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 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

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 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

It has been hypothesized that blood infusion of reconstituted HDL (rHDL) is a possible therapeutic strategy for the treatment of coronary artery disese. To compare short-term anti-inflammatory activity of wildtype (WT) apoA-I and point mutants, each rHDL containing WT, V156K, or R173C was infused into apo-E deficient atherosclerotic mice. Each rHDL was injected via the tail vein at a dosage of 120 mg/kg of body weight in 0.4 ml of tris-buffered saline (TBS), and blood was then collected at 24 and 48 h post-injection. Although regression activity was observed in each of the rHDL infused groups, a 30% reduction in the lipid-stained area of the aortic sinus was observed in the V156K and R173C-rHDL groups when compared to that of the WT-rHDL group, and this reduction was well correlated with an approximately 60% reduction in the accumulation of macrophages in the lesion area. Additionally, the groups that received the V156K and R173C-rHDL treatments showed smaller increases in the GOT, GPT, interleukin-6, myeloperoxidase (MPO) and lipid hydroperoxide (LPO) serum levels than those that received the WT-rHDL treatment. In addition, the strongest serum paraoxonase and ferric reducing ability was observed in the V156K and R173C-rHDL groups. In vitro nitration and chlorination of apoA-I by MPO treatment revealed that V156K-rHDL and R173C-rHDL were less susceptible to chlorination. Furthermore, rHDL treatment inhibited cellular uptake of oxidized LDL by macrophage cells and the production of proatherogenic species in culture media. In conclusion, blood infusions of the rHDLs exerted in vivo regression activity with anti-inflammatory and antioxidant activity in apo-E deficient mice and THP-1 cells, especially in those that were treated with V156K and R173C apoA-I.
Animals ; Anti-Inflammatory Agents/immunology/*therapeutic use ; Apolipoprotein A-I/blood/genetics/immunology/*therapeutic use ; Apolipoproteins E/genetics ; Aryldialkylphosphatase/blood/metabolism ; Atherosclerosis/*drug therapy ; Cell Line ; Cell Membrane Permeability ; Cholesterol/blood/metabolism ; Humans ; Lipoproteins, HDL/genetics/immunology/*therapeutic use ; Lipoproteins, LDL/metabolism ; Macrophages/cytology ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Oxidation-Reduction/*drug effects ; Peroxidase/blood/metabolism ; Point Mutation

OBJECTIVETo establish ELISAs based on rLipL32/1-LipL21-OmpL1/2 fusion antigen of Leptospira interrogans for detecting specific IgG and IgM in serum of patients with leptospirosis.

METHODSMicroscope agglutination test(MAT) was performed to detect serum specimens from leptospirosis patients and to determine titers of rabbbit antiserum agaist rLipL32/1-LipL21-OmpL1/2 to reference standard strains of L. interrogans. By using rLipL32/1-LipL21-OmpL1/2, rLipL32/1, rLipL21 and rOmpL1/2 as the coated antigens, ELISAs for detecting specific serum IgM and IgG were established. The established ELISAs were applied to MAT-positive serum specimens from 107 patients with leptospirosis.

RESULTThe results of MAT confirmed that 66% (71/107) of the patients were infected with L.interrogans serogroup Icterohaemorrhagiae, and the rLipL32/1-LipL21-OmpL1/2 antiserum were able to agglutinate all 15 reference standard L.interrogans strains with 1 : 20approximate, equals1 : 160 titers. The positive rates of ELISAs using rLipL32/1-LipL21-OmpL1/2, rLipL32/1, rLipL21 or rOmpL1/2 as the antigen were 89.7%, 75.7%, 85.1% and 79.4% for detecting IgM, respectively, while 99.1%, 99.1%, 94.4% and 86.0% for detecting IgG, respectively. The positive detection rate of rLipL32/1-LipL21-OmpL1/2-IgM-ELISA was higher than those of the other three IgM detection ELISAs (P<0.05). The positive detection rate of rLipL32/1-LipL21-OmpL1/2-IgG-ELISA was higher than that of rOmpL1/2-IgG-ELISA (P<0.05), while there was no significant differnce with that of rLipL21-IgG-ELISA and rLipL32/1-IgG-ELISA (P>0.05).

CONCLUSIONThe ELISAs using rLipL32/1-LipL21-OmpL1/2 as the antigen can be applied as a sensitive,specific and universal serological method for diagnosis of leptospirosis.rLipL32/1-LipL21-OmpL1/2-IgM-ELISA shows a definite value for early diagnosis of leptospirosis compared with the other ELISAs used in this study.

Agglutination Tests ; Animals ; Antibodies, Bacterial ; blood ; Antigens, Bacterial ; genetics ; metabolism ; Bacterial Outer Membrane Proteins ; genetics ; metabolism ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; genetics ; metabolism ; Humans ; Immunoglobulin G ; blood ; Immunoglobulin M ; blood ; Leptospira interrogans ; genetics ; immunology ; Leptospirosis ; diagnosis ; immunology ; Lipoproteins ; genetics ; metabolism ; Rabbits ; Recombinant Fusion Proteins ; genetics ; immunology ; metabolism ; Sensitivity and Specificity ; Serologic Tests ; methods

OBJECTIVETo construct the eukaryotic expression system of L.interrogans lipL32/1-ompL1/1 fusion gene and to identify the immunoreactivity of expression products.

METHODSPCR with linking primer was used to construct the fusion gene lipL32/1-ompL1/1. The P.pastoris eukaryotic expression system of the fusion gene, pPIC9K-lipL32/1-ompL1/1-P. pastorisGS115, was constructed after the fusion gene was cloned and sequenced. Colony with phenotype His(+)Mut(+) was isolated by using MD and MM plates and His(+) Mut(+) transformant with high resistance to G418 was screened out by using YPD plate. Using lysate of His(+) Mut(+) colony with high copies of the target gene digested with yeast lyase as the template and 5'AOX1 and 3'AOX1 as the primers, the target fusion gene in chromosome DNA of the constructed P. pastoris engineering strain was detected by PCR. Methanol in BMMY medium was used to induce the target recombinant protein rLipL32/1-rOmpL1/1 expression. rLipL32/1-rOmpL1/1 in the medium supernatant was extracted by using ammonium sulfate precipitation and Ni-NTA affinity chromatography. Output and immunoreactivity of rLipL32/1-rOmpL1/1 were measured by SDS-PAGE and Western blot methods, respectively.

RESULTSAmplification fragments of the obtained fusion gene lipL32/1-ompL1/1 was 1794 bp in size. The homogeneity of nucleotide and putative amino acid sequences of the fusion gene were as high as 99.94 % and 100 %, respectively, compared with the sequences of original lipL32/1 and ompL1/1 genotypes. The constructed eukaryotic expression system was able to secrete rLipL32/1-rOmpL1/1 with an output of 10 % of the total proteins in the supernatant, which located the expected position after SDS-PAGE. The rabbit anti-rLipL32/1 and anti-rOmpL1/1 sera could combine the expressed rLipL32/1-rOmpL1/1.

CONCLUSIONAn eukaryotic expression system with high efficiency in P.pastoris of L.interrogans lipL32/1-ompL1/1 fusion gene was successfully constructed in this study. The expressed fusion protein shows specific immunoreactivity, which can be used as a potential antigen for developing a novel vaccine of L.interrogans.

Amino Acid Sequence ; Antigens, Bacterial ; genetics ; immunology ; Bacterial Outer Membrane Proteins ; genetics ; Base Sequence ; Cloning, Molecular ; Eukaryotic Cells ; metabolism ; Gene Expression Regulation, Bacterial ; Genes, Bacterial ; genetics ; Humans ; Leptospira interrogans ; genetics ; Leptospirosis ; immunology ; microbiology ; Lipoproteins ; genetics ; Molecular Sequence Data ; Polymerase Chain Reaction ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Vaccines, Synthetic ; immunology

OBJECTIVEThe purpose of this study is to construct plant expression plasmid containing the gene encoding chimera SBR-CT delta A1.

METHODSThe target gene fragment P2, including the gene-encoded chimera SBR-CT delta A1 (3,498-5,378 bp), was obtained by standard PCR amplification. The PCR products were ligated with pGEM-easy vector through TA clone to form plasmid pTSC. The plasmid pTSC and plasmid pPOKII were digested by restricted endonuclease BamHI and KpnI, and the digested products were extracted and purified for recombination. Then the purified P2 and plasmid pPOKII were recombined by T4 DNA ligase to form recombinant plasmid pROSC; inserting bar gene into the plasmid and form pROSB plasmid. The recombined plasmids were isolated and identified by restricted endonuclease cutting and Sanger dideoxy DNA sequencing.

RESULTSP2 gene was linked to pPOKII plasmid and formed recombinant plasmid pROSC. The DNA sequence and orientation were corrected. And bar gene was inserted into pPOSC and form recombinant plasmid pROSB.

CONCLUSIONPlant expression vector pROSC and pROSB containing the gene encoding chimera SBR-CT delta A1, which may provide useful experiment foundation for further study on edible vaccine against caries have been successfully constructed.

Adhesins, Bacterial ; genetics ; Animals ; Chimera ; genetics ; Cloning, Molecular ; Dental Caries ; prevention & control ; Gene Transfer Techniques ; Genetic Vectors ; Lipoproteins ; genetics ; Lycopersicon esculentum ; genetics ; metabolism ; Plasmids ; genetics ; Rabbits ; Recombinant Proteins ; biosynthesis ; genetics ; Recombination, Genetic ; Streptococcal Vaccines ; genetics ; immunology ; Streptococcus mutans ; genetics ; immunology ; Vaccines, DNA ; 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

Outer membrane proteins of Pasteurella (P.) multocida have been known to be protective immunogens. Pasteurella lipoprotein E (PlpE) has been reported to be an important cross reactive outer membrane protein in P. multocida. The gene encoding the PlpE of P. multocida serotypes A: 3, B: 2 and D: 1 was amplified from the genomic DNA. The amplified products were cloned and the nucleotide sequence was determined. Sequence analysis of the recombinant clones revealed a single open reading frame of 1,011 bp, 1,008 bp and 1,017 bp encoding a protein with a calculated molecular mass of 37.829 kDa, 37.389 kDa and 37.965 kDa for serotypes A: 3, B: 2 and D: 1 respectively. The comparison of the plpE sequence in different capsular types revealed a high degree (>90%) of homology. Furthermore, the plpE gene of Haemorhhagic septicaemia causing serotype (B: 2) was expressed in E. coli and recombinant PlpE was strongly immunostained by antiserum against whole cell antigen, indicating that the protein is expressed in vivo.
Animals ; Bacterial Outer Membrane Proteins/*genetics/immunology/metabolism ; Base Sequence ; Blotting, Western ; Cattle ; Cattle Diseases/*microbiology ; Cloning, Molecular ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; *Genetic Variation ; Hemorrhagic Septicemia/microbiology/*veterinary ; India ; Lipoproteins/*genetics/immunology/metabolism ; Molecular Sequence Data ; Open Reading Frames/genetics ; Pasteurella multocida/*genetics/immunology ; Sequence Analysis, DNA ; Sequence Homology ; Serotyping ; Species Specificity