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Recombinant bacillus subtilis and application thereof

A technology of Bacillus subtilis and spores, which is applied in the field of recombinant Bacillus subtilis and its application, can solve problems such as expression and display, and achieve good virus replication ability and the effect of inhibiting virus replication ability

Active Publication Date: 2021-08-10
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, so far, there have been no studies showing feline coronavirus-associated polypeptides expressed on the surface of Bacillus subtilis spores

Method used

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  • Recombinant bacillus subtilis and application thereof
  • Recombinant bacillus subtilis and application thereof
  • Recombinant bacillus subtilis and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: Design, expression and in vitro inhibitory effect determination of HR2-derived peptides

[0028]The HR2 region of S protein FCoV was predicted by the computer software LearnCoil-VMF. Downstream of the HR2 domain, including the transmembrane region and cytoplasmic tail. The HR2 domains of different strains were selected for sequence comparison, including type I (strain SB22, GenBank Accession No.MH817484.1), (strain NTU / 2R / 2003, GenBank Accession No.DQ160294.1), (strain Black, GenBank Accession No.EU186072.1) and Type II (strain NTU156P2007, GenBank Accession No.GQ152141.1), FECV (strain 79-1683, GenBank Accession No.X80799.1), FIPV (strain FCoVWSU791146_P50, GenBank Accession No.KC461237. ), FIPV (strain HRBXF17, GenBank Accession No.MK987175.1), and found that the HR2 domain is highly conserved between different strains of FCoV, including serotypes I and II, between FECV and FIPV ( figure 1 ). It was demonstrated that peptides designed for the FCoV HR2 do...

Embodiment 2

[0034] Example 2: Preparation of mouse polyantiserum for HR2P and detection of neutralizing antibodies

[0035] Mouse anti-HR2P sera were prepared by immunizing mice with soluble HR2P-His protein expressed in E. coli and hypothesized that it could also block viral entry, and the resulting antisera were tested by enzyme-linked immunosorbent assay (ELISA) The reactivity of HR2P-His was used as the antigen, and the coating concentration was 1:2000. In addition, the neutralizing ability of the antisera was tested to determine whether neutralizing antibodies against FCoV were generated by immunization with HR2P-His.

[0036] Results: The serum of unimmunized mice could not neutralize FCoV infection, while the antiserum of mice immunized with HR2P-His could effectively inhibit FCoV infection, and the primary antibody was TGEVN protein antibody with high homology to FCoV ( Figure 4 ). The results showed that the antisera inhibited 50% of FCoV infection between 1:64 and 1:32 diluti...

Embodiment 3

[0037] Example 3: Construction and identification of HR2P recombinant Bacillus subtilis displayed on the surface

[0038] B. subtilis was selected as a candidate vector for antigen delivery in animals, and a recombinant B. subtilis strain expressing HR2P on the surface was constructed. First, extract the Bacillus subtilis genome as a template to amplify the CotB gene (the gene sequence is shown in SEQ ID No.3), and the constructed plasmid pET32a-HR2P is used as a template to amplify HR2P. PCR) ligation, and then digested by Hind III and EcoRI (TaKaRa, Japan), using the backbone plasmid pDG364 to construct the shuttle plasmid pDG364-CotB-HR2P (the vector can transfer the exogenous gene CotB-HR2P through the homology arm of the amy E region into the genome of wild-type Bacillus subtilis), and linearized by AvrII enzyme digestion, electroporated Bacillus subtilis competent cells, and the exogenous gene (CotB-HR2P) was integrated into the starch in the Bacillus subtilis chromosome...

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Abstract

The invention discloses a recombinant bacillus subtilis and an application thereof. According to the recombinant bacillus subtilis, a gene for coding antiviral polypeptide and a gene for coding spore coat protein in bacillus subtilis are fused, exogenous fusion expression is carried out in the bacillus subtilis, and the recombinant bacillus subtilis capable of displaying the antiviral polypeptide on the spore surface of the bacillus subtilis is obtained. The antiviral polypeptide is derived from feline coronavirus, and the amino acid sequence of the antiviral polypeptide is as shown in SEQ ID No.2. The spores generated by the recombinant bacillus subtilis immunize mice by means of intragastric administration and nasal drip, can activate strong mucosal immune response in intestinal mucosa and specific and systemic immune response in mice, can resist adverse environment of gastrointestinal tracts, and are effectively colonized, so the spore of the recombinant bacillus subtilis is a potential excellent carrier.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a recombinant bacillus subtilis and its application. Background technique [0002] Feline Coronavirus (FCoV) infection is extremely common in cats. There is currently no effective treatment. Peptides corresponding to the predicted heptad repeat2 domain of the feline coronavirus spike protein are potent inhibitors of viral infection .PLoS One 8(12):e82081.2013). [0003] Bacillus subtilis is a safe probiotic, which is widely added to food, medicine and feed. Bacillus subtilis Spore Surface Display Technology (BSSD) technology is considered to be one of the most promising methods for expressing heterologous proteins with high activity and stability. Expression of exogenous antigens or proteins on the surface of Bacillus subtilis spores has been practiced for more than a decade with remarkable success. [0004] For example, the invention application with the publication number CN10...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/50C12N15/75A61K39/215A61P31/14C12R1/125
CPCC07K14/005C12N15/75A61K39/12A61P31/14C12N2770/20022C12N2770/20034
Inventor 黄耀伟陈楚徐令东吕方理
Owner ZHEJIANG UNIV
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