人乳头瘤病毒18型病毒样颗粒的特征分析

Characteristics of virus-like particles of human papilloma virus 18

目的对人乳头瘤病毒18型(human papilloma virus 18,HPV18)病毒样颗粒(virus-like particles,VLPs)进行特征分析。方法采用质谱技术分析HPV18 VLPs(重组大肠埃希菌表达系统表达L1蛋白,自组装成为VLPs)蛋白质一级结构,圆二色谱技术分析蛋白质二级结构,高效液相色谱、透射电镜和动态光散射技术分析蛋白质三级和四级结构;同时采用假病毒中和试验分析HPV18 VLPs的免疫原性,SDS-PAGE法分析样品纯度。结果HPV18 VLPs蛋白翻译后修饰主要包括氧化和去酰胺化,完整L1单体分子量约56000;二级结构中螺旋、折叠、转角、不规则卷曲的比例分别为8.4%、46.1%、18.5%、30.2%;颗粒纯度达99%以上,且形态饱满完整,大小均一,直径约60 nm。HPV18 VLPs诱导小鼠产生中和抗体滴度的几何均值达3000以上。HPV18 VLPs中完整L1单体含量达95%以上。结论HPV18 VLPs结构完整,颗粒大小均一,纯度高,免疫原性良好,本研究为宫颈癌疫苗安全性和有效性的评价奠定了基础。

Objective To analyze the characteristics of virus-like particles(VLPs)of human papilloma virus(HPV)18.Methods HPV18 VLPs were obtained by self-assembly of L1 protein expressed by recombinant E.coli expression system and analyzed for primary structure by mass spectrometry,for secondary structure by circular dichroism technique,and for tertiary and quaternary structures by high performance liquid chromatography,transmission electronic microscopy and dynamic light scattering technique.Meanwhile,the immunogenicity of HPV18 VLPs in animals was analyzed by pseudovirusbased neutralization assay,while the purity by SDS-PAGE.Results The post-translational modification of HPV18 VLPs mainly included oxidation and deamidation,while the relative molecular mass of intact L1 monomer was about 56000.The proportions of helix,fold,angle and irregular coil in secondary structure were 8.4%,46.1%,18.5%and 30.2%respectively.The VLPs,with a purity of more than 99%,were full and intact in shape,even in size and at a diameter of about 60 nm,by which the GMT of neutralizing antibody induced in mice reached more than 3000.However,the content of intact L1 monomer in HPV18 VLPs was more than 95%.Conclusion HPV-18 VLPs were intact in structure and even in size,which reached a high purity and showed good immunogenicity.It laid a foundation of evaluation on safety and effectiveness of cervical cancer vaccine.