Preparation method of anti-periodontitis functional milk

Abstract

The invention discloses a preparation method of anti-periodontitis functional milk, and belongs to the technical field of prevention and functional health care products, the preparation method of the anti-periodontitis functional milk comprises the following steps: a, preparing a DNA vaccine aiming at key antigens or virulence factors of main pathogenic bacteria of periodontitis; b, the prepared DNA vaccine is used for constructing a periodontitis animal model, and the immune effect and safety of vaccine plasmids are analyzed and verified; c, preparing functional buffalo milk by taking the periodontitis DNA vaccine as an immunogen; and d, analyzing the effectiveness and applicability of the prepared cow milk. The functional cow milk for preventing periodontitis is prepared by immunizing buffalo with the DNA vaccine for periodontitis, so that the development bonus of a new transgenic technology is utilized, the risk of long-term safety problem is avoided through a bioreactor, and compared with direct application of the DNA vaccine, the method is more convenient and easier to accept, and is more beneficial to popularization.

Description

Technical Field

[0001] The invention relates to the technical field of preventive and functional health products, in particular to a method for preparing functional cow's milk for resisting periodontitis. Background Art

[0002] Periodontitis has a high incidence and is very harmful. It seriously affects human oral health and is closely related to systemic diseases. The fourth national oral epidemiological survey showed that among residents aged 35 to 44, 96.7% of the population had dental calculus in their mouths and 87.4% had symptoms of gingival bleeding, which was 10.1 percentage points higher than 10 years ago. Periodontitis is a chronic nonspecific inflammation, and the damage to periodontal tissues is persistent and irreversible. Therefore, in terms of incidence, early prevention of periodontitis is particularly important. At the same time, with people's attention to aesthetics, more and more people are undergoing orthodontic treatment. When the periodontal condition of patients with periodontal disease is not well controlled, orthodontic treatment cannot be performed. In the process of orthodontic treatment for patients without periodontal disease using fixed appliances, the orthodontic attachments are bonded to the tooth surface, making oral cleaning more difficult, causing plaque to accumulate more easily under the arch wire, and the patient's chance of suffering from periodontal disease is significantly increased. At present, the clinical treatment of periodontal disease is mainly based on periodontal basic treatment combined with periodontal surgical treatment. Current methods cannot interfere with bacterial recolonization, so clinical periodontal treatment cannot quickly restore periodontal tissue. Therefore, from a clinical treatment perspective, an effective method for preventing periodontal disease is urgently needed.

[0003] DNA vaccine is a new vaccine developed from the field of gene therapy in the 1990s. Due to its advantage of highly efficient induction of immune response, it has become a hot topic in the research of disease prevention and treatment in recent years. It also provides new ideas for the prevention of periodontal disease and is expected to become an effective way to prevent periodontal disease. With the continuous deepening of periodontal disease DNA vaccine research, its potential application has been widely recognized. However, the development of periodontal disease DNA vaccine still faces many problems and challenges, such as difficulty in approval and people's concerns about the long-term safety of transgenic technology.

[0004] In order to solve this problem, we innovatively utilized the advantages of bioreactors, used periodontitis DNA vaccine to immunize buffaloes, and prepared functional cow milk for preventing periodontitis. This not only took advantage of the development dividends of new transgenic technologies, but also avoided the risks of long-term safety issues through bioreactors. It is also more convenient and easier to accept than direct application of DNA vaccines, which is conducive to promotion. Summary of the invention

[0005] The purpose of the present invention is to overcome the above-mentioned background technical difficulties and provide a method for preparing anti-periodontitis functional milk.

[0006] To achieve the above-mentioned purpose, the technical scheme adopted is: a method for preparing functional cow's milk against periodontitis, comprising the following steps: a. preparing a DNA vaccine against key antigens or virulence factors of the main pathogenic bacteria of periodontitis, the method for preparing the DNA vaccine comprising ① construction of a gene vaccine of Porphyromonas gingivalis ② detection of the mRNA expression level of the recombinant plasmid in eukaryotic cells ③ detection of the IL-15 protein expression level of the recombinant plasmids pVAX1-HA2-fimA / IL-15, pVAX1-HA2 / IL-15, and pVAX1-fimA / IL-15 in eukaryotic cells; b. using the prepared DNA vaccine to construct a periodontitis animal model, analyzing and verifying the immune effect and safety of the vaccine plasmid; c. preparing functional buffalo milk using the periodontitis DNA vaccine as an immunogen; d. analyzing the effectiveness and applicability of the prepared cow's milk.

[0007] Furthermore, in the step b, multiple female SD rats are selected for control group experiments to construct a periodontitis animal model, and a periodontitis model is constructed for the rats in each control group. The plasmid immune effects in multiple control groups are tested separately, and the periodic body weight, heart, liver, spleen, lung and kidney pathological staining of the control group rats are tested separately.

[0008] Furthermore, the step b also includes using the DNA vaccine in step a to immunize rats, and then measuring and analyzing the IgG index in rat serum and rat colostrum.

[0009] Furthermore, in the preparation of the functional buffalo milk in step c, the buffalo is vaccinated four times in total, the second injection is injected one week after the first injection, the third injection is injected two weeks after the second injection, and the fourth injection is injected one week after the third injection. During this period, the first injection site of the buffalo is a subcutaneous multi-point injection on the back of the buffalo, the second and third injections are intramuscular injections, and the fourth is intravenous injection. After the injection, the original immune milk is obtained every seven days as a cycle, and the antibody titer is measured after each acquisition of the original immune milk.

[0010] Furthermore, the original immune milk is centrifuged at 1000 rpm for 30-40 min, and the precipitate is collected and mixed with conventional milk. The ratio of the original immune milk to conventional milk is 1:50-100. After mixing, the milk is sterilized by pasteurization to obtain drinkable immune functional milk.

[0011] Furthermore, the construction of the Porphyromonas gingivalis gene vaccine in step a uses pIRES2-EGFP, Porphyromonas gingivalis W83 type, and cDNA reverse transcribed from SD rat total RNA as templates, and pVAX1 plasmid as a vector. Primers are synthesized according to the reported gene sequence, and the target genes HA2, fimA, IRES, and IL-15 are amplified in vitro to construct recombinant expression plasmids pVAX1-HA2-fimA / IL-15, pVAX1-HA2 / IL-15, pVAX1-HA2-fimA, pVAX1-fimA / IL-15, and pVAX1-HA2.

[0012] Furthermore, the detection of the mRNA expression level of the recombinant plasmid in eukaryotic cells in step a is that the recombinant plasmid constructed with the Porphyromonas gingivalis gene vaccine is transiently transfected into 293T cells by liposomes, and the total cell RNA is extracted after 24 hours, reverse transcribed into cDNA, and RT-PCR is used to verify the expression of its mRNA level.

[0013] Furthermore, the detection of IL-15 protein expression levels in eukaryotic cells by the recombinant plasmids pVAX1-HA2-fimA / IL-15, pVAX1-HA2 / IL-15, and pVAX1-fimA / IL-15 in step a is based on the pVAX1-HA2-fimA / IL-15, pVAX1-HA2 / IL-15, and pVAX1-fimA / IL-15 plasmids constructed by the Porphyromonas gingivalis gene vaccine, transiently transfecting 293T cells, and detecting the expression of IL-15 protein levels after 24 hours.

[0014] The beneficial effects of the above scheme are as follows: First, the method for preparing functional cow's milk against periodontitis requires the construction of plasmids using key antigens or virulence factors of Porphyromonas gingivalis, the main pathogenic bacteria of periodontitis, and secondly, the construction of an animal model of periodontitis to analyze and verify the immune effect and safety of the vaccine plasmid. Finally, buffaloes are immunized with a DNA vaccine for periodontitis to prepare functional cow's milk, and its effectiveness and applicability are analyzed.

[0015] Finally, the applicant took advantage of the bioreactor and used the periodontitis DNA vaccine to immunize buffaloes and prepare functional milk for preventing periodontitis. This not only took advantage of the development dividends of new transgenic technologies, but also avoided the risks of long-term safety issues through the bioreactor. It is also more convenient and easier to accept than direct application of DNA vaccines, and is more conducive to promotion. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is the physical map of plasmid pIRES2-EGFP and the sequence map of IRES gene.

[0017] Figure 2The figure shows the construction flow chart of pVAX1-HA2 plasmid.

[0018] Figure 3 Flowchart for the construction of pVAX1-HA-fimA / IL-15.

[0019] Figure 4 This is the plasmid structure diagram of pVAX1-HA2-fimA / IL-15, pVAX1-HA2 / IL-15, pVAX1-HA2-fimA, pVAX1-HA2, and pVAX1-fimA / IL-15.

[0020] Figure 5 The plasmid maps are pVAX1-HA2-fimA / IL-15, pVAX1-HA2 / IL-15, and pVAX1-fimA / IL-15.

[0021] Figure 6 This is a graph showing the titer of rat-specific antibodies determined using the ELISA method.

[0022] Figure 7 This is a graph showing the average specific IgG level in the serum of buffalo immunized experimental group and control group for 4 weeks measured by indirect ELISA.

[0023] Figure 8 The graph shows the average specific IgG level in colostrum and different dilutions after buffalo delivery determined by indirect ELISA.

[0024] Fig. 9 The figure shows the construction flow chart of pVAX1-HA2 / IL-15.

[0025] Fig.10 Flowchart for the construction of pVAX1-HA2-fimA.

[0026] Fig.11 Flowchart for construction of pVAX1-fimA / IL-15. DETAILED DESCRIPTION

[0027] The technical scheme of the present invention is clearly and completely described below in conjunction with the specific embodiments of the present invention. The described embodiments are only part of the embodiments of the present invention, not all. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0028] Embodiment 1 A method for preparing anti-periodontitis functional milk, characterized by comprising the following steps: a. Prepare a DNA vaccine targeting the key antigens or virulence factors of the main pathogenic bacteria of periodontitis, and the preparation method of the DNA vaccine includes ① construction of a Porphyromonas gingivalis gene vaccine ② detection of the mRNA expression level of the recombinant plasmid in eukaryotic cells ③ detection of the IL-15 protein expression level of the recombinant plasmids pVAX1-HA2-fimA / IL-15, pVAX1-HA2 / IL-15, and pVAX1-fimA / IL-15 in eukaryotic cells; b. Use the prepared DNA vaccine in a constructed animal model of periodontitis to analyze and verify the immune effect and safety of the vaccine plasmid; c. Prepare functional buffalo milk using the periodontitis DNA vaccine as an immunogen; d. Analyze the effectiveness and applicability of the prepared buffalo milk.

[0029] Embodiment 2 The construction steps of ① construction of Porphyromonas gingivalis gene vaccine in step a of Example 1 are as follows: using pIRES2-EGFP, Porphyromonas gingivalis W83 type, and cDNA reverse transcribed from SD rat total RNA as templates, and pVAX1 plasmid as vector, primers are synthesized according to the reported gene sequence, and target genes HA2, fimA, IRES, and IL-15 are amplified in vitro to construct recombinant expression plasmids pVAX1-HA2-fimA / IL-15, pVAX1-HA2 / IL-15, pVAX1-HA2-fimA, pVAX1-fimA / IL-15, and pVAX1-HA2.

[0030] The bacterial species and plasmids used are: (1) plasmid pVAX1 (kept by the Key Laboratory of Oral Disease Research in Guizhou Province); (2) plasmid pIRES2-EGFP, kindly donated by Professor Yue Changwu of the Microbial Resources and Drug Development Laboratory; (3) E. coli DH5a, competent cells from Shanghai Bioengineering Co., Ltd.; (4) Porphyromonas gingivalis W83 type, kept by the Key Laboratory of Oral Disease Research in Guizhou Province. The main experimental reagents used are: pfu enzyme, dNTPmixture solution, 25 mM, 10×pfuBuffer, defibrinated sheep blood, high-purity plasmid small-scale rapid extraction kit, restriction endonucleases: Nhe I, Xho I, double enzyme digestion buffer: FD buffer, T4 DNA Ligase, T4 DNA Ligasebuffer, AxyPrepPCR purification kit, AxyPrep gel recovery kit, reverse transcription kit, Trizol lysis buffer, 2% SDS, Tris-balanced phenol, DEPC water, lysozyme, phenol-chloroform-isoamyl alcohol. The main reagents used are: BHI blood agar solid medium, BHI liquid medium, LB liquid medium, LB solid medium, 1% (1.2%) agarose gel. The main antibiotics used are: kanamycin sulfate, ampicillin.

[0031] The physical map of plasmid pIRES2-EGFP and the IRES gene sequence are as follows Figure 1 shown.

[0032] The construction of pVAX1-HA2 gene includes: Design of specific primers: The primers for amplification fragments were designed based on the gene sequence reported in GenBank (HA2 GI: U15282) and were designed and synthesized by Shanghai Shenggong Biotechnology Co., Ltd., as shown in Table 1 below: Table 1: Primer sequences of HA2 gene Among them, the boxed parts in Table 1 are protective bases; the underlined parts are restriction endonuclease cleavage sites, which are Nhe I: GCTAGC and Xho I: CTCGAG; the bold parts are the complementary sequences of the added initiator and terminator; and the italic parts are the kozak sequence.

[0033] Cultivation of Porphyromonas gingivalis and extraction of genomic DNA: (1) Take out the milk-preserved bacteria Porphyromonas gingivalis W83 from the -80℃ refrigerator and resuscitate it on a BHI blood agar plate. Resuscitate it in an anaerobic chamber (80% N2, 10% H2, 10% CO2) away from light for 4-7 days. Observe that the black colonies grow to about 1 mm. (2) Pick a single colony for routine Gram staining and morphological identification to determine whether it is Porphyromonas gingivalis. (3) Pick a single colony and inoculate it into a 15ml centrifuge tube containing 5ml BHI liquid culture medium. Place it in an anaerobic incubator and culture it for 1-2 days. Observe that the liquid becomes turbid and a small amount of bacterial precipitation occurs. (4) Use the phenol-chloroform method to extract bacterial genomic DNA: ① 10 ml of bacterial solution, 3000 rpm, 10 min, collect the bacteria; ② Add SinⅠ to wash the bacteria, 3000 rpm, discard the supernatant; ③ Add SinⅠ according to the amount of bacteria, add 1 / 10 lysozyme, and leave at 37℃ overnight; ④ Add SDS with a final concentration of 2%, leave at 55℃-60℃ for 1h to clarify; ⑤ Add an equal volume of Tris-balanced phenol, mix upside down, leave for a few minutes, 12000 rpm, 10 min; ⑥ Take the supernatant, add an equal volume of phenol-chloroform-isoamyl alcohol, mix upside down, leave for a few minutes, 12000 rpm, 10 min, repeat once; ⑦ Take the supernatant, add 1 / 10 SInⅢ, add an equal volume of isopropanol, place at -20℃ for 10min, 12000rpm, 10min;⑧WB rinse twice, 12000rpm, 2min, place in a 56℃ oven for 3min, EB elution, electrophoresis and sequencing verification. Measure OD260 / 280 and concentration under UV spectrophotometer, and store at -80℃.

[0034] PCR amplification of target gene: using total DNA of Porphyromonas gingivalis as template and primers as shown in Table 1, PCR amplification of HA2 was performed according to the reaction system shown in Table 2: Table 2: PCR reaction system Reactants System (50 ul) template 1ul F-terminal primer 2ul R-terminal primer 2ul dNTP 1ul(25mM each) 10×pfu Buffer 5ul Pfu 0.4ul(5u / ul) ddH2O Make up to 50ul The primer concentration was 1 OD dissolved in 400ul ddH2O.

[0035] Reaction conditions: pre-denaturation at 94 ℃ for 3 min, denaturation at 94 ℃ for 20 sec×29, annealing at 58 ℃ for 20 sec×29, extension at 72 ℃ for 2 min×29, extension at 72 ℃ for 6 min×29, 4 ℃ ∞.

[0036] After the reaction is completed, the target band is verified by electrophoresis and purified and recovered.

[0037] Amplification of pVAX1 and pIRES-EGFP and extraction of plasmids: (1) Take out the glycerol bacteria E. coli JM109 carrying the pVAX1 or pIRES2-EGFP plasmid from the -80℃ freezer, revive it in LB liquid medium (no resistance), and then culture it on solid plates (Kana 100ug / ml) (37℃, 12-16h) until the colony diameter reaches about 1mm.

[0038] (2) Pick a single colony and inoculate it into a 50 ml centrifuge tube containing 10 ml LB liquid culture medium (Kana 100 ug / ml), and culture it in a 37°C constant temperature shaker overnight until OD600 = 0.8-1.0.

[0039] (3) Use a high-purity plasmid small-volume rapid extraction kit to extract the plasmid: ① Collect bacteria: bacterial solution, 5000rpm, 10min, discard the supernatant, add 6mlddH2O, 5000rpm, 10min, wash the bacteria, and discard the supernatant; ② Add 500ul of solution P1 to suspend the precipitate, mix on an oscillator for 15 seconds, and let stand at room temperature for 5 minutes; ③Add 500ul of solution P2, invert repeatedly 7-10 times, and let stand at room temperature for 5 minutes ④ Add 700ul of solution P3, invert repeatedly until the flocculent precipitate no longer increases, 12000rpm, 15min; ⑤ Collect the supernatant into the centrifugal column, 10000rpm, 1min, and discard the filtrate; ⑥ Add 500ul of PE deproteinizing solution, 10000rpm, 1min, deproteinize; ⑦ Add 700ul of rinsing solution WB, 10000rpm, 1min, repeat rinsing once, empty once, put the adsorption column into a clean EP tube, and let it stand at 56℃ for 5min; ⑧ Add 30ul of eluent EB (preheated at 56℃), let stand for 2min, collect (12000rpm, 1min). Measure OD260 / OD280 and concentration under UV spectrophotometer, and store at -80℃.

[0040] PCR product and vector digestion: Table 3: PCR products and pVAX1 plasmid restriction enzyme digestion system (50ul) Reactants system Reactants system PCR products 20ul pVAX1 1.5ug XOt 1ul(10u / ul) XOt 1ul(10u / ul) Nhe I 1ul(10u / ul) NeA 1ul(10u / ul) FD Buffer 5ul FD Buffer 10ul ddH2O Make up to 50ul <![CDATA[ddH 2 The]]> Make up to 50ul The above system was placed in a 37°C constant temperature water bath for 3 h, and the enzyme cleavage products were detected by 1% agarose gel electrophoresis.

[0041] Purification of target fragment and vector fragment: Recover the purified product according to the instructions of the AxyPrep PCR purification kit, as follows: (1) Add three times the volume of PCR product and Buffer PCR-A, mix well, and pass through the column at 12000 rpm for 1 min; (2) Add 700ul of Buffer W2 to the column at 10000rpm for 1min, repeat once; (3) Leave in an empty oven for 1 min and place in a 56°C oven until the ethanol evaporates. Add ddH2O (preheated at 56°C) and place in an oven for 5 min. 12000rpm, 1min, -20℃ for standby use.

[0042] Target fragment and vector connection: Table 4: Target fragment and vector connection system Reactants System (20ul) Enzyme digestion target fragment 8ul Enzyme digestion vector 8ul 10X T4 DNAligase Buffer 3ul T4 DNAligase 1ul(5u / ul) ddH2O Make up to 20ul The above ligation mixture was ligated in a 16°C water bath overnight.

[0043] Recombinant plasmids transform competent cells: Transform the connected recombinant plasmid into competent DH5α cells as follows: (1) Take out two competent cells from -80℃ and place them in an ice bath; (2) Add 10 μl of the ligation product to each competent cell, gently swirl to mix the contents, and place in an ice bath for 30 min; (3) Place the centrifuge tube in a 42°C water bath for 90 seconds, quickly transfer to an ice bath and cool for 2 minutes, leaving it as still as possible; (4) Add 500 μl of sterile, antibiotic-free LB medium into the centrifuge tube in a clean bench, mix well, and place in a 37°C constant temperature shaker for 45 min to revive the bacterial solution; (5) Spin the bacterial solution at 3000 rpm for 10 min, collect the cells, add 50 μl of the solution and remix the cells; (6) Inoculate the bacterial suspension onto LB medium plates containing Amp resistance in a clean bench and culture at 37°C overnight.

[0044] (7) Select a single colony and place it in a 1.5 ml centrifuge tube with 500 μl of resistant liquid culture medium. Incubate at 37°C in a constant temperature shaker for 4-6 hours, observe the growth, and select the amplified bacteria for bacterial liquid PCR.

[0045] Bacterial liquid PCR and recombinant screening: Select the amplified bacteria for bacterial liquid PCR to screen the recombinants. The bacterial liquid PCR system is as follows: Table 5: Bacterial liquid PCR system Reactants System (10μl) Bacterial liquid 1μl Upstream primer (10pmol / μl) 0.5μl Downstream primer (10pmol / μl) 0.5μl 2X Taq PCR MasterMix 5μl ddH2O 3μl Reaction conditions: 94 ℃ pre-denaturation 5min×29, 94 ℃ denaturation 20sec×29, 65 ℃ annealing 20sec×29, 72 ℃ extension 2min×29, 72 ℃ extension 6min×29, 4 ℃ ∞.

[0046] The PCR products were subjected to 1.2% agarose gel electrophoresis at 100V for 30min. The results were analyzed using a gel electrophoresis imaging system. The recombinants were screened for bacterial preservation and amplification. Eight recombinants were selected for sequencing. The sequencing work was completed by Shanghai Yingwei Jieji Trading Co., Ltd.

[0047] The pVAX1-HA2 plasmid construction process is as follows Figure 2 shown.

[0048] Construction of pVAX1-HA2-fimA / IL-15 co-expression plasmid: Primer design and synthesis: Primers for amplification fragments were designed based on the gene sequences reported in GenBank (HA2GI: U15282; fimAGI: 34539880; IL-15GI: AF015719) and were designed and synthesized by Shanghai Shenggong Biotechnology Co., Ltd.

[0049] Table 6: Primer design list Note: The boxed part is the protected base; the underlined part is the restriction site: Nhe I: GCTAGC, Xho I: GAGCTC; the slashed part is the kozak sequence. IRES1 is the IRES sequence between HA2 and fimA, and IRES2 is the IRES sequence between fimA and IL-15.

[0050] Preparation of target gene template: (1) Cultivation of Porphyromonas gingivalis type W83 and extraction of genomic DNA: Same as the cultivation and extraction of genomic DNA of Porphyromonas gingivalis.

[0051] (2) Extraction and reverse transcription of total RNA from SD rats a: Take a healthy female SD rat of 6-8 weeks old, remove its liver, place it in a 1.5 ml enzyme-free EP tube, and weigh (weigh the weight of the EP tube in advance) M (liver) = M (total amount after placing the liver) - M (EP tube without liver). The amount of liver in each EP tube should not exceed 1g. b: Add 1 ml of Trizol to the tissue and incubate at -80℃ overnight. c: Use the Trizol method to extract total RNA from the liver of SD rats. The UV spectrophotometer measures OD260 / 280 = 1.90, and the concentration = 1308.8ng / μl. (1) Reverse transcription using a reverse transcription kit using the tailing method: (the total amount of RNA is less than 1 μg, i.e. 1000 ng) Table 7: 20ul reverse transcription reaction system Reactants Volume (μl) 1 4 2 1 3 1 4 1 RNA 0.7 DEPC water 12.3 Reverse transcription reaction conditions: 37℃ 15min, 85℃ 5sec, 4℃ ∞. The obtained reverse transcription product was stored at -80℃.

[0052] PCR amplification of target gene (1) HA2, fimA and IL-15 were amplified by PCR using total DNA of Porphyromonas gingivalis, pIRES2-EGFP and cDNA of SD rats as templates and primers as shown in Table 7.

[0053] Table 8: Reaction system of each target gene (50 μl) Reactants System (50 μl) template 1μl Upstream primer 2μl Downstream primer 2μl dNTP 1μl (25mM each) 10×pfu Buffer 5μl Pfu 0.4μl(5μ / μl) ddH2O Make up to 50 μl The reaction conditions are as follows: ① HA2 PCR reaction conditions: 94℃ pre-denaturation for 3min; 94℃ denaturation for 20sec; 58℃ annealing for 20sec; 72℃ extension for 30sec; 29 cycles; 72℃ extension for 6min; 4℃ ∞.

[0054] ②fimA PCR reaction conditions: 94℃ pre-denaturation for 3min; 94℃ denaturation for 30sec; 60℃ annealing for 40sec; 72℃ extension for 1min; 29 cycles; 72℃ extension for 6min; 4℃ ∞.

[0055] ③ IL-15 PCR reaction conditions: 94℃ pre-denaturation for 3min; 94℃ denaturation for 30sec; 55℃ annealing for 30sec; 72℃ extension for 1min; 29 cycles; 72℃ extension for 6min; 4℃ ∞.

[0056] ④IRES PCR reaction conditions: 94℃ pre-denaturation for 3min; 94℃ denaturation for 30sec; 56℃ annealing for 30sec; 72℃ extension for 1min; 29 cycles; 72℃ extension for 6min; 4℃ ∞.

[0057] (2) The target fragments were recovered by gel electrophoresis: The PCR products of HA2, fimA, IL-15, and IRES target genes were verified by 1% agarose gel electrophoresis to verify whether the expected size of the gene was obtained. The purified products were recovered according to the instructions of the AxyPrepPCR purification kit, and the steps were the same as the PCR product and vector enzyme digestion method.

[0058] Secondary splicing between target genes: The previously obtained target genes HA2, IRES1 (spliced ​​between HA2 and fimA), fimA, IRES2 (spliced ​​between fimA and IL-15), and IL-15 were used as templates for pairwise secondary splicing: (1) Splicing between HA2 and IRES1: The F end of HA2 was used as the upstream primer and the R end of IRES1 was used as the downstream primer. Primers, using HA2 and IRES1 as templates, were used for PCR amplification: Table 9: Amplification reaction system and reaction conditions: Reactants system Template (HA2) 1μl Template (IRES1) 1μl Upstream primer 2μl Downstream primer 2μl dNTP 1μl (25mM each) 10×pfu Buffer 5μl Pfu 0.4μl(5μ / μl) ddH2O Make up to 50 μl Reaction conditions: 94 ℃ pre-denaturation for 3 min, 94 ℃ denaturation for 20 sec×29, 55 ℃ annealing for 20 sec×29, 72 ℃ extension for 2 min×29, 72 ℃ extension for 6 min, 4 ℃ ∞, product recovery and preparation for splicing with fimA fragment.

[0059] (2) Secondary splicing of HA2-IRES1 fragment and fimA: the upstream primer is the HA2 F-terminal primer, and the downstream primer is the fimA downstream primer.

[0060] Table 10: PCR amplification system and reaction conditions Reactants system Template (HA2-IRES1) 1μl Template (fimA) 1μl Upstream primer 2μl Downstream primer 2μl dNTP 1μl (25mM each) 10×pfu Buffer 5μl Pfu 0.4μl(5μ / μl) ddH2O Make up to 50 μl Reaction conditions: 94 ℃ pre-denaturation for 3 min, 94 ℃ denaturation for 30 sec×29, 56 ℃ annealing for 2 min×29, 72 ℃ extension for 2 min×29, 72 ℃ extension for 6 min×29, 4 ℃ ∞, the product was recovered and finally spliced ​​with the lower amplification product.

[0061] (3) Secondary splicing of IRES2 and IL-15: the upstream primer is the IRES2 F-terminal primer, and the downstream primer is the IL-15 downstream primer.

[0062] Table 11: PCR amplification system and reaction conditions Reactants system Template (IRES2) 1μl Template (IL-15) 1μl Upstream primer 2μl Downstream primer 2μl dNTP 1μl (25mM each) 10×pfu Buffer 5μl Pfu 0.4μl(5μ / μl) ddH2O Make up to 50 μl Reaction conditions: 94°C pre-denaturation for 3 min, 94°C denaturation for 40 sec×29, 55°C annealing for 80 sec×29, 72°C extension for 80 sec×29, 72°C extension for 6 min, 4°C ∞. The reaction product was recovered and spliced ​​with the fragment obtained in (2).

[0063] (4) Obtaining HA2-fimA / IL-15 fragment: Table 12: PCR amplification system and reaction conditions Reactants system Template (IRES2 / IL-15) 1μl Template (HA2-IRES-fimA) 1μl Upstream primer (HA2 F end) 2μl Downstream primer (fimA R end) 2μl dNTP 1μl (25mM each) 10×pfu Buffer 5μl Pfu 0.4μl(5μ / μl) ddH2O Make up to 50 μl Reaction conditions: pre-denaturation at 94 ℃ for 3 min, denaturation at 94 ℃ for 1 min×29, annealing at 55 ℃ for 2 min×29, extension at 72 ℃ for 2 min×29, extension at 72 ℃ for 6 min, 4 ℃ ∞. The reaction products were purified and recovered, and the products were detected by 1% agarose gel electrophoresis. Some of the products were sequenced. The sequencing work was carried out by Shanghai Bioengineering Co., Ltd.

[0064] Double restriction digestion of target gene and pVAX1: Reaction conditions of enzyme digestion: the same as those in Table 3 and Table 4 of the enzyme digestion method of PCR products and vectors.

[0065] The reaction was carried out in a 37°C water bath for 4 hours. The reaction steps were as follows: loading the enzyme cleavage product onto the prepared 1% agarose gel, electrophoresing at 100V for 30 minutes, rapidly cutting out the HA2 fragment under the UV gel imaging analysis system, weighing the gel volume, and performing gel recovery. The recovery process was as follows: ① After weighing the gel volume, add 3 volumes (1 mg = 1 μl) of Buffer DE-A, mix well, and heat intermittently at 75°C until completely melted; ②Add 0.5 volume of Buffer DE-B to Buffer DE-A and mix well; ③ Transfer to DNA preparation tube, 12000rpm, 1min, discard the filtrate; ④ Add 500 μl Buffer W1 to the centrifugal column, 12000 rpm, 1 min, and discard the filtrate; ⑤ Add 700 μl Buffer W2, 12000 rpm, 1 min, discard the filtrate, repeat once; ⑥12000rpm idling for 1min; ⑦ Place the centrifugal column in a 1.5 ml collection tube, add 25 μl ddH2O to the center of the adsorption membrane, let stand at room temperature for 3 min, 12000 rpm for 1 min. Store briefly at 4°C for later use.

[0066] The target fragment is connected to the vector, and the connection system is such as the system for connecting the target fragment to the vector. The method for transforming the recombinant plasmid into competent cells is such as the method for transforming the recombinant plasmid into competent cells. The screening method of recombinants is such as bacterial liquid PCR and the screening method of recombinants. The positive clones are screened for sequencing, and the sequencing work is completed by Shanghai Bioengineering Co., Ltd.

[0067] The construction flow chart of pVAX1-fimA / IL-15 is shown in Figure 3 shown.

[0068] Construction of pVAX1-HA2 / IL-15 plasmid: It includes amplification of IRES fragment (using IRES1 F end as upstream primer; IRES2 R end as downstream primer, using the constructed pIRES2-EGFP as template, amplifying the sequence IRES, temporarily named IRES3), secondary splicing of IRES3 and IL-15 fragment (using fragments IRES3 and IL-15 fragments as templates, using IRES1 F end as upstream primer, using IL-15 R end as downstream primer, secondary splicing), splicing of HA2 / IL-15 fragments (using the purified product in the previous step and HA2 fragment as template, selecting HA2 F end as upstream primer, and selecting IL-15 R end as downstream primer, for PCR amplification), double restriction digestion of target gene and pVAX1, connection of target gene and pVAX1 (same as connection of target fragment and vector), transformation of recombinant plasmid into competent cells (same as transformation of recombinant plasmid into competent cells), screening of recombinants (bacterial liquid PCR and screening of recombinants) Next, the construction of pVAX1-HA2 / IL-15 was carried out. The flow chart is as follows: Fig. 9 As shown, the construction of pVAX-HA2-fimA was then performed, and the flow chart is as follows Fig.10 Then, pVAX1-fimA / IL-15 was constructed, as shown in Fig.11 shown.

[0069] According to the above method, the applicant analyzed the results after obtaining them. 1. Selection of genes and determination of key regions: Gingipin is an arginine-lysine-specific cysteine ​​protease on the surface of P. gingivalis cells, and the encoding genes are rgpA, rgpB and kgp. The hemagglutinin gene hagA, which is located at the terminal region of rgpA and kgpA, encodes the hemagglutinin / adhesion domain, which is particularly important for P. gingivalis to degrade hemoglobin and take up heme. Because its coding sequence is long, it is difficult to insert into the plasmid, and it also affects the expression effect. Therefore, choosing a conservative core coding region becomes the key to simplifying this experiment. The translation product of rgpA is a 227aa propeptide region (Met1-Arg227), of which the N-terminus includes a recognized signal sequence (Met1 - Ala23), a 492aa protein catalytic domain (Tyr228- Arg719), the remaining 543aa is adhesin / hemagglutinin (ᵝ domain) (Ser720- Arg1262), a 444aa C-terminal extension (Phe1263-Lys1706), and the ᵞᵝ domain contains several peptide repeat sequences, which may be an important adhesion region. Literature shows that the core region of the hemagglutinin gene is divided into four segments, namely HA1, HA2, HA3, and HA4. The four segments are similar in size, and the gene sequences of the four segments are similar, with a repetition rate of more than 90%, which significantly increases the difficulty of replicating the entire gene. Among the four sequences, HA2 plays a relatively large role. It binds to hemoglobin and is a multifunctional protein region that aggregates red blood cells, dissolves red blood cells to obtain lysine from hemoglobin, and absorbs iron, heme or porphyrin

[16] . At the same time, when comparing the genes of the hemagglutinin superfamily, the coding region of HA2 is highly homologous. Some scholars have analyzed the hemagglutinin binding site of the three polypeptide segments, and the results show that HA2 is the receptor binding region, so it is feasible to select this gene sequence. The fimbrial protein gene is a relatively mature and common gene at this stage. Among them, fimA is the main component. Various vaccines targeting fimA have emerged in an endless stream. At the same time, there is a wide range of genetic polymorphism in the fimA encoding gene. According to the different nucleotide sequences of its open reading frame, it can be divided into 5 types (Ⅰ~Ⅴ). However, since there is no immune cross-talk between different subtypes of FimA protein, a vaccine targeting one subtype cannot guarantee that it is effective against all Porphyromonas gingivalis. Because the coding region of Rgp-Kgp is adjacent to that of fimA in the total DNA, and some articles have pointed out that there is a synergistic effect between HA2 and fimA, fimA was selected as the auxiliary gene in this experiment to enhance the immune effect.2. Selection of gene concatenation method: The co-expression plasmid constructed in this experiment is a non-fusion protein obtained by gene concatenation. Each gene is linked by the IRES sequence. IRES can recruit ribosomes to translate mRNA. After IRES is fused with exogenous cDNA, ribosomes can independently start translation after finding IRES. Therefore, independently expressed proteins can be obtained, thereby reducing the impact of weakened immunogenicity of fusion proteins. Therefore, when multi-gene co-expression plasmids need to express independent proteins rather than fusion proteins, IRES sequences are selected. At the same time, since the HA2 gene is a gene fragment and has no initiator, the ATG initiator is added before the HA2 gene during design. At the same time, due to the particularity of pVAX1, the kozak sequence with the sequence of GCCACC is added before ATG to increase the translation efficiency of eukaryotic cells. 3. Selection of splicing method: The experiment adopts a small segment amplification of the target fragment, and then splices it in pairs, so as to connect the target gene in series to form a complete target gene sequence. Therefore, when designing primers, head-to-tail splicing is required, that is, the F end needs to add the tail of the previous sequence, and the R end needs to add the complementary sequence at the beginning of the next sequence. The splicing length is between 12-20bp. 4. Selection of high-fidelity enzyme pfu enzyme: In the process of constructing plasmids in this experiment, pfu high-fidelity enzyme is used. Compared with Taq enzyme, pfu high-fidelity enzyme can automatically modify mutated genes in PCR amplification, thereby reducing the mutation probability of genes during amplification and ensuring the accuracy of target gene amplification. At the same time, when designing primers, attention should be paid to the length of primers to be extended accordingly. Therefore, the primers designed in this experiment when amplifying the target band are all 35-45bp, so as to ensure the accuracy of the experiment.

[0070] Embodiment 3 The method for detecting the mRNA expression level of the recombinant plasmid in step a of Example 1 in eukaryotic cells is as follows: according to the recombinant plasmid constructed in Experiment 1, 293T cells are transiently transfected with liposomes, and total cell RNA is extracted after 24 hours, reverse transcribed into cDNA, and RT-PCR is used to verify the expression of its mRNA level.

[0071] The plasmids pVAX1-HA2-fimA / IL-15, pVAX1-HA2 / IL-15, pVAX1-HA2-fimA, pVAX1-HA2, pVAX1-fimA / IL-15 in the detection method were constructed in the early stage of the experiment. Figure 4 shown.

[0072] The experimental method includes: extraction of five plasmids (same as the extraction step of pVAX1 plasmid in Example 2), culture of 293T cells, transfection of 293T cells with five plasmids, extraction of total RNA from 293T cells, reverse transcription of total RNA, and PCR verification of HA2, fimA and IL-15 genes.

[0073] The applicant analyzed the experimental results obtained from the above experiments: Lip2000 is one of the well-known transfection products, and its main function is to transiently carry plasmids into cells, and replicate and express them in the cells. In this experiment, 293T cells were transiently transfected with Lip2000, and total RNA was extracted. The stem-loop method was used to reversely transcribe the cDNA, and the target band was specifically amplified by designing specific amplification primers. At the same time, 293T cells that were not transfected with any plasmids were treated in the same way. The results showed that the transfected cells could amplify the corresponding bands, while the untransfected cells could not amplify the corresponding bands, and its successful expression was detected from the mRNA level.

[0074] There are two methods of reverse transcription: tailing method and stem-loop method. Tailing method is to add polyA tail to miRNA on the basis of the obtained miRNA through tailing primer design and then reverse transcribe it, and screen and classify the tailed mRNA through single base anchor method, which is more suitable for detecting a large number of miRNA samples; while the stem-loop method is to effectively extend the length of mature miRNA through primers with universal stem-loop structure, form complementary cDNA under the action of reverse transcriptase, and is not interfered by the precursor. At the same time, the specifically designed primers can avoid binding with other homologous genes and reduce the probability of non-specific amplification. Therefore, the stem-loop method is suitable for experiments that fail to reverse transcription by tailing method and require strong specificity and high sensitivity. At the same time, the experimental steps of stem-loop method are simple, and only one amplification is needed to obtain the corresponding cDNA using the kit, but the cost is higher than that of tailing method. The stem-loop method requires corresponding tailing and then secondary reverse transcription. In subsequent experiments, the influence of polyA tail on experimental results must also be considered, but the cost is lower. This experiment used the stem-loop method for reverse transcription to eliminate the influence of polyA on the experiment and simplify the next experimental steps.

[0075] Fluorescence quantitative PCR (Real-Time PCR) is a method of labeling and tracking PCR products through fluorescent dyes or specific probes labeled with fluorescent markers. The reaction process is monitored online in real time through software. At the same time, the products can be analyzed and the initial concentration of the sample template to be tested can be calculated. Real-Time PCR is one of the faster and more accurate methods for quantitative nucleic acid and a powerful means of biological quantitative analysis. The RT-PCR used in this experiment is a qualitative analysis of the expression of its mRNA level, but it cannot be quantitatively analyzed. Since it cannot be guaranteed that all cells can be successfully transfected after transfection, the transfection efficiency cannot be detected. At the same time, there are systematic errors when extracting RNA from each group, and the reverse transcription efficiency cannot be detected. Therefore, although Real-Time PCR requires the amount of total RNA in each control group to be consistent, it cannot guarantee that the amount of the target gene can be accurately measured, so it is a relative quantification. Therefore, in subsequent experiments, a more rigorous method can be explored to quantitatively analyze its mRNA level.

[0076] Embodiment 4 The method for detecting the expression level of IL-15 protein in eukaryotic cells of the recombinant plasmids pVAX1-HA2-fimA / IL-15pVAX1-HA2 / IL-15 and pVAX1-fimA / IL-15 in step a of Example 1 is as follows: based on the construction of pVAX1-HA2-fimA / IL-15, pVAX1-HA2 / IL-15 and pVAX1-fimA / IL-15 plasmids according to Example 2, 293T cells are transiently transfected, and the expression level of IL-15 protein is detected after 24 hours.

[0077] The plasmids in the detection method: pVAX1-HA2-fimA / IL-15, pVAX1-HA2 / IL-15, pVAX1-fimA / IL-15, are constructed as in the previous Example 1. The plasmid maps of pVAX1-HA2-fimA / IL-15, pVAX1-HA2 / IL-15, and pVAX1-fimA / IL-15 are as follows: Figure 5 shown.

[0078] The experimental method is as follows: extraction of five plasmids (same as the extraction step of pVAX1 plasmid in Example 2), culture of 293T cells, plasmid transfection of 293T cells (including seeding, transfection and five plasmid transfection of 293T cells for standby), enzyme-linked immunosorbent assay (ELISA) to determine the content of IL-15 protein in transfected cells (extraction of total protein from 293T cells, preparation of IL-15 standard curve and determination of IL-15 ELISA).

[0079] According to the above experimental results, the applicant analyzed that 293T cells are derived from 293 cells and are human renal epithelial cells transfected with adenovirus E1A gene. Transient transfection of 293T cells is a convenient way to obtain intracellular and extracellular overexpressed proteins (secreted or membrane proteins), and is one of the cell lines that are easier to successfully transfect among all cell lines. The protein expression level is high, and the expressed protein can be easily detected by alkaline phosphatase analysis 2-3 days after transfection. Since 293T cells have poor wall adhesion, careful operation should be taken during cell passage and digestion. During operation, try to let the liquid flow along the side wall of the culture dish or culture bottle instead of dripping directly into the cells, so as to avoid uneven cell growth after the cells are dispersed and re-adhered to the wall. At the same time, during cell passage, try to reduce the digestion time when using trypsin digestion to avoid over-digestion.

[0080] The expression of exogenous proteins in 293T cells reached the highest level on the 2nd and 3rd days, so the expression of IL-15 protein was detected at 24h, 48h, and 72h in the preliminary experiment. According to the results of the preliminary experiment, 48h was finally selected as the detection time point. The results of the ELISA experiment showed that the protein concentration of IL-15 in the experimental group was higher than that in the control group, and the difference was statistically significant, proving that the target gene IL-15 can be expressed in 293T cells.

[0081] Since there are no commercial antibodies for HA2 and FimA proteins, it is not possible to directly prove the target gene HA2 and fimA The recombinant plasmid constructed in this experiment contains only one promoter and can only transcribe one mRNA. Due to the presence of the IRES sequence, the ribosome automatically recognizes and binds to the sequence during the translation process. Therefore, the recombinant plasmid can theoretically express a single target protein rather than a fusion protein. The IL-15 gene is the last gene in series on the plasmid, so the expression of the IL-15 protein can indirectly prove the expression of the target proteins HA2 and FimA. At present, the prokaryotic expression plasmid has been constructed and the corresponding protein has been successfully induced for the next step of production. Study on the expression of target genes HA2 and fimA detected by specific antibodies.

[0082] Embodiment 5 According to Examples II to IV. 1. Successfully construct pVAX1-HA2-fimA / IL-15, pVAX1-HA2 / IL-15, pVAX1-HA2-fimA, pVAX1-HA2, and pVAX1-fimA / IL-15 co-expression plasmids. Using pIRES2-EGFP, Porphyromonas gingivalis W83 type, and cDNA reverse transcribed from SD rat total RNA as templates, pVAX1 plasmid as vector, and synthesized primers according to the reported gene sequence, amplified target genes HA2, fimA, IRES, and IL-15 in vitro, and constructed five recombinant expression plasmids pVAX1-HA2-fimA / IL-15, pVAX1-HA2 / IL-15, pVAX1-HA2-fimA, pVAX1-fimA / IL-15, and pVAX1-HA2. PCR, double enzyme digestion, and sequencing confirmed that the recombinant plasmids were correctly constructed. 2. The target genes HA2, fimA and IL-15 can be correctly expressed at the mRNA level in eukaryotic cells. Five eukaryotic plasmids were transiently transfected into 293T cells. After 24 hours, the total cell RNA was extracted and reverse transcribed into cDNA. RT-PCR was used to verify the expression of its mRNA level. At the same time, the product was sequenced and BLAST comparison was performed to confirm that the target gene can be correctly expressed at the mRNA level. 3. IL-15 can be expressed at the protein level in the recombinant plasmids pVAX1-HA2-fimA / IL-15, pVAX1-HA2 / IL-15 and pVAX1-fimA / IL-15. Recombinant plasmids pVAX1-HA2-fimA / IL-15, pVAX1-HA2 / IL-15 and pVAX1-fimA / IL-15 were transiently transfected into 293T cells. After 48 hours, ELISA was used to detect IL-15 protein and statistical analysis was performed. The results showed that the IL-15 gene can be expressed at the protein level.

[0083] Therefore, the applicant once again used the above results to prepare a DNA vaccine, which was used to construct an animal model of periodontitis and analyze and verify the immune effect and safety of the vaccine plasmid.

[0084] The applicant selected multiple female SD rats for control group experiments in step b to construct a periodontitis animal model. The vaccine was inoculated into multiple rats, and a periodontitis model was constructed for rats in each control group (blank group, periodontitis model group + pVAX1-HA2-fimA, periodontitis model + vector group, periodontitis model group). At the same time, the plasmid immune effects in multiple control groups were detected, and the periodic body weight, heart, liver, spleen, lung and kidney pathological staining of the control group rats were detected. It was found that the body weight of rats vaccinated with the vaccine prepared in Example 5 showed an increasing trend, and no pathological changes were found in the heart, liver, spleen, lung and kidney of the rats by visceral HE staining. At the same time, the alveolar bone absorption of the maxillary second molar of the vaccinated rats was less than that of the empty vector. This shows that vaccination has a significant effect on periodontitis and is safe.

[0085] Next, the applicant used ELISA to measure the specific antibody titers of the multiple control groups, such as Figure 6 As shown, it can be seen that the DNA vaccine immune stimulation significantly increased the specific IgG in rat serum and milk, so it can be seen that the vaccination was effective.

[0086] Embodiment 6 On the basis of Example 5, the applicant prepared functional buffalo milk using periodontitis DNA vaccine as an immunogen. In the preparation of the functional buffalo milk in step c, the buffalo was vaccinated four times. The second injection was given one week after the first injection, the third injection was given two weeks after the second injection, and the fourth injection was given one week after the third injection. During this period, the first injection site of the buffalo was a multiple-point subcutaneous injection on the back of the cow, the second and third injections were intramuscular injections, and the fourth was intravenous injections. After the injections, the original immune milk was obtained every seven days as a cycle, and the antibody titer was measured after each acquisition of the original immune milk.

[0087] like Figure 7 and Figure 8 As shown, the applicant used indirect ELISA to determine the average specific IgG level in the serum of the buffalo immunized experimental group and the control group for four weeks, and used indirect ELISA to determine the average specific IgG level in the colostrum and different dilutions after the buffalo gave birth. It can be seen that the serum specific IgG index was the highest between three and four weeks, and the average specific IgG level in the colostrum and different dilutions after delivery was the highest. At the same time, the applicant subsequently conducted a control group analysis on the levels of antimicrobial peptides in the immune system after immunization and found that the antimicrobial peptide level reached the highest level at the ninth week. The antimicrobial peptides have broad-spectrum antimicrobial activity, which inhibits the pathogenic bacteria of periodontitis to a certain extent. When used in combination with probiotics in subsequent cow's milk, it can also have a good nutritional effect.

[0088] The above experiments all indicate that the DNA vaccine prepared in Example 5 has an immune effect on periodontitis in buffalo milk, and can achieve a functional effect of preventing periodontitis.

[0089] Embodiment 7 On the basis of Example 6, the applicant obtained the original immune cow's milk and centrifuged it at 1000 rpm for 30-40 minutes, collected the precipitate and mixed it with regular cow's milk, the ratio of the original immune cow's milk to regular cow's milk was 1:50-100, and after mixing, it was sterilized by pasteurization to obtain drinkable immune functional cow's milk.

[0090] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

[0091] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A method for preparing anti-periodontitis functional milk, characterized in that: The following steps are involved: a. A DNA vaccine is prepared for the key antigens or virulence factors of the main pathogenic bacteria of periodontitis. The preparation method of the DNA vaccine includes ① construction of a gene vaccine of Porphyromonas gingivalis ② detection of the mRNA expression level of the recombinant plasmid in eukaryotic cells ③ detection of the IL-15 protein expression level of the recombinant plasmids pVAX1-HA2-fimA / IL-15, pVAX1-HA2 / IL-15, and pVAX1-fimA / IL-15 in eukaryotic cells; b. Use the prepared DNA vaccine to construct a periodontitis animal model, and analyze and verify the immune effect and safety of the vaccine plasmid; c. Preparation of functional buffalo milk using periodontitis DNA vaccine as immunogen; d. Analyze the effectiveness and applicability of preparing milk.

2. The method for preparing anti-periodontitis functional milk according to claim 1, characterized in that: In the step b, multiple female SD rats are selected for control group experiments to construct a periodontitis animal model, and a periodontitis model is constructed for the rats in each control group. The plasmid immune effects in multiple control groups are tested respectively, and the periodic body weight, heart, liver, spleen, lung and kidney pathological staining of the control group rats are tested respectively.

3. The method for preparing anti-periodontitis functional milk according to claim 1, characterized in that: The step b also includes using the DNA vaccine in step a to immunize rats, and then measuring and analyzing the IgG index in rat serum and rat colostrum.

4. The method for preparing the anti-periodontitis functional milk according to claim 1, characterized in that: In the preparation of the functional buffalo milk in step c, the buffalo is vaccinated four times in total, the second injection is injected one week after the first injection, the third injection is injected two weeks after the second injection, and the fourth injection is injected one week after the third injection. During this period, the first injection site of the buffalo is a subcutaneous multi-point injection on the back of the buffalo, the second and third injections are intramuscular injections, and the fourth is intravenous injection. After the injection, the original immune milk is obtained every seven days as a cycle, and the antibody titer is measured after each acquisition of the original immune milk.

5. The method for preparing anti-periodontitis functional milk according to claim 1, characterized in that: The original immune milk is obtained and centrifuged for 30-40 minutes in a centrifuge at 1000 rpm. The precipitate is collected and mixed with regular milk. The ratio of the original immune milk to the regular milk is 1:50-100. After mixing, the milk is sterilized by pasteurization to obtain drinkable immune functional milk.

6. The method for preparing anti-periodontitis functional milk according to claim 1, characterized in that: The construction of the Porphyromonas gingivalis gene vaccine in step a uses pIRES2-EGFP, Porphyromonas gingivalis W83 type, and cDNA reversely transcribed from SD rat total RNA as templates, pVAX1 plasmid as vector, and primers are synthesized according to the reported gene sequence to amplify the target genes HA2, fimA, IRES, and IL-15 in vitro to construct recombinant expression plasmids pVAX1-HA2-fimA / IL-15, pVAX1-HA2 / IL-15, pVAX1-HA2-fimA, pVAX1-fimA / IL-15, and pVAX1-HA2.

7. The method for preparing anti-periodontitis functional milk according to claim 1 or 6, characterized in that: The detection of the mRNA expression level of the recombinant plasmid in eukaryotic cells in step a is to use the recombinant plasmid constructed with the Porphyromonas gingivalis gene vaccine, transiently transfect 293T cells with liposomes, extract total cell RNA after 24 hours, reverse transcribe it into cDNA, and verify the expression of its mRNA level by RT-PCR.

8. The method for preparing anti-periodontitis functional milk according to claim 1 or 6, characterized in that: The detection of IL-15 protein expression levels in eukaryotic cells by the recombinant plasmids pVAX1-HA2-fimA / IL-15, pVAX1-HA2 / IL-15, and pVAX1-fimA / IL-15 in step a is based on the pVAX1-HA2-fimA / IL-15, pVAX1-HA2 / IL-15, and pVAX1-fimA / IL-15 plasmids constructed by the Porphyromonas gingivalis gene vaccine, transiently transfecting 293T cells, and detecting the expression of IL-15 protein levels after 24 hours.

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