Construction method of broad-spectrum antiviral recombinant salmonella

A Salmonella and construction method technology, applied in antiviral agents, microbial-based methods, recombinant DNA technology, etc., can solve the problems of body cell damage, increased risk of drug administration, and difficulty in being accepted by the market

Inactive Publication Date: 2021-06-29
YANGZHOU UNIV
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  • Abstract
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  • Claims
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Problems solved by technology

This material is difficult to be metabolized and excreted in the body, and has certain cytotoxicity, which increases the risk of administration
Injury to body cells when STING agonists are presented using cationic adjuvants
As far as the gene gun is concerned, the threshold of gene gun injection technology is high, and the cost of drug delivery is high
Poor applicability when combined with STING agonists
Especially in the field of veterinary medicine, the high cost is hardly accepted by the market
In addition, it is difficult to target innate immune cells with cationic adjuvants or gene guns. Most of these STING agonists are transported to the injection site or administration site, and the STING protein is mainly present in dendritic cells and macrophages, two natural immune cells. In immune cells, the above two drug delivery methods cannot target these two natural immune cells
This makes STING agonists much less effective

Method used

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  • Construction method of broad-spectrum antiviral recombinant salmonella
  • Construction method of broad-spectrum antiviral recombinant salmonella
  • Construction method of broad-spectrum antiviral recombinant salmonella

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Implementation 1 Construction and identification of rSC0120(pS-DacA) and rSC0119(pS-DacA);

[0031] 1.1 Construction and identification of pS-DacA plasmid;

[0032] The complete gene sequence of Listeria monocytogenes NH1 strain (Genbank ID: 1358004970) was used as a template. Design and synthesize DacA base fragments without "ACA" base sequence. The above fragment and pYA3493 plasmid were digested by EcoR I and Hind III at the same time, and the digested products were ligated by T4 ligase. The ligation product was transformed into engineering bacteria Escherichia coli rSC0002, the clone was picked, and identified by double enzyme digestion and sequencing. The double enzyme digestion identification results were as follows: figure 2 .

[0033] The correct clone was identified by double enzyme digestion and sequencing as a positive clone, named rSC0002(pS-DacA), and the pS-DacA plasmid in rSC0002(pS-DacA) was extracted for future use.

[0034] 1.2 Construction and id...

Embodiment 2

[0037] rSC0120(pS-DacA) synthesizes c-di-AMP(CDA) in vitro and releases CDA to the extracellular space The ability to induce the body to produce mucosal immunity, humoral immunity and cellular immunity has been widely used as a carrier to deliver protective antigens and nucleic acid vaccines to prevent certain infectious diseases. However, it is difficult for the protective antigen and nucleic acid recombined into the bacterial vector vaccine to pass through the bacterial cell wall and release into the host cell to play a role. In order to solve this problem, our laboratory developed a Salmonella choleraesuis lysis vector rSC0120 that can lyse and release intracellular substances in a programmed manner. In this part, the in vitro synthesis of the Salmonella choleraesuis lysis vector rSC0120 (pS-DacA) was evaluated by in vitro passage experiments. And unleash the power of CDA.

[0038] to rSC0119(ΔrelA::araC P BAD lacIΔP mazE ::TT araC P BAD mazEΔpmiΔasdA) and rSC0120(ΔrelA...

Embodiment 3

[0041] rSC0120(pS-DacA) activates the STING pathway in 3D4 / 21 cells;

[0042] 3.1 rSC0120(pS-DacA) up-regulates the expression of STING in the cGAS-knockdown porcine lung macrophage cell line 3D4 / 21CDA can activate the STING pathway and up-regulate the expression of STING protein. When Salmonella infects cells, its own DNA may up-regulate the expression of cGAS and activate the subsequent STING pathway. Therefore, in order to prove that it is the influence of the CDA-activated STING pathway catalyzed by the DacA expressed by rSC0120 (pS-DacA), rather than the effect of cGAS, the present invention knocks down the cGAS of the porcine macrophage cell line 3D4 / 21 cells by lentivirus infection , to explore the characteristics of rSC0120(pS-DacA) in activating the STING pathway. 3D4 / 21 cells knocked down cGAS were plated in a 24-well plate, 2.5×10 per well 6 cells. After the cells adhered tightly to the wall, use 1 MOI of recombinant Salmonella choleraesuis rSC0120(pS-DacA) and r...

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Abstract

The invention relates to a construction method of broad-spectrum antiviral recombinant salmonella. The construction method comprises the following steps: constructing a recombinant plasmid pS-DacA carrying adenylate cyclase, and introducing the plasmid into a salmonella choleraesuis cracking vector rSC0120 to form a recombinant salmonella choleraesuis cracking vector rSC0120 (pS-DacA). When the DacA is expressed in the salmonella choleraesuis cracking vector, the ATP is catalyzed to form c-di-AMP. After oral administration, the recombinant salmonella choleraesuis cracking vector rSC0120 (pS-DacA) enters host immune cells, c-di-AMP is released, and a host STING pathway is activated to achieve a broad-spectrum antiviral effect. The recombinant strain rSC0120 (pS-DacA) is proved to be capable of activating the STING pathway in macrophages and mice, causing the antiviral state of the macrophages and mice, and resisting the attack of PRRSV and PRV. The recombinant strain has broad-spectrum antiviral efficacy and is suitable for preventing and treating various animal viruses clinically.

Description

technical field [0001] The invention relates to a method for constructing a broad-spectrum antiviral recombinant Salmonella, belonging to the technical field of recombinant strains. Background technique [0002] STING (stimulator of interferon genes) is a very popular target for drug development. STING agonists refer to compounds that can bind to STING protein and activate its downstream natural immune response. Such compounds are usually two molecules of nucleotide or adenine acid polymers, such as: c-di-GMP, c-di- AMP etc. In recent years, STING agonists have been widely used in the field of anti-tumor. Emily P et al. showed that the human and mouse STING agonist ADU-S100 reduced local and distal tumor burden in mice bearing pancreatic ductal adenocarcinoma (PDA). Although STING agonists have been widely studied in the field of tumor therapy, the research and application of STING agonists in the field of antiviral are rare. Tina M. Sali et al. used the artificially syn...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/74C12N15/66C12N15/60C12N1/21A61K47/46A61K45/00A61P31/12C12R1/42
CPCC12N15/74C12N9/88A61K47/46A61K45/00A61P31/12C12Y406/01001
Inventor 石火英李玉安孙燕妮付杨
Owner YANGZHOU UNIV
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