人乳头瘤病毒58型病毒样颗粒的表达、纯化及免疫原性评价

Expression,purification and immunogenicity evaluation of recombinant HPV58 virus-like particles

目的 利用毕赤酵母重组表达人乳头瘤病毒(human papillomavirus,HPV)58型L1蛋白病毒样颗粒(virus-like particles,VLPs),纯化后免疫小鼠,检测制备的HPV58 L1 VLPs的免疫原性。方法 构建HPV58 L1多拷贝表达质粒,转化KM71巴斯德毕赤酵母,筛选高表达毕赤酵母工程菌,5 L发酵罐发酵,分离纯化HPV58 L1 VLPs。纯化后的HPV58L1 VLPs以不同剂量经腹腔免疫雌性BALB/c小鼠80只,4周后采用半数有效剂量(median effective dose,ED_(50))评价其免疫效果。结果HPV58 L1表达质粒经酶切鉴定构建正确,Western blot分析、氨基酸序列测定和透射电镜观察结果显示,HPV58 L1蛋白可在毕赤酵母中成功表达并可自发组装成直径约50 nm的VLPs,经离子交换和体积排阻层析纯化,可获得纯度95%以上的HPV58 L1 VLPs。纯化后的HPV58 L1 VLPs在小鼠模型上的ED_(50)为0.009μg。结论 利用毕赤酵母成功制备了具有良好免疫原性的HPV58 L1 VLPs。

Objective To prepare recombinant human papillomavirus 58 L1 protein virus-like particles(HPV58 LI VLPs)in Pichia pastoris and immunize mice with the purified product to evaluate the immunogenicity of HPV58 L1 VLPs.Methods The HPV58 L1 multi-copy expression plasmid was constructed and transformed into Pichia pastoris KM71.The highly expressed Pichia pastoris engineering strain was screened and cultured in 5 L fermenter.The expressed HPV58 L1 VLPs were isolated and purified,which were then used to immunize 80 female BALB/c mice at different doses by intraperitoneal injection.After four weeks,the immune effect was evaluated by median effective dose(ED_(50)).Results Restriction analysis results showed that HPV58 L1 expression vectors were constructed correctly.Western blot,amino acid sequencing and transmission electron microscopy(TEM) results showed that HPV58 L1 protein was correctly expressed in Pichia pastoris and could self-assemble into VLPs at a diameter of about 50 nm.After purification by ion exchange and size exclusion chromatography,the HPV58 L1 VLPs reached a purity above 95%,and the ED_(50) in mouse model was 0.009 μg.Conclusion Recombinant HPV58 L1 VLPs with good immunogenicity were successfully prepared by using Pichia pastoris.