Functionalized cationic polymer as well as preparation method and application thereof

Abstract

The invention discloses a functionalized cationic polymer as well as a preparation method and application thereof. The functionalized cationic polymer comprises a cationic polymer coupled with targeted polypeptide, the amino acid sequence of the targeted polypeptide is as shown in SEQ ID NO. 2; the cationic polymer includes a polyamide-amine. The functionalized cationic polymer provided by the invention combines the cationic polymer PAA and the targeting polypeptide CPE30, can specifically target M cells in mucosa tissues, accelerates the immune response speed of an organism to vaccines, remarkably improves the antibody titer, and can be widely used as a vaccine adjuvant in the field of biological medicines.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to a functionalized cationic polymer and a preparation method and application thereof. Background Art

[0002] Vaccination is one of the important means to prevent and control infectious diseases. Vaccine injection can stimulate the human body to produce antibodies, thereby preventing the occurrence of infectious diseases. There are many types of vaccines, including live attenuated vaccines, inactivated vaccines, subunit vaccines, etc. Among them, the antigen protein of subunit vaccines is formed by genetic engineering expression, and its preparation is relatively simple, low cost, and easy to scale production. Therefore, protein vaccines are currently a hot topic in vaccine research. Subunit vaccines still have disadvantages such as weak immunogenicity and weak cellular immunity. However, extensive research on vaccine adjuvants has significantly improved the weak immunogenicity of subunit vaccines, not only improving the body's humoral immunity, but also generating endogenous antigen immune responses and enhancing cellular immunity through cross-presentation, which provides new possibilities for the preparation of subunit vaccines. Summary of the invention

[0003] 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 provides a functionalized cationic polymer, which can be used as a vaccine adjuvant and target M cells to enhance the body's mucosal immune response to the vaccine.

[0004] The invention also provides a method for preparing the functionalized cationic polymer.

[0005] The present invention also proposes the application of the functionalized cationic polymer.

[0006] The present invention also provides a vaccine.

[0007] The invention also provides a method for preparing the vaccine.

[0008] The present invention also provides the application of the above vaccine.

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

[0010] In some embodiments of the present invention, the chemical structural formula of the polyamidoamine is as follows:

[0011]

[0012] The polyamidoamine (PAA) can break the disulfide bonds contained in it by biological reduction, directly release the antigen protein by reduction in antigen presenting cells, promote MHC class I antigen presentation, and enhance cellular immune response.

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

[0014] The CPE30 is a 30-amino acid residue from the C-terminus of the enterotoxin protein of Clostridium perfringens, which can specifically bind to the receptor claudin 4 of the special antigen presenting cell M cell (Microfold cell) present in the mucosal tissue. Therefore, the use of CPE30 in vaccine adjuvants can greatly improve the efficiency of vaccine activation of mucosal immunity.

[0015] In some embodiments of the present invention, the targeting polypeptide and the cationic polymer are coupled via an amidation reaction between a carboxyl group in the targeting polypeptide and an amino group in the cationic polymer.

[0016] According to the second aspect of the present invention, a method for preparing the functionalized cationic polymer as described in the first aspect of the present invention is proposed, and the preparation method comprises the following steps: mixing polyamide-amine, targeting polypeptide and cross-linking agent, and reacting to obtain the functionalized cationic polymer.

[0017] In some embodiments of the present invention, the cross-linking agent includes 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS).

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

[0019] In some preferred embodiments of the present invention, the mixing mass ratio of the polyamidoamine and the targeting polypeptide is 5:3.

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

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

[0022] In some embodiments of the present invention, the reaction time is 12 to 36 hours.

[0023] In some preferred embodiments of the present invention, the reaction time is 18 to 30 hours.

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

[0025] According to the third aspect of the present invention, a use of the functionalized cationic polymer as described in the first aspect of the present invention in the preparation of a vaccine adjuvant is proposed.

[0026] According to a fourth aspect of the present invention, a vaccine is provided, which comprises the functionalized cationic polymer and a fusion protein as described in the first aspect of the present invention; the amino acid sequence of the fusion protein is shown in SEQ ID NO.2.

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

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

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

[0030] In some embodiments of the present invention, 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 protein expressed by the fusion of PiuA and PiaA; wherein PiuA is a PiuA protein (1-32AA) without a signal peptide encoded in the piuA gene (spd_1652) of Streptococcus pneumoniae, PiaA is a membrane permease involved in the iron uptake of pneumococcus, and the piaA gene is a PiaA protein (1-26AA) without a 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 the body, thereby better inducing an immune response.

[0032] According to the fifth aspect of the present invention, a method for preparing the vaccine as described in the fourth aspect of the present invention is proposed, and the preparation method comprises the following steps: mixing the fusion protein solution and the functionalized cationic polymer solution.

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

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

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

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

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

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

[0039] 1) The functionalized cationic polymer provided by the present invention combines the cationic polymer PAA with the targeting polypeptide CPE30, which can specifically target the M cells in the mucosal tissue, accelerate the body's immune response to the vaccine, and significantly increase the antibody titer, and can be widely used as a vaccine adjuvant in the field of biomedicine;

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

[0041] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0042] Figure 1 and Figure 2 This is a characterization diagram of the intermediate product of the functionalized cationic polymer PAA-CPE30 in Example 1 of the present invention;

[0043] Figure 3 Schematic diagram of the synthesis route of the functionalized cationic polymer PAA-CPE30 in Example 1 of the present invention;

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

[0045] Figure 5This is a gel electrophoresis diagram for verifying the fusion protein PiuA-PiaA in Example 2 of the present invention; wherein, "M" is the protein Mrmarker, lane 1 is the mixed protein sample before enzyme cleavage, lane 2 is the GST-PiuA-PiaA fusion protein before enzyme cleavage, lane 3 is the PiuA-PiaA fusion protein and GST tag protein fragment after thrombin enzyme cleavage, and lane 4 is the target protein PiuA-PiaA fusion protein obtained by GST affinity chromatography;

[0046] Figure 6 This is a graph showing the cytotoxicity test results of the functionalized cationic polymer PAA-CPE30 at different concentrations on DC2.4 cells in the test examples of the present invention;

[0047] Figure 7 The results of HE staining of the main organ sections of mice after nasal immunization with different vaccine preparations in the test examples of the present invention are shown in Figure 1, wherein the magnification of the result diagram is 200 times;

[0048] Figure 8 The confocal microscopy results of treating dendritic cells with different vaccine preparations at different incubation times in the test examples of the present invention are shown, wherein Figure a is the result of co-incubation for 2 hours, and Figure b is the result of co-incubation for 6 hours, and the scale bar is 20 μm;

[0049] Fig. 9 The figures are the results of in vivo imaging of small animals obtained after immunizing mice with different vaccine preparations in the test examples of the present invention; wherein, Figure A is the imaging result, Figure B is the corresponding fluorescence intensity statistical graph, and Figure C is the statistical graph of the ratio of the fluorescence intensity of each group of mice at different time points to the fluorescence intensity at 0h;

[0050] Fig.10 This is a statistical graph of serum IgG antibody titers detected after mice were immunized with different vaccine preparations by intranasal drops in the test examples of the present invention; wherein, "**" indicates p<0.01, and "***" indicates p<0.001;

[0051] Fig.11 The statistical graph of serum IgG1 and IgG2a antibody titers detected after mice were immunized with different vaccine preparations by intranasal drops in the test examples of the present invention; wherein, "*" indicates p<0.05, and "****" indicates p<0.0001;

[0052] Fig.12 This is a statistical graph of serum IgA antibody titers detected after mice were immunized with different vaccine preparations by intranasal drops in the test examples of the present invention; where "*" indicates p<0.05, and "***" indicates p<0.001;

[0053] Fig.13This is a curve diagram of the survival rate of mice after being challenged with Streptococcus pneumoniae after mice were immunized with different vaccine preparations by intranasal drops in the test examples of the present invention;

[0054] Fig.14 This is a diagram showing the H&E staining results of lung sections of mice after being challenged with Streptococcus pneumoniae after mice were immunized with different vaccine preparations by intranasal drops in the test examples of the present invention. DETAILED DESCRIPTION

[0055] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0056] Example 1

[0057] This 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 Dissolve 2.5 g of cystamine dihydrochloride in 11.1 mL of pure water to obtain a cystamine dihydrochloride solution; dissolve 3.304 mL of acryloyl chloride in 3.33 mL of dichloromethane (DCM) to obtain an acryloyl chloride solution; dissolve 1.7763 g of sodium hydroxide in 4.441 mL of pure water to obtain a sodium hydroxide solution;

[0060] ② Alternately add the sodium hydroxide solution and acryloyl chloride solution obtained in step ① to the pre-cooled cystamine dihydrochloride solution, react at room temperature for 7 hours (6 to 8 hours), extract with dichloromethane and remove impurities with water-saturated sodium bicarbonate and deionized water, rotary evaporate and vacuum dry to obtain BAC.

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

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

[0063] ② Dissolve 0.6424 g of BAC synthesized in step 1) in the CaCl2 methanol solution obtained in step ①, add CaCl2 aqueous solution after it is fully dissolved, and when the temperature rises slowly to 50°C, add 160 μL of 1-(2-aminoethyl)piperazine (AEPZ) and react at 50°C for 36 hours;

[0064] ③ After the reaction is completed, 345 μL AEPZ is added to the reaction system for 4-8 hours, and the product PAA is collected, dialyzed and freeze-dried for later use.

[0065] After the preparation, the BAC and PAA prepared in the above steps were characterized by nuclear magnetic resonance spectroscopy. The characterization results are as follows: Figure 1 and Figure 2 shown.

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

[0067] The -NH2 on the surface of PAA and the -COOH in the structure of the peptide CPE30 were amidated by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS), and the reaction was stirred at room temperature for 24 hours to obtain PAA-CPE30. The schematic diagram of the synthesis route of PAA-CPE30 is shown in Figure 3 shown.

[0068] Example 2

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

[0070] 1) Plasmid construction:

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

[0072]

[0073] The primer sequences used to amplify the piuA-piaA gene fragment are:

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

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

[0076] ② Randomly pick 3 single colonies on the LB plate grown after the recombinant plasmid pGEX-4T-piuA-piaA was transformed and cultured in LB liquid culture medium for 6 to 8 hours. Take 2 μL of the bacterial solution as a template for PCR identification. The PCR identification results are as follows: Figure 4 The primer sequences used for PCR identification are shown in SEQ ID NO.2 and SEQ ID NO.3;

[0077] Depend on Figure 4 It can be seen that monoclonal colonies No. 1 to 3 all amplified DNA fragments with the same size as the target gene. The three recombinant plasmid bacterial cultures were sent to Shanghai Biotechnology Co., Ltd. for sequencing. The sequencing results were 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] ① The fusion protein GST-PiuA-PiaA obtained by inducing expression in Escherichia 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 reduced glutathione (10 mM, pH 8.0), concentrated by an ultrafiltration tube with a molecular weight cutoff of 10000 kDa, and 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] Amino acid sequence of the fusion protein PiuA-PiaA: ETSSSAPTEITIKSSLDEVKLSKVPEKIVTFDL (SEQ ID NO. 4);

[0081] ② Gel electrophoresis was used to detect the unpurified mixed protein in step ①, the fusion protein GST-PiuA-PiaA before purification and enzyme digestion, the fusion protein PiuA-PiaA+GST tag protein after enzyme digestion, and the fusion protein PiuA-PiaA after affinity chromatography. The results are as follows: Figure 5 As shown;

[0082] Depend on Figure 5 It can be seen that this example successfully prepared PiuA-PiaA fusion protein with a purity of >95%, which can be used for further vaccine preparation.

[0083] 3) Vaccine preparation:

[0084] Add 1 mL of PiuA-PiaA fusion protein solution (0.15 mg / mL) to 1 mL of PAA-CPE30 solution (0.75 mg / mL), mix by pipetting, and allow to react at room temperature for 30 min to obtain the PAA-CPE30@PiuA-PiaA vaccine; the “@” in this scheme indicates the electrostatic adsorption relationship between PAA and the fusion protein.

[0085] Comparative Example 1

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

[0087] Comparative Example 2

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

[0089] Test example

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

[0091] 1. Detection of cytotoxicity of PAA-CPE30@PiuA-PiaA by CCK-8 method:

[0092] 1×10 4 After DC2.4 cells were inoculated at a density of 100 cells / well and cultured overnight, the culture medium was discarded, and then different concentrations of PAA-CPE30@PiuA-PiaA solution (wherein the concentrations of PAA-CPE30 were 0, 100, 250, 500, 600, 750, 800, 900, and 1000 μg / mL, respectively, and the mass ratio of PAA-CPE30:PiuA-PiaA=5:1 was mixed) were added to each well and cultured for 24 h. The live cell ratio was detected by CCK-8 kit (DOJINDO, Lot.SX748) and detected by multifunctional microplate reader at a wavelength of 450 nm. The results are shown in the figure. Figure 6 shown.

[0093] Depend on Figure 6It can be seen that compared with the control group, no obvious cytotoxicity was detected when the concentration of PAA-CPE30 was less than 800 μg / mL, indicating that the PAA-CPE30 provided by the present invention has good biocompatibility.

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

[0095] In the mouse model, PBS buffer (control group), PBS + fusion protein PiuA-PiaA, vaccine PAA@PiuA-PiaA, vaccine PAA-CPE30@PiuA-PiaA and vaccine CTB+PiuA-PiaA were used for 3 times of nasal drip (each time with an interval of 14 days, each time 200 μL of vaccine was dripped into the nose of each mouse, corresponding to 30 μg of antigen), and then the heart, liver, spleen, lung and kidney tissues of the mice were taken for H&E staining to observe the pathological damage of each vaccine preparation to the main organs of the body. The results are shown in the figure. Figure 7 shown.

[0096] Depend on Figure 7 It can be seen that there was no obvious pathological damage in each treatment group compared with the control group, indicating that PAA-CPE30 loaded with fusion protein PiuA-PiaA will not cause obvious organ damage, and its biocompatibility is high and suitable for wide application.

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

[0098] DC is a type of professional antigen presenting cell (APC) that can recognize, capture, process and present antigens to naive T cells, thereby inducing the body to produce antigen-specific immune responses. It is the key to connecting the body's innate and adaptive immune responses. The DC's uptake efficiency and presentation pathway for exogenous antigens play a decisive role in the effectiveness of vaccination and the type of immune response. Therefore, this experimental example used confocal laser scanning microscopy (CLSM) to test the DC's antigen uptake efficiency with or without the assistance of vaccine adjuvant PAA-CPE30 at the in vitro cell level and the lysosomal escape effect of the antigen after the vaccine preparation entered the DC.

[0099] In this test example, fluorescently labeled chicken ovalbumin OVA-Cy5 was used as a model antigen instead of the fusion protein PiuA-PiuA. 5 DC2.4 cells per well were incubated with PBS / OVA-Cy5, PAA@OVA-Cy5, and PAA-CPE30@OVA-Cy5 preparation solutions (final antigen concentration: 0.15 mg / mL, final adjuvant concentration: 0.75 mg / mL) for 2 h or 6 h, respectively. After the incubation, DC2.4 cells were observed by CLSM. The results are shown in the figure. Figure 8shown.

[0100] Depend on Figure 8 It can be seen that when DC2.4 was incubated with different preparations for 2 hours, PAA-CPE30 can assist the antigen protein to be taken up by DC2.4 more quickly and efficiently, 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 preparations for 6 hours, it can be found that compared with the antigen alone group, after the addition of polymer PAA or PAA-CPE30, more antigens were taken up by DC2.4, and the lysosomal 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 of PAA-CPE30:

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

[0103] Depend on Fig. 9 It can be seen that when the cationic polymer PAA or PAA-CPE30 is introduced, the antigen protein in the nasal cavity of the corresponding immunized group mice can stay longer, and the amount of antigen protein in the nasal cavity of the PAA or PAA-CPE30 treated mice is greater than that of the control group in the same time period, indicating that its antigen delivery and targeting are stronger.

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

[0105] Balb / c mice were immunized by intranasal drops with PBS+PiuA-PiaA, PAA@PiuA-PiaA, CTB+PiuA-PiaA and PAA-CPE30@PiuA-PiaA, respectively. The control group was injected with 1×PBS. Each mouse was given 30 μg of fusion protein PiuA-PiaA. There were 6 mice in each group. They were immunized 3 times with an interval of 14 days between each immunization. The antibody titer of PiuA-PiaA in the mice was detected by sampling 7 days after the third immunization.

[0106] 1) Generally, the immune response caused by protein vaccines is humoral immunity. During the humoral immunity process, the body produces IgG antibodies in the serum to resist the invasion of pathogens through different mechanisms. Therefore, the antibody titer of IgG in the serum reflects the strength of the humoral immune response caused by the antigen. The results of this test example for detecting the IgG antibody titer in the serum after the immune response are as follows: Fig.10 shown.

[0107] Depend on Fig.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 induce strong humoral immune responses, manifested as higher serum IgG antibody titers.

[0108] 2) The immune response to the antigen is generated through the interaction between the antigen and the immune system cells. The immune response generated can be divided into two major categories, namely Th1 type immune response (cell-mediated immune response) and Th2 type immune response (humoral immune response). The production of IgG1 and IgG2a subtypes in mice is related to Th2 and Th1 type immune responses, respectively. Therefore, this test example detected the antibody titers of IgG1 and IgG2a in mouse serum, and the results were as follows: Fig.11 shown.

[0109] Depend on Fig.11 It can be seen that PAA-CPE30@PiuA-PiaA can stimulate higher IgG1 and IgG2a antibody titers, 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 infection by pathogens in the mucosal area. PiuA-PiaA-specific SIgA antibodies were detected in the nasal mucosal washes of mice after vaccination. The results are as follows Fig.12 shown.

[0111] Depend on Fig.12 It can be seen that after three immunizations, a higher SIgA antibody titer was detected in the nasal mucosal washes of mice in the PAA-CPE30@PiuA-PiaA group, and this result was similar to that of the IgG2a antibody.

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

[0113] In this experiment, a mouse pneumococcal infection model was constructed to evaluate the protective effect of PAA-CPE30@PiuA-PiaA vaccine against pneumococcal infection. Balb / c mice were immunized with PBS+PiuA-PiaA, PAA@PiuA-PiaA, CTB+PiuA-PiaA and PAA-CPE30@PiuA-PiaA by intranasal drops, respectively. The control group was injected with 1×PBS. Each mouse was given 30 μg of fusion protein PiuA-PiaA. There were 11 mice in each group. The mice were immunized 3 times with an interval of 14 days between each immunization. Seven days after the third immunization, 1×10 9 CFU of Streptococcus pneumoniae. After 48 hours, three mice were randomly selected from each group for pathological examination, and the survival of the remaining mice was observed every day. The survival rate results were as follows: Fig.13 The results of H&E staining of pathological sections are shown in Fig.14 shown.

[0114] Depend on Fig.13 It can be seen that the survival rate of mice in the PAA@PiuA-PiaA vaccine immunization group and the PAA-CPE30@PiuA-PiaA vaccine immunization group was significantly higher than that in the control group, and was statistically significant, indicating that the vaccine provided by the present invention can effectively play a protective role in pneumococcal infection.

[0115] Depend on Fig.14 It can be seen that the alveolar structure of mice immunized with PAA-CPE30@PiuA-PiaA vaccine was intact and the alveoli were relatively thin; a large number of inflammatory cell infiltrations and unformed alveoli were observed in the lung sections of mice in the control group.

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

[0117] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention 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 invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A functionalized cationic polymer, characterized in that The functionalized cationic polymer includes a cationic polymer coupled to a targeting polypeptide; The amino acid sequence of the targeting polypeptide is shown in SEQ ID NO.3; The cationic polymer includes polyamidoamine.

2. The functionalized cationic polymer according to claim 1, characterized in that The chemical structural formula of the polyamidoamine is as follows:

3. A method for preparing a functionalized cationic polymer as claimed in claim 1 or 2, characterized in that: The preparation method comprises the following steps: mixing polyamide-amine, targeting polypeptide and cross-linking agent, and reacting them to obtain functionalized cationic polymer.

4. The preparation method according to claim 3, characterized in that: The crosslinking agents include 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 polyamidoamine and the targeting polypeptide is (4-6):

3.

6. Use of the functionalized cationic polymer according to claim 1 or 2 in the preparation of vaccine adjuvants.

7. A vaccine, characterized in that The vaccine comprises the functionalized cationic polymer as claimed in claim 1 or 2 and an immunogen; the immunogen comprises a fusion protein; the amino acid sequence of the fusion protein is shown in SEQ ID NO.

2.

8. The vaccine according to claim 7, characterized in that The nucleotide sequence of the nucleic acid molecule encoding the fusion protein is shown in SEQ ID NO.

1.

9. A method for preparing the vaccine according to claim 7 or 8, characterized in that: The preparation method comprises the following steps: mixing the fusion protein solution and the functionalized cationic polymer solution.

10. Use of the vaccine according to claim 7 or 8 in the preparation of a product for preventing and / or treating pneumonia.

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