Preparation method and application of pseudomonas aeruginosa DNA vaccine combined with CpG-ODN adjuvant

Abstract

The invention relates to the technical field of vaccine preparations, in particular to a preparation method and application of a pseudomonas aeruginosa DNA vaccine combined with a CpG-ODN adjuvant. The invention discloses a pseudomonas aeruginosa DNA vaccine combined with a CpG-ODN adjuvant. The pseudomonas aeruginosa DNA vaccine is prepared from the following raw materials: CpG oligonucleotide and a pseudomonas aeruginosa DNA vaccine. According to the technical scheme, the technical problems that an existing DNA vaccine is insufficient in immunogenicity, and effective immune response is difficult to induce can be solved. A synergistic effect is generated between the adjuvant and the vaccine, the secretion of cell factors is promoted, the antibody titer is improved, and the immune response effect is improved in the aspect of inducing the immune response of Th1 / Th2 / Th17 mixed cells. According to the technical scheme, a small-dose adjuvant is innovatively combined to enhance the immunogenicity of the vaccine, the immune efficacy of the vaccine and the safety of the vaccine are improved, and the novel DNA vaccine is simple in preparation method, remarkable in effect and ideal in application and popularization prospect.

Description

Technical Field

[0001] The invention relates to the technical field of vaccine preparations, and in particular to a preparation method and application of a Pseudomonas aeruginosa DNA vaccine combined with a CpG-ODN adjuvant. Background Art

[0002] DNA vaccines have obvious advantages, such as high safety, good stability, suitability for mass production, and low cost. DNA vaccines can continuously express antigens in the body, do not require frequent booster immunization, and provide long-term protection. The production process of DNA vaccines is relatively simple, and does not require the cultivation of pathogens or large-scale purification of antigens, which reduces manufacturing costs and is also easy to mass produce. DNA vaccines are relatively stable at room temperature, easy to store and transport, and do not require a cold chain, which is especially important in resource-limited or remote areas. DNA vaccines do not contain live viruses or bacterial components, reducing the risk of disease caused by the vaccine itself, and there will be no problem of virulence recovery that may occur in attenuated vaccines. DNA technology allows for rapid adjustment of vaccine design to respond to viral mutations, and multiple antigens can be easily combined to prepare multivalent vaccines.

[0003] Although DNA vaccines have the above advantages, no DNA vaccines for human use have been approved for marketing. DNA vaccines need to be delivered to the cell nucleus to work effectively. Compared with mRNA vaccines that can work in the cytoplasm, it is more difficult to achieve an effective immune response with DNA vaccines. DNA molecules are large and negatively charged, and it is difficult to pass through the cell membrane, which is also negatively charged. This means that without an effective delivery mechanism, it is difficult for DNA vaccines to enter the cell, let alone reach the cell nucleus for transcription and expression. Even if DNA successfully enters the cytoplasm, it still needs to pass through the nuclear pore complex to enter the cell nucleus, which is another major obstacle. The cell nucleus has a selective barrier that only allows molecules of a certain size and nature to pass through, and natural DNA molecules usually have difficulty directly penetrating this barrier. Therefore, for a long time, DNA vaccines have been difficult to induce effective immune responses in clinical trials. The effects of DNA vaccines on small animals are relatively ideal, but it is difficult to produce the same level of immune protection response in human experiments, and the effectiveness of DNA vaccines in large animals is difficult to guarantee. Therefore, the DNA vaccines currently on the market are all animal vaccines, and DNA vaccines used in humans, such as melanoma vaccines, HIV virus, influenza virus and Zika virus vaccines, are still in the clinical trial stage and have not been approved. Therefore, how to effectively improve the delivery efficiency of DNA vaccines and achieve an effective immune response has been a problem that the field has been committed to solving for a long time. Summary of the invention

[0004] The purpose of the present invention is to provide a Pseudomonas aeruginosa DNA vaccine combined with a CpG-ODN adjuvant to solve the technical problems of insufficient immunogenicity of existing DNA vaccines and difficulty in inducing effective immune responses.

[0005] In order to achieve the above object, the present invention adopts the following technical scheme:

[0006] A Pseudomonas aeruginosa DNA vaccine combined with a CpG-ODN adjuvant, the raw materials of which include a CpG-ODN (CpG oligonucleotide) adjuvant and a Pseudomonas aeruginosa DNA vaccine.

[0007] Furthermore, the Pseudomonas aeruginosa DNA vaccine includes pVAX1-OprF plasmid and / or pVAX1-PcrV plasmid.

[0008] Furthermore, the pVAX1-OprF plasmid is formed by integrating the OprF gene into the pVAX1 empty vector; the pVAX1-PcrV plasmid is formed by integrating the PcrV gene into the pVAX1 empty vector; the GenBank ID number of the OprF gene is 878442; and the GenBank ID number of the PcrV gene is NC_002516.2.

[0009] Furthermore, the mass ratio of CpG-ODN adjuvant, pVAX1-OprF plasmid and pVAX1-PcrV plasmid was 20:10:10.

[0010] Furthermore, the Pseudomonas aeruginosa DNA vaccine is combined with a transfection reagent, and the usage ratio of the transfection reagent to the Pseudomonas aeruginosa DNA vaccine is 2 μL:1 μg.

[0011] The technical solution also provides a method for preparing a Pseudomonas aeruginosa DNA vaccine combined with a CpG-ODN adjuvant, wherein an in vivo transfection reagent is mixed with the Pseudomonas aeruginosa DNA vaccine and incubated, and then the CpG-ODN adjuvant is added, and after dilution, the Pseudomonas aeruginosa DNA vaccine combined with the CpG-ODN adjuvant is obtained.

[0012] Furthermore, the in vivo transfection reagent was mixed with the P. aeruginosa DNA vaccine and incubated for 15 min at room temperature, and then the CpG-ODN adjuvant was added.

[0013] Furthermore, in the Pseudomonas aeruginosa DNA vaccine combined with CpG-ODN adjuvant, the contents of CpG-ODN adjuvant, pVAX1-OprF plasmid and pVAX1-PcrV plasmid were 20 μg / 200 μL, 10 μg / 200 μL and 10 μg / 200 μL, respectively.

[0014] Further, the in vivo transfection reagent is Entranster TM-in vivo, the usage ratio of in vivo transfection reagent and Pseudomonas aeruginosa DNA vaccine was 2μL:1μg.

[0015] The technical solution also provides an application of a Pseudomonas aeruginosa DNA vaccine combined with a CpG-ODN adjuvant in the preparation of a drug for preventing and treating diseases caused by Pseudomonas aeruginosa infection.

[0016] Furthermore, the drug is used to induce a mixed Th1 / Th2 / Th17 cellular immune response.

[0017] The principles and beneficial effects of this technical solution are as follows:

[0018] This technical solution mainly solves the problem of insufficient immunogenicity of DNA vaccines, and uses a small dose of CpG-ODN adjuvant to enhance the immunogenicity of the vaccine, thereby enhancing the immune efficacy of the vaccine. The CpG-ODN adjuvant and DNA vaccine produce a synergistic effect, which promotes the secretion of cytokines under antigen stimulation, increases antibody titers, and improves the immune response effect in inducing Th1 / Th2 / Th17 mixed cellular immune responses. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The in vivo biosafety evaluation results of Example 2 (tissue pathology sections).

[0020] Figure 2 The in vivo biosafety evaluation results of Example 2 (blood routine heat map).

[0021] Figure 3 The in vivo immune response experiment results of Example 2 (photo of typical colony formation).

[0022] Figure 4 The in vivo immune response experiment results of Example 2 (statistical results of the number of colonies formed).

[0023] Figure 5 This is the result of detecting the cytokine expression level of spleen lymphocytes in Example 3 (OprF antigen stimulation).

[0024] Figure 6 This is the result of detecting the cytokine expression level of spleen lymphocytes in Example 3 (PcrV antigen stimulation). DETAILED DESCRIPTION

[0025] The present invention is further described in detail below with reference to the examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art; the experimental methods used are conventional methods; the materials, reagents, etc. used can all be obtained from commercial sources. Among them, the molecular cloning methods used (such as DNA vaccine plasmids) are all conventional methods and can be completed according to the described recombinant technology (see Molecular Cloning, Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York).

[0026] Embodiment 1:

[0027] CpG oligonucleotide (CpG ODN) is a special type of DNA oligonucleotide, usually with a length of 10-30 bases, where "CpG" means a cytosine and a guanine are arranged together in a linked manner. In this technical solution, the CpG oligonucleotide is specifically CpG ODN 1826 adjuvant, which comes from MCE (manufacturer); CAS number: 202668-42-6; item number: #HY-146245 / CS-0530626.

[0028] The vaccine used in this technical solution is a Pseudomonas aeruginosa DNA vaccine (including two parts: the recombinant plasmid of pVAX1-PcrV and the recombinant plasmid of pVAX1-PcrV). The details are as follows:

[0029] The recombinant plasmid of pVAX1-PcrV was synthesized by Shanghai Genomics, Inc. The PcrV gene (GenBank: NC_002516.2) was inserted into the pVAX1 vector by conventional molecular cloning methods to construct the pVAX1-PcrV recombinant plasmid. pVAX1-OprF can also be commissioned to be synthesized by conventional methods of existing technology. The construction method of the recombinant plasmid can refer to the literature (X. Yu, Y. Wang, Y. Xia, L. Zhang, Q. Yang, J. Lei, A DNA vaccine encoding VP22 of herpes simplex virus type I (HSV-1) and OprF confers enhanced protection from Pseudomonas aeruginosa in mice, Vaccine 34 (37) (2016) 4399–4405.). Among them, the GenBank number of the OprF gene is Gene ID: 878442, and it is inserted into the pVAX1 empty vector by conventional molecular cloning methods of existing technology. For pVAX1-OprF and pVAX1-PcrV plasmids, bacterial genomic DNA was extracted using a bacterial genomic DNA extraction kit (Tiangen Biotechnology Co., Ltd., Beijing, China), and pVAX1-OprF plasmid and pVAX1-PcrV plasmid were separated and purified by conventional means for subsequent vaccine preparation. The pVAX1-OprF plasmid and pVAX1-PcrV plasmid can also be found in the inventor's paper (Yating Zhang, Linxia Tian, ​​Xuan Zhao, XiaoFengJiang, Jiaglei Qin, Yong Wang, Xian Yu*. Enhanced protective efficacy of anOprF / PcrV bivalent DNA vaccine against Pseudomonas aeruginosa using ahydrogel delivery system. Biomedicine & Pharmacotherapy, 172 (2024) 116264), which is a DNA vaccine disclosed by the inventor in the paper.

[0030] If CpG ODN is added, in vivo transfection reagent (Entranster TM-in vivo, catalog number: 18668-11-2) and plasmid were combined at room temperature for 15 minutes, and then CpG ODN was added. The mass ratio of the sum of the mass of pVAX1-OprF and pVAX1-PcrV plasmid vectors to CpGODN was 1: 1. For example, the system of Pseudomonas aeruginosa DNA vaccine combined with CpG-ODN adjuvant is 200 μL in total (the dose for one immunization per mouse), which contains 10 μg pVAX1-OprF, 10 μg pVAX1-PcrV, and 20 μg CpG-ODN adjuvant.

[0031] Example 2

[0032] The preparation of Example 1 was used to immunize mice (6-8 weeks old mice), and the experimental groups and experimental methods were as follows:

[0033] PBS group: PBS was used for immunization of mice. The specific method was: subcutaneous injection of 200 μl PBS per mouse.

[0034] OP group: En / (pVAX1-OprF+pVAX1-PcrV) was used for immunization of mice. The specific method was as follows: the in vivo transfection reagent and plasmid were mixed at room temperature in a ratio of 2:1 (the plasmids included 10 μg of pVAX1-OprF and pVAX1-PcrV, respectively), allowed to stand for 15 min, and finally diluted with sterile enzyme-free water to 200 μl / mouse system, and injected subcutaneously. Immunization was performed once every 2 weeks, for a total of three times.

[0035] CpG group: mice were immunized with CpG ODN. The specific method was as follows: Similarly, 0.1 μg / μl CpG ODN, 200 μl (20 μg CpG ODN in total) was subcutaneously injected once every 2 weeks for a total of three immunizations.

[0036] COP group: En / (pVAX1-OprF+pVAX1-PcrV)+CpG was used for immunization of mice. The specific method was as follows: the in vivo transfection reagent and plasmid (10 μg of pVAX1-OprF and pVAX1-PcrV, respectively) were mixed at a ratio of 2:1 at room temperature, allowed to stand for 15 min, and 20 μg of CpG ODN was added and mixed evenly. Finally, the mice were diluted with sterile enzyme-free water to a system of 200 μl / mouse, and injected subcutaneously. Immunization was carried out once every 2 weeks for a total of three times.

[0037] In vivo biosafety: After three immunizations, important organs of mice (heart, liver, lungs, and kidneys) were taken for histopathological sections to observe the changes in important organs of mice after three immunizations and evaluate the safety of the vaccine. The results showed that there was no significant difference compared with the PBS control group, and no significant structural changes or inflammation were observed. The results show (see Figure 1), the vaccine preparation of this scheme has good biocompatibility in vivo.

[0038] After three immunizations, the mice were tested for blood routine to evaluate the safety of the vaccine. Figure 2 ), the blood routine test results of each group were similar to those of the PBS group, indicating that the preparation had good biosafety.

[0039] In vivo immune response experiments were conducted on the mice treated as above, using the lung colony counting method. The specific process was as follows: 40 hours after the three immunizations, the lung tissues of the mice were collected and the bacterial load in the lungs was detected. More specifically, 2 weeks after the three immunizations, the BALB / C mice were anesthetized and fixed on a 60° inclined wooden board. A non-lethal dose (9×10 7 CFU / mL) of PA01 bacteria (Pseudomonas aeruginosa), hang the mouse incisors with a thin rope, gently pull out the mouse tongue with tweezers, aim the transmitted light at the mouse neck, adjust the animal tilt angle until the glottic fissure can be clearly seen through the mouth, then insert the indwelling needle (the needle consists of an internal needle core and an external hose) into the airway, then withdraw the needle core, and push 50μl of the above concentration of bacterial solution inward, and keep the mouse head high and foot bottom position, rotate it left and right, so that the bacterial solution is evenly distributed in the left and right lungs of the mouse. 40h after bacterial attack, dissect the mouse, remove the mouse lungs, and homogenize them in 1ml sterile PBS, then inoculate the homogenate on the LB plate and culture it at 37℃ overnight, and calculate the number of colonies in the lung tissue of each group of mice.

[0040] Figure 3 The typical colony formation after different treatments of lung tissue is shown. Figure 4 Statistical images of the number of colony formations in different treatments are shown. Compared with the control group, there was no significant difference in the bacterial load in the lungs of mice in the PBS and CpG groups, and the bacterial load in the lungs of mice in the COP group was significantly reduced. Compared with the CpG group or the OP group, the bacterial load in the lungs of mice in the COP group was significantly reduced. In the COP group, there was a significant synergistic effect between CpG ODN and DNA vaccines (pVAX1-OprF and pVAX1-PcrV plasmids).

[0041] Among them, Figure 4The bacterial load in the lungs of mice in the PBS group was 247.67±9.02 CFU / mL; the bacterial load in the lungs of mice in the CpG group was 164.67±83.55 CFU / mL; the bacterial load in the lungs of mice in the OP group was 146±13.87 CFU / mL; and the bacterial load in the lungs of mice in the COP group was 33±4.36 CFU / mL (n=3, mean±standard deviation). Immunizing mice with CpG ODN resulted in a reduction in the bacterial load in the mice's lungs of 247.67 CFU / mL-164.67 CFU / mL = 83 CFU / mL; immunizing mice with En / (pVAX1-OprF+pVAX1-PcrV) resulted in a reduction in the bacterial load in the mice's lungs of 247.67 CFU / mL-146 CFU / mL = 101.67 CFU / mL; immunizing mice with En / (pVAX1-OprF+pVAX1-PcrV)+CpG ODN resulted in a reduction in the bacterial load in the mice's lungs of 247.67 CFU / mL-33 CFU / mL = 214.67 CFU / mL > 83 + 101.67 CFU / mL. This shows that the combined use of CpG oligonucleotides and the DNA vaccine of this scheme has a synergistic effect in inhibiting lung infection caused by Pseudomonas aeruginosa.

[0042] Example 3

[0043] The embodiment studied the effect of the preparation on cytokine secretion, and the experimental grouping and experimental methods were the same as in Example 2. 14 days after the three immunizations of mice, the mouse spleen lymphocytes were taken by conventional means of the prior art, and the supernatant was collected after stimulation with the corresponding protein (OprF antigen or PcrV antigen), and the corresponding cytokine levels were detected. The ELISA kit was used to detect the levels of Th1 cytokines (TNF-α), Th2 cytokines (IL-4) and Th17 cytokines (IL-17) secreted by the spleen lymphocytes of the immunized mice. More specifically, the concentration of the prepared spleen lymphocyte suspension was adjusted to 2×10 6 cells / mL, 100 μL of the cell suspension was plated in a 96-well plate, and spleen lymphocytes were stimulated with OprF antigen and PcrV antigen (final concentration 10 μg / mL), respectively, and incubated for 72 h. The supernatant was collected by centrifugation at 1000 r / min for 5 min, and the levels of TNF-α, IL-4, and IL-17 in the supernatant of spleen lymphocytes were determined by ELISA commercial kits.

[0044] The experimental results can be found in Figure 5 and Figure 6, under the stimulation of OprF or PcrV protein, compared with the CpG group and the OP group, the spleen lymphocytes of the mice in the OPC group induced higher levels of TNF-α, IL-4, and IL-17. The results suggest that OPC (En / (pVAX1-OprF+pVAX1-PcrV)+CpG) is a better DNA vaccine for inducing OprF and PcrV-specific cell responses, and the vaccine induces a mixed Th1 / Th2 / Th17 cellular immune response. In the prior art, although CpG oligonucleotides and other types of DNA vaccines are used in combination, these combined uses do not show the synergistic effect of stimulating the secretion of Th1 / Th2 / Th17 type cytokines at the same time. The combined use of CpG oligonucleotides and the special DNA vaccine of this scheme can enhance the secretion of cytokines in the above three aspects, and all produce synergistic effects. This shows that the effect of CpG oligonucleotides on the immune effect enhancement of the specific DNA vaccine of this scheme is particularly obvious, and the immune effect can be guaranteed through multiple pathways.

[0045] After OprF protein stimulation, the TNF-α levels in the PBS group, CpG group, OP group, and COP group were 202.21±17.86, 226.41±14.08, 272.1±11.38, and 314.69±16.64 (n=6, pg / mL), respectively. The COP group used a combination of CpG oligonucleotides + DNA vaccines, which resulted in an increase in TNF-α levels of 314.69pg / mL-202.21pg / mL = 112.48pg / mL compared to the blank control PBS; the CpG group used only CpG oligonucleotides, which resulted in an increase in TNF-α levels of 226.41pg / mL-202.21pg / mL = 24.2pg / mL; the OP group used only DNA vaccines, which resulted in an increase in TNF-α levels of 272.1pg / mL-202.21pg / mL = 69.89pg / mL. TNF-α is a Th1 cytokine secreted by mouse spleen lymphocytes, reflecting the immune response of the organism. The effect of increasing TNF-α levels in the COP group was greater than the sum of the effects of increasing TNF-α levels in the CpG group and the OP group, indicating that the combination of CpG oligonucleotide and DNA vaccine produced a synergistic effect in the immune response effect (stimulating the secretion of Th1 cytokines) when responding to OprF antigen stimulation.

[0046] After OprF protein stimulation, the IL-4 levels of the PBS group, CpG group, OP group, and OPC group were 71.44±5.06, 84.41±2.07, 134.89±2.58, and 148.16±0.73 (n=6, pg / mL), respectively. IL-4 is a Th2-type cytokine secreted by mouse spleen lymphocytes, which reflects the immune response effect of the organism. The effect of increasing the IL-4 level in the COP group was greater than the sum of the effects of increasing the IL-4 levels in the CpG group and the OP group, indicating that the combination of CpG oligonucleotides and DNA vaccines produced a synergistic effect in the immune response effect (stimulating the secretion of Th2-type cytokines) in response to OprF antigen stimulation.

[0047] After OprF protein stimulation, the IL-17 levels of the PBS group, CpG group, OP group, and OPC group were 13.75±0.58, 14.38±0.77, 33.56±1.26, and 38.43±2.7 (n=6, pg / mL), respectively. IL-17 is a Th17-type cytokine secreted by mouse spleen lymphocytes, which reflects the immune response effect of the organism. The effect of increasing the IL-17 level in the COP group was greater than the sum of the effects of increasing the IL-17 levels in the CpG group and the OP group, indicating that the combination of CpG oligonucleotides and DNA vaccines produced a synergistic effect in the immune response effect (stimulating the secretion of Th17-type cytokines) in response to OprF antigen stimulation.

[0048] After PcrV protein stimulation, the TNF-α levels of PBS group, CpG group, OP group, and OPC group were 130.8±7.61, 156.68±12.97, 373.53±10.11, and 439.52±40.33 (n=6, pg / mL). The COP group used a combination of CpG oligonucleotides + DNA vaccines, and the TNF-α level improvement effect relative to the blank control PBS was: 439.52pg / mL-130.8pg / mL=308.2pg / mL; the CpG group only used CpG oligonucleotides, and the TNF-α level improvement effect was: 156.68pg / mL-130.8pg / mL=25.88pg / mL; the OP group only used DNA vaccines, and the TNF-α level improvement effect was: 373.53pg / mL-130.8pg / mL=242.73pg / mL. TNF-γ is a Th1 cytokine secreted by mouse spleen lymphocytes, reflecting the immune response of the organism. The effect of increasing the TNF-α level in the COP group was greater than the sum of the effects of increasing the TNF-α levels in the CpG group and the OP group, indicating that the combination of CpG oligonucleotides and DNA vaccines produced a synergistic effect in the immune response effect (stimulating the secretion of Th1 cytokines) in response to PcrV antigen stimulation.

[0049] After PcrV protein stimulation, the IL-4 levels of PBS group, CpG group, OP group, and OPC group were 64.73±2.6, 62.42±3.68, 104.98±3.56, and 124.24±3.38 (n=6, pg / mL). The COP group used a combination of CpG oligonucleotides + DNA vaccines, and the IL-4 level improvement effect relative to the blank control PBS was: 124.24pg / mL-64.73pg / mL=59.51pg / mL; the CpG group only used CpG oligonucleotides, and the IL-4 level improvement effect was: 62.42pg / mL-64.73pg / mL=-2.31pg / mL; the OP group only used DNA vaccines, and the IL-4 level improvement effect was: 104.98pg / mL-64.73pg / mL=40.25pg / mL. IL-4 is a Th2 cytokine secreted by mouse spleen lymphocytes, reflecting the immune response of the organism. The effect of increasing the IL-4 level in the COP group was greater than the sum of the effects of increasing the IL-4 levels in the CpG group and the OP group, indicating that the combination of CpG oligonucleotides and DNA vaccines produced a synergistic effect in the immune response effect (stimulating the secretion of Th2 cytokines) in response to PcrV antigen stimulation.

[0050] After PcrV protein stimulation, the IL-17 levels of the PBS group, CpG group, OP group, and OPC group were 12.89±0.95, 15.18±0.7, 26.99±0.81, and 31.16±1.05 (n=6, pg / mL), respectively. IL-17 is a Th17-type cytokine secreted by mouse spleen lymphocytes, which reflects the immune response effect of the organism. The effect of increasing the IL-17 level in the COP group was greater than the sum of the effects of increasing the IL-17 levels in the CpG group and the OP group, indicating that the combination of CpG oligonucleotides and DNA vaccines produced a synergistic effect in the immune response effect (stimulating the secretion of Th17-type cytokines) in response to PcrV antigen stimulation.

[0051] The above is only an embodiment of the present invention, and the common knowledge such as the known specific technical solutions and / or characteristics in the solution is not described in detail here. It should be pointed out that for those skilled in the art, without departing from the technical solution of the present invention, several modifications and improvements can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. A Pseudomonas aeruginosa DNA vaccine combined with a CpG-ODN adjuvant, characterized in that: Its raw materials include CpG-ODN adjuvant and Pseudomonas aeruginosa DNA vaccine.

2. The Pseudomonas aeruginosa DNA vaccine combined with CpG-ODN adjuvant according to claim 1, characterized in that: The Pseudomonas aeruginosa DNA vaccine includes pVAX1-OprF plasmid and / or pVAX1-PcrV plasmid.

3. The Pseudomonas aeruginosa DNA vaccine combined with CpG-ODN adjuvant according to claim 2, characterized in that: The pVAX1-OprF plasmid is formed by integrating the OprF gene into the pVAX1 empty vector; the pVAX1-PcrV plasmid is formed by integrating the PcrV gene into the pVAX1 empty vector; the GenBank ID number of the OprF gene is 878442; the GenBank ID number of the PcrV gene is NC_002516.

2.

4. The Pseudomonas aeruginosa DNA vaccine combined with CpG-ODN adjuvant according to claim 3, characterized in that: The mass ratio of CpG-ODN adjuvant, pVAX1-OprF plasmid and pVAX1-PcrV plasmid was 20:10:

10.

5. The Pseudomonas aeruginosa DNA vaccine combined with CpG-ODN adjuvant according to claim 4, characterized in that: The Pseudomonas aeruginosa DNA vaccine is combined with a transfection reagent, and the usage ratio of the transfection reagent to the Pseudomonas aeruginosa DNA vaccine is 2 μL:1 μg.

6. A method for preparing a Pseudomonas aeruginosa DNA vaccine combined with a CpG-ODN adjuvant according to any one of claims 1 to 5, characterized in that: The in vivo transfection reagent and the Pseudomonas aeruginosa DNA vaccine are mixed and incubated, and then a CpG-ODN adjuvant is added, and after dilution, a Pseudomonas aeruginosa DNA vaccine combined with a CpG-ODN adjuvant is obtained.

7. The method for preparing a Pseudomonas aeruginosa DNA vaccine combined with a CpG-ODN adjuvant according to claim 6, characterized in that: The in vivo transfection reagent was mixed with the P. aeruginosa DNA vaccine and incubated for 15 min at room temperature, and then the CpG-ODN adjuvant was added.

8. The method for preparing a Pseudomonas aeruginosa DNA vaccine combined with a CpG-ODN adjuvant according to claim 7, characterized in that: In the Pseudomonas aeruginosa DNA vaccine combined with CpG-ODN adjuvant, the contents of CpG-ODN adjuvant, pVAX1-OprF plasmid and pVAX1-PcrV plasmid were 20 μg / 200 μL, 10 μg / 200 μL and 10 μg / 200 μL, respectively.

9. The method for preparing a Pseudomonas aeruginosa DNA vaccine combined with a CpG-ODN adjuvant according to claim 8, characterized in that: The in vivo transfection reagent is Entranster TM -in vivo, the usage ratio of in vivo transfection reagent and Pseudomonas aeruginosa DNA vaccine was 2μL:1μg.

10. Use of a Pseudomonas aeruginosa DNA vaccine combined with a CpG-ODN adjuvant according to any one of claims 1 to 5 in the preparation of a drug for preventing and treating diseases caused by Pseudomonas aeruginosa infection, characterized in that: The drug is used to induce a Th1 / Th2 / Th17 mixed cellular immune response.

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