摘要
目的研究4型戊型肝炎病毒（hepatitis E virus，HEV）开放阅读框架2（open reading frame2，ORF2）编码的多肽二聚体特性和抗原性特征。方法从患者血清克隆HEV ORF2基因，在大肠杆菌中表达不同长度的ORF2多肽，并表达点突变多肽，纯化后在SDS—PAGE不同条件下分析二聚体特性，同时用Western blot分析其抗原性特征。结果由459～607位氨基酸构成的ORF2多肽能形成二聚体，而在氨基端截短13个氨基酸或在羧基端截短13个氨基酸均不形成二聚体，562位天冬酰胺、595位异亮氨酸分别突变后在SDS作用下仅以单体存在。Western blot显示，抗-HEV仅与二聚体的ORF2多肽反应，而不与单体ORF2多肽或截短肽反应。结论4型HEV ORF2多肽形成二聚体必须含有459—607位氨基酸，其中562位和595位氨基酸突变能影响二聚体的稳定性。该多肽的抗原性取决于二聚体结构。
Objective To characterize the dimerization and the antigenieity of the ORF2 polypeptide of hepatitis E virus （ HEV, genotype 4）. Methods HEV ORF2 gene was cloned from the serum of a patient with hepatitis E. The genotype was determined by sequencing. Three ORF2 polypeptides differing in size and other polypeptides with point mutations were produced in E. coll. The recombinant polypeptides were purified and analyzed by SDS-PAGE and Western blot. Results The ORF2 polypeptide containing 459-607 amino acid formed homodimer even in 8 mol/L urea. The truncated polypeptides containing amino acid 472-607 or 459-594 formed monomer only. The mutations at amino acid 562 or 595 disrupted the homodimer, whereas the mutations at amino acid 476 or 580 did not. Anti-HEV from hepatitis E patients only reacted with the homodimer form of the polypeptide 459-607 and did not react with monomer or truncated polypeptides. Conclusion The amino acid 459-607 of HEV ORF2 is essential for dimerization of the ORF2 polypeptide. Residues at amino acid 562 and 595 are critical for the dimerization. The antigenicity of the polypeptide 459-607 mainly depends on its homodimer form.
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2008年第11期993-997,共5页
Chinese Journal of Microbiology and Immunology
基金
江苏省医学重点人才项目（RC2007005）
南京市回国人员科技活动择优资助
