A truncated mutant of the Open Reading Frame 2 (ORF2, aa384-606) was amplified from cDNA of genotype IV hepatitis E virus (HEV) by polymerase chain reaction (PCR), subcloned to expression plasmid pTO-T7, and expressed in Escherichia coli. SDS-PAGE and Western blotting were used to detect and identify the recombinant protein, namely rP24. After washing of inclusion bodies, dissolving in denaturing agents, refoldeding by dialysis, ion exchange chromatography and gel chromatography, dynamic light scatter was used to study the hydrated radius of rP24. Western blotting was applied to detect the immunoreactivity of rP24, and mouse immunity test and indirect enzyme linked immunosorbent assay (ELISA) were applied to evaluate the immunogenicity and the detection rate of HEV positive and negative serum. SDS-PAGE and Western blotting show that rP24 was highly expressed in the form of inclusion bodies after induction, and had strong immunoreactivity to monoclonal antibody (McAb) 15B2. After a multi-step purification of rP24, Western blotting indicated that the purified rP24 also had strong immunoreactivity to neutralizing McAb 8C11 and HEV positive serum, suggesting that rP24 simulated the nature structure of HEV capsid protein. Dynamic light scatter demonstrated that the average hydration radius of purified rP24 was 7.48 nm. The mouse immunity test showed that the purified rP24 also had good immunogenicity, and the period of serum antibodies converted from negative to positive was very short, but the antibodies maintained more than 20 weeks. Indirect ELISA tests showed that the detection rate of was the same as anti-HEV-IgG diagnostic kit (Wan Tai corporation). Taken together, the rP24 simulated the neutralizing epitopes of natural HEV, and had strong immunoreactivity and immunogenicity. It provided a basis for the further investigation of the difference of infection mechanism between genotype I and genotype IV HEV.
Animals ; Antibodies, Monoclonal ; Antibodies, Neutralizing ; Blotting, Western ; Capsid Proteins ; biosynthesis ; Electrophoresis, Polyacrylamide Gel ; Enzyme-Linked Immunosorbent Assay ; Epitopes ; Genotype ; Hepatitis E virus ; Mice ; Mutant Proteins ; biosynthesis ; Open Reading Frames ; Recombinant Proteins ; biosynthesis

OBJECTIVETo perform a comparative analysis of the reactivation rate of hepatitis B virus (HBV) infection and related risk factors after treatment of HBV-related hepatocellular carcinoma (HCC) by radiofrequency ablation (RFA) or hepatic resection.

METHODSWe retrospectively analyzed the HBV reactivation rate and related risk factors of a cohort of 218 patients treated for HBV-related HCC between August 2008 and August 2011; the study population consisted of 125 patients who received RFA and 93 patients who received hepatic resection. Comparisons were made using the unpaired Student's t-test for continuous variables and the x2-test and Fisher's exact test for categorical variables. Univariate and multivariate logistic regression analysis was used to assess risk factors.

RESULTSTwenty patients showed HBV reactivation following treatment, but the incidence was significantly lower in the RFA group than in the hepatic resection group (5.6% vs. 14.0%, 7/125 vs. 13/93, x2 = 4.492, P = 0.034). The univariate and multivariate analysis indicated that no antiviral therapy (OR = 11.7; 95% CI: 1.52-90.8, P = 0.018) and the treatment type (i.e. RFA or hepatic resection) (OR = 3.36; 95% CI: 1.26-8.97, P = 0.016) were significant risk factors of HBV reactivation. Subgroup analysis showed that the incidence of HBV reactivation was lower in patients who received antiviral therapy than in those who did not for both the RFA group and the hepatic resection group but the difference was not significant in the former group (1/68 vs. 19/150, x2=7.039, P = 0.008 and 0/33 vs. 7/92, x2 = 2.660, P = 0.188, respectively). However, the incidence of HBV reactivation in patients who did not receive antiviral therapy was higher than in those who did receive antiviral therapy in the hepatic resection group (12/58 vs. 1/35, x2 = 5.773, P = 0.027).

CONCLUSIONThe incidence of HBV reactivation was lower in patients who received RFA than in those who received hepatic resection to treat HBV-related HCC. Antiviral therapy prior to the hepatic resection treatment may be beneficial for reducing the incidence of HBV reactivation.

Adult ; Aged ; Aged, 80 and over ; Carcinoma, Hepatocellular ; surgery ; virology ; Catheter Ablation ; adverse effects ; Female ; Hepatectomy ; adverse effects ; Hepatitis B virus ; physiology ; Humans ; Incidence ; Liver Neoplasms ; surgery ; virology ; Male ; Middle Aged ; Retrospective Studies ; Virus Activation ; Young Adult

Hepatitis B virus (HBV) infection can cause the severe threat to the health of the people around the world. It depends upon the development of efficient diagnostic reagent and vaccine to prevent the prevalence of HB. In this study, we constructed the high expression recombinant Pichia pastoris and performed the screening tests in shake flasks to obtain the optimal values of several key fermentation parameters. Based on their effects on the growth and expression level of recombinant strains, FBS was the optimal industrial medium. The optimal values for the dissolve oxygen (DO), the final concentrations of methanol and the pH values were 50 mL, 1% (V/V) and 5.4-6.0, respectively. The optimal values of the parameters simulated in shake flasks were successfully scaled up to 10 L bioreactors to achieve high-throughput production: 310 OD600 in biomass and 27 mg/L in recombinant HBsAg. The expressed recombinant HBsAg in P. Pastoris was confirmed by SDS-PAGE and Western blotting. Electron microscopy examination showed that the purified protein could be self-assembled to 22 nm virus-like particles. The results provided a basis for industrial scale-up production of diagnostic reagent and vaccine of next generation against HB.
Bioreactors ; Fermentation ; Hepatitis B Surface Antigens ; biosynthesis ; genetics ; Humans ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics