Determination method of multivalent antigen sequence of anti-periodontitis red complex

Abstract

The invention belongs to the technical field of biology, and discloses a method for determining a multivalent antigen sequence of an anti-periodontitis red complex, which comprises the following steps: firstly, screening out key virulence factor antigens (FimA-II, RgpA, Msp and BspA) from three core pathogenic bacteria of the red complex: porphyromonas gingivalis, treponema denticola and fusisterone; then, independently cloning each antigen gene to a pUC57 cloning vector, constructing a single antigen recombinant plasmid, and avoiding intergene interference by designing a specific enzyme cutting site; and finally, through plasmid transformation, positive clone screening and double enzyme digestion verification, the plasmid quality is ensured. The obtained single antigen plasmid can be flexibly combined and used, is beneficial to subsequent multivalent vaccine development, and has the advantages of wide antigen coverage, high expression reliability, flexible construction strategy, easiness in industrialization and the like.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to a method for determining the multivalent antigen sequence of the red complex against periodontitis. Background Technology

[0002] Periodontitis is a chronic infectious disease mediated by dental plaque biofilm and involving a host immune inflammatory response. Its incidence remains high in the adult population, making it the leading cause of tooth loss in adults. It can also trigger systemic diseases such as cardiovascular disease, diabetes, and premature birth, posing a serious threat to human health. The pathogenesis of periodontitis is closely related to the imbalance of the subgingival microbiota. The red complex, composed of *Forsythia suspensa*, *Porphyromonas gingivalis*, and *Treponema denticulatum*, is recognized as the core pathogenic flora. This flora promotes the destruction of periodontal tissues through the secretion of virulence factors, mediating the host inflammatory response, and forming biofilms.

[0003] Currently, the clinical treatment of periodontitis mainly involves debridement (supragingival scaling, subgingival curettage, and root surface smoothing) and the use of antibiotics. However, these methods have obvious technical limitations. Therefore, the development of vaccines based on pathogenic bacterial antigens has become a research hotspot for the specific prevention and treatment of periodontitis. While existing technologies include antigen research and plasmid construction targeting single bacterial species and single virulence factors of the periodontitis red complex, many problems remain to be solved in core aspects such as antigen screening and plasmid construction. Current research mainly focuses on single virulence factors of single bacterial species, resulting in narrow antigen coverage, failure to break down the pathogenic network among bacterial communities, and limited immune protection. Furthermore, the construction of multi-antigen plasmids often employs fusion cloning or physical mixing. Fusion cloning inserts multiple antigen genes into the same vector, which can easily lead to problems such as gene sequence interference, expression competition, and protein folding errors, resulting in low antigen expression levels and loss of biological activity. Physical mixing suffers from uncontrollable plasmid ratios and poor stability, failing to meet the needs of standardized research and application. In addition, existing technologies often directly clone antigen genes into expression vectors. If a change in expression system (prokaryotic / eukaryotic) is required later, gene sequences and restriction enzyme sites must be redesigned, which is cumbersome and inefficient. Therefore, there is an urgent need to develop a method for screening multi-target antigen sequences of periodontitis red complexes that is precise in target selection, flexible in construction strategy, high in sequence fidelity, and highly adaptable to vectors, so as to promote the research and development and industrial application of multivalent periodontitis vaccines. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention proposes a method for determining the multivalent antigen sequence against the red periodontitis complex. This method focuses on antigen screening, design of specific restriction enzyme sites, modular independent cloning, and standardized verification and preservation. Four key virulence factor antigens are screened from the three core pathogenic bacteria of the red periodontitis complex. Four independent recombinant plasmids are constructed through directed cloning, and a standardized plasmid combination is obtained through transformation, screening, and verification.

[0005] To achieve the above objectives, the present invention provides a method for determining the multivalent antigen sequence of the red complex against periodontitis, comprising the following steps: 1) Initial screening of target antigens: Antigen amino acid sequences were screened from three pathogenic bacteria of the periodontitis red complex: Porphyromonas gingivalis, Forsythia stomatitis and Treponema denticulatum. 2) Gene design and cloning vector construction: The antigen amino acid sequence from step 1) is reverse-translated into a DNA sequence, and specific restriction endonuclease recognition sites are designed on both sides of each antigen gene. Then, each antigen gene is independently cloned into a cloning vector to construct a recombinant vector of a single antigen gene. 3) Plasmid transformation and verification: The recombinant plasmids from step 2) were transformed into competent cells, and positive clones were obtained by antibiotic screening. Plasmids were extracted and double enzyme digestion was performed on the positive clones to ensure that each antigen gene was correctly inserted and there was no sequence interference. 4) Application of plasmids: Combine and apply the recombinant plasmids of each antigen that have been verified as correct in step 3).

[0006] Preferably, the antigenic amino acids screened in step 1) include the fimin protein FimA-II antigen and arginine gingival protease RgpA antigen of Porphyromonas gingivalis ATCC 33277, the outer sheath protein Msp antigen of Treponema denticulata ATCC 35405, and the surface protein BspA antigen of Forsythia stomatis 92A2.

[0007] Preferably, the amino acid sequence of the FimA-II antigen is shown in SEQ ID NO:1; the amino acid sequence of the RgpA antigen is shown in SEQ ID NO:2; the amino acid sequence of the Msp antigen is shown in SEQ ID NO:3; and the amino acid sequence of the BspA antigen is shown in SEQ ID NO:4.

[0008] Preferably, the cloning vector in step 2) is the pUC57 vector.

[0009] Preferably, in step 2), the restriction enzyme sites of the FimA-II antigen gene are NcoI and EcoRI; the restriction enzyme sites of the RgpA antigen gene are SmaI and EcoRI; the restriction enzyme sites of the Msp antigen gene are HindIII and EcoRI; and the restriction enzyme sites of the BspA antigen gene are SmaI and NdeI.

[0010] Preferably, the competent cells in step 3) are E. coli DH5α; the transformation specifically includes heat shock at 42℃±0.5℃ for 60-90 seconds, followed by immediate placement on ice for 2-3 minutes, then addition of LB liquid medium, and culture at 37℃ and 180-200 rpm for 60 minutes with shaking.

[0011] Preferably, the double enzyme digestion verification in step 3) specifically includes: using restriction endonucleases corresponding to the antigen genes on both sides of each recombinant plasmid for enzyme digestion, and confirming by agarose gel electrophoresis that the digestion products simultaneously contain vector bands and antigen gene insertion fragment bands of the expected size.

[0012] A multivalent antigen gene combination for combating red periodontitis complex is also provided, the multivalent antigen gene combination comprising two or more antigen recombinant plasmids prepared by the above-described method.

[0013] A multivalent vaccine development kit for combating red periodontitis complex is also provided, the kit containing a combination of multivalent antigen genes for combating red periodontitis complex.

[0014] Compared with the prior art, the present invention has the following advantages and technical effects: 1) This invention is the first to screen four key virulence factor antigens covering the three core pathogens based on the synergistic pathogenicity characteristics of the three pathogens in the red complex. Each antigen meets the criteria of strong immunogenicity, high conservation, and direct correlation with the pathogenic mechanism. It can induce the body to produce a broad-spectrum immune response against the red complex, break the synergistic pathogenicity network of the pathogens, and solve the problems of narrow antigen coverage and limited protective effect in the prior art.

[0015] 2) This invention clones four antigen genes into independent cloning vectors to construct four single-antigen recombinant plasmids, which reduces sequence interference, expression competition and protein folding errors among multiple genes and ensures independent amplification and expression of each antigen gene. At the same time, each plasmid can be used alone, combined arbitrarily and its ratio can be adjusted to adapt to different immunization strategies, reducing the problems of unreasonable construction strategies and poor flexibility.

[0016] 3) The antigen combination of the present invention covers the three core pathogenic bacteria of the red complex of periodontitis, which can induce the body to produce a broad-spectrum immune response against the red complex, effectively breaking the synergistic pathogenic network of pathogenic bacteria. Compared with existing single antigen vaccines, the protective effect is qualitatively improved, providing a feasible technical solution for the specific radical treatment of periodontitis.

[0017] 4) The plasmid construction method of the present invention has a clear operation process, well-defined parameters, and strict quality control standards. All operations adopt conventional molecular biology techniques, requiring no special instruments or reagents. It is easy to carry out industrialization in biopharmaceutical companies and has broad market application prospects.

[0018] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0019] Figure 1 This is a flowchart illustrating the screening process for multi-target antigen sequence genes according to the present invention. Detailed Implementation

[0020] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0021] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0022] Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards.

[0023] Unless otherwise defined or stated, all technical and scientific terms used in this invention have the same meaning as those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein can be applied to the methods of this invention. It should be noted that, unless otherwise specified, the embodiments and features described in this invention can be combined with each other.

[0024] Unless otherwise stated, all experimental instruments, equipment, and reagents used in the following examples are commercially available raw materials.

[0025] In this example, E. coli DH5α competent cells were purchased from Beijing Solarbio Science & Technology Co., Ltd. Storage conditions: -80℃, thawed slowly on ice before use; Quality control: Transformation efficiency of each batch of competent cells ≥1×10⁻⁶. 6 -1×10 7 cfu / μg.

[0026] Example 1 I. Screening of antigen sequences.

[0027] 1) Screening for FimA-II type antigen sequences: The source strain is *Porphyromonas gingivalis* (… Porphyromonas gingivalis ATCC 33277, accessed via NCBI database with accession number BAA04623.1, the functional structural region of this protein was selected as amino acids 2-348 (as shown in SEQ ID NO.1). This region is a typical type II fimbriae major subunit, containing key structural domains for fimbriae assembly and host adhesion, and has strong immunogenicity and conservation.

[0028] 2) RgpA antigen sequence screening: The source strain is *Porphyromonas gingivalis* (… Porphyromonas gingivalis ATCC 33277, accessed through the NCBI database with accession number WP_114621325.1, selected the amino acid sequence from position 228 to 1704 containing the complete catalytic domain (excluding positions 1-227 of the leader peptide) (as shown in SEQ ID NO.2). This sequence has a naturally repeating structure and can trigger a strong immune response in the body.

[0029] 3) Msp antigen sequence screening: The source strain is *Treponema denticulatum* (…). Treponema denticola ATCC35405, retrieved from the NCBI database, accession number WP_002681434.1, the amino acid sequence of positions 2-543 corresponding to the CDS region was selected (as shown in SEQ ID NO.3), this region contains multiple predicted B cell linear epitopes, and the immunogenicity is significant.

[0030] 4) BspA antigen sequence screening: The source strain is Forsythia stomatologica (… Tannerella forsythia )92A2, retrieved from the NCBI database, accession number WP_157755308.1, selected the complete sequence of amino acids 2-1077 containing the typical LRR (leucine-rich repeat) domain (as shown in SEQ ID NO.4), which is the core virulence region of Fussystana and a key target for immune recognition.

[0031] II. Gene design and construction of pUC57 cloning vector.

[0032] 1) Gene design: The amino acid sequences of the four antigens identified in Example 1 were reverse translated into their respective DNA sequences. Based on the multiple cloning site of the pUC57 vector, specific restriction endonuclease recognition sites were designed on both sides of each gene.

[0033] Specifically, the restriction sites for FimA-II are NcoI and EcoRI; the restriction sites for RgpA are SmaI and EcoRI; the restriction sites for Msp are HindIII and EcoRI; and the restriction sites for BspA are SmaI and NdeI. The design of these restriction sites ensures that the gene can be accurately inserted into the vector and effectively expressed.

[0034] 2) Gene synthesis and cloning: The four gene sequences optimized in step 1) above were entrusted to a professional biotechnology company (Nanjing Genscript Biotech Co., Ltd.) for synthesis. During the synthesis process, designed double restriction sites were introduced at both ends of the genes. The restriction endonuclease digestion-ligation method was used to directionally clone the four antigen genes into the corresponding restriction sites of the pUC57 vector to avoid mutual interference between genes.

[0035] 3) Obtaining cloning vectors: Four recombinant plasmids were synthesized and cloned, namely pUC57-FimAII (containing NcoI and EcoRI restriction sites), pUC57-RgpA (containing SmaI and EcoRI restriction sites), pUC57-Msp (containing HindIII and EcoRI restriction sites), and pUC57-BspA (containing SmaI and NdeI restriction sites).

[0036] III. Transformation and validation of pUC57 recombinant plasmid into competent cells.

[0037] 1) Preparations before conversion: Four recombinant pUC57 plasmids (pUC57-FimAII, pUC57-RgpA, pUC57-Msp, and pUC57-BspA) constructed by Nanjing Genscript Biotech Co., Ltd. were diluted with sterile water to a working concentration of 10 ng / μL. E. coli DH5α competent cells were selected (E. coli DH5α competent cells were taken out from -80℃ and immediately placed on ice to thaw for 5-10 min). Subsequently, five sterile 1.5 mL centrifuge tubes were taken and labeled as T-01, T-02, T-03, T-04 and control tube, where T-01, T-02, T-03 and T-04 correspond to the recombinant plasmids pUC57-FimAII, pUC57-RgpA, pUC57-Msp and pUC57-BspA, respectively. Add 100 μL of E. coli DH5α competent cells to each tube. Add 2 μL of the corresponding recombinant plasmid to T-01, T-02, T-03 and T-04 respectively. Add 2 μL of sterile ddH2O to the control tube. Then, each group gently tapped the walls of the centrifuge tubes to mix them, and then let them stand on ice for 30 minutes (this step must be done gently to avoid violent shaking).

[0038] 2) Heat shock treatment: Preheat the water bath to 42°C, place 5 centrifuge tubes into the water bath at the same time, heat shock precisely for 60-90 seconds, and then immediately transfer the 5 centrifuge tubes to ice and let them stand on ice for 2-3 minutes.

[0039] 3) Resuscitation culture: Add 500 μL of preheated LB liquid medium (without antibiotics) to T-01, T-02, T-03, T-04 and control tubes, and then incubate at 37℃ and 180-200 rpm for 60 min with shaking.

[0040] 4) Plate screening and monoclonal isolation: Prepare LB agar plates containing 100 μg / mL kanamycin, air dry them beforehand, and store them in an incubator. Take out 5 LB plates and label them as: FimAII plate, RgpA plate, Msp plate, BspA plate, and control plate.

[0041] Take 100-300 μL of the revived bacterial culture and spread it onto LB agar plates containing 100 μg / mL kanamycin (T-01 corresponds to FimAII plate, T-02 corresponds to RgpA plate, T-03 corresponds to Msp plate, T-04 corresponds to BspA plate, and the control tube corresponds to the control plate). Incubate the plates at room temperature for 5-10 min to allow the bacterial culture to be absorbed. Finally, incubate at 37°C upside down for 12-16 h and obtain positive clones through antibiotic screening.

[0042] Among them, FimAII, RgpA, Msp and BspA plates showed a large number of single colonies; control plates showed no colony growth.

[0043] 5) Positive clone screening and glycerol bacteria preservation: Single colony selection: Select 6 morphologically robust independent single colonies from each positive plate (FimAII, RgpA, Msp and BspA plates) and inoculate them into test tubes containing 2 mL of LB (kanamycin 100 μg / mL) and incubate at 37℃ and 200 rpm for 12-16 h with shaking.

[0044] Bacterial culture preservation (establishment of working strain library): Select 6 morphologically plump, free of contaminants and uniform in size single colonies from each positive plate, take 1 mL of bacterial culture from each, add an equal volume of 50% sterile glycerol (final concentration 25%), mix well, and dispense into 2 cryovials (1 mL each), label with information, and store at -80℃.

[0045] 6) Conversion efficiency calculation.

[0046] The formula for calculating conversion efficiency is: ; The total volume after resuscitation is in μL, the plate volume is in μL, and the amount of DNA added is in μg.

[0047] Quality control standard: Conversion efficiency ≥ 1×10 4 cfu / μg.

[0048] The transformation efficiencies of the four recombinant plasmids (FimAII, RgpA, Msp, and BspA) were calculated to be 8.99 × 10⁻⁶. 4 cfu / μg, 9×10 4 cfu / μg, 9.54×10 4 cfu / μg and 9.375×10 4 cfu / μg.

[0049] Furthermore, the transformation efficiency of the four plasmids differed by ≤10 times, ensuring the consistency and stability of plasmid transformation.

[0050] Example 2 Colony validation method.

[0051] 1) Plasmid extraction.

[0052] Six clones of each of the four recombinant plasmids cultured in Example 1 were taken and extracted using the Novizan Plasmid Mini-Prep Kit. The procedure was strictly followed according to the kit instructions, with the addition of protein and RNA removal steps to ensure plasmid purity. The specific procedures are as follows: S1. Take 1 mL of bacterial culture and add it to a 1.5 mL centrifuge tube. Centrifuge at 12000×g for 1 min, discard the supernatant, and collect the bacterial cells. Place the adsorption column in the centrifuge tube, add 100 μl of Buffer QB to the adsorption column, centrifuge at 12000 rpm for 1 min, and discard the filtrate (the adsorption column must be processed on the same day). S2. Add 250 μL of Buffer P1 (RNase A Solution has been added) to the centrifuge tube, vortex until the bacterial cells are completely resuspended, ensuring no bacterial cell precipitation; S3. Add 250 μL Buffer P2, gently invert and mix 8-10 times, and let stand at room temperature for 3 minutes to allow the cells to fully lyse. Avoid vigorous shaking to prevent genomic DNA contamination. S4. Add 125 μL Buffer P5 and immediately gently invert the container 12-15 times. At this point, a white flocculent precipitate will appear (which is a genomic DNA and protein complex). Centrifuge at 12000×g for 10 min. S5. Transfer 600 μL of supernatant to a new 1.5 mL centrifuge tube, add an equal volume of Buffer ERB, and mix by inverting the tube 10 times to remove residual protein and impurities. S6. Transfer the mixture to the adsorption column of the kit, centrifuge at 12000 rpm for 1 min, and discard the filtrate. S7. Add 500 μL Buffer ERW to the adsorption column, centrifuge at 12000 rpm for 1 min, discard the filtrate, and repeat once to enhance the protein removal effect. S8. Add 500 μL Buffer PW1 to the adsorption column, centrifuge at 12000 rpm for 1 min, and discard the filtrate. S9. Add 700 μL of Buffer PW2 (with anhydrous ethanol added) to the adsorption column, centrifuge at 12000 rpm for 1 min, discard the filtrate, repeat once to remove residual salt ions. S10. Place the adsorption column back into the collection tube and centrifuge at 12000 rpm for 2 min to completely remove residual reagents from the adsorption column and avoid affecting subsequent enzyme digestion and sequencing. S11. Place the adsorption column in a new 1.5 mL sterile centrifuge tube, add 30 μL of sterile water preheated to 65 °C to the center of the membrane of the adsorption column, let it stand at room temperature for 3 min to allow the plasmid to be fully eluted, then centrifuge at 12000 rpm for 2 min, discard the adsorption column, collect the elution buffer, which is the extracted plasmid DNA, and store it at -20 °C.

[0053] 2) Double enzyme digestion verification.

[0054] The extracted plasmids were subjected to specific double enzyme digestion verification. Based on the combination of enzyme digestion sites for each plasmid, the corresponding restriction endonuclease was selected. The digestion system was 20 μL, and the specific procedures were as follows: Prepare the double enzyme digestion reaction system according to the formula (as shown in Table 1). Prepare six clones of each plasmid separately, and set up a blank control (vector only, without insert fragment).

[0055] Table 1. Preparation formula of the double enzyme digestion reaction system

[0056] Gently mix the prepared reaction system and place it in a 37°C constant temperature water bath for 30 minutes to allow the enzyme digestion reaction to proceed fully.

[0057] 2) Agarose gel electrophoresis verification: After the enzyme digestion reaction is complete, add 2 μL of 6× DNA loading buffer to the reaction system, mix well, and then perform 1% agarose gel electrophoresis. Electrophoresis conditions: 120V, 30min, using DL2000 DNA Marker as molecular weight standard; Results criteria: After agarose gel electrophoresis, the enzyme digestion products show clear bands, one of which is the pUC57 vector band and the other is the antigen gene insertion fragment band. The size of the insertion fragment is consistent with the expectation, and there are no extraneous bands or tails. This indicates that the clone has passed the enzyme digestion verification. The blank control only shows the vector band and no insertion fragment band.

[0058] In summary, this invention provides a systematic, efficient, and industrially scalable method for determining the sequences of multivalent antigens against the red complex of periodontitis. First, based on a synergistic pathogenicity network, this method accurately screens four key virulence factor antigens (FimA-II, RgpA, Msp, and BspA) covering three core pathogenic bacteria, ensuring the immunogenicity, conservation, and pathogenicity of the antigens. Second, by independently cloning each antigen gene into the pUC57 vector and designing non-interfering specific restriction enzyme sites, four single-antigen recombinant plasmids were successfully constructed, effectively reducing sequence interference, expression competition, and protein folding errors during multi-gene co-expression. Subsequently, a rigorous plasmid transformation, positive clone screening, and double enzyme digestion verification process ensured the correctness of each plasmid construction and high transformation efficiency. Finally, these validated single-antigen plasmids can serve as standardized, modular components, which can be used individually or in any proportion to meet different research or application needs, laying a reliable technical foundation for developing multivalent vaccines capable of breaking the synergistic network of pathogenic bacteria and providing broad-spectrum immune protection. This method has a clear process, strict quality control, and high flexibility, and has good repeatability and industrial application prospects.

[0059] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for determining the multivalent antigen sequence of the red complex against periodontitis, characterized in that, Includes the following steps: 1) Initial screening of target antigens: Antigen amino acid sequences were screened from three pathogenic bacteria of the periodontitis red complex: Porphyromonas gingivalis, Forsythia stomatitis and Treponema denticulatum. 2) Gene design and cloning vector construction: The antigen amino acid sequence described in step 1) is reverse translated into a DNA sequence, and specific restriction endonuclease recognition sites are designed on both sides of each antigen gene. Then, each antigen gene is independently cloned into a cloning vector to construct a recombinant vector of a single antigen gene. 3) Plasmid transformation and verification: The recombinant plasmids described in step 2) are transformed into competent cells respectively. Positive clones are obtained by antibiotic screening. Plasmids are extracted and double enzyme digestion is performed on the positive clones to ensure that each antigen gene is correctly inserted and there is no sequence interference. 4) Application of plasmids: Combine and apply the recombinant plasmids of each antigen that have been verified as correct in step 3).

2. The determination method according to claim 1, characterized in that, The antigenic amino acids screened in step 1) include the fimbriae protein FimA-II antigen and arginine gingival protease RgpA antigen of Porphyromonas gingivalis ATCC 33277, the outer sheath protein Msp antigen of Treponema denticulata ATCC 35405, and the surface protein BspA antigen of Forsythia stomatis 92A2.

3. The determination method according to claim 2, characterized in that, The amino acid sequence of the FimA-II antigen is shown in SEQ ID NO:1; the amino acid sequence of the RgpA antigen is shown in SEQ ID NO:2; the amino acid sequence of the Msp antigen is shown in SEQ ID NO:3; and the amino acid sequence of the BspA antigen is shown in SEQ ID NO:

4.

4. The determination method according to claim 1, characterized in that, The cloning vector mentioned in step 2) is the pUC57 vector.

5. The determination method according to claim 1, characterized in that, In step 2), the restriction enzyme sites of the FimA-II antigen gene are NcoI and EcoRI; the restriction enzyme sites of the RgpA antigen gene are SmaI and EcoRI; the restriction enzyme sites of the Msp antigen gene are HindIII and EcoRI; and the restriction enzyme sites of the BspA antigen gene are SmaI and NdeI.

6. The determination method according to claim 1, characterized in that, The competent cells mentioned in step 3) are E. coli DH5α; the transformation specifically includes heat shock at 42℃±0.5℃ for 60-90 seconds, followed by immediate placement on ice for 2-3 minutes, then addition of LB liquid medium, and culture at 37℃ and 180-200 rpm for 60 minutes with shaking.

7. The determination method according to claim 1, characterized in that, The double enzyme digestion verification described in step 3) specifically includes: performing enzyme digestion using restriction endonucleases corresponding to the antigen genes on both sides of each recombinant plasmid, and confirming by agarose gel electrophoresis that the digestion products simultaneously contain vector bands and antigen gene insertion fragment bands of the expected size.

8. A multivalent antigen gene combination for combating the red complex of periodontitis, characterized in that, The multivalent antigen gene combination comprises two or more recombinant antigen plasmids prepared by the determination method described in any one of claims 1-7.

9. A multivalent vaccine development kit for combating red complexes of periodontitis, characterized in that, The kit contains the multivalent antigen gene combination as described in claim 8.

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