基于HEV中和表位的基因免疫you导特异性抗体及中和作用研究.docVIP

　　基于HEV中和表位的基因免疫诱导特异性抗体及中和作用研究 作者：王春玲,杨书才,孟继鸿,王立新 【摘要】 　　目的：观察基于戊型肝炎病毒中和表位(p179)的基因免疫是否能在小鼠体内诱导具有中和作用的抗体应答。方法：构建含HEV中和表位p179编码基因的真核表达质粒pVAX179,以肌肉注射法对BALB/c小鼠实施基因免疫，实验组每只小鼠给pVAX179 100 μg,并设立空质粒pVAX和HEVp179蛋白两个对照组。定期采集小鼠血清,ELISA法检测其特异性抗HEVIgG抗体及其亲合力；以基于PCR的体外中和实验检测抗体的中和活性。结果：pVAX179质粒基因免疫诱导小鼠体内产生了特异性抗HEVIgG抗体,抗体水平于第10周达高峰,OD值为0.55±0.13,与空质粒pVAX组(0.15±0.07)比较差异有统计学意义(P＜0.001)。第6周实验组抗体亲合力(ED50为2.5 mol·L-1)高于蛋白对照组（ED50为2.0 mol·L-1),基因免疫促进了抗体亲合力的成熟；体外中和实验证实产生的特异性抗体能够阻止HEV感染人肝癌细胞7721,具有中和HEV的活性。结论：基于戊型肝炎病毒中和表位(p179)的基因疫苗pVAX179能够在小鼠体内诱导产生具有中和作用的抗体应答。 【关键词】 戊型肝炎病毒； 中和表位； 基因疫苗； 抗体应答； 中和作用 　　［Abstract］ Objective：To investigate whether the DNA vaccine based on HEV neutralization epitope(s) HEVp179 could induce neutralizing antibody response in mice.Methods：The neutralization epitope(s) HEVp179coding gene was subcloned into a eukaryotic expression vector pVAX to construct recombinant plasmid pVAX179. BALB/c mice were immunized i.m. with 100 μg DNA of pVAXp179 plasmid, mock DNA,and 1 μg protein of HEVp179,respectively.Sera collected at the indicated times from two groups were determined the levels and avidity of specific antiHEV IgG antibody by ELISA.The neutralizing activity of induced antiHEV antibody was detected in a PCRbased cell culture neutralization assay.Results:Immunizations with pVAX179 successfully induced antiHEV specific antibody response and accelerated the avidity maturation of antibodies.The antiHEV level reached the hightest values about 10 weeks after first vaccination.The avidity of antibodies from pVAX179 immunized mice was higher than that from HEVp179 protein immunized group.In addition,the postimmune sera obtained from pVAXp179 vaccinated mice were found the protective neutralizing activity against HEV infection.Conclusion：These results indicate that the DNA vaccine based on HEV neutralization epitope(s)HEVp179 could induce neutralizing antibody response against HEV in mice． 　　［Key word］ hepatitis E virus； neutralization epit