大肠杆菌表达的戊型肝炎病毒ORF2多肽对恒河猴的免疫保护研究  

The Immuno-protect Study of a Hepatitis E Virus ORF2 Peptide Expressed in E.coli

作　　者：葛胜祥[1] 张军[1] 黄果勇[2] 逄淑强[1] 周开姣[2] 夏宁邵[1] 

机构地区：[1]厦门大学细胞生物学与肿瘤细胞工程教育部重点实验室,厦门361005 [2]广西壮族自治区卫生防疫站,南宁430021

出　　处：《微生物学报》2003年第1期35-42,共8页Acta Microbiologica Sinica

摘　　要：在大肠杆菌中表达的一段戊型肝炎病毒 (HEV)结构蛋白NE2 ,纯化后以弗氏佐剂 ,按0d ,1 0d ,30d的方案 1 0 μg 针的剂量免疫 3只恒河猴 ,在第 2周抗体阳转 ,第 6周时 1只滴度达 1∶1 0 0 0 0 0 ,另 2只滴度 1∶2 0 0 0 0 ,此时以 1 0 6PCR滴度的HEV病毒粪悬液攻击。对照组 3只均出现血清转氨酶 (ALT)升高 ,抗体阳转 ,粪便持续排毒 1月以上 ;疫苗组无一发病 ,未检出非疫苗来源的抗体 ,其中 1只始终未检出粪便排毒 ,另 2只仅出现短暂排毒。以一份NE2免疫后猴血清 (滴度 1∶2 0 0 0 0 )与 1 0 3PCR滴度的病毒混匀后感染 2只恒河猴 ,结果对照组 2只均持续排毒 3周以上 ,抗体阳转 ,1只ALT明显升高 ;而抗体中和组 2只猴始终未检出粪便排毒 ,抗NE2抗体缓慢下降 ,ALT正常。这些结果表明NE2具有良好的免疫原性和免疫保护性 ,有可能成为有效的戊肝疫苗。A hepatitis E virus (HEV) ORF2 peptide expressed in an unfused pattern in E. coli, NE2, can self-assembly into homodimers and oligomers, and the immuno-reactivity of the dimers or oligomers to HEV infected serum is much stronger than monomer, which suggested that some important conformational epitopes be better exposed in dimer or oligomer form. Three Rhesus monkeys were vaccinated with three doses (10μg/dose) purified NE2 in Freud's adjuvant under a schedule of 0d, 10d, and 30d. Specific antibodies can be detected on second week, and on sixth week while virus challenge were performed with 106 PCR titer of virus positive stool suspention, the antibody titer of one monkey was 1∶100 000, the other two were both 1∶20 000. Three monkeys in control group presented typical acute hepatitis E manifestation: increased seral amino transferase (ALT), antibody conversion, and continuous virus excretion in stool. In contrast, the ALT of monkeys in vaccinated group continued to be normal, stool virus had not been detected in one monkey, and presented only a short duration in another two. One NE2-vaccinated monkey serum with antibody titer 1: 20 000 was first incubated with HEV (103 PCR titer) for neutralization, then the mixture were used to challenge two monkeys, the results showed that two monkeys in control group continued to excrete virus for more than three weeks, sera antibody conversion, and one monkey presented obvious ALT increase. Both two monkeys challenged with antibody neutralized virus had not detected virus in stool, the antibodies decreased slowly, and ALT continued to be normal. These results suggested that the prokaryotic expression recombinant protein NE2 have good immunogenicity and immunoprotectivity, and should be a good candidate for an effective hepatitis E vaccine.

关 键 词：大肠杆菌 基因表达 戊型肝炎病毒 ORF2多肽 恒河猴 免疫保护 

分 类 号：R512.6[医药卫生—内科学]