Functional cationic polymer and preparation method and application thereof

Abstract

The application discloses a kind of functional cationic polymer and its preparation method and application, the functional cationic polymer includes the cationic polymer coupled with targeting polypeptide;The amino acid sequence of the targeting polypeptide is as shown in SEQ ID NO.2;The cationic polymer includes polyamide-amine.The functional cationic polymer provided in the application combines cationic polymer PAA and targeting polypeptide CPE30, can specifically target M cell in mucosa tissue, accelerates the immune response speed of vaccine in organism, and significantly improves antibody titer, and can be used as vaccine adjuvant and widely used in biological medicine field.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a functional cationic polymer and a preparation method and application thereof. BACKGROUND

[0002] Vaccination is one of the important means for preventing and controlling infectious diseases. By injecting vaccines, the human body can be stimulated to produce antibodies, thereby preventing the occurrence of infectious diseases. There are many types of vaccines, including attenuated live vaccines, inactivated vaccines, subunit vaccines, etc. The antigen protein of the subunit vaccine is formed by gene engineering expression, and the preparation thereof is relatively more simple, low in cost, and easy to scale up. Therefore, protein vaccines are currently a hot spot in vaccine research, and subunit vaccines still have the shortcomings of weak immunogenicity and weak cell immunity. However, extensive research on vaccine adjuvants has significantly improved the weak immunogenicity of subunit vaccines, not only improving the humoral immunity of the body, but also producing endogenous antigen immune responses through cross-presentation, thereby enhancing cell immunity, which provides new possibilities for the preparation of subunit vaccines. SUMMARY

[0003] The present application aims to at least solve one of the technical problems existing in the prior art. To this end, the present application proposes a functional cationic polymer, which can be used as a vaccine adjuvant and target M cells, thereby improving the mucosal immune response of the body to vaccines.

[0004] The present application also proposes a preparation method of the functional cationic polymer.

[0005] The present application also proposes an application of the functional cationic polymer.

[0006] The present application also proposes a vaccine.

[0007] The present application also proposes a preparation method of the vaccine.

[0008] The present application also proposes an application of the vaccine.

[0009] According to a first aspect of the present application, a functional cationic polymer is provided, which comprises a cationic polymer coupled with a targeting polypeptide; the amino acid sequence of the targeting polypeptide is shown in SEQ ID NO. 3; and the cationic polymer comprises a polyamide-amine.

[0010] In some embodiments of the present application, the chemical structural formula of the polyamide-amine is as follows:

[0011]

[0012] The poly(amido amine) (PAA) can release the antigen protein directly in antigen-presenting cells by reduction, promote MHC class I antigen presentation, and enhance cellular immune response through bioreduction of the disulfide bond contained therein.

[0013] In some embodiments of the present application, the targeting polypeptide is CPE30.

[0014] The CPE30 is 30 amino acids from the C-terminal of Clostridium perfringens enterotoxin protein, which can specifically bind to the receptor claudin 4 of M cells (Microfold cells) present in mucosal tissues. Therefore, the use of CPE30 in vaccine adjuvants can greatly improve the activation efficiency of mucosal immunity of vaccines.

[0015] In some embodiments of the present application, the targeting polypeptide and the cationic polymer are coupled by an amide reaction between the carboxyl group in the targeting polypeptide and the amino group in the cationic polymer.

[0016] According to a second aspect of the present application, a preparation method of the functionalized cationic polymer is provided, which comprises the following steps: mixing the poly(amido amine), the targeting polypeptide, and the crosslinking agent, and reacting to obtain the functionalized cationic polymer.

[0017] In some embodiments of the present application, the crosslinking agent comprises 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS).

[0018] In some embodiments of the present application, the mixing mass ratio of the poly(amido amine) and the targeting polypeptide is (4-6):3.

[0019] In some preferred embodiments of the present application, the mixing mass ratio of the poly(amido amine) and the targeting polypeptide is 5:3.

[0020] In some embodiments of the present application, the reaction temperature is 20-30°C.

[0021] In some preferred embodiments of the present application, the reaction temperature is 25°C.

[0022] In some embodiments of the present application, the reaction time is 12-36h.

[0023] In some preferred embodiments of the present application, the reaction time is 18-30h.

[0024] In some more preferred embodiments of the present application, the reaction time is 24h.

[0025] According to a third aspect of the present application, there is provided use of the functionalized cationic polymer according to the first aspect of the present application in the preparation of a vaccine adjuvant.

[0026] According to a fourth aspect of the present application, there is provided a vaccine comprising the functionalized cationic polymer according to the first aspect of the present application and a fusion protein; the amino acid sequence of the fusion protein is shown as SEQ ID NO. 2.

[0027] In some embodiments of the present application, the mass ratio of the functionalized cationic polymer and the fusion protein is (3-7): 1.

[0028] In some preferred embodiments of the present application, the mass ratio of the functionalized cationic polymer and the fusion protein is (4-6): 1.

[0029] In some more preferred embodiments of the present application, the mass ratio of the functionalized cationic polymer and the fusion protein is 5: 1.

[0030] In some embodiments of the present application, the nucleotide sequence of the nucleic acid molecule encoding the fusion protein is shown as SEQ ID NO. 1.

[0031] The fusion protein is a PiuA and PiaA fusion expressed protein; wherein the PiuA is a PiuA protein (1-32AA) without signal peptide encoded in the piuA gene (spd_1652) derived from Streptococcus pneumoniae, and the PiaA is a membrane permease involved in the iron uptake of Streptococcus pneumoniae, the piaA gene is a PiaA protein (1-26AA) without signal peptide encoded in spd_0915; the PiuA-PiaA fusion protein can provide two different immunogens, and the fusion protein can prolong the half-life of the immunogen in vivo, thereby better inducing immune response.

[0032] According to a fifth aspect of the present application, there is provided a preparation method of the vaccine according to the fourth aspect of the present application, the preparation method comprising the following steps: mixing a fusion protein solution with a functionalized cationic polymer solution to obtain.

[0033] In some embodiments of the present application, the fusion protein solution and the functionalized cationic polymer solution are mixed in equal volume. In some embodiments of the present application, the concentration of the fusion protein solution is 0.1-0.3 mg / mL.

[0034] In some embodiments of the present application, the concentration of the fusion protein solution is 0.15 mg / mL.

[0035] In some embodiments of the present application, the concentration of the functionalized cationic polymer solution is 0.5-1 mg / mL.

[0036] In some embodiments of the present application, the concentration of the functionalized cationic polymer solution is 0.75 mg / mL.

[0037] According to a sixth aspect of the present application, there is provided use of the vaccine as described in the third aspect of the present application in the preparation of a product for preventing and / or treating pneumonia.

[0038] The present application has at least the following beneficial effects:

[0039] 1) The functionalized cationic polymer provided by the present application combines cationic polymer PAA and targeting polypeptide CPE30, can specifically target M cells in mucosal tissue, accelerate the immune response speed of the body to the vaccine, and significantly improve the antibody titer, and can be widely used as a vaccine adjuvant in the field of biological medicine.

[0040] 2) The vaccine provided by the present application combines the above-mentioned functionalized cationic polymer for targeting mucosal immunity with PiuA-PiaA fusion protein, and a vaccine preparation is prepared through electrostatic interaction, the obtained vaccine can significantly improve the IgG and IgA titers in the pneumonia model mice and improve the survival rate; the vaccine prepared by the present application provides a theoretical basis and technical support for pneumonia prevention, and is suitable for being widely applied in actual production. BRIEF DESCRIPTION OF DRAWINGS

[0041] The present application will be further described below in combination with the drawings and examples, in which:

[0042] Figure 1 and Figure 2 FIG. 1 is an intermediate product characterization diagram of the functionalized cationic polymer PAA-CPE30 in Example 1 of the present application;

[0043] Figure 3 FIG. 2 is a schematic diagram of the synthesis route of the functionalized cationic polymer PAA-CPE30 in Example 1 of the present application;

[0044] Figure 4 FIG. 3 is an agarose gel electrophoresis diagram of the recombinant plasmid pGEX-4T-piuA-piaA in Example 2 of the present application; wherein, “M” refers to DL5000 DNA marker; lanes 1-3 are three randomly selected monoclonal colony samples;

[0045] Figure 5Gel electrophoresis chart for verifying the fusion protein PiuA-PiaA in Example 2 of the present application; wherein, "M" is a protein Mr marker, lane 1 is a mixed protein sample before enzyme cutting, lane 2 is a GST-PiuA-PiaA fusion protein before enzyme cutting, lane 3 is a PiuA-PiaA fusion protein and a GST tag protein fragment after thrombin enzyme cutting, and lane 4 is a target protein PiuA-PiaA fusion protein obtained through GST affinity chromatography;

[0046] Figure 6 A graph of the cytotoxicity detection results of the functionalized cationic polymer PAA-CPE30 in different concentrations on DC2.4 cells in the test example of the present application;

[0047] Figure 7 A graph of HE staining results of main organ sections of mice after intranasal immunization with different vaccine formulations in the test example of the present application, wherein the magnification of the results graph is 200 times;

[0048] Figure 8 A confocal microscope result graph of using different vaccine formulations to treat dendritic cells for different incubation times in the test example of the present application, wherein a graph is the result of co-incubation for 2h, b graph is the result of co-incubation for 6h, and the scale bar is 20μm;

[0049] Figure 9 A small animal live imaging result graph obtained after mice were immunized with different vaccine formulations in the test example of the present application; wherein, A graph is the imaging result, B graph is the corresponding fluorescence intensity statistical graph, and C graph is the fluorescence intensity ratio statistical graph of each group of mice at different time points to the fluorescence intensity at 0h;

[0050] Figure 10 A serum IgG antibody titer statistical graph detected after mice were immunized with different vaccine formulations in the test example of the present application; wherein, "**" indicates p<0.01, and "***" indicates p<0.001;

[0051] Figure 11 A serum IgG1 and IgG2a antibody titer statistical graph detected after mice were immunized with different vaccine formulations in the test example of the present application; wherein, "*" indicates p<0.05, and "****" indicates p<0.0001;

[0052] Figure 12 A serum IgA antibody titer statistical graph detected after mice were immunized with different vaccine formulations in the test example of the present application; wherein, "*" indicates p<0.05, and "***" indicates p<0.001;

[0053] Figure 13Fig. 1 is a graph showing the survival rate of mice after being challenged by S. pneumoniae after being immunized by different vaccine formulations in the experimental example of the present application;

[0054] Figure 14 Fig. 4 is a graph showing the H&E staining results of lung sections of mice after being challenged by S. pneumoniae after being immunized by different vaccine formulations in the experimental example of the present application. DETAILED DESCRIPTION

[0055] The concept and technical effects of the present application will be described in detail below in combination with examples, so as to fully understand the purpose, features and effects of the present application. Obviously, the described examples are only a part of the examples of the present application, but not all the examples. Based on the examples of the present application, other examples obtained by those skilled in the art without creative labor are within the protection scope of the present application.

[0056] Example 1

[0057] The present embodiment provides a functionalized cationic polymer PAA-CPE30, and the preparation method of the functionalized cationic polymer specifically comprises the following steps:

[0058] 1) Synthesis of double bond cystamine (BAC):

[0059] ① 2.5 g of cystamine dihydrochloride was dissolved in 11.1 mL of pure water to obtain a cystamine dihydrochloride solution; 3.304 mL of acryloyl chloride was dissolved in 3.33 mL of dichloromethane (DCM) to obtain an acryloyl chloride solution; 1.7763 g of sodium hydroxide was dissolved in 4.441 mL of pure water to obtain a sodium hydroxide solution;

[0060] ② The sodium hydroxide solution and the acryloyl chloride solution obtained in step ① were alternately added dropwise into the pre-cooled cystamine dihydrochloride solution, and reacted at room temperature for 7 h (6-8 h can be used), extracted with dichloromethane and saturated with sodium bicarbonate and deionized water, and then vacuum dried after rotary evaporation to obtain BAC.

[0061] 2) Synthesis of reduction-responsive polyamide-amine (PAA):

[0062] ① 0.444 g of CaCl2 was dissolved in 10 mL of methanol to obtain a CaCl2 methanol solution; 0.148 g of CaCl2 was dissolved in 3.3 mL of deionized water to obtain a CaCl2 aqueous solution;

[0063] ② 0.6424 g of BAC synthesized in step 1) was dissolved in the CaCl2 methanol solution obtained in step ①, and after being fully dissolved, the CaCl2 aqueous solution was added, and when the temperature was gently increased to 50℃, 160 μL of 1-(2-aminoethyl)piperazine (AEPZ) was added, and reacted at 50℃ for 36 h;

[0064] After the reaction was completed, 345 μL of AEPZ was added to the reaction system, and the reaction was continued for 4-8 h. The product PAA was collected, dialyzed and lyophilized for standby use.

[0065] After preparation, the BAC and PAA prepared in the above steps were respectively subjected to chemical structure characterization using a nuclear magnetic resonance instrument, and the obtained characterization results are shown in Figure 1 and Figure 2 .

[0066] 3) Synthesis of functionalized polyamide-amine (PAA-CPE30):

[0067] The amidation reaction of -NH2 on the surface of PAA and -COOH in the structure of polypeptide CPE30 was catalyzed by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS), and PAA-CPE30 was obtained by stirring at room temperature for 24 h. The synthesis route of PAA-CPE30 is shown in Figure 3 .

[0068] Example 2

[0069] This example provides a vaccine PAA-CPE30@PiuA-PiaA, which uses the functionalized cationic polymer PAA-CPE30 prepared in Example 1 as a vaccine adjuvant, and uses the fusion protein PiuA-PiaA as an immunogen. The specific preparation method is as follows:

[0070] 1) Plasmid construction:

[0071] ① The piuA-piaA gene fragment was amplified by PCR, the nucleotide sequence of the piuA-piaA gene fragment is shown in SEQ ID NO. 1, and the piuA-piaA gene fragment was cloned into the pGEX-4T-1 plasmid vector (purchased from Shanghai Yingjun Biotechnology Co., Ltd.) to construct the recombinant plasmid pGEX-4T-piuA-piaA;

[0072]

[0073] The primer sequence for amplifying the piuA-piaA gene fragment is as follows:

[0074] F: 5'-GAGACCAGTAGCTCTGCTCC-3' (SEQ ID NO. 2);

[0075] R: 5'-TTATTTCGCATTTTTGCATGC-3' (SEQ ID NO. 3);

[0076] ②Randomly pick 3 single colonies on the LB plate grown after transformation of the recombinant plasmid pGEX-4T-piuA-piaA, and place them in LB liquid medium for culture for 6-8 h, and take 2 μL of the bacterial liquid as a template for PCR identification, and the PCR identification result is shown in Figure 4 The primer sequence used in the PCR identification is shown in SEQ ID NO. 2 and SEQ ID NO. 3;

[0077] It can be known from Figure 4 that the No. 1-3 single colonies all amplified the DNA fragment with the same size as the target gene, and the three recombinant plasmid bacterial liquids were sent to Shanghai Eng Bioengineering Co., Ltd. for sequencing, and the sequencing result was completely matched with the target gene sequence by BLASTn comparison, indicating that the recombinant plasmid pGEX-4T-piuA-piaA was successfully constructed.

[0078] 2) Preparation of fusion protein PiuA-PiaA:

[0079] ①The fusion protein GST-PiuA-PiaA obtained by inducing expression in E. coli BL21 was purified by GST affinity chromatography, and the fusion protein GST-PiuA-PiaA bound to the GST affinity chromatography column was eluted by reducing glutathione (10 mM, pH 8.0), concentrated by an ultrafiltration tube with a molecular weight cut-off of 10000 kDa, and then the GST tag was cut off by thrombin, and the target fusion protein PiuA-PiaA was further obtained by GST affinity chromatography; the amino acid sequence of the fusion protein PiuA-PiaA is shown in SEQ ID NO. 4;

[0080] The amino acid sequence of the fusion protein PiuA-PiaA is: ETSSSAPTEITIKSSLDEVKLSKVPEKIVTFDLGAADTIRALGFEKNIVGMPTKTVPTYLKDLVGTVKNVGSMKEPDLEAIAALEPDLIIASPRTQKFVDKFKEIAPTVLFQASKDDYWTSTKANIESLASAFGETSTQKAKEELAKLDKSIQEVATKNESSDKKALAILLNEGKMAAFGAKSRFSFLYQTLKFKPTDTKFEDSRHGQEVSFESVKEINPDILFVINRTLAIGGDNSSNDGVLENALIAETPAAKNGKIIQLTPDLWYLSGGGLESTKLMIEDIQKALKNEENTSKEHAPDKIVLDHAFGQTILDKKPERVATIAWGNHDVALALGIVPVGFSKANYGVSADKGVLPWTEEKIKELNGKANLFDDLDGLNFEAISNSKPDVILAGYSGITKEDYDTLSKIAPVAAYKSKPWQTLWRDMIKIDSKALGMEKEGDELIKNTEARISKELEKHPEIKGKIKGKKVLFTMINAADTSKFWIYTSKDPRANYLTDLGLVFPESLKEFESEDSFAKEISAEEANKINDADVIITYGDDKTLEALQKDPLLGKINAIKNGAVAVIPDNTPLAASCTPTPLSINYTIEEYLNLLGNACKNAK (SEQ ID NO. 4);

[0081] ②The mixed protein which was not purified in step ①, the fusion protein GST-PiuA-PiaA before enzyme cutting after purification, the fusion protein PiuA-PiaA+GST tag protein after enzyme cutting, and the fusion protein PiuA-PiaA after affinity chromatography were detected by gel electrophoresis, and the results are shown in Figure 5

[0082] As shown in Figure 5 It can be seen that the PiuA-PiaA fusion protein with a purity of >95% is successfully prepared in the embodiment, and can be used for further vaccine preparation.

[0083] 3) Vaccine preparation:

[0084] ​PAA-CPE30@PiuA-PiaA vaccine was prepared by adding 1 mL of PiuA-PiaA fusion protein solution (0.15 mg / mL) into 1 mL of PAA-CPE30 solution (0.75 mg / mL), mixing by blowing, and reacting at room temperature for 30 min; the "@" in this scheme represents the electrostatic adsorption relationship between PAA and the fusion protein.

[0085] Comparative Example 1

[0086] A vaccine PAA@PiuA-PiaA was prepared in this comparative example, which used the cationic polymer PAA prepared in Example 1 as a vaccine adjuvant and the fusion protein PiuA-PiaA as an immunogen, and the specific preparation method was the same as that in Example 2.

[0087] Comparative Example 2

[0088] A vaccine CTB+PiuA-PiaA was prepared in this example, which used the non-toxic B subunit of cholera toxin (CTB) as a vaccine adjuvant and the fusion protein PiuA-PiaA as an immunogen, and the specific preparation method was the same as that in Example 2.

[0089] Test Example

[0090] This test example tested the cytotoxicity and dendritic cell (DC) targeting of the functionalized cationic polymer PAA-CPE30 provided in Example 1, and also tested the toxicity, effect on survival rate, and immune response induction effect of the vaccines obtained in Examples 2 and Comparative Examples 1-2. The specific detection methods and results are as follows:

[0091] 1. CCK-8 method for detecting the cytotoxicity of PAA-CPE30@PiuA-PiaA:

[0092] DC2.4 cells were inoculated at a density of 1×10 4 After the cells were inoculated at a density of 1×10 Figure 6

[0093] The results are shown in Table 1. Figure 6 ​It can be seen that compared with the control group, no obvious cytotoxicity was detected when PAA-CPE30 was less than 800 μg / mL, indicating that the PAA-CPE30 provided by the application has good biocompatibility.

[0094] 2. Evaluation of the biocompatibility of PAA-CPE30@PiuA-PiaA vaccine:

[0095] After the mice were intranasally administered PBS buffer (control group), PBS + fusion protein PiuA-PiaA, vaccine PAA@PiuA-PiaA, vaccine PAA-CPE30@PiuA-PiaA, and vaccine CTB + PiuA-PiaA, respectively, three times with an interval of 14 days each time, 200 μL of vaccine was administered to each mouse each time, corresponding to 30 μg of antigen, the heart, liver, spleen, lung, and kidney tissues of the mice were taken for H&E staining, and the pathological damage of the main organs of the body caused by each vaccine preparation was observed, and the results are shown in Figure 7 .

[0096] From Figure 7 it can be seen that compared with the control group, no obvious pathological damage was observed in each treatment group, indicating that PAA-CPE30 loaded with fusion protein PiuA-PiaA does not cause obvious organ damage, and has high biocompatibility, which is suitable for wide application.

[0097] 3. In vitro verification of the DC targeting property of functional cationic polymer PAA-CPE30:

[0098] DC is a kind of professional antigen presenting cell (APC) that can recognize, capture, process and present antigens to initial T cells, thereby inducing the body to produce antigen-specific immune response, and is the key to connecting the innate and adaptive immune responses of the body. The uptake efficiency and presentation pathway of DC for exogenous antigens play a decisive role in the effect of immunization and the type of immune response. Therefore, in this test example, laser confocal microscopy (CLSM) was used to test the antigen uptake efficiency of DC with / without the assistance of vaccine adjuvant PAA-CPE30 and the lysosome escape effect of the vaccine preparation after entering the DC.

[0099] In this test example, fluorescently labeled chicken egg white albumin OVA-Cy5 was used as a model antigen instead of fusion protein PiuA-PiuA. Briefly, 1 × 10 5 DC2.4 cells were co-incubated with PBS / OVA-Cy5, PAA@OVA-Cy5, and PAA-CPE30@OVA-Cy5 preparation solutions (antigen final concentration 0.15 mg / mL, adjuvant final concentration 0.75 mg / mL) for 2 h or 6 h, respectively, and then the DC2.4 cells were observed by CLSM, and the results are shown in Figure 8as shown.

[0100] As can be seen from Figure 8 When DC2.4 was incubated with different formulations for 2h, PAA-CPE30 could assist the more efficient uptake of antigen protein by DC2.4, and the intracellular red fluorescence signal in the PAA-CPE30@OVA treatment group was significantly higher than that in the other two treatment groups. When DC2.4 was incubated with different formulations for 6h, it was found that compared with the antigen alone group, more antigen was taken up by DC2.4 after the addition of polymer PAA or PAA-CPE30, and the lysosome escape effect of the antigen protein was effectively mediated (the red signal in the figure is the antigen protein, and the green signal is the lysosome).

[0101] 4. In vivo verification of the M cell targeting property of PAA-CPE30:

[0102] Mice (n=6) were immunized by intranasal administration of PBS, PBS+OVA-Cy5, PAA@OVA-Cy5 and PAA-CPE30@OVA-Cy5 formulation solutions (200μL vaccine per mouse), and the amount and residence time of the antigen protein in the nasal cavity were observed by a small animal live imaging instrument at different time intervals (0.5, 1, 2 and 4h) before (0h) and after intranasal administration. The results are shown in Figure 9 as shown.

[0103] As can be seen from Figure 9 It can be seen that in the presence of cationic polymer PAA or PAA-CPE30, the antigen protein in the nasal cavity of the immunized mice can stay longer, and the amount of antigen protein in the nasal cavity of the mice in the PAA or PAA-CPE30 treatment group is more than that in the control group at the same time interval, indicating that the antigen delivery and targeting property is stronger.

[0104] 5. Detection of antibody titer induced by PAA-CPE30@PiuA-PiaA vaccine in mice:

[0105] Balb / c mice were immunized by intranasal administration of PBS+PiuA-PiaA, PAA@PiuA-PiaA, CTB+PiuA-PiaA and PAA-CPE30@PiuA-PiaA respectively, and the control group was injected with 1×PBS. Each mouse was given 30μg of fusion protein PiuA-PiaA, and each group had 6 mice. A total of 3 immunizations were performed, with an interval of 14 days between each immunization. Sampling was performed 7 days after the third immunization to detect the antibody titer of PiuA-PiaA in the mice.

[0106] 1) Typically, the immune response induced by protein vaccines is humoral immunity. During humoral immunity, the body produces IgG antibodies in the serum to defend against pathogen invasion through various mechanisms. Therefore, the antibody titer of IgG in the serum reflects the strength of the humoral immune response induced by the antigen. The results of detecting the IgG antibody titer in the serum after the immune response in this experimental case are as follows: Figure 10 As shown.

[0107] Depend on Figure 10 It can be seen that after three immunizations, the PAA@PiuA-PiaA, CTB+PiuA-PiaA, and PAA-CPE30@PiuA-PiaA groups were able to elicit a strong humoral immune response, as evidenced by high serum IgG antibody titers.

[0108] 2) The interaction between antigens and immune system cells generates an immune response against the antigen. This immune response can be broadly classified into two categories: Th1 immune responses (cell-mediated immune responses) and Th2 immune responses (humoral immune responses). In mice, the production of IgG1 and IgG2a subtypes is associated with Th2 and Th1 immune responses, respectively. Therefore, this experiment measured the antibody titers of IgG1 and IgG2a in mouse serum, and the results are as follows: Figure 11 As shown.

[0109] Depend on Figure 11 It is known that PAA-CPE30@PiuA-PiaA can stimulate high titers of IgG1 and IgG2a antibodies, indicating that PAA-CPE30@PiuA-PiaA has significant effects on both (Th2) humoral immunity and (Th1) cellular immunity.

[0110] 3) IgA antibodies secreted after mucosal vaccination can protect the host from pathogen infection at the mucosal site. PiuA-PiaA specific SIgA antibodies were detected in mouse nasal mucosal washings after vaccination, as shown in the following results. Figure 12 As shown.

[0111] Depend on Figure 12 It was found that after three immunizations, a high SIgA antibody titer was detected in the nasal mucosal wash of mice in the PAA-CPE30@PiuA-PiaA group, a result similar to that of IgG2a antibody.

[0112] 6. Evaluate the effect of PAA-CPE30@PiuA-PiaA vaccine on survival rate in a mouse pneumonia model:

[0113] The test example constructs a mouse S. pneumoniae infection model for evaluating the protection effect of the PAA-CPE30@PiuA-PiaA vaccine on S. pneumoniae infection; Balb / c mice are immunized by intranasal injection of PBS+PiuA-PiaA, PAA@PiuA-PiaA, CTB+PiuA-PiaA and PAA-CPE30@PiuA-PiaA respectively, the control group is injected with 1×PBS, each mouse is given 30 μg of the fusion protein PiuA-PiaA, each group has 11 mice, a total of 3 immunizations, with an interval of 14 days between each immunization, 7 days after the third immunization, 1×10 9 CFU of S. pneumoniae is dropped into the nasal cavity of the mouse, 48 h later, 3 mice are randomly selected from each group for pathological examination, and the survival of the remaining mice is observed every day, and the survival rate is as shown in Figure 13 , the H&E staining results of the pathological sections are as shown in Figure 14 .

[0114] As can be seen from Figure 13 , the survival rate of the mice in the PAA@PiuA-PiaA vaccine immunization group and the PAA-CPE30@PiuA-PiaA vaccine immunization group is significantly higher than that of the control group, and has statistical significance, which shows that the vaccine provided by the application can effectively play a protective role in S. pneumoniae infection.

[0115] As can be seen from Figure 14 , the alveolar structure of the mice immunized with the PAA-CPE30@PiuA-PiaA vaccine is complete and the alveolar is relatively thin; a large number of inflammatory cell infiltrates and unshaped alveoli are observed in the lung sections of the control group mice.

[0116] The above results show that in the mouse S. pneumoniae infection model, immunization with the PAA-CPE30@PiuA-PiaA vaccine can significantly improve the survival rate of mice in the pneumonia model and reduce lung inflammation.

[0117] The above embodiments of the application are described in detail in combination with the drawings, but the application is not limited to the above embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the purpose of the application. In addition, the embodiments of the application and the features in the embodiments can be combined with each other without conflict.

Claims

1. A vaccine comprising, The vaccine comprises a functional cationic polymer and an immunogen; the immunogen comprises a fusion protein; the amino acid sequence of the fusion protein is shown as SEQ ID NO. 4; the functional cationic polymer comprises a cationic polymer coupled with a targeting polypeptide; the targeting polypeptide is CPE30; the cationic polymer comprises a polyamide-amine; the chemical structural formula of the polyamide-amine is as follows: 。 2. The vaccine of claim 1, characterized in that, The nucleotide sequence of the nucleic acid molecule encoding the fusion protein is shown as SEQ ID NO.

1.

3. A method of preparing a vaccine as claimed in claim 1 or 2, characterized in that, The preparation method comprises the following steps: uniformly mixing a fusion protein solution with a functional cationic polymer solution to obtain the vaccine. The functional cationic polymer is prepared by the following preparation method: uniformly mixing a polyamide-amine, a targeting polypeptide and a crosslinking agent, and then reacting to obtain the functional cationic polymer.

4. The production method according to claim 3, characterized by, The crosslinking agent comprises 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide.

5. The preparation method according to claim 3, characterized in that, The mixing mass ratio of the polyamide-amine and the targeting polypeptide is (4-6):

3.

6. Use of the vaccine of claim 1 or 2 in the preparation of a product for preventing pneumonia caused by Streptococcus pneumoniae infection.

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