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CN-121971653-A - Enteritis targeting type probiotic delivery system and preparation method and application thereof

CN121971653ACN 121971653 ACN121971653 ACN 121971653ACN-121971653-A

Abstract

The invention belongs to the technical field of biological medicine, and discloses an enteritis targeting type probiotics delivery system and a preparation method and application thereof, wherein the system comprises i) a recombinant plasmid for encoding anti-inflammatory functional protein, wherein the plasmid comprises an anti-inflammatory peptide segment, surface protein modification and an antibiotic switch; ii) genetically engineered Escherichia coli chassis bacteria with high outer membrane vesicle productivity, and iii) recombinant engineering bacteria obtained by introducing the recombinant plasmid into the Escherichia coli chassis bacteria. The delivery system can target the intestinal inflammation part, deliver the anti-inflammatory protein through the outer membrane vesicle, specifically release the anti-inflammatory factor in the inflammation microenvironment, thereby reducing the intestinal inflammation reaction, realizing the accurate targeting of the intestinal inflammation, effectively controlling the remote delivery and continuous secretion of the anti-inflammatory protein in space and time, having the advantages of strong targeting, lasting effect, small side effect and the like, and providing a new strategy for the safe and efficient treatment of the intestinal inflammatory diseases.

Inventors

  • SUN JIAN
  • Gong Liufei
  • LUO YANG
  • ZHANG XIAOJING

Assignees

  • 华南农业大学

Dates

Publication Date
20260505
Application Date
20260114

Claims (10)

  1. 1. An enteritis targeted probiotic delivery system, the system comprising: A genetically engineered escherichia coli Nissle 1917 strain, from which yrbE gene was knocked out, resulting in a Δ yrbE strain; And a recombinant expression plasmid transferred into the Δ yrbE strain, the recombinant expression plasmid comprising an inducible promoter, a coding sequence for an outer membrane vesicle surface protein ClyA, a coding sequence for an anti-inflammatory protein IL-37b, and a tag sequence for purification, the recombinant expression plasmid being capable of expressing a ClyA-IL-37b fusion protein in the Δ yrbE strain and causing the fusion protein to be loaded into outer membrane vesicles secreted by the Δ yrbE strain.
  2. 2. The enteritis targeted probiotic delivery system of claim 1, wherein the inducible promoter is a anhydrotetracycline responsive promoter pTet and the tag sequence is a 6xHis tag.
  3. 3. The enteritis targeted probiotic delivery system according to claim 1 or 2, characterized in that the amino acid sequence encoding anti-inflammatory protein IL-37b is shown in SEQ ID No.1 and the amino acid sequence of fusion protein ClyA-IL-37b is shown in SEQ ID No. 2.
  4. 4. A recombinant expression plasmid is characterized in that the plasmid comprises a fusion protein pTet-ClyA-IL-37b, and the fusion protein pTet-ClyA-IL-37b comprises: (a) A anhydrotetracycline responsive promoter (pTet); (b) A ClyA-IL-37b fusion protein comprising an outer membrane vesicle surface protein ClyA, a linker peptide Linke, an anti-inflammatory protein IL-37b, and a purification tag; the amino acid sequence of the fusion protein pTet-ClyA-IL-37b is shown as SEQ ID NO. 3; The recombinant expression plasmid comprises an amino acid sequence shown as SEQ ID NO. 4 or a nucleotide sequence encoding the amino acid sequence shown as SEQ ID NO. 3.
  5. 5. A genetically engineered bacterium is characterized in that the genetically engineered bacterium is a yrbE gene knocked out escherichia coli Nissle 1917 strain, and the strain carries a recombinant expression plasmid capable of expressing a ClyA-IL-37b fusion protein.
  6. 6. A method of preparing a enteritis targeted probiotic delivery system according to any one of claims 1-3, or a recombinant expression plasmid according to claim 4, or a genetically engineered bacterium according to claim 5, comprising the steps of: S1, constructing a recombinant expression plasmid, namely synthesizing a gene fragment containing an inducible promoter, a ClyA coding sequence, an IL-37b coding sequence and a tag sequence, and cloning the gene fragment into a vector to obtain the recombinant expression plasmid; S2, constructing a delta yrbE strain, namely targeting and knocking out yrbE genes of escherichia coli Nissle 1917 by utilizing a CRISPR/Cas9 gene editing system to obtain a delta yrbE strain; s3, constructing engineering bacteria, namely converting the recombinant expression plasmid obtained in the step S1 into the delta yrbE strain obtained in the step S2, and screening to obtain positive clones, namely obtaining the enteritis targeted probiotic delivery system.
  7. 7. The preparation method according to claim 6, wherein the plasmid comprising the Cas9 protein expression element in step S2 is pCasKP/Apr plasmid comprising the amino acid sequence as shown in SEQ ID No.5 or the corresponding coding nucleotide sequence.
  8. 8. The preparation method according to claim 6, wherein the sgRNA expression plasmid of the target yrbE gene in the step S2 is a pSGKP-arr-TyrbE plasmid constructed by taking a pSGKP-arr plasmid as a framework, and the pSGKP-arr plasmid comprises an amino acid sequence shown as SEQ ID NO. 6 or a corresponding coding nucleotide sequence.
  9. 9. An outer membrane vesicle preparation, wherein the outer membrane vesicle is secreted by the genetically engineered bacterium of claim 5 and loaded with a ClyA-IL-37b fusion protein.
  10. 10. Use of the enteritis targeted probiotic delivery system of any one of claims 1-3, the recombinant expression plasmid of claim 4, the genetically engineered bacterium of claim 5, or the outer membrane vesicle formulation of claim 9 in the manufacture of a medicament for the prevention and/or treatment of an inflammatory disease of the intestinal tract.

Description

Enteritis targeting type probiotic delivery system and preparation method and application thereof Technical Field The invention belongs to the technical field of biological medicine, and particularly relates to a enteritis targeting type probiotic delivery system, a preparation method and application thereof. Background Inflammatory bowel disease, such as Inflammatory Bowel Disease (IBD), crohn's disease, ulcerative colitis, etc., is an autoimmune disease that causes repeated chronic inflammation of the intestinal tract as one of public health events affecting the world. The existing treatment methods mainly comprise anti-inflammatory drugs, immunosuppressants, biological agents and the like, but the treatment methods often have the problems of large side effects, easy recurrence, high price and the like. In recent years, treatment of chronic enteritis, which occurs repeatedly, has been dependent on systemic administration of anti-inflammatory drugs or biological agents for a long period of time. Such as anti-tumor necrosis factor-alpha monoclonal antibodies. Although effective, systemic side effects such as infection, immunosuppression and the like are often accompanied by the administration mode of the system, and high concentration enrichment of focus parts cannot be realized, so that the therapeutic index is low. To address such problems, oral delivery of specific anti-inflammatory proteins is also an attractive topical therapeutic strategy, but proteins are susceptible to degradation in the gastrointestinal tract, difficult to penetrate the mucus layer and are efficiently taken up by epithelial cells, which is the primary biological delivery barrier they face. Still further, bacterial Outer Membrane Vesicles (OMVs) are of interest as a new type of biogenic nano-delivery vehicle. Delivery of therapeutic molecules using OMVs can theoretically solve the problem of protein instability. However, OMVs produced by naturally wild-type strains suffer from significant drawbacks such as extremely low yields, difficulty in meeting therapeutic dose requirements, complex and uncontrollable endoluminal components, the possibility of encapsulating endogenous bacterial components rather than the desired therapeutic proteins, and lack of targeting to specific cells or inflammatory sites of the gut remain an unsolved key technical challenge in this field. As a protein drug with high pharmacological activity and low side effect, the human specific cytokine IL-37b belongs to the IL-1 cytokine family, and researches show that the IL-37b can play a strong anti-inflammatory role at the concentration of pg level. With the development of technologies such as synthetic biology and genetic engineering, therapies based on probiotics have received much attention. Probiotics can play a therapeutic role through various mechanisms of regulating intestinal flora balance, enhancing intestinal barrier function, regulating immune response and the like. These superior properties make the probiotic delivery protein system a very potential choice for application. Disclosure of Invention The invention utilizes the escherichia coli Nissle 1917 to engineer and transform the escherichia coli Nissle, integrates the anti-inflammatory fusion protein pTet-ClyA-IL-37 b into an escherichia coli chassis, and obtains a functional probiotic system which can target the inflammation part of the intestinal tract and efficiently deliver anti-inflammatory substances. In order to achieve the above purpose, the technical scheme adopted by the invention is as follows: An enteritis targeted probiotic delivery system, the system comprising: A genetically engineered escherichia coli Nissle 1917 strain, from which yrbE gene was knocked out, resulting in a Δ yrbE strain; And a recombinant expression plasmid transferred into the Δ yrbE strain, the recombinant expression plasmid comprising an inducible promoter, a coding sequence for an outer membrane vesicle surface protein ClyA, a coding sequence for an anti-inflammatory protein IL-37b, and a tag sequence for purification, the recombinant expression plasmid being capable of expressing a ClyA-IL-37b fusion protein in the Δ yrbE strain and causing the fusion protein to be loaded into outer membrane vesicles secreted by the Δ yrbE strain. Preferably, the inducible promoter is a anhydrotetracycline responsive promoter pTet, and the tag sequence is a 6XHis tag. Preferably, the amino acid sequence of the encoding anti-inflammatory protein IL-37b is shown as SEQ ID NO.1, and the amino acid sequence of the fusion protein ClyA IL-37b is shown as SEQ ID NO. 2. The invention also protects a recombinant expression plasmid, wherein the plasmid comprises a fusion protein pTet-ClyA-IL-37b, and the fusion protein pTet-ClyA-IL-37b comprises: (a) A anhydrotetracycline responsive promoter (pTet); (b) A ClyA-IL-37b fusion protein comprising an outer membrane vesicle surface protein ClyA, a linker peptide Linke, an anti-inflammatory prot