Induced by 42 degrees C, the recombinant engineering bacterial pBV/cpa408 was highly expressed. After having been pelleted by 80% (NH4)2 SO4 and dialysised, the expressed protein was isolated and purified by the gel filtration choromatography. Then according to an amount of 1.0 mg/kg, the Kunming Mice (body weighted 18g) were immuned with the purified protein by intraperitoneal inoculation. One week after the first enhanced immunization, the Kunming Mice were attacked with an amount of 1.0MLD alpha-toxin, in which the eight mice immuned all survive and the control group all died. During the period of immunization, the titre of the mouse's serum antibody was measured by ELISA. One week after the first immunization, the titre of the mice's serum antibody was 1:800, but that of one week after the first enhanced immunization reached to 1:6400.
Animals ; Antibodies, Bacterial ; blood ; Antigens, Bacterial ; biosynthesis ; immunology ; Bacterial Toxins ; immunology ; Calcium-Binding Proteins ; immunology ; Female ; Immunization ; Male ; Mice ; Recombinant Proteins ; biosynthesis ; immunology ; isolation & purification ; Type C Phospholipases ; immunology

OBJECTIVETo construct prokaryotic expression systems of ltB/ctB-ompL1/1 fusion genes and to determine the L.interrogans carrying status in leptospirosis patients with the expression products.

METHODSThe fusion genes ltB-ompL1/1 and ctB-ompL1/1 were constructed using linking primer PCR method. SDS-PAGE was used to examine expression of the target recombinant proteins rLTB-rOmpL1/1 and rCTB-rOmpL1/1. Western blot and GM1-ELISA were used to measure the immunogenic and GM(1)-binding activities of rLTB-rOmpL1/1 and rCTB-rOmpL1/1, respectively. PCR and MAT were performed to detect the expression of ompL1 gene in 97 wild L.interrogans strains. Antibodies against ompL1 gene products in serum samples of 228 leptospirosis patients were detected with ELISA method.

RESULTSThe homogeneity of nucleotide and putative amino acid sequence of ltB-jompL1/1 and ctB-ompL1/1 fusion genes were 99.7 % - 99.9 % and 99.5 % - 100 %, in comparison with the reported corresponding sequences. Expression outputs of both rLTB-rOmpL1/1 and rCTB-rOmpL1/1, mainly present in inclusion body, accounted for 10% of the total bacterial protein. Both rLTB-rOmpL1/1 and rCTB-rOmpL1/1 could combine to rabbit anti-rOmpL1/1 serum and bovine GM(1). 89.7 % of L.interrogans wild strains had ompL1 gene. 87.6% of the wild L.interrogans strains presented positive results for MAT (titers :1:4-1:256) with the rabbit anti-rOmpL1/1 or anti-rOmpL1/2 sera. 86.8% and 88.6% of the patients' serum samples were positive for rOmpL1/1 and rOmpL1/2 antibodies, respectively.

CONCLUSIONThe fusion proteins, rLTB-rOmpL1/1 and rCTB-rOmpL1/1, showed high immunogenic and GM(1)-binding activities. ompL1 gene is extensively distributed and frequently expressed in different serogroups of L.interrogans and its products expressed by different genotypes exhibit extensive cross-antigenicity.

Bacterial Outer Membrane Proteins ; genetics ; immunology ; Bacterial Toxins ; genetics ; Bacterial Vaccines ; genetics ; Cloning, Molecular ; Enterotoxins ; genetics ; Escherichia coli Proteins ; genetics ; Genes, Bacterial ; genetics ; Humans ; Leptospira interrogans ; genetics ; immunology ; Prokaryotic Cells ; metabolism ; Recombinant Fusion Proteins ; genetics ; immunology ; Vaccines, Synthetic ; genetics

Designed a pair of primers through modifying N-terminal bases (5bps) of gene after ATG but not changing amino acid, and amplified a smaller mutated gene sequnce (468bp) containing two protective antigenic determinants from pBlue-BoNTaHc, N-terminal codon of mutated gene fragment is changed from low to high frenqence in E. coli. Mutated gene was ligated into pGEM-T vector and sequenced, then, cloned into a expression plasmid pBV220. As a result, cloned gene was expressed in insoluble form by temperature inducing (from 30 degrees C to 42 degrees C) in E. coli. Expression product is 40% of total proteins and is of specific binding activity to antibody in ELISA. The successful modification and high level expression of protective fragment of botulinum neurotoxin serotype A(BoNTaHc468) gene is conducive to further study on antitoxin and vaccine.
Bacterial Vaccines ; immunology ; Botulinum Toxins, Type A ; genetics ; immunology ; Clostridium botulinum type A ; immunology ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; genetics ; Plasmids ; Polymerase Chain Reaction ; Recombinant Proteins ; biosynthesis

An expression plasmid carrying anthrax protective antigen (PA) gene was constructed, which has an OmpA signal sequence attached to the 5' end of PA gene. The plasmid was transformed into E. coli and induced to express recombinant PA (rPA) . The recombinant protein, about 10% of the total bacterial protein in volume, was secreted to the periplasmic space of the cell. After a purification procedure including ion-exchange, hydrophobic interaction chromatography, and gel filtration, about 15 mg of 95 % pure rPA was obtained from 1-liter culture. The bioactivity of rPA was proved by in vitro cytotoxicity assay. The polyclonal antiserum from rabbits immunized with rPA could inhibit the action of anthrax lethal toxin in vitro, which suggests that antibodies against rPA can provide high passive protection against anthrax. The results reported here may be helpful to develop a safe and efficacious recombinant PA vaccine against anthrax.
Amino Acid Sequence ; Animals ; Anthrax Vaccines ; immunology ; Antigens, Bacterial ; chemistry ; genetics ; immunology ; toxicity ; Bacterial Toxins ; chemistry ; genetics ; immunology ; toxicity ; Base Sequence ; Mice ; Molecular Sequence Data ; Plasmids ; Rabbits ; Recombinant Proteins ; biosynthesis ; immunology ; Vaccines, Synthetic ; immunology

Escherichia coli heat-labile enterotoxin (LT), which causes a characteristic diarrhea in humans and animals, is a strong mucosal immunogen and has powerful mucosal adjuvant activity towards coadministered unrelated antigens. Here we report the different mucosal adjuvanticity of nontoxic LT derivatives, LTS63Y and LTdelta110/112, generated by immunizing through two different mucosal routes. Intragastric (IG) immunization with Helicobacter pylori urease alone resulted in poor systemic IgG and IgA responses and no detectable local secretory IgA, but IG co-immunization with urease and LTdelta110/112 induced high titers of urease-specific local secretory IgA and systemic IgG and IgA, comparable to those induced by wild-type LT. LTS63Y showed far lower adjuvant activity towards urease than LTdelta110/112 in IG immunization, but was more active than LTdelta110/112 in inducing immune responses to urease by intranasal (IN) immunization. LTdelta110/112 predominantly enhanced the induction of urease-specific IgG1 levels following IG immunization, whereas LTS63Y induced high levels of IgG1, IgG2a and IgG2b following IN immunization. In addition, quantitative H. pylori culture of stomach tissue following challenge with H. pylori demonstrated a 90-95% reduction (p < 0.0002) in bacterial burden in mice immunized intranasally with urease using either mutant LT as an adjuvant. These results indicate that the mechanism(s) underlying the adjuvant activities of mutant LTs towards coadmnistered H. pylori urease may differ between the IN and IG mucosal immunization routes.
Adjuvants, Immunologic/administration & dosage* ; Administration, Intranasal ; Animal ; Bacterial Toxins/immunology* ; Bacterial Toxins/genetics ; Bacterial Toxins/administration & dosage ; Enterotoxins/immunology* ; Enterotoxins/genetics ; Enterotoxins/administration & dosage ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli* ; Feces ; Female ; Gastric Mucosa/microbiology ; Gastric Mucosa/immunology* ; Helicobacter pylori ; Human ; IgA, Secretory/immunology* ; IgG/immunology ; Mice ; Mice, Inbred BALB C ; Mutagenesis, Site-Directed ; NAD+ ADP-Ribosyltransferase/immunology ; NAD+ ADP-Ribosyltransferase/genetics ; Nasal Mucosa/immunology* ; Point Mutation ; Urease/immunology* ; Urease/administration & dosage ; Vaccination*

Escherichia coli heat-labile enterotoxin (LT) is composed of catalytic A and non-catalytic homo-pentameric B subunits and causes diarrheal disease in human and animals. In order to produce a nontoxic LT for vaccine and adjuvant development, two novel derivatives of LT were constructed by a site-directed mutagenesis of A subunit; Ser63 to Tyr63 in LTS63Y and Glu110, Glu112 were deleted in LT delta 110/112. The purified mutant LTs (mLTs) showed a similar molecular structural complex as AB5 to that of wild LT. In contrast to wild-type LT, mLTs failed to induce either elongation activity, ADP-ribosyltransferase activity, cAMP synthesis in CHO cells or fluid accumulation in mouse small intestine in vivo. Mice immunized with mLTs either intragastrically or intranasally elicited high titers of LT-specific serum and mucosal antibodies comparable to those induced by wild-type LT. These results indicate that substitution of Ser63 to Tyr63 or deletion of Glu110 and Glu112 eliminate the toxicity of LT without a change of AB5 conformation, and both mutants are immunogenic to LT itself. Therefore, both mLTs may be used to develop novel anti-diarrheal vaccines against enterotoxigenic E. coli.
Amino Acid Substitution ; Animal ; Bacterial Toxins/toxicity* ; Bacterial Toxins/metabolism ; Bacterial Toxins/immunology* ; Bacterial Toxins/genetics ; CHO Cells ; Cyclic AMP/metabolism ; Enterotoxins/toxicity* ; Enterotoxins/metabolism ; Enterotoxins/immunology* ; Enterotoxins/genetics ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli*/metabolism ; Escherichia coli*/genetics ; Female ; Hamsters ; IgA, Secretory/blood ; Ileum/metabolism ; Immunity, Mucosal ; Mice ; Mice, Inbred BALB C ; Mutagenesis, Site-Directed ; NAD+ ADP-Ribosyltransferase/metabolism ; Recombinant Proteins/toxicity ; Recombinant Proteins/metabolism ; Recombinant Proteins/immunology ; Recombinant Proteins/chemistry

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

ApxI is one of the most important virulence factors of Actinobacillus pleuropneumoniae (APP). To study the immunogenicity of the ApxI, the complete coding sequence (3146bp) and its 5'-terminal 1140 bp fragment of the apxIA gene were separately cloned into the prokaryotic expression vector pET-28a, and expressed in the E. coli BL21 (DE3) with induction by IPTG. The expression products, rApxIA and rApxIAN, were present in a form of inclusion bodies and showed the same immunological reactivity as natural ApxI (nApxI) in Western-blot analysis. BALB/c mice were intraperitoneally immunized with the rApxIA, rApxIAN and nApxI respectively. The serum antibody levels of the rApxIAN immunized mice were significantly lower than those immunized with rApxIA or nApxI in an ApxI-specific ELISA, but serum neutralization test demonstrated that immunized mice with rApxIAN, rApxIA and nApxI could generate similar levels of antibodies neutralizing the hemolytic activity of the natural ApxI. The rApxIAN was able to elicite 80% protection rate against APP serovar 1 and 100% against serovar 2 when challenged at a dose of one LD50 after 2 weeks of boost immunization.
Actinobacillus Infections ; prevention & control ; Actinobacillus pleuropneumoniae ; genetics ; immunology ; Animals ; Antibodies ; blood ; Bacterial Proteins ; genetics ; immunology ; Bacterial Toxins ; genetics ; immunology ; Bacterial Vaccines ; immunology ; Cytotoxins ; genetics ; immunology ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Hemolysin Proteins ; genetics ; immunology ; Inclusion Bodies ; genetics ; immunology ; Mice ; Mice, Inbred BALB C ; Peptides ; genetics ; immunology ; Recombinant Proteins ; genetics ; immunology

Clostridium (C.) difficile is a common cause of nosocomial diarrhea in horses. Vancomycin and metronidazole have been used as standard treatments but are only moderately effective, which highlights the need for a novel alternative therapy. In the current study, we prepared antiserum of equine origin against both C. difficile toxins A and B as well as whole-cell bacteria. The toxin-neutralizing activities of the antibodies were evaluated in vitro and the prophylactic effects of in vivo passive immunotherapy were demonstrated using a conventional mouse model. The data demonstrated that immunized horses generated antibodies against both toxins A and B that possessed toxin-neutralizing activity. Additionally, mice treated with the antiserum lost less weight without any sign of illness and regained weight back to a normal range more rapidly compared to the control group when challenged orally with 10(7) C. difficile spores 1 day after serum injection. These results indicate that intravenous delivery of hyperimmune serum can protect animals from C. difficile challenge in a dose-dependent manner. Hence, immunotherapy may be a promising prophylactic strategy for preventing C. difficile infection in horses.
Animals ; Antibodies, Bacterial/blood/*immunology/therapeutic use ; Bacterial Proteins/immunology/therapeutic use ; Bacterial Toxins/immunology/therapeutic use ; Clostridium Infections/microbiology/prevention & control/*veterinary ; Clostridium difficile/*immunology ; Enterotoxins/immunology/therapeutic use ; Female ; Horse Diseases/microbiology/*prevention & control ; Horses ; Immune Sera/*immunology ; Immunization, Passive/*veterinary ; Mice ; Mice, Inbred C57BL ; Spores, Bacterial/immunology

BACKGROUND: Enzyme immunoassay (EIA) capable of detecting both toxin A and toxin B is strongly recommended for the diagnosis of Clostridium difficile associated disease. Therefore, we evaluated two different EIAs for the detection of C. difficile toxin A/B. METHODS: For a total of 228 stool specimens we performed bacteriologic cultures for C. difficile and examined for toxin A and toxin B using enzyme linked fluorescent immunoassay (ELFA; VIDAS CDAB, Bio-Merieux sa, France) and ELISA (C.DIFFICILE TOX A/B II, TECHLAB, USA). We also performed PCR assays for toxin A and B genes in 117 C. difficile isolates that grew from the stool cultures and compared the results with those obtained with the two different EIAs. RESULTS: The concordance rate between ELFA and ELISA was 85.5% (195/228). Using the culture and PCR results as the standard, the sensitivity/specificity of the ELFA and ELISA were 65.0%/72.1% and 71.8%/70.3%, and for positive/negative predictive values were 78.4%/69.6% and 71.8%/70.3%, respectively (P value >0.05). No differences were observed between the results of ELFA and ELISA with toxin A- toxin B+ strains of C. difficile. CONCLUSIONS: The sensitivity of the ELISA was slightly higher than that of ELFA for toxin A and toxin B, but the specificity and positive predictive value of the ELFA were rather higher than those of the ELISA, although no statistical differences were observed. A bacteriologic culture and PCR assay for toxin genes are recommended in case the both EIAs are negative.
Bacterial Proteins/*analysis/genetics/immunology ; Bacterial Toxins/*analysis/genetics/immunology ; Clostridium difficile/genetics/isolation & purification/*metabolism ; Enterotoxins/*analysis/genetics/immunology ; Enzyme-Linked Immunosorbent Assay/*methods ; Feces/microbiology ; Fluorescent Dyes/chemistry ; Humans ; Reagent Kits, Diagnostic