CAJ | 학술논문

目的研究4型戊型肝炎病毒(hepatitis E virus,HEV)开放阅读框架2(open readingframe 2,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Westem 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.Westem 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.
목적 연구4형무형간염병독(hepatitis E virus,HEV)개방열독광가2(open readingframe 2,ORF2)편마적다태이취체특성화항원성특정.방법 종환자혈청극륭HEV ORF2기인,재대장간균중표체불동장도적ORF2다태,병표체점돌변다태,순화후재SDS-PAGE불동조건하분석이취체특성,동시용Westem blot분석기항원성특정.결과 유459～607위안기산구성적ORF2다태능형성이취체,이재안기단절단13개안기산혹재최기단절단13개안기산균불형성이취체,562위천동선알、595위이량안산분별돌변후재SDS작용하부이단체존재.Westem blot현시,항-HEV부여이취체적ORF2다태반응,이불여단체ORF2다태혹절단태반응.결론 4형HEV ORF2다태형성이취체필수함유459～607위안기산,기중562위화595위안기산돌변능영향이취체적은정성.해다태적항원성취결우이취체결구.
Objective To characterize the dimerization and the antigenicity of the ORF2 polypep-tide 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. coli. The recombinant polypeptides were purified and analyzed by SDS-PAGE and Western blot. Results The ORF2 polypeptide containing 459-607 amino acid formed homedimer 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 ho-modimer, 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 tnmcated pol-ypeptides. 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.