Polypeptides, nucleic acid molecules and uses in the preparation of anti-streptococcus drugs

Abstract

The application provides a polypeptide against Streptococcus mutans, a nucleic acid molecule against Streptococcus mutans and an application of the polypeptide against Streptococcus mutans in preparing a medicine against Streptococcus mutans, wherein the amino acid sequence of the polypeptide against Streptococcus mutans is shown as SEQ ID NO. 7, and the nucleotide sequence of the nucleic acid molecule against Streptococcus mutans is shown as SEQ ID NO: 8. The nucleic acid molecule against Streptococcus mutans provided by the application can inhibit the proliferation of Streptococcus, and thus can be used for preparing a medicine against Streptococcus mutans.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and relates to a polypeptide against Streptococcus mutans, a nucleic acid molecule and application in preparing a medicine against Streptococcus mutans. BACKGROUND

[0002] Dental caries is a chronic infectious disease of the oral cavity characterized by oral microecological imbalance caused by various factors, mainly bacterial factors, and is the most common bacterial disease in humans, with a high incidence and a low treatment rate, which seriously endangers oral and systemic health. Strengthening and improving the standardized treatment of dental caries is a key problem in the whole population and whole life cycle management of dental caries. At present, the focus of dental caries prevention is 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 the problems of tooth tissue damage and bacterial drug dependence. Microecological preparations and antibacterial peptides emerge as the times require, which can inhibit oral pathogenic bacteria to achieve the purpose of regulating oral microecological balance, and are 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. Therefore, research on Streptococcus mutans plays a key role in the prevention and treatment of dental caries. Therefore, it is necessary to develop an anti-Streptococcus mutans drug.

[0004] The research and development team of the present application obtained two monoclonal antibodies through immunization of mice, fusion and subcloning screening experiments. One of them is monoclonal antibody KLH336 (patent 2024106236628, publication number CN 118638220 A). It is found through experimental analysis that the polypeptide with the amino acid sequence GFTFSDFYSKNKFNGYTTARGNWDVDFDY (referred to as an anti-Streptococcus mutans polypeptide) can inhibit the proliferation of Streptococcus, and thus can be used for preparing an anti-Streptococcus mutans medicine. The nucleic acid molecule sequence encoding the anti-Streptococcus mutans polypeptide is

[0005] GGGTTCACCTTCAGTGATTTCTACAGTAAAAACAAATTTAATGGTTATACAACAGCAAGAGGCAACTGGGACGTAGACTTTGACTAC. The other is monoclonal antibody KLH348 (disclosed in the present application). It is found through experimental analysis that the polypeptide with the amino acid sequence shown in SEQ ID NO: 7 (referred to as an anti-Streptococcus mutans polypeptide) can inhibit the proliferation of Streptococcus, and thus can be used for preparing an anti-Streptococcus mutans medicine. The nucleic acid molecule encoding the anti-Streptococcus mutans polypeptide is shown in SEQ ID NO: 8;

[0006] GGATACACATTCACTGACTACAACATTAATCCTAACAATGGTGATATTGCTCGAGGCAATTACGCCTTTGACTAT. The present application provides the process of obtaining anti-streptococcus nucleic acid molecules, polypeptides and their application in the preparation of anti-streptococcus drugs around another monoclonal antibody KLH348. SUMMARY

[0007] In order to solve the technical problem, the present application provides anti-streptococcus polypeptides, nucleic acid molecules and their application in the preparation of anti-streptococcus drugs, which can inhibit streptococcus proliferation and thus can be used for the preparation of anti-streptococcus drugs.

[0008] The present application adopts the following technical solutions:

[0009] In the first aspect of the present application, an anti-streptococcus polypeptide is provided, and the amino acid sequence of the anti-streptococcus polypeptide is shown in SEQ ID NO. 7.

[0010] Further, the polypeptide further comprises:

[0011] The polypeptide has the same function after the amino acid sequence of the polypeptide is substituted, deleted and / or increased by one or more amino acids;

[0012] Or the amino acid sequence has at least 80% homology with the polypeptide.

[0013] In the second aspect of the present application, an anti-streptococcus nucleic acid molecule encoding the polypeptide is provided.

[0014] Further, the nucleotide sequence of the anti-streptococcus nucleic acid molecule is shown in SEQ ID NO: 8.

[0015] Further, the anti-streptococcus nucleic acid molecule further comprises: at least one of the nucleic acid molecules is modified, and the modification comprises at least one of phosphorylation, methylation, amination, sulfhydrylation, substitution of oxygen with sulfur, substitution of oxygen with selenium or isotopic substitution.

[0016] Further, the anti-streptococcus nucleic acid molecule further comprises: at least one of the nucleic acid molecules is connected to a substance for labeling or treatment; and the substance for labeling or treatment comprises at least one of a fluorescent marker, a radioactive substance, a therapeutic substance, biotin, digoxin, a nanoluminescent material and a small peptide.

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

[0018] In a fourth aspect of the present application, an engineered bacterium or eukaryotic host cell comprising the expression vector is provided.

[0019] In a fifth aspect of the present application, the polypeptide or the nucleic acid molecule or the expression vector or the engineered bacterium or eukaryotic host cell is used in the preparation of a medicine for resisting Streptococcus mutans.

[0020] The one or more technical solutions in the embodiments of the present application have at least the following technical effects or advantages:

[0021] The nucleic acid molecule for resisting Streptococcus mutans, the polypeptide and the use of the polypeptide in the preparation of a medicine for resisting Streptococcus mutans provided by the present application can inhibit the proliferation of Streptococcus, and the bacteriostatic efficiency is 50% or above compared with a control group. BRIEF DESCRIPTION OF DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed in the embodiment description will be briefly introduced. Obviously, the drawings in the following description are some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative effort.

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

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

[0025] Figure 3 The colony PCR electrophoresis diagram, MW: DL10000, and the identified positive clone is selected for sequencing analysis.

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

[0027] The advantages and various effects of the present application will be more clearly presented by the following specific embodiments and examples. Those skilled in the art should understand that these specific embodiments and examples are used to illustrate the present application, not to limit the present application.

[0028] Throughout this specification, unless otherwise specified, the terminology used herein should be understood as having the meaning commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the event of any conflict, this specification shall prevail.

[0029] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0030] The overall concept of this invention is as follows:

[0031] A monoclonal antibody, KLH348, was obtained through mouse immunization, fusion, and subcloning screening.

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

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

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

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

[0036] (DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNR FSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHLPLTFGVGTKLELK); The mouse fixed region was removed, and the light chain antibody variable region sequence is shown in SEQ ID NO: 6.

[0037] QSIVHSNGNTYKVSFQGSHLPLT;

[0038] Further analysis revealed:

[0039] The heavy chain variable region removes the mouse fixed region, and the amino acid sequence is shown in SEQ ID NO: 7, and the sequence is GYTFTDYNINPNNGDIARGNYAFDY. It is found through experiments that the polypeptide shown in SEQ ID NO: 7 (referred to as an anti-streptococcus mutans polypeptide) can inhibit the proliferation of streptococcus, and thus can be used for preparing an anti-streptococcus mutans drug.

[0040] The nucleic acid molecule encoding the anti-streptococcus mutans polypeptide is shown in SEQ ID NO: 8.

[0041] GGATACACATTCACTGACTACAACATTAATCCTAACAATGGTGATATTGCTCGAGGCAA

[0042] TTACGCCTTTGACTAT

[0043] The antibody of the present application and the preparation method and application effect thereof will be described in detail below in combination with examples and experimental data. The specific experimental conditions and methods not specified in the following examples are generally according to the conditions described in the books such as: J. Sambrook et al., Science Press, 1992, Molecular Cloning Laboratory Manual (3rd Edition); D. L. Specter et al., Science Press, 2001, Cell Experimentation Guide, or according to the conditions suggested by the manufacturer.

[0044] The bacteria used in the experiment are international reference strains S. mutans Ingbritt (serotype C), which are purified in a streptococcus selective culture medium MSB, and single typical colonies are picked and cultured in TSB at 37 degrees under microaerobic conditions for 24 hours (95% N2, 5% CO2), total protein is extracted and identified by SDS-PAGE.

[0045] Example 1, obtaining of the anti-streptococcus mutans polypeptide and nucleic acid molecule

[0046] 1. Antigen preparation

[0047] The bacteria used in the experiment are international reference strains S. mutans Ingbritt (serotype C), which are purified in a streptococcus selective culture medium MSB, and single typical colonies are picked and cultured in TSB at 37 degrees under microaerobic conditions for 24 hours (95% N2, 5% CO2), total protein is extracted and identified by SDS-PAGE. Figure 4

[0048] 2. Five mice are immunized with the antigen, a total of four times, as shown in Table 1.

[0049] Table 1

[0050] ​

[0051]

[0052] 3. Fusion and subclone screening

[0053] Select 1-2 mice with good serum immune results and good verification results, take the spleen for cell fusion, and obtain positive hybridoma cells by HAT medium screening. The positive hybridoma cells are subcloned by limited dilution method to obtain monoclonal cell strains. 2-3 rounds of indirect ELISA screening are performed during each subcloning, and the positive monoclonal cell strains that meet the requirements are established.

[0054] 4. Single cell strain sequencing

[0055] Total RNA is extracted from hybridoma cell strains, and cDNA is amplified by reverse transcription PCR. The variable region sequence of the antibody is amplified by PCR with subtype-specific primers, and then RT-PCR modified by SMART (RNA transcript 5' end conversion mechanism) technology. This technology is based on the intrinsic properties of Moloney mouse leukemia virus (MMLV) reverse transcriptase, and the application of a custom sequence template switch oligo containing 3 riboguanine (rGrGrG) at its 3' end upstream of the primer. In order to amplify the antibody variable region, RT-PCR reverse primers were designed for highly conserved sequences of mouse antibody kappa, lambda and IgG heavy chain constant region (Table 2 and Table 3)

[0056] Table 2 - Mouse IgG reverse transcription primers

[0057] Primer Name Sequence (5'-3') Template Switching 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 - Mouse IgG reverse transcription primers

[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 first strand synthesis, it adds several nucleotides, usually deoxycytidines, to the 3' end of the cDNA transcript. These added bases allow the template switch oligonucleotide to bind upon annealing.

[0064] Step 3, When base pairing occurs between the 3' riboguanine of the template switch oligonucleotide and the deoxycytidine of the cDNA, the MMLV reverse transcriptase switches templates and continues polymerization. Note: At this point, the template switch oligonucleotide is being used as a template for polymerization, not the hybridoma RNA.

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

[0066] Step 5, The final result is a single-stranded cDNA molecule containing a starting universal sequence.

[0067] Step 6, The single-stranded cDNA is used as a template to synthesize double-stranded cDNA.

[0068] Step 7, PCR amplification using the added universal sequence. The forward PCR primer (Table 3) has the same sequence as the template switch oligonucleotide; the reverse PCR primer (Table 3) is specific for the constant region of each chain (at the 2nd highly conserved sequence) and is nested within the cDNA sequence to increase amplification specificity.

[0069] The products were subcloned into T vectors and sequenced. The primary structure of the cDNA encoding the antibody is shown schematically in Figure 1 .

[0070] 6, Antibody subtype identification

[0071] The antibody subtypes were analyzed using Anti-mouse IgG Ab-coated Elisa plates, supernatants were added to the ELISA plates, and different HRP-labeled anti-mouse subtype secondary antibodies were used for detection. The results are shown in Table 4:

[0072] Table 4. Antibody subtype determination results

[0073]

[0074]

[0075] 7, Sequencing method

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

[0077] Table 5 Primer of light chain variable region fragment

[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 Primer of heavy chain variable region light chain variable region fragment

[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, and the obtaining of the polypeptide against Streptococcus mutans

[0082] The polypeptide against Streptococcus mutans can be obtained by directly synthesizing the amino acid sequence shown in SEQ ID NO: 7, which is GYTFTDYNINPNNGDIARGNYAFDY.

[0083] In other embodiments, a nucleic acid molecule with the nucleotide sequence shown in 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 effect of resistance

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

[0086] 2. Preparation of culture medium: MRS liquid medium (MRS broth MRS broth) is used, and the main components (g / L) are as follows: peptone 10 g, beef powder 5 g, glucose 20 g, Tween 80 1 ml, potassium hydrogen phosphate 2 g, sodium acetate 5 g, yeast powder 4 g, magnesium sulfate 0.2 g, magnesium sulfate 0.05 g, and tricine citric acid 2 g. 48 g of MRS broth is added to 1000 ml of distilled water, and the mixture is autoclaved in a high-pressure sterilization pot.

[0087] 3. Experimental method: 15 50ml centrifuge tubes were taken, 10ml MRS liquid medium was added to each tube and 100ul streptococcus bacteria was inoculated. They were divided into two groups (PBS group, experimental group), 50ug, 200ug, 500ug, 1000ug and 2000ug of the anti-streptococcus polypeptide solution prepared in Example 1 (PBS configuration, 100ul) were added to the experimental group respectively, 100ul of PBS solution was added to the PBS group respectively, and they were placed in a 37℃, 120r / min shaker overnight culture, and the next day the absorbance value of each centrifuge tube at 600nm wavelength was measured.

[0088] Table 7 absorbance detection data

[0089]

[0090] As can be seen from Table 7, as the amount of antibody added increases, the concentration of streptococcus 8148 OD value becomes lower, which can prove that the anti-streptococcus polypeptide of the application can effectively inhibit the growth of streptococcus, and compared with the control group, the bacteriostatic efficiency is 50% and above.

[0091] Finally, it should be noted that the terms "comprising", "including" or any other variant thereof are intended to cover non-exclusive inclusion, so that processes, methods, articles or equipment including a series of elements not only include those elements, but also include other elements not explicitly listed, or inherent to such processes, methods, articles or equipment.

[0092] Although the preferred embodiments of the application have been described, those skilled in the art can make further changes and modifications to these embodiments once they have been informed of the basic inventive concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications falling within the scope of the application.

[0093] Obviously, those skilled in the art can make various modifications and variations to the present application without departing from the spirit and scope of the present application. Thus, if these modifications and variations of the present application fall within the scope of the claims of the present application and their equivalents, the present application 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 anti-S. mutans polypeptide is shown as SEQ ID NO.

7.

2. An anti-S. mutans nucleic acid molecule encoding the polypeptide of claim 1.

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

8.

4. An expression vector comprising the nucleic acid molecule of any one of claims 2-3. The expression vector is capable of expressing the nucleic acid molecule in prokaryotic or eukaryotic host cells.

5. An engineered bacterium comprising the expression vector of claim 4.

6. Use of the polypeptide of claim 1 or the nucleic acid molecule of any one of claims 2-3 or the expression vector of claim 4 or the engineered bacterium of claim 5 in the preparation of an anti-S. mutans medicament.

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