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>

The immunization effects of HBV core antigen and surface antigen fusion protein in mice / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To evaluate the immunization effects of HBV core antigen and surface antigen fusion protein.</p><p><b>METHODS</b>The DNA fragments encoding HBsAg 100-162 aa; HBcAg 1-78 aa and HBcAg 83-144 aa were PCR-amplified, and then cloned into pcDNA3 plasmid. The chimeric gene was subcloned into the prokaryotic vector, pRSET-B. The E.coli expressed recombinant protein purified. BALB/c mice were immunized with recombinant protein or eukaryotic expression plasmid, humoral response and cellular response were examined.</p><p><b>RESULTS</b>The plasmid containing the chimeric gene of HBsAg and HBcAg induced effective anti-HBs antibody response and strong HBcAg specific lymphocyte proliferative response, but could not induce anti-HBc antibody response. Fusion protein induced strong anti-HBs and anti-HBc antibody response, and it also caused significant HBcAg specific lymphocyte proliferation. Compared to the recombinant fusion protein, the plasmid containing the chimeric gene of HBsAg and HBcAg can induce more effective cellular response but weaker humoral response.</p><p><b>CONCLUSION</b>Compared to the recombinant fusion protein, the plasmid containing the chimeric gene of HBsAg and HBcAg is a more effective vaccine.</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>

Distribution of hepatitis B virus genotypes in Hubei province and its clinical significance / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the distribution of hepatitis B virus genotype in Hubei province (China) and its clinical significance.</p><p><b>METHODS</b>Serum samples from 190 HBV DNA positive patients with chronic HBV infection,including 52 asymptomatic HBV carriers (ASC), 56 chronic hepatitis (CH), 32 fulminant hepatic failure (FHF), 22 liver cirrhosis (LC), and 28 hepatocellular carcinoma (HCC) patients were collected and tested for HBV genotypes by type-specific primers.</p><p><b>RESULTS</b>A simple and precise genotyping system based on PCR using type-specific primers was developed for the determination of genotypes of hepatitis B virus (HBV). Of the 190 patients, 140 (73.7%) were genotype B and 42 (22.1%) were genotype C. Genotype B was more prevalent in the FHF and HCC patients than in the ASC patients; the ALT value was significantly higher in genotype B than in genotype C patients. The rate of anti-HBe was significantly higher in genotype B than in genotype C except in the patients of the ASC group.</p><p><b>CONCLUSION</b>The system we used seems to be a useful tool for the molecular diagnosis of HBV infection and for large-scale surveys. Genotype B, genotype C and BC combination exist in Hubei province, and genotype B is the major genotype in this area especially in FHF and HCC patients.</p>

Infection of tupaia hepatocytes with hepatitis C virus in vitro / 中华肝脏病杂志

<p><b>OBJECTIVE</b>Tupaia belangeri (tree shrew) has a close phylogenetic relationship with primates and has been shown to be susceptible to a variety of human viruses. This study was conducted to investigate whether or not hepatitis C virus (HCV) could infect primary tupaia hepatocytes (PTHs) in vitro.</p><p><b>METHODS</b>Serum-derived HCV was cultivated with PTHs, and then positive and negative strand HCV RNA in PTHs, as well as the encapsidated HCV RNA in the culture medium were detected to evaluate the infection. Virus from the culture medium of the infected PTHs was passed to naïve PTHs, and the quasispecies of HCV were compared among the inoculum and PTHs after infection and passage.</p><p><b>RESULTS</b>Both positive and negative strand HCV RNA were detected in PTHs after infection. The negative strand RNA was detectable from day 5 to day 10 after infection, while the positive strand RNA was positive up to day 14. HCV RNA, which was RNase resistant, could be detected from the culture medium of the infected PTHs from day 3 to day 14. Production of infectious virons of PTH were demonstrated by passage HCV to naïve PTHs. Compared analysis of HCV quasispecies after infection and passage showed that PTHs were selectively infected with defined HCV quasispecies, and new quasispecies emerged in PTHs after passage.</p><p><b>CONCLUSION</b>The present study strongly indicates that PTHs could be infected by HCV and support HCV replication in vitro. Our results would be helpful for the establishment of a tupaia model of HCV infection.</p>