Protection against Escherichia coli O157:H7 challenge by immunization of mice with purified Tir proteins.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 infections cause serious public health problems worldwide. The translocation intimin receptor (Tir) is responsible for adhesion and attaching and effacing lesions. In the current study, we used a mitomycin-treated mouse model to evaluate the efficacy of subcutaneous vs intranasal administration of the recombinant Tir as vaccine. Following immunization, mice were infected with E. coli O157:H7 and faces were monitored for shedding. Mice immunized intrasally with purified Tir proteins produced higher IgG and IgA titers in serum and feces, resulting in significant reductions in fecal shedding of EHEC O157 and higher a survival rate (92.9%), compared with subcutaneous or control immunizations. These results demonstrate the potential for the use of Tir proteins in mucosal vaccine formulations to prevent colonization and shedding of E. coli O157:H7. Therefore, purified Tir protects mice against EHEC challenge after intranasal immunization and is worth further clinical development as a vaccine candidate.

Screening test for anti-Helicobacter pylori activity of traditional Chinese herbal medicines.

AIM: To evaluate the anti-Helicobacter pylori (H. pylori) activity of 50 traditional Chinese herbal medicines in order to provide the primary evidence for their use in clinical practice. METHODS: A susceptibility test of water extract from 50 selected traditional Chinese herbal medicines for in vitro H. pylori Sydney strain 1 was performed with broth dilution method. Anti-H. pylori activity of the selected Chinese herbal medicines was evaluated according to their minimum inhibitory concentration (MIC). RESULTS: The water extract from Rhizoma Coptidis, Radix Scutellariae and Radix isatidis could significantly inhibit the H. pylori activity with their MIC less than 7.8 mg/mL, suggesting that traditional Chinese herbal medicines have anti-inflammatory and antibacterial effects and can thus be used in treatment of H. pylori infection. CONCLUSION: Rhizoma Coptidis, Radix Scutellariae and Radix isatidis are the potential sources for the synthesis of new drugs against H. pylori.

[Construction of a recombinant Lactobacillus acidophilus expressing high levels of Helicobacter pylori adhesin Hp0410].

OBJECTIVE: To construct a recombinant Lactobacillus acidophilus that expresses high levels of Helicobacter pylori (Hp) adhesin Hp0410. METHODS: The 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. RESULTS: A 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. CONCLUSIONS: The recombinant Lactobacillus acidophilus with high constitutive expression of Hp0410 has been constructed successfully.

[Effects of Newcastle disease virus on the expression of survivin and cell cycle in human tongue squamous carcinoma TSCCa cells].

OBJECTIVE: To investigate the effects of Newcastle disease virus (NDV) infection on the expression of survivin and cell cycle in human tongue squamous carcinoma TSCCa cells. METHODS: The proliferation of TSCCa cells infected with NDV in vitro was evaluated by means of MTT assay, and survivin expression in the infected cells was detected using RT-PCR and Western blotting. Flow cytometry was performed to assess the changes in the cell apoptosis, cell cycle and cell proliferation index (PI) of the cells. RESULTS: NDV infection resulted in decreased survivin expression and increased apoptosis of TSCCa cells, with reduced cell percentage in G2/M and S phases and lowered PI of the cells, showing significant differences from those of the negative control cells (P<0.05). CONCLUSION: NDV infection can inhibit survivin expression, affect the cell cycle of TSCCa cells and induce their apoptosis.

[Clone, expression and immunoreaction of napA gene of Helicobacter pylori].

AIM: To construct a prokaryotic expression system of Helicobacter pylori(Hp) (neutrophil-activating protein) napA gene, analyze nucleic acid sequence and study its immunity and inflammation. METHODS: napA fragment was amplified from Hp NCTC11639 chromosomal DNA by PCR. Its T-A was cloned, sequenced and compared with other Hp strains on the GenBank. Then the gene was cloned into pGEX-4T-1 fusion expression vector, expressed in E.coli and purified by GST-affinity chromatography. The purified product was used to screen 29 stains of mouse anti Hp monoclonal antibodies(mAb) and its immunity and inflammation analyzed with sera of Hp-infected patients by Western blot. RESULTS: napA fragment was composed of 435 base pairs (GenBank No.DQ341279) and the nucleotide homology with other Hp strains on the GenBank was 94%-98%. The molecular weight of the recombinant napA-pGEX-4T-1 expressed in E.coli was 44 kDa. 3 of 29 anti-Hp mAbs were against NAP. Western blot analysis proved that the recombinant NAP was specifically recognized by the sera of Hp-infected patients. CONCLUSION: The recombinant NAP has original immunoreaction. It is of great value to clinical sero-diagnosis and vaccine study of Hp.

Selection of SARS-coronavirus-specific B cell epitopes by phage peptide library screening and evaluation of the immunological effect of epitope-based peptides on mice.

Antibodies to SARS-Coronavirus (SARS-CoV)-specific B cell epitopes might recognize the pathogen and interrupt its adherence to and penetration of host cells. Hence, these epitopes could be useful for diagnosis and as vaccine constituents. Using the phage-displayed peptide library screening method and purified Fab fragments of immunoglobulin G (IgG Fab) from normal human sera and convalescent sera from SARS-CoV-infected patients as targets, 11 B cell epitopes of SARS-CoV spike glycoprotein (S protein) and membrane protein (M protein) were screened. After a bioinformatics tool was used to analyze these epitopes, four epitope-based S protein dodecapeptides corresponding to the predominant epitopes were chosen for synthesis. Their antigenic specificities and immunogenicities were studied in vitro and in vivo. Flow cytometry and ELISPOT analysis of lymphocytes as well as a serologic analysis of antibody showed that these peptides could trigger a rapid, highly effective, and relatively safe immune response in BALB/c mice. These findings might aid development of SARS diagnostics and vaccines. Moreover, the role of S and M proteins as important surface antigens is confirmed.

[Variability analysis of S2 gene of SARS-CoV].

OBJECTIVE: To determine the sequence of S2 gene of SARS-associated coronavirus (SARS-CoV) GD322 and analyze the phyletic evolution of S2 gene. METHOD: S2 gene fragment was amplified from SARS-CoV GD322 genome with RT-PCR and ligated to pGEM-T vector for sequence analysis after transformation of the plasmid into E. coli DH5a. The variability of S2 genes and S2 proteins from 12 strains isolated in the early, intermediate and advanced stages of the SARS outbreak were analyzed and the phylogenetic tree was constructed with Lasergene, Clustal X, DNAman and Treeview. T cell antigen epitopes of S2 protein were predicted on the basis of Internet database. RESULT: With the epidemic spread of SARS-CoV, the S2 genes of the virus tended to become stable. Homology of S2 genes of SARS-CoV isolated in advanced stage of the outbreak reached 99.9%. Prediction of T cell antigen epitope showed that mutation at the 57th amino acid effected T cell antigen epitope. CONCLUSION: S2 gene of GD322 SARS-CoV is relatively stable during the epidemic spread of the virus, and mutation at the 57th amino acids of S2 protein may affect the T cell antigen epitope.