In view of the potential of replicase protein as a diagnostic reagent for human caliciviruses (HuCVs), we have cloned and over-expressed this gene from the Snow Mountain-like Korean strains in Escherichia coli as a fusion protein with glutathione S-transferase (GST, and described the preliminary antigenic characterization of the recombinant products. Each 470bp fragment corresponding to highly conserved region of RNA-dependent RNA polymerase was generated by RT-PCR from stools of two diarrheal children, cloned in pMOSBlue T-vector, and subcloned between the EcoRI and SalI restriction sites of pGEX-47-3, a GST gene fusion vector, yielding pGCVpol. This construct expressed a Snow Mountain-like HuCV replicate under the control of the IPTG-inducible pac promoter. An extract prepared by sonication of the E. coli cell inclusion bodies bearing pGCVpol products was purified and analyzed by SDS-PAGE. After Coomassie blue staining, it was shown that the recombinant replicase migrated on the gels with an approximate molecular mass of 46.5 kDa, that was subsequently cleaved into a 26 kDa GST fragment and a 20.5 kDa replicase protein upon digestion with thrombin protease. The replicase was recognized on immunoblotting with the sera from symptomatic children with the HuCV-associated diarrhea but not by asymptomatic sera from adults. The results presented the first biological activity of individually expressed HuCV replicase subunit and provided important reagents for diagnosis of HuCV infection.
Adult ; Child ; Clone Cells ; Diagnosis ; Diarrhea ; Digestion ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; Gels ; Gene Fusion ; Glutathione Transferase ; Humans ; Immunoblotting ; Inclusion Bodies ; Indicators and Reagents ; RNA Replicase ; Snow* ; Sonication ; Staphylococcal Protein A ; Thrombin

Most of the current licenced hepatitis B vaccines are being produced by recombinant DNA technology in large fermentation cultures of Saccharomyces cerevisiae of yeast cells which carry the gene coded for hepatitis B virus surface antigen. These vaccines are proved very effective clinically and the immunogenicity of vaccines could be maintained for a long time under refrigeration. To develope the stabilizer that could increase the stability of hepatitis B virus vaccine which could be stored for a long period at room temperature or higher conditions, glucose, lactose and sucrose solutions in phosphate buffered saline were added into hepatitis B vaccine respectively to make 2.5%, 5%, 7.5% and 10% final concentration in vaccines. These sugar-vaccine mixtures were stored at room temperature for one month, two months and three months respectively and then inoculated into ICR mice intramuscularly. On the fourteenth day after inoculation, mice were bled and sera were tested for the evaluation of efficacies of vaccines. The results showed that 5% glucose, 7.5% lactose and sucrose increased the stability of vaccines in some degree and this method could be applied for the production of other viral vaccines and bacterial vaccines.
Animals ; Antigens, Surface ; Bacterial Vaccines ; Carbohydrates* ; DNA, Recombinant ; Fermentation ; Glucose ; Hepatitis B Vaccines ; Hepatitis B virus* ; Hepatitis B* ; Hepatitis* ; Lactose ; Mice ; Mice, Inbred ICR ; Refrigeration ; Saccharomyces cerevisiae ; Sucrose ; Vaccines ; Viral Vaccines ; Yeasts

We evaluated Immunochromatographic assay kit to screen HBsAg in human serum. When the reference HBsAg was applyed to ICA, HA and EIA kits, the limit of detection for HBsAg were found out to be 4, 2 and 0.25 ng/ml respectively. But ICA kit required 5 minutes to read the result whereas HA and EIA kit more than one hour. The sensitivity was 97% (29 of 30 samples) and the specificity 100% (45 samples) compared with conventional EIA. The ICA kit needs no instrument or machine to perform the test contrary to the conventional methods. Therefore, this rapid and sensitive ICA kit can be used for HBsAg-screening, especially in the emergency room and in the scene of the accident.
Emergency Service, Hospital ; Hepatitis B Surface Antigens* ; Hepatitis B* ; Hepatitis* ; Humans ; Immunochromatography* ; Limit of Detection ; Sensitivity and Specificity

Serum samples from 123 males and 123 females collected by age in 1996 were analyzed for antibodies against surface antigen of Hepatitis B virus and C22-3, C200 antigens of Hepatitis C virus. Sera from the children under the age of 10 showed 30% seropositivity to the surface antigen of Hepatitis B virus, 33.3% in 10~19 year group, 20% in 20~29 year group, 17.6% in 30~39 year group, 3.3% in 40~49 year group, 5.9% in 50~59 year group, 8,3% in 60~69 year group, 2.9% in 70~79 year group, but antibody could not found in 80~86 year group. 12 out of 123 male sera were positive, 19 out of 123 female sera were positive and overall rate of positivity of antibody against surface antigen of Hepatitis B virus was 12.6%. Serum samples from peoples under the age of 30 had not antibody against C22-3, C200 antigens of Hepatitis C virus. The positivity rate was 2.9% in 30~39 year group. 5 out of 30 sera from 40~49 year age group were positive, and 3 positive sera showed extremely high titer (1:524,288) but the titers of two remaining sera were 1:32, 1:8,192 respectively. 5.9% was positive in 50~59 year group, 8.3% in 60~69 year group, 11.8% in 70~79 year group but all negative in 80~86 yea. group. 6 out of 123 male sera were positive (4.9%), 9 out of 123 female sera were positive (7.3%). Overall .ate of positivity of antibody against C22-3, C200 antigen of Hepatitis C virus was 6.1%. None out of 246 sera had both antibodies against Hepatitis B virus and Hepatitis C virus.
Antibodies* ; Antigens, Surface ; Child ; Female ; Hepacivirus* ; Hepatitis B virus* ; Hepatitis B* ; Hepatitis C* ; Hepatitis* ; Humans* ; Male

Three cDNA fragments located within NS5 region of HCV were synthesized by RT using viral RNA extracted from blood sample of Korean patient as a template. The cDNAs were amplified by PCR, cloned into the T-vector, and the nucleotide sequences were determined. Comparative a analysis of the nucleotide and amino acid sequence of NS5 cDNAs showed that it is closely related with HCV type 1b. The cloned NS5 cDNA showed 91-94% homology at the nucleotide sequence level and 96-98% homology at the amino acid sequence level with several strains of the HCV type 1b. The NS5 cDNAs were subcloned into E. coli expression vectors to construct pRSETA5-1, pTHAN5-1, pRSETC5-2, pRSETBB1, pRESTCB1 and PRSETB-H3. Expression of the NS5 proteins was achieved by inducing the promoter with isopropyl-thio-P-D-galactoside (IPTG) and confirmed by SDS-polyacrylamide gel electrophoresis. The NS5 proteins were immunoreactive against sera from Korean hepatitis C patients in Western blot analysis. Among the recombinant NS5 proteins, pRSETA5-1 plasmid derived protein, coded from aa2022 to aa2521 of HCV polyprotein, showed the strongest immunoreactivity against sera from Korean hepatitis C patients in immunoblot analysis. These results suggest that NS5 proteins would be useful as an antigen for detection of antibody against HCV in the blood samples.
Amino Acid Sequence ; Base Sequence ; Blotting, Western ; Clone Cells* ; Cloning, Organism* ; DNA, Complementary ; Electrophoresis ; Hepacivirus* ; Hepatitis C* ; Hepatitis* ; Humans ; Plasmids ; Polymerase Chain Reaction ; RNA, Viral

The truncated E1192-283 and E2384-649 genes of hepatitis C virus (HCV) linked to the gene for glutathione 5-transferase (GST) were constructed and their expressions were analyzed. The GST-E1192-283 fusion gene overexpressed the fusion protein in E. coli as a soluble form, while the GST-E1192-383 plasmid did not express expected fusion protein. The purified GST-E1192-283 fusion protein was efficiently cleaved by thrombin. More than 90% pure, HCV E1192-283 protein was obtained by GST-agarose chromatography. The truncated GST-E2384-649 fusion gene expressed the fusion protein mainly as an insoluble form, whereas the GST-E2384-740 did not express the fusion protein. The truncated GST-E1 182-283 and GST-E2384-649 fusion proteins reacted specifically with an HCV patient serum. In addition, mice immunized with either the purified E1192-283 or GST-E2384-649 proteins generated specific antibodies to each antigen. The results suggested that hydrophobic carboxyl portions of the E1 and E2 proteins might affect expression levels as well as the solubility of each fusion protein in bacteria. Also, the truncated E1 protein with Tyr-192 to Ser-283 contained antigenic epitope(s) which could be specifically recognized by an HCV patient serum.
Animals ; Antibodies ; Bacteria ; Chromatography ; Glutathione ; Hepacivirus* ; Hepatitis C* ; Hepatitis* ; Humans ; Mice ; Plasmids ; Solubility ; Thrombin

No Abstract Available.
HIV-1* ; Transcription Factors*

No Abstract Available.
Animals ; Hantavirus* ; Indonesia* ; Murinae* ; Thailand*

No Abstract Available.
Animals ; Base Sequence* ; Cattle* ; Serotyping*

No Abstract Available.
Adenoviridae* ; Coinfection* ; Herpesvirus 3, Human* ; Humans ; Synovial Fluid* ; Synovitis*