Silicon-based nano vaccine for targeting M cells as well as preparation method and application of silicon-based nano vaccine

Abstract

The invention relates to a silicon-based nano vaccine for targeting M cells as well as a preparation method and application of the silicon-based nano vaccine. The invention relates to a preparation method of a silicon-based nano vaccine for targeting M cells. The preparation method comprises the following steps: (1) preparing mesoporous silica; and (2) sequentially carrying out amination modification, responsive material modification and brucella surface antigen OMP31-Col modification on the mesoporous silica, so as to obtain the silicon-based nano vaccine for targeting M cells. According to the silicon-based nano vaccine for targeting M cells as well as the preparation method and the application of the silicon-based nano vaccine, a ligand Col sequence capable of targeting M cells is connected to a Brucella surface antigen OMP31 by using a linker, efficient packaging and delivery are realized through a macroporous / mesoporous silica delivery system, and the stability of the antigen and the target cell uptake efficiency are ensured; on the basis, a responsive group of a natural macromolecule is introduced, so that the expression level of the antigen is enhanced.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of nanocarrier vaccines, and particularly relates to a silicon-based nanovaccine targeting M cells and a preparation method and application thereof. BACKGROUND

[0002] Brucellosis is a disease caused by Brucella, which is a class A infectious disease listed by the World Organization for Animal Health and a class B infectious disease in China. The disease is globally distributed. Since the late 1980s, brucellosis has rapidly spread. It can not only infect various domestic animals and wild mammals, causing significant economic losses in the livestock industry, but also infect humans through contact transmission, digestive tract transmission and other routes, causing typical symptoms such as undulant fever, excessive sweating and joint pain, which seriously affects public health safety.

[0003] There are several vaccines for the prevention and control of brucellosis, such as attenuated live vaccines, vector vaccines, subunit vaccines, DNA vaccines, etc. At present, attenuated live vaccines are the most effective vaccines for controlling animal brucellosis, which are cheap and effective, and can induce immune responses through humoral and cell-mediated responses. However, attenuated live vaccines for brucellosis have some defects, such as antibiotic resistance, interference with serological diagnostic tests, and residual toxicity to animals and humans. Subunit vaccines are promising candidates due to their safety, clear non-infectivity, inability to revert to virulent strains, non-viability and operability different from attenuated vaccines. However, the poor antigenicity, instability and short half-life of the vaccine seriously limit its use, so the vaccine host immune response is generally enhanced by adding adjuvants, immunomodulators, antigen delivery systems and other ligands.

[0004] In addition, traditional vaccines cannot control the release amount of the vaccine and recognize the target tissue, resulting in the non-targeted distribution of the vaccine in the body and the passive acceptance of a certain amount of drug by cells due to their different positions.

[0005] Therefore, the application provides a silicon-based nanovaccine targeting M cells and a preparation method and application thereof. The method combines mesoporous silica, natural polymer materials and modified brucella antigens that can target M cells to improve the immunogenicity of the antigens and can be used for immune prevention and treatment of brucellosis. SUMMARY

[0006] The application aims to provide a preparation method of a silicon-based nanovaccine targeting M cells. The method combines silicon-based nanomaterials and brucella surface antigens, and then modifies a responsive material to prepare a silicon-based nanovaccine targeting M cells.

[0007] To achieve the above-mentioned purposes, the technical solutions adopted are as follows:

[0008] The application discloses a preparation method of a silicon-based nano vaccine targeting M cells, and comprises the following steps.

[0009] (1) preparing mesoporous silica;

[0010] (2) sequentially performing amino modification, responsive material modification and Brucella surface antigen OMP31-Col modification on the mesoporous silica to obtain the silicon-based nano vaccine targeting M cells.

[0011] Further, in the step (1), the mesoporous silica is spherical mesoporous silica.

[0012] Further, in the step (1), the spherical mesoporous silica is prepared by the following method: dissolving CTAT in water containing TEA, stirring at 75-85 DEG C for 1-2 h, then adding TEOS, reacting at 75-85 DEG C for 2-3 h, centrifuging and washing, vacuum drying, removing the template, and obtaining the spherical mesoporous silica.

[0013] Further, the Brucella surface antigen OMP31-Col is obtained by a prokaryotic expression technology, and the operation is as follows:

[0014] Plasmid construction: performing double enzyme digestion on the target gene OMP31-Col and the expression vector pET-22b(+) respectively, purifying the enzyme digestion products, then performing ligation by using a seamless cloning technology, and constructing a recombinant plasmid pET-22b(+)-OMP31-Col;

[0015] OMP31-Col protein expression: transforming the recombinant plasmid pET-22b(+)-OMP31-Col into BL21 (DE3) competent cells, inducing expression by IPTG, collecting bacterial bodies and lysing, and obtaining a lysate containing the target protein;

[0016] Purification: purifying the lysate containing the target protein by using an affinity chromatography method, and obtaining high-purity OMP31-Col protein, namely the Brucella surface antigen OMP31-Col.

[0017] Further, in the plasmid construction, the volume ratio of the target gene to the vector to MIX in a ligation system is 1:1:2.

[0018] The concentration of the inducer IPTG is 1 mmol / L, and the dosage of the inducer added is 1 ‰.

[0019] Further, in the step (2), the modification process is as follows:

[0020] dispersing the mesoporous silica in a mixed solution of ethanol and 3-aminopropyl triethoxysilane, stirring at 75-85 DEG C for 20-28h, centrifugal washing, drying, and collecting product 1;

[0021] dispersing the responsive material carboxymethyl chitosan in an aqueous solution containing EDC and NHS, adding the product 1 after stirring and activation, stirring and reacting for 20-28h, centrifugal washing, drying, and collecting product 2;

[0022] adding the Brucella surface antigen OMP31-Col to a PBS solution containing EDC and NHS, adding the product 2 after stirring and activation, stirring for 20-28h, and centrifugal washing.

[0023] Furthermore, the volume ratio of the ethanol and 3-aminopropyl triethoxysilane is 50-100:1.

[0024] The mass ratio of the product 1 to the responsive material carboxymethyl chitosan is 2.5-3.5:2.

[0025] The mass ratio of the product 2 to the Brucella surface antigen OMP31-Col is 3-5:1.

[0026] The mass ratio of EDC to NHS in the aqueous solution is 0.20-0.30:0.12-0.18.

[0027] The mass ratio of EDC to NHS in the PBS buffer is 170-190:140-160, and the pH of the PBS buffer is 6-8.

[0028] Furthermore, the volume ratio of the ethanol and 3-aminopropyl triethoxysilane is 50:1.

[0029] The mass ratio of the product 1 to the responsive material carboxymethyl chitosan is 3:2.

[0030] The mass ratio of the product 2 to the Brucella surface antigen OMP31-Col is 4:1.

[0031] The mass ratio of EDC to NHS in the aqueous solution is 0.25:0.15.

[0032] The mass ratio of EDC to NHS in the PBS buffer is 180:150.

[0033] Another object of the present application is to provide a silicon-based nano-vaccine targeting M cells, which is prepared by the above method, and can be orally taken, so that the antigen is not destroyed by digestive enzymes, the delivery efficiency is improved, the intestinal mucosal immunity is efficiently initiated, and multiple protections are realized.

[0034] The application also has an application purpose of providing the application of the above-mentioned silicon-based nanovaccine in drugs for immunoprophylaxis and treatment of brucellosis bacteremia. The carrier vaccine has the properties of high utilization of outer membrane protein antigen, sensitive response to the environment and high biocompatibility, has the effect of target recognition of ligands on M cells, can start the immune response of the body faster, and can slowly release protein antigens, thereby providing an effective way for the treatment effect of brucellosis prevention and the bacteremia stage, i.e. the period when Brucella proliferates in blood or lymph.

[0035] Compared with the prior art, the application has the beneficial effects that:

[0036] The silicon-based nanovaccine targeting M cells, the preparation method and the application thereof are a silicon-based nanovaccine for immunoprophylaxis and treatment of the bacteremia stage of brucellosis targeting M cells. Through systematic optimization of subunit vaccine design, the introduction of a Col targeting sequence, the construction of a Brucella surface antigen targeting M cells, and the modification of a natural high molecular responsive material, the expression level of the antigen is enhanced. Through a mesoporous silica delivery system, efficient loading and delivery are achieved, the stability of the antigen and the uptake efficiency of target cells are ensured, and a silicon-based nanovaccine with a targeting effect is obtained. The specific advantages are as follows:

[0037] 1. In the technical scheme of the application, the nanomaterial delivery system is used to solve the technical problem that traditional vaccines cannot control the release amount of the vaccine and recognize the target tissue. This system can protect the wrapped antigen protein from being enzymatically degraded in the body and can maintain slow and continuous release, thereby helping to improve the immunostimulatory property of the vaccine. The antigen can be wrapped inside the nanocarrier or modified on the surface of the material. Further modification with a targeting molecule such as a ligand of a cell surface receptor can deliver the vaccine to specific cell populations, increase the delivery efficiency of the nanoparticles to antigen-presenting cells, and thereby induce natural and acquired immune responses.

[0038] 2. In the technical scheme of the application, the prepared nanodelivery system has a large-pore spherical structure, can load part of the antigen, reduces the waste of protein, maintains the activity of the protein, is suitable for stable storage of protein antigens, and can be excreted out of the body through the kidney after a certain period of time, has high biocompatibility, controllable particle size and pore structure, can be synthesized in batches, and is easy to industrialize.

[0039] 3. In the technical scheme of the application, M cells have specific recognition ability for particulate antigens, can specifically bind to Col on the surface of the antigen through the surface receptor C5aR, make the antigen efficiently enriched in the Peyer's collection lymph node area, thereby more efficiently and quickly delivering the antigen to macrophages or dendritic cells, and inducing a stronger immune response of the body.

[0040] 4. In the technical scheme of the present application, by modifying the natural high molecular responsive material carboxymethyl chitosan, the antigenicity of the protein antigen can be improved, and a stronger specific immune response can be induced, and it is a pH-sensitive polymer that can be released in response to an acidic environment.

[0041] 5. In the technical scheme of the present application, the modification of carboxymethyl chitosan and Brucella surface antigen has high universality, and the amidation reaction of carboxyl and amino is used in the modification process, the catalyst is used to make the reaction efficient and fast, and the connection between the materials will not change the activity of the protein. BRIEF DESCRIPTION OF DRAWINGS

[0042] Figure 1 SEM image of the silicon-based nanovaccine in the modification process in Example 1;

[0043] Figure 2 TEM image of the silicon-based nanovaccine in the modification process in Example 1;

[0044] Figure 3 PCR amplification result and bacterial liquid PCR identification result of Omp31-Col gene in Example 1;

[0045] Figure 4 SDS-PAGE diagram of Omp31-Col recombinant protein induced expression, Western Blot analysis and affinity chromatography purification in Example 1;

[0046] Figure 5 Surface potential change of the silicon-based nanovaccine in the modification process in Example 1;

[0047] Figure 6 Thermogravimetric change of the silicon-based nanovaccine in the modification process in Example 1;

[0048] Figure 7 Infrared spectrum analysis of the silicon-based nanovaccine in the modification process in Example 1;

[0049] Figure 8 SDS-PAGE gel diagram of the silicon-based nanovaccine in the modification process in Example 1 after connecting OMP31-Col protein;

[0050] Figure 9 Blood compatibility evaluation result diagram of the silicon-based nanovaccine in the modification process in Example 1;

[0051] Figure 10 Toxicity result evaluation diagram of different carrier concentrations of the silicon-based nanovaccine before and after modification in Example 1. DETAILED DESCRIPTION

[0052] In order to further illustrate the application of the silicon-based nanovaccine targeting M cells and the preparation method and application thereof, and achieve the intended application purposes, the silicon-based nanovaccine targeting M cells and the preparation method and application thereof according to the application, the specific implementation, structure, features and functions thereof are described in detail as follows. In the following description, different 'an embodiment' or 'embodiments' do not necessarily refer to the same embodiment. In addition, the specific features, structures or characteristics in one or more embodiments can be combined in any suitable form.

[0053] The application of the silicon-based nanovaccine targeting M cells and the preparation method and application thereof will be further described in detail as follows by combining specific embodiments:

[0054] The application first prepares mesoporous silica spheres by a soft template method, prepares a Brucella surface antigen connected with a Col targeting ligand by prokaryotic expression technology, and then connects the Brucella surface antigen with natural high molecular carboxymethyl chitosan by amidation reaction to prepare a silicon-based nanovaccine.

[0055] The technical scheme of the application is as follows:

[0056] The preparation method of the silicon-based nanovaccine targeting M cells comprises the following steps:

[0057] (1) preparing macroporous mesoporous silica;

[0058] (2) sequentially performing amino modification, responsive material modification and modification of Brucella surface antigen OMP31-Col on the mesoporous silica to obtain the silicon-based nanovaccine targeting M cells.

[0059] Preferably, in the step (1), the mesoporous silica is spherical mesoporous silica.

[0060] Further preferably, in the step (1), the preparation method of the spherical mesoporous silica is as follows: CTAT is dissolved in water containing TEA, after stirring at 75-85 DEG C for 1-2 h, TEOS is added, and after reaction at 75-85 DEG C for 2-3 h, centrifugal washing and vacuum drying are performed to remove the template to obtain the spherical mesoporous silica.

[0061] Preferably, the Brucella surface antigen OMP31-Col is obtained by prokaryotic expression technology, and the operation is as follows:

[0062] Plasmid construction: the target gene OMP31-Col and the expression vector pET-22b(+) are double-digested respectively, the digested products are purified, and then the seamless cloning technology is used for ligation to construct the recombinant plasmid pET-22b(+)-OMP31-Col; the reaction product is transformed into TOP10 competent cells, positive clones are screened, plasmid extraction and sequencing verification are performed, and the Brucella surface antigen OMP31-Col is obtained.

[0063] Expression of OMP31-Col protein: the recombinant plasmid pET-22b(+)-OMP31-Col is transformed into BL21(DE3) competent cells, expression is induced by IPTG, and the bacterial cells are collected and lysed to obtain a lysate containing the target protein;

[0064] Purification: the lysate containing the target protein is purified by affinity chromatography, and high-purity OMP31-Col protein, i.e., the Brucella surface antigen OMP31-Col, is obtained through the steps of bacteria removal by filtration, nickel column equilibration, nickel column and protein binding, and protein elution.

[0065] Further preferably, in the plasmid construction, the volume ratio of the target gene to the vector to MIX is 1:1:2.

[0066] The concentration of the inducer IPTG is 1 mmol / L, and the dosage is 1 ‰.

[0067] Preferably, in step (2), the modification process is:

[0068] Disperse mesoporous silica in a mixed solution of ethanol and 3-aminopropyl triethoxysilane, stir at 75-85°C for 20-28h, centrifuge and wash, dry, and collect product 1;

[0069] Disperse the responsive material carboxymethyl chitosan in an aqueous solution containing EDC and NHS, add product 1 after stirring and activation, stir and react for 20-28h, centrifuge and wash, dry, and collect product 2;

[0070] Add the Brucella surface antigen OMP31-Col to a PBS solution containing EDC and NHS, stir and activate, then add product 2, and stir for 20-28h.

[0071] Further preferably, the volume ratio of ethanol to 3-aminopropyl triethoxysilane is 50-100:1.

[0072] The mass ratio of product 1 to the responsive material carboxymethyl chitosan is 2.5-3.5:2.

[0073] The mass ratio of product 2 to the Brucella surface antigen OMP31-Col is 3-5:1.

[0074] The mass ratio of EDC to NHS in the aqueous solution is 0.20-0.30:0.12-0.18.

[0075] The mass ratio of EDC and NHS in the PBS buffer is 170-190: 140-160; the pH of the PBS buffer is 6-8.

[0076] Further preferably, the volume ratio of ethanol and 3-aminopropyl triethoxysilane is 50:1.

[0077] The mass ratio of product 1 and carboxymethyl chitosan is 3:2;

[0078] The mass ratio of product 2 and Brucella surface antigen OMP31-Col is 4:1;

[0079] The mass ratio of EDC and NHS in the aqueous solution is 0.25g: 0.15g.

[0080] The mass ratio of EDC and NHS in the PBS buffer is 180:150.

[0081] Example 1.

[0082] The specific operation steps are as follows:

[0083] (1) Preparation of mesoporous silica nanospheres

[0084] Dissolve 0.96g CTAT in 50ml water containing 120μl TEA, stir at 80℃ for 1.5h, then quickly add 7.8ml TEOS, react at 80℃ for 2h, then wash with water and ethanol for three times respectively, vacuum dry, and remove the template by high temperature calcination in a muffle furnace to obtain mesoporous silica nanospheres (MSN).

[0085] (2) Preparation of Brucella surface antigen OMP31-Col targeting M cells by prokaryotic expression technology

[0086] The Brucella surface antigen OMP31-Col targeting M cells is purified by prokaryotic expression technology, and the process is as follows:

[0087] ① Construction of pET-22b(+)-OMP31-Col recombinant expression vector

[0088] The OMP31-Col gene synthesized by Shengong Company was cloned, and the gel was recovered and purified using a DNA recovery kit. The purified target gene and the vector pET-22b(+) plasmid were double-digested with Nde I and Xho I restriction enzymes, and were placed in a 37°C water bath for 1 h. The enzyme-digested products were identified using 1% agarose gel, and the target gene and the vector were ligated. The ligation system included 5 μl of the target gene and 5 μl of the vector, and 10 μl of MIX. The two recombinant plasmids were transformed into the prepared competent cells TOP10, and were coated on LB solid medium containing ampicillin and were cultured overnight in a 37°C incubator. Positive single colonies were picked from the plate after the culture and were inoculated into LB liquid medium (Amp+) at 37°C and 200 rpm, and were sent for sequencing.

[0089] 2. Induction expression of the recombinant OMP31-Col protein

[0090] The recombinant expression plasmid pET-22b(+)-OMP31-Col with correct gene sequence verified by sequencing was transformed into E. coli BL21(DE3) competent cells, which were coated on LB solid medium containing Amp and were cultured overnight. Positive single colonies were picked, and the bacterial liquid was identified by PCR. The recombinant strain with correct identification was named pET-22b(+)-OMP31-Col-BL21(DE3), which was inoculated into 20 mL of liquid LB medium containing Amp+ and was cultured at 37°C and 180 rpm until the OD600 reached 0.6. 1‰ of inducer IPTG was added in proportion, so that the final concentration reached 1 mmol / L. The control sample was not added with inducer, and was continued to be cultured at 37°C and 200 rpm for 6 h. 600nm = 0.6. 1‰ of inducer IPTG was added in proportion, so that the final concentration reached 1 mmol / L. The control sample was not added with inducer, and was continued to be cultured at 37°C and 200 rpm for 6 h.

[0091] 3. Solubility identification of the recombinant protein

[0092] The pET-22b (+) -OMP31-Col-BL21 (DE3) was expanded and induced with IPTG. After completion of induction, the bacteria were collected by centrifugation at 8000 rpm for 5 min at 4°C, and the supernatant was discarded. Then, 35 mL of lysis buffer was added to the bacteria, which were lysed at 4°C overnight. The centrifuge tube containing the overnight lysate was frozen in liquid nitrogen for 5 min, and then thawed in a 37°C water bath by repeatedly shaking. This freezing and thawing was repeated three times, and the bacteria should be in a viscous state. Then, the bacteria were sonicated using an ultrasonic disrupter, and the program was set as follows: sonication for 3 s and pause for 2 s. The centrifuge tube was placed in an ice-water mixture during sonication. After sonication, 1 mL of the lysate was centrifuged at 12000 rpm for 10 min to separate the supernatant (soluble protein) and the precipitate (inclusion body protein). After boiling treatment with protein loading buffer, the soluble expression of the target protein was identified by SDS-PAGE.

[0093] IV. Affinity purification of recombinant protein

[0094] To the precipitate, 25 mL of 8 mol / L urea solution was added, and the mixture was dissolved at room temperature overnight on a shaker. The solution was filtered using a 0.45 μm filter. The target protein was purified using a His-tag protein purification pre-packed column HisTrap™ FF Crude from GE. The basic steps are as follows: 5 times the column bed volume of distilled water was drawn into a disposable syringe and slowly pushed into the protein purification pre-packed column to wash away the 20% ethanol solution in the column. The recommended flow rate was about 2 mL / min. Column equilibration: 5 times the column bed volume of binding buffer was used to equilibrate the column at a flow rate of about 2 mL / min. Sample loading: 5 mL of filtered protein solution was slowly pushed into the protein purification column at a flow rate of less than 1 mL / min. After 2 mL of protein solution was pushed in, the purification column was placed on ice for 3-5 min. During this step, the sample loading speed should be slowed down to reduce the loss of target protein due to insufficient binding of the target protein to nickel in the purification column, which would affect the purification efficiency. Impurity elution: 10 times the column bed volume of binding buffer was used to elute the impurities at a flow rate of about 2 mL / min. Target protein elution: 10 times the column bed volume of elution buffer was used to elute the target protein at a flow rate of about 1 mL / min, and the first 20 mL of eluate was collected.

[0095] V. Desalting, renaturation, and concentration

[0096] The 3 kDa molecular cut-off dialysis bag was boiled in EDTA-NaHCO3 solution for 10 min, washed with distilled water, and then loaded with the protein solution. The dialysis bag was sealed and dialyzed in the dialysis solution. The dialysis solution was sequentially 8 mol / L urea, 6 mol / L urea, 4 mol / L urea, 2 mol / L urea, and 1x PBS buffer, and each dialysis lasted for 6 h. The used dialysis bag was washed with distilled water and then stored in 20% ethanol solution. After the protein solution was dialyzed in 1x PBS buffer, sucrose was scattered on the outside of the dialysis bag, and when the residual drop volume in the dialysis bag was appropriate, the protein solution (containing the Brucella surface antigen OMP31-Col) was collected and then stored in a sterile EP tube at -80°C.

[0097] (3) The modification process of the mesoporous silica with the responsive material carboxymethyl chitosan and the Brucella surface antigen OMP31-Col is as follows:

[0098] 0.5 g of mesoporous silica was dispersed in a mixed solution of 50 ml of ethanol and 1 ml of 3-aminopropyl triethoxysilane, stirred at 80°C for 24 h, centrifuged and washed with water and ethanol three times each, and the product was collected and vacuum dried at 60°C to obtain the amino-modified mesoporous silica (i.e., MSN-NH2).

[0099] 0.1 g of the responsive material carboxymethyl chitosan was dispersed in a 10 ml aqueous solution containing 0.25 g of EDC and 0.15 g of NHS, stirred and activated, and then 10 ml of water containing 0.15 g of the product MSN-NH2 was added. After stirring and reacting for 24 h, the product was collected by centrifugation and washing with water five times, and vacuum dried at 60°C to obtain the mesoporous silica modified with the responsive material (i.e., MSN-CMCS).

[0100] The protein solution (containing 15 mg of the Brucella surface antigen protein OMP31-Col) obtained in step (2) was added to a PBS solution (the PBS buffer had a pH of 7.2-7.4) containing 0.18 g of EDC and 0.15 g of NHS, stirred and activated, and then 10 ml of PBS (the PBS buffer had a pH of 7.2-7.4) containing 60 mg of MSN-CMCS was added. After stirring for 24 h, the product was collected by centrifugation and washing with water three times, and freeze-dried to obtain the responsive silica-based nano-vaccine that can target M cells (i.e., MSN-CMCS-OMP31-Col).

[0101] Example 2.

[0102] The SEM test results are as follows: the MSN and MSN-CMCS-OMP31-Col prepared in Example 1 are dissolved in water and dispersed by ultrasonic for 20 min, 20 μl of the sample is dropped on a silicon wafer, the silicon wafer is attached to a conductive glue to prepare a sample and is labeled, the conductive property of the material is enhanced by gold plating to facilitate obtaining better morphology characteristics in SEM, after the sample is found at a low magnification, a suitable area is selected for observation and photographing at a high magnification.

[0103] The results are shown in Figure 1 Figure 1 a is a scanning electron microscope (SEM) picture of MSN 1 μm, the MSN presents a spherical structure; 1b is a scanning electron microscope (SEM) picture of MSN-CMCS-OMP31-Col 500 nm, the surface channel is covered, but still maintains a spherical structure.

[0104] Example 3.

[0105] The TEM test results are as follows: a small amount of MSN and MSN-CMCS-OMP31-Col prepared in Example 1 is dissolved in water and dispersed by ultrasonic, 20 μl of the sample is dropped on a copper net, and scanning observation is performed by a TEM transmission electron microscope.

[0106] The results are shown in Figure 2 Figure 2 a is a transmission electron microscope (TEM) picture of MSN 200 nm, the dendritic structure can be obviously seen; 2b is a transmission electron microscope (TEM) picture of MSN-CMCS-OMP31-Col 100 nm, the dendritic channel is covered, indicating that the organic matter is successfully coated on the surface of the silicon ball.

[0107] Example 4.

[0108] The results of the target gene amplification and bacterial liquid PCR in Example 1 are shown in Figure 3 , indicating that the target peptide sequence is inserted on the basis of the gene OMP31 sequence, the target gene OMP31-Col is obtained by PCR amplification, agarose gel electrophoresis is used for verification, a bright single band of about 723 bp is obtained, which is consistent with the expected size of the target gene, indicating that the target gene is successfully cloned. After the target gene is connected with the vector, it is transformed into the competent cells of Escherichia coli DH5α, is coated on an LB solid plate containing ampicillin, a number of single colonies are randomly selected for colony PCR, and are identified by agarose gel, and finally the target fragment of about 723 bp can be amplified.

[0109] Example 5.

[0110] The results of the Omp31-Col recombinant protein induced expression, Western Blot analysis and affinity chromatography purification in Example 1 are shown in Figure 4 ​​As shown, this protein is expressed in both the supernatant and the precipitate, but expressed in greater quantities in the precipitate; Western blot results show that this protein is antigenic; and finally, the pure recombinant protein can be purified by affinity chromatography.

[0111] Example 6.

[0112] The surface charge changes of the nanoparticles MSN-CMCS-OMP31-Col, MSN, MSN-NH2, and MSN-CMCS in Example 1 were characterized by dispersing the nanoparticles in 5 mL of water during the preparation process, sonicating for 15 min, and then characterizing the surface charge changes of the mixed solution.

[0113] The results are as follows Figure 5 As shown, the introduction of CMCS makes it positively charged after being linked to amino groups. After modifying the protein, since the number of negatively charged amino acids in the protein is greater than the number of positively charged amino acids, the protein still carries a negative charge after modification. The continuous change of the zeta potential further proves the successful modification and alteration of mesoporous silica.

[0114] Example 7.

[0115] Thermogravimetric analysis was performed on the nanoparticles MSN-CMCS-OMP31-Col, MSN, and MSN-CMCS in Example 1. The method was as follows: the nanoparticles in the preparation process were subjected to thermogravimetric testing at a rate of 10 K / min between 25-800°C under a N2 atmosphere.

[0116] The results are as follows Figure 6 As shown, MSN exhibits a thermogravimetric loss of only 4.49% between 25℃ and 800℃. However, after modification with CMCS and OMP31-Col, the thermogravimetric loss rates reach 36.56% and 57.97%, respectively. This is because both CMCS and OMP31-Col are organic compounds, and the increase in the content of organic components leads to an increase in the carbon content lost, thus resulting in a continuous increase in the thermogravimetric loss rate. This also demonstrates the successful modification of carboxymethyl chitosan with Brucella surface antigen.

[0117] Example 8.

[0118] The nanoparticles MSN-CMCS-OMP31-Col, MSN, OMP31-Col, and MSN-CMCS in Example 1 were analyzed by infrared functional group testing. The method was as follows: the nanoparticle powder and potassium bromide powder were mixed and ground in a mass ratio of approximately 1:100, and then pressed into tablets for infrared testing.

[0119] The results are as follows Figure 7As shown, compared with MSN, MSN-CMCS appeared the infrared characteristic absorption peak of CMCS at the marked position, and MSN-CMCS-OMP31-Col appeared the infrared characteristic absorption peaks of both CMCS and OMP31-Col.

[0120] Example 9.

[0121] SDS-PAGE analysis was performed on the nanomaterials in Example 1, and the results are shown in Figure 8 As shown, the bands of MSN-CMCS-OMP31-Col and OMP31-Col appeared at the marked position after connection, indicating that the recombinant protein was successfully connected to the material.

[0122] Example 10.

[0123] The blood compatibility of the nanoparticles MSN-CMCS-OMP31-Col, MSN, and MSN-CMCS in Example 1 was evaluated by the following method: Fresh sheep blood (sterile) was centrifuged at 2000 rpm for 10 min at 4°C, and red blood cells (RBCs) were obtained after washing 5 times with PBS solution. Subsequently, a 5% RBCs suspension was obtained by diluting with PBS solution. Then, 0.5 mL of nanoparticle sample solution (2 mg / mL) was added to 0.5 mL of 5% RBCs suspension, and the same volume of PBS and 0.1% Triton X-100 mixed RBCs were used as negative and positive controls, respectively. The mixed solution was vortexed and incubated in a constant temperature shaking incubator at 37°C for 2 h. Subsequently, all samples were centrifuged at 2000 rpm for 10 min at 4°C, and the absorbance value of the supernatant at 540 nm was measured to calculate the hemolysis rate.

[0124] The results are shown in Figure 9 As shown, according to the standard of the International Organization for Standardization, when the hemolysis rate exceeds 5%, the nanomaterial is determined to have a hemolysis risk. Although the hemolysis rate of MSN is 72.572%, the hemolysis rates of the mesoporous silica after final modification are all lower than 5%, indicating good biocompatibility.

[0125] Example 11.

[0126] Cytotoxicity analysis of the nanoparticles MSN and MSN-CMCS-OMP31-Col in Example 1 at different carrier concentrations: Macrophages were seeded in a 96-well plate at 5000 cells / well, and after incubation with different concentrations of MSN and MSN-CMCS-OMP31-Col (20, 40, 60, 80, 100 μg / mL) for 24 h, cell viability was detected by CCK-8 method, and the toxicity of different pure carriers to cells was judged by comparing the cell viability. The cell survival rate was calculated according to the formula:

[0127] Cell survival rate (%) = [(OD1-OD0) / (OD2-OD0)]x100

[0128] In the formula: OD1 is the absorbance of the experimental group; OD2 is the absorbance of the control group; OD0 is the absorbance of the blank group.

[0129] Dosing group: cells + nanoparticles + medium + CCK-8

[0130] Control group: cells + medium + CCK-8

[0131] Blank group: medium + CCK-8

[0132] The results are shown in Figure 10 As shown, the modified carrier material itself has weaker toxicity to macrophages than before modification, indicating that the final modified nanomedicine carrier has lower cytotoxicity, avoiding the toxic side effects of the nanomedicine carrier itself on normal human tissue cells.

[0133] Example 12.

[0134] The specific operation steps are as follows:

[0135] (1) Preparation of mesoporous silica nanospheres

[0136] Dissolve 0.96g CTAT in 50ml water containing 120μl TEA, stir at 75℃ for 2h, then quickly add 7.8ml TEOS, react at 75℃ for 3h, wash with water and ethanol each three times, vacuum dry, calcine at high temperature in a muffle furnace to remove the template, and obtain mesoporous silica nanospheres (MSN).

[0137] (2) Preparation of Brucella surface antigen OMP31-Col targeting M cells by prokaryotic expression technology, which is the same as step (2) in Example 1.

[0138] (3) The modification process of mesoporous silica with responsive material carboxymethyl chitosan and Brucella surface antigen OMP31-Col is as follows:

[0139] Disperse 0.5g of mesoporous silica in a mixture of 80ml ethanol and 1ml 3-aminopropyl triethoxysilane, stir at 75℃ for 28h, centrifuge and wash, collect the product by washing with water and ethanol each three times, and vacuum dry at 60℃ to obtain amino-modified mesoporous silica (i.e. MSN-NH2).

[0140] The 0.1 g of responsive material carboxymethyl chitosan was dispersed in 10 ml of aqueous solution containing 0.20 g of EDC, 0.12 g of NHS, after stirring and activation, 10 ml of water containing 0.125 g of product MSN-NH2 was added, after stirring for 20 h, centrifugal washing, water washing for five times, the product was collected, vacuum drying at 60°C, to obtain the responsive material modified mesoporous silica (i.e. MSN-CMCS).

[0141] The protein solution (containing 15 mg of Brucella surface antigen protein OMP31-Col in the protein solution) obtained in step (2) was added to the PBS solution (the PBS buffer solution pH was 6) containing 0.17 g of EDC, 0.14 g of NHS, after stirring and activation, 10 ml of PBS (the PBS buffer solution pH was 6) containing 45 mg of MSN-CMCS was added, stirring for 20 h, centrifugal washing, water washing for three times, freeze-drying, to obtain the M cell targeted responsive silica-based nano vaccine (i.e. MSN-CMCS-OMP31-Col).

[0142] Example 13.

[0143] The specific operation steps are as follows:

[0144] (1) Preparation of mesoporous silica nanospheres

[0145] 0.96 g of CTAT was dissolved in 50 ml of water containing 120 μl of TEA, after stirring at 85°C for 1 h, 7.8 ml of TEOS was quickly added, after reaction at 85°C for 2.5 h, water and ethanol were washed for three times respectively, vacuum drying, high temperature calcination in the muffle furnace to remove the template, to obtain mesoporous silica nanospheres (MSN).

[0146] (2) Preparation of M cell targeted Brucella surface antigen OMP31-Col by prokaryotic expression technology, the operation steps are the same as step (2) in example 1.

[0147] (3) The modification process of mesoporous silica with responsive material carboxymethyl chitosan and Brucella surface antigen OMP31-Col is as follows:

[0148] 0.5 g of mesoporous silica was dispersed in 100 ml of ethanol and 1 ml of 3- aminopropyl triethoxysilane mixed solution, stirring at 85°C for 20 h, centrifugal washing, water and ethanol were washed for three times respectively to collect the product, vacuum drying at 60°C, to obtain the amino-modified mesoporous silica (i.e. MSN-NH2).

[0149] The 0.1 g responsive material carboxymethyl chitosan was dispersed in 15 ml aqueous solution containing 0.30 g EDC, 0.18 g NHS, after stirring and activation, 10 ml water containing 0.175 g product MSN-NH2 was added, after stirring and reaction for 28 h, centrifugal washing, water washing for five times, the product was collected, vacuum drying at 60℃, to obtain responsive material modified mesoporous silica (i.e. MSN-CMCS).

[0150] The protein solution (the protein solution contains 15 mg Brucella surface antigen protein OMP31-Col) obtained in step (2) was added to PBS solution (PBS buffer pH 8) containing 0.19 g EDC, 0.16 g NHS, after stirring and activation, 10 ml PBS (PBS buffer pH 8) containing 75 mg MSN-CMCS was added, stirring for 28 h, centrifugal washing, water washing for three times, freeze drying, to obtain responsive silicon-based nano vaccine targeting M cells (i.e. MSN-CMCS-OMP31-Col).

[0151] The above is only the preferred embodiment of the present application, and does not limit the present application in any form. Any simple modification, equivalent change and modification of the above embodiment according to the technical essence of the present application are still within the scope of the technical solution of the present application.

Claims

1. A method for preparing a silicon-based nanovaccine targeting M cells, characterized in that, Includes the following steps: (1) Preparation of mesoporous silica; (2) The mesoporous silica was sequentially modified by amination, responsive material modification and Brucella surface antigen OMP31-Col to obtain the silicon-based nanovaccine targeting M cells.

2. The preparation method according to claim 1, characterized in that, In step (1), the mesoporous silica is spherical mesoporous silica.

3. The preparation method according to claim 2, characterized in that, In step (1), the method for preparing spherical mesoporous silica is as follows: CTAT is dissolved in water containing TEA, stirred at 75-85℃ for 1-2 hours, TEOS is added, reacted at 75-85℃ for 2-3 hours, centrifuged and washed, vacuum dried, and the template is removed to obtain spherical mesoporous silica.

4. The preparation method according to claim 1, characterized in that, The Brucella surface antigen OMP31-Col was obtained through prokaryotic expression technology, the operation of which is as follows: Plasmid construction: The target gene OMP31-Col and the expression vector pET-22b(+) were double-digested with enzymes, the digestion products were purified and then ligated using seamless cloning technology to construct the recombinant plasmid pET-22b(+)-OMP31-Col; Expression of OMP31-Col protein: The recombinant plasmid pET-22b(+)-OMP31-Col was transformed into BL21(DE3) competent cells, expression was induced by IPTG, the cells were collected and lysed to obtain a lysate containing the target protein; Purification: The lysate containing the target protein was purified by affinity chromatography to obtain high-purity OMP31-Col protein, namely Brucella surface antigen OMP31-Col.

5. The preparation method according to claim 4, characterized in that, In the plasmid construction process, the target gene:vector:MIX volume ratio in the ligation system is 1:1:

2. The concentration of the inducer IPTG is 1 mmol / L, and the dosage added is 1‰.

6. The preparation method according to claim 1, characterized in that, In step (2), the modification process is as follows: Mesoporous silica was dispersed in a mixed solution of ethanol and 3-aminopropyltriethoxysilane, stirred at 75-85℃ for 20-28 h, centrifuged, washed, dried, and product 1 was collected. The responsive material carboxymethyl chitosan was dispersed in an aqueous solution containing EDC and NHS. After stirring and activation, product 1 was added. The mixture was stirred and reacted for 20-28 hours. After centrifugation, washing, and drying, product 2 was collected. Brucella surface antigen OMP31-Col was added to a PBS solution containing EDC and NHS, and after stirring and activation, product 2 was added. The mixture was stirred for 20-28 hours and then centrifuged and washed.

7. The preparation method according to claim 6, characterized in that, The volume ratio of ethanol to 3-aminopropyltriethoxysilane is 50-100:1; The mass ratio of product 1 to the responsive material carboxymethyl chitosan is 2.5-3.5:2; The mass ratio of product 2 to Brucella surface antigen OMP31-Col is 3-5:1; The mass ratio of EDC to NHS in the aqueous solution is 0.20-0.30:0.12-0.18; The mass ratio of EDC to NHS in the PBS buffer is 170-190:140-160; the pH of the PBS buffer is 6-8.

8. The preparation method according to claim 7, characterized in that, The volume ratio of ethanol to 3-aminopropyltriethoxysilane is 50:1; The mass ratio of product 1 to the responsive material carboxymethyl chitosan is 3:2; The mass ratio of product 2 to Brucella surface antigen OMP31-Col is 4:1; The mass ratio of EDC to NHS in the aqueous solution is 0.25:0.

15. The mass ratio of EDC to NHS in the PBS buffer is 180:

150.

9. A silicon-based nanovaccine targeting M cells, characterized in that, It is prepared by the preparation method according to any one of claims 1-8.

10. The application of the silicon-based nanovaccine of claim 9 in the immunoprophylaxis and treatment of brucellosis bacteremia.

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