ABSTRACT
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.
Subject(s)
Animals , Antibodies , Bacteria , Chickens , Hemagglutination , HN Protein , Homologous Recombination , Immunoglobulin A, Secretory , Immunoglobulin G , Lymphocytes , Methods , Newcastle disease virus , Newcastle Disease , Plasmids , Poultry , Salmonella , Suicide , VaccinesABSTRACT
The orexin system was discovered in 1998 with two G-protein coupled receptors (GPCRs):orexin-1 (OX1R) and orexin-2 (OX2R) receptors that bind the neuropeptides orexin-A (OX-A) and orexin-B (OX-B). The causal link between the orexin system and obesity, anxiety, and sleep/wake disorders as a potential therapeutic target has drawn much attention in the field of pharmaceuticals. The developments of dual antagonism of the receptors by small molecules are clinically efficacious in the treatment of insomnia, where the most advanced molecule suvorexant has been approved by FDA in August, 2014. The small molecule orexin receptor antagonists (ORA) between January 2010 and August 2017 are summarized in this review and we focus on their chemical structures, mechanism and human clinical trials.
ABSTRACT
Objective:To develop a host-vector balanced lethal system of attenuated Salmonella typhinurium secreted effector K1 mutant,and an live vaccine vector which stably carries exogenous genes. Methods:We constructed SL1344ΔsseK1Δasd mutant strain by recombinant suicide plasmid( pREΔasd) ,and screened by two-step method,transformed pYA3493 plasmid containing the asd gene without resistance electric into the mutant strain of SL1344ΔsseK1Δasd, then the recombinant strain SL1344ΔsseK1Δasd (pYA3493) was constructed successfully. Results:The results of PCR and sequencing showed that SL1344ΔsseK1Δasd(pYA3493)was constructed successfully. Further studies had shown that the serotype of the recombinant strain was identical to the parent SL1344ΔsseK1 and wild SL1344 strains,and the mutant was stable with the recombinant Δasd gene in vitro. It was found that the re-combinant strain had displayed identical growth profile and biochemical characteristics compared with the parent SL1344ΔsseK1 strain and wild SL1344 strain. The oral challenge of bacteria in mice revealed that LD50 of the recombinant strain was 5. 24×108 CFU,and the toxicity reduced to about 0. 048%;the immunoprotective effect assay showed that the protection rate infected with wild strain of Salmonella typhimurium was 62. 5% on the 17th day post-immunization,which was identical to the parent SL1344ΔsseK1. Conclusion:These results show that the secreted effector K1 gene deleted mutant host-vector balanced lethal system of Salmonella typhimurium SL1344 strain has been successfully constructed,and genetic stability,significantly reduced virulence,which has laid a foundation for developing potential oral live vaccin vector to express foreign genes.
ABSTRACT
Sucrose non-fermenting 1-related protein kinase 2(SnRK2) plays a key role in abiotic stress signaling in plants. In this study, we cloned a SmSnRK2.4 gene belonging to subclass I of SnRK2 from Salvia miltiorrhiza by screening its transcriptome database. The SmSnRK2.4 gene contains 8 introns and 9 exons, with a 1 068 bp open reading frame encoding a polypeptide of 355 amino acids, the predicted molecular mass of which is 40.63 kDa. Prokaryotic expression of SmSnRK2.4 protein using pMAL-c2X as the expression vector displayed that the recombinant protein of SmSnRK2.4 gene in E. coli was consistent with the predicted size. A 3 000 bp promoter sequence of SmSnRK2.4 contained some stress-responsive elements and hormone-responsive elements. Quantitative real-time PCR analysis revealed that the expression of SmSnRK2.4 in root was much higher than that in stem and leaf, SmSnRK2.4 was strongly induced by PEG stress, weakly induced by ABA stress. This research provided a basis for further study of the SmSnRK2.4 gene playing the role in accumulate mechanism of secondary metabolites in S. miltiorrhiza under drought.
ABSTRACT
Objective:To develop a host-vector balanced lethal system of attenuated Salmonella typhinurium secreted effector K1 mutant,and an live vaccine vector which stably carries exogenous genes. Methods:We constructed SL1344ΔsseK1Δasd mutant strain by recombinant suicide plasmid( pREΔasd) ,and screened by two-step method,transformed pYA3493 plasmid containing the asd gene without resistance electric into the mutant strain of SL1344ΔsseK1Δasd, then the recombinant strain SL1344ΔsseK1Δasd (pYA3493) was constructed successfully. Results:The results of PCR and sequencing showed that SL1344ΔsseK1Δasd(pYA3493)was constructed successfully. Further studies had shown that the serotype of the recombinant strain was identical to the parent SL1344ΔsseK1 and wild SL1344 strains,and the mutant was stable with the recombinant Δasd gene in vitro. It was found that the re-combinant strain had displayed identical growth profile and biochemical characteristics compared with the parent SL1344ΔsseK1 strain and wild SL1344 strain. The oral challenge of bacteria in mice revealed that LD50 of the recombinant strain was 5. 24×108 CFU,and the toxicity reduced to about 0. 048%;the immunoprotective effect assay showed that the protection rate infected with wild strain of Salmonella typhimurium was 62. 5% on the 17th day post-immunization,which was identical to the parent SL1344ΔsseK1. Conclusion:These results show that the secreted effector K1 gene deleted mutant host-vector balanced lethal system of Salmonella typhimurium SL1344 strain has been successfully constructed,and genetic stability,significantly reduced virulence,which has laid a foundation for developing potential oral live vaccin vector to express foreign genes.
ABSTRACT
Neurodegenerative disease is common and frequently occurs in elderly patients. Previous studies have shown that ginsenoside Rg1 was able to inhibit senescent of brain, but the mechanism on the brain during the treatment remains elucidated. To study the mechanism of ginsenoside Rg1 in the process of anti-aging of brain, forty male SD rats were randomly divided into normal group, Rg1 normal group, brain aging model group and Rg1 brain aging model group, each group with 10 rats (brain aging model group: subcutaneous injection of D-galactose (120 mg kg(-1)), qd for 42 consecutive days; Rg1 brain aging model group: while copying the same test as that of brain aging model group, begin intraperitoneal injection of ginsenosides Rg1 (20 mg x kg(-1)) qd for 27 d from 16 d. Rg1 normal group: subcutaneous injection of the same amount of saline; begin intraperitoneal injection of ginsenosides Rg1 (20 mg x kg(-1)) qd for 27 d from 16 d. Normal: injected with an equal volume of saline within the same time. Perform the related experiment on the second day after finishing copying the model or the completion of the first two days of drug injections). Learning and memory abilities were measured by Morris water maze. The number of senescent cells was detected by SA-beta-Gal staining while the level of IL-1 and IL-6 proinflammatory cytokines in hippocampus were detected by ELISA. The activities of SOD, contents of GSH in hippo- campus were quantified by chromatometry. The change of telomerase activities and telomerase length were performed by TRAP-PCR and southern blotting assay, respectively. It is pointed that, in brain aging model group, the spatial learning and memory capacities were weaken, SA-beta-Gal positive granules increased in section of brain tissue, the activity of antioxidant enzyme SOD and the contents of GSH decreased in hippocampus, the level of IL-1 and IL-6 increased in hippocampus, while the length of telomere and the activity of telomerase decreased in hippocampus. Rats of Rg1 brain aging group had their spatial learning and memory capacities enhanced, SA-beta-Gal positive granules in section of brain tissue decreased, the activity of antioxidant enzyme SOD and the contents of GSH increased in hippocampus, the level of IL-1 and IL-6 in hippocampus decreased, the length contraction of telomere suppressed while the change of telomerase activity increased in hippocampus. Compared with that of normal group, the spatial learning and memory capacities were enhanced in Rg1 normal group, SA-beta-Gal positive granules in section of brain tissue decreased in Rg1 normal group, the level of IL-1 and IL-6 in hippocampus decreased in Rg1 normal group. The results indicated that improvement of antioxidant ability, regulating the level of proinflammatory cytokines and regulation of telomerase system may be the underlying anti-aging mechanism of Ginsenoside Rg1.
Subject(s)
Animals , Male , Rats , Aging , Brain , Ginsenosides , Pharmacology , Rats, Sprague-DawleyABSTRACT
HPV16 E7 fusion protein was expressed in E. coli BL21, and its applied value for HPV was evaluated. HPV16 E7 gene was amplified by PCR, and cloned into prokaryotic expression vector pGEX6p-1. The recombinant plasmid was transformed into E. coli BL21, and HPV16 E7 fusion was expressed through IPTG induction. The expressed product was analyzed by SDS-PAGE and Western blot, subsequently purified according to Glutathione Sepharose 4B purification procedure. An indirect ELISA with the purified fusion protein as the coating antigen was then established to detect E7 serum antibodies from mice immunized with recombinant Listeria monocytogenes delivering HPV16 E7. The results demonstrated that the soluble fusion protein was highly expressed at 25 degrees C after induction with 0.5 mM IPTG. Furthermore, the result of Western blot analysis showed that the fusion protein had good specific reaction with an anti-E7 monoclonal antibody. Indirect ELISA result confirmed that the fusion protein could detect the serum antibodies against E7 with a titer of 1:200. The expressed GST-E7 fusion protein was immunocompetent, which was useful in the research of E7 biological function and therapeutic vaccine.