【摘要】 目的　研究乙肝病毒“a”决定簇 12 9Leu变异株免疫原性低下的机理及其临床意义。方法　用野毒株质粒DNA(p3.8Ⅱ )及用p3.8Ⅱ组建的 12 9Leu、12 9His、14 5Arg(分别称为p3.8Ⅱ 12 9L、p3.8Ⅱ 12 9H、p3.8Ⅱ 14 5R)重组质粒DNA转染HepG2细胞 ,检测转染细胞上清HBsAg、HBeAg水平及胞内HBV复制情况。以重组质粒DNA免疫BALB/c及C5 7BL/ 6鼠 ,比较各重组质粒DNA免疫鼠后诱生的抗体水平及脾细胞特异性IFN γ水平。对各重组质粒DNA表达S基因“a“决定簇作亲 /疏水性分析。在 134例HBsAg阳性慢性乙肝患者中用PCR SSCP筛选后测序方法 ,寻找“a”决定簇变异株。结果p3.8Ⅱ 12 9L重组质粒免疫鼠血清的抗 HBs效价与脾细胞诱生的IFN γ在 2种品系鼠中均低于野毒型或p3.8Ⅱ 12 9H免疫鼠 (P <0 .0 5 )。在 134名HBsAg阳性的慢性乙肝患者血清中 ,仅有 9/ 134(6 .9% )出现“a”决定簇氨基酸变异 ,但其中 1例为 12 9Leu变异。结论　 12 9Leu变异株的T、B细胞免疫原性低下主要是由于该氨基酸变为亮氨酸所致 ,可能与该位点变为亮氨酸后疏水性增高有关。该变异株检出率虽然不高 ,但确实存在于慢性乙肝患者中。更多还原

【Abstract】 Objective To study the mechanism and clinical significance of low immunogenicity of HBV "a" determinant 129Leu variant. Methods Four recombinant plasmids containing S gene of wild type(p3.8Ⅱ) and 129Leu (p3.8Ⅱ129L), 129His(p3.8Ⅱ129H), 145Arg(p3.8Ⅱ145R) variants were constructed and separately used to transfect HepG2 cells. HBsAg, HBeAg in supernatant and HBV DNA replication in intracellular were assayed. These plasmids were also used to immunize BALB/c and C57BL/6 mice. Antibodies and cytokines induced in splenocytes of immunized mice were examined. The hydrophobicity of residue 129 was compared between the wild type and mutated "a" determinant by computer based analysis. Mutations of "a" determinant were detected in 134 HBsAg positive hepatitis B patients by PCR SSCP and sequencing technology. Results The titer of anti HBs and the level of IFN γ induced by splenocytes in two strains of mice immunized with p3.8Ⅱ129L plasmid DNA were both lower than those immunized with wild type and p3.8Ⅱ129H DNA ( P <0.05). Totally, nine serum samples from 134 HBsAg positive hepatitis B patients showed "a" determinant amino acid substitutions (6.9%) , and 1/9 was 129Leu variant. Conclusions The cellular and humoral low immunogenicity of variant 129Leu was most likely caused by the substitution of 129 amino acid from glutamine to leucine, because when 129 amino acid was substituted to leucine, the hydrophobicity of this residue was markedly increased. This 129Leu variant was detected in chronically infected hepatitis B patients. [更多还原