In order to develop a recombinant attenuated Salmonella typhimurium as oral live vaccine vector, we constructed recombinant plasmid pYA-sopENt100 by replacing the trc promoter with the sopE promoter and secretion signal sequence sopENt100 of Salmonella typhimurium on the basis of plasmid pYA3493. Then, the complementary plasmid pYA-sopENt100 was transformed into ΔcrpΔasdSL1344 by electroporation to generate attenuated Salmonella typhimurium type III secretion system ΔcrpΔasdSL1344 (pYA-sopENt100). We further characterized ΔcrpΔasdSL1344 (pYA-sopENt100). We also constructed a recombinant strain ΔcrpΔasdSL1344 (pYA-sopENt100-egfp) that harbored the reporter gene-enhanced green fluorescent protein (egfp) gene. Vero cells were infected with ΔcrpΔasdSL1344 (pYA-sopENt100-egfp) and the ability of delivery foreign antigens was tested via Western blotting analysis. The results of PCR, enzyme digestion and sequencing showed that the ΔcrpΔasdSL1344 (pYA-sopENt100) type III secretion system was constructed successfully. The serotype of ΔcrpΔasdSL1344 (pYA-sopENt100) was identical to ΔcrpΔasdSL1344 and SL1344. Compared with wild strain SL1344, the biochemical characteristics of ΔcrpΔasdSL1344 (pYA-sopENt100) had obvious change, but it was basically the same with ΔcrpΔasdSL1344. The growth speed was much slower than that of the wild strain SL1344. The chicken virulence test (LD₅₀) showed that the virulence of ΔcrpΔasdSL1344 (pYA-sopENt100) was 7×10⁴ times lower than SL1344. In addition, we observed the 37 kDa SopENt100-egfp protein in the cultured supernatant of ΔcrpΔasdSL1344 (pYA-sopENt100-egfp) strain by Western blotting analysis. However, both the 37 kDa SopENt100-egfp protein and 27 kDa EGFP protein were detected in ΔcrpΔasdSL1344 (pYA-sopENt100-egfp)-infected Vero cells. These results demonstrated that the recombinant Salmonella typhimurium type III secretion system ΔcrpΔasdSL1344 (pYA-sopENt100) was successfully constructed, and it should be used as a live vaccine vector for expressing foreign genes.
Animals ; Bacterial Proteins ; genetics ; Cercopithecus aethiops ; Mice ; Plasmids ; Promoter Regions, Genetic ; Salmonella typhimurium ; genetics ; Type III Secretion Systems ; genetics ; Vaccines, Attenuated ; genetics ; Vero Cells ; Virulence

This study is designed to evaluate antioxidant and antigenotoxic activities of corn tassel extracts (CTTs). The major bioactive components of CTTs include flavonoid, saponin and polysaccharide. The antioxidant properties of the three bioactive components of CTTs were investigated by Ferric Reducing Antioxidant Property (FRAP) and 1, 1-diphenyl-2-picrylhydrazyl (DPPH) assays. The activities of the extracts were determined by assessing the inhibition of mutagenicity of the direct-acting mutagen fenaminosulf, sodium azide, and indirect-acting mutagen 2-aminofluorene using the Ames test (strains TA98 and TA100). The results showed that the extraction rates of flavonoid, saponin, and polysaccharide from the dried corn tassels were 1.67%, 2.41% and 4.76% respectively. DPPH and FRAP assay strongly demonstrated that CTTs had antioxidant properties. CTTs at doses of 625, 1250 and 2500 μg per plate reduced 2-aminofluorene mutagenicity by 12.52%, 28.76% and 36.49% in Salmonella typhimurium TA98 strain assay respectively and by 10.98%, 25.27% and 37.83%, at the same doses in Salmonella typhimurium TA100 assay system, respectively. 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay showed that the different concentrations of CTTs inhibited the proliferation of MGC80-3 cells in a dose-dependent manner (P<0.01). It is concluded that these integrated approaches to antioxidant and antigenotoxicity assessment may be useful to study corn tassel as a natural herbal material.
Antimutagenic Agents ; pharmacology ; Antioxidants ; pharmacology ; Biphenyl Compounds ; antagonists & inhibitors ; metabolism ; Cell Line, Tumor ; Cell Survival ; drug effects ; Flavonoids ; pharmacology ; Fluorenes ; pharmacology ; Free Radical Scavengers ; pharmacology ; Humans ; Inflorescence ; chemistry ; Mutagens ; pharmacology ; Picrates ; antagonists & inhibitors ; metabolism ; Plant Extracts ; pharmacology ; Polysaccharides ; pharmacology ; Salmonella typhimurium ; drug effects ; genetics ; Saponins ; pharmacology ; Zea mays ; chemistry

OBJECTIVETo study the effection of suppression murine melanoma growth by Intratumor injection of recombinant attenuated salmonella carrying heat shock protein 70 and herpes simplex virus thymidine kinase genes.

METHODSPlasmids PCMV-mtHSP70-IRES-TK were electro-transferred into salmonella typhimurium SL7207 to construct recombinant salmonella typhimurium. In vivo, Recombinant bacteria were injected into the mouse melanoma and the antitumor effection was observed. The survival period was recorded and safety analysis for this vaccine in each group.

RESULTSIn vivo, the mtHSP70/HSV-tk recombinant bacteria can suppress tumor growth significantly and extend survival. After recombinant Salmonella, 10(9) CFU/mL, was administered as an intratumoral injection, No diarrhea were observed. During therapy, body weight did not change markedly.

CONCLUSIONResults of the animal experiment suggests intratumor injection of recombinant attenuated salmonella typhimurium containing mtHSP70 and HSV-tk genes, has targeting ability against B16 tumor cell and could significantly inhibit tumor growth .

Animals ; Bacterial Proteins ; genetics ; immunology ; Cancer Vaccines ; genetics ; immunology ; pharmacology ; Genetic Therapy ; methods ; HSP70 Heat-Shock Proteins ; genetics ; immunology ; Melanoma, Experimental ; microbiology ; pathology ; therapy ; Mice ; Mice, Inbred C57BL ; Mycobacterium tuberculosis ; genetics ; Salmonella typhimurium ; genetics ; immunology ; Simplexvirus ; enzymology ; genetics ; Skin Neoplasms ; therapy ; Thymidine Kinase ; genetics ; immunology ; Vaccines, Attenuated ; genetics ; immunology ; pharmacology ; Vaccines, DNA ; genetics ; immunology ; pharmacology

Th-2-biased immune responses are known to play a key role in the pathogenesis of atopic dermatitis. In particular, the macrophage-derived chemokine CCL22 is directly implicated in Th-2-associated skin inflammatory reactions, and its levels are significantly elevated in serum and are correlated with disease severity in atopic dermatitis. In this study, we tested the development of genetic therapeutic options to treat atopic dermatitis using bacteria expressing miRNA. We constructed a recombinant strain of Salmonella typhimurium expressing CCL22 miRNA (ST-miRCCL22) for the in vivo knockdown of CCL22. The CCL22 gene was downregulated with CCL22 miRNA in activated lymphocytes. In mice with a cutaneous disease similar to atopic dermatitis, interleukin-4 was inhibited and interferon-gamma was induced after treatments with ST-miRCCL22. Furthermore, CCL22 levels were suppressed in the atopic mice treated with ST-miRCCL22. These results suggest that ST-miRCCL22 may be an effective genetic agent for treating atopic dermatitis.
Animals ; Cell Line ; *Chemokine CCL22/genetics ; Cytokines/blood ; Dermatitis, Atopic/pathology ; Disease Models, Animal ; Female ; Gene Expression Regulation/drug effects ; Gene Silencing ; Immunoglobulin E/blood ; Mice ; *MicroRNAs/genetics/pharmacology ; *Organisms, Genetically Modified/genetics ; *Salmonella typhimurium/genetics/metabolism ; Skin/*drug effects/pathology

The fusion protein (F) gene of Newcastle disease virus was amplified by polymerase chain reaction (PCR) from the recombinant plasmid pVAX1-F, and subcloned into eukaryotic expression vector pmcDNA3. 1+. The F gene was identified by sequencing. The recombinant plasmid was transformed into attenuated Salmonella typhimurium SL7207, and the recombinant was designated as SL7207 (pmcDNA3. 1-F). In vitro and in vivo experiments showed that the plasmid stability of pmcDNA3. 1-F was apparently higher than that of pcDNA3. 1-F in SL7207. In order to compare the immune response induced by these two re combinant bacteria, BALB/c mice were immunized orally with them at the dosage of 2 x 10(9) CFU respectively. Both SL7207(pcDNA3. 1-F) and SL7207(pmcDNA3. 1-F) initiated F-specific serum and mucosal antibodies in immunized mice. Furthermore, 4-day-old SPF chickens were immunized with SL7207(pcDNA3. 1-F) and SL7207(pmcDNA3. 1-F) at the dosage of 5 x 10(9) CFU and boosted two weeks later with the same dosage. Humoral and intestinal mucosal immune responses were observed and their levels were significantly higher than that of negative and positive controls. The result of protective efficacy showed that the chickens immunized with SL7207(pmcDNA3. 1-F) had the protective rate of 70.0%, higher than that of the SL7207 (pcDNA3. 1-F) with 50.0%. In summary, the DNA vaccine delivered by attenuated Salmonella typhimurium has good immunogenicity. A novel mucosal DNA vaccine has been developed and could be useful for controlling the infection and epidemic of Newcastle disease in the poultry.
Animals ; Chickens ; Female ; Immunization ; Mice ; Mice, Inbred BALB C ; Newcastle disease virus ; immunology ; Plasmids ; Salmonella typhimurium ; genetics ; Vaccines, Attenuated ; immunology ; Vaccines, DNA ; immunology ; Viral Vaccines ; immunology

Cells, Cultured ; Coal ; toxicity ; Dust ; Humans ; In Vitro Techniques ; Mutagenicity Tests ; Mutagens ; toxicity ; Mutation ; drug effects ; Salmonella typhimurium ; drug effects ; genetics ; Sister Chromatid Exchange ; drug effects

Hepatitis C virus (HCV) core protein is considered to be an attractive candidate for development of protective HCV vaccines. However, this protein may attenuate the induction of systemic immune responses due to its immunomodulatory properties. In this study, we constructed a HCV core gene-containing eukaryotic expression plamid pCI-C, and an in vivo-inducible prokaryotic expression plasmid pZW-C, and transformed the recombinant plasmids into an attenuated Salmonella typhimurium aroA strain SL7207. The resulting bacterial strains SL7207/pCI-C and SL7207/pZW-C were used to orally immunize BALB/c mice, and the immune responses specific to HCV core protein were assessed. Immunization with the recombinant bacteria SL7207/pCI-C led to a persistent drop in percentage of CD3 CD4 T cells, and induced a weak anti-core IgG production. Splenocytes from SL7207/pCI-C immunized mice developed a relatively weak proliferation response and inferior cytotoxic activity compared to those from the mice immunized with bacteria SL7207/pZW-C. Boost immunization with SL7207/ pCI-C yielded limited improvement in immune strength, while the boost with bacteria SL7207/pZW-C significantly enhanced the immune response. These results suggest that de novo synthesis of native HCV core protein may blunt the induction of immune responses. Attenuated S. typhimurium carrying HCV core protein could efficiently activate systemic cellular and humoral responses, and may be a promising strategy for the development of core-based HCV vaccines.
Animals ; Hepacivirus ; genetics ; immunology ; Mice ; Plasmids ; genetics ; Salmonella typhimurium ; genetics ; metabolism ; Vaccines, DNA ; immunology ; Viral Core Proteins ; genetics ; immunology ; Viral Hepatitis Vaccines ; genetics ; immunology

OBJECTIVETo investgate the metabolism of terephthalic acid (TPA) in rats and its mechanism. Methods Metabolism was evaluated by incubating sodium terephthalate (NaTPA) with rat normal liver microsomes, or with microsomes pretreated by phenobarbital sodium, or with 3-methycholanthrene, or with diet control following a NADPH-generating system. The determination was performed by high performance liquid chromatography (HPLC), and the mutagenic activation was analyzed by umu tester strain Salmonella typhimurium NM2009. Expression of CYP4B1 mRNA was detected by RT-PCR. Results The amount of NaTPA (12.5-200 micromol x L(-1)) detected by HPLC did not decrease in microsomes induced by NADPH-generating system. Incubation of TPA (0.025-0.1 mmol x L(-1)) with induced or noninduced liver microsomes in an NM2009 umu response system did not show any mutagenic activation. TPA exposure increased the expression of CYP4B 1 mRNA in rat liver, kidney, and bladder.

CONCLUSIONLack of metabolism of TPA in liver and negative genotoxic data from NM2009 study are consistent with other previous short-term tests, suggesting that the carcinogenesis in TPA feeding animals is not directly interfered with TPA itself and/or its metabolites.

Animals ; Aryl Hydrocarbon Hydroxylases ; genetics ; metabolism ; Gene Expression Regulation, Enzymologic ; drug effects ; Genes, Bacterial ; genetics ; Kidney ; enzymology ; Liver ; enzymology ; Male ; Microsomes, Liver ; drug effects ; enzymology ; Mutagenicity Tests ; Phthalic Acids ; pharmacokinetics ; toxicity ; RNA, Messenger ; metabolism ; Rats ; Rats, Sprague-Dawley ; Salmonella typhimurium ; genetics ; Urinary Bladder ; enzymology ; beta-Galactosidase ; metabolism

OBJECTIVETo evaluate the feasibility of oral cytokine gene therapy against tumor using live attenuated Salmonella as a vector.

METHODSA live attenuated AraA- autotrophic mutant of Salmonella typhimurium (SL3261) was used as carrier for eukaryotic expression vectors EGFPN1 and pCMVmIL-12 administered orally in BALB/c and C57BL/6 mice. After 6 weeks, the mice were challenged with 4T1 or Lewis tumor cells, respectively, and flow cytometry and confocal microscopy were used to detect the expression of green fluorescence protein (GFP) in the tissues. PCR and ELISA were performed to detect the integration and expression of mIL-12 gene, and the survival time of the mice was also recorded.

RESULTSGFP expression and mIL-12 gene integration could be detected in the liver, spleen, intestinal, kidney and tumor tissues of the mice. The serum level of mIFN-gamma, mIL-12 increased significantly in mice with oral mIL-12 administration (P<0.05), which resulted in the survival prolongation of the mice as compared with the control mice (P<0.05).

CONCLUSIONOral gene therapy using live attenuated Salmonella can be potentially a simple, effective and above all, safe means for tumor treatment.

Administration, Oral ; Animals ; Carcinoma, Lewis Lung ; therapy ; Flow Cytometry ; Genetic Therapy ; methods ; Genetic Vectors ; administration & dosage ; genetics ; metabolism ; Green Fluorescent Proteins ; genetics ; metabolism ; Interleukin-12 ; genetics ; metabolism ; Mammary Neoplasms, Animal ; therapy ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Microscopy, Confocal ; Neoplasms, Experimental ; therapy ; Salmonella typhimurium ; genetics ; immunology ; metabolism ; Vaccines, Attenuated ; immunology

OBJECTIVETo increase the immune effect of gene vaccine, T7 RNA polymerase was used to establish a system of cytoplasmic expression.

METHODS(1) The plasmid pT7 EMCVP1, including T7 promoter sequence, 5'-untranslated sequence of encephalomyocarditis (EMC) virus, VP1 sequence of coxsackievirus B3 (CVB3), was cotransfected with the plasmid pAR 3132, which codes for the T7 RNA polymerase, into HeLa cells and murine peritoneal macrophages. (2) The plasmid pT7 EMCVP1 and pAR 3132 were respectively transformed into the attenuated Salmonella typhimurium SL 7207. The two kinds of transformed bacteria were coinfected into murine peritoneal macrophages.

RESULTS(1) The target antigen VP1 in the cytoplasm was about 2-4-fold higher than that of pcDNA3 VP1 singly transfected. (2) After the murine peritoneal macrophages were coinfected by two kinds of transformed bacteria, the target antigen VP1 could also be detected.

CONCLUSIONThe pT7 EMCVP1 and pAR 3132 could be expressed in the cytoplasm of HeLa cells and murine peritoneal macrophages and the amount of the antigen VP1 increased remarkably as compared with that of pcDNA3 VP1 singly transfected.

Animals ; Bacteriophage T7 ; genetics ; Capsid Proteins ; genetics ; metabolism ; Cytoplasm ; metabolism ; Electrophoresis, Polyacrylamide Gel ; Enterovirus B, Human ; genetics ; Gene Expression ; HeLa Cells ; Humans ; Macrophages, Peritoneal ; cytology ; metabolism ; Mice ; Plasmids ; genetics ; Promoter Regions, Genetic ; genetics ; Salmonella typhimurium ; genetics ; Transfection