OBJECTIVETo construct recombinant BCG against leptospirosis.

METHODSWe amplified the entire open reading frame of the OmpL1 gene from the genome of the leptospire serovar Lai strain 017. Two recombinant plasmids pBQ1 and pBQ2 were constructed by oriented ligation based on the E. coli-BCG shuttle plasmids pMV261 and pMV361 respectively. The recombinant plasmids were transformed into BCG by electroporation. The rBCGs bearing pBQ1 and pBQ2 were induced by high temperature of 45 degrees C.

RESULTSThe expressed product, a 35kD protein was detected by SDS-PAGE. The result indicates that pBQ1 and pBQ2 can express OmpL1 in rBCG.

CONCLUSIONThe technical methods in this study may help detect the immunogenicity and immunoprotection of OmpL1 and develop more safe, highly effective rBCG bearing leptospiral antigen with long-lasting protection.

BCG Vaccine ; genetics ; Bacterial Outer Membrane Proteins ; biosynthesis ; genetics ; DNA, Bacterial ; genetics ; Gene Expression ; Genes, Bacterial ; Leptospira interrogans ; genetics ; Open Reading Frames ; genetics ; Plasmids ; Recombinant Proteins ; biosynthesis

This study was aimed to construct a prokaryotic expressing vector of Hap gene from Nontypeable Haemophilus influenzae, and express and identify the fusion proteins of Hap-His in E. coli. The gene encoding protein Hap was amplified from Nontypeable Haemophilus influenzae ATCC49247 chromosomal DNA by PCR, then it was cloned into prokaryotic expression plasmid pET-32a (+). The recombinant plasmid pET-32a(+)-Hap was transformed into E. coli BL21 and expression was induced by Isopropy-beta-D-thiogalatoside(IPTG). The Hap-His fusion protein expressed so was analyzed by SDS-PAGE and Western-blot. The results showed that the recombinant expressive plasmid pET-32a (+)-Hap was constructed successfully, and the recombinant plasmid expressed Hap-His fusion protein with relative molecule mass 176 000 and mainly existed in inclusion body. This fusion protein could combine with anti-His monoclonal antibody specifically through Western blot analysis. Successful expression of Hap-His fusion protein in prokaryotic cell could lay a basis for further study of immunocompetence of Hap protein and development of NTHi vaccine.
Bacterial Outer Membrane Proteins ; biosynthesis ; genetics ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Haemophilus influenzae ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Serine Endopeptidases ; biosynthesis ; genetics

OBJECTIVETo express a fusion protein of Neisseria gonorrhoeae with a mucosal adjuvant.

METHODSThe gene coding Loop VI-VIII(PL678) of porin, an out-membrane protein of Neisseria gonorrhoeae, was obtained by PCR. It was inserted into a plasmid fused with subunit B of heat labile enterotoxin. The recombinant was transformed in E. coli. The expression of fusion protein was analysed by ELISA, SDS-PAGE and Western-blot.

RESULTFusion protein with LTB was successfully expressed, and displayed both the ability of binding GM1 and the reactogenicity with polyclonal antibodies against Neisseria gonorrhoeae.

CONCLUSIONThe expression of fusion protein laid a foundation for the study of the intramolecular vaccine against Neisseria gonorrhoeae.

Bacterial Outer Membrane Proteins ; biosynthesis ; Bacterial Toxins ; biosynthesis ; Bacterial Vaccines ; immunology ; Enterotoxins ; biosynthesis ; Escherichia coli ; genetics ; Escherichia coli Proteins ; Neisseria gonorrhoeae ; chemistry ; immunology ; Polymerase Chain Reaction ; Recombinant Fusion Proteins ; biosynthesis ; Vaccines, Synthetic ; 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

This study was conducted to potentiate the expression of outer membrane protein OmpL17 of the strong virulent L. interrogans serovar Lai and investigate its immunogenicity in rabbits. The OmpL17 was cloned into prokaryotic expression vector pGEX-1lambdaT. The recombination expression plasmid pGEX-OmpL17 was transformed into E. Coli JM109. The GST fused protein GST-OmpL17 was expressed after induction by IPTG, then GST-tag was by thrombin and purified using Bulk GST purification Modules. SDS-PAGE and Western blotting analysis indicated that the molecular weight of GST-OmpL17 and OmpL17 was about 54 KDa and 28 KDa respectively. The outer membrane protein OmpL17 was subcutaneously injected into rabbits and high titre anti-OmpL17 antibody was obtained (1:4896) which could conjugate specifical with OmpL17. In conclusion, OmpL17 and specifical anti-OmpL17 antibody were obtained, which provided an experimental basis for researching pathogenic effect and immunity functions of OmpL17.
Bacterial Outer Membrane Proteins ; biosynthesis ; genetics ; immunology ; Bacterial Proteins ; biosynthesis ; genetics ; immunology ; Cloning, Molecular ; Humans ; Leptospira interrogans ; genetics ; immunology ; Porins ; biosynthesis ; genetics ; immunology ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Virulence

BACKGROUND: Despite increasing importance of Acinetobacter baumannii in nosocomial infections and rapid development of multi-antimicrobial resistance in this strain, the resistance mechanisms of beta-lactam antimicrobials in A. baumannii were not clearly defined. In order to observe the resistance mechanisms against beta-lactams and carbapenem, we characterized the production of beta-lactamases and outermembrane protein (OMP) profiles for the 44 clinical isolates of A. baumannii. METHODS: The MICs of antimicrobials were determined by agar dilution test. The secondary beta-lactamases were characterized by isoelectric focusing, polymerase chain reactions and nucleotide sequencing, and the production of chromosomal beta-lactamases was quantitated by spectrophotometric method. For two strains with an elevated MIC of carbapenem, outermembrane protein (OMP) profile was analyzed by ultracentrifugation of the sonicated bacteral cells and SDS-PAGE. RESULTS AND CONCLUSION: Twenty two or 4 of 44 strains produced TEM-1-like beta-lactamase or PER-1 extended-spectrum beta-lactamase, respectively. However, when we analyzed the MICs of several beta-lactams with the beta-lactamase production, the resistance level of beta-lactam was mainly determined by the production of chromosomal beta-lactamase, not by the secondary beta-lactamases in the clinical isolates of A. baumannii. In two strains with an elevated MIC of imipenem, a decrease or loss of about 35 kDa and 22 kDa proteins in OMP was observed, which suggested that the change of OMP played a role in carbapenem resistance.
Acinetobacter/*drug effects/isolation & purification/metabolism ; Acinetobacter Infections/drug therapy/microbiology ; Antibiotics, Lactam/*pharmacology ; Bacterial Outer Membrane Proteins/biosynthesis ; Carbapenems/pharmacology ; Cross Infection/drug therapy/microbiology ; Drug Resistance, Bacterial ; Human ; beta-Lactamases/biosynthesis

OBJECTIVETo investigate the immune-functional epitopes and inflammation-inducing effects of the major outer envelope proteins of Leptospira interrogans.

METHODSNi-NTA affinity chromatography was established to extract the target recombinant proteins rOmpL1/1 and OmpL1/2, LipL32/1 and rLipL32/2, LipL41/1 and rLipL41/2 expressed by the different genotypes. By using Signal P-NN software in Signal P3.0 prediction server, EMBOSS software in propred MHC class-II binding peptide prediction-ProPred prediction server, the possible signal peptides, MHC-II binding peptides and lymphocyte B epitopes were analyzed. The IL-1, IL-8 and TNF-alpha secretion in human umbilical vein endothelial cell line EVC-304 induced by target recombinant proteins were measured by ELISA.

RESULTSUnder the inducement of IPTG, the constructed prokaryotic systems efficiently expressed rOmpL1/1 and rOmpL1/2, rLipL32/1 and rLipL32/2, and rLipL41/1 and rLipL41/2 with outputs of 30% and 15%, 40% and 35%, and 15% and 10% of the total bacterial proteins, respectively. Each of the purified target recombinant proteins showed a single protein band in SDS-PAGE. The signal peptides of OmpL1s, LipL32/1 and LipL32/2, and LipL41s were located at the N ends of 1-24, 1-21 and 1-24, and 1-24 amino acid residuals, respectively. OmpL1s, LipL32s and LipL41s displayed 2,2 and 1 same major epitopes of MHC-II binding peptides and lymphocyte B and OmpL1/2 had another one (59-78). The different dosages of rOmpL1s, rLipL32s and rLipL41s increased the secretion of IL-1alpha , IL-8 and TNF-alpha (P<0.05) in EVC-304 cells. The IL-1alpha levels reached the highest at the 24 h and then declined,while the IL-8 and TNF-alpha levels after 48 h treatment were higher that those after 24 h.

CONCLUSIONThe expression products in ompL1/1, lipL32 or lipL41 genotypes of L.interrogans contain similar immune functional epitopes. rOmpL1/1 and rOmpL1/2, rLipL32/1 and rLipL32/2, and rLipL41/1 and rLipL41/2 are able to directly induce inflammatory reaction in EVC-304 cells.

Bacterial Outer Membrane Proteins ; immunology ; pharmacology ; Cells, Cultured ; Endothelial Cells ; cytology ; Epitopes ; Genotype ; Humans ; Inflammation ; etiology ; Interleukin-1 ; biosynthesis ; Leptospira interrogans ; genetics ; immunology ; Lipoproteins ; immunology ; pharmacology ; Recombinant Proteins ; immunology ; Tumor Necrosis Factor-alpha ; biosynthesis ; Umbilical Veins ; cytology

A 21-kDa leptospiral lipoprotein (LipL21) was evaluated for its diagnostic potential to detect bovine leptospirosis by ELISA. Both native LipL21 (nLipL21) and recombinant LipL21 (rLipL21) proteins were tested and compared regarding diagnostic efficiency, and no statistically significant difference was observed. The sensitivity of rLipL21 ELISA for 62 microscopic agglutination test (MAT) positive sera was 100% and the specificity with 378 MAT negative sera was 97.09%. Thus, rLipL21 protein-based ELISA could be used as an alternative to MAT for the diagnosis of bovine leptospirosis.
Animals ; Antibodies, Bacterial/blood ; Antigens, Bacterial/biosynthesis/*chemistry/genetics ; Bacterial Outer Membrane Proteins/biosynthesis/*chemistry/genetics ; Cattle ; Cattle Diseases/blood/*microbiology ; Enzyme-Linked Immunosorbent Assay/veterinary ; Leptospira interrogans/*isolation & purification ; Leptospirosis/blood/microbiology/*veterinary ; Lipoproteins/biosynthesis/*chemistry/genetics ; Recombinant Proteins/biosynthesis/chemistry/genetics ; Sensitivity and Specificity

OBJECTIVETo establish an efflux pump inhibitor screening model with the out-membrane protein OprM in Pseudomonas aeruginosa efflux pump system as the target point.

METHODSEfflux pump out-membrane protein gene oprM was obtained from standard Pseudomonas aeruginosa PA01 strain. Expression of OprM protein was induced in E. coli strain HS151 with T-easy vector as the cloning vector, and pMMB67EH as the expression vector. In order to evaluate the function of OprM protein, we measured intracellular tetracycline concentrations with liquid scintillation counter, measured the diameters of bacteriostatic circles with paper disc, and then established a screening model accordingly.

RESULTSOprM protein was highly expressed. Using Pseudomonas aeruginosa as the main detecting bacteria, we established a drug screening model acting on OprM. A total of 1 600 microbial fermentation samples were screened with this model, among which 56 positive strains were found, with a positive rate of 3.5%.

CONCLUSIONOprM plays an important role in drug efflux. The established model has good specificity and maneuverability.

Anti-Bacterial Agents ; metabolism ; pharmacology ; Bacterial Outer Membrane Proteins ; biosynthesis ; drug effects ; genetics ; Bacterial Proteins ; genetics ; Drug Evaluation, Preclinical ; methods ; Drug Resistance, Microbial ; Drug Resistance, Multiple ; genetics ; Escherichia coli ; genetics ; Humans ; Membrane Transport Proteins ; biosynthesis ; drug effects ; genetics ; Plasmids ; genetics ; Pseudomonas aeruginosa ; drug effects ; genetics

OBJECTIVETo construct lipL32/1-lipL41/1 fusion gene and its prokaryotic expression system and to determine frequencies of carrying and expression of lipL32 and lipL41 genes in L.interrogans wild strains and specific antibody levels in sera from leptospirosis patients.

METHODSlipL32/1-lipL41/1 fusion gene was constructed using linking primer PCR method and the prokaryotic expression system of the fusion gene done with routine techniques. SDS-PAGE was used to examine expression of the target recombinant protein rLipL32/1-rLipL41/1. Immunogenicity of rLipL32/1-rLipL41/1 was identified by Western blot. PCR and MAT were performed to detect carrying and expression of lipL32 and lipL41 genes in 97 wild L.interrogans strains. Antibodies against products of lipL32 and lipL41 genes in serum samples from 228 leptospirosis patients were detected by ELISA method.

RESULTSThe homogeneity of nucleotide and putative amino acid sequence of lipL32/1-lipL41/1 fusion gene were 99.9 % and 99.8 % in comparison with the reported sequences. Expression output of the target recombinant protein rLipL32/1-rLipL41/1, mainly present in inclusion body, accounted for 10 % of the total bacterial proteins. Both the rabbit antisera against rLipL32/1 and rLipL41/1 could combine to rLipL32/1-rLipL41/1. 97.9 % and 87.6 % of the L.interrogans wild strains had lipL32 and lipL41 genes, respectively. 95.9 % and 84.5 % of the wild strains were positive for MAT with titers of 1:4 - 1:128 using rabbit anti-rLipL32s or anti-rLipL41s sera, respectively. 94.7 % - 97.4 % of the patients'serum samples were positive for rLipL32s antibodies, while 78.5 % - 84.6 % of them were rLipL41s antibodies detectable.

CONCLUSIONlipL32/1-jlipL41/1 fusion gene and its prokaryotic expression system were successfully constructed. The expressed fusion protein had qualified immunogenicity. Both the lipL32 and lipL41 genes are extensively carried and frequently expressed by different serogroups of L.interrogans, and their expression products exhibit cross-antigenicity.

Antibodies, Bacterial ; blood ; Antigens, Bacterial ; genetics ; immunology ; Bacterial Outer Membrane Proteins ; genetics ; Cloning, Molecular ; Enzyme-Linked Immunosorbent Assay ; Gene Expression Regulation, Bacterial ; Genes, Bacterial ; genetics ; Humans ; Leptospira interrogans ; genetics ; Leptospirosis ; immunology ; microbiology ; Lipoproteins ; genetics ; Prokaryotic Cells ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology