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Construction and expression of recombinant uricase?expressing genetically engineered bacteria and its application in rat model of hyperuricemia.

ABSTRACT: At present, the treatment of hyperuricemia is designed primarily to decrease the production of uric acid using xanthine oxidase inhibitors; however, the therapeutic effect is not satisfactory. Therefore, the key to the successful treatment of hyperuricemia is to increase the excretion of uric acid. The aim of present study was to construct uricase?expressing genetically engineered bacteria and analyze the effects of these engineered bacteria on the lowering of uric acid levels in a rat model of hyperuricemia. The uricase expression vector was constructed by gene recombination technology and transfected into Escherichia coli. The expression and activity of uricase were analyzed by SDS?PAGE analysis and Bradford assay. The water consumption, food intake, body weight, eosinophil count and intestinal histology, in addition to the levels of serum uric acid (SUA) and allantoin in the feces of the rats, were assessed. The intestinal contents of the rats were analyzed by 16S rDNA sequencing technology. The results demonstrated that uricase?expressing genetically engineered bacteria secreted active uricase. All rats exhibited a natural growth trend during the entire experiment, and the SUA of hyperuricemic rats treated with uricase?expressing engineered bacteria was significantly decreased. In conclusion, these results indicate that uricase secreted by recombinant uricase?expressing genetically engineered bacteria served an important role in decreasing SUA levels in a rat model of hyperuricemia.

SUBMITTER: Cai L

PROVIDER: S-EPMC7138262 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature

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Construction and expression of recombinant uricase‑expressing genetically engineered bacteria and its application in rat model of hyperuricemia.

Cai Liming L Li Qin Q Deng Yongbing Y Liu Xianjun X Du Weihong W Jiang Xue X

International journal of molecular medicine 20200224 5

At present, the treatment of hyperuricemia is designed primarily to decrease the production of uric acid using xanthine oxidase inhibitors; however, the therapeutic effect is not satisfactory. Therefore, the key to the successful treatment of hyperuricemia is to increase the excretion of uric acid. The aim of present study was to construct uricase‑expressing genetically engineered bacteria and analyze the effects of these engineered bacteria on the lowering of uric acid levels in a rat model of ...[more]

PMID: 32323736

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Project description:BackgroundPorcine reproductive and respiratory syndrome virus (PRRSV) continues to be an important problem for the swine industry. Inactivated vaccines and modified-live virus vaccines are widely used in the field; however, the efficacy of these PRRSV vaccines is suboptimal due to poor immunogenicity. Granulocyte-macrophage colony stimulating factor (GM-CSF) has been extensively used as an effective genetic and protein adjuvant to enhance the efficiencies vaccines expressing tumor or pathogen antigens. The purpose of this study was to determine if GM-CSF could increase the efficiency of PRRSV vaccine.MethodsThe GM-CSF gene was inserted in the HuN4-F112 vaccine strain by overlap PCR. The expression of GM-CSF by the recombinant virus was confirmed with methods of indirect immunofluorescent assay (IFA) and Western blotting. The stability of recombinant virus was assessed by cDNA sequence and IFA after 20 passages. To detect the biological activity of GM-CSF expressed by the recombinant virus, bone marrow-derived dendritic cells (BMDCs) were isolated and co-cultured with the recombinant virus or parental virus and the surface phenotypes of BMDCs were examined by flow cytometric analysis. The cytokines secreted by BMDCs infected with PRRSV, or treated with LPS, GM-CSF or medium alone were evaluated by ProcartaPlexTM Multiplex Immunoassays and qRT-PCR.ResultsA novel modified-live PRRSV vaccine strain expressing GM-CSF (rHuN4-GM-CSF) was successfully constructed and rescued. The GM-CSF protein was stable expressed in recombinant virus-infected cells after 20 passages. Analysis of virus replication kinetics showed that the novel vaccine strain expressing GM-CSF had a similar replication rate as the parental virus. In vitro studies showed that infection of porcine BMDCs with rHuN4-GM-CSF resulted in increased surface expression of MHCI+, MHCII + and CD80/86+ that was dependent on virus expressed GM-CSF. The expression of representative cytokines was significantly up-regulated when BMDCs were incubated with the recombinant GM-CSF expressing virus.ConclusionsOur results indicated that the expression of GM-CSF during infection with a vaccine strain could enhance the activation of BMDCs and increase cytokine response, which is expected to result in higher immune responses and may improve vaccine efficacy against PRRSV infection.

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Construction and expression of recombinant uricase?expressing genetically engineered bacteria and its application in rat model of hyperuricemia.

Publications

Construction and expression of recombinant uricase‑expressing genetically engineered bacteria and its application in rat model of hyperuricemia.

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