Antigens, Bacterial ; immunology ; Antigens, Surface ; immunology ; China ; epidemiology ; Diarrhea ; microbiology ; Escherichia coli Infections ; epidemiology ; microbiology ; Escherichia coli O157 ; classification ; immunology ; isolation & purification ; Humans

OBJECTIVETo establish an improved procedure for isolation and identification of epitopes from a random peptide library displayed on the bacterial surface.

METHODSEpitopes were screened from FliTrx random peptide library by a monoclonal antibody 3B9 against HBV-PreS2 protein. The enrichment was monitored in each round. Higher affinity clones were obtained by increasing the washing strength and randomly selected for sequencing and Western blot analysis.

RESULTSClones specifically binding to antibody were enriched in each round. Ten sequences were obtained from sixteen sequenced clones, seven of them contained the common motif RXRGXY with high homogeneity to 135-140 amio acids in HBV-PreS protein and have positive results in Western blot analysis. The other three sequences have no typical motif RXRGXY and showed different Western blot results.

CONCLUSIONSIt's easy and quick to drive epitopes from a random peptide library displayed on the bacterial surface.

Amino Acid Sequence ; Animals ; Antigens, Viral ; immunology ; Bacterial Proteins ; immunology ; Flagellin ; immunology ; Hepatitis B Surface Antigens ; genetics ; immunology ; isolation & purification ; Immunodominant Epitopes ; immunology ; Molecular Sequence Data ; Peptide Library ; Protein Precursors ; genetics ; immunology ; isolation & purification

Three different chemical carcinogens, 2-acetylaminofluorene (AAF), diethylnitrosamine(DENA), and 3'-methyl-4dimethylaminoazobenzene (3'-Me DAB) were used to induce hepatomas in rats. Plasma membrane surface proteins of normal rat liver cells and rat hepatomas were extracted with 3M KCI. From the analysis of the proteins of normal rat liver and rat hepatoma induced by 3'-Me DAB by discontinuous polyacrylamide gel electrophoresis(Disc-PAGE), under nonreducing and nondenaturing conditions polyacrylamide gel electrophoresis in the presence of SDS and 2-mercaptoethanol (SDS-PAGE), Sephadex G-200 gel permeation chromatography, DEAE-A50 ion-exchange chromatography and two-dimensional gel electrophoresis, at least three tumor specific antigens were identified. One had a molecular weigh of 66,000 (pl=6.79) while the other two had the same molecular weight 73,000 but differed in their isoelectric points (7.58 and 7.81). For immunological analysis of tumor specific antigens, the absorbed antiserum was prepared. Plasma membrane surface proteins of rat hepatoma induced by 3'-Me DAB were used to obtain New Zealand White male rabbit antiserum. Rabbit antiserum was then reacted with the proteins isolated from the plasma membrane surface of normal rat liver and the absorbed antiserum reacting specifically with the tumor specific antigens derived by 3'-Me DAB was obtained. Using the absorbed antiserum, the immunoreactivities of plasma membrane surface proteins isolated from rat hepatomas induced by 3'-Me DAB, AAF, and DENA were compared by Ouchterlony double immunodiffusion analysis and immunoelectrophoresis. To characterize the proteins reacting to the absorbed antiserum, immunoglobulin G was separated from the absorbed antiserum and coupled to cyanogen bromide-activated Sepharose CL-4B. The isolated proteins from the plasma membrane surface proteins of 3'-Me DAB-induced hepatoma using this immunoaffinity chromatography had molecular weights of 66,000 and 73,000. The localization of these proteins on surface plasma membranes of rat hepatomas induced by 3'-Me DAB was confirmed by an immunofluorescence technique. The experimental results revealed the existence of cross-reacting common antigens on the plasma membrane surface of rat hepatomas induced by different hepatocarcinogens.
2-Acetylaminofluorene ; Animal ; Antigens, Neoplasm/*isolation and purification ; Antigens, Surface/isolation and purification ; Diethylnitrosamine ; Liver Neoplasms, Experimental/chemically induced/*immunology ; Methyldimethylaminoazobenzene ; Rats ; Rats, Inbred Strains ; Support, Non-U.S. Gov't

In summarizing the results of the experimental studies up to the present, it is conjectured that the pathogenicity of Entamoeba histolytica or establishment of amoebiasis is not unique but differs by strain and age of Entamoeba histolytica and the age of the host. A non-virulent strain is more likely adapt to as low a temperature as 32 . This is not so in the strains which originated form clinical cases. Iso-enzyme patterns may roughly characterize pathogenic strains from non-pathogenic, Red blood cells may contribute as nutrients for growth of Entamoeba histolytica only after they have been hemolysed, but they are toxic to the amoebae as long as they remains intact. A low protein diet and stress may facilitate the establishment of amoebiasis; male sex hormones or previous infection by enteric bacteria provide a more advantageous condition than the female; and hepatotoxic agents will accelerate amoebic hepatitis.
2-Acetylaminofluorene ; Animal ; Antigens, Neoplasm/*isolation and purification ; Antigens, Surface/isolation and purification ; Diethylnitrosamine ; Liver Neoplasms, Experimental/chemically induced/*immunology ; Methyldimethylaminoazobenzene ; Rats ; Rats, Inbred Strains ; Support, Non-U.S. Gov't

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

OBJECTIVETo develop a technology for production of recombinant SAG1 of Toxoplasma gondii (T.g) in batches.

METHODSThe rSAG1 of T.g was expressed in E.coli by high-density fermentation and purified by Sephadex G-75 column chromatography after Ni-NTA agarose at native condition. The activity of rSAG1 and its efficacy in T.g diagnosis were identified by Western blotting and ELISA, respectively.

RESULTSThe optical density (OD) of the bacteria reached 20.21 after induction, and 300 g bacteria were harvested from 11.5 L broth. The rSAG1 was highly expressed in E.coli as a fusion protein, accounting for about 25.82% of the total bacterial protein. The purity of rSAG1 reached 98.54% after purification by Ni-NTA combined with Sephadex G-75 column chromatography. Western blotting revealed a distinct band reacting with the sera of rabbits vaccinated by T.g. Twenty-four of the 25 sera of mice infected with T.g and 36 of the 38 sera of human subjects with IgG antibody against T.g were detected by rSAG1-ELISA.

CONCLUSIONA large-scale production of immunoreactive SAG1 of T.g is developed by high-density fermentation and purification with Ni-NTA combined with Sephadex G-75 column chromatography.

Animals ; Antigens, Protozoan ; biosynthesis ; genetics ; immunology ; Antigens, Surface ; immunology ; Blotting, Western ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; genetics ; metabolism ; Fermentation ; Protozoan Proteins ; biosynthesis ; genetics ; immunology ; Recombinant Fusion Proteins ; biosynthesis ; immunology ; isolation & purification ; Toxoplasma ; immunology

OBJECTIVETo investigate the presentation of a neutralization epitope-containing peptide antigen of hepatitis E virus (HEV) on chimeric virus-like particles (VLPs) of hepatitis B surface antigen (HBsAg).

METHODSThe gene fragment corresponding to amino acids (aa) 551-607 (HEnAg) of HEV capsid protein, which contains the only neutralization epitope identified to date, was fused via a synthetic glycine linker in frame with the gene of HBsAg. The resulted fusion gene was then integrated through transformation into the genome of Pichia pastoris under the control of a methanol-induced alcohol oxidase 1 (AOX1) promoter and expressed intracellularly. The expression products in the soluble cell extracts were characterized by Western blot, ELISA, CsCl density gradient analysis, and electron microscopic visualization.

RESULTSThe novel fusion protein incorporating HBsAg and the neutralization epitope-containing HEnAg was expressed successfully in Pichia pastoris with an expected molecular weight of approximately 32 kD. It was found to possess the ability to assemble into chimeric HBV/HEV VLPs with immunological physical and morphological characteristics akin to HBsAg particles. Not only did the chimeric VLPs show high activity levels in a HBsAg particle-specific ELISA but they were also strongly immunoreactive with hepatitis E (HE) positive human serum in a HEV specific ELISA, indicating that HEnAg peptide fragments were exposed on VLP surfaces and would be expected to be readily accessible by cells and molecules of the immune system. Similarity between chimeric VLPs to highly immunogenic HBsAg particles may confer good immunogenicity on surface-displayed HEnAg.

CONCLUSIONThe chimeric HBV/HEV VLPs produced in this study may have potential to be a recombinant HBV/HEV bivalent vaccine candidate.

Epitopes ; Genetic Engineering ; Hepatitis Antigens ; biosynthesis ; genetics ; immunology ; Hepatitis B Surface Antigens ; biosynthesis ; genetics ; immunology ; Hepatitis B virus ; genetics ; immunology ; Hepatitis E virus ; genetics ; immunology ; Pichia ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification ; Vaccines, Synthetic

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

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

Human hepatitis B virus surface antigen (HBsAg) binding protein(SBP) shows a specific binding ability to HBV surface antigen HBsAg. Previous work proved an ability of SBP to enhance the immune response of HBsAg vaccine. To investigate the function and mechanism of this protein, we constructed SBP-expression strains with Pichia pastoris expression system. We screened these strains and have got an expression strain with high protein expression quantity. Fermentation product was collected and purified to gain a large amount of purified protein. Identification of purified SBP with SDS-PAGE, High performance liquid chromatography, Western blotting and mass spectrometry suggested that the protein was highly purified and with a good integrity. ELISA test of purified SBP showed a significant binding ability to HBsAg, suggesting a good protein activity. This work offers a solid foundation to the research of SBP function and mechanism of immune enhancement.
Binding Sites ; Genetic Vectors ; genetics ; metabolism ; Hepatitis B Surface Antigens ; metabolism ; Humans ; Pichia ; genetics ; metabolism ; Receptors, Virus ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; isolation & purification