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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 N-glycosylation mutations in major hydrophilic region (MHR) of S gene in hepatitis B virus (HBsAg) -positive HBs double positive patients and to explore the pathogenesis and clinical significance of double positive for HBsAg + anti-HBs. Methods 284 cases of HBsAg + anti-HBs double positive and 314 cases of HBsAg single positive chronic HBV infection S gene sequencing analysis. A sample of patients with chronic hepatitis B carrying novel N-glycosylation mutations in MHR region were collected and studied. Construction of double N-glycosylation mutation and control pre-S / S gene recombinant plasmid, transfected HepG2 cells, analysis of the mutation on the viral replication and antigenicity. Results The positive rate of N-glycosylation mutation in MHR region was 11.3% (32/284) in HBsAg + anti-HBs double positive patients, which was significantly higher than 2.9% (9/314) in HBsAg single positive patients (P <0.01) . Among the HBsAg + anti-HBs double positive group, 72 cases of hepatocellular carcinoma (HCC) accounted for 46.9% (15/32) and 22.6% (57/252) of the N-glycosylation mutation-positive and negative patients, respectively <0.01). The mutations of the new strains detected in one patient were s116-118TST → NST + s131-133TSM → NST, combined with sP120 deletion + G145D mutation, which accounted for 98.0% and 2.0% of the three follow-up samples, respectively 2.5%. In the second sample, s130-132GTS → NSS single N-glycosylation was detected, accounting for 17.6%, but no s120 deletion + G145D mutation. Compared with the wild-type strain, the new mutant strain replication increased by 31%, but a quantitative reduction of HBsAg 99%. The results of immunofluorescence showed that the double N-glycosylation site-directed mutagenesis could partially restore the detection level of HBsAg, suggesting that the combination of sP120 deletion + G145D mutation also had a significant effect on HBsAg antigenicity in addition to the double N-glycosylation mutation. Conclusions The novel N-glycosylation mutation in MHR region of HBV S gene is correlated with double positive of HBsAg + anti-HBs, which may be a high risk factor for HCC. The addition of double N-glycosylation + sP120 deletion in MHR region of S gene + sG145D combined mutations affect the antigenicity of HBsAg.