A recombinant pentameric protein, polynucleotide, expression vector, host cell, method of making and use thereof

Abstract

This invention relates to the field of biopharmaceutical technology, specifically to a recombinant pentamer protein, polynucleotide, expression vector, host cell, preparation method, and application. The fusion protein is formed by fusing LTB or its immunogenic functional variant with the pentamer domain of COMP via a flexible linker peptide. This invention constructs the gene of this fusion protein in a eukaryotic expression vector, transfects it into CHO-K1 cells, obtains stable high-expression cell lines through pressure selection, and performs efficient secretory expression using serum-free suspension culture. The resulting protein forms a pentamer under non-reducing conditions. The sIgA level in mouse nasal lavage fluid is significantly higher than that of the antigen alone, confirming its effective activation of local immunity in the respiratory mucosa. This invention utilizes the CHO expression system to achieve large-scale preparation of structurally correct, homogeneous, and endotoxin-free LTB pentamers, which has significant application value in the development of mucosal vaccines, targeted delivery, and diagnostic reagents.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and medicine, specifically to a recombinant pentamer protein, polynucleotide, expression vector, host cell, preparation method, and application. Background Technology

[0002] The thermostable enterotoxin B subunit (LTB) of *Escherichia coli* is a non-toxic subunit of the enterotoxin secreted by enterotoxin-producing *E. coli* (ETEC), and it has attracted much attention due to its strong mucosal immunogenicity and its ability as a targeting carrier. Naturally occurring LTB exists in pentamer form, and this spatial structure is the structural basis for its specific and high-affinity binding to the GM1 ganglioside receptor on the host cell surface and its function as an immune adjuvant. Therefore, obtaining structurally correct and highly pure LTB pentamers is a key prerequisite for the development of related mucosal vaccines and targeted drugs.

[0003] Traditional methods for producing LTB primarily rely on prokaryotic expression systems (such as E. coli). However, the LTB produced by this method has significant limitations: ① Prokaryotic cells lack the ability to perform post-translational modifications in eukaryotic cells, affecting the correct folding and immunogenicity of LTB; ② Exogenous proteins easily form insoluble inclusion bodies, requiring complex refolding steps, but the pentamer structure is heterogeneous and the active component is lost after refolding; ③ Endotoxins are byproducts of prokaryotic systems and are difficult to completely remove, their residues can interfere with immunoassay and pose safety risks.

[0004] Therefore, there is an urgent need to develop a structurally homogeneous, immunogenic, and low-endotoxin LTB pentamer. Summary of the Invention

[0005] To address the above problems, this invention provides a recombinant pentamer protein, polynucleotide, expression vector, host cell, preparation method, and application.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A recombinant pentamer fusion protein, the recombinant pentamer fusion protein comprising: a) The amino acid sequence of the heat-labile enterotoxin B subunit of *Escherichia coli*, or a functional variant thereof having GM1 binding activity and / or immunogenicity, preferably with sequence homology >95%; the amino acid sequence of the heat-labile enterotoxin B subunit of *Escherichia coli* is shown in SEQ ID NO.3; b) The amino acid sequence of the pentamerizing domain of the cartilage oligomeric matrix protein, or a functional variant thereof capable of spontaneously forming pentamers; the amino acid sequence of the pentamerizing domain of the cartilage oligomeric matrix protein is shown in SEQ ID NO.4; Wherein, element a) and element b) are directly or through a flexible linker peptide to maintain the independence of their respective domains, so that the recombinant pentamer fusion protein can form a homopentamer; SEQ ID NO.3: MRDCADVYQAGFNKSGIYTIYINNMPEPKKVFCNMDVNGGGWTVIQHREDGSLDFQRGWKEYKMGFGNPSGEYWLGNEFIFAITSQRQYMLRIELMDWEGNRAYSQYD RFHIGNEKQNYRLYLKGHTGTAGKQSSLILHGADFSTKDADNDNCMCKCALMLTGGWWFDACGPSNLNGMFYTAGQNHGKLNGIKWHYFKGPSYSLRSTTMMIRPLDF.

[0007] SEQ ID NO.4: DLAPQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMECDACG.

[0008] Preferably, the amino acid sequence of the flexible linker peptide is (Gly-Gly-Gly-Gly-Ser)n, where n is an integer from 1 to 5.

[0009] Preferably, the N-terminus or C-terminus of the recombinant pentamer fusion protein further includes a tag for purification; the tag is a histidine tag.

[0010] Preferably, the label is a 6×His label located at the C end.

[0011] Preferably, the heat-labile enterotoxin B subunit of Escherichia coli can be located at the N-terminus or C-terminus of the pentamerization domain of the cartilage oligomeric matrix protein.

[0012] A polynucleotide, the base sequence of which is shown in SEQ ID NO.1; encoding the recombinant pentamer fusion protein.

[0013] An expression vector comprising the aforementioned polynucleotide; the polynucleotide is cloned into a mammalian expression vector (e.g., pWTY(+)), placed downstream of a strong promoter (e.g., a CMV promoter), and comprises a signal peptide sequence (e.g., a bee venom signal peptide), a selection marker (e.g., an isoprothiolane resistance gene), and a polyA signal, thereby constituting a recombinant expression vector.

[0014] A host cell containing the expression vector or having the polynucleotides integrated into its genome.

[0015] Preferably, the host cell is a Chinese hamster ovary cell; Preferably, the Chinese hamster ovary cells are CHO-K1 cells; A method for preparing a recombinant pentamer fusion protein includes the following steps: (1) Provide the host cell; (2) The host cells are cultured under conditions suitable for protein expression; (3) Harvest and purify the recombinant pentamer fusion protein from the culture.

[0016] Preferably, the culture in step (2) is a suspension culture in a serum-free medium; Preferably, the purification in step (3) is performed using histidine-tagged metal chelate affinity chromatography.

[0017] The purified protein was comprehensively identified by non-reducing SDS-PAGE (130kDa), size exclusion chromatography (SEC-HPLC, single symmetric peak), Western blotting (anti-His tag antibody), and transmission electron microscopy (TEM, fivefold symmetric structure).

[0018] The application of the recombinant pentamer fusion protein in the preparation of vaccines or immune adjuvants for the prevention or treatment of enterotoxigenic Escherichia coli infection.

[0019] The application of the recombinant pentamer fusion protein in the preparation of diagnostic reagents for ETEC infection.

[0020] Compared with the prior art, the present invention has the following beneficial effects: 1. Precise structure and excellent activity: The COMP scaffold forces LTB to form a pentamer in its natural active conformation, ensuring its high affinity binding to the GM1 receptor and preserving its complete biological and immunological functions.

[0021] 2. Advanced expression system: The CHO expression system ensures the correct folding of the protein and the necessary glycosylation modification, making the product closer to the natural conformation, with stronger immunogenicity, and fundamentally eliminating endotoxin contamination.

[0022] 3. Highly efficient and easily scaled up: Through vector engineering and host cell screening, high-level secretory expression of the protein (>50 mg / L) was achieved. The C-terminal His tag facilitates one-step affinity chromatography to obtain high-purity (>95%) products, and the downstream process is simple and suitable for large-scale production.

[0023] 4. Broad application prospects: The obtained uniform and stable pentamer can be used as an ideal antigen for the development of novel mucosal vaccines; its excellent targeting properties can also be used for targeted delivery of drugs or nucleic acids; at the same time, it is an ideal raw material for high-sensitivity diagnostic reagents. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present 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 only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 The diagram shows the DNA sequence structure of the target protein synthesized in this invention and the plasmid map of the recombinant expression vector. A represents the DNA sequence structure of the target protein, which sequentially shows: CMV promoter—signal peptide coding sequence—LTB gene—(Gly4Ser)3 Linker coding sequence—COMP pentamerization domain gene—6×His tag coding sequence—PolyA signal; B represents the plasmid map of the recombinant expression vector pWTY-LTB-COMP.

[0026] Figure 2 This is an indirect immunofluorescence (IFA) identification image of the CHO-K1 cell line stably transfected according to the present invention.

[0027] Figure 3 The image shows the Western Blot results of the purified LTB-COMP fusion protein of this invention; where A is the cell viability density graph; and B is the WB graph showing the protein supernatant expression on the second day.

[0028] Figure 4 The images show size exclusion chromatography (SEC-HPLC) analysis of the purified LTB-COMP fusion protein of this invention; where A is the elution peak of the LTB protein; B is the weak exposure peak of the purified protein; and C is the strong exposure peak of the purified protein.

[0029] Figure 5 These are negative staining transmission electron microscope (TEM) images of the LTB-COMP pentamer of this invention; where A is a spatial structure diagram of the recombinant protein; and B is a TEM image of the structure characterization.

[0030] Figure 6 This is a graph showing the results of mucosal sIgA antibody titer detection in this invention. Detailed Implementation

[0031] To facilitate understanding of the present invention, a more comprehensive description is provided below, along with preferred embodiments. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0032] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this invention and in its specification is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0033] Chinese hamster ovary (CHO) cells are currently the "gold standard" mammalian expression system for the production of recombinant therapeutic proteins. Their advantages lie in their ability to perform complex protein folding and precise post-translational modifications, achieving high-level secretory expression of recombinant proteins without endotoxin contamination. Suspension-acclimated cell lines such as CHO-K1 are more suitable for serum-free, high-density culture, facilitating large-scale industrial production.

[0034] The inventive concept of this invention is as follows: The heat-labile enterotoxin B subunit (LTB) of *Escherichia coli* has significant applications in vaccine adjuvants and drug delivery due to its strong mucosal immunogenicity and receptor targeting ability. The biological function of natural LTB is strictly dependent on its homopentamer structure, which is the basis for its specific binding to the GM1 ganglioside receptor and induction of mucosal immune responses. Therefore, obtaining structurally correct, homogeneous, and stable LTB pentamers is a core prerequisite for developing related products.

[0035] Currently, LTB production mainly relies on prokaryotic expression systems such as *E. coli*. However, this method has significant limitations: ① Prokaryotic cells lack eukaryotic post-translational modification capabilities, potentially affecting the correct folding and immunogenicity of LTB; ② Exogenous proteins easily form insoluble inclusion bodies, requiring complex refolding steps, but the resulting pentamer structure is heterogeneous and the active component is lost; ③ Endotoxins are byproducts of prokaryotic systems and are difficult to completely remove, their residues interfering with immunoassay and posing safety risks. The common consequence of these problems is that current technologies struggle to provide structurally intact, high-purity, endotoxin-free LTB pentamer products with excellent immunogenicity.

[0036] The N-terminal pentamerizing domain of chondrocyte oligomeric matrix protein (COMP) possesses the ability to spontaneously assemble into stable pentamers and has been used as a molecular scaffold to enhance the structural stability and functional activity of fusion proteins. Fusing LTB with the COMP pentamerizing domain holds promise for forcing LTB to form a native conformational pentamer through the scaffold effect of COMP, while maintaining its GM1 binding capacity. However, there are currently no reports on the expression of LTB-COMP fusion proteins in eukaryotic systems and their large-scale preparation utilizing their natural endotoxin-free and modifiable advantages.

[0037] This invention constructs a novel recombinant pentamer protein by fusing the LTB and COMP pentamer domains via a flexible linker peptide, and efficiently expresses it in CHO cells. As a mammalian expression system, CHO cells possess the inherent advantage of being free from endotoxin contamination—endotoxins are a byproduct of prokaryotic expression systems such as E. coli, and their complete removal is widely acknowledged; eukaryotic cells, however, do not produce endotoxins. Therefore, recombinant proteins expressed through the CHO system naturally avoid endotoxin contamination, allowing for the acquisition of a safer product without complex endotoxin removal processes. Furthermore, CHO cells possess excellent post-translational modification capabilities, which helps maintain the correct folding and immunogenicity of the LTB pentamer.

[0038] The purified protein exhibited a pentamer band of approximately 130 kDa under non-reducing conditions. Transmission electron microscopy revealed a clear five-fold symmetry structure, and it effectively induced the production of sIgA in the respiratory mucosa of mice, confirming its excellent immunomodulatory activity. This invention leverages the inherent advantages of the CHO system—naturally free of endotoxins and capable of proper modification—to successfully solve the technical challenges of endotoxin residues and structural inhomogeneity in existing LTB pentamer products, providing an ideal candidate molecule for mucosal vaccines, immune adjuvants, and targeted delivery systems.

[0039] The beneficial effects of the present invention will be illustrated below through specific embodiments.

[0040] Example 1: Construction of recombinant expression vector 1. Gene Design and Synthesis: Based on the codon preference of CHO cells, the DNA sequence encoding the target protein was optimized and synthesized. The sequence structure is: 5'-meal venom signal peptide-LTB (GenBank: AAA24818.1)-(Gly4Ser)3 Linker-COMP pentamer domain (aa 21-83, UniProt: P49747)-6×His tag-3', as shown below. Figure 1 As shown in Figure A, the whole genome synthesis was commissioned to GenScript Biotech Co., Ltd.

[0041] The optimized DNA sequence encoding the bee venom signal peptide -LTB-(G4S)3-COMP pentamer domain-6×His is shown in SEQ ID NO.1: Atgaaatttctggtgaacgtggcgctggtgtttatggtggtgtatattagctatatttatgcgggcagcggcatgcgcgattgcgcggatgtgtatcaggcgggctttaacaaaagcggcatttataccatttatattaacaacatgccggaaccgaaaaaagtgttttgcaacatggatgtgaacggcggcggctggaccgtgattcagcatcgcgaagatggcagcctggattttcagcgcggctggaaagaatataaaatgggctttggcaacccgagcggcgaatattggctgggcaacgaatttatttttgcgattaccagccagcgccagtatatgctgcgcattgaactgatggattgggaaggcaaccgcgcgtatagccagtatgatcgctttcatattggcaacgaaaaacagaactatcgcctgtatctgaaaggccataccggcaccgcgggcaaacagagcagcctgattctgcatggcgcggattttagcaccaaagatgcggataacgataactgcatgtgcaaatgcgcgctgatgctgaccggcggctggtggtttgatgcgtgcggcccgagcaacctgaacggcatgttttataccgcgggccagaaccatggcaaactgaacggcattaaatggcattattttaaaggcccgagctatagcctgcgcagcaccaccatgatgattcgcccgctggattttggcggcggcggcagcggcggcggcggcagcggcggcggcggcagcgatctggcgccgcagatgctgcgcgaactgcaggaaaccaacgcggcgctgcaggatgtgcgcgaactgctgcgccagcaggtgaaagaaattacctttctgaaaaacaccgtgatggaatgcgatgcgtgcggc。

[0042] The amino acid sequence of the recombinant pentamer fusion protein (LTB-COMP fusion protein) is shown in SEQ ID NO.2 and is as follows: MKFLVNVALVFMVVYISYIYAGSGMRDCADVYQAGFNKSGIYTIYINNMPEPKKVFCNMDVNGGGWTVIQHREDGSLDFQRGWKEYKMGFGNPSGEYWLGNEFIFAITSQRQYMLRIELMDWEGNRAYSQYDRFHIGNEKQNYRLYLKGH TGTAGKQSSLILHGADFSTKDADNDNCMCKCALMLTGGWWFDACGPSNLNGMFYTAGQNHGKLNGIKWHYFKGPSYSLRSTTMMIRPLDFGGGGSGGGGSGGGGSDLAPQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMECDACG.

[0043] 2. Vector Construction: The synthesized gene fragment and the mammalian expression vector pWTY(+) (Beijing BGI Genomics) were double-digested using restriction endonucleases HindIII and XhoI (purchased from NEB). Ligation was performed overnight at 16°C using T4 DNA ligase (NEB). The ligation product was transformed into *E. coli* DH5α competent cells, plated on LB agar plates containing ampicillin (100 μg / mL), and incubated overnight at 37°C.

[0044] 3. Screening and Verification of Positive Clones: Single colonies were picked and cultured to expand the culture, and plasmids were extracted. PCR verification was performed using universal primers and internal specific primers. The positive clone plasmid was sent to Beijing Ruiboxingke Biotechnology Co., Ltd. for Sanger sequencing, confirming that the gene sequence was 100% correct. The correct plasmid was named pWTY-LTB-COMP. Figure 1 As shown in B in the diagram.

[0045] Example 2: Construction of a CHO-K1 cell line stably expressing LTB-COMP pentameric protein 1. Cell culture: CHO-K1 cells (ATCC CCL-61) were cultured in suspension using CD CHO medium (Gibco) and placed in a constant temperature shaker at 37°C, 8% CO2, and 130 rpm.

[0046] 2. Cell transfection: CHO-K1 cells in logarithmic growth phase with a viability >95% were harvested and their density adjusted to 1×10⁻⁶ cells / cells. 6 cells / mL. Take 1 mL of cell suspension and mix it with 4 μg of pWTY-LTB-COMP plasmid and 8 μL of Lipofectamine 3000 transfection reagent (Invitrogen), following the instructions. Replace with fresh complete culture medium 6 hours after transfection.

[0047] 3. Pressure Selection and Monoclonalization: Forty-eight hours after transfection, blast fungicide (Blasticidin S, Invivogen) was added to the culture medium to a final concentration of 15 μg / mL for selection. Selection continued for 3 weeks, with the medium containing the selection drug being changed periodically. After the resistant cell pool was formed, cells were seeded into 96-well plates using a limiting dilution method, with an average of 0.5 cells per well, and continued to be cultured in medium containing the selection drug to obtain monoclonal cell lines.

[0048] 4. High-yield clone screening: The culture supernatant of single-clonal cell lines was collected by centrifugation, and the protein expression levels of each clone were initially detected using a sandwich ELISA method (using anti-LTB antibody as the capture antibody and HRP-labeled anti-His tag antibody as the detection antibody). The results are as follows: Figure 2 As shown, positive clones exhibit a clear fluorescent signal, indicating successful expression of the fusion protein. The three clones with the highest expression levels were selected for amplification.

[0049] Example 3: Serum-free suspension culture and harvesting of LTB-COMP pentameric protein 1. Shake flask culture: The selected high-yielding clones were inoculated into 250 mL shake flasks containing 50 mL of CD CHO medium, with an initial viable cell density of 3 × 10⁻⁶ cells / mL. 5 cells / mL. Cultured at 37°C, 8% CO2 (v / v), and 130 rpm.

[0050] 2. Process Monitoring: Samples were taken daily, and cell density and viability were measured using a Countstar cell counter. The concentration of the target protein in the supernatant was measured using the ELISA method described above. Culture was terminated when cell viability dropped below 70% or the culture reached a plateau. In this example, protein expression reached its peak at approximately 65 mg / L on day 7 of culture.

[0051] 3. Harvesting: Centrifuge the culture at 3000g for 20 minutes at 4℃ and collect the supernatant. Filter the supernatant through a 0.22μm PES membrane filter (Millipore) to remove residual cell debris, obtaining a clear harvest solution, which can be stored at -80℃ or used immediately for purification.

[0052] Example 4: Purification and Physicochemical Identification of Recombinant Protein 1. Metal chelate affinity chromatography: All steps were performed in a 4°C cold chamber. The clarified harvest solution was loaded onto a 5 mL HisTrap HP pre-packed column (Cytiva) equilibrated with equilibration buffer (20 mM Tris-HCl, 300 mM NaCl, 20 mM imidazole, pH 8.0). The loading flow rate was 1 mL / min. After loading, unbound contaminants were washed with 10 column volumes of equilibration buffer. Weakly bound contaminants were then eluted with wash buffer containing 50 mM imidazole. Finally, the target protein was eluted with elution buffer containing 250 mM imidazole at a flow rate of 1 mL / min, and the elution peak was collected.

[0053] 2. Desalting and Concentration: The eluent was loaded into an ultrafiltration centrifuge tube (Millipore) with a molecular weight cutoff of 10 kDa. The solution was changed and concentrated with PBS (pH 7.4) at 4°C and 4000g to remove imidazole.

[0054] 3. Concentration determination: The protein concentration was determined using a NanoDrop 2000 spectrophotometer (Thermo Fisher) based on the absorbance of the protein at 280 nm (calculated using the theoretical extinction coefficient).

[0055] 4. SDS-PAGE and Western Blot Analysis: 10 μg of purified protein was mixed with a loading buffer free of β-mercaptoethanol and subjected to 12% non-reducing SDS-PAGE. After electrophoresis, half of the gel was stained with Coomassie Brilliant Blue R250, and the other half was transferred to a PVDF membrane using a wet transfer method. After blocking with 5% skim milk for 1 hour, the membrane was incubated with mouse anti-6×His-tagged monoclonal antibody (1:5000 dilution, Abcam) at room temperature for 1 hour. After washing, it was incubated with HRP-labeled goat anti-mouse secondary antibody (1:10000 dilution) at room temperature for 45 minutes. Development was performed using ECL luminescent substrate (Thermo) and observed under a chemiluminescence imaging system (Tanon). Results are as follows: Figure 3 As shown, a single, clear band appears at approximately 130 kDa, consistent with the theoretical molecular weight of the pentamer.

[0056] 5. Size exclusion chromatography: An Agilent 1260 Infinity II HPLC system equipped with a TSKgel G3000SWxl column (7.8 mm × 300 mm) was used. The mobile phase was PBS (pH 7.4), the flow rate was 0.5 mL / min, and the detection wavelength was 280 nm. 50 μg of purified protein was loaded for analysis. Results are as follows: Figure 4As shown, the elution spectrum presents a single, symmetrical main peak with a retention time of approximately 12.5 minutes, which is consistent with the relative position of the pentamer protein standard (Thyroglobulin, 669 kDa). This indicates that the sample is highly homogeneous, mainly exists in pentamer form, has high purity (>95%), and exhibits high-level secretory expression of the protein (>50 mg / L).

[0057] Example 5: Structural Characterization TEM Images The protein was further purified by gel filtration chromatography. The protein from the main peak portion of the gel filtration chromatography curve was collected. TEM negative staining samples were selected for observation. 10 µL of the protein was dropped onto a coated copper grid, incubated for 5 min, rinsed three times with distilled water, and then stained with 5 µL of uranium acetate for 5 min. After drying, the samples were observed under a transmission electron microscope. The images showed that the protein particles were uniformly distributed, with a clear five-fold symmetry structure (indicated by white arrows), a high central electron density, and a lower outer electron density, consistent with the designed COMP core-LTB periphery structure model. No obvious aggregation was observed. Figure 5 As shown.

[0058] Example 6: Functional validation as a candidate adjuvant for PRRSV mucosal vaccine This embodiment aims to verify the ability of LTB-COMP pentameric protein as a mucosal adjuvant to enhance the immunogenicity of porcine reproductive and respiratory syndrome virus (PRRSV) GP5 protein antigen. Using a BALB / c mouse model, mice were divided into a PBS control group, a GP5 antigen-only group (10 μg), and a mixed group of GP5 antigen (10 μg) and LTB-COMP adjuvant (5 μg). Immunization was administered twice via the nasal route on days 0 and 14. Serum and nasal lavage fluid were collected on day 14 after the last immunization. The level of secretory IgA (sIgA) in the nasal lavage fluid of the adjuvant group was significantly higher than that in the antigen-only group, confirming its effective activation of local immunity in the respiratory mucosa. Figure 6 As shown.

[0059] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0060] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. Those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of this invention should be determined by the appended claims.

Claims

1. A recombinant pentamer fusion protein, characterized in that, The recombinant pentamer fusion protein comprises: a) The amino acid sequence of the heat-labile enterotoxin B subunit of *Escherichia coli*, or a functional variant thereof having GM1 binding activity and / or immunogenicity; the amino acid sequence of the heat-labile enterotoxin B subunit of *Escherichia coli* is shown in SEQ ID NO.3; b) The amino acid sequence of the pentamerizing domain of the cartilage oligomeric matrix protein, or a functional variant thereof capable of spontaneously forming pentamers; the amino acid sequence of the pentamerizing domain of the cartilage oligomeric matrix protein is shown in SEQ ID NO.4; Wherein, element a) and element b) are directly or through a flexible linker peptide, so that the recombinant pentamer fusion protein can form a homopentamer.

2. The recombinant pentamer fusion protein according to claim 1, characterized in that, The amino acid sequence of the flexible linker peptide is (Gly-Gly-Gly-Gly-Ser)n, where n is an integer from 1 to 5.

3. The recombinant pentamer fusion protein according to any one of claims 1 to 2, characterized in that, The recombinant pentamer fusion protein also contains a tag for purification at its N-terminus or C-terminus; the tag is a histidine tag.

4. A polynucleotide, characterized in that, The base sequence of the polynucleotide is shown in SEQ ID NO: 1; Its encoding is the recombinant pentamer fusion protein as described in any one of claims 1 to 3.

5. An expression carrier, characterized in that, It contains the polynucleotide as described in claim 4.

6. A host cell, characterized in that, It comprises the expression vector of claim 5, or the polynucleotide of claim 4 integrated into the genome.

7. The host cell according to claim 6, characterized in that, The host cells were Chinese hamster ovarian cells.

8. A method for preparing a recombinant pentamer fusion protein, characterized in that, Includes the following steps: (1) Providing the host cell as described in claim 7; (2) The host cells are cultured under conditions suitable for protein expression; (3) Harvest and purify the recombinant pentamer fusion protein from the culture.

9. The use of the recombinant pentamer fusion protein as described in any one of claims 1 to 3 in the preparation of vaccines or immune adjuvants for the prevention or treatment of enterotoxigenic Escherichia coli infection.

10. The use of the recombinant pentamer fusion protein as described in any one of claims 1 to 3 in the preparation of a diagnostic reagent for ETEC infection.

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