and high specificity.The purified antibody with a titer up to 1∶6 400 was successfully obtained and the anti-CVN antibody-HRP conjugate was achieved after labeling the purified antibody with HRP.CONCLUSION： The purified antibody against CVN has been purified and further coniugated with HRP,with can be used for future research.