The effects of attenuated Salmonella carrying IL-2/NK4 double gene on immune function in mice / 中国生物制品学杂志

@#Objective To study the effects of attenuated Salmonella(Ty21a-pIRES-IL-2-NK4,TPIN)carrying interleukin-2(IL-2)/4-kringle antagonist of hepatocyte growth factor(NK4)double gene on humoral and cellular immune function.Methods Eighteen BALB/c mice,half male and half female,were randomly divided into control group(1. 5 mL 10%NaHCO3 by gastric tube feeding),Ty21a group(0. 1 mL Ty21a by gastric tube feeding)and TPIN group(0. 1 mL TPIN by gastric tube feeding),with 6 mice in each group. The immunization was boosted twice 7 d after the initial immunization. At 21d after administration,the blood samples were collected from eyeballs and the serum was separated,which was detected for the serum IgG antibody level by ELISA. The thymus and spleen of mice were isolated aseptically,and the spleen cells were stimulated by Ty21a and TPIN respectively in vitro. After 72 h,the proliferation ability of spleen cells was measured by CCK-8 assay,and the expression level of cytokines in spleen cells was detected by ELISA. The spleen and thymus were weighed,the spleen and thymus indexes were analyzed,and HE staining was performed.Results Compared with the control group and Ty21a group,the serum IgG level(F = 111. 74,P < 0. 01)and the contents of IFNγ,IL-4 and IL-10 in spleen cell supernatant(F = 38. 21,11. 37 and 26. 92,respectively,each P < 0. 05)increased significantly,as well as the spleen and thymus indexes(F = 10. 419 and 5. 859,respectively,each P < 0. 05)showed significant increase. In mice of Ty21a and TPIN group,the thymus cortex widened,lymphocytes increased,and there was mild inflammatory reaction;the white pulp and lymphocytes in spleen increased with neutrophil infiltration.Conclusion TPIN has a good immune protective effect,and can significantly stimulate the body to produce humoral immunity and cellular immunity,which may have a good therapeutic effect on tumors.

Therapeutic effect of recombinant Salmonella SGN1 on melanoma in mice and its mechanism of action / 中国生物制品学杂志

@#Objective To investigate the therapeutic effect of recombinant Salmonella SGN1 on murine melanoma model and the action mechanism,and to provide reference for clinical treatment of melanoma with SGN1.Methods The recombinant Salmonella SGN1 and control strain VNP-V were co-cultured with mouse melanoma B16F10 cells,and the effect of SGN1 on the proliferation of B16F10 cells in vitro was detected by cell counting method. The B16F10 subcutaneous tumor transplantation model in mice was constructed. SGN1,VNP-V and PBS(control group)were injected into the tumor at a single dose,with five mice in each group,and the inhibitory effect of SGN1 on tumor growth was observed. The pathological changes of tumor tissues in mice were observed by HE staining. The distribution of SGN1 in tumor tissues was observed by plate counting. Flow cytometry was used to evaluate the proportion of infiltrating T lymphocyte subsets. The expression of T-cell marker CD3 was detected by immunofluorescence and immunohistochemical staining.Results Compared with the control group,recombinant Salmonella SGN1 significantly inhibited the proliferation of mouse melanoma B16F10 cells in vitro(t = 6. 935,P < 0. 01). In tumor-bearing mice,SGN1 was tumor-targeted and significantly inhibited the growth of B16F10 subcutaneous transplanted melanoma in mice(t = 7. 566,P < 0. 001). HE staining showed that SGN1 significantly induced the necrosis of subcutaneous transplanted melanoma cells in mice. The results of immunofluorescence,immunohistochemistry and flow cytometry confirmed that SGN1 significantly increased tumor-infiltrating CD3~+ T-cells(t = 11. 91,8. 873 and 5. 300,respectively,each P < 0. 01).Conclusion The recombinant Salmonella SGN1 can significantly inhibit the proliferation of B16F10 cells and exert anti-tumor effects by promoting tumor cell necrosis and elevating CD3~+ tumor-infiltrating T-cells,providing a theoretical basis for the clinical treatment of melanoma with SGN1

Prevention and inhibition of nasopharyngeal carcinoma growth by attenuated salmonella SGN1 / 中国药理学通报

Aim To study the inhibitory effect of attenuated salmonella SGN1, overexpressing methioninase, on nasopharyngeal carcinoma (NPC) and the underlying mechanism. Methods The cell proliferation, cell cycle, cell apoptosis, clony formation and migration a-bility of 5-8F, HNE-2, CNE-2 cells were measured u-sing flow cytometry assay, clone formation assay, and wound assay after the methionine restriction treatment. 5-8F, HNE-2, CNE-2 cells were infected with SGN1 at the multiplicity of infection (MOI) of 1: 100 for 5 hours, followed with the measurement of cell growth. A xenograft model was constructed by subcutaneous injection of 5-8F cells in mice to observe the inhibitory effect of SGN1 on nasopharyngeal carcinoma. Results Compared with the control group, methionine restriction significantly inhibited the proliferation, migration ability, and clone formation of nasopharyngeal carcinoma cells and blocked the G

Recombinant-attenuated Salmonella Pullorum strain expressing the hemagglutinin-neuraminidase protein of Newcastle disease virus (NDV) protects chickens against NDV and Salmonella Pullorum challenge

Newcastle disease virus (NDV) and Salmonella Pullorum have significant damaging effects on the poultry industry, but no previous vaccine can protect poultry effectively. In this study, a recombinant-attenuated S. Pullorum strain secreting the NDV hemagglutinin-neuraminidase (HN) protein, C79-13ΔcrpΔasd (pYA-HN), was constructed by using the suicide plasmid pREasd-mediated bacteria homologous recombination method to form a new bivalent vaccine candidate against Newcastle disease (ND) and S. Pullorum disease (PD). The effect of this vaccine candidate was compared with those of the NDV LaSota and C79-13ΔcrpΔasd (pYA) strains. The serum hemagglutination inhibition antibody titers, serum immunoglobulin G (IgG) antibodies, secretory IgA, and stimulation index in lymphocyte proliferation were increased significantly more (p 0.05). Moreover, the novel strain provides 60% and 80% protective efficacy against the NDV virulent strain F48E9 and the S. Pullorum virulent strain C79-13. In summary, in this study, a recombinant-attenuated S. Pullorum strain secreting NDV HN protein was constructed. The generation of the S. Pullorum C79-13ΔcrpΔasd (pYA-HN) strain provides a foundation for the development of an effective living-vector double vaccine against ND and PD.

Inhibiting effect of IL-24 combined with targeting attenuated Salmonella vector SL 7207/pBud-Vp3 on growth of gastric cancer cells / 重庆医学

Objective To investigate the inhibiting effect of interleukin (IL)‐24 combined with targeted attenuated Salmonella typhimurium vector SL7207/pBud‐VP3 on the growth of gastric cancer cells .Methods The co‐expression eukaryotic expression plasmid pBud‐VP3‐IL‐24 was constructed .The plasmid pBud‐VP3‐IL‐24 was transformed into attenuated Salmonella typhimurium SL7207 by using the high voltage electroporation for constructing the SL 7207/pBud‐VP3‐IL‐24 strain .The mouse gastric cancer transplantation tumor model was established and randomly divided into the normal saline control group ,SL7207/pBud group , SL7207/pBud‐VP3 group and SL7207/pBud‐VP3‐IL‐24 group .The tumor‐bearing mice were fed by oral administration of bacterial strain .The tumor volume was measured and the tumor inhibition rate was calculated .The expression of IL‐24 was detected by Western blotting .The levels of IFN‐γ,IL‐6 and TNF‐αin tumor tissue were detected by using RT‐PCR .The expression of Caspase‐3 and VEGF were detected by using immunohistochemistry .Results The plasmids attenuated Salmonella typhimurium vector carrying the gene IL‐24 was successfully constructed .The IL‐24 protein expression was detected in gastric cancer tissue after 14 d treatment .The tumor volume after 28 d treatment in the SL7207/pBud‐VP3‐IL‐24 group was reduced compared with the other groups ,moreover the tumor growth was significantly inhibited ,and the differences were statistically significant (P<0 .05) .RT‐PCR and immunohistochemistry results showed that IL‐24 combined with SL7207/bBud‐VP3 could significantly increase the expression levels of immune factor IL‐6 ,IFN‐γ and TNF‐αin tumor tissue ,.in addition ,up‐regulated the expression of Caspase‐3 and down‐regulated the VEGF expression(P<0 .05) .Conclu‐sion IL‐24 combined with SL7207/pBud‐VP3 can synergically play the inhibitory effect on the growth of gastric cancer cells ,its mecha‐nism is related with the tumor apoptosis promotion ,tumor vessel inhibition and immune regulation .

Construction and characterization of type III secretion system of attenuated Salmonella typhimurium / 生物工程学报

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.

Attenuated Salmonella carrying pGRIM-19-si-survivin co-expression plas-mid inhibits growth of prostate cancer subcutaneous xenografts in vivo / 中国病理生理杂志

AIM:To explore the effects of pGRIM-19-si-survivin co-expression plasmid carried by human at-tenuated Salmonella on prostate cancer subcutaneous xenograft growth in nude mice .METHODS: Prostate cancer xeno-graft model was established in nude mice .Co-expression plasmids carried by attenuated Salmonella were introduced by in-traperitoneal injection .The xenograft volumes were monitored timely .Immunohistochemical staining , RT-PCR and TUNEL assay were applied to investigate the related mechanisms that pGRIM-19-si-survivin inhibited tumor growth in vivo.RE-SULTS:Compared with psi-survivin and pGRIM-19 carried by attenuated Salmonella ( control groups ) , the tumor volumes were reduced markedly in pGRIM-19-si-survivin plasmid group .The mean shrinkage rates were 2.36 and 3.02 times. pGRIM-19-si-survivin co-expression plasmid carried by attenuated Salmonella inhibited survivin expression but strengthened GRIM-19 expression obviously (P<0.05).The mRNA expression of apoptosis-related proteins such as Bcl-xL, Stat3, cy-clin D1 and c-Myc was inhibited , and the vascular endothelial growth factor ( VEGF) mRNA and Ki67 protein were also in-hibited, but the caspase-3 mRNA expression was up-regulated ( P<0.05 ) with significant cell apoptosis .CONCLU-SION:pGRIM-19-si-survivin co-expression plasmid carried by human attenuated Salmonella inhibits the growth of prostate cancer subcutaneous xenografts in nude mice by promoting cell apoptosis and inhibiting prostatic cancer proliferation .

Construction and identification of an asdA mutant attenuated Salmonella ty phimurium / 中国人兽共患病学报

In this study ,a wild type Salmonella typhimurium (S .typhimurium) strain was isolated and identified in Hong Kong (S129) ,then the asdA gene was knocked out and replaced with kanamycin resistant gene in a Salmonella typhi-murium strain S129 using the λ RED-mediated recombination method .The constructed mutant asdAΔS129 was validated by culturing in the presence or absence of 2 ,6-diaminopimelic acid (DAP) growth in vitro and evaluating its virulence in BALB/c mice challenge assay .Therefore ,this study has demonstrated that an asdA mutant Salmonella typhimurium has been success-fully constructed .

Application of attenuated salmonella in the gene treatment of tumor / 国际肿瘤学杂志

Salmonella is a kind of intracellular invasive facultative anaerobe,maintaining its antigenicity after being attenuated.Now,it has been widely used as a transporter of DNA vaccines.Salmonella carrying related antigen gene can inhibit multiple tumors,and salmonella carrying repair gene has a clear repair effect for the organism injury after chemoradiotherapy.

Inhibitory effects of attenuated Salmonella typhimurium-mediated TRAIL and VP3 gene against gastric cancer cells / 第二军医大学学报

Objective: To investigate the inhibitory effect of eukaryotic expression vector (attenuated salmonella typhimurium) carrying tumor necrosis factor-related apoptosis inducing ligand (TRAIL) and Chicken anemia virus VP3 gene on gastric cancer cells in vitro and in vivo. Methods: The cloning vectors pBud-TRAIL, pBud-VP3, and pBud-TRAIL-VP3 were transformed into attenuated Salmonella typhimurium by electric transformation technique. The S. typhimurium-based carriers were then transfected into gastric cancer cells, line SGC-7901 after stability assay. The expression of fusion green fluorescent protein was examined using fluorescent microscopy after 24 h. MTT assay was used to examine the inhibition of cell growth. Flow cytometry was used to detect cycle distribution and apoptosis rates of cells. The expression of caspase-3 and caspase-9 was assayed by immunohistochemistry method. Salmonella typhimurium carrying recombinant plasmid was administrated orally in sarcoma-bearing mice; 8 weeks later RT-PCR was used to detect the expression of cloning vectors in tumor tissue. Meanwhile, the sizes of tumors were also determined. Results: The recombinant plasmids were stably transformed into attenuated Salmonella typhimurium, and the plasmids was satisfactorily expressed in gastric cancer cells via attenuated Salmonella typhimurium. TRAIL and VP3 inhibited the proliferation of gastric cancer cells after 48 h. Flow cytometry analysis showed that the pBud-TRAIL-VP3 obviously enhanced apoptosis rates of gastric cancer cells. TRAIL and VP3 jointly increased the expression of caspase-3 and caspase-9. In vivo study showed that TRAIL and VP3 genes were expressed in tumor tissue and could inhibit the tumor growth(P<0.05). Conclusion: Attenuated Salmonella typhimurium-mediated TRAIL and VP3 transfection of gastric cancer cells can inhibit cell growth in vitro and in vivo. The joint effect of TRAIL and VP3 is correlated with the increase of caspase-3 and caspase-9 expression.

Live-attenuated Salmonella typhimurium as a Oral Delivery Vehicle for SARS-CoV N DNA Vaccine / 中国生物工程杂志

Objective:To investigate the immunogenicity of SARS-CoV N DNA vaccine and the feasibility of live-attenuated Salmonella typhimurium as the carrier to deliver the N DNA vaccine.Method:The recombinant attenuated salmonella strain CS022 harboring the pcDNA-N DNA vaccine was constructed.And mouse was immunized with the recombinant strain via intranasal and oral routes.Cellular and humoral immune responses were assessed by ELISA,lymphocyte proliferation assays,ELISPOT and FACS.Result:The oral immunization with the transformed salmonellae elicited strong immune responses mainly including high level of N-specific antibody,a dramatic activation of IFN-?-secreting cells,a high level of lymphocyte proliferation,and a high level of activated CD8+ T cells.Conclusion:Live-attenuated Salmonella typhimurium could effectively deliver the SARS-CoV N DNA vaccine in vivo.These encouraging pre-clinical data provide a rational basis for undertaking a new immune style to investigate SARS vaccine.

Recombinant Attenuated salmonella typhimurium Haboring TIP30 and Human IFN-? Genes Inhibits the Growth of Adenoid Cystic Carcinoma in vivo / 中国肿瘤生物治疗杂志

Objective: To construct attenuated salmonella typhimurium haboring an eukaryotic co-expression plasmid encoding TIP30 and human IFN-? gene and observing its effect on the growth of adenoid cystic carcinoma. Methods: The TIP30 and human IFN-? genes were amplified by PCR and inserted into eukaryotic expression vector pCI-neofor the construction of expression plasmids pCI-TIP30 and pCI-IFN, respectively. A co-expression plasmid pCI-TIP30/IFN was constructed by linking TIP30 and human IFN-? gene using the sequence of internal ribosome binding sequence (IRES). Three recombinant expression plasmids were transformed into an attenuated AroA'autotrophic mutant of salmonella typhimurium SL7207, the resultant bacteria were used to infected murine macrophage in vitro and the expressed products were detected by Western blot and ELISA, respectively. Tumor growth was observed by oral administration of the recombinant salmonella typhimuriums to the nude mouse with adenoid cystic carcinoma. Results: Murine macrophage infected with recombinant salmonella transformed with both plasmids pCI-TIP30 and pCI-TIP30/IFN could express TIP30 protein, and murine macrophage infected with recombinant salmonella transformed with pCI-IFN or pCI-TIP30/IFN could secret human IFN-? in the culture supernatant. Attenuated salmonella typhimurium and three constructed recombinant salmonella typhimuriums all had evident inhibition onthe tumor growth in nude mouse with adenoid cystic carcinoma. Conclusion: The recombinant attenuated salmonella typhimuriums haboring plasmid pCI-TIP30, plasmidpCI-IFN and co-expressing plasmidp-CI-TIP30/IFN were successfully constructed, which could inhibit the growth of adenoid cystic carcinoma in nude mouse.

Construction of eukaryotic expression vector of attenuated Salmonella typhimurium containing hCTLA4Ig gene and expression in COS-7 cells / 第三军医大学学报

Objective To construct eukaryotic expression vector of attenuated Salmonella typhimurium containing hCTLA-4Ig cDNA and identify its expression in COS-7 cells for the further study of function in SLE models. Methods Touchdown PCR was used to amplify hCTLA-4Ig cDNA.The PCR product was ligated into the multiple clone site of eukaryotic expression vector pcDNA3.1(+) by gene recombination technique.Then the recombinated plasmid pcDNA3.1(+)-CTLA-4Ig was transfected into COS-7 cells using DOTAP.The expression of interest protein in the supernatant of the cell disruption was detected with SDS-PAGE and Western blotting.Results Restriction analysis and DNA sequence analysis showed that the CTLA-4Ig cDNA had been successfully inserted into pcDNA3.1(+) eukaryotic expression vector.The interest protein could be detected in the supernatant of cell disruption 48h after the transfection of pcDNA3.1(+)-CTLA-4Ig.This protein specifically bound with human CTLA-4 monoclonal antibody.Conclusion The eukaryotic expression vector containing hCTLA-4Ig gene was successfully constructed and bioactive interest protein could be successfully expressed in mammalian cells.