Responsive self-splitting engineering bacterium based on TNF-alpha as well as construction method and application of responsive self-splitting engineering bacterium

Abstract

The invention discloses a response type self-splitting engineering bacterium based on TNF-alpha as well as a construction method and application of the self-splitting engineering bacterium. An induction-splitting and induction-imaging coupling module is formed by fusing a TNF-alpha nano antibody (targeted recognition of inflammation / tumor markers) and fluorescent protein or splitting protein capable of specifically initiating splitting of escherichia coli. Compared with a traditional administration way, the design has the advantages of precise targeting, dynamic regulation and monitoring, improvement of treatment efficiency and safety, multifunctional integration and production feasibility.

Claims

1. A TNF-a-responsive self-cleaving engineered bacterium, characterized in that, Co-expression of TNF-α recognition protein and cleavage protein; It includes an identification module and a splitting module; The recognition module contains a protein capable of recognizing TNF-α or a gene encoding the protein; The cleavage module contains cleavage proteins or genes that can encode cleavage proteins.

2. The TNF-a-responsive self-cleaving engineered bacteria according to claim 1, characterized in that, The protein that can recognize TNF-α is a TNF-α nanobody.

3. The TNF-α-responsive self-lytic engineered bacteria according to claim 1, characterized in that, The lysed protein is φX174 protein.

4. The TNF-α-responsive self-lytic engineered bacteria according to claim 1, characterized in that, It also includes a response module containing a fluorescent protein or a fluorescent protein coding frame encoding a fluorescent protein, the fluorescent protein including green fluorescent protein GFP and its derivatives, and the red fluorescent protein RFP family.

5. The TNF-α-responsive self-lytic engineered bacteria according to claim 1, characterized in that, The identification module contains a gene encoding a TNF-α nanobody, the nucleotide sequence of which is at least one of the sequences shown in SEQ ID NO: 1, SEQ ID NO: 14 to SEQ ID NO:

18.

6. The method for constructing TNF-α-responsive self-lytic engineered bacteria according to any one of claims 1 to 5, characterized in that, Includes the following steps: Step 1, Construction of pET-IR plasmid: Using pUC-IR plasmid as a template, and with IR-NdeI-F and IR-XhoI-R as upstream and downstream primers respectively, an IR target gene fragment with NdeI and XhoI double restriction sites was obtained. This fragment was then ligated to pET28a plasmid using T4 DNA Ligase to obtain the pET-IR recombinant plasmid. The pUC-IR plasmid sequence is listed as SEQ ID NO: 1, and the IR-NdeI-F and IR-XhoI-R sequences are listed as SEQ ID NO: 2 and SEQ ID NO: 3, respectively. Step 2, Construction of mutant plasmids: Using the pET-IR recombinant plasmid as a template, and with IR-SM2A-F, IR-SM2A-R, IR-YM4A-F, and IR-YM4A-R as upstream and downstream primers, the plasmid was amplified by reverse PCR. The PCR product was ligated into E. coli DH5α competent cells via T4 DNA Ligase and T4 polynucleotide kinase to obtain pUC-IM2R and pUC-IM4R plasmids. The sequence listings of IR-SM2A-R, IR-SM2A-F, IR-YM4A-F, and IR-YM4A-R are SEQ ID NO: 4~SEQ ID NO: 7, respectively, and the sequence listings of pUC-IM2R and pUC-IM4R are SEQ ID NO: 15 and SEQ ID NO: 17, respectively. Step 3, Construction of pUC-IE, pUC-IM2E, and pUC-IM4E self-cleaving plasmids: Using pUC-IR, pUC-IM2R, and pUC-IM4R plasmids as templates, and pUC-HindIII-F and pUC-BamHI-R as upstream and downstream primers, respectively, vectors containing HindIII and BamI double restriction sites and partial target gene fragments were obtained. Using [E]-pTD103 plasmid as a template, and [E]-BamHI-F and [E]-HindIII-R as upstream and downstream primers, respectively, the phage lysis protein φX174 fragment containing HindIII and BamI double restriction sites was obtained. The PCR products were ligated with T4 DNA Ligase and transformed into E. coli DH5α competent cells. The sequence listings for pUC-HindIII-F and pUC-BamHI-R are SEQ ID NO: 8 and SEQ ID NO: 9, respectively; the sequence listing for [E]-pTD103 is SEQ ID NO: 20; and the sequence listings for [E]-BamHI-F and [E]-HindIII-R are SEQ ID NO:

9. NO: 10, SEQ ID NO:

11.

7. The use of the TNF-α-responsive self-lysing engineered bacteria according to any one of claims 1 to 5 in the preparation of reagents for detecting diseases characterized by elevated TNF-α levels.

8. A probiotic composition, characterized in that, It comprises the TNF-α-responsive self-lytic engineered bacteria and drug-loaded strain as described in any one of claims 1 to 5; the drug-loaded strain expresses a protein with anti-inflammatory activity.

9. The probiotic composition according to claim 8, characterized in that, The drug-loaded strain contains sea cucumber peptide SCP protein or a gene expressing sea cucumber peptide SCP protein, and the amino acid sequence of the SCP protein is SEQ ID NO:

27.

10. Use of the probiotic composition of claim 8 in the preparation of a medicament for treating inflammatory bowel disease or tumors.

Images