A pH-responsive anti-caries vaccine

Abstract

The present invention belongs to the technical field of vaccines, and discloses a pH-responsive anti-caries vaccine, comprising ZIF-8 and a mutans streptococcus surface protein antigen. The pH-responsive anti-caries vaccine has the characteristics of being released in response to acidic pH, and has a significant anti-caries effect, and has broad application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of vaccines, and in particular relates to a pH-responsive anti-caries vaccine. Background Art

[0002] Streptococcus mutans (S. mutans) is one of the main cariogenic bacteria, and its main surface antigens are surface protein antigens (PAc) and glucosyltransferases (GTF). Therefore, in recent years, the anti-caries vaccine for Streptococcus mutans has gradually changed from the early vaccine types to DNA vaccines and antigen protein subunit vaccines.

[0003] However, the existing anti-caries vaccines still face the defects of weak immunogenicity and insufficient anti-caries effect. Therefore, the present invention hopes to propose a new vaccine product with more significant anti-caries effect. Summary of the invention

[0004] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention proposes a pH-responsive anti-caries vaccine. The pH-responsive anti-caries vaccine has the characteristics of being released in response to acidic pH, and has a significant anti-caries effect, and has broad application prospects.

[0005] The invention provides a pH-responsive anti-caries vaccine, comprising ZIF-8 and mutans streptococcus surface protein antigen (PAc).

[0006] ZIF-8 (zeolitic imidazolate framework) is a metal-organic framework material (MOFs) composed of Zn 2+ It is coordinated with 2-methylimidazole and has been used as an exoskeleton to encapsulate and protect various biological entities, such as proteins, nucleic acids and carbohydrates. The present invention proposes to use ZIF-8 as a vaccine adjuvant, which has a good electrostatic adsorption ability for surface protein antigens of Streptococcus mutans and can disintegrate and release antigens under acidic pH conditions, thereby inducing a high level of immune response, so it has outstanding potential and advantages for preparing vaccines.

[0007] Preferably, the mutans streptococcus surface protein antigen is a recombinant antigen, and the recombinant antigen consists of antigen fragment 1 shown in SEQ ID NO: 1, antigen fragment 2 shown in SEQ ID NO: 2, and a connecting peptide.

[0008] More preferably, the amino acid sequence of the connecting peptide is as shown in SEQ ID NO: 3. When the amino acid sequence of the connecting peptide is SEQ ID NO: 3, the amino acid sequence of the mutans streptococcus surface protein antigen is as shown in SEQ ID NO: 4.

[0009] Preferably, the mass ratio of ZIF-8 to mutans streptococcus surface protein antigen in the pH-responsive anti-caries vaccine is (5-40):1.

[0010] More preferably, the mass ratio of ZIF-8 to mutans streptococcus surface protein antigen in the pH-responsive anti-caries vaccine is (5-20):1.

[0011] Further preferably, the mass ratio of ZIF-8 to mutans streptococcus surface protein antigen in the pH-responsive anti-caries vaccine is 10:1.

[0012] Preferably, the preparation method of ZIF-8 is: placing zinc nitrate hexahydrate and 2-methylimidazole in an alcohol solution for reaction, collecting the precipitate, washing with alcohol, and drying.

[0013] More preferably, the alcohol solution is a methanol solution.

[0014] Preferably, the pH-responsive anti-caries vaccine further comprises at least one of a preservative, an emulsifier, a stabilizer or a diluent.

[0015] Preferably, the pH-responsive anti-caries vaccine comprises a subcutaneous injection vaccine or a nasal drop vaccine.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0017] The present invention combines ZIF-8 and PAc protein as an anti-caries vaccine, exhibits the characteristics of high loading amount and release in response to acidic pH changes, and has a significant anti-caries effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The electrophoresis results of each stage in the process of recombining the surface protein PAc of Streptococcus mutans. A is the PCR amplification result of the pac gene; B is the bacterial solution PCR verification test, No. 1-8 are different monoclones, and No. 3 is a negative clone; C is the pac sequencing verification; D is the electrophoresis results of Marker, 1: whole protein, 2: impurity protein, 3: 10mM imidazole, 4: 25mM imidazole, 5: 50mM imidazole, 6: 100mM imidazole, 7: 500mM imidazole; E is Marker, 1: HisA-PAc fusion protein, 2-3: PAc protein electrophoresis results after HisA tag protein was cleaved by enterokinase and purified by column.

[0019] Figure 2The characterization results of ZIF-8 and ZIF-8@PAc. A and B are the particle sizes of ZIF-8 nanomaterials and ZIF-8@PAc measured by SEM, respectively; CD is the internal situation of ZIF-8 measured by TEM; EF is the internal situation of ZIF-8@PAc measured by TEM; H is the adsorption degree of PAc protein and ZIF-8 nanomaterials at different mass ratios; I is the release capacity of ZIF-8@PAc at different pH.

[0020] Figure 3 The results are the antibody titer, bacterial content change and organ index of rats. Among them, A is the IgG antibody titer; B is the IgA antibody titer; C is the statistical chart of bacterial content change; DH are the statistical charts of the heart, liver, spleen, lung and kidney organ indexes.

[0021] Figure 4 The anti-caries results of PBS, PBS+PAc, and ZIF-8@PAc on rats. DETAILED DESCRIPTION

[0022] In order to make those skilled in the art more clearly understand the technical solution of the present invention, the following examples are listed for illustration. It should be pointed out that the following examples are only preferred embodiments of the present invention and do not constitute a limitation on the protection scope of the present invention. Any modification, substitution, and combination made without violating the spirit and principle of the present invention are included in the protection scope of the present invention.

[0023] Unless otherwise specified, the raw materials, reagents or devices used in the following examples can be obtained from conventional commercial sources or by existing known methods.

[0024] Example 1: Synthesis of ZIF-8 nanomaterials

[0025] 1. Dissolve zinc nitrate hexahydrate / Zn(NO3)2·6H2O (1.195 g, 4 mmol) and 2-methylimidazole / 2-MIM (2.463 g, 30 mmol) in 50 mL of methanol solution respectively.

[0026] 2. Add the methanol solution containing zinc nitrate hexahydrate dropwise to the methanol solution of 2-methylimidazole under stirring conditions (1000 rpm), and stir the reaction at room temperature for 1 hour. During the reaction, the formation of a white precipitate can be observed.

[0027] 3. After the reaction, the mixture was evenly divided into four tubes for centrifugation (8000 rpm, 10 min) to collect the white precipitate, which was washed three times with 50 mL of methanol and dried in an oven at 60°C overnight to obtain a white solid ZIF-8.

[0028] Example 2: Preparation of mutans streptococcus surface protein antigen (PAc)

[0029] This embodiment provides a mutans streptococcus surface protein antigen (PAc), which is composed of antigen fragment 1 shown in SEQ ID NO: 1, antigen fragment 2 shown in SEQ ID NO: 2, and a connecting peptide shown in SEQ ID NO: 3. The amino acid sequence of the mutans streptococcus surface protein antigen finally obtained is shown in SEQ ID NO: 4.

[0030] VKTAEEAVQKETEIKEDYTKQAEDIKKTTDQYKSDVAAHEAEVAKIKAKNQATKEQYEKDMAAHKAEVERINAANAASKTAYEAKLAQYQADLAAVQKTNAANQAAYQKALAAYQAELKRVQEANAAAKAAYDTAVAANNAKNTEIAAANEEIRKRNATAKAEYETKLAQYQA ELKRVQEANAANEADYQAKLTAYQTELARVQKANADAKAAYEAAVAANNAKNAALTAENTAIKQRNENAKATYEAALKQYEADLAAVKKANAANEADYQAKLTAYQTELARVQKANADAKAAYEAAVAANNAANAALTAENTAIKKRNADAKADYEAKLAKYQADLAKY(SEQ ID NO:1);

[0031] VNVPKVTKEKPTPPVKPTAPTKPTYETEKPLKPAPVAPNYEKEPTPPTRTPDQAEPNKPTPPTYETEKPLEPAPVEPSYEAEPTPPTRTPDQAEPNKPTPPTYETEKPLEPAPVEPSYEAEPTPPTPTPDQPEPNKPVEPTYEVIPTPPTDPVYQDLPTPPSVPTVHF(SEQ ID NO:2);

[0032] STSTGSTSTG (SEQ ID NO: 3);

[0033] VKTAEEAVQKETEIKEDYTKQAEDIKKTTDQYKSDVAAHEAEVAKIKAKNQATKEQYEKDMAAHKAEVERINAANAASKTAYEAKLAQYQADLAAVQKTNAANQAAYQKALAAYQAELKRVQEANAAAKAA YDTAVAANNAKNTEIAAANEEIRKRNATAKAEYETKLAQYQAELKRVQEANAANEADYQAKLTAYQTELARVQKANADAKAAYEAAVAANNAKNAALTAENTAIKQRNENAKATYEAALKQYEADLAAVKKA NAANEADYQAKLTAYQTELARVQKANADAKAAYEAAVAANNAANAALTAENTAIKKRNADAKADYEAKLAKYQADLAKYSTSTGSTSTGVNVPKVTKEKPTPPVKPTAPTKPTYETEKPLKPAPVAPNYEK EPTPPTRTPDQAEPNKPTPPTYETEKPLEPAPVEPSYEAEPTPPTRTPDQAEPNKPTPPTYETEKPLEPAPVEPSYEAEPTPPTPTPDQPEPNKPVEPTYEVIPTPPTDPVYQDLPTPPSVPTVHF(SEQID NO:4).

[0034] The preparation method of the mutans streptococcus surface protein antigen (PAc) is as follows:

[0035] (1) The A region + P region gene sequence of the pac gene of Streptococcus mutans SM UA159 strain was obtained according to the GenBank database, and the nucleotide sequence of TCCACCTCCACCGGATCCACCTCCACCGGA was designed between the A region and the P region, and sent to Sangon Biotech (Shanghai) Co., Ltd. for synthesis (1563 bp). It was then ligated with pGEX-4T-1 to obtain the pGEX-4T-pac recombinant vector.

[0036] (2) Using the pGEX-4T-pac recombinant vector as a template, the corresponding pac upstream and downstream primers (as shown in SEQ ID NO: 5-6) were designed to amplify the target gene. The PCR reaction system was: Prime STARHS (Premix): 25 μL, upstream and downstream primers 2 μL each, template 2 μL, ddH2O 19 μL. The PCR reaction conditions were: 95°C pre-denaturation for 5 min; 94°C heat denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 90 s, a total of 30 cycles; and finally 72°C extension for 5 min.

[0037] Upstream primer: 5′-CGATGGGGATCCGAGCTCGTTAAAACACCTGAAGAAGC-3′ (SEQ ID NO: 5);

[0038] Downstream primer: 5'-TCGAATTCCCATATGTTAGAAATGAACAGTTGGATCAG-3' (SEQ ID NO: 6).

[0039] (3) The target gene obtained by PCR amplification was recovered by gel extraction, and then the empty plasmid pBAD / HisA was double-digested with restriction endonucleases SacⅠ and KpnⅠ (37°C, 30 min). The PCR product and double-digested product were recovered using a gel extraction kit, respectively, and the concentration was measured. The purified PCR amplified target fragment and the linear vector pBAD / HisA were connected using a seamless cloning kit, and the molar ratio of the target fragment to the vector was between 2:1-3:1 (reacted at 50°C for 20 min). The ligation product (i.e., the recombinant plasmid pBAD / HisA-pac) was transformed into E. coli Top10 competent cells, spread on LB plates containing 100 μg / mL ampicillin, and cultured at 37°C overnight. Eight single colonies were randomly picked from the LB plate and placed in LB liquid culture medium (containing 100 μg / mL ampicillin) for culture for a certain period of time. The bacterial liquid was taken for PCR identification, and the positive clones were sent to Sangon Biotechnology (Shanghai) Co., Ltd. for sequencing. The sequencing results were compared with the previously synthesized pac by BLAST.

[0040] (4) The bacterial suspension containing the recombinant plasmid pBAD / HisA-pac that was correctly identified by BLAST was inoculated into 10 mL of LB liquid medium (containing 100 μg / mL ampicillin) and cultured overnight at 37°C at 200 r / min. The next day, the culture was expanded according to the inoculum size of 2%-5% and cultured in a shaking incubator at 37°C at 200 r / min until the OD 600When the concentration of arabinose was 0.6-0.8, arabinose was added to a final concentration of 0.04%, and the expression was induced at 37°C and 200r / min for 4-6h. Centrifuge at 4°C and 4000r / min for 10min, discard the supernatant, and collect the bacteria. Add an appropriate amount of PBS to resuspend the bacteria, freeze and thaw repeatedly at liquid nitrogen and 37°C for 3 times, and ultrasonically disrupt for 30min. Centrifuge at 4°C and 10000r / min for 30min, collect the supernatant for SDS-PAGE analysis and pass through Ni-NTA affinity chromatography purification column. The fusion protein HisA-PAc bound to the Ni-NTA affinity chromatography purification column was eluted with different concentrations of imidazole (10mM, 25mM, 50mM, 100mM, 500mM), concentrated by ultrafiltration tube, and HisA tag was cut off with enterokinase (enzyme cleavage overnight), and then further passed through Ni-NTA affinity chromatography purification column to obtain the target protein PAc.

[0041] Figure 1 The figure shows the electrophoresis of each stage in the process of recombinant mutans streptococcus surface protein PAc. A is the PCR amplification result of pac gene; B is the bacterial solution PCR verification test, No. 1-8 are different monoclones, and No. 3 is a negative clone; C is the pac sequencing verification; D is the electrophoresis results of Marker, 1: whole protein, 2: impurity protein, 3: 10mM imidazole, 4: 25mM imidazole, 5: 50mM imidazole, 6: 100mM imidazole, 7: 500mM imidazole; E is Marker, 1: HisA-PAc fusion protein, 2-3: PAc protein electrophoresis results after HisA tag protein was cleaved by enterokinase and purified by column.

[0042] Example 3: pH-responsive anti-caries vaccine

[0043] The present embodiment provides a pH-responsive anti-caries vaccine, comprising ZIF-8 and a mutans streptococcus surface protein antigen, and the preparation method thereof is as follows: the ZIF-8 nanomaterial in Example 1 and the mutans streptococcus surface protein antigen (PAc) in Example 2 are added to a PBS buffer solution, and the ZIF-8@PAc is obtained by combining them through electrostatic adsorption.

[0044] The particle sizes of ZIF-8 nanomaterials and ZIF-8@PAc were determined by SEM. The test results are as follows: Figure 2 As shown in A and B; the internal conditions of ZIF-8 and ZIF-8@PAc were determined by TEM. The detection results of ZIF-8 nanomaterials are shown in Figure 2 As shown in Figure CD, the detection results of ZIF-8@PAc are as follows Figure 2 The potentials of ZIF-8, PAc and ZIF-8@PAc were measured by a laser nanoparticle size analyzer. Figure 2 As shown in G.

[0045] In order to obtain the optimal adsorption conditions of PAc protein and ZIF-8 nanomaterials, the concentration of ZIF-8 nanomaterials was changed by controlling the concentration of PAc protein. The mass ratios of ZIF-8 nanomaterials and PAc were 0:1, 0.5:1, 1:1, 2:1, 5:1, 10:1, 20:1, 40:1 (n=3), respectively. The two were combined through the principle of electrostatic adsorption, at 25°C, shaken for 60 minutes, centrifuged at 8000rpm for 2 minutes, and the residual protein concentration in the supernatant was measured to obtain the adsorption ratio of PAc protein and ZIF-8 nanomaterials. Figure 2 It can be seen from Figure 3 that when the mass ratio of ZIF-8 to PAc is about (5-40):1, the two have good adsorption and binding effects.

[0046] ZIF-8@PAc was prepared under the optimal adsorption conditions (i.e., the mass ratio of ZIF-8 to PAc was 10:1), and ZIF-8@PAc was placed in PBS buffer for PAc protein release. Three different pH conditions were set, namely pH 7.2, pH 6.0, and pH 5.0. Samples were taken at time periods of 1h, 2h, 3h, 6h, 9h, 12h, and 24h, and the supernatant was centrifuged to determine the residual protein concentration and calculate the release rate (n=3). Figure 2 It can be seen from Figure I that ZIF-8@PAc has stronger release ability in an acidic environment and is pH responsive, which is more conducive to the vaccine to function in an acidic cell environment.

[0047] Example 4: Rat dental caries experiment

[0048] Female SD rats aged 18 days were used as experimental animals. They were initially fed with Keyes 2000diet dental caries diet without antibiotics. On the 20th day, chloramphenicol, ampicillin and carbenicillin (all 1g / kg) were added to the dental caries diet, and 200μg / mL penicillin and 1500μg / mL streptomycin were added to the drinking water for 3 consecutive days. Immunization began on the 23rd day, and three groups (PBS, PBS+PAc, ZIF-8@PAc) (n=5) were set up. In the PBS group, each rat was immunized with 100μL PBS, while in the PBS+PAc and ZIF-8@PAc groups, each rat was immunized with 50μg PAc, and the total volume was diluted to 100μL with PBS. Subcutaneous multi-point immunization was used for a total of 3 immunizations, each with an interval of 14 days. Mutans Streptococcus (UA159) (1×10 9CFU / mL, 3 times a day, 200μL each time, 30min interval). Every week after the first immunization, the rats were weighed and 10μL of saliva was taken and diluted 100 times to apply THYE solid culture medium plates, and the number of colonies was counted, which lasted for 5 weeks. 14 days after the third immunization, saliva and serum were collected to determine the antibody titer, the rats were anesthetized and killed, the heart, liver, spleen, lungs and kidneys were taken out and weighed, the organ index was calculated, and the upper and lower jaws were separated, and the enamel and dentin were scored and counted. Through the above experiments, the anti-caries effect of ZIF-8@PAc protein vaccine on rats can be determined.

[0049] No rats immunized with ZIF-8@PAc died during the experiment. The 3D-H organ index in the figure shows that ZIF-8@PAc has good safety. Figure 3 Changes in AC antibody titer and bacterial content in Figure 4 The results of rat caries showed that the caries prevention effect of ZIF-8@PAc was significantly stronger than that of PAc antigen alone.

[0050] The embodiments of the present application are described in detail above in conjunction with the accompanying drawings, but the present application is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present application. In addition, the embodiments of the present application and the features in the embodiments can be combined with each other without conflict.

Claims

1. A pH-responsive anti-caries vaccine, characterized in that: It includes ZIF-8 and mutans streptococcus surface protein antigen bound by electrostatic adsorption; The preparation method of ZIF-8 is as follows: zinc nitrate hexahydrate and 2-methylimidazole are placed in an alcohol solution for reaction, and the precipitate is collected, washed with alcohol, and dried; The mutans streptococcus surface protein antigen is a recombinant antigen, which consists of an antigen fragment 1 shown in SEQ ID NO: 1, an antigen fragment 2 shown in SEQ ID NO: 2 and a connecting peptide, the amino acid sequence of the connecting peptide is shown in SEQ ID NO: 3, and the amino acid sequence of the mutans streptococcus surface protein antigen is shown in SEQ ID NO: 4; the mass ratio of ZIF-8 to the mutans streptococcus surface protein antigen in the pH-responsive anti-caries vaccine is (5-40): 1; the pH-responsive anti-caries vaccine is a subcutaneous injection vaccine or a nasal drop vaccine.

2. The pH-responsive anti-caries vaccine according to claim 1, characterized in that The mass ratio of ZIF-8 to mutans streptococcus surface protein antigen in the pH-responsive anti-caries vaccine is (5-20):

1.

3. The pH-responsive anti-caries vaccine according to claim 2, characterized in that The mass ratio of ZIF-8 to mutans streptococcus surface protein antigen in the pH-responsive anti-caries vaccine is 10:

1.

4. The pH-responsive anti-caries vaccine according to claim 1, characterized in that The alcohol solution is a methanol solution.

5. The pH-responsive anti-caries vaccine according to claim 1, characterized in that The pH-responsive anti-caries vaccine also includes a preservative.

6. The pH-responsive anti-caries vaccine according to claim 1, characterized in that The pH-responsive anti-caries vaccine also includes an emulsifier.

7. The pH-responsive anti-caries vaccine according to claim 1, characterized in that The pH-responsive anti-caries vaccine also includes a stabilizer.

8. The pH-responsive anti-caries vaccine according to claim 1, characterized in that The pH-responsive anti-caries vaccine also includes a diluent.

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