Positive clones from Pichia pastoris strain GS115-47-4 and KM71-47-1 were grown in 1200 ml BMGY/BMMY medium into 2 liters fermentor and 50 ml innoculum with high density was added, temperature was 30oC, agitation speed was 300 rpm and pressure was 0.5 kg/cm2. The biomass was harvested after 72 h of induction in case of GS115-47-4 strain and 48 h for KM71-47-1 strain. The density was 3,9 x 109 cells/ml. After cell lysis, the total HBsAg contents were obtained approximately 30 mg in both strains. 2 consecutive isopycnic KBr gradient ultracentifugations following one rate-zonal sucrose were performed.
hepatitis B vaccines ; isolation & purification ; Hepatitis B Surface Antigens

China ; epidemiology ; Hepatitis B ; classification ; epidemiology ; virology ; Hepatitis B Surface Antigens ; blood ; Hepatitis B virus ; genetics ; isolation & purification ; Humans ; Mutation

Adult ; Female ; Hepatitis B Core Antigens ; analysis ; Hepatitis B Surface Antigens ; analysis ; Hepatitis B virus ; isolation & purification ; Hepatitis B, Chronic ; virology ; Humans ; Ovary ; virology

In order to produce recombinant human anti-HBsAg Fab antibody in Pichia pastoris, the recombinant yeast was fermented using fed-batch system in a 30 L bioreactor. The fermentation temperature was 30 degrees C, the pH was 5.0 approximately 5.3, and the DO was 20% approximately 30%. The recombinant Fab antibody was purified from crude culture supernatant by ion exchange and analyzed by SDS-PAGE and western blot and ELISA. When the absorbance (OD600) of broth reach 300 at the end of fed-batch phase, the induced phase was initiated. The results showed that recombinant human anti-HBsAg Fab antibody was high-level expressed in recombinant Pichia pastoris using a fed-batch fermentation system. Both chains of the Fab were successfully expressed upon methanol induction. After 192 h of induction, the expression level of recombinant Fab (soluble) reached 412 mg/L. The recombinant Fab antibody was purified effectively by ion-exchange chromatography from the fermentation supernatant to a purity of 95%. And the affinity activities of the purified recombinant Fab antibdy and fermentation supernatant were detected, and both of them showed high affinity activities. The results demonstrated that recombinant human anti-HBsAg Fab antibody could be high level produced by fed-batch fermentations in Pichia pastoris. Which can be efficiently used in industrial production.
Fermentation ; Hepatitis B Antibodies ; biosynthesis ; isolation & purification ; Hepatitis B Surface Antigens ; immunology ; Humans ; Immunoglobulin Fab Fragments ; biosynthesis ; isolation & purification ; Pichia ; genetics ; Recombinant Proteins ; biosynthesis ; isolation & purification

<b>OBJECTIVEb>To investigate the viral contamination of invasive medical instruments in dentistry and to provide health administrative institutions with surveillance data.

<b>METHODSb>Sterilized samples were randomly collected from the department of dentistry to detect HBV-DNA, HCV-RNA, HIV-RNA and HBsAg.

<b>RESULTSb>Of the invasive medical instruments that were sterilized with 2% glutaraldehyde, one of the samples was positive for HBV-DNA, and another sample was positive for HBsAg.

<b>CONCLUSIONb>Though massive virus contamination of invasive medical instruments in dentistry has been reduced to a low level, the occurrence of contamination still remains.

DNA, Viral ; analysis ; Dental Instruments ; virology ; Equipment Contamination ; HIV ; isolation & purification ; Hepacivirus ; isolation & purification ; Hepatitis B Surface Antigens ; analysis ; Hepatitis B virus ; isolation & purification ; Humans ; RNA, Viral ; analysis

Thirty turtles (15 Clemys mutica and 15 Geoclemys reevesii) which were inoculated with human sera those were positive for hepatitis B surface antigen (HBsAg) and hepatitis B "e" antigen (HBeAg) were found to be infected with hepatitis B virus (HBV). The levels of HBV infection markers, such as HBsAg and antibody to HBsAg (anti-HBsAg), were retinely monitored in the turtles' serum for 46 weeks. Within two weeks of the inoculation, 42% of the turtles tested were positive for HBsAg, and their reciprocal titers as measured by reverse passive hemagglutination (RPHA) and enzyme linked immunoabsorbance assay (ELISA) ranged from 16 to 96. Within 20 weeks, the remaining turtles tested HBsAg positive, as confirmed by ELISA. At 20 weeks, all but one of the turtles exhibited changes in HBV blood marker from HBsAg to anti-HBs; the one exception was positive for both HBsAg and anti-HBs. At the 47th week, 7 animals were killed and their organs were examined for HBV infected cells utilizing an immunofluorescent technique. Numerous fluorescent cells which reacted with human anti-HBs nad anti-HBc were observed in the following organs: pancreas, liver, kidney, and brain. Histopathologically, edematous changes in hepatocytes and minor cellular infiltration attributed to an inflammatory response were noted. Liver and kidney cells from the infected animals were cultured, and HBV antigen positive cells for HBsAg and HBcAg were detected in the cultures. Throughout the experiment, HBsAg was detected in the supernatant by ELISA. Virus particles which were indistinguishable from Dane particles were seen in the cytoplasmic vacuoles of the cultured cells by electron microscopy. Finally, the presence of HBV DNA was established by molecular hybridization techniques in the culture supernatants of kidney cells from the infected turtles.
Animal ; Hepatitis B/microbiology/transmission/*veterinary ; Hepatitis B Antibodies/isolation and purification ; Hepatitis B Core Antigens/isolation and purification ; Hepatitis B Surface Antigens/isolation and purification ; Human ; Kidney/microbiology ; Liver/microbiology/pathology ; Turtles/*microbiology

BACKGROUNDS/AIMS: Occult HBV infection is characterized by the presence of HBV infection with undetectable HBsAg. This study was carried out to find out the frequency of HBV infection in HBsAg- negative patients. METHODS: Fifty-six HBsAg-negative patients including 17 anti-HCV positive patients were evaluated. Patients were grouped according to their serological status; group A (anti-HBc+, anti-HBs-, n=16), B (anti-HBc+, anti-HBs+, n=26), and C (anti-HBc-, anti-HBs+/-, n=14). DNA was extracted from frozen liver biopsy specimen, and HBV DNA level was measured with real-time PCR. RESULTS: Overall frequency of detectable intrahepatic HBV DNA was 34% (19/56). The frequency was 56% (9/16) in group A, 31% (8/26) in group B and 14% (2/14) in group C (P=0.01). Intrahepatic HBV DNA levels were as follows; 2,010 +/- 6,660 copies/mg in group A, 6,180 +/- 29,530 copies/mg in group B and 350 +/- 1,220 copies/mg in group C. The frequency of occult HBV infection was not increased in anti-HCV positive patients. CONCLUSIONS: Intrahepatic HBV DNA is frequently detected in anti-HBc positive, HBsAg-negative patients, although the concentration is low.
Middle Aged ; Male ; Liver/*virology ; Humans ; Hepatitis B virus/*genetics/isolation & purification ; Hepatitis B Surface Antigens/*analysis ; Hepatitis B/*diagnosis/immunology/virology ; Female ; DNA, Viral/*analysis ; Aged ; Adult

<b>BACKGROUNDb>To establish a new assay for detecting the quantity of HBV DNA with PCR and enzyme-linked immunosorbent assay(ELISA).

<b>METHODSb>The products of PCR using primers pre-labeled with biotin were hybridized with the capture probes that were immobilized on the microtiter strips and then bond with Sav-Ap. The quantity of DNA was detected by measuring the yellow color at 405 nm wave length.

<b>RESULTSb>Totally 125 sera from patients with hepatitis B were tested for HBV DNA by this method,the sera were also tested for HBV immunological markers by solid phase radio immuno-assay (SPRIA). The HBV positive rate with PCR-ELISA was 64.9% (24/37) in samples which were positive for HBsAg, HBeAg and HBcAb; and 34.2% (13/38) in sera which were positive for HBsAg, HBeAb and HBcAb; in sera positive for HBsAg and HBcAb or only HBcAb, the positive rate was 6.7% (1/15) and 5.9% (2/34) respectively.

<b>CONCLUSIONSb>The PCR ELISA assay is simple and suitable for clinical laboratory in quantitative determination of HBV DNA.

DNA Probes ; DNA, Viral ; analysis ; biosynthesis ; genetics ; Enzyme-Linked Immunosorbent Assay ; Hepatitis B ; virology ; Hepatitis B Antibodies ; blood ; Hepatitis B Surface Antigens ; blood ; Hepatitis B e Antigens ; blood ; Hepatitis B virus ; genetics ; isolation & purification ; Humans ; Polymerase Chain Reaction ; Sensitivity and Specificity

DNA, Viral ; blood ; Female ; Hepatitis B ; immunology ; virology ; Hepatitis B Antibodies ; blood ; Hepatitis B Core Antigens ; immunology ; Hepatitis B Surface Antigens ; immunology ; Hepatitis B virus ; genetics ; isolation & purification ; Humans ; Male ; Polymerase Chain Reaction ; methods

DNA, Viral ; blood ; Drugs, Chinese Herbal ; therapeutic use ; Hepatitis B Surface Antigens ; blood ; Hepatitis B e Antigens ; blood ; Hepatitis B virus ; isolation & purification ; Hepatitis B, Chronic ; drug therapy ; Humans ; Phytotherapy