隐匿性乙型肝炎病毒感染者血清表面抗体性质鉴定

Characterization of HBsAbs in occult hepatitis B virus infection

目的了解隐匿性HBV感染者抗-HBs的特性及其与HBsAg的结合能力。方法对2例抗HBs阳性的隐匿性HBV感染者进行长期随访，使用多种试剂盒对患者血清进行多次HBsAg检测，利用不同血清型HBsAg对患者血清进行中和反应，了解血清中抗体亚型情况。PCR扩增S基因构建真核表达质粒，分析S基因变异情况，并将质粒转染HepG2细胞，取培养上清液及转染细胞分别混合进行HBsAg检测。使用患者血清及抗-HBs阳性血清（对照组）对部分HBsAg阳性克隆上清液进行中和反应。不同组问数据比较采用f检验。结果多种试剂盒进行的多次检测结果均表明患者血清HBsAg为阴性；HBsAg的3种不同血清型（adr、adw、ay）均能够中和患者血清中大部分抗-HBs（82．1％～100．0％）。S基因序列分析表明核苷酸同源性和氨基酸同源性分别为95．13％～97．79％和92．04％～95．58％；培养上清液和转染细胞裂解液中HBsAg定量分别为对照组的48．1％和59．3％，上清液／细胞裂解液比值分别为0．85和0．38。中和试验结果显示转染上清液中HBsAg定量较混合前有所降低，但是仍然可以检测到，而对照组检测不到HBsAg，差异均有统计学意义（F值分别为353．6和645．2，P值均〈0．01）。结论抗-HBs阳性隐匿性HBV感染者体内HBsAg的抗原性及分泌能力有所下降，抗HBs主要针对不同血清型HBsAg的共同表位，但可能与疫苗注射产生的抗HBs有所不同。

Objective To investigate the properties of HBsAb in occult hepatitis B virus infection and its affinity to different serotypes of hepatitis B virus surface antigen （HBsAg）. Methods Long-term follow-up was conducted in 2 HBsAb positive patients with occult hepatitis B virus infection. HBsAg was detected using multiple diagnostic kits and the HBsAb subtype was determined by performing neutralization experiments with different serotypes of HBsAg. The viral S gene was PCR-amplified and mutation analysis was conducted. Plasmids expressing HBsAgs were constructed by inserting these PCR products into an eukaryotic expression vector and were then transfected into HepG2 cells. The cell culture supernatant and cellular extracts were detected for HBsAg respectively. Neutralization experiments were carried out in the cell culture supernatant from HBsAg plasmids transfected HepG2 cells and serum samples from these patients and others who had been confirmed to be positive for HBsAb. Results Multiple tests using various diagnostic kits showed that the 2 patients were negative for HBsAg and the three different serotypes of HBsAg （adr, adw, ay） could neutralize 82.1%-100% of HBsAb existed in the 2 patients. Sequence analysis of S gene cloned from these patients revealed that the homology to reference strain were 95.13%-97.79% and 92.04%- 95.58% respectively at the nucleotide and amino acid levels. Quantitation of HBsAg showed that the expression levels of HBsAg from the two patients were 41.1% and 22.6% respectively of that of control HBsAg in cell culture supernatant and 48.1% and 59.3% respectively in cellular extract, and the supernatant/cell lysate ratios were 0.85 and 0.38 respectively. In neutralization experiments, HBsAg could be totally absorbed by control serum, whereas could only be partially neutralized by HBsAbs from the two patients （F = 353.6 and 645.2, P 〈 0.01）. Conclusion Both the antigenicity and the ability of HBsAg secreted outside of the cells are decresed in these HBsAb-positive patients with occult HBV infection. The HBsAbs are mainly specific for common epitopes among different serotypes of HBsAg and are probably different as compared with those produced by vaccine inoculation.