Streptococcus mutans bacteriophage gp15 protein and application thereof in rapid diagnosis of streptococcus mutans

Abstract

The invention discloses a streptococcus mutans bacteriophage gp15 protein and an application thereof in rapid diagnosis of streptococcus mutans, the gp15 protein in the streptococcus mutans bacteriophage is found and confirmed to be a novel RBP for the first time, and construction of a gp15-mNG fusion protein system confirms that the protein can efficiently and specifically recognize and combine a streptococcus mutans surface receptor, so that the streptococcus mutans bacteriophage can be used for rapidly diagnosing streptococcus mutans. The binding rate reaches up to 99% or above, and an ideal molecular tool is provided for developing a rapid diagnosis technology of streptococcus mutans.

Description

Technical Field

[0001] This invention belongs to the fields of microbiology, immunology and clinical diagnostic technology, and specifically relates to a Streptococcus mutans phage gp15 protein and its application in the rapid diagnosis of Streptococcus mutans. Background Technology

[0002] Streptococcus mutans is a common pathogenic bacterium in the human oral cavity and is closely related to the occurrence and development of dental caries. Currently, detection methods for Streptococcus mutans mainly include traditional culture methods, PCR molecular detection, and immunological detection. Traditional culture methods are time-consuming and have low sensitivity; PCR detection relies on specialized equipment and technicians and is easily affected by nucleic acid contamination, impacting accuracy; immunological methods such as ELISA have sensitivity and specificity limited by the quality of antibody preparation. Therefore, developing a rapid, sensitive, and easy-to-operate method for detecting Streptococcus mutans is of great significance.

[0003] Bacteriophages are a class of viruses that specifically infect bacteria and other microorganisms. Their infection process begins with the specific adsorption of the host bacterial surface. This adsorption process depends on the specific recognition and binding of the receptor-binding protein (RBP) at the tail of the bacteriophage to receptors on the surface of the host bacteria. As the core functional molecule for bacteriophage recognition of the host, RBP naturally possesses significant advantages such as strong binding specificity, high affinity, small molecular weight, good stability, and ease of genetic engineering, making it a highly promising new recognition element in the field of rapid microbial diagnostics.

[0004] However, current research on RBPs from Streptococcus mutans bacteriophages remains incomplete: existing literature has not clearly identified any functional RBP molecules from Streptococcus mutans bacteriophages. This technological gap results in a lack of highly specific and high-affinity single-molecule recognition tools for Streptococcus mutans, leading to time-consuming and low-sensitivity detection methods. This severely limits the breakthrough and clinical translation of rapid diagnostic technologies for Streptococcus mutans, necessitating the discovery of novel functional RBPs and the establishment of related application systems. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a Streptococcus mutans phage gp15-mNG fusion protein, clarify the biological function of gp15 protein as a receptor-binding protein (RBP), and achieve intuitive characterization of protein binding activity by utilizing the fluorescence properties of mNG; at the same time, a rapid diagnostic method for Streptococcus mutans based on this protein is established, which is applicable to the rapid screening and diagnosis of Streptococcus mutans in clinical samples, oral health monitoring and food safety fields.

[0006] The present invention achieves the above objectives using the following technical solution: The first aspect of the present invention provides a Streptococcus mutans phage receptor binding protein gp15, the amino acid sequence of which is shown in SEQ ID NO:1.

[0007] The *Streptococcus mutans* bacteriophage involved in this invention is a type of virus that depends on *Streptococcus mutans* for growth and belongs to the family Longtail Phageidae. Its head is an isometric icosahedral capsid, and its tail is a non-contractile long tail. The estimated capsid diameter is (67±0.4) nm, and the estimated length and width of the non-contractile tail are (283±7) nm and (8.3±0.1) nm, respectively. Bacteriophages are a type of virus that specifically infects bacteria. Their host recognition depends on the high affinity and high specificity of the interaction between the tail receptor-binding protein (RBP) and bacterial surface receptors. The functional RBP molecule of *Streptococcus mutans* bacteriophages has not yet been clearly identified in the prior art. In this invention, *Streptococcus mutans* bacteriophages include reported bacteriophages such as M102, UA140, and UA159. APCM01, SM1, etc., also include unreported bacteriophages such as 1-7 in the embodiments of the present invention.

[0008] The second aspect of the present invention provides a nucleic acid molecule encoding the receptor-binding protein gp15 described in the first aspect, the nucleotide sequence of which is shown in SEQ ID NO:2 or has more than 90% identity with SEQ ID NO:2.

[0009] The term "identity," also known as "homology," refers to a sequence that is at least 80% identical in amino acid or nucleotide sequence to the sequence provided in this invention. To determine sequence identity, sequence alignment can be performed using various methods known to those skilled in the art, such as BLAST, BLAST-2, ALIGN, NEEDLE, Megalign (DNASTAR), Snapgene, or DNAMAN software. Those skilled in the art can determine appropriate parameters for alignment, including any algorithms required to achieve optimal alignment across the full-length sequences being compared.

[0010] A third aspect of the present invention provides a fusion protein gp15-mNG, wherein the fusion protein gp15-mNG is composed of the receptor-binding protein gp15 described in the first aspect and the fluorescent protein mNeonGreen.

[0011] In some embodiments, the amino acid sequence of the fusion protein gp15-mNG is shown in SEQ ID NO:3.

[0012] In some embodiments, the fusion protein gp15-mNG of the present invention is obtained through artificial synthesis. Methods for artificially synthesizing fusion proteins are known in the art, for example, the fusion protein of the present invention is obtained through direct amino acid synthesis. In some embodiments, the fusion protein of the present invention is obtained through genetic engineering expression. Genetic engineering expression systems include prokaryotic cell expression systems, eukaryotic cell expression systems, and cell-free expression systems. Examples of prokaryotic cell expression systems include Escherichia coli expression systems. Eukaryotic cell expression systems include enzyme expression systems, insect cell expression systems, and mammalian cell expression systems.

[0013] The fourth aspect of the present invention provides a recombinant expression vector encoding the fusion protein gp15-mNG, wherein the recombinant expression vector is pET-28a::gp15-mNG, which is composed of a gp15 gene fragment, a fluorescent protein mNeonGreen and a linearized pET-28a vector.

[0014] In some embodiments, the nucleotide sequence of the recombinant expression vector is as shown in SEQ ID NO:4 or has more than 90% identity with SEQ ID NO:4.

[0015] In this invention, the term "nucleic acid molecule" or "nucleic acid" refers to a polymeric form of nucleotides of any length containing deoxyribonucleotides, ribonucleotides, and / or their analogues, including DNA, RNA, and DNA / RNA hybrids, and also including DNA or RNA analogues, such as those containing a modified backbone (e.g., peptide nucleic acid (PNA) or phosphate thioester) or modified bases. Therefore, the nucleic acids of this invention include DNA, cDNA, mRNA, recombinant nucleic acids, etc. Once the coding sequence of the receptor-binding protein gp15 or the fusion protein gp15-mNG described in this invention, or a sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it, can be obtained in large quantities using recombinant technology.

[0016] In this invention, the term "expression vector" or "vector" refers to an artificial construct capable of delivering and preferably expressing one or more target genes or sequences in a host cell. Vectors can be known vectors or self-constructed vectors. Known vectors include plasmid vectors, lentiviral vectors, adenovirus vectors, AAV viral vectors, herpesvirus vectors, and other viral vectors. Other viral vectors may include bacteriophage vectors, baculovirus vectors, animal viral vectors, plant viral vectors, and may include papillomavirus vectors, herpesvirus vectors, poxvirus vectors, RNA virus vectors, bovine papillomavirus vectors, EB virus vectors, retroviral vectors, etc.

[0017] The fifth aspect of the present invention provides a recombinant host cell comprising the fusion protein gp15-mNG described in the third aspect and / or the recombinant expression vector described in the fourth aspect.

[0018] In this invention, the term "recombinant cell" or "recombinant host cell" refers to any cell type suitable for transformation, transfection, transduction, etc., using an expression vector containing nucleic acid molecules provided by this invention. Recombinant cells include any progeny of the parent cell that differs from the parent cell due to mutations occurring during replication. The recombinant cells include prokaryotic cells and eukaryotic cells; the prokaryotic cells include bacteria, actinomycetes, cyanobacteria, mycoplasma, chlamydia, and rickettsiae; the eukaryotic cells include mammalian cells, insect cells, plant cells, and yeast cells; preferably, the recombinant host cell is an immune cell; most preferably, the immune cell includes T cells, NK cells, iNKT cells, B cells, CTL cells, monocytes, myeloid cells, dendritic cells, macrophages, or any combination thereof.

[0019] The sixth aspect of the present invention provides a colloidal gold-gp15 conjugate comprising the receptor-binding protein gp15 described in the first aspect and colloidal gold.

[0020] In some embodiments, the colloidal gold-gp15 conjugate is prepared by conjugating receptor-binding protein gp15 with colloidal gold.

[0021] The seventh aspect of the present invention provides for any of the following applications of the receptor-binding protein gp15 described in the first aspect, the fusion protein gp15-mNG described in the third aspect, and / or the colloidal gold-gp15 conjugate described in the sixth aspect: 1) application in the preparation of products for the specific detection of Streptococcus mutans; 2) application in the preparation of products for dental caries risk assessment or oral cariogenic bacteria screening.

[0022] In some embodiments, the product includes diagnostic reagents, reagent kits, test strips, microfluidic chips, biosensors, systems, devices, and readable media.

[0023] In some embodiments, the diagnostic reagent is labeled with a detectable marker.

[0024] In some embodiments, the detectable marker is selected from colloidal gold, fluorescent protein, enzyme, quantum dot, magnetic bead, or radioactive isotope.

[0025] In some embodiments, the kit further includes at least one of a diluent, a quality control, a buffer solution, and an instruction manual.

[0026] In some embodiments, the test strip uses the colloidal gold-gp15 conjugate described in the sixth aspect as a conjugate, comprising a detection line coated with a receptor-binding protein gp15-specific antibody, a control line coated with a polyclonal antibody against the receptor-binding protein gp15, and a chromatography carrier composed of a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad.

[0027] In some embodiments, the test strip is a colloidal gold immunochromatographic test strip.

[0028] The eighth aspect of the present invention provides a method for preparing the fusion protein gp15-mNG described in the third aspect, the method comprising the steps of: amplifying the receptor-binding protein gp15 described in the first aspect, recombining it with fluorescent protein mNeonGreen and linearized pET-28a vector via a Gibson ligation reaction, and transforming it into competent cells.

[0029] In some embodiments, the competent cells are competent Escherichia coli.

[0030] In some embodiments, the competent cells are competent Escherichia coli BL21(DE3).

[0031] The ninth aspect of the present invention provides a product comprising any one of the following: 1) a colloidal gold immunochromatographic kit for the specific detection of Streptococcus mutans, the colloidal gold immunochromatographic kit comprising the colloidal gold-gp15 conjugate described in the sixth aspect, a detection line coated with a receptor-binding protein gp15-specific antibody, a control line coated with a polyclonal antibody against the receptor-binding protein gp15, and a chromatography carrier composed of a sample pad, a conjugation pad, a nitrocellulose membrane, and an absorbent pad; 2) a fluorescent detection kit for the specific detection of Streptococcus mutans, the fluorescent detection kit comprising the fusion protein gp15-mNG described in the third aspect, and at least one of a diluent, a quality control, a buffer, and an instruction manual.

[0032] In some embodiments, the fluorescence detection kit further includes a microplate, glass slide, or microfluidic chip for carrying the sample.

[0033] In this invention, the product may comprise a solid substrate such as a chip, a glass slide, an array, etc., having reagents capable of detecting and / or quantifying one or more subject-derived samples immobilized at predetermined locations on the substrate. As an illustrative example, reagents immobilized at discrete predetermined locations may be provided to the chip for the specific detection of Streptococcus mutans in a sample.

[0034] In some embodiments, the sample is a sample containing the oral microbiota of the subject.

[0035] In this invention, the term "oral flora" refers to the collection of microorganisms that reside in the human oral cavity, and its components include bacteria, fungi, viruses, etc.

[0036] In the context of this invention, the term "sample" as used refers to a composition obtained from or derived from a subject (e.g., an individual of interest) that contains cells and / or other molecular entities to be characterized and / or identified based on, for example, physical, biochemical, chemical, and / or physiological characteristics. For example, a sample refers to any sample derived from a subject of interest that is expected or known to contain cells and / or molecular entities to be characterized. Samples include, but are not limited to, tissue samples, primary or cultured cells or cell lines, cell cultures, cell supernatants, cell lysates, platelets, serum, plasma, vitreous fluid, lymph, synovial fluid, follicular fluid, semen, amniotic fluid, emulsion, whole blood, blood-derived cells, urine, cerebrospinal fluid, sputum, tears, sweat, mucus, saliva, dental plaque, caries plaque, supragingival plaque, subgingival plaque, periimplantation submucosal plaque, root canal plaque, dorsum of the tongue plaque, and other mucosal surface plaque samples, tissue culture fluids, tissue extracts, homogenized tissues, cell extracts, and combinations thereof.

[0037] In the context of this invention, a subject refers to any individual of interest, preferably a live organism for which Streptococcus mutans needs to be detected, including humans, other mammals, preferably primates, and particularly preferably humans.

[0038] Advantages and beneficial effects of the present invention: The present invention is the first to identify and prepare a novel functional RBP: the gp15 protein of Streptococcus mutans bacteriophage is identified as a novel RBP, and the protein is expressed in a high-efficiency prokaryotic environment and purified to a high purity by constructing a fusion expression vector. It is also confirmed that the protein can specifically bind to the surface receptor of Streptococcus mutans (with a binding rate of over 99%), filling the relevant research gap and laying the foundation for the innovation of rapid diagnostic technology for Streptococcus mutans.

[0039] Based on the above findings, this invention uses the gp15 protein as the core recognition element and innovatively develops two detection platforms: colloidal gold immunochromatography (rapid qualitative analysis) and fluorescent probes (high-sensitivity quantitative analysis). The two systems can be used alone or in combination to achieve the "initial screening-confirmation" process, which is suitable for multiple scenarios such as on-site screening in dental clinics, community screening, clinical diagnosis, and food safety monitoring. The system is easy to operate, cost-controllable, and easy to industrialize and promote, helping to improve the early screening rate of Streptococcus mutans infection. Attached Figure Description

[0040] Figure 1 The figure shows the results of flow cytometry analysis of the specific binding ability of the gp15-mNG fusion protein. Detailed Implementation

[0041] The reagents, raw materials, and experimental consumables used in this invention are readily available to those skilled in the art and, unless otherwise specified, can be obtained commercially. Experimental methods not specifying particular conditions in this invention are typically performed under conventional conditions or according to the manufacturer's recommendations. In particular, the following examples are for illustrative purposes only and should not be construed as limiting the scope of the invention in any way. It should be noted that the experimental conditions and results described in the following examples are for illustrative purposes only and should not, and will not, limit the invention as described in the claims.

[0042] The present invention will be further illustrated below with reference to specific embodiments. These specific embodiments are for illustrative purposes only and should not be construed as limiting the invention. Those skilled in the art will understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the invention. The scope of the invention is defined by the claims and their equivalents. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains.

[0043] Example I. Experimental Methods 1. Cloning and expression of gp15-mNG fusion protein: Using the genome of Streptococcus mutans phage M102 as a template, specific primers were designed based on the gp15 gene sequence (Table 1), and the full-length gp15 gene fragment was obtained by PCR amplification.

[0044] Table 1. Primer sequence listing

[0045] The prokaryotic expression vector pET-28a::gp15-mNG was constructed. The amplified gp15 gene fragment, fluorescent protein mNeonGreen (mNG), and linearized pET-28a vector were recombined via Gibson ligation and transformed into competent Escherichia coli BL21 (DE3).

[0046] Table 2. Reagents and Product Numbers

[0047] Positive clones were selected for sequencing verification. The engineered bacteria with correct sequencing were inoculated into LB medium (containing 50 μg / mL kanamycin) and cultured at 37°C until OD600 = 0.6-0.7. Then, IPTG was added to a final concentration of 0.2 mM and expression was induced at 16°C for 20 hours.

[0048] The expression product was purified using a Ni-NTA affinity chromatography column, and the protein purity was verified by SDS-PAGE electrophoresis. The protein concentration was determined by Nanodrop, and high-purity gp15-mNG protein was obtained.

[0049] 2. Verification of gp15-mNG fusion protein binding activity: Each experimental strain was inoculated into BHI liquid medium and incubated statically at 37℃ and 5% CO2 for 16-18 hours until the stationary phase. 1 mL of bacterial suspension was centrifuged at 6000×g for 1 minute, and the supernatant was discarded. The cells were gently resuspended in pre-chilled PBS buffer and washed twice to remove medium components and reduce non-specific adsorption. The bacterial concentration of each strain was adjusted to approximately 1×10⁻⁶ cells / mL using PBS-BSA. 8 CFU / mL (corresponding to OD600≈0.6-0.8), place on ice for later use.

[0050] Target bacterium: Streptococcus mutans standard strain UA159 ( Figure 1 159), Streptococcus mutans UA140 ( Figure 1 140), laboratory isolation of Streptococcus mutans 1-7 ( Figure 1 (1-7)

[0051] Negative control bacteria: Common non-target bacteria in the oral cavity, such as Streptococcus Gordonii ( Figure 1 (s_g) and Streptococcus sanguinis ( Figure 1 (s_s).

[0052] Binding activity was verified using single-resolution microscopy and flow cytometry, respectively. 1) Single-resolution microscopy detection: The purified gp15-mNG protein (diluted to a final concentration of 0.9 mg / mL with PBS buffer at pH 7.4) was mixed with the bacterial culture at a volume ratio of 1:100, incubated at 37°C with shaking for 10 minutes, and washed twice before the binding efficiency was analyzed by a single-molecule detection system.

[0053] 2) Flow cytometry: The purified gp15-mNG protein (final concentration 0.9 mg / mL) was mixed with the bacterial culture at a volume ratio of 1:20 and incubated with the bacterial culture. After washing twice, the proportion of fluorescent positive bacteria was detected by flow cytometry to verify the binding specificity.

[0054] ①The experiment was set up with the following groups: Experimental group (gp15-mNG group): 100 μL of prepared Streptococcus mutans UA159, UA140 and 1-7 bacterial suspensions were mixed with 5 μL of gp15-mNG protein working solution (final concentration approximately 0.9 mg / mL).

[0055] Negative control group 1 (bacterial autofluorescence control): 100 μL of Streptococcus mutans UA159 bacterial culture was taken and only 5 μL of PBS-BSA buffer was added to detect the bacterial autofluorescence background.

[0056] Specific control group (non-target bacteria group): 100 μL of Streptococcus sanguinis or Streptococcus salivarius bacterial suspension was mixed with 5 μL of gp15-mNG protein working solution to verify the specificity of gp15 protein binding.

[0057] ② Incubation and Detection: Gently vortex the above mixed sample and incubate in a 37℃ constant temperature shaker for 10 minutes in the dark. After incubation, add 1 mL of pre-chilled PBS to each tube, centrifuge at 6000×g for 1 minute, and discard the supernatant to remove unbound free proteins. Repeat this washing step twice. Resuspend the bacterial cells in 500 μL of pre-chilled PBS and immediately perform instrumental detection.

[0058] ③ Flow cytometry: Flow cytometry was used for detection. Forward scattering (FSC) and side scattering (SSC) parameters were set for unlabeled bacteria to delineate bacterial phylogenetic groups. The mNG fluorescence signal was detected using a fluorescence channel (e.g., FITC channel, excitation light 488 nm, detector 530 / 30 nm bands). First, the negative control was used to establish the position of the negative control group, ensuring its fluorescence signal was primarily below the set threshold. Then, all experimental samples were detected under the same voltage settings.

[0059] ④ Data Analysis: Data were analyzed using the flow cytometry software. The percentage of bacteria in each sample whose fluorescence signal intensity was higher than the negative control threshold (usually the 99th percentile of the "bacterial autofluorescence control") was recorded as the binding positivity rate. Data are expressed as mean ± standard deviation.

[0060] II. Experimental Results 1. Flow cytometry analysis results as follows Figure 1 As shown, the specific binding ability of the gp15-mNG fusion protein is clearly demonstrated.

[0061] High binding efficiency: For the target strains Streptococcus mutans UA159, UA140 and 1-7, the binding positivity rate of the gp15-mNG experimental group was higher than 99.5% (P<0.001), indicating that the gp15 protein can bind efficiently and in large quantities to the surface of almost every Streptococcus mutans cell.

[0062] Binding specificity: After incubation with non-target bacteria (Streptococcus sanguinis, Streptococcus salivarius) in the "gp15-mNG group", the positive binding rate was less than 5%, which was not significantly different from the "bacterial autofluorescence control" group (P>0.05), proving that the binding of gp15 protein has high species specificity and is limited to Streptococcus mutans.

[0063] In summary, this embodiment quantitatively confirmed by flow cytometry that the gp15 protein derived from *Streptococcus mutans* phage is a highly efficient and specific receptor-binding protein (RBP), with a binding rate of nearly 100% to *Streptococcus mutans* and no significant binding to the non-target oral streptococci tested. This result provides direct and strong experimental evidence for developing highly specific rapid diagnostic kits for *Streptococcus mutans* using the gp15 protein as a core recognition element.

[0064] 2. Construction of a rapid diagnostic system: Using the gp15 protein of Streptococcus mutans phage as the core recognition element, a dual-complementary rapid diagnostic system is constructed to replace the existing culture-isolation, nucleic acid amplification, or antibody recognition mechanisms. The technical solution is as follows: 1) Cloning, expression, and purification of gp15 protein: Using the genome of Streptococcus mutans phage M102 as a template, the full-length gp15 gene fragment was amplified by PCR, and the pET-28a::gp15-mNG fusion expression vector was constructed and transformed into Escherichia coli BL21(DE3). After IPTG induction, high-purity gp15 protein was obtained by Ni-NTA affinity chromatography. Flow cytometry and other methods confirmed that the protein specifically binds to Streptococcus mutans with a binding rate of over 99%. The relevant sequences are shown in Table 3.

[0065] Table 3. Sequence List

[0066] 2) Construction of a dual rapid diagnostic system: ① Colloidal gold immunochromatographic assay system: The gp15 protein is conjugated with colloidal gold to prepare a conjugate. The gp15-specific antibody (detection line) and goat anti-gp15 polyclonal antibody (control line) are coated on a nitrocellulose membrane and assembled into a test strip. During detection, Streptococcus mutans in the sample binds to the colloidal gold-gp15 conjugate, migrates with the chromatography solvent to the detection line and is captured, forming a red band, thus achieving rapid qualitative analysis.

[0067] ② Fluorescent probe detection system: The purified gp15-mNG fusion protein is used directly as a fluorescent probe (no additional fluorescent dye is required). The reaction conditions such as incubation temperature and time are optimized, and a standard curve is plotted by detecting the correlation between the fluorescence intensity of mNG and the bacterial concentration to achieve highly sensitive quantitative detection of Streptococcus mutans. Other fluorescent dyes can also be selected to prepare probes according to detection needs to adapt to different detection scenarios.

[0068] Complementary application modes: the colloidal gold system is used for rapid on-site screening, while the fluorescent probe system is used for accurate quantitative confirmation of positive samples, taking into account the needs of different scenarios.

[0069] The above solution solves the problems of existing technologies such as long processing time, reliance on specialized equipment, insufficient specificity and sensitivity, and limited application scenarios. At the same time, the mNG fusion system simplifies the characterization process of protein binding activity, enabling efficient and accurate detection.

[0070] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. A Streptococcus mutans phage receptor-binding protein gp15, characterized in that, Its amino acid sequence is shown in SEQ ID NO:

1.

2. A nucleic acid molecule encoding the receptor-binding protein gp15 of claim 1, characterized in that, Its nucleotide sequence is as shown in SEQ ID NO:2 or has more than 90% identity with SEQ ID NO:

2.

3. A fusion protein gp15-mNG, characterized in that, The fusion protein gp15-mNG is composed of the receptor-binding protein gp15 as described in claim 1 and the fluorescent protein mNeonGreen; Preferably, the amino acid sequence of the fusion protein gp15-mNG is shown in SEQ ID NO:

3.

4. A recombinant expression vector encoding the fusion protein gp15-mNG, characterized in that, The recombinant expression vector is pET-28a::gp15-mNG, which consists of a gp15 gene fragment, fluorescent protein mNeonGreen and a linearized pET-28a vector; Preferably, the nucleotide sequence of the recombinant expression vector is as shown in SEQ ID NO:4 or has more than 90% identity with SEQ ID NO:

4.

5. A recombinant host cell, characterized in that, The recombinant host cell comprises the fusion protein gp15-mNG of claim 3 and / or the recombinant expression vector of claim 4.

6. A colloidal gold-gp15 coupling compound, characterized in that, The colloidal gold-gp15 conjugate comprises the receptor-binding protein gp15 as described in claim 1 and colloidal gold; Preferably, the colloidal gold-gp15 conjugate is prepared by conjugating receptor-binding protein gp15 with colloidal gold.

7. Any of the following applications of the receptor-binding protein gp15 of claim 1, the fusion protein gp15-mNG of claim 3, and / or the colloidal gold-gp15 conjugate of claim 6: 1) Application in the preparation of products for the specific detection of Streptococcus mutans; 2) Application in the preparation of dental caries risk assessment or oral cariogenic bacteria screening products.

8. The application according to claim 7, characterized in that, The products include diagnostic reagents, reagent kits, test strips, microfluidic chips, biosensors, systems, devices, and readable media. Preferably, the diagnostic reagent is labeled with a detectable marker; Preferably, the detectable marker is selected from colloidal gold, fluorescent protein, enzyme, quantum dot, magnetic bead or radioactive isotope; Preferably, the kit further includes at least one of the following: diluent, quality control sample, buffer solution, and instruction manual; Preferably, the test strip uses the colloidal gold-gp15 conjugate as described in claim 6 as a conjugate, comprising a detection line coated with a specific antibody against the receptor-binding protein gp15, a control line coated with a polyclonal antibody against the receptor-binding protein gp15, and a chromatography carrier composed of a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad. Preferably, the test strip is a colloidal gold immunochromatographic test strip.

9. A method for preparing the fusion protein gp15-mNG according to claim 3, characterized in that, The method includes the following steps: amplifying the receptor-binding protein gp15 as described in claim 1, recombining it with fluorescent protein mNeonGreen and linearized pET-28a vector via a Gibson ligation reaction, and transforming it into competent cells; Preferably, the competent cells are competent Escherichia coli; Preferably, the competent cells are competent Escherichia coli BL21(DE3).

10. A product characterized in that, The product includes any one of the following: 1) A colloidal gold immunochromatographic kit for the specific detection of Streptococcus mutans, wherein the colloidal gold immunochromatographic kit comprises the colloidal gold-gp15 conjugate as described in claim 6, a detection line coated with a specific antibody against the receptor-binding protein gp15, a control line coated with a polyclonal antibody against the receptor-binding protein gp15, and a chromatographic carrier composed of a sample pad, a conjugation pad, a nitrocellulose membrane, and an absorbent pad; 2) A fluorescent detection kit for the specific detection of Streptococcus mutans, wherein the fluorescent detection kit comprises the fusion protein gp15-mNG as described in claim 3, and at least one of the following: diluent, quality control, buffer, and instructions for use; Preferably, the fluorescence detection kit further includes a microplate, glass slide, or microfluidic chip for carrying the sample; Preferably, the sample is a sample containing the oral microbiota of the subject.

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