Proliferative enteropathy was reproduced in IFN-gamma receptor knockout (IFN-gamma R-) mice by experimental infection with Lawsonia intracellularis (L. intracellularis). The cecum and the colon of the infected mice were evidently enlarged 2 weeks post infection. The presence of L. intracellularis was identified in the stool and the cecum of the mice after infection. However, high levels of IFN-gamma were detected in the sera of the infected mice 2 weeks PI. These data indicated that the IFN-gamma produced in the infected mice should have been utilized by it's receptor to elicit protective immune responses against L. intracellularis infections.
Animals ; DNA, Viral ; Desulfovibrionaceae Infections/*immunology/microbiology ; Interferon Type II/*immunology ; Intestinal Diseases/*immunology/microbiology ; Intestinal Mucosa/immunology ; Lawsonia Bacteria/*immunology/isolation&purification ; Mice ; Mice, Knockout ; Polymerase Chain Reaction ; Receptors, Interferon/physiology

OBJECTIVES: DNA polymerase (pol) of Hepatitis B virus (HBV) includes 3 different domains such as terminal protein (TP), reverse transcriptase (RT) and RNase H. Humoral immune responses to each of these proteins have not been well documented previously, although antibody to pol was detected in serum of patients with chronic hepatitis B. We have constructed TP (amino acids 1-182), RT (amino acids 346-685) and RNase H (amino acids 690-832). METHODS: By ELISA using each protein expressed in E. coli as antigens, the corresponding antibodies were tested in serum from 40 patients with type B viral chronic liver diseases. (20 HBeAg-positive and 20 HBeAg-negative). As negative controls, sera from 3 healthy young men were used. With the mean values of the OD, which were tested 4 times per each test sample and 3 times per each control sample, we considered to be positive if the mean OD of each test sample is 2-fold or higher than that of controls. RESULTS: Five of 40 sera (12.5%) contained one or two different antibodies detectable by this method: 4 of 20 HbeAg-positive sera (20%) and 1 of 20 HbeAg-negative sera (5%). Anti-TP, anti-RT and anti-RNase H antibodies were detected in 2.5% (1/40), 10% (4/40) and 7.5% (3/40), respectively. Among 4/20 HbeAg-positive ELISA-positive sera, anti-TP, anti-RT and anti-RNase H were positive in 5% (1/20), 20% (4/20) and 10% (2/20), respectively, while 1 HBeAg-negative ELISA-positive sera were positive only for anti-RNase H. CONCLUSIONS: These results suggest that the corresponding antibody responses to individual recombinant peptides derived from 3 domains of DNA polymerase may tend to be detected more frequently in HBeAg-positive sera than in HBeAg-negative sera from various patients with type B viral chronic liver diseases.
Adult ; Antibodies, Antinuclear/analysis* ; Biological Markers/analysis ; DNA Polymerase II/immunology* ; Enzyme-Linked Immunosorbent Assay ; Female ; Hepatitis B Surface Antigens/analysis* ; Hepatitis B Virus/immunology* ; Hepatitis B, Chronic/immunology* ; Human ; Male ; Middle Age ; Odds Ratio ; Reference Values ; Substances: DNA Polymerase II ; Substances: Hepatitis B Surface Antigens ; Substances: Biological Markers ; Substances: Antibodies, Antinuclear

OBJECTIVETo develop a simple method for genotyping of hepatitis E virus (HEV) and to investigate HEV genotype distribution in Nanjing area.

METHODSTwenty-seven full HEV sequences currently-available in GenBank were analyzed with MegAlign and MapDraw programs of DNA STAR software. Degenerate primers were designed and applied to amplify a fragment in HEV ORF1 region. HEV genotypes were determined by the size of the PCR products and by single restriction endonuclease analysis.

RESULTSThe PCR products of HEV genotype 1 and 2 were 275 bp and 269 bp in size. Distinctively, the PCR products of genotype 3 and 4 were 317 bp and 314 bp in size. Moreover, the PCR products of genotype 1 could be digested by Nae 1, but the products of genotype 2 could not. Distinctively, the PCR products of HEV genotype 3 could be digested by Not 1, but the products of genotype 4 could not. Six HEV reference strains standing for different HEV genotypes were clustered into their own types as predicted. Within 43 HEV IgM-positive clinical specimens collected in Nanjing, 19 were HEV PCR-positive and identified as genotype 4.

CONCLUSIONA simple method of PCR combined with single restriction endonuclease analysis is developed for HEV genotyping. This assay allows rapid identification of a large number of HEV isolates directly from clinical specimens. Among patients with hepatitis E in Nanjing, most were infected with HEV genotype 4.

DNA Restriction Enzymes ; metabolism ; DNA, Complementary ; genetics ; metabolism ; Deoxyribonucleases, Type II Site-Specific ; metabolism ; Genotype ; Hepatitis E ; blood ; genetics ; immunology ; Hepatitis E virus ; genetics ; Humans ; Polymerase Chain Reaction ; methods ; RNA, Viral ; genetics ; Reverse Transcriptase Polymerase Chain Reaction

Human Prostate Specific Membrane Antigen(PSMA) cDNA was amplified using total RNA extracted from prostate carcinoma tissue by RT-PCR. The cDNA fragment of extracellular domain of PSMA(edPSMA) gene was amplified by PCR and cloned into expression vector pMAL-c2x. Sequence analysis of both PSMA and edPSMA revealed identity to the GenBank reported. The edPSMA was expressed in E. coli as part of a fusion protein with MBP as the induction of IPTG. Western blot analysis showed the recombinant protein could react with PSMA monocloned antibodies 4G5. MBP-edPSMA fusion protein were purified by amylose resin affinity chromatography and showed to be homogeneity in SDS-PAGE(120 kD). BALB/C mice were immunized with the purified protein for the preparation of polyclonal antibody. The polyclonal antibody, which had a title of 1:12,800, were indicated the specificity to prostate tissue.
Animals ; Antibodies ; immunology ; Antibody Formation ; Antigens, Surface ; Carboxypeptidases ; biosynthesis ; genetics ; immunology ; isolation & purification ; Chromatography, Affinity ; methods ; Cloning, Molecular ; DNA, Complementary ; genetics ; Escherichia coli ; genetics ; Gene Expression ; Genetic Vectors ; Glutamate Carboxypeptidase II ; Humans ; Mice ; Mice, Inbred BALB C ; Protein Structure, Tertiary ; genetics ; physiology ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; isolation & purification ; Reverse Transcriptase Polymerase Chain Reaction ; instrumentation