HCV中和抗原表位与HBV S抗原嵌合病毒样颗粒的制备及纯化  

Preparation and purification of chimeric virus-like particles of HCV neutralizing epitopes and HBV S antigen

作　　者：舒放[1] 王晓岩[1] 雷迎峰[2] 林芳[1] 王希[1] 李斌[1] 刘冲[1] 张惠中[1] 韦三华[1] 

机构地区：[1]第四军医大学唐都医院临床实验与检验输血科,陕西西安710038 [2]第四军医大学基础部微生物学教研室,陕西西安710032

出　　处：《中国生物制品学杂志》2015年第3期257-260,265,共5页Chinese Journal of Biologicals

基　　金：国家自然科学基金项目(81072500)

摘　　要：目的制备丙型肝炎病毒(hepatitis C virus,HCV)中和抗原表位与HBV S抗原嵌合病毒样颗粒(virus-like particles,VLPs),并进行纯化和鉴定。方法采用脂质体法分别将4个HCV中和抗原表位与HBV S抗原嵌合重组表达载体p CI-HBSE1、p CI-HBSE2、p CI-HBSE3、p CI-HBSE4转染HEK293T细胞,48 h后收集培养上清,获得自我装配的嵌合HCV中和表位的HBV VLPs,分别命名为VLPs-SE1、VLPs-SE2、VLPs-SE3、VLPs-SE4,蔗糖密度梯度离心,透析浓缩后,电镜观察VLPs,电化学发光法进行HBs Ag定量,ELISA法检测嵌合VLPs作为包被抗原与HCV感染患者血清的反应。结果电镜观察可见4种嵌合VLPs,大小约为22 nm;浓度最高的VLPs-SE2 HBs Ag含量达5.51×103ng/ml,该浓度可满足后续试验的要求;用混合VLPs作为包被抗原检测的HCV感染患者血清中和抗体水平稍高于单一VLPs作为包被抗原检测的水平。结论成功制备了HCV中和抗原表位与HBV S抗原嵌合VLPs,为进一步评价VLPs体内诱导的中和抗体及中和抗体的保护作用奠定了基础。Objective To prepare, purify and identify the chimeric virus-like particles（VLPs） of HCV neutralizing epitopes and HBV S antigen. Methods Four recombinant plasmids for chimeric expression of HCV neutralizing epitopes and HBV S antigen, p CI-HBSE1, p CI-HBSE2, p CI-HBSE3 and p CI-HBSE4, were transfected to HEK293 T cells in mediation of liposome respectively, and the culture supernatants were collected 48 h later. The obtained self-assembled HBV VLPs chimerized with HCV neutralizing epitopes, named as VLPs-SE1, VLPs-SE2, VLPs-SE3 and VLPs-SE4 respectively,were purified by sucrose density gradient centrifugation, concentrated by dialysis, observed by electron microscopy, deter-mined for HBs Ag content by electrochemical luminescence assay, and tested for reactions with the sera of patients infected with HCV by ELISA. Results Four kinds of chimeric VLPs were observed by electron microscopy, each at an size of about 22 nm. The HBs Ag content in VLPs-SE2 with the highest concentration reached 5. 51 × 103 ng / ml, which met the needs for further study. The serum neutralizing antibody levels of patients infected with HCV determined using pooled VLPs as coating antigen were slightly higher than those using single VLPs. Conclusion The chimeric VLPs of HCV neutralizing epitopes and HBV S antigen was prepared successfully, which laid a foundation of further evaluation of the neu-tralizing antibody and its protective effect induced by VLPs in vivo.

关 键 词：丙型肝炎病毒 中和抗原表位 乙型肝炎病毒 S抗原 嵌合 病毒样颗粒 

分 类 号：R373.2[医药卫生—病原生物学] R392-33[医药卫生—基础医学]