OBJECTIVETo construct a recombinant Lactobacillus acidophilus that expresses high levels of Helicobacter pylori (Hp) adhesin Hp0410.

METHODSThe gene fragment encoding Hp0410 was amplified by PCR from the DNA of H. pylori NCTC11639 strain and cloned into the shuttle plasmid pMG36e to construct pMG36e-Hp0410, which was transformed into Lactobacillus acidophilus by electroporation. The target protein was confirmed with SDS-PAGE and silver nitrate staining and analyzed by Western blotting. The stability of the recombinant plasmid was assessed by drawing the growth curve of the recombinant Lactobacillus acidophilus.

RESULTSA 750-bp fragment was inserted into the pMG36e plasmid and transformed into Lactobacillus lactis. The transformed bacterium expressed the target protein with a relative molecular mass of about 34 kD. Western blotting confirmed that the expressed proteins could be recognized by the serum of patients with Hp infection. The recombinant plasmid pMG36e-Hp0410 exhibited good stability in the presence or absence of erythromycin.

CONCLUSIONSThe recombinant Lactobacillus acidophilus with high constitutive expression of Hp0410 has been constructed successfully.

Adhesins, Bacterial ; biosynthesis ; genetics ; immunology ; Bacterial Vaccines ; biosynthesis ; Helicobacter Infections ; prevention & control ; Humans ; Lactobacillus acidophilus ; genetics ; metabolism ; Plasmids ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Vaccines, Attenuated ; biosynthesis

An Escherichia coli strain (SEPT13) isolated from the liver of a hen presenting clinical signs of septicaemia had a LD(50) of 4.0 x 10(5) CFU/ml in one-day-old chickens, expressed Ia, Ib, E1, E3, K and B colicins and aerobactin. The strain was ampicillin and streptomycin resistant, and found to have fimA, csgA and tsh DNA related sequences; it could adhere to and invade HEp-2 and tracheal epithelial cells, expressed fimbriae (observed by electron microscopy), and had five plasmids of 2.7, 4.7, 43, 56, and 88 MDa. Transposon mutagenesis of strain SEPT13, with transposon TnphoA, resulted in a mutant strain named ST16 that had a LD(50) of 1.2 x 10(12) CFU/ml. All other biological characteristics of strain ST16 were the same as those detected for strain SEPT13 except for the migration of an 88 MDa plasmid to the 93 MDa position indicating the insertion of the transposon into the 88 MDa plasmid. The 93 MDa plasmid of strain ST16 was transferred, by electroporation assay, to non-pathogenic receptor strains (E. coli strains K12 MS101 and HB101), resulting in transformant strains A and B, respectively. These strains exhibited adhesion properties to in vitro cultivated HEp-2 cells but did not have the capacity for invasion. The adherence occurred despite the absence of fimbriae; this finding suggests that the 88 MDa plasmid has afimbrial adhesin genes.
Adhesins, Escherichia coli/*genetics/*metabolism ; Animals ; Chickens ; Escherichia coli/genetics/*metabolism ; Gene Expression Regulation, Bacterial ; Plasmids/*genetics/metabolism ; Poultry Diseases/*microbiology ; Sepsis/*microbiology

OBJECTIVETo detect and compare the activity and intensity of gingipain K (Kgp)-caspase like subdomain in culture medium and cell extract of Porphyromonas gingivalis (Pg) isolates in puberty gingivitis and to reveal the possible association of Kgp with puberty gingivitis.

METHODSThirty-six children of 14 to 17 years old were enrolled in this study. Clinical parameters including gingival index (GI), sulcus bleeding index (SBI) and probing depth (PD) were evaluated. Subgingival plaque samples were collected and Pg isolates were obtained. 16S rRNA PCR was used to confirm Pg clinical isolates. Bacteria were grown in batches of BHI base and harvested at the end of log-phase growth. Culture fractions (culture medium and cell extract) of 10 Pg isolates were performed with SDS-PAGE and Western blot technique using primary antibody against specific Kgp-caspase like subdomain. Activity of Kgp in both samples was detected as well. The data were statistically analyzed using SPSS 11.5 software. The relationship between the Kgp intensity/activity of Kgp and the clinical parameters was statistically analyzed using Spearman correlation coefficient.

RESULTSThere was positive correlation between the intensity/activity of Kgp and the clinical parameters (P < 0.05).

CONCLUSIONSThe Kgp in clinical isolates of Pg from puberty gingivitis is in complicated forms. Caspase-like molecules with low molecular weight may exist as intracellular functional protein molecules which can affect the interaction between Pg and host. Kgp was contributes in certain degree to the pathogenesis of puberty gingivitis.

Adhesins, Bacterial ; genetics ; metabolism ; Adolescent ; Cysteine Endopeptidases ; genetics ; metabolism ; Dental Plaque ; microbiology ; Female ; Gingivitis ; enzymology ; microbiology ; Humans ; Male ; Porphyromonas gingivalis ; genetics ; isolation & purification ; metabolism

The cp36 gene encoding an adhesive protein was amplified by PCR from genomic DNA of rabbit P. multocida C51-3 strain, and cloned into the pMD18-T vector and then sequenced. The mature adhesive protein without a signal peptide of cpm36 gene was amplified by PCR from the recombinant plasmid pMD18-cp36, then cloned into the prokaryotic expression vector pQE30 to provide a recombinant plasmid pQE30-cpm36. The recombinant protein of CPM36 was produced in Escherichia coli M15 harboring the recombinant plasmid pQE30-cpm36 by IPTG induction, and the recombinant protein purified by the affinity chromatography with Ni(2+)-NTA resin. The sequence analyses showed that the ORF of cp36 gene was 1032 bp in length, and DNA homology of the cp36 genes between the C51-3 strain and the previously reported different serotype strains of P. multocida in GenBank was 76.9 to 100%. The SDS-PAGE analyses revealed a single fusion protein band with a molecular weight of 37 kD, and the Western blotting analysis demonstrated that the recombinant protein CPM36 and native 36 kD protein of C51-3 were recognized specifically by an antiserum against the recombinant protein, suggesting that the recombinant protein is an antigenic protein.
Adhesins, Bacterial ; biosynthesis ; genetics ; immunology ; Animals ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Pasteurella multocida ; chemistry ; Rabbits ; microbiology ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology

To construct a non-resistance and attenuated Salmonella typhimurium strain which expresses conservative region of adhesin(AB) of Helicobacter pylori(Hp). The AB gene was amplified by PCR and inserted into the expression vector pYA248 containing asd gene and was introduceded into the delta Cya, delta Crp, delta Asd attenuated Salmonella typhimurium strain by twice transformations, which is a balanced lethal recombinant. Bridged ELISA method was used to measure AB expressed in sonicate and culture supernatant. According to Meacock's way and growth curve, stability of the recombinant is evaluated. Semi-lethal capacity test was used to evaluate the safty of recombinant. Results showed S. typhimurium X4072(pYA248-AB) was constructed successfully, recombinant X4072(pYA248- AB) content of supernatant serum was higher than that of thallus lytic liquor confirmed by bridged ELISA, and after recombinant pYA248- AB cultured 100 generation without selection pressure, all the recombinant germ selected randomly can grow, and the AB antigen was positive by ELISA detection. The growth curve of the recombinant germ showed that the growth state of X4072(pYA248) and X4072(pYA248- AB) were coincidence on the whole, and the survival rate of C57BL/6 was still 100%, 30 days after taking X4072(pYA248- AB) 1.0 x 10(10)cfu. orally. Non-resistance S. typhimurium X4072(pYA248- AB) was constructed successfully. The recombinant plasmid was stable indicated by in vitro experiment. And the recombinant strain was safe confirmed by animal experiment. This live vaccine strain is worthy to be considered as a new live oral vaccine candidate against Hp infection.
Adhesins, Bacterial ; genetics ; metabolism ; Animals ; Enzyme-Linked Immunosorbent Assay ; Genetic Vectors ; genetics ; Helicobacter pylori ; genetics ; Male ; Mice ; Mice, Inbred C57BL ; Plasmids ; genetics ; Polymerase Chain Reaction ; Salmonella typhimurium ; genetics ; growth & development ; metabolism

OBJECTIVETo obtain highly purified intimin encoded by the eae gene and study its adhesion activity.

METHODSThe eae gene was amplified from enterohemorrhagic Escherichia coli O157:H7 (EHEC) chromosome by PCR and cloned into pMD19-T vector. The eae gene was cut from pMD19-T vector and subcloned into the prokaryotic expression plasmid pET28a(+), and expressed in E.coli BL21(DE3). The recombinant protein was purified with Ni(2+)-chelating affinity chromatography followed by identification with SDS-PAGE and Western blotting. The purified intimin was detected by immunofluorescence staining to test its adhesion.

RESULTSThe 2805-bp eae gene fragment was obtained, and the recombinant expression plasmid pET28a(+)-eae was successfully expressed in E.coli BL21 (DE3). The molecular weight of the recombinant protein was 97 000. Purified recombinant intimin was recognized by rabbit anti-O157 antiserum, and bound to the surface of HEp-2 cells as revealed by immunofluorescence staining.

CONCLUSIONHighly purified and immunoreactive intimin has been successfully obtained, which can adhere to the surface of HEp-2 cells. The acquisition of recombinant intimin provides the basis for studying its interaction with the host receptors during EHEC O157:H7 infection.

Adhesins, Bacterial ; biosynthesis ; genetics ; isolation & purification ; metabolism ; Animals ; Blotting, Western ; Cell Adhesion ; Cell Line ; Cloning, Molecular ; Escherichia coli ; genetics ; Escherichia coli O157 ; Escherichia coli Proteins ; biosynthesis ; genetics ; isolation & purification ; metabolism ; Gene Expression ; Plasmids ; genetics

OBJECTIVETo determine the effects of lactose inducing on the expression of recombinant Helicobacter pylori rUreB and rhpaA, and Escherichia coli rLTB and rLTKA63.

METHODSBIO-RAD gel image analysis system was applied to detect the outputs of the recombinant proteins. SDS-PAGE was performed to measure the target protein expression of recombinant genes at various periods of growth, different lactose concentrations, various inducing temperatures and times. The results of the target protein expression induced by lactose were compared to those by isopropyl-beta-D-thiogalactoside (IPTG).

RESULTSLactose showed higher efficiency to induce the expression of rHpaA, rUreB, rLTB and rLTKA63 than IPTG. The expression outputs of target recombinant proteins induced at 37 degrees C were remarkably higher than those at 28 degrees C. The optimal expression parameters were 0.8 of OD600 value, 50 g/L of lactose, 4 hours of inducing time for rHpaA, and 1.2 of OD600 value, 100 g/L of lactose, 5 hours of inducing time for both the rUreB and rLtB,and 0.8 of OD600 value, 100 g/L of lactose, 4 hours for rLTKA63.

CONCLUSIONLactose, a sugar with non-toxicity and low cost, is able to induce the recombinant genes to express the target proteins with higher efficiency than IPTG.

Adhesins, Bacterial ; biosynthesis ; genetics ; Bacterial Toxins ; biosynthesis ; genetics ; Bacterial Vaccines ; biosynthesis ; genetics ; Enterotoxins ; biosynthesis ; genetics ; Escherichia coli ; genetics ; metabolism ; Escherichia coli Proteins ; biosynthesis ; genetics ; Genetic Engineering ; Helicobacter Infections ; prevention & control ; Helicobacter pylori ; genetics ; metabolism ; Humans ; Lactose ; pharmacology ; Recombinant Proteins ; biosynthesis ; genetics ; Urease ; genetics ; Vaccines, Synthetic ; biosynthesis ; genetics

In this study, we showed the direct interaction between Mycobacterium avium subsp. paratuberculosis fibronectin attachment protein (FAP) and toll-like receptor4 (TLR4) via co-localization and binding by using confocal microscopy and co-immunoprecipitation assays. FAP triggered the expression of pro- and anti-inflammatory cytokines in a TLR4-dependent manner. In addition, FAP-induced cytokine expression in bone marrow-derived dendritic cells (BMDCs) was modulated in part by glycogen synthase kinase-3 (GSK-3). FAP-induced expression of CD80, CD86, major histocompatibility complex (MHC) class I, and MHC class II in TLR4+/+ BMDCs was not observed in TLR4-/- BMDCs. Furthermore, FAP induced DC-mediated CD8+ T cell proliferation and cytotoxic T lymphocyte (CTL) activity, and suppressed tumor growth with DC-based tumor vaccination in EG7 thymoma murine model. Taken together, these results indicate that the TLR4 agonist, FAP, a potential immunoadjuvant for DC-based cancer vaccination, improves the DC-based immune response via the TLR4 signaling pathway.
*Adhesins, Bacterial/genetics/metabolism ; Animals ; CD8-Positive T-Lymphocytes/metabolism ; *Cancer Vaccines/therapeutic use ; Cell Proliferation ; Cytokines/metabolism ; Dendritic Cells/*cytology ; Disease Models, Animal ; Gene Expression Regulation ; Glycogen Synthase Kinase 3/metabolism ; Humans ; Mice ; Mice, Inbred C57BL ; Mycobacterium avium/genetics/metabolism ; Paratuberculosis/metabolism ; Protein Binding ; Signal Transduction ; T-Lymphocytes, Cytotoxic/metabolism ; *Thymoma/genetics/metabolism ; *Toll-Like Receptor 4/agonists/genetics/metabolism

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

The 987P fimbriae of enterotoxigenic Escherichia coli (ETEC) mediates adhesive interactions with brush border vesicle (BBV) of the intestinal epithelial cells from the neonatal piglets. By adhering to intestinal epithelial cells, producing localized multiplication, the 987P ETEC can progress to mucosal surface colonization and concomitant effective enterotoxin delivery. To identify the receptors for the 987P, BBV proteins from piglet intestinal villous epithelial cells were separated by SDS-PAGE and analyzed by Ligand blot, protein bands with a set of 32-35 kD recognized by the 987P fimbriae were subjected to in gel proteolysis with trypsin. The tryptic fragments were separated by microbore reversed phase HPLC(RP-HPLC), samples shown to contain one major peak by MALDI-MS were submitted to Edman sequencing, three peptides were sequenced successfully and the all of three peptides matched the sequences of human or porcine histone H1 proteins. Porcine histone H1 proteins isolated from both piglet intestinal epithelial cells and BBV demonstrated the same SDS-PAGE migration pattern and 987P-binding properties as the 987P-specific protein receptors from piglet intestinal brush border did. The above results indicated that the 987P protein receptors are piglet BBV-derived Histone H1 proteins.
Adhesins, Escherichia coli ; metabolism ; Amino Acid Sequence ; Animals ; Enterotoxigenic Escherichia coli ; metabolism ; pathogenicity ; Escherichia coli Infections ; microbiology ; veterinary ; Fimbriae Proteins ; metabolism ; Fimbriae, Bacterial ; chemistry ; Histones ; genetics ; metabolism ; Host-Pathogen Interactions ; Intestinal Mucosa ; metabolism ; Molecular Sequence Data ; Receptors, Cell Surface ; genetics ; metabolism ; Swine