重组人细小病毒B19 VP2病毒样颗粒的制备及免疫原性初步研究

Preparation and immunological characterization of recombinant human parvovirus B19 virus-like particle assembled from VP2 protein

目的利用汉逊酵母系统重组表达人细小病毒B19(human parvovirus B19,B19V)主要衣壳蛋白VP2,并对VP2病毒样颗粒(virus-like particles,VLP)进行理化性质研究及免疫效果评价。方法重组表达B19V VP2蛋白,并使其自组装成VLP;通过高密度发酵和系列层析纯化步骤获得VP2-VLP;SDS-PAGE和凝胶过滤高效液相色谱检测蛋白质纯度,动态光散射和透射电镜观察颗粒形态,并检测VP2-VLP磷脂酶活性。采用ELISA和红细胞凝集抑制试验检测小鼠体内VP2-VLP特异性IgG抗体和中和抗体水平。结果本研究制备的重组VP2-VLP纯度大于95%,颗粒均一且形态良好,大小近似天然病毒颗粒;VLP可与特异性单克隆抗体结合,磷脂酶A2活性为阴性;吸附佐剂后的VP2-VLP能够诱导小鼠产生高滴度的特异性IgG抗体和中和抗体。结论汉逊酵母表达系统制备的B19V VP2-VLP可作为候选靶抗原用于B19V预防性疫苗的开发。

Objective To express the recombinant major capsid protein VP2 of human parvovirus B19(B19V)using Hansenula polymorpha expression system and analyze the physicochemical properties as well as the immunogenicity of the virus-like particle(VLP)assembled from the recombinant VP2.Methods The recombinant VP2 was expressed and self-assembled into VP2-VLP.B19V VP2-VLP was obtained after high-density fermentation and chromatographic purification.SDS-PAGE and size exclusion chromatography-high performance liquid chromatography were used to detect the purity.The morphology of VP2-VLP was observed using dynamic light scattering and transmission electron microscopy.The phospholipase activity of VP2-VLP was also detected.Specific IgG antibody levels and neutralizing antibody titers in mice after immunization were measured by ELISA and hemagglutination inhibition assay.Results The purity of the recombinant VP2-VLP was greater than 95%.It was similar to the wild-type viral particle in size with good uniformity and morphology.The recombinant VP2-VLP could bind to the specific monoclonal antibody and possessed no phospholipase A2 activity.After being absorbed to adjuvant,the recombinant VP2-VLP could induce the production of specific IgG and neutralizing antibodies at high titers in mice.Conclusions The recombinant B19V VP2-VLP expressed by the Hansenula polymorpha expression system could serve as a candidate vaccine antigen for developing vaccines against B19V.