A light chain polypeptide against Streptococcus mutans, the nucleic acid molecule encoding it, and its applications

Abstract

This invention belongs to the field of biotechnology and discloses a light chain polypeptide against Streptococcus mutans, the nucleic acid molecule encoding it, and its applications. The amino acid sequence of the polypeptide is shown in SEQ ID NO: 2. This invention originates from the light chain variable region of the anti-Streptococcus mutans monoclonal antibody PAC008. This polypeptide can be used to prepare drugs against Streptococcus mutans, providing a new candidate molecule for the prevention and treatment of dental caries.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically a light chain polypeptide against Streptococcus mutans, the nucleic acid molecule encoding it, and its applications. Background Technology

[0002] Dental caries is a chronic oral infectious disease characterized by an imbalance of the oral microecology caused primarily by bacterial factors. It is the most common bacterial disease in humans, characterized by high incidence and low treatment rates. Streptococcus mutans, a resident flora in the oral cavity, plays a crucial role in the early stages of caries development and is a key target for caries prevention and treatment. Traditional methods for preventing or treating caries cannot fundamentally address issues such as tooth tissue damage and bacterial resistance. Therefore, it is necessary to develop novel drugs against Streptococcus mutans, particularly small molecule peptides based on antibody variable regions. Summary of the Invention

[0003] The purpose of this invention is to overcome the shortcomings of the prior art and provide a light chain polypeptide against Streptococcus mutans, the nucleic acid molecule encoding it, and its applications, thereby solving the problems mentioned in the above-mentioned technical background. This polypeptide can inhibit the proliferation of Streptococcus, and thus can be used to prepare drugs against Streptococcus mutans.

[0004] To achieve the above objectives, the present invention provides the following technical solution: This invention is achieved using the following technical solution: In a first aspect of the invention, a light chain polypeptide against Streptococcus mutans is provided, the amino acid sequence of which is shown in SEQ ID NO: 2 (sequence: QCLMYQILCGLNQGLEPSLREGG).

[0005] In a second aspect of the invention, a nucleic acid molecule encoding the light chain polypeptide is provided, the nucleotide sequence of which is shown in SEQ ID NO: 3.

[0006] In a third aspect of the invention, an expression vector comprising the nucleic acid molecule is provided, the expression vector being capable of expressing the nucleic acid molecule in a prokaryotic or eukaryotic host cell.

[0007] In a fourth aspect of the invention, an engineered bacterium or eukaryotic host cell comprising the expression vector is provided.

[0008] In a fifth aspect of the invention, the use of the light chain polypeptide, nucleic acid molecule, expression vector, engineered bacteria or eukaryotic host cell in the preparation of a medicament against Streptococcus mutans is provided.

[0009] Preferably, the drug is used to prevent or treat dental caries caused by Streptococcus mutans.

[0010] Compared with the prior art, the beneficial effects of the present invention are: 1. This invention provides a novel anti-Streptococcus mutans active peptide sequence. Existing technologies typically focus on intact antibody molecules or the variable region of the antibody heavy chain. However, this invention innovatively extracts a short peptide sequence (QCLMYQILCGLNQGLEPSLREGG) with independent antibacterial activity from the variable region of the light chain of the anti-Streptococcus mutans monoclonal antibody PAC008. Experiments have confirmed that this light chain peptide can effectively inhibit the growth of Streptococcus mutans, and the antibacterial effect significantly increases with increasing peptide concentration, providing a novel candidate molecule for the development of anti-caries drugs.

[0011] 2. Extremely small molecular weight, with potentially high biofilm penetration. Compared to complete antibody macromolecules or even heavy-chain peptides, the light-chain peptides provided by this invention have shorter sequences and extremely low molecular weights. This physicochemical property enables them to potentially have stronger penetration and diffusion capabilities when facing dense dental plaque biofilms, allowing them to act more deeply on the underlying microenvironment of Streptococcus mutans colonization and improve the actual antibacterial effect.

[0012] 3. It possesses both targeted antibacterial and microecological regulation functions. The light chain polypeptide of this invention is derived from the antibody region that specifically recognizes Streptococcus mutans, enabling it to specifically inhibit core cariogenic bacteria. This targeted inhibition avoids the microecological imbalance side effects caused by traditional "one-size-fits-all" sterilization, aligning with the current research and development concepts of precision oral medicine and microecological preparations.

[0013] 4. Facilitates synthesis and combination drug development. This invention clarifies the amino acid and nucleic acid sequences of the light chain peptide, which can be obtained at low cost through mature synthetic biology techniques. More importantly, this light chain peptide can be used in combination with heavy chain peptides derived from the same parent antibody, providing a solid material basis and application expansion space for the development of novel compound anti-mutagenic streptococcal preparations with synergistic effects (such as heavy-light chain dipeptide conjugates). Attached Figure Description

[0014] Figure 1 A schematic diagram of the antibody-encoding gene structure (including the 5' and 3' non-coding regions); Figure 2 Electrophoresis image of PAC008VL PCR amplification; MW: DL2000; Figure 3 The results are those of total protein extracted and identified by SDS-PAGE during antigen preparation in Example 1. Detailed Implementation

[0015] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that, unless otherwise specified, the following embodiments and features described therein can be combined with each other.

[0016] The overall concept of this invention is as follows: A monoclonal antibody, PAC008, was obtained through mouse immunization, fusion, and subcloning screening. The antibody includes a heavy chain variable region and a light chain variable region: Furthermore, the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 1: DVLMTQTPLSLPVSLGDQASISCRSSQCLMYQILCGLLEWYLQKPGQSPKLLIYNQGNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCLEPSLREGGFGGGTKLE; The mouse fixed region was removed, and the amino acid sequence of the variable region of the light chain antibody is shown in SEQ ID NO: 2, which is QCLMYQILCGLNQGLEPSLREGG; Experiments showed that the polypeptide with the amino acid sequence SEQ ID NO: 2 (called the light chain polypeptide against Streptococcus mutans) can inhibit the proliferation of Streptococcus mutans, and can therefore be used to prepare drugs against Streptococcus mutans.

[0017] Further analysis revealed: The nucleic acid molecule encoding a light chain polypeptide against Streptococcus mutans is shown in SEQ ID NO: 3; CAGTGTTTGATGTATCAAATCCTCTGTGGATTAAATCAAGGGTTAGAACCAAGTCTCCGGGAAGGCGGC (encoding light chain polypeptide amino acid SEQ ID NO: 2) The antibodies, their preparation methods, and application effects of this application will be described in detail below with reference to examples and experimental data. Specific experimental conditions and methods not specified in the following examples are generally in accordance with conventional conditions such as those described in books like J. Sambrook et al. (eds.), *Molecular Cloning: A Laboratory Manual* (3rd edition), Science Press, 1992; and DL Spector et al., *Cellular Laboratory Manual*, Science Press, 2001, or as recommended by the manufacturer.

[0018] Example 1: Obtaining peptides and nucleic acid molecules against Streptococcus mutans 1. Antigen preparation The bacteria used in this experiment was the international reference strain *S. mutans Ingbritt* (serotype C), purified in selective culture medium MSB for *Streptococcus mutans*. Single typical colonies were picked and cultured in TSB at 37°C with microaergy for 24 hours (95% N2, 5% CO2). Total protein was extracted and identified by SDS-PAGE (results are shown in the figure). Figure 3 (As shown).

[0019] 2. Five mice were immunized with the antigen described above, for a total of four immunizations, as shown in Table 1.

[0020] Table 1

[0021] 3. Fusion and subcloning screening Select 1-2 mice with good serum immunization and validation results, take spleens for cell fusion, screen for positive hybridoma cells using HAT medium, subclone the positive hybridoma cells using limiting dilution to obtain monoclonal cell lines, perform 2-3 rounds of indirect ELISA screening during each subcloning period, and establish cell lines after obtaining positive monoclonal cell lines that meet the requirements.

[0022] 4. Single-cell sequencing Total RNA was extracted from hybridoma cell lines and cDNA was amplified by reverse transcription PCR. The variable region sequence of the antibody was amplified by PCR using subtype-specific primers, followed by RT-PCR modified with SMART (5' RNA transcript switching mechanism) technology. This technique is based on the intrinsic characteristics of Moloney mouse leukemia virus (MMLV) reverse transcriptase and the application of template-switch oligos to a custom sequence template containing 3-riboguanine (rGrGrG) at its 3' end. To amplify the antibody variable region, RT-PCR reverse primers targeting highly conserved sequences in the constant regions of mouse antibody kappa, lambda, and IgG heavy chains were designed (Tables 2 and 3). Table 2. Mouse IgG reverse transcription primers

[0023] Table 3. Mouse IgG reverse transcription primers

[0024] 5. The RT-PCR amplification process for the antibody variable region is as follows: Step 1: After the primers in Table 2 specifically bind to the highly conserved constant region on the hybridoma RNA, MMLV reverse transcriptase initiates polymerization.

[0025] Step 2: During the synthesis of the first strand, MMLV reverse transcriptase reaches the 5' end of the RNA template and adds several nucleotides, usually deoxycytosine, to the 3' end of the cDNA transcript. These added bases allow for template binding and conversion of oligonucleotides after annealing.

[0026] Step 3: When base pairing occurs between the 3' riboguanine of the template-converting oligonucleotide and the deoxycytosine of the cDNA, the MMLV reverse transcriptase converts the template and continues polymerization. Note: At this stage, the template-converting oligonucleotide is used as the template for polymerization, not hybridoma RNA.

[0027] Step 4: MMLV reverse transcriptase initiates polymerization until the template is converted to the 5' end of the oligonucleotide. The final result is a single-stranded cDNA molecule containing the universal initiation sequence.

[0028] Step 5: The final result is a single-stranded cDNA molecule containing the universal start sequence.

[0029] Step 6: Using the single-stranded cDNA as a template, synthesize double-stranded cDNA.

[0030] Step 7: Perform PCR amplification using the added universal sequences. The forward PCR primers (Table 3) have the same sequence as the template-converting oligonucleotides; the reverse PCR primers (Table 3) are specific to the constant region (the second highly conserved sequence) of each strand and are nested within the cDNA sequence to improve amplification specificity.

[0031] The products were subcloned into T vectors and sequenced for analysis. A schematic diagram of the primary structure of the cDNA encoding the antibody is shown below. Figure 1 As shown.

[0032] 6. Antibody subtype identification The culture supernatant of the ELISA plates coated with Anti-mouse IgG Ab was added separately to the plates, and the antibody subtypes were analyzed using different HRP-labeled anti-mouse subtype secondary antibodies. The results are shown in Table 4. Table 4. Results of Antibody Subtype Determination

[0033] 7. Sequencing methods Total RNA was extracted from hybridoma cell lines, and total cDNA was amplified by RT-PCR using oligo-dT primers. Using cDNA as a template, nested-PCR amplification was performed on the light chain (VL) variable region fragment using forward degenerate primers (designed according to the V or L gene) and reverse constant region-specific primers (the primer sequences are shown in the table below). Figure 2 The PCR product was ligated into a T vector, transformed, and colony PCR was performed. Positive clones were selected for sequencing analysis.

[0034] Table 5 Primers for the light chain variable region fragment

[0035] The antibody light chain sequencing results are shown in SEQ ID NO: 1. 8. Obtaining light chain polypeptides against Streptococcus mutans The amino acid sequence shown in SEQ ID NO: 2 can be directly synthesized. QCLMYQILCGLNQGLEPSLREGG was used to obtain a light chain polypeptide that is effective against Streptococcus mutans.

[0036] In other embodiments, a nucleic acid molecule with the nucleotide sequence shown in SEQ ID NO: 3 can be used to construct an expression plasmid for expression to obtain a light chain polypeptide against Streptococcus mutans. Example 2: Investigation into the actual resistance effect of light chain polypeptides against Streptococcus mutans 1. Experimental strain: Streptococcus mutans 8148 2. Preparation of Culture Medium: MRS liquid culture medium (MRS broth) was used, with the following main components (g / L): peptone 10g, beef meal 5g, glucose 20g, Tween 80 1ml, dipotassium hydrogen phosphate 2g, sodium acetate 5g, yeast extract 4g, magnesium sulfate 0.2g, magnesium sulfate 0.05g, and eucalyptol citrate 2g. 48g of MRS broth was added to 1000ml of distilled water and sterilized in an autoclave.

[0037] 3. Experimental Methods: Take 10 50ml centrifuge tubes, add 10ml of MRS liquid culture medium to each tube and inoculate with 100ul of Streptococcus mutans. Divide the centrifuge tubes into a control group and an experimental group, with 5 tubes in each group. Control group: Add 10μl, 20μl, 50μl, 100μl, and 200μl of PBS buffer, respectively; Experimental group: Add 10μl, 20μl, 50μl, 100μl, and 200μl of the anti-Streptococcus mutans light chain polypeptide solution prepared in Example 1 (the concentration of the light chain polypeptide solution is 10 mg / ml, that is, the final mass of the added light chain polypeptide is 100μg, 200μg, 500μg, 1000μg, and 2000μg, respectively).

[0038] The centrifuge tubes were incubated overnight at 37°C and 120 rpm in a shaker. The absorbance of the bacterial cells in each centrifuge tube was measured at 600 nm the next day. Inhibition rate (%) = (A0-A) / A0×100%; where A is the absorbance value after treatment with different concentrations of anti-Streptococcus mutans light chain peptides, and A0 is the absorbance value of the PBS group.

[0039] Table 6 Absorbance Detection Data

[0040] As shown in Table 6, as the amount of light chain polypeptide added increases, the concentration OD value of Streptococcus 8148 decreases, which proves that the polypeptide of the present invention that inhibits the growth of Streptococcus mutans can effectively inhibit the growth of Streptococcus. Compared with the control group, the antibacterial efficiency reaches 30% or more.

[0041] The embodiments described above are merely illustrative of specific implementations of the present invention, and while the descriptions are detailed, they should not be construed as limiting the scope of protection of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A light chain polypeptide against Streptococcus mutans, characterized in that, The amino acid sequence of the light chain polypeptide is shown in SEQ ID NO:

2.

2. A nucleic acid molecule encoding the light chain polypeptide of claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that, The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:

3.

4. An expression vector comprising the nucleic acid molecule of claim 3, characterized in that, The expression vector can express the nucleic acid molecule in prokaryotic or eukaryotic host cells.

5. An engineered bacterium or eukaryotic host cell comprising the expression vector of claim 4.

6. The use of the light chain polypeptide of claim 1, the nucleic acid molecule of claim 2 or 3, the expression vector of claim 4, or the engineered bacteria or eukaryotic host cell of claim 5 in the preparation of a drug against Streptococcus mutans.

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