Polypeptide and nucleic acid molecule for resisting streptococcus mutans and application of polypeptide and nucleic acid molecule in preparation of medicine for resisting streptococcus mutans

Abstract

The invention provides an anti-streptococcus mutans polypeptide, a nucleic acid molecule and application in preparation of an anti-streptococcus mutans drug. The amino acid sequence of the anti-streptococcus mutans polypeptide is as shown in SEQ ID NO.7, and the nucleotide sequence of the anti-streptococcus mutans nucleic acid molecule is as shown in SEQ ID NO.8. The invention further provides an application of the anti-streptococcus mutans polypeptide and the nucleic acid molecule in preparation of the anti-streptococcus mutans drug. The anti-streptococcus mutans nucleic acid molecule provided by the invention can inhibit streptococcus proliferation, so that the anti-streptococcus mutans nucleic acid molecule can be used for preparing anti-streptococcus mutans drugs.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to a polypeptide and a nucleic acid molecule against Streptococcus mutans and their application in preparing drugs against Streptococcus mutans. Background Art

[0002] Dental caries is an oral chronic infectious disease characterized by an oral microecological imbalance caused by multiple factors mainly bacterial factors. It is the most common bacterial disease in humans 1, 2, with characteristics such as a high incidence rate and a low treatment rate, seriously endangering oral and general health. Strengthening and improving the standardized treatment of dental caries is a key issue faced by the whole population and the whole-life cycle dental caries management. At present, the focus of dental caries prevention is concentrated on the removal of dental plaque, and dental plaque is composed of a large number of symbiotic microorganisms. Therefore, the concept of "removing or killing all" plaque microorganisms creates an open and non-competitive environment for pathogenic microorganisms. Traditional methods for preventing or treating dental caries cannot fundamentally solve problems such as dental tissue damage and bacterial drug resistance. Microecological preparations and antimicrobial peptides have emerged, aiming to regulate the oral microecological balance by inhibiting oral pathogenic bacteria, which is a new method for preventing and treating dental caries.

[0003] Streptococcus mutans is a resident flora in the oral cavity and plays an important role in the early stage of dental caries occurrence. Therefore, the research on Streptococcus mutans plays a key role in the prevention and treatment of dental caries. Therefore, it is necessary to develop a drug against Streptococcus mutans.

[0004] The R & D team of the present invention obtained two monoclonal antibodies through immunizing mice, fusion and subcloning screening experiments. One of them is monoclonal antibody KLH336 (Patent 2024106236628, publication number CN 118638220 A). Through experimental analysis, it was found that a polypeptide with the amino acid sequence GFTFSDFYSKNKFNGYTTARGNWDVDFDY (referred to as the polypeptide against Streptococcus mutans) can inhibit the proliferation of Streptococcus, and thus can be used to prepare drugs against Streptococcus mutans. The nucleic acid molecule sequence encoding the polypeptide against Streptococcus mutans is

[0005] GGGTTCACCTTCAGTGATTTCTACAGTAAAAACAAATTTAATGGTTATACAACAGCAAGAGGCAACTGGGACGTAGACTTTGACTAC. The other is monoclonal antibody KLH348 (disclosed in the present invention). Through experimental analysis, it was found that a polypeptide with the amino acid sequence shown in SEQ ID NO: 7 (referred to as the polypeptide against Streptococcus mutans) can inhibit the proliferation of Streptococcus, and thus can be used to prepare drugs against Streptococcus mutans. The nucleic acid molecule encoding the polypeptide against Streptococcus mutans is as shown in SEQ ID NO: 8;

[0006] GGATACACATTCACTGACTACAACATTAATCCTAACAATGGTGATATTGCTCGAGGCAATTACGCCTTTGACTAT. The present invention provides a nucleic acid molecule and a polypeptide against Streptococcus mutans, and the process for obtaining the same, as well as their use in the preparation of medicaments against Streptococcus mutans, centered around another monoclonal antibody KLH348. SUMMARY OF THE INVENTION

[0007] To solve the above technical problems, the present invention provides a polypeptide and a nucleic acid molecule against Streptococcus mutans, and their use in the preparation of medicaments against Streptococcus mutans. The polypeptide against Streptococcus mutans can inhibit the proliferation of Streptococcus, and thus can be used in the preparation of medicaments against Streptococcus mutans.

[0008] The present invention is achieved by the following technical solutions:

[0009] In the first aspect of the present invention, a polypeptide against Streptococcus mutans is provided, and the amino acid sequence of the polypeptide against Streptococcus mutans is as shown in SEQ ID NO.7.

[0010] Furthermore, the polypeptide further includes:

[0011] an antibody with the same function obtained by substituting, deleting and / or adding one or more amino acids to the amino acid sequence of the polypeptide;

[0012] or includes an amino acid sequence having at least 80% homology with the polypeptide.

[0013] In the second aspect of the present invention, a nucleic acid molecule against Streptococcus mutans encoding the polypeptide is provided.

[0014] Furthermore, the nucleotide sequence of the nucleic acid molecule against Streptococcus mutans is as shown in SEQ ID NO:8.

[0015] Furthermore, the nucleic acid molecule against Streptococcus mutans further includes: at least one modification of the nucleic acid molecule, and the modification includes at least one of phosphorylation, methylation, amination, thiolation, substitution of oxygen with sulfur, substitution of oxygen with selenium or isotopic labeling.

[0016] Furthermore, the nucleic acid molecule against Streptococcus mutans further includes: at least one substance for labeling or treatment is linked to the nucleic acid molecule; the substance for labeling or treatment includes at least one of a fluorescent label, a radioactive substance, a therapeutic substance, biotin, digoxin, a nano-luminescent material and a small peptide.

[0017] In the third aspect of the present invention, an expression vector containing the nucleic acid is provided, and the expression vector can express the nucleic acid in a prokaryotic or eukaryotic host cell.

[0018] In the fourth aspect of the present invention, there is provided an engineered bacterium or eukaryotic host cell comprising the expression vector described above.

[0019] In the fifth aspect of the present invention, there is provided the use of the polypeptide, or the nucleic acid molecule, or the expression vector, or the engineered bacterium or eukaryotic host cell in the preparation of a drug against Streptococcus mutans.

[0020] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0021] The nucleic acid molecule and polypeptide against Streptococcus mutans provided by the present invention and their use in the preparation of a drug against Streptococcus mutans can inhibit the proliferation of Streptococcus, and the bacteriostatic efficiency is 50% or more compared with the control group. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following drawings are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0023] Figure 1 Schematic diagram of the antibody-encoding gene structure (including 5' and 3' non-coding regions);

[0024] Figure 2 VH and VL PCR amplification electrophoresis diagram, where VL1-VL2 and VH1-VH2 fragments are amplified with different primer systems; MW: DL10000;

[0025] Figure 3 Colony PCR electrophoresis diagram, MW: DL10000, and the identified positive clones are selected for sequencing analysis.

[0026] Figure 4 SDS-PAGE results of total protein extraction and expression identification in antigen preparation in Example 1; from right to left, lane M: Protein Marker; lanes 1-2: expressed total protein; lanes 3-5: eluted and purified protein. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0027] The present invention will be specifically described below in combination with the specific embodiments and examples, and the advantages and various effects of the present invention will be presented more clearly therefrom. Those skilled in the art should understand that these specific embodiments and examples are for illustrating the present invention, rather than limiting the present invention.

[0028] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as having the meanings as commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as the general understanding of those skilled in the art to which the present invention pertains. In case of any contradiction, the present specification shall prevail.

[0029] Unless otherwise specifically stated, various raw materials, reagents, instruments, and equipment used in the present invention can be obtained through market purchase or can be prepared by existing methods.

[0030] The overall idea of the present invention is as follows:

[0031] By immunizing mice, fusing, and subcloning and screening, a monoclonal antibody KLH348 is obtained.

[0032] The antibody includes a heavy chain variable region and a light chain variable region:

[0033] The heavy chain variable region has three complementary determining regions with the amino acid sequences shown in SEQ ID NO: 1 - SEQ ID NO: 3; GYTFTDYN (SEQ ID NO: 1); INPNNGDI (SEQ ID NO: 2); ARGNYAFDY (SEQ ID NO: 3).

[0034] Furthermore, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 4 (EVKLQESGPSLVKPGASVKIPCKASGYTFTDYNIDWVRQSHGKSLEWIGDINPNNGDIL YNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARGNYAFDYWGQGTTLTVSS);

[0035] The amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 5:

[0036] (DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNR FSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHLPLTFGVGTKLELK); After removing the murine constant region, the light chain antibody variable region sequence is as shown in SEQ ID NO: 6, and the sequence is

[0037] QSIVHSNGNTYKVSFQGSHLPLT;

[0038] Further analysis reveals that:

[0039] The constant region of the heavy chain of the mouse is removed, and the amino acid sequence is shown in SEQ ID NO: 7, and the sequence is GYTFTDYNINPNNGDIARGNYAFDY; through experiments, it is found that the polypeptide shown in the amino acid sequence SEQ ID NO: 7 (referred to as the polypeptide against Streptococcus mutans) can inhibit the proliferation of Streptococcus, and thus can be used to prepare drugs against Streptococcus mutans.

[0040] The nucleic acid molecule encoding the polypeptide against Streptococcus mutans is shown in SEQ ID NO: 8;

[0041] GGATACACATTCACTGACTACAACATTAATCCTAACAATGGTGATATTGCTCGAGGCAA

[0042] TTACGCCTTTGACTAT

[0043] Next, the antibodies of the present application, their preparation methods and application effects will be described in detail in combination with examples and experimental data. The specific experimental conditions and methods not specified in the following examples are usually in accordance with conventional conditions such as: edited by J. Sambrook et al., Science Press, 1992, Molecular Cloning Experiment Guide (Third Edition); D.L. Spector et al., Science Press, 2001, Cell Experiment Guide and other conditions described in the books, or in accordance with the conditions recommended by the manufacturer.

[0044] The bacteria used in this experiment were the international reference strain S. mutans Ingbritt (serotype C), which was purified in the Streptococcus mutans selective medium MSB. A single typical colony was picked and cultured microaerobically at 37 °C in TSB for 24 hours (95% N2, 5% CO2), and the total protein was extracted and identified by SDS-PAGE.

[0045] Example 1. Obtaining of the polypeptide and nucleic acid molecule against Streptococcus mutans

[0046] 1. Antigen preparation

[0047] The bacteria used in this experiment were the international reference strain S. mutans Ingbritt (serotype C), which was purified in the Streptococcus mutans selective medium MSB. A single typical colony was picked and cultured microaerobically at 37 °C in TSB for 24 hours (95% N2, 5% CO2), and the total protein was extracted and identified by SDS-PAGE (the results are as Figure 4 shown).

[0048] 2. Immunize 5 mice with the above antigen, a total of four immunizations, as shown in Table 1 specifically.

[0049] Table 1

[0050]

[0051]

[0052] 3. Fusion and subcloning screening

[0053] Select 1 - 2 mice with good serum immunization results and good verification results, take spleens for cell fusion, screen positive hybridoma cells through HAT medium, subclone the positive hybridoma cells by limiting dilution method to obtain monoclonal cell lines, and perform 2 - 3 rounds of indirect ELISA screening during each subcloning. After obtaining positive monoclonal cell lines that meet the requirements, establish the cell lines.

[0054] 4. Single - cell line sequencing

[0055] Extract total RNA from the hybridoma cell line and perform reverse transcription PCR to amplify cDNA. The variable region sequences of the antibody are amplified by PCR using subtype - specific primers, and then perform RT - PCR improved by SMART (Switching Mechanism at the 5' - end of RNA Transcripts) technology. This technology is based on the intrinsic properties of Moloney murine leukemia virus (MMLV) reverse transcriptase and the application of a custom sequence template - switch oligo upstream primer containing 3 riboguanines (rGrGrG) at its 3' - end. To amplify the antibody variable region, RT - PCR reverse primers targeting highly conserved sequences in the constant regions of murine antibody kappa, lambda, and IgG heavy chains were designed (Tables 2 and 3).

[0056] Table 2 - Reverse transcription primers for murine IgG

[0057] Primer Name Sequence (5’-3’) Template Switch Oligonucleotide Universal Forward Primer aagcagtggtatcaacgcagagtacatgrgrgr (SEQ ID NO: 9) mIGKRT Reverse Primer for kappa chain ttgtcgttcactgccatcaatc (SEQ ID NO: 10) mIGKRT Reverse Primer for lambda chain ggggtaccatctaccttccag (SEQ ID NO: 11) mIGHGRT Reverse Primer for heavy chain agctgggaaggtgtgcacac (SEQ ID NO: 12)

[0058] Table 3 - Reverse transcription primers for murine IgG

[0059]

[0060]

[0061] 5. The RT - PCR amplification process of the antibody variable region is as follows:

[0062] 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.

[0063] Step 2. After the MMLV reverse transcriptase reaches the 5' end of the RNA template during the synthesis of the first strand, several nucleotides, usually deoxycytidines, are added to the 3' end of the cDNA transcript. These added bases allow the template-switching oligonucleotide to bind after annealing.

[0064] Step 3. When base pairing occurs between the 3'-riboguanine of the template-switching oligonucleotide and the cDNA deoxycytidine, the MMLV reverse transcriptase switches templates and continues polymerization. Note: At this time, polymerization is carried out using the template-switching oligonucleotide as the template, rather than the hybridoma RNA.

[0065] Step 4. The MMLV reverse transcriptase initiates polymerization until the 5' end of the template-switching oligonucleotide. Finally, a single-stranded cDNA molecule containing the starting universal sequence is obtained.

[0066] Step 5. Finally, a single-stranded cDNA molecule containing the starting universal sequence is obtained.

[0067] Step 6. Using this single-stranded cDNA as a template, double-stranded cDNA is synthesized.

[0068] Step 7. PCR amplification is carried out using the added universal sequence. The forward PCR primer (Table 3) has the same sequence as the template-switching oligonucleotide; the reverse PCR primer (Table 3) is specific for the constant region (at the second highly conserved sequence) of each strand and is nested in the cDNA sequence to improve amplification specificity.

[0069] The products are subcloned into the T vector respectively and subjected to sequencing analysis. The schematic diagram of the primary structure of the cDNA encoding the antibody is as Figure 1 shown.

[0070] 6. Antibody subtype identification

[0071] The culture supernatants are added to the ELISA plates coated with Anti-mouse IgG Ab respectively, and detected with different HRP-labeled anti-mouse subtype secondary antibodies to analyze the antibody subtypes. The results are shown in Table 4:

[0072] Table 4 Results of antibody subtype determination

[0073]

[0074]

[0075] 7. Sequencing method

[0076] Total RNA was extracted from the hybridoma cell line, and oligo-dT was used as a primer for RT-PCR amplification of total cDNA. Using the cDNA as a template, nested-PCR amplification was performed on the VH or VL variable region fragments using a forward degenerate primer (designed according to the V or L gene) and a reverse constant region-specific primer (the primer sequences used are shown in the following table) (see Figure 2 ). The PCR product was ligated to a T vector, and after transformation, colony PCR was performed (see Figure 3 ), and positive clones were selected for sequencing analysis.

[0077] Table 5 Primers for light chain variable region fragments

[0078] Name Sequence (5’-3’) Primer 1 GGTGATAICGTGAT(A / G)AC(C / A)CA(G / A)GATGAACTCTC (SEQ ID NO: 17) Primer 2 GGTGATATC(A / T)TG(A / C)TGACCCAA(A / T)CTCCACTCTC (SEQ ID NO: 18) Primer 3 GGTGATATCGT(G / T)CTCAC(C / T)CAA / G)TCTCCAGCAAT (SEQ ID NO: 19)

[0079] Table 6 Primers for heavy chain variable and light chain variable region fragments

[0080] Name Sequence (5’-3’) Primer 1 GACGTGAAGCTGCAGGAGTCAGGAOCTAGCCTGGTG (SEQ ID NO: 20) Primer 2 AGGT(C / G)(A / C)AACTGCAG(C / G)AGTC(A / T)GG (SEQ ID NO: 21) Primer 3 AGGT(C / G)(AC)AGCTGCAG(C / G)AGTC(A / T)GG (SEQ ID NO: 22) Primer 4 AGGT(C / G)CAGCTGCAG(C / G)AGTC(A / T)GG (SEQ ID NO: 23)

[0081] The sequencing result of the antibody heavy chain is shown as SEQ ID NO: 4. The sequencing result of the antibody light chain is shown as SEQ ID NO: 5. 6. Obtaining the polypeptide against Streptococcus mutans

[0082] The polypeptide against Streptococcus mutans can be directly synthesized with the amino acid sequence shown as SEQ ID NO: 7, and the sequence is GYTFTDYNINPNNGDIARGNYAFDY.

[0083] In other embodiments, a nucleic acid molecule with the nucleotide sequence shown as SEQ ID NO: 8 can be used to construct an expression plasmid for expression to obtain the polypeptide against Streptococcus mutans.

[0084] Example 2. Exploration of the actual resistance effect

[0085] 1. Experimental strain: The strain is Streptococcus mutans 8148

[0086] 2. Preparation of the culture medium: MRS liquid medium (MRS broth) was used, and the main components (g / L) were: peptone 10 g, beef powder 5 g, glucose 20 g, Tween 80 1 ml, dipotassium hydrogen phosphate 2 g, sodium acetate 5 g, yeast powder 4 g, magnesium sulfate 0.2 g, manganese sulfate 0.05 g, and trisodium citrate 2 g. 48 g of MRS broth was added with 1000 ml of distilled water and sterilized in an autoclave.

[0087] 3. Experimental method: Take 15 50-ml centrifuge tubes, add 10 ml of MRS liquid medium to each tube and inoculate 100 μl of Streptococcus mutans strain. Divide them into two groups (PBS group, experimental group). The experimental group was respectively added with 50 μg, 200 μg, 500 μg, 1000 μg, 2000 μg of the polypeptide solution against Streptococcus mutans prepared in Example 1 (configured with PBS, 100 μl). The PBS group was respectively added with 100 μl of PBS solution, placed in a shaker at 37°C and 120 r / min for overnight culture, and the absorbance values of the bacteria in each centrifuge tube were measured at a wavelength of 600 nm the next day.

[0088] Table 7 Absorbance detection data

[0089]

[0090] As can be seen from Table 7, as the amount of antibody added increases, the OD value of the concentration of Streptococcus 8148 becomes lower, which can prove that the polypeptide against Streptococcus mutans of the present invention can effectively inhibit the growth of Streptococcus. Compared with the control group, the antibacterial efficiency reaches 50% and above.

[0091] Finally, it should also be noted that the term "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion, so that a process, method, article or device comprising a series of elements not only includes those elements, but also includes other elements not expressly listed, or further includes elements inherent to such process, method, article or device.

[0092] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications once they know the basic creative concept. Therefore, the appended claims are intended to be construed to include the preferred embodiments as well as all changes and modifications falling within the scope of the present invention.

[0093] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these modifications and variations.

Claims

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

7.

2. A nucleic acid molecule against Streptococcus mutans encoding the polypeptide according to claim 1.

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

8.

4. An expression vector comprising the nucleic acid molecule according to any one of claims 2-3, characterized in that, The expression vector can express the nucleic acid molecule in prokaryotic or eukaryotic host cells.

5. An engineered bacterium comprising the expression vector according to claim 4.

6. Use of the polypeptide according to claim 1, or the nucleic acid molecule according to any one of claims 2-3, or the expression vector according to claim 4, or the engineered bacterium according to claim 5 in the preparation of a medicament against Streptococcus mutans.

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