Orientia tsutsugamushi, a causative pathogen of Scrub typhus, is a gram-negative intracellular bacterium. Outer membrane vesicles (OMVs) are produced from the membrane of bacteria and play many roles related to the survival of the pathogen. However, there have been no reports confirming whether O. tsutsugamushi indeed produce OMVs. O. tsutsugamushi boryong was cultured in ECV-304 cells for the purification of OMVs. Western blot analysis and immunoenrichment using anti-O. tsutsugamushi monoclonal antibody and electron microscopy were employed for identification and characterization of OMVs. We confirm the presence of OMVs derived from O. tsutsugamushi, and also found that those OMVs contain a major surface antigen of 56-kDa protein and variant immunogenic antigens.
Antibodies, Monoclonal/*immunology ; Antigens, Bacterial/*immunology ; Antigens, Surface/*immunology ; Cell Line ; Cell Membrane/immunology ; Humans ; Microscopy, Electron ; Orientia tsutsugamushi/*immunology/metabolism ; Scrub Typhus/diagnosis/microbiology ; Secretory Vesicles/*immunology

OBJECTIVETo determine the location on outer envelope and natural antibody response and types of genus-specific lipoprotein antigen LipL41s in patients with Leptospira interrogans.

METHODSMicroscope agglutination test (MAT) was used to examine leptospirosis patients' serum samples from Sichuan area, China. Ni-NTA affinity chromatography was performed to extract the target recombinant rLipL41/1 and rLipL41/2 products that expressed under inducement of IPTG. Western blot assay was performed to detect the immunoreactivity between the sera from the patients infected with different serogroups of L. interrogans and rLipL41s. Immune aurosol electron microscopy was selected to locate the position of LipL41s on leptospiral envelope. ELISA based on rLipL41s was established to confirm the level and types of specific antibody.

RESULTSL. interrogans serogroup icterohaemorrhagiae remained to be the most dominant leptospiral serogroup in Sichuan area. All the sera from patients infected with different serogroups of L. interrogans could efficiently recognize the LipL41s which were the protein molecular that located on the external surface of leptospiral envelope. In the 156 serum samples from MAT positive leptospirosis patients, the positive rates for rLipL41/1 or rLipL41/2 specific IgM appeared to be 84.6%-87.8% and 78.2%-83.3%, respectively, while for rLipL41/1 or rLipL41/2 specific IgG they were 69.2%-81.4% and 75.0%-80.1%, respectively.

CONCLUSIONLipL41s were the leptospiral superficial protein antigen of L. interrogans. Both the LipL41/1 and LipL41/2 could induce serum antibodies IgM and IgG with extensive antigenic-cross reaction during natural infection of L. interrogans in general populations. Hence, rLipL41/1 or rLipL41/2 could be used as the antigen candidate for developing universal genetic engineering vaccine and detection kit.

Antibodies, Bacterial ; blood ; immunology ; Antigens, Bacterial ; metabolism ; Bacterial Outer Membrane Proteins ; metabolism ; Cell Membrane ; metabolism ; Cross Reactions ; Humans ; Immunoglobulin G ; blood ; immunology ; Immunoglobulin M ; blood ; immunology ; Leptospira interrogans ; metabolism ; ultrastructure ; Leptospirosis ; immunology ; Species Specificity

OBJECTIVETo investigate the antigenicity of recombinant Chlamydia trachomatis (Ct) OmcBc protein and search for the new target for early diagnosis of Chlamydia infection and Chlamydia vaccine development.

METHODSThe C fragment of OmcB encoding the amino acids from T270 to T553 was amplified from Chlamydia serovar D genomic DNA. The pGEX-6p-Ct OmcBc expression plasmid was constructed and transformed into E.coli XL-1blue. The expression of recombinant Ct OmcBc protein was induced by IPTG. Serum samples were collected from 120 patients with urogenital Chlamydia infection. The antiserum samples were collected from 7 New Zealand white rabbits and 5 Balb/C mice immunized subcutaneously and intraperitoneally with Ct serovar D inactivated EB, respectively, and from 9 Balb/C mice intranasally infected with Ct serovar D live EB. The anti-Chlamydia specific antibody were titrated by an immunofluorescence assay (IFA). The reactivity of the recombinant OmcBc protein with all the above antisera was detected by ELISA.

RESULTSThe pGEX-6p-Ct OmcBc expression plasmid was successfully constructed. DNA sequencing showed that the inserted OmcBc was about 852 bp, encoding a protein with 284 amino acids. The expression of the recombinant GST-OmcBc was induced by IPTG, producing a fusion protein with a molecular weight of about 57 kD. The titer of the specific antibodies to Chlamydia in all the antisera was high. ELISA results showed strong reactivities of the recombinant GST-OmcBc fusion protein with all the above antisera.

CONCLUSIONSOmcBc protein is an immunodominant protein of Chlamydia. The recombinant GST-OmcBc with strong antigenicity may provide a basis for further study of early diagnosis of chlamydia infection and development of Chlamydia vaccine.

Animals ; Antibodies, Bacterial ; blood ; Antigens, Bacterial ; immunology ; metabolism ; Bacterial Outer Membrane Proteins ; genetics ; immunology ; Chlamydia trachomatis ; genetics ; immunology ; metabolism ; Cloning, Molecular ; Genes, Bacterial ; Humans ; Immune Sera ; immunology ; Mice ; Mice, Inbred BALB C ; Plasmids ; Rabbits

OBJECTIVETo clone pIA and pIB genes of Neisseria gonorrhoeae,and to construct pIA-pIB fusion gene and its prokaryotic expression system, and to establish enzyme linked immunosorbent assay (ELISA) based on rPIA-PIB for detecting serum and pus samples from gonorrhea patients and to evaluate the sensitivity and specificity of the ELISA.

METHODSpIA-pIB fusion gene was constructed by polymerase chain reaction (PCR) using linking primers and a prokaryotic expression system of the fusion gene was constructed by using routine molecular biological methods. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) plus BioRad Gel Image Analyzer was used to measure the expression of the target recombinant protein rPIA-PIB. Ni-NTA affinity chromatography was performed to extract and purify rPIA-PIB. An ELISA by using rPIA-PIB as the coated antigen for detecting the specific IgG against rPIA and/or rPIB in gonorrhea patients' sera as well as another ELISA by using rPIA-PIB antiserum as the first antibody for detecting the rPIA and/or rPIB in gonorrhea patients' pus samples were established. In these experiments, ELISAs associated with rPIA, rPIB and their antisera were applied as the controls.

RESULTS100% similarities of the nucleotide and putative amino acid sequences of the pIA-pIB fusion gene were confirmed when compared with the original sequences. The output of rPIA-PIB was 29.8% of the total bacterial proteins. The purified rPIA-PIB only showed a single target protein segment in gel after SDS-PAGE. Using a positive rate (98.3%) of rPIA-PIB-IgG-ELISA to detect 119 cases of gonorrhea patients' serum samples was remarkably higher than that of rPIA-IgG-ELISA (30.3%) or rPIB-IgG-ELISA (66.4%) (P<0.01). The positive rate (91.6%) of rPIA-PIB-ELISA to detect 119 cases of gonorrhea patients' pus samples was also significantly higher than that of rPIA-IgG-ELISA (27.7%) or rPIB-IgG- ELISA (62.2%) (P<0.01).

CONCLUSIONIn this study we successfully constructed pIA-pIB fusion gene of N. gonorrhoeae and its prokaryotic expression system while rPIA-PIB showed obvious superiority used as the antigen in gonorrhea associated detection kits compared to both the rPIA and rPIB.

Antigens, Bacterial ; immunology ; Bacterial Outer Membrane Proteins ; genetics ; immunology ; metabolism ; Base Sequence ; Enzyme-Linked Immunosorbent Assay ; methods ; Gene Expression Regulation, Bacterial ; Humans ; Neisseria gonorrhoeae ; genetics ; immunology ; metabolism ; Recombinant Fusion Proteins ; genetics ; immunology ; metabolism

OBJECTIVETo determine expression changes of major outer membrane protein(OMP) antigens of Leptospira interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai during infection of human macrophages and its mechanism.

METHODSOmpR encoding genes and OmpR-related histidine kinase (HK) encoding gene of L.interrogans strain Lai and their functional domains were predicted using bioinformatics technique. mRNA level changes of the leptospiral major OMP-encoding genes before and after infection of human THP-1 macrophages were detected by real-time fluorescence quantitative RT-PCR. Effects of the OmpR-encoding genes and HK-encoding gene on the expression of leptospiral OMPs during infection were determined by HK-peptide antiserum block assay and closantel inhibitive assays.

RESULTSThe bioinformatics analysis indicated that LB015 and LB333 were referred to OmpR-encoding genes of the spirochete, while LB014 might act as a OmpR-related HK-encoding gene. After the spirochete infecting THP-1 cells, mRNA levels of leptospiral lipL21, lipL32 and lipL41 genes were rapidly and persistently down-regulated (P <0.01), whereas mRNA levels of leptospiral groEL, mce, loa22 and ligB genes were rapidly but transiently up-regulated (P<0.01). The treatment with closantel and HK-peptide antiserum partly reversed the infection-based down-regulated mRNA levels of lipL21 and lipL48 genes (P <0.01). Moreover, closantel caused a decrease of the infection-based up-regulated mRNA levels of groEL, mce, loa22 and ligB genes (P <0.01).

CONCLUSIONExpression levels of L.interrogans strain Lai major OMP antigens present notable changes during infection of human macrophages. There is a group of OmpR-and HK-encoding genes which may play a major role in down-regulation of expression levels of partial OMP antigens during infection.

Antigens, Bacterial ; genetics ; metabolism ; Bacterial Outer Membrane Proteins ; genetics ; metabolism ; Cell Line ; Chaperonin 60 ; genetics ; metabolism ; Humans ; Leptospira interrogans ; genetics ; immunology ; pathogenicity ; Lipoproteins ; genetics ; metabolism ; Macrophages ; microbiology

OBJECTIVETo reconstruct the nucleotide sequence of Leptospira interrogans lipL21 gene for increasing the output of prokaryotic expression and to understand the changes on immunogenicity of the expression products before and after reconstruction, and to determine the position of envelope lipoprotein LipL21 on the surface of leptospiral body.

METHODSAccording to the preferred codons of E.coli, the nucleotide sequence of lipL21 gene was designed and synthesized, and then its prokaryotic expression system was constructed. By using SDS-PAGE plus BioRad agarose image analysor, the expression level changes of lipL21 genes before and after reconstruction were measured. A Western blot assay using rabbit anti-TR/Patoc I serum as the first antibody was performed to identify the immunoreactivity of the two target recombinant proteins rLipL21s before and after reconstruction. The changes of cross agglutination titers of antisera against two rLipL21s before and after reconstruction to the different leptospiral serogroups were demonstrated using microscope agglutination test (MAT). Immuno-electronmicroscopy was applied to confirm the location of LipL21s.

RESULTThe expression outputs of original and reconstructed lipL21 genes were 8.5 % and 46.5 % of the total bacterial proteins, respectively. Both the two rLipL21s could take place immune conjugation reaction with TR/Patoc I antiserum. After immunization with each of the two rLipL21s in rabbits, the animals could produce specific antibody. Similar MAT titers with 1:80 - 1:320 of the two antisera against rLipL21s were present. LipL21 was confirmed to locate on the surface of leptospiral envelope.

CONCLUSIONLipL21 is a superficial antigen of Leptospira interrogans. The expression output of the reconstructed lipL21 gene is remarkably increased. The expression rLipL21 maintains fine antigenicity and immunoreactivity and its antibody still shows an extensive cross immunoagglutination activity. The high expression of the reconstructed lipL21 gene will offer a favorable condition to use its product for further developing a novel universal vaccine as well as detection kit of leptospirosis.

Amino Acid Sequence ; Animals ; Antigens, Bacterial ; genetics ; immunology ; metabolism ; Bacterial Outer Membrane Proteins ; genetics ; immunology ; metabolism ; Bacterial Vaccines ; immunology ; Base Sequence ; Blotting, Western ; Cloning, Molecular ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; genetics ; Immune Sera ; immunology ; Leptospira interrogans ; genetics ; immunology ; ultrastructure ; Lipoproteins ; genetics ; immunology ; metabolism ; Microscopy, Immunoelectron ; Molecular Sequence Data ; Rabbits ; Recombinant Fusion Proteins ; genetics ; immunology ; metabolism ; Sequence Analysis, DNA ; Vaccines, DNA ; immunology

OBJECTIVETo construct lipL32/1-lipL21-OmpL1/2 fusion gene of Leptospira interrogans and its prokaryotic expression system, and to identify the immunogenicity of its products.

METHODSPCR using linking primers was applied to construct lipL32/1-lipL21-OmpL1/2 fusion gene and a prokaryotic expression system of the fusion gene was then established using routine genetic engineering technique. SDS-PAGE was used to examine output of the target recombinant protein rLipL32/1-LipL21-OmpL1/2. Double immunodiffusion and Western Blot assay were applied to identify immunogenicity of rLipL32/1-LipL21-OmpL1/2.

RESULTlipL32/1-lipL21-OmpL1/2 fusion gene with correct sequence and its prokaryotic expression system E.coli BL21DE3pET42a-lipL32/1-lipL21-ompL1/2 was obtained in this study. The output of rLipL32/1-LipL21- OmpL1/2 after optimisation was 37.78 mg/L. The immunodiffusion titer of rabbit antiserum against rLipL32/1-LipL21-OmpL1/2 was 1:4. The rLipL32/1-LipL21-OmpL1/2 antiserum was able to recognize rLipL32/1-LipL21-OmpL1/2, rLipL32/1, rLipL21 and rOmpL1/2. Positive Western hybridization signals were found among rLipL32/1-LipL21-OmpL1/2 and rabbit antiserum against whole cell of strain 56601 and serum from patients infected with L.interrogans serogroups Icterohaemorrhagiae, Grippotyphosa, Autumnalis and Pomona.

CONCLUSIONThe fusion gene lipL32/1-lipL21-OmpL1/2 and its prokaryotic expression system were successfully constructed in this study. The expressed fusion protein can be used as the antigen for developing universal genetic engineering vaccine and universal serological tests of leptospirosis.

Animals ; Antigens, Bacterial ; biosynthesis ; genetics ; Bacterial Outer Membrane Proteins ; biosynthesis ; genetics ; Bacterial Vaccines ; immunology ; Escherichia coli ; genetics ; metabolism ; Humans ; Leptospira interrogans ; genetics ; immunology ; Lipoproteins ; biosynthesis ; genetics ; Rabbits ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Vaccines, Synthetic ; immunology

The antigenicity of Photobacterium damselae (Ph. d.)subsp. piscicida, cultured in four different growth media[tryptone soya broth (TSB), glucose-rich medium (GRM),iron-depleted TSB (TSB+IR-), and iron-depleted GRM(GRM+IR-)] was compared by enzyme-linked immuno-sorbent assay (ELISA) and Western blot analysis usingsera obtained from sea bass (Dicentrarchus labrax) raisedagainst live or heat-killed Ph. d. subsp. piscicida. Theantigenic expression of Ph. d. subsp. piscicida was found todiffer depending on the culture medium used. A significantlyhigher antibody response was obtained with iron-depletedbacteria by ELISA compared with non-iron depletedbacteria obtained from the sera of sea bass raised againstlive Ph. d. subsp. piscicida. The sera from sea bass raisedagainst live bacteria showed a band at 22kDa in bacteriacultured in TSB+IR- or GRM+IR- when bacteria thathad been freshly isolated from fish were used for thescreening, while bands at 24 and 47kDa were observedwith bacteria cultured in TSB or GRM. When bacteriawere passaged several times on tryptic soya agar prior toculturing in the four different media, only bands at 24 and47kDa were recognized, regardless of the medium used toculture the bacteria. It would appear that the molecularweight of Ph. d. subsp. piscicida antigens change in thepresence of iron restriction, and sera from sea bassinfected with live bacteria are able to detect epitopes onthe antigens after this shift in molecular weight.
Animals ; Antibodies, Bacterial/blood ; Antigens, Bacterial/immunology/*metabolism ; Bass/blood/*immunology ; Blotting, Western/veterinary ; Cell Count/methods ; Culture Media ; Enzyme-Linked Immunosorbent Assay/veterinary ; Fish Diseases/immunology/*microbiology ; Molecular Weight ; Pasteurella Infections/immunology/microbiology/*veterinary ; Photobacterium/*immunology

BACKGROUNDTreponema pallidum (T. pallidum) subsp. pallidum is the causative agent of syphilis. Analysis of recombinant antigens of T. pallidum led to the identification of potential candidate antigens for vaccine development and syphilis serodiagnosis. Tp0965 was predicted to be a membrane fusion protein and was found to be reactive with infected human sera in previous studies, but the results were controversial. In this research, the antigenicity and immunoreactivity of recombinant protein Tp0965 were assessed.

METHODST. pallidum subsp. pallidum (Nichols strain) was propagated and isolated and the genomic DNA was extracted. The Tp0965 gene was amplified by polymerase chain reaction (PCR). Then the recombinant protein Tp0965 was expressed in Escherichia coli and purified by nickel-nitrilotriacetic acid (Ni-NTA) purification system. The reactivities of protein Tp0965 were examined by immunoblot analysis and indirect enzyme-linked immunosorbent assay. The antisera against protein Tp0965 were obtained by immune rabbits and the immunogenicity of antisera were detected by indirect enzyme-linked immunosorbent assay.

RESULTSRecombinant protein Tp0965 was expressed successfully in vitro. Immunoblot assay showed that the recombinant protein Tp0965 could be recognized by human syphilitic sera of all stages. Indirect enzyme-linked immunosorbent assay showed there were only 4 of 74 human syphilitic sera that failed to show reactivity to recombinant antigen Tp0965, and lack of reactivity of Tp0965 to all 28 uninfected sera. A low titer of antiserum against Tp0965 in immune rabbits could be detected after the third time of immunization.

CONCLUSIONSThe recombinant antigen Tp0965 shows excellent sensitivity for the reactivity with sera from syphilitic individuals at all stages. The results also demonstrate a potential application for the serodiagnosis of syphilis.

Animals ; Antigens, Bacterial ; genetics ; immunology ; Bacterial Proteins ; genetics ; immunology ; Enzyme-Linked Immunosorbent Assay ; Humans ; Membrane Proteins ; genetics ; immunology ; Polymerase Chain Reaction ; Rabbits ; Syphilis ; immunology ; microbiology ; Treponema pallidum ; immunology ; metabolism

OBJECTIVETo detect the expression of Mycobacterium tuberculosis secreted protein Ag85B in paraffin-embedded tissues by immunohistochemistry (IHC), and to evaluate its application in the pathological diagnosis of tuberculosis.

METHODSOne hundred and five tuberculosis specimens (54 pulmonary tuberculosis, 51 lymph nodal tuberculosis) and 51 specimens of other diseases (8 lung cancer, 10 pulmonary abscess, 10 bronchiectasis, 7 lymphoma, 5 necrotizing lymphadenitis, 4 reactive hyperplasia lymphoid, and 7 sarcoidosis) were collected from January 2012 to July 2013 from Beijing Chest Hospital, Capital Medical University. One-step IHC was performed on paraffin-embedded tissues using antibody directed against Ag85B.

RESULTSIHC and Ziehl-Neelsen (ZN) acid-fast staining showed that distribution and intensity of Ag85B expression were concordant with the distribution and number of acid-fast bacilli. IHC showed significantly higher sensitivity than ZN staining (50.5%, 53/105 vs. 31.4%, 33/105; χ² = 7.877, P = 0.005). The combined sensitivity of IHC and ZN staining was 59.0%. Moreover, oil immersion was not necessary for IHC, allowing more rapid diagnosis.

CONCLUSIONIHC detection of Ag85B is a simple method with higher sensitivity than ZN staining, and demonstrated good value in the pathological diagnosis of tuberculosis.

Acyltransferases ; metabolism ; Antigens, Bacterial ; metabolism ; Biomarkers ; metabolism ; Bronchiectasis ; diagnosis ; immunology ; Humans ; Immunohistochemistry ; Lymphadenitis ; diagnosis ; immunology ; Mycobacterium tuberculosis ; immunology ; Sarcoidosis ; diagnosis ; Staining and Labeling ; Tuberculosis, Lymph Node ; diagnosis ; immunology ; Tuberculosis, Pulmonary ; diagnosis ; immunology