ABSTRACT
Serovars of 22 leptospiral field isolates from rats trapped in Korea were identified by cross-agglutinin absorption test (CAAT). Genomic characteristics of 7 selected isolates and 6 antigenically closely related reference serovars of lai, yeonchon, birkini, gem, mwogolo, and canicola were differentiated by arbitrarily primed PCR (AP-PCR) and southern blot hybridization using 16S rRNA gene probe from Borrelia burgdorferi. Among the 22 isolates, 21 strains were identified as serovar lai by CAAT, while the serological reactivity of NR13 did not accord with that of serovar lai. Results of AP-PCR using primers RSP, KF and PB-1 were in general agreement with those obtained by serological identification, and all 7 isolates including NR13 showed the same profile with serovar lai or yeonchon. In the southern blot hybridization with 16S rRNA gene probe, the isolates were divided into two ribotype groups when HindIII and BamHI digests were employed: isolates NR4, NR13, and serovar lai showed the same profile, and isolates JR34, JR57, KR48, JR77, and JR82 were classified as the another ribotype group. Isolate NR13 and serovar yeonchon, which were isolated in Korea and showed serological differences with serovar lai, were indistinguishable from serovar lai in this DNA study using AP-PCR and ribotyping. These results demonstrate that Korean leptospiral isolates were closely related in DNA level, and ribotyping would be useful for subgrouping of field isolates.
Subject(s)
Animals , Rats , Absorption , Blotting, Southern , Borrelia burgdorferi , DNA , Genes, rRNA , Korea , Leptospira , Polymerase Chain Reaction , RibotypingABSTRACT
BACKGROUND: We developed a Pseudomonas aeruginosa outer membrane protein(OMP) vaccine, CFC-101, and the prophylactic efficacy of which has been demonstrated in animal models. In order to evaluate the safety and immunogenicity of the P. aeruginosa vaccine, we carried out a phase I/IIa clinical trial in healthy male volunteers. METHODS: Groups of eight volunteers, including two placebo subjects, were vaccinated intramuscularly with three doses of 0.25, 0.5 or 1.0 mg of the vaccine at one week intervals. Signs of systemic and local reactions observed after vaccination were recorded for each vaccinee for 5 days. Physical examinations were performed on days 0, 1, 7, 8, 14, 15, 21, and 42, and clinical laboratory tests were done on days 0, 3, and 21. Blood samples for assay of serum antibody levels were obtained up to 42 days after the first vaccination. RESULTS: The vaccine was generally well tolerated by all vaccinees, showing no significant side effects. In the three dosage groups, all vaccinees, except one receiving the 0.25 mg dose, showed significant elevation in serum IgG antibody titers against the vaccine proteins, indicating 100% seroconversion in 0.5 and 1.0 mg groups. The human antibodies induced by the vaccine were specific for P. aeruginosa OMPs, as confirmed by western blot analysis and immunoprecipitation assays. The capacity of the human antisera to enhance opsonophagocytic killing activity by polymorphonuclear leukocytes and to confer protection against P. aeruginosa infections indicates that the antibodies elicited by the vaccine have protective efficacy. CONCLUSION: We conclude that the P. aeruginosa OMP vaccine is safe and effective for human use and its optimal dose to be 0.5 or 1.0 mg.
Subject(s)
Humans , Male , Antibodies , Blotting, Western , Homicide , Immune Sera , Immunoglobulin G , Immunoprecipitation , Membrane Proteins , Membranes , Models, Animal , Neutrophils , Physical Examination , Pseudomonas aeruginosa , Pseudomonas , Vaccination , VolunteersABSTRACT
Human papillomavirus (HPV) 16, E7 proteins derived from the prototype (Bac73) and natural variant (Bac101) E7 open reading frame were produced in Sf9 insect cells. The variant E7 gene occurred naturally by substitution mutation at the position of 88 nucleotide, resulting serine instead of asparagine. Using E7 specific monoclonal antibody (VD6), both E7 proteins were identified in recombinant baculovirus infected SF9 cells. Radiolabelling and immunoprecipitation analysis revealed that both E7 proteins were phosphoproteins. Immunostaining result showed that E7 proteins were mainly localized in the cytoplasm. Nuclear form of E7 proteins was also detected after a sequential fractionation procedure for removing chromatin structure. Considering that the VD6 recognition site in E7 protein is located within 10 amino acid at the N-terminus, this region appears to be blocked by the nuclear component. Western blot analysis revealed that nuclear form was more abundant than cytoplasmic E7 proteins. Time course immunostaining showed that the primary location of E7 protein was the nucleus and exported to the cytoplasm as proteins were accumulated. These events occurred similarly in both Bac73 and Bac101 infected Sf9 cells, suggesting that these two proteins may have similar biological functions.
Subject(s)
Humans , Asparagine , Baculoviridae , Blotting, Western , Chromatin , Cytoplasm , Immunoprecipitation , Insecta , Open Reading Frames , Phosphoproteins , Serine , Sf9 CellsABSTRACT
The type-specific PCR and the sequence analysis of 56 kDa gene of Orientia tsutsugamushi infected in field rodents specimens have shown intratypic genetic heterogeneity in genotype Karp. In sequence comparison, this genetic heterogeneity was mainly due to insertion or deletion of a repeated unit in variable domain I (VDI) region. These results suggested that genetic duplication or deletion of the specific sequence rnight be involved in intratypic genetic heterogeneity of Orientia tsutsugamushi.
Subject(s)
Genetic Heterogeneity , Genotype , Orientia tsutsugamushi , Polymerase Chain Reaction , Rodentia , Sequence AnalysisABSTRACT
The 86 strains of field rodents captured in Chollanamdo were analyzed its infection rates of Orientia tsutsugamushi by nested PCR. The detection rate of O. tsutsugamushi was 14.3 % in A. agrarius whereas O. tsutsugamushi could not be detected in C. lasiura. In locality, the rodents captured in the mountainous area showed higher detection rate than suburban area. In the case of detection rate by organs, the spleen was most appropriate specimen, but in two cases, O. tsutsugamushi could be detected in only kidney specimens. The major serotype of detected O. tsutsugamushi was serotype Karp, but four cases were serotype Boryong. These serotypes were confirmed by nucleotide sequence determination of amplified PCR products. In conclusion, the serotype Karp was the major O. tsutsugamushi in Chollanamdo.
Subject(s)
Base Sequence , Kidney , Orientia tsutsugamushi , Polymerase Chain Reaction , Rodentia , SpleenABSTRACT
The gene encoding E7 oncoprotein of human papillomavirus type 16 was cloned and expressed in Escherichia coli, and the monoclonal antibodies (Mabs) against this expressed protein were generated. For the efficient immunization, two kind of recombinant E7 protein in fusion form were produced. One was maltose binding protein (MBP) fusion type (MBP-E7) and the other was T7 phage gene 10 product fusioa type (gene 10-E7). Immunization with these two fusion protein to mice, finally two Mabs (VD6 and IB10) were obtained. VD6 and IB10 showed reactivities with E7 protein in CaSki cell but not in HeLa by Western blot analysis. In addition, the Mab, VD6, reacted with COS-7 cell transfected with E7 gene majorly in cytoplasm by immunofluorescence test. Also VD6 could detect E7 protein in cytoplasm and nucleus of CaSki ceU by immunogold electron microscopy. Based on these results, the Mab VD6 was could be used for various E7 detection system such as Western blot analysis and immunohistochemical methods.
Subject(s)
Animals , Humans , Mice , Antibodies, Monoclonal , Bacteriophage T7 , Blotting, Western , Clone Cells , COS Cells , Cytoplasm , Escherichia coli , Fluorescent Antibody Technique , Immunization , Maltose-Binding Proteins , Microscopy, ElectronABSTRACT
No abstract available.
Subject(s)
Humans , Animals , Antibodies, Bacterial/blood , Antigens, Bacterial/blood , Mites/microbiology , Orientia tsutsugamushi/classification , Scrub Typhus/diagnosisABSTRACT
No abstract available.
Subject(s)
Humans , Antibodies , Chlamydia trachomatis , Chlamydia , Neisseria gonorrhoeae , Neisseria , PrevalenceABSTRACT
No abstract available.
Subject(s)
Humans , Antibodies , Chlamydia trachomatis , Chlamydia , Neisseria gonorrhoeae , Neisseria , PrevalenceABSTRACT
Twenty-four monoclonal antibodies were produced by immunizing BALB/c mice with Rickettsia tsutsugamushi Boryong strain and used for the analysis of antigenic characteristics of R.tsutsugamushi Boryong strain and antigenic heterogeneity of R.tsutsugamushi by indirect immunofluorescent(IF) test. R. tsutsugamushi Kato, Karp, Gilliam, TA686, TA716, TA763, TC586, TH1817, and Boryong were used for the analysis of antigenic heterogeneity of R.tsutsugamushi. Five monoclonal antibodies were reactive with 27-kDa protein, four monoclonal antibodies were reactive with 47-kDa protein, and eight monoclonal antibodies were reactive with 56-kDa protein of R.tsutsugamushi Boryong strain. The reactive protein of seven monoclonal antibodies could not be identified by immunoblotting method. All monoclonal antibodies to 27-kDa protein and three monoclonal antibodies to 47-kDa protein, and five monoclonal antibodies to 56-kDa protein were reactive with three to eight strains among nine strains of R. tsutsugamushi tested. One monoclonal antibody reactive to 47-kDa protein(KI18) and two monoclonal antibodies reactive to 56-kDa protein(KI36, and KI37) reacted with all the strains of R. tsutsugamushi tested. Strain-specific monoclonal antibody(KI58) could be found among antibodies which were reactive with 56-kDa protein. There was no strain which showed same reactivity pattern to these 24 monoclonal antibodies among nine strains. From this results, it could be concluded that Boryong strain is antigenically different from other strains of R.tsutsugamushi and antigenic heterogeneity of R.tsutsugamushi is due to the antigenic diversity of several proteins of R. tsutsugamushi including 56-kDa protein.
Subject(s)
Animals , Mice , Antibodies, Monoclonal/immunology , Antigens, Bacterial/analysis , Bacterial Proteins/analysis , Mice, Inbred BALB C , Orientia tsutsugamushi/immunology , Species SpecificityABSTRACT
No abstract available.
Subject(s)
Borrelia burgdorferi , Borrelia , Ixodes , Korea , Lyme Disease , TicksABSTRACT
No abstract available.