Immunomodulatory bacterial protein containing cytokine motif and application of immunomodulatory bacterial protein

Abstract

According to the immunomodulatory bacterial protein containing the cytokine motif and the application of the immunomodulatory bacterial protein, the immunomodulatory bacterial protein containing the cytokine motif can specifically activate and promote immune cell proliferation, so that the anti-tumor immune response of a host is enhanced, and the biosafety is good. High-efficiency expression, purification and delivery of the protein can be realized through a genetic engineering strain system, the production process is stable, large-scale implementation is facilitated, the protein is suitable for developing immunotherapy drugs and tumor adjuvant therapy preparations, and a new strategy is provided for clinical treatment.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to an immunomodulatory bacterial protein containing cytokine motifs and its applications. Background Technology

[0002] Gut microorganisms produce a variety of metabolites that regulate the host's immune and inflammatory responses and maintain gut homeostasis. The biomolecules derived from the gut microbiota, such as DNA, RNA, proteins, and polysaccharides, exhibit significant diversity and participate in the formation of "pathogen-associated molecular patterns" (PAMPs), which also have a profound impact on the host's immune system.

[0003] Bacteria contain immunomodulatory proteins similar to those in eukaryotes, providing a new perspective for those skilled in the art to explore the interaction between the microbiome and the host. Human cytokines play a crucial role in regulating innate and adaptive immunity, cell growth, differentiation, and death. However, many human cytokines face challenges in bacterial expression, including low expression levels, low solubility, and a lack of post-transcriptional modification.

[0004] Cytokine motif-containing immunomodulatory bacterial proteins (CMCPs), encoded by bacteria, play crucial roles in host immune regulation and disease development. CMCPs not only retain immunomodulatory activity but are also compatible with bacterial expression systems, theoretically solving the problem of heterologous cytokine expression and promoting the development of bacteria-based immunotherapies. By combining CMCPs with different cytokines or immune checkpoint inhibitors, broader applications are possible in various diseases, including autoimmune diseases, metabolic diseases, and cancer. The identification and development of CMCPs will provide important insights into the role of the gut microbiome in immune regulation and enrich the theoretical knowledge of microbial molecular biology. However, current research on CMCPs is still in its early exploratory stages, and more efficient, controllable, and safe CMCPs need to be developed to create novel biopharmaceutical formulations. Summary of the Invention

[0005] To address the aforementioned problems in the prior art, this invention provides an immunomodulatory bacterial protein containing cytokine motifs and its applications.

[0006] This invention utilizes a hidden Markov model based on relevant metagenomic data from cancer patients and healthy individuals. Through abundance calculation and differential analysis, two disease-associated CMCPs, bIL-1 and bIL-33, were identified. This invention achieves heterologous soluble expression of CMCPs using genetically engineered strains, and uses ELISA to determine their effects on immune cells, demonstrating their ability to stimulate immune responses. Further, it utilizes *E. coli* Nissle 1917 (… E. coli Nissle Using 1917, EcN) as the engineered bacterial substrate, CMCP is induced and delivered directly to the intestinal tumor lesions of animals, thereby inhibiting the proliferation of intestinal tumors and enhancing the immune response.

[0007] Specifically, the present invention provides the following technical solutions: In a first aspect, the present invention provides an immunomodulatory bacterial protein containing a cytokine motif, comprising any one of the following amino acid sequences: (1) the amino acid sequence shown in SEQ ID NO:1; (2) the amino acid sequence shown in SEQ ID NO:2; (3) the amino acid sequence of the amino acid sequence shown in (1) by substitution, insertion or deletion of one or more amino acids to obtain an amino acid sequence of the same function; (4) the amino acid sequence of the amino acid sequence shown in (2) by substitution, insertion or deletion of one or more amino acids to obtain an amino acid sequence of the same function.

[0008] Secondly, the present invention provides a nucleic acid molecule that encodes the immunomodulatory bacterial protein containing the cytokine motif.

[0009] According to the present invention, a nucleic acid molecule preferably comprises any one of the following nucleotide sequences: (1) a nucleotide sequence as shown in SEQ ID NO:5 or SEQ ID NO:8; (2) a completely complementary sequence of a nucleotide sequence as shown in (1); (3) a nucleotide sequence of a nucleotide sequence as shown in (1) obtained by substitution, deletion or insertion of one or more nucleotides, which can encode a protein having the same function.

[0010] In a specific embodiment of the present invention, when the nucleic acid molecule encodes the immunomodulatory bacterial protein containing the cytokine motif as shown in SEQ ID NO:1, the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO:5, the completely complementary sequence of the nucleotide sequence shown in SEQ ID NO:5, or the nucleotide sequence shown in SEQ ID NO:5 obtained by substitution, deletion, or insertion of one or more nucleotides, which can encode a protein with the same function.

[0011] In a specific embodiment of the present invention, when the nucleic acid molecule encodes the immunomodulatory bacterial protein containing the cytokine motif as shown in SEQ ID NO:2, the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO:8, the completely complementary sequence of the nucleotide sequence shown in SEQ ID NO:8, or the nucleotide sequence shown in SEQ ID NO:8 obtained by substitution, deletion, or insertion of one or more nucleotides, which can encode a protein with the same function.

[0012] Thirdly, the present invention provides a conjugate comprising the immunomodulatory bacterial protein containing the cytokine motif, and a chemical portion conjugated thereto.

[0013] Fourthly, the present invention provides a composition comprising the immunomodulatory bacterial protein containing the cytokine motif.

[0014] The composition provided according to the present invention preferably further comprises other antitumor drugs. More preferably, the composition provided according to the present invention is an anti-intestinal tumor drug.

[0015] Fifthly, the present invention provides a biological material containing the nucleic acid molecule, wherein the biological material is recombinant DNA, expression cassette, transposon, vector, microorganism or cell.

[0016] According to the biological material provided by the present invention, preferably, the expression cassette, transposon, or vector contains a nucleic acid molecule encoding a tag protein. More preferably, the tag protein is DsbA or MBP.

[0017] According to the present invention, the biomaterial preferably uses pET28a or pCRCTS1-I as a backbone. More preferably, the vector comprises an induction module, a signaling molecule, and a cleavage module; the induction module includes a promoter P. vioA532 and cviR532 Gene; the signaling molecule includes N-hexanoyl-L-homoserine lactone; the cleavage module includes E. coli E protein of bacteriophage Phi X174.

[0018] More preferably, in the carrier, the promoter P tet r start up cviR532 Gene expression is mediated by the promoter P. vioA532 The expression of the X174 coding gene and the nucleic acid molecule is initiated sequentially.

[0019] More preferably, the vector also contains a gene encoding a fluorescent protein.

[0020] More preferably, in the carrier, the promoter P tet r start up cviR532 Gene expression is mediated by the promoter P. vioA532 The expression of the X174 coding gene, the fluorescent protein coding gene, and the nucleic acid molecule is initiated sequentially.

[0021] More preferably, the fluorescent protein is a red fluorescent protein, namely mCherry.

[0022] According to the present invention, the biological material preferably includes *Escherichia coli*. More preferably, the strain of *Escherichia coli* is... E. coli BL21(DE3), E. coli C43(DE3) or Nissle 1917 ( E. coli Nissle 1917, EcN).

[0023] In a sixth aspect, the present invention provides the use of the immunomodulatory bacterial protein containing the cytokine motif, the nucleic acid molecule, the conjugate, the composition, or the biomaterial in the preparation of a medicament for treating tumors.

[0024] According to the present invention, the use of the immunomodulatory bacterial protein containing cytokine motifs, the nucleic acid molecule, the conjugate, the composition, or the biomaterial in the preparation of a medicament for treating tumors, preferably, the tumor includes intestinal tumors. More preferably, the intestinal tumor includes any one or more of small intestinal tumors, colon tumors, and rectal tumors.

[0025] According to the present invention, the use of the immunomodulatory bacterial protein containing cytokine motifs, the nucleic acid molecule, the conjugate, the composition, or the biomaterial in the preparation of a medicament for treating tumors, preferably, the treatment of tumors includes any one or more of reducing the number of tumors, reducing the tumor area, enhancing the immune response, and alleviating weight loss in tumor patients.

[0026] In a seventh aspect, the present invention provides the use of the immunomodulatory bacterial protein containing the cytokine motif, the nucleic acid molecule, the conjugate, the composition or the biomaterial in the preparation of a drug, wherein the drug is any one or more of the following (1) to (4): (1) a drug for reducing the number of tumors; (2) a drug for reducing the area of ​​tumors; (3) a drug for enhancing the immune response; (4) a drug for alleviating weight loss in cancer patients.

[0027] Eighthly, the present invention provides a biological agent comprising the immunomodulatory bacterial protein containing cytokine motifs, the nucleic acid molecule, the conjugate, the composition, or the biological material.

[0028] In a ninth aspect, the present invention provides a method for preparing the immunomodulatory bacterial protein containing the cytokine motif, wherein the microorganism or cell is cultured under conditions that allow the expression of the immunomodulatory bacterial protein containing the cytokine motif, and the immunomodulatory bacterial protein containing the cytokine motif is obtained from the resulting culture.

[0029] The present invention has the following beneficial effects: This invention provides two immunomodulatory bacterial proteins containing specific cytokine motifs that can specifically activate and promote the proliferation of immune cells, thereby enhancing the host's anti-tumor immune response, with good biosafety. The proteins can be efficiently expressed, purified, and delivered using a genetically engineered bacterial strain system. The production process is stable and easy to scale up, making it suitable for developing immunotherapeutic drugs and adjuvant cancer treatment agents, providing a new strategy for clinical treatment. Attached Figure Description

[0030] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0031] Figure 1 This is a plasmid map of the Escherichia coli soluble expression vector pET28a-DsbA-bIL-1-6×His-Tag in Example 2 of the present invention.

[0032] Figure 2 This is a plasmid map of the Escherichia coli soluble expression vector pET28a-MBP-bIL-33-6×His-Tag in Example 2 of the present invention.

[0033] Figure 3 The image shows SDS-PAGE electrophoresis diagrams of purified proteins from the four strains in Example 2 of this invention; U represents the purified protein after ultrafiltration; E represents the purified protein after endotoxin removal.

[0034] Figure 4 The following are examples of the effects of two CMCPs on mouse spleen-derived T cells in Example 3 of this invention: a) the effect of the two CMCPs on T cells secreting IFN-γ; b) the effect of the two CMCPs on T cells secreting IL-6; c) the effect of the two CMCPs on T cells secreting IL-10; d) the effect of the two CMCPs on T cells secreting IL-17A; and e) the effect of the two CMCPs on T cells secreting TNF-α.

[0035] Figure 5This is a schematic diagram of the structure of the expression lysis vector for delivering cleaved expression of CMCP in Embodiment 4 of the present invention.

[0036] Figure 6 The results show the mCherry fluorescence intensity of the four engineered strains (EcN-I-bIL-1, EcN-I-bIL-33, EcN-I-vector and Neg) in Example 4 of this invention.

[0037] Figure 7 This is a schematic diagram of the mouse experiment process in Example 5 of the present invention, and shows the effects of different engineered strains on mouse weight maintenance and intestinal tumor inhibition; a is a schematic diagram of the mouse experiment process; b is... APC min / + Changes in body weight percentage in mice after treatment with engineered bacteria; c represents APC min / + Statistical results of the number of intestinal tumors in mice after treatment with engineered bacteria; d is APC min / + Statistical results of intestinal tumor area in mice after treatment with engineered bacteria.

[0038] Figure 8 The results of gene set enrichment analysis (GSEA) of different engineered strains on the mouse duodenum transcriptome in Example 5 of this invention are shown; a is the EcN-I-bIL-1 group; b is the EcN-I-bIL-33 group.

[0039] Figure 9 The following are the pathological results and scores of the small intestine of mice by different engineered strains in Example 5 of the present invention: a is a representative illustration of the small intestine pathological section of each group of mice; b is the comprehensive intestinal pathological score of each group of mice; c is the pathological score of the proximal small intestine of each group of mice; d is the pathological score of the middle small intestine of each group of mice; e is the pathological score of the distal small intestine of each group of mice.

[0040] Figure 10 This invention, in Example 5, illustrates the effects of multiplex fluorescent staining on the infiltration of immune cells in the mouse intestinal tract, showing the effects of different engineered bacterial strains. a) is a representative image of multiplex fluorescent staining of the intestines of mice in each group; b) shows CD8+ within the tumor. + T cell and NKT cell infiltration; c represents CD8+ in adjacent tissue. + The infiltration of T cells and NKT cells; d represents the infiltration of immune cells, T cells, B cells, and NK cells within the tumor; e represents the infiltration of immune cells, T cells, B cells, and NK cells in the tissue adjacent to the tumor.

[0041] Figure 11 This is Example 6 of the present invention, showing the direct killing effect of CMCP on tumor cells. Detailed Implementation

[0042] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0043] Unless otherwise specified, the experimental methods involved in the following embodiments are conventional methods in the art. For example, you can refer to the experimental manual in the art or follow the conditions recommended in the manufacturer's instructions.

[0044] Unless otherwise specified, all experimental materials and reagents used in the following examples are commercially available. The colorectal cancer metagenomic database involved in this invention is published in a publicly available database. Animal experiments were conducted with the approval of the ethics committees of the Institute of Microbiology and the Institute of Zoology, Chinese Academy of Sciences. The *E. coli* strains used in the examples... E. coli BL21(DE3), E. coli C43(DE3), Nissle 1917 ( E. coli Nissle 1917 (EcN) is deposited in the Wang Jun research group at the Institute of Microbiology, Chinese Academy of Sciences. The public can obtain this biological resource from the applicant.

[0045] Example 1: Mining CMCP from a CRC-related metagenomic database This invention, based on a Hidden Markov Model (HMM), identifies two CMCPs (colonial cancer progenitor cells) with significantly different abundances in the intestinal metagenomics of colorectal cancer patients and healthy individuals from a colorectal cancer-related metagenomic database. These two progeny cells are bIL-1 and bIL-33, and their sequence information is as follows: The amino acid sequence of bIL-1 is: TTENTAAHNDIDPTVWVETVDGEEHRYLAWGNTLYYVCELNEDMTSVKDINGDGIVDKKDVKLQTINNLPAGLGYTEAPWIYRRTDENGNYTGKYYLFAAFGWREQMAYAT SDTMWGPWEFGGVLMPPTATSNTNHPSVIDFKGKTYFIYHNGSKPWGSGFRRVVCAEEFTINDDGTIDPIQETSTGLLTGTKSAIMQNGGYIYHDNFVNPSDDASYPLVKNVYFGKRYSASKDTQW EIVAGKADTANENYVSIAAVNKPGLYIAARSDNSVILTQDSKQNDTSMQKAMTFKTVKGLNGQENAVSFESVLKPGYYLSTKDSAITLISCSDIDRETSSFEIGAALGDDSISGMMADISGAWI KNGSSVRFYLNNAALYQNVNAYVAEYKDGMLIGVGAVNNIEINSSIQSIEIPYERKDNDSELKIFVWNSMLPATEPVNVTVMENPYAMPTGYTSYFNFDENINDTQTEAQGSLVGAKITDS (SEQ ID NO:1); The amino acid sequence of bIL-33 is: EIANEYALDYLSETKLRRIELEMKSLSTFNDTRIMFITPSGDVILDTNDSSTDKSNDDRVLFSINDFDYGDLKGKHDILWDFYGLFSEPALSVFSPISNSFEIKGYVVINIPESAIVERVYDTFNTNYLTLAIVLIL (SEQ ID NO: 2).

[0046] Example 2: Heterologous expression of CMCP in Escherichia coli The inventors' previous research found that the two CMCPs originated from the gut metagenomics, the species were unknown, and their heterologous expression in E. coli was mainly as inclusion bodies, which were difficult to express in soluble form, thus limiting their downstream applications.

[0047] This embodiment provides a method for successfully achieving extracellular secretion and soluble expression of CMCP in Escherichia coli. The method involves ultrasonic disruption to obtain a signal peptide, vector, and E. coli genetically engineered strain containing CMCP supernatant. The specific steps are as follows: 1. Construction of bIL-1 soluble expression vector and engineered strain in Escherichia coli This invention uses DsbA-Tag (SEQ ID NO:3) to assist bIL-1 expression. Using Gibson seamless ligation technology, the coding sequences of DsbA-Tag (SEQ ID NO:4) and bIL-1 (SEQ ID NO:5) are sequentially ligated to the pET28a vector and inserted between the RBS and 6×His-Tag after the T7 promoter, resulting in the following expression. Figure 1 The bIL-1 soluble expression vector for Escherichia coli shown is denoted as pET28a-DsbA-bIL-1-6×His-Tag.

[0048] The coding sequence of DsbA-Tag (SEQ ID NO:4) was ligated into the pET28a vector and inserted between the RBS and 6×His-Tag after the T7 promoter to obtain the soluble expression vector of DsbA-Tag for Escherichia coli, denoted as pET28a-DsbA-6×His-Tag.

[0049] pET28a-DsbA-bIL-1-6×His-Tag was transferred into the chemical conversion method. E. coli BL21 (DE3) strain was used to obtain an engineered strain that solublely expresses bIL-1, named the bIL-1 strain; pET28a-DsbA-6×His-Tag was then transformed into... E. coli A strain expressing DsbA-Tag in soluble form was obtained from BL21 (DE3) strain, and it is designated as strain DsbA.

[0050] 2. Construction of bIL-33 soluble expression vector and engineered strain in Escherichia coli This invention uses MBP-Tag (SEQ ID NO:6) to assist in the expression of bIL-33. The coding sequences of MBP-Tag (SEQ ID NO:7) and bIL-33 (SEQ ID NO:8) are ligated into the pET28a vector using Gibson seamless ligation technology, inserted between the RBS and 6×His-Tag after the T7 promoter, resulting in a soluble E. coli expression vector for bIL-33, denoted as pET28a-MBP-bIL-33-6×His-Tag.

[0051] The coding sequence of the MBP-Tag (SEQ ID NO:7) was ligated to the pET28a vector and inserted between the RBS and 6×His-Tag after the T7 promoter, resulting in the following: Figure 2 The soluble expression vector for MBP in Escherichia coli shown is denoted as pET28a-MBP-6×His-Tag.

[0052] The pET28a-MBP-bIL-33-6×His-Tag strain was transformed into E. coli BL21 (DE3) strain via chemical transformation to obtain an engineered strain expressing bIL-33 in soluble form, designated as the bIL-33 strain; the pET28a-MBP-6×His-Tag strain was then transformed into... E. coli The BL21 (DE3) strain was used to obtain an engineered strain that solublely expresses MBP, which is denoted as the MBP strain.

[0053] 3. Cultivation of engineered strains and enrichment of proteins Table 1. Cultivation, induction of expression, and lysis conditions of the strain

[0054] bIL-1, DsbA, bIL-33, and MBP strains were inoculated into LB medium containing kanamycin at a final concentration of 50 μg / mL and cultured for 3–4 h under the conditions shown in Table 1 until the culture OD... 600 The pH was set to 0.6–0.8, and IPTG, the inducing agent, was added for further culturing. After culturing, the cultures of each strain were collected into suitable containers, centrifuged at 12,000 rpm at 4°C for 2–10 min, and the supernatant was discarded to obtain the bacterial cells of each strain. The cells were then placed on ice for later use. The bacterial cells of each strain were lysed by ultrasonic disruption, centrifuged at 12,000 rpm for 5 min at 4°C, and the supernatant was collected, filtered, and subjected to protein affinity chromatography. After elution, the protein was examined by SDS-PAGE.

[0055] SDS electrophoresis results are as follows Figure 3 As shown, using DsbA-Tag and MBP-Tag, this invention successfully obtained soluble expression and purified four proteins (DsbA-bIL1, MBP-bIL33, DsbA-Tag, and MBP-Tag). The expression vector and engineered strains provided by this invention can assist in the soluble expression of CMCP fusion, but are not limited to the CMCP mentioned in this invention.

[0056] Example 3: Detection of the effect of CMCP on immune cells derived from mouse spleen This embodiment is based on DsbA-bIL1, MBP-bIL33, DsbA-Tag and MBP-Tag obtained in Example 2. The CMCP was determined by co-incubating with mouse spleen cells and bone marrow-derived immune cells and detecting the cytokines produced by the immune cells by ELISA.

[0057] 1. Co-incubation of CMCP with mouse spleen cells (1) Experimental methods First, four C57BL / 6J mice were subjected to cervical dislocation and immediately placed in a metal lunchbox containing 75% ethanol for sterilization for 15 minutes. Next, RPMI 1640 medium containing 10% fetal bovine serum and 1% penicillin-dextrose antibiotics was prepared. In a clean bench, cell culture dishes, a 70 μm cell filter, and a grinding rod were prepared, and the filter was rinsed with 2 mL of culture medium.

[0058] Subsequently, the right abdomen of the mouse was opened, the intestinal segment was freed to locate the spleen, the spleen was separated and cut off and placed in a filter, and the remaining part was properly disposed of. The spleen was crushed using a grinding stick, rinsed with 2 mL of culture medium, the filter was discarded, and the cells were collected into 15 mL centrifuge tubes.

[0059] Next, add twice the volume of erythrocyte lysis buffer, mix gently, and let stand for 10 minutes. Then, centrifuge at 1000 rpm for 6 minutes at room temperature and discard the supernatant. Resuspend the cells in 2 mL of culture medium and collect them by filtering through a flow cytometry tube with a filter cap. After removing the filter cap, seal the flow cytometry tube with film and centrifuge again at 1000 rpm for 6 minutes at room temperature. Discard the supernatant to finally collect mouse spleen cells.

[0060] For the culture of mouse spleen cells, 2 mL of culture medium was added to each cell pellet and transferred to a 50 mL centrifuge tube. 20 mL of culture medium was then added and mixed thoroughly. Subsequently, 5 µg / mL anti-CD3 (Essential Biotech 108567-T08) and 5 µg / mL anti-CD28 (Essential Biotech 50103-RP01) antibodies were added to stimulate T cell differentiation. Two CMCPs (DsbA-bIL1 and MBP-bIL33) or their corresponding tags (DsbA-Tag and MBP-Tag) were added to a final concentration of 100 μg / mL and incubated for 48 hours. Finally, the cell supernatant was collected, and cytokines were detected using ELISA.

[0061] (2) Experimental results like Figure 4 As shown in a to e, both types of CMCP have a strong stimulatory effect on T cells, promoting the secretion of IFN-γ, IL-6, IL-10, IL-17A and TNF-α by T cells. This suggests that bIL1 and -bIL33 may play an important role in the regulation of inflammation, and can activate T cells and enhance the body's immune response (especially the immune response of T cells).

[0062] Example 4: Construction of expression lysis vector and engineered strain for delivering CMCP lysis expression This invention provides the utilization E. coli Nissle 1917 (EcN) method for delivering expression lysing vectors and engineered strains for lysing CMCP expression.

[0063] 1. Construction of CMCP expression lysis vector The expression lysis vector constructed in this embodiment is as follows: Figure 5 As shown, the system was constructed using the pCRCTS1 vector (a gift from Researcher Li Jin'e of the Institute of Microbiology, Chinese Academy of Sciences) as the backbone, referencing existing technologies such as "A visualization reporter system for characterizing antibiotic biosynthetic gene clusters expression with high-sensitivity" (doi.org / 10.1038 / s42003-022-03832-9) and "Synchronized cycles of bacterial lysis for in vivo delivery" (doi.org / 10.1038 / nature18930). It includes induction, signaling molecule, and cleavage modules.

[0064] The induction module is derived from Chromobacterium violaceum CV31532 ( Chromobacterium violaceum CV31532, C. violaceum The swarm sensing system (CV31532) is powered by the promoter P. vioA532 and cviR532 Genome composition (“532” is a marker carried over from CV31532). The signaling molecule is N-hexanoyl-L-homoserine lactone (C6-HSL), a six-carbon hydrocarbon chain isolated from Gram-negative bacteria (G-). It is an N-acylhomoserine lactone (AHL) that induces the P-phase of the module. vioA532 It can be done cviR532 Encoding the AHL receptor CviR, it initiates downstream gene expression in response to C6-HSL signaling. The cleavage module originates from... E. coli The E protein (cleavage protein, X174E) of bacteriophage Phi X174.

[0065] This invention uses P tet r promoter expression cviR532 Gene, P vioA532 Promoter tandem expression X174E (from) (X174 phage lysis gene) CMCP and mCherry(Encoding red fluorescent protein) Three genes, during C6-HSL induction, CviR532 protein acts on P vioA532 Startup, Start X174E, CMCP and mCherry The tandem expression of the system. The success of the system can be assessed by the lysis of bacterial cells (OD). 600 The red fluorescence emitted by the mCherry protein confirmed this.

[0066] This invention utilizes Gibson seamless connection technology to first... mCherry The gene was inserted downstream of the X174E gene in the pCRCTS1-I-Neg vector to obtain the pCRCTS1-I-mCherry vector. Subsequently, the coding sequence of bIL-1 (SEQ ID NO:5) was inserted into the pCRCTS1-I-mCherry vector. X174E and mCherry Between these steps, the expression cleavage vector of bIL-1 was obtained, denoted as pCRCTS1-I-bIL-1. Following the same method, the coding sequence of bIL-33 (SEQ ID NO:8) was inserted into the pCRCTS1-I-mCherry vector to obtain the expression cleavage vector of bIL-33, denoted as pCRCTS1-I-bIL-33.

[0067] 2. Construction of engineered strains for delivering lysed expression of CMCP By electroporation, pCRCTS1-I-bIL-1 was transformed into EcN strain to obtain an engineered strain that could respond to C6-HSL molecule expression and release bIL-1 (SEQ ID NO:1), denoted as EcN-I-bIL-1; pCRCTS1-I-bIL-33 was transformed into EcN strain to obtain an engineered strain that could respond to C6-HSL molecule expression and release bIL-33 (SEQ ID NO:2), denoted as EcN-I-bIL-33; pCRCTS1-I-mCherry vector was transformed into EcN strain to obtain an experimental control engineered strain, denoted as EcN-I-vector; and pCRCTS1-I-Neg vector was transformed into EcN strain to obtain a negative control engineered strain, denoted as Neg.

[0068] 3. Cultivation and detection of engineered bacteria for CMCP delivery Each strain (EcN-I-bIL-1, EcN-I-bIL-33, EcN-I-vector and Neg) was cultured in LB medium containing 34 μg / mL chloramphenicol and 0.1 μM C6-HSL at 37℃ and 180 rpm under complete darkness.

[0069] Culture supernatants of each strain were collected into black 96-well plates at 4 h, 8 h, 12 h, 24 h, and 48 h of culture, respectively. The mCherry fluorescence intensity (RFU) was detected using a microplate reader with an excitation wavelength of 587 nm and an emission wavelength of 610 nm. A higher RFU indicates that the corresponding strain expressed more lysed protein.

[0070] like Figure 6 As shown, after the addition of C6-HSL, except for the non-fluorescent control group EcN-Neg (carrying the pCRCTS1-I-Neg plasmid), the other three strains all produced significant red fluorescence, indicating that CMCP can not only be synthesized and expressed intracellularly, but also effectively released into the culture medium after cell lysis. This finding provides important experimental evidence for subsequent research, demonstrating that the engineered strain constructed in this invention can effectively synthesize, express, and release CMCP in a soluble manner, and has good application potential.

[0071] The expression lysis vector and engineered strains provided by this invention can induce expression and lysis to release bIL-1 and bIL-33, but are not limited to these two CMCPs mentioned in this invention.

[0072] Example 5: Tumor inhibitory effect of CMCP on colorectal tumor-bearing mice 1. Experimental Methods (1) APC min / + Construction and feeding management of mouse tumor-bearing models With C57BL / 6J- APC min / + A mouse model of spontaneous colorectal cancer precancerous lesions (denoted as ) APC min / + Mice were housed in an animal facility under constant temperature (23℃±0.5℃) and humidity (50%±5%). Animal lighting was provided from 7:00 AM to 7:00 PM, with a 12-hour light-dark cycle. Mice were allowed free access to food and water unless otherwise required.

[0073] like Figure 7 As shown in a, APC min / + After mice reached 4 weeks of age and were weaned, they were separated into different cages and fed a high-fat diet of Rodent Diet (60% kcal% fat) produced by Research Diets to induce tumor formation. Intestinal adenomas generally began to appear in mice at 8-9 weeks of age and persisted until the end of the experiment. Throughout the feeding period, the mice were weighed and their condition was monitored weekly.

[0074] (2) Engineered bacteria therapy Will APC min / +Mice were randomly divided into three groups: EcN-I-vector group (control group), EcN-I-bIL-1 group and EcN-I-bIL33 group, with eight mice in each group.

[0075] like Figure 7 As shown in a, antibiotic sweep was performed at 8 weeks of age. 0.25 g / L neomycin, 0.25 g / L penicillin, 0.25 g / L metronidazole and 0.125 g / L vancomycin were added to the drinking water of mice in each group. Fecal samples were collected and smeared one week later to test the sweep effect.

[0076] The day before the formal start of the engineered strain treatment (the last day of 8 weeks of age), 0.05 g / L chloramphenicol and 50 μM C6-HSL were added to the drinking water of each group of mice, and the engineered bacteria were activated and induced to grow, as described in Example 4.

[0077] Mice were treated with engineered bacterial strains via gavage. The entire gavage procedure was performed in a clean bench. After restraint, 100 μL of each engineered bacterial strain was drawn from a 12-gauge gavage syringe (dose was 10 μL). 8 Mice were administered a PBS suspension containing CFU / mouse via gavage. After gavage, each group of mice was wiped with alcohol and sterilized with UV light for at least 20 minutes in a clean bench to prevent cross-contamination.

[0078] After gavage, the cages of mice from different groups were dispersed to avoid the possibility of the engineered bacteria being affected by aerosol transmission. Generally, one day after gavage, each mouse was restrained and subcutaneously injected with 100 μL of 50 μM C6-HSL solution through the back of the neck to enhance induction.

[0079] During the experimental period, mice in each group were administered the corresponding engineered strain by gavage every two weeks, and 50 μM C6-HSL solution was injected subcutaneously every three days to enhance the induction of the engineered strain.

[0080] Mice were weighed weekly during administration, and feces were collected periodically to confirm the colonization of engineered bacteria.

[0081] The experiment was stopped when the mice met any of the following criteria: (1) their weight dropped to less than 30% of the previous week; (2) the mice were in extremely poor condition (including limited activity, difficulty eating, etc.); (3) the experiment was conducted up to 20 weeks.

[0082] (3) Mouse intestinal anatomy, detection and transcriptome sequencing The mice were fasted the day before the experiment ended. At the end of the experiment, the mice in each group were anesthetized and their cervical spine was dislocated to separate the intestinal tissue.

[0083] The small intestine and colon were separated from the cecum, and the cecum was discarded. The small intestine was divided into three equal segments (proximal, middle, and distal). Three sample blocks of approximately 5 mm were cut from the proximal duodenum and placed in EP tubes, then flash-frozen in liquid nitrogen for RNA extraction. RNA extraction, library preparation, and sequencing were performed by Novogene.

[0084] The small intestine was longitudinally cut into segments, fixed to paraffin plates, stained with 0.1% methylene blue solution, and then rinsed with pre-cooled sterile PBS. The segments were examined and photographed using a stereomicroscope, and the size and number of tumors in the intestine were recorded. The tumors were then rolled into Swiss rolls using wooden sticks and pinned, soaked in paraformaldehyde for 48 hours, then replaced with alcohol for preservation, and embedded for tissue sectioning, HE staining, and multiplex fluorescent staining. The antibodies used for staining are shown in Table 2, and the pathological scores are shown in Table 3.

[0085] Table 2. Antibodies used for multiplex fluorescent staining

[0086] Table 3 Scoring criteria for mouse intestinal pathology

[0087] 2. Experimental Results The weight gain curves of each group of mice are as follows: Figure 7 As shown in b, in the later stages of the experiment, especially at weeks 19 and 20, the body weight of mice in the EcN-I-bIL-1 group and the EcN-I-bIL-33 group was significantly higher than that of the control group, indicating that EcN-I-bIL-1 and EcN-I-bIL-33 can prevent colorectal cancer from causing weight loss in mice and have good biosafety.

[0088] The statistical results of the number and area of ​​intestinal tumors in each group of mice are as follows: Figure 7 As shown in c and d, both EcN-I-bIL-1 and EcN-I-bIL-33 significantly reduced the number of tumors, and in terms of tumor area, both EcN-I-bIL-1 and EcN-I-bIL-33 significantly reduced the tumor area. This indicates that EcN-I-bIL-1 and EcN-I-bIL-33 have a significant inhibitory effect on tumor growth.

[0089] The transcriptome sequencing analysis results of each group of mice are as follows: Figure 8 As shown in a and b, in the EcN-I-bIL-1 group, pathways related to adaptive immune responses were significantly upregulated, particularly those involving B cells and T cells, which contribute to tumor elimination; conversely, pathways significantly downregulated included those related to energy metabolism and synaptic translation, which may help suppress tumor-related energy metabolism and enteroneurinic signaling; the EcN-I-bIL-33 group showed similar transcriptomic features to the EcN-I-bIL-1 group.

[0090] HE staining and intestinal histopathological scores of mice in each group are as follows: Figure 9 As shown in a to e, compared with the control group, the pathological scores of the proximal, middle and distal small intestines of mice in the EcN-I-bIL-1 group were significantly reduced, which has a promoting effect on maintaining the normal morphology of the intestine (especially the small intestine) and reducing the tumor burden.

[0091] Multiplex immunofluorescence staining of the intestines of mice in each group, as shown below Figure 10 As shown in a~e, immune cells (CD45) in the intestinal tumor region of mice in the EcN-I-bIL-1 group and the EcN-I-bIL-33 group. + The number of T cells (CD45+ cells) in the EcN-I-bIL-1 group was significantly higher than that in the control group, especially in the EcN-I-bIL-1 group, where intratumoral T cells (CD45+ cells) were observed. + CD3 + The infiltration of CD8 cells was significantly increased, especially at the tumor periphery. Furthermore, the significant tumor elimination effect in the EcN-I-bIL-1 group can be attributed to CD8+. + A significant increase in T cells was observed. Compared to the EcN-I-bIL-33 group, the EcN-I-bIL-1 group tended to concentrate CD8+ cells in the tumor region. + T cells. The EcN-I-bIL-33 group also showed a higher degree of B cell infiltration.

[0092] The above results indicate that the two CMCPs provided by this invention have excellent anti-tumor effects. After being delivered into the body by engineered bacteria, they can significantly inhibit tumor growth and significantly increase the abundance of anti-tumor immune cells, especially T cells in tumors, which helps in the anti-tumor immune response.

[0093] Example 6: In vitro antitumor activity of CMCP 1. Experimental Methods This experiment investigated the in vitro antitumor effects of two CMCPs, namely their direct effects on the human colon adenocarcinoma cell line Caco-2. The proliferation of Caco-2 cells under CMCP treatment was mainly studied using the Cell Counting Kit-8 (CCK8) kit.

[0094] When Caco-2 cells reached 80% confluence in the culture dish, they were passaged using 100 μL per well at a concentration of 10... 3 Cells / well ~10 5Cells were seeded per well in 96-well plates. After overnight culture to allow Caco-2 cells to adhere, the medium was changed and treated with MBP-bIL33 at working concentrations of 0.1 μg / mL, 1 μg / mL, and 10 μg / mL, respectively. MBP-Tag at the same concentration was used as a control. The treatment time was 24 h.

[0095] When the treatment is terminated, change the medium, add the prepared 10% CCK-8 reaction solution, and treat for 1 h to 4 h according to the remaining cell volume to allow OD to be adjusted. 450 The absorbance was measured at around 1, and the results were statistically analyzed after detection using a spectrophotometer.

[0096] 2. Experimental Results In the CCK-8 experiment, such as Figure 11 As shown, compared with MBP-Tag, different concentrations of MBP-bIL33 inhibited Caco-2 cell proliferation in a concentration-dependent manner after 24 h of treatment, with the most significant inhibitory effect observed at a concentration of 10 μg / mL. This indicates that bIL-33 has a certain direct killing effect on tumor cells in vitro. bIL-1 also showed a similar in vitro tumor cell killing effect.

[0097] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An immunomodulatory bacterial protein containing cytokine motifs, characterized in that, It includes any of the following amino acid sequences: (1) The amino acid sequence as shown in SEQ ID NO:1; (2) The amino acid sequence as shown in SEQ ID NO:2; (3) The amino acid sequence of a protein with the same function obtained by substituting, inserting or deleting one or more amino acids as shown in (1). (4) The amino acid sequence of a protein with the same function obtained by replacing, inserting or deleting one or more amino acids as shown in (2).

2. A nucleic acid molecule, characterized in that, It encodes the immunomodulatory bacterial protein containing the cytokine motif as described in claim 1.

3. A fusion protein, characterized in that, It includes the immunomodulatory bacterial protein containing the cytokine motif as described in claim 1, and a protein tag fused thereto.

4. A composition, characterized in that, It comprises the immunomodulatory bacterial protein containing cytokine motifs as described in claim 1; preferably, it also comprises other antitumor drugs.

5. A biomaterial, characterized in that, The biological material contains the nucleic acid molecule of claim 2, wherein the biological material is recombinant DNA, expression cassette, transposon, vector, microorganism or cell.

6. The use of the immunomodulatory bacterial protein containing cytokine motifs of claim 1, the nucleic acid molecule of claim 2, the conjugate of claim 3, the composition of claim 4, or the biomaterial of claim 5 in the preparation of a medicament for treating tumors.

7. The application according to claim 6, characterized in that, The tumors include intestinal tumors.

8. The use of the immunomodulatory bacterial protein containing cytokine motifs of claim 1, the nucleic acid molecule of claim 2, the conjugate of claim 3, the composition of claim 4, or the biomaterial of claim 5 in the preparation of a drug, characterized in that, The drug is any one or more of the following (1) to (4): (1) Drugs used to reduce the number of tumors; (2) Drugs used to reduce tumor size; (3) Drugs used to enhance immune responses; (4) Drugs used to alleviate weight loss in cancer patients.

9. A biological agent, characterized in that, The invention comprises the immunomodulatory bacterial protein containing cytokine motifs as described in claim 1, the nucleic acid molecule as described in claim 2, the conjugate as described in claim 3, the composition as described in claim 4, or the biomaterial as described in claim 5.

10. The method for preparing the immunomodulatory bacterial protein containing cytokine motifs as described in claim 1, characterized in that, Under conditions that allow the expression of the immunomodulatory bacterial protein containing the cytokine motif as described in claim 1, the microorganism or cell described in claim 5 is cultured to obtain the immunomodulatory bacterial protein containing the cytokine motif from the resulting culture.

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