[Show abstract][Hide abstract] ABSTRACT: To investigate the surface display of the anthrax protective antigen (PA) on attenuated Bacillus anthracis, a recombinant B. anthracis strain, named AP429 was constructed by integrating into the chromosome a translational fusion harboring the DNA fragments encoding the cell wall-targeting domain of the S-layer protein EA1 and the anthrax PA. Crerecombinase action at the loxP sites excised the antibiotic marker. Western blot analysis, fluorescence-activated cell sorting and immunofluorescence analysis confirmed that PA was successfully expressed on the S-layer of the recombinant antibiotic marker-free strain. Notwithstanding extensive proteolytic degradation of the hybrid protein SLHs-PA, quantitative ELISA revealed that approximately 8.1 × 106 molecules of SLHs-PA were gained from each Bacillus cell. Moreover, electron microscopy assay indicated that the typical S-layer structures could be clearly observed from the recombinant strain micrographs.
Full-text · Article · Dec 2014 · World Journal of Microbiology and Biotechnology
[Show abstract][Hide abstract] ABSTRACT: To investigate the association between the tag single nucleotide polymorphisms (TagSNPs) of NOD1 and NOD2 and the risk of developing gastric cancer.
We conducted a hospital-based case-control study including 296 incident gastric cancer patients and 160 gastritis controls. Eight TagSNPs in the NOD1 and NOD2 genes were selected from the Hapmap database using the haploview software and genotyped by the Sequenom MassArray system. The serum levels of anti-Helicobacter pylori (H. pylori) IgG were measured by enzyme-linked immunosorbent assay to indicate H. pylori infection. The odds ratios (OR) and 95% confidence intervals (CI) were calculated by unconditional logistic regression, including sex and age as confounding factors.
The NOD1 rs2907749 GG genotype showed a decreased risk for gastric cancer (OR 0.50, 95% CI: 0.26-0.95, P = 0.04) while the rs7789045 TT genotype showed an increased risk (OR 2.14, 95% CI: 1.20-3.82, P = 0.01). An elevated susceptibility to gastric cancer was observed in the subjects with H. pylori infection and the NaOD1 rs7789045 TT genotype (OR 2.05, 95% CI: 1.07-3.94, P = 0.03) or the NOD2 rs7205423 GC genotype (OR 2.52, 95% CI: 1.05-6.04, P = 0.04). Haplotype analysis suggested that the distribution of AGT (rs2907749, rs2075820 and rs7789045) in NOD1 between the cases and control groups was significantly different (P corrected: 0.04), and the diplotype AGT/AGT was associated with an elevated gastric cancer risk (OR 1.98, 95% CI: 1.04-3.79, P = 0.04). The association of the NOD1 rs7789045 TT genotype and the diplotype AGT/AGT was significant with H. pylori-related diffuse-type gastric cancer (OR 3.00, 95% CI: 1.38-6.53, P = 0.01; OR 4.02, 95% CI: 1.61-10.05, P < 0.01, respectively).
Genetic polymorphisms in NOD1 and NOD2 may interact with H. pylori infection and may play important roles in promoting the development of gastric cancer in the Chinese population.
Full-text · Article · May 2012 · World Journal of Gastroenterology
[Show abstract][Hide abstract] ABSTRACT: Extracellular antigen 1 (EA1), a major component of the Bacillus anthracis surface layer (S-layer), was used as a fusion partner for the expression of heterologous antigen. A recombinant B. anthracis strain was constructed by integrating a translational fusion harboring the DNA fragments encoding the cell wall–targeting
domain of the S-layer protein EA1 and the 20-kDa N-terminal fragment of anthrax protective antigen (PA20) into the chromosome.
A thermosensitive plasmid expressing Cre recombinase was introduced at a permissive temperature to remove the antibiotic marker.
Cre recombinase action at the loxP sites excised the spectinomycin resistance cassette. The final derivative strains were
analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, Western blot analysis, and immunofluorescence analysis.
PA20 was successfully expressed on the S-layer of the recombinant antibiotic marker-free strain. Guinea pigs were immunized
with the attenuated recombinant B. anthracis strain, and the bacilli elicited a humoral response to PA20. This antibiotic marker-free strain and the correlative experiment
method may have potential applications for the generation of a live attenuated anthrax vaccine.
–Surface display–Cre–loxP system–Vaccine vehicle
Full-text · Article · Nov 2011 · World Journal of Microbiology and Biotechnology
[Show abstract][Hide abstract] ABSTRACT: To identify linear B-cell epitopes of urease B (UreB), a series of 19 partially overlapping fragments of the UreB gene were expressed. Three MAbs against UreB of Helicobacter pylori (H. pylori), A1H10, A3C10, and B3D9, were tested for their reactivity to the truncated proteins by Western blot and enzyme-linked immunosorbent assay (ELISA). Three linear B-cell epitopes were identified covering a stretch of 15 amino acid (aa) residues and localized in the aa regions 158-172, 181-195, and 349-363 of UreB. ELISA also showed that the three synthetic peptides containing epitope sequences (UP32: GGGTGPADGTNATTI, UP35: WMLRAAEEYSMNLGF, and UP38: TLHDMGIFSITSSDS) were recognized by the corresponding MAbs and H. pylori positive sera from H. pylori infected patients. Mice immunized with glutathione S-transferase (GST) fusion peptides showed that epitope-specific antibodies were capable of inhibiting urease enzymatic activity. These results should be useful in clinical applications and highlight the potential importance of these epitopes as the targets for development of epitope-based vaccines against H. pylori.
[Show abstract][Hide abstract] ABSTRACT: AIM: To construct a CS3 fimbria-displayed random peptide library on the Escherichia coli cell surface. METHODS: Firstly, a new display vector with double restriction sites was reconstructed, and the ability of the new vector to form CS3 fim- briae on E. coli surface was confirmed by West - ern blot and transmission electron microscopy. Then two oligonucleotides were synthesized, one of which contained a random oligonucle- otide encoding region (NNK)10 and the other was its complement. The two synthesized oligo- nucleotides was annealed and then extended by Klenow Fragment. The double-stranded oligo- nucleotides were digested by XhoⅠ and BamH Ⅰ and purified by PAGE, then inserted into the digested display vector. The ligation product was purified and electroporated into XL1-Blue. Ten randomly selected clones were sequenced and the sequences were analyzed. RESULTS: A library with diversity of 1.8×10 6
Preview · Article · Jan 2006 · World Chinese Journal of Digestology
[Show abstract][Hide abstract] ABSTRACT: To construct and evaluate a polyvalent recombinant vaccine strain Shigella flexneri 2a T32 against enterotoxigenic E.coli (ETEC).
By using a host-plasmid balanced lethal system based on asd gene, a polyvalent recombinant strain was constructed to highly express CS3 and regularly express fusion enterotoxin of LTB subunit and mutant ST (LTB/STm) in a vaccine strain Shigella flexneri 2a T32 with specific deletion of asd gene. Fimbria CS3 was observed by immunofluorescence and electron microscopy assay. The security of LTB/STm was examined by ileal loop assay and suckling mouse assay. To evaluate this new candidate vaccine, it was compared with a previous vaccine strain in plasmid and protein level, growth assay and immunogenicity in Balb/c mice.
The newly constructed vaccine expressed CS3 and grew better than the previously constructed vaccine except for the lower expression of LTB/STm. Serum IgG and mucosal IgA against CS3, LTB, ST, and host lipopolysaccharide (LPS) were produced after immunization of Balb/c mice by oral route with the new strain. The titers were not significantly different from the Balb/c mice with the previous strain.
This novel candidate diarrheal vaccine can effectively induce serum and mucosal antibody responses against ETEC and Shigella.
Preview · Article · Jul 2005 · World Journal of Gastroenterology
[Show abstract][Hide abstract] ABSTRACT: Bacillus anthracis is the causative organism of the potentially fatal disease anthrax, and the used vaccines have some disadvantages. There are new developments appeared for the Bacillus anthracis in recent years, such as anti-PA antibody kills the spore of Bacillus anthracis, mucosal immunization induces immune responses in both systemic and secretory immune compartments, Poly (gamma-D-PGA) protein induce IgG antibodies to the vegetative bacteria, new pathogens were found by genomic analysis. The DNA vaccine and live vector vaccine will be the next generation vaccines for anthrax. It will have a shorter immunization schedule and will be greater protective efficacy than before.
No preview · Article · Mar 2005 · ACTA MICROBIOLOGICA SINICA
[Show abstract][Hide abstract] ABSTRACT: Among the known colonization factors of enterotoxigenic Escherichia coli (ETEC), CFA/I and CS6 (the common antigen in the CFA/IV fimbrial antigens ) are two of the most prevalent fimbriae found in clinical isolates but are never expressed by the same wild-type strains. In this study, CFA/I and CS6 of ETEC were co-expressed in Shigella flexneri 2a T32 derivative strain FWL01 by using a host-plasmid lethal balancing system based on asd gene. The results indicate that the recombinant plasmid carrying CFA/I and CS6 could be stably integrated in FWL01. Expression of the two antigens did not interfere the host growth. The results of immunofluorescence analysis showed that CFA/I and CS6 were localized on the surface of the strain FWL01. In Balb/c mice orally immunized with the recombinant strain, the immune responses against CFA/I and CS6 were observed. Those observations show the feasibility of a multivalent vaccine expressing different fimbrial antigens in attenuated Shigella flexneri.
No preview · Article · Dec 2003 · Sheng wu hua xue yu sheng wu wu li xue bao Acta biochimica et biophysica Sinica