Characterization of HBsAbs in occult hepatitis B virus infection / 中华肝脏病杂志

<p><b>OBJECTIVE</b>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).</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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 is less than 0.01).</p><p><b>CONCLUSION</b>Both the antigenicity and the ability of HBsAg secreted outside of the cells are decreased 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.</p>

S gene mutations in HBsAg/HBsAb double positive chronic hepatitis B patients / 中华肝脏病杂志

<p><b>OBJECTIVES</b>To investigate S gene mutations in HBsAg/HBsAb double positive chronic hepatitis B patients.</p><p><b>METHODS</b>HBV S gene from 8 patients (Group A) with HBsAg (+)/HBsAb (+) and 9 patients (Group B) with HBsAg (+)/HBsAb (-)was amplified by polymerase chain reaction (PCR) and sequencing. Both the distribution of genotype and serotype and the rate of MHR region were compared by Fisher's exact test. The mutation rate of both the DNA level and amino acid level was compared by t test.</p><p><b>RESULTS</b>No significant difference in distribution of genotypes was found between the two groups (P=0.153). In group A, 2 were genotype B, 6 were genotype C; In group B, 6 were genotype B, 3 were genotype C. No significant difference in distribution of serotypes was found between the two groups, either (P=0.218). In group A, 2 were adw, 5 were adr, 1 was ayr; In group B, 6 were adw, 3 were adr. The mutation rate of Pre-S1 region at both the DNA level (2.29% vs 1.80%, t=2.66, P more than 0.05) and the amino acid level (2.66% vs 1.59%, t=1.39, P>0.05) was not significantly different between these two groups; the mutation rate of Pre-S2 region in group A patients was significantly higher than that in group B at the DNA level (1.74% vs 0.91%, t=4.68, P<0.01), but not higher at the amino acid level (3.18% vs 2.05%, t=1.85, P>0.05), the mutation rate of S region in group A patients was significantly higher than that in group B at both the DNA level (2.13% vs 0.81%, t=6.00, P<0.01) and the amino acid level (4.37% vs 1.52%, t=5.32, P<0.01). Amino acid substitutions were found both within and beyond the MHR region. The rate of "a" determinant mutations in these two groups was also found to be significantly different (P<0.05).</p><p><b>CONCLUSION</b>Higher HBV S gene mutation rate exists in HBsAg/HBsAb double positive patients than that in HBsAg (+)/HBsAb (-) patients.</p>

Common linear B-cell epitopes in human hepatitis B virus core protein and woodchuck hepatitis virus core protein / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To search for and verify some common B cell epitopes in the core proteins of woodchuck hepatitis virus and human hepatitis B virus.</p><p><b>METHODS</b>Monoclonal antibodies against both core proteins of woodchuck hepatitis virus (WHV) and human hepatitis B virus (HBV) were prepared by inoculating Balb/c mice with denatured recombination WHV and HBV core proteins. ELISA and immunoblotting assays for WHcAg and HBcAg were carried out by using these antibodies. Immunohistochemistry was carried out with liver tissue sections of both WHV-infected woodchucks and chronic HBV-infected patients. The epitopes were mapped with the mouse mAbs (6D1 and 1H4) by using a panel of 24 16mer overlapping peptides covering the entire WHcAg. The amino acid sequences of WHcAg and HBcAg were compared.</p><p><b>RESULTS</b>Cross-reactions were observed between mAbs (6D1 and 1H4) and WHcAg and between Mabs and HBcAg/HBcAg in ELISA and immunoblotting assay. Liver tissue sections of both WHV-infected woodchucks and chronic HBV-infected patients could be stained specifically by mAbs. The epitopes were mapped at aa1-8 (6D1) and aa125-140 (1H4) of the core proteins of both WHV and HBV by using ELISA assay. WHcAg and HBcAg share similar amino acids sequences at aa1-8 and aa125-140 respectively.</p><p><b>CONCLUSION</b>The core proteins of woodchuck hepatitis virus and human hepatitis B virus share common linear B cell epitopes which span aa1-8 and aa125-140 respectively.</p>

Cloning, prokaryotic expression and polyclonal antibody preparation of HCCR: a new biomarker for hepatocellular carcinoma / 中华肝脏病杂志

<p><b>OBJECTIVES</b>To construct a prokaryotic plasmid expressing truncated human cervical cancer oncogene (HCCR-1(167-360)), to express and purify the recombinant protein, and to develop the polyclonal antibody against HCCR.</p><p><b>METHODS</b>HCCR-1(167-360) was amplified by RT-PCR from HepG2 cells and cloned into vector pRSET-B, then expressed in E.coli BL21(DE3) pLysS, which was induced by IPTG. The recombinant protein was purified using Ni-NTA spin column and acrylamide gel electrophoresis. A polyclonal antibody was developed by immunizing BALB/c mice with the purified recombinant protein, and their sensitivity and specificity were tested using enzyme-linked immunosorbent assay, immunohistochemical staining and Western blot analysis.</p><p><b>RESULTS</b>Recombinant plasmid expressing truncated HCCR-1167-360 was constructed. A protein of 2.70 x 10(4) was successfully expressed and purified. High titer polyclonal antibody with a high specificity was obtained by immunizing BALB/c mice with the purified recombinant protein.</p><p><b>CONCLUSIONS</b>The truncated recombinant HCCR-1(167-360) developed in this study is highly purified and shows strong antigenecity; the polyclonal antibody against this HCCR protein was generated by regular immunization method, showing both high sensitivity and specificity. The protein and the antibody can be used for further clinical examination and research of HCCR.</p>