摘要
目的分析乙型肝炎病毒(HBV)HBsAg+抗HBs双阳性患者S基因主要亲水区(MHR)新增N-糖基化突变的特点,探讨HBsAg+抗HBs双阳性的发生机制和临床意义。方法对284例HBsAg+抗HBs双阳性和314例HBsAg单阳性的慢性HBV感染者S基因进行测序分析。对1例携有MHR区双N-糖基化新型突变株的慢性乙肝患者样本进行随访收集和研究。构建双N-糖基化突变和对照前S/S基因重组质粒,转染HepG2细胞,分析突变对病毒复制力和抗原性的影响。结果 HBsAg+抗HBs双阳性患者MHR区新增N-糖基化突变的检出率为11.3%(32/284),显著高于HBsAg单阳性患者的2.9%(9/314)(P<0.01)。HBsAg+抗HBs双阳性组中,72例肝细胞癌(HCC)在N-糖基化突变阳性和阴性患者中所占比例分别为46.9%(15/32)和22.6%(57/252)(P<0.01)。从1例患者中检出的新型株突变形式为s116-118TST→NST+s131-133TSM→NST,并联合sP120缺失+G145D突变,在3份随访样本中该突变株分别占98.0%、2.0%和2.5%,第2份样本中检出s130-132GTS→NSS单N-糖基化突变株,占17.6%,但无s P120缺失+G145D联合突变。与野生株相比,新型突变株复制力提高31%,但HBsAg定量降低99%。免疫荧光结果显示,双N-糖基化定点回复突变株可部分恢复HBsAg检出水平,提示除双N-糖基化突变外,sP120缺失+G145D联合突变对HBsAg抗原性减弱也有明显影响。结论 HBV S基因MHR区新增N-糖基化突变与HBsAg+抗HBs双阳性相关,两者同时出现可能是HCC发生的高风险因素;S基因MHR区新增双N-糖基化+sP120缺失+sG145D联合突变共同影响HBsAg的抗原性。
Objective To analyze the characteristics of newly added N-glycosylation mutation in major hydrophilic region（MHR） of HBV S gene in patients with coexistence of HBsAg and anti HBs, and reveal the generation mechanism and clinical implications of the coexistence. Methods HBV S genes from 284 patients with HBsAg＋anti HBsand 314 patients with single HBsAg were amplified respectively for sequence analysis. A chronic hepatitis B（CHB） patient with HBsAg＋anti HBsin MHR was found to harbor a novel double N-glycosylation mutation and selected for further study. Recombinant vectors harboring the novel mutant or control Pre S/S genes were constructed and transfected in Hep G2 cells respectively for phenotypic analysis, and the effects of the mutations on HBV duplication and antigenicity were investigated. Results The detection rate of MHR N-glycosylation mutation was significantly higher in HBsAg＋anti HBsgroup than in single HBsAg group（11.3% vs. 2.9%, P0.01, respectively）. In HBsAg＋anti HBscohort, the proportion of hepatocellu lar carcinoma（HCC） patients accounted for 46.9%（15/32） in patients with N-glycosylation mutation at the time of testing; by contrast, the number was 22.6%（57/252） in patients with non-N-glycosylation mutation（P0.01）. N-glycosylation mutational pattern of the novel strain was s116-118TST→NST＋s131-133TSM→NST concomitant with s P120 deletion＋G145D mutation. The novel mutants accounted for 98.0%, 2.0% and 2.5%, respectively, of viral clones in three sequential serum samples. Mutants with single N-glycosylation mutation s130-132GTS→NSS without s P120 deletion＋G145D were detected in sample 2, accounting for 17.6% of viral clones. Compared to the wild-type, the novel mutant had an increase of 31% in replication capacity, but a decrease of 99% in HBsAg level. Immunofluorescence showed that elimination of the two additional N-glycosylation mutations only partly restored HBsAg detection by anti HBs, suggesting that s P120 deletion＋G145D mutation also attenuated
出处
《解放军医学杂志》
CAS
CSCD
北大核心
2016年第5期351-357,共7页
Medical Journal of Chinese People's Liberation Army
基金
国家自然科学基金(81373136,81271847,81572010)~~
