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

OBJECTIVEYersinia enterocolitica is an extracellular pathogen and its related antigens interact with the host immune system. We investigated the difference in immunological characteristics between a highly pathogenic and poorly pathogenic strain of Y. enterocolitica.

METHODSWe used SDS-PAGE and western blotting to characterize lipopolysaccharide (LPS), Yersinia outer membrane proteins (Yops), membrane proteins, and whole-cell proteins from poorly pathogenic Y. enterocolitica bio-serotype 2/O:9, isolated from China, and highly pathogenic bio-serotype 1B/O:8, isolated from Japan.

RESULTSThese two strains of Y. enterocolitica had different LPS immune response patterns. Comparison of their Yops also showed differences that could have accounted for their differences in pathogenicity. The membrane and whole-cell proteins of both strains were similar; immunoblottting showed that the 35 kD and perhaps the 10 kD proteins were immunogens in both strains.

CONCLUSIONThe major antigens of the two strains eliciting the host immune response were the LPS and membrane proteins, as shown by comparing protein samples with reference and purified preparations.

Animals ; Antigens, Bacterial ; genetics ; metabolism ; Bacterial Proteins ; genetics ; metabolism ; Blotting, Western ; Electrophoresis, Polyacrylamide Gel ; Female ; Gene Expression Regulation, Bacterial ; physiology ; Lipopolysaccharides ; metabolism ; Rabbits ; Yersinia enterocolitica ; classification ; metabolism

K88ac-LT(B) gene derived from pQE30-K88ac-LT(B) was cloned into the expression vector pLA and then the recombinant vector was transformed into the competent cells Lactobacillus casei 525. The recombinant bacteria were grown at 37 degrees C, in MRS broth. Western blotting analysis with rabbit-anti-K88ac-LT(B) polyclonal serum indicated that the recombinant protein reacted with the specific antibodies. The results showed that the molecular weight of the recombinant protein was about 71.2 kD. The K88ac-LT(B) fusion protein on the cell surface was confirmed by immunofluorescence mciroscopy and flow cytometric analysis. In addition, the survival of recombinant Lactobacillus casei 525 was studied in imitative gastrointestinal environments such as artificial gastro fluid (pH 1.5-5.5), artificial intestinal fluid, bile(0.3-3.0 g/L). The results indicated that the recombinant strain survived well in artificial gastric fluids at pH 2.5-4.5 in 5 h. The recombinant Lactobacillus casei 525 could slowly grow in the artificial intestinal fluid for different time, and could survive in 0.3% bile.
Antigens, Bacterial ; genetics ; Bacterial Toxins ; genetics ; metabolism ; Enterotoxigenic Escherichia coli ; genetics ; metabolism ; Enterotoxins ; genetics ; metabolism ; Escherichia coli Proteins ; genetics ; metabolism ; Fimbriae Proteins ; genetics ; Gastric Juice ; Lactobacillus casei ; genetics ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Recombination, Genetic

To begin with, we constructed cfp10-esat6 fusion gene and its prokaryotic expression vector and had it express in E. coli. By GeneSOEing techniques, a fusion gene was constructed by splicing cfpl0 gene and esat6 gene, and then was cloned into pGEX-4T-1 plasmid. Secondly, we constructed the prokaryotic expression recombinant plasmid pGcfp10-esat6. After identification with restriction enzyme analysis, PCR and nucleotide sequencing analysis, The E. coli BL21 containing the recombinant plasmid was induced by IPTG (Isopropy-beta-D-thiogalatoside). The fusion protein CFP10-ESAT6 with GST-tag about 42 kDa was expressed and purified with GST-fusion protein purification kit,The expression of cfp10-esat6 fusion gene was subsequently detected by SDS-polyacrylamine gel electrophoresis and Western-blot analysis. The sequence of cfp10 and esat6 in recombinant plasmid was consistent with that of GenBank report. The fusion protein existed in cytoplasm in soluble form and represented about 40% total bacterial protein of E. coil. The fusion protein was purified and the purity reached 90%. Its antigenicity was confirmed by Western-blotting. The prokaryotic expression vector (pGcfp1o-esat6) was constructed successfully, and the fusion protein CFP10-ESAT6 was obtained. This study provided an experimental basis for potential application of the recombinant CFP10-ESAT6 in the diagnosis of tuberculosis.
Antigens, Bacterial ; biosynthesis ; genetics ; Bacterial Proteins ; biosynthesis ; genetics ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Mycobacterium tuberculosis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification

This study was conducted to amplify the cfpl0-esat6 fusion gene by SOE and insert into the integrating shuttle plasmid pMV361 to form the recombinant plasmid. Then another recombinant plasmid was constructed by insertinga-A g signal sequence of BCG. The two recombinant plasmids were introduced into BCG and the induced products from recombinant BCG were analyzed. In conclusion,the successful construction of rBCG expressing the fusion protein CFP10-ESAT6 will be the base of the development of novel Mycobacterium tuberclosis vaccines.
Antigens, Bacterial ; biosynthesis ; genetics ; Bacterial Proteins ; biosynthesis ; genetics ; Mycobacterium bovis ; genetics ; metabolism ; Mycobacterium tuberculosis ; genetics ; Plasmids ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Tuberculosis Vaccines ; biosynthesis

For studying the effects of Mycobacterium tuberculosis secretory protein ESAT-6 on the related functions of macrophages, RAW264.7 cells were transfected with pEGFP-C1-ESAT-6 and pEGFP-C1 by liposome respectively. After screening with a high level of G418, the macrophage cell lines that stably expressed EGFP-ESAT-6 fusion protein or EGFP were established. The gene and protein expression levels were further analyzed by RT-PCR, fluorescence microscopy and Western blotting. The results indicated that the EGFP-ESAT6 fusion gene was integrated into the chromosome and the protein could be stably expressed in the selected macrophage cell line. These results gave us a tool for the future study in the mechanisms of ESAT-6 protein in modulating the macrophage cells.
Animals ; Antigens, Bacterial ; biosynthesis ; genetics ; Bacterial Proteins ; biosynthesis ; genetics ; Cell Line ; Green Fluorescent Proteins ; biosynthesis ; genetics ; Macrophages ; cytology ; metabolism ; Mice ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Transfection

Antigens, Bacterial ; genetics ; metabolism ; Bacterial Proteins ; genetics ; metabolism ; Child ; Child, Preschool ; DNA, Bacterial ; genetics ; Gastritis ; pathology ; Gene Expression Regulation, Bacterial ; Helicobacter Infections ; microbiology ; pathology ; Helicobacter pylori ; genetics ; Humans ; Immunohistochemistry ; Peptic Ulcer ; microbiology ; pathology ; Polymerase Chain Reaction

OBJECTIVETo investigate whether the presence of structured CagA proteins in Western- and Eastern-type Helicobacter pylori (H. pylori) induces different incidences of gastric diseases.

METHODSCagA and phosphorylated CagA were expressed in AGS gastric epithelial cells infected with wild type and mutant strains. The ability of individual CagA was determined by immunoprecipitation and Western blot assay. Morphological changes of these cells were observed under microscope to evaluate the appearance of elongation hummingbird phenotype.

RESULTSThe sizes of CagA proteins in different strains were different, and no phosphorylated CagA proteins were detected in wild-type strains. Meanwhile, the kinetics of CagA status in AGS infected with H. pylori was detected. The molecular weight of phosphorylated CagA with the same size of CagA proteins in H. pylori was different in infections with different wild-type strains. CagA and phosphorylated CagA increased in a time-dependent manner after the infection. The hummingbird phenotype with H. pylori for time-course was observed under microscope. Instead of HPK5 strain, the wild-type 26695 strain induced hummingbird phenotype in a time-dependent manner.

CONCLUSIONTranslocation and phosphorylation of CagA are necessary, but not sufficient, for the induction of hummingbird phenotype in AGS cells.

Amino Acid Sequence ; Antigens, Bacterial ; metabolism ; Bacterial Proteins ; metabolism ; Cell Line ; Helicobacter Infections ; metabolism ; microbiology ; Helicobacter pylori ; Humans ; Interleukin-1 ; genetics ; metabolism ; Protein Transport

OBJECTIVE: To determine the effect of CagA(+) Helicobacter pylori(H.pylori)strain and anti-H.pylori drugs on the expression of connexin 43(Cx43) and cell proliferation of BGC-823 cells in vitro,and to investigate the relation between the changes of Cx43 expression, cell proliferation of BGC-823 cells and CagA(+)H.pylori. METHODS: BGC-823 cells were co-cultured with CagA(+) H.pylori strain(NCTC J99) or CagA(-) H.pylori strain(NCTC 12908)at bacteria/cells ratio of 20:1,100:1 and 500:1 for 24 hours and 48 hours respectively. anti-H.pylori drugs was given in the group co-cultured at bacteria/cells ratio of 100:1 after 16 hours. In the control group, BGC-823 cells were cultured for 24 hours and 48 hours respectively,but without H.pylori or antij H.pylori drugs. Immunocytochemical SABC method and the image analysis of the computer were applied to detect the changes of Cx43 expression in BGC-823 cells. The cell proliferation was examined by methyl tetrazolium (MTT) method. RESULTS: (1)The expression of Cx43 in the control group after cultivation for 48 hours was higher than that for 24 hours (P< 0.05). The expression of Cx43 in the groups co-cultured with CagA(+) H.pylori strain after cultivation for 48 hours was lower than that co-cultured for only 24 hours, and that of the groups co-cultured with CagA(+) H.pylori strain was lower than that of the control group for both 24 hours and 48 hours (P< 0.05). The expression of Cx43 in the groups at bacteria/cells ratio of 500:1 was lower than that at bacteria/cells ratio of 20:1 and 100:1 for both 24 and 48 hours (P< 0.05),and that at bacteria/cells ratio of 100:1 was lower than that at bacteria/cells ratio of 20:1 for 48 hours (P< 0.05).However, there was no significant difference in Cx43 expression between 24 and 48 hours in the groups co-cultured with CagA(-) H.pylori strain (P>0.05). Cx43 expression in the groups co-cultured with CagA(-) H.pylori strain at the ratio of 100:1 and 500:1 was lower than that in the control group, and Cx43 expression at the ratio of 500:1 was lower than that at the ratio of 20:1 for 24 hours and 48 hours. Cx43 expression increased after the intervention with anti-H.pylori drugs for 48 hours. (2) In the groups co-cultured with CagA(+)H.pylori strain, the optical density value of MTT indicated that the cell proliferation at the bacteria/cells ratio of 100:1 was higher than that in the control group, but no significant difference was found in other two groups co-cultured for 24 hours. After co-culturing for 48 hours, the cell proliferation at the bacteria/cells ratio of 20:1 and 100:1 was significantly accelerated, while the cell proliferation at 500:1 was inhibited. In the groups co-cultured with CagA(-) H.pylori strain,there was no change in the cell proliferation. Intervention with anti-H.pylori drugs could suppress the cell proliferation. CONCLUSION CagA(+) H.pylori can down-regulate the expression of Cx43 in BGC-823 cells,which is related to the reaction time and the density of H.pylori. Low density of CagA(+)H.pylori suspensions can accelerate the proliferation of BGC-823 cells, while high density can suppress the cell proliferation. The CagA(-) H.pylori has no effect on the cell proliferation. Intervention with anti-H.pylori drugs can up-regulate the expression of Cx43,and suppress the cell proliferation of BGC-823 cells.
Anti-Bacterial Agents ; pharmacology ; Antigens, Bacterial ; genetics ; metabolism ; Bacterial Proteins ; genetics ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Connexin 43 ; biosynthesis ; Helicobacter pylori ; drug effects ; genetics ; metabolism ; Humans ; Immunohistochemistry ; Stomach Neoplasms ; metabolism ; microbiology ; pathology

OBJECTIVETo construct a lipL32/1-lipL21-OmpL1/2 fusion gene and its prokaryotic expression system, and to establish an enzyme-linked immunosorbent assay (ELISA) using the rLipL32/1-LipL21-OmpL1/2 fusion antigen of Leptospira interrogans for sensitive and specific detection of IgM in the serum of patients with leptospirosis.

METHODSlipL32/1-lipL21-OmpL1/2 fusion genes were constructed using a primer-linking PCR. The target recombinant protein antigens, rLipL32/1, rLipL21, rOmpL1/2 and rLipL32/1-LipL21-OmpL1/2, were expressed and the purified antigens were then immobilized to the surface of microplate wells for ELISA-based detection of IgM in the sera of leptospirosis patients.

RESULTSOf 493 acute leptospirosis patients, 95.7% and 97.8% were positive by rLipL32/1-LipL21- OmpL1/2-IgM-ELISA using different serum dilutions, which was higher than the rLipL32/1-IgM-ELISA (93.1% and 90.3%), rLipL21-IgM-ELISA (90.3% and 87.0%), and rOmpL1-IgM-ELISA (85.6% and 81.1%) (P<0.01). All IgM-ELISAs tested negative against 56 non-leptospirosis patients with typhoid fever, hemorrhagic fever or dengue fever.

CONCLUSIONTrigeminal fusion antigen increases ELISA sensitivity and the rLipL32/1-LipL21-OmpL1/2- IgM-ELISA is a sensitive and specific serological diagnostic method for clinical leptospirosis.

Antigens, Bacterial ; genetics ; metabolism ; Bacterial Outer Membrane Proteins ; genetics ; metabolism ; Enzyme-Linked Immunosorbent Assay ; methods ; Humans ; Immunoglobulin M ; immunology ; Leptospirosis ; diagnosis ; metabolism ; Lipoproteins ; genetics ; metabolism ; Recombinant Fusion Proteins ; genetics ; metabolism