A vaccine against pseudomonas aeruginosa, preparation method and application

Abstract

The application belongs to the technical field of biology and particularly relates to a vaccine against pseudomonas aeruginosa, a preparation method and application. The vaccine against pseudomonas aeruginosa comprises a recombinant protein. The preparation method comprises the following steps: introducing a PA1304 gene into an expression plasmid through gene recombination to construct a recombinant plasmid, introducing the recombinant plasmid into a competent cell to obtain a recombinant bacterium, culturing the recombinant bacterium in an LB culture medium, inducing expression by using IPTG, collecting the bacterium, crushing the bacterium, collecting supernatant, purifying, obtaining the recombinant protein, mixing the recombinant protein with Freund's adjuvant, and obtaining the vaccine. The vaccine obtained by the application is used for preventing and / or treating pseudomonas aeruginosa infection, has a high effective value, effectively activates B cell differentiation and antibody secretion, and has a better effect of resisting pseudomonas aeruginosa infection.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a vaccine against Pseudomonas aeruginosa, its preparation method, and its application. Background Technology

[0002] Pseudomonas aeruginosa (PA) is a non-capsulated, non-spore-forming, motile, obligate aerobic Gram-negative bacterium widely distributed in water, soil, air, and on the skin and intestines of humans and animals, thriving particularly well in moist environments. It is an opportunistic pathogen, typically not infecting healthy individuals, but can cause severe infections in immunocompromised patients, making it one of the most important pathogens causing hospital-acquired infections. Pseudomonas aeruginosa infections can occur in multiple sites and tissues of the body, leading to acute infectious diseases such as severe pneumonia, endocarditis, gastroenteritis, and osteomyelitis.

[0003] In recent years, with the widespread use of antibiotics, drug resistance in *Pseudomonas aeruginosa* has become increasingly serious. The continuous emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains poses a severe challenge to clinical anti-infective treatment. *Pseudomonas aeruginosa* exhibits resistance to a variety of antibiotics, including β-lactams, aminoglycosides, and quinolones. Of particular concern is the surge in carbapenem-resistant strains (CRPA), which are often used as a last resort for treating multidrug-resistant *Pseudomonas aeruginosa* infections, worldwide.

[0004] Given the severity of drug resistance in Pseudomonas aeruginosa and the extreme difficulty in developing new antibiotics, the World Health Organization (WHO) has listed Pseudomonas aeruginosa as one of the three key priority pathogens. Controlling Pseudomonas aeruginosa infection and preventing the spread of drug-resistant strains have become critical challenges that urgently need to be addressed. Although the development of a Pseudomonas aeruginosa vaccine has spanned decades, no commercially available product has yet been launched. While several candidate vaccines have entered clinical trials, they have not yet achieved success due to insufficient protective efficacy or safety issues. Summary of the Invention

[0005] One of the technical problems to be solved by the present invention is to provide a vaccine against Pseudomonas aeruginosa for the prevention and / or treatment of Pseudomonas aeruginosa infection.

[0006] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows:

[0007] A vaccine against Pseudomonas aeruginosa, comprising a recombinant protein, the amino acid sequence of which is shown in SEQ ID NO:2.

[0008] Preferably, the vaccine against Pseudomonas aeruginosa provided by the present invention also includes an adjuvant.

[0009] In the anti-Pseudomonas aeruginosa vaccine provided by the present invention, the adjuvant is further preferably Freund's complete adjuvant or Freund's incomplete adjuvant.

[0010] Another problem to be solved by the present invention is to provide a method for preparing a vaccine against Pseudomonas aeruginosa, comprising the following steps:

[0011] S1. The target gene and expression plasmid pET22b(+) were double-digested with Xho I and EcoRI at 37℃.

[0012] S2. Recover the enzyme digestion product and ligate it with T4 DNA ligase at 16°C for 10 hours to obtain the ligation solution.

[0013] S3. The ligation solution was heat-shocked at 42℃ for 45s to transform E. coli DH5α competent cells, and positive clones of recombinant plasmids were screened.

[0014] S4. The positive clone recombinant plasmid was heat-shocked at 42℃ for 45s and introduced into Escherichia coli BL21 competent cells to obtain recombinant bacteria.

[0015] S5. Inoculate the recombinant bacteria into 100 mL of LB liquid medium containing ampicillin sodium and culture until OD500. 600 The seed culture was obtained when the OD value was 0.5. The seed culture was then inoculated into 2000 mL of LB liquid medium containing 100 μg / mL ampicillin sodium for fermentation. After 1 hour of culture, selenium disulfide was added to the medium at a rate of 0.002–0.008 g / L, and the culture was continued until the OD value reached 0.5. 600 The value was 0.4. IPTG was added at a concentration of 0.5 mmol / L to induce expression, and the cells were cultured at 30°C for 5 hours after induction.

[0016] S6. Centrifuge to collect bacterial cells, add PBS buffer to the bacterial cells at a weight ratio of 1:5, sonicate to disrupt the bacterial cells, and centrifuge to collect the supernatant.

[0017] S7. Filter the supernatant through a filter membrane with a pore size of 0.22 μm, then purify the filtrate with a nickel column and collect the target protein;

[0018] S8. Dialyze the target protein in PBS dialysate at 4°C for 24 hours using a dialysis membrane with a molecular cutoff of 20 kDa. Change the medium every 8 hours to obtain a recombinant protein solution.

[0019] S9. Mix the recombinant protein solution with Freund's adjuvant and emulsify by ultrasonication to obtain the vaccine.

[0020] In the method for preparing a vaccine against Pseudomonas aeruginosa provided by the present invention, preferably, the target gene is the PA1304 gene.

[0021] In the method for preparing the anti-Pseudomonas aeruginosa vaccine provided by the present invention, it is further preferred that the final concentration of ampicillin sodium in step S5 is 100 μg / mL.

[0022] In the preparation method of the anti-Pseudomonas aeruginosa vaccine provided by the present invention, preferably, the process of mixing the recombinant protein solution with Freund's adjuvant is to first adjust the recombinant protein with PBS buffer to a recombinant protein solution with a recombinant protein content of 1 mg / mL, and then mix the recombinant protein solution with Freund's complete adjuvant at a volume ratio of 1:1.

[0023] In the preparation method of the anti-Pseudomonas aeruginosa vaccine provided by the present invention, preferably, the process of mixing the recombinant protein solution with Freund's adjuvant is to first adjust the recombinant protein with PBS buffer to a recombinant protein solution with a recombinant protein content of 1 mg / mL, and then mix the recombinant protein solution with Freund's incomplete adjuvant at a volume ratio of 1:1.

[0024] Another problem to be solved by the present invention is the use of recombinant protein in the preparation of medicaments for the prevention and / or treatment of Pseudomonas aeruginosa infection, wherein the amino acid sequence of said recombinant protein is shown in SEQ ID NO:2.

[0025] The use of the recombinant protein provided by this invention in the preparation of a medicament for the prevention and / or treatment of Pseudomonas aeruginosa infection, preferably, the medicament is a vaccine.

[0026] Using the above technical solution, the vaccine titer of the recombinant protein and adjuvant reaches over 1:128000, effectively activating B cell differentiation and antibody secretion, inducing a strong humoral immune response, effectively alleviating the damage and stress response caused by Pseudomonas aeruginosa, and exhibiting good immunoprotective effects, including protection against pneumonia caused by Pseudomonas aeruginosa. It can simultaneously activate Th1, Th2, and Th17 immune responses, effectively regulating the body's cellular and humoral immune levels, and demonstrating good anti-Pseudomonas aeruginosa infection effects. The addition of 0.002–0.008 g / L selenium disulfide during fermentation effectively increased the yield of the recombinant protein, reaching over 30.64 mg / L. Attached Figure Description

[0027] Figure 1 The plasmid map of pET22b(+)-PA1304;

[0028] Figure 2The image shows the results of recombinant plasmid enzyme digestion identification. In the image: 1 is Plasmid, 2 is Plasmid Digested with XhoI-EcoRI, and M is DNA Marker.

[0029] Figure 3 The images show SDS-PAGE images of the supernatant and recombinant protein solution. In the images, M is the protein marker, 1 is the supernatant, and 2 is the recombinant protein solution.

[0030] Figure 4 The antibody titer diagram for the recombinant protein;

[0031] Figure 5 SDS-PAGE image of purified total IgG from mouse immune serum (under reduction conditions). In the image: M is the protein marker, 1-3 are the purified antibody, and 4 is the serum (after 10-fold dilution).

[0032] Figure 6 This is a graph showing the changes in mouse body weight after challenge with the virus.

[0033] Figure 7 This is a graph showing the changes in food intake of mice after challenge with the virus;

[0034] Figure 8 This is a graph showing the changes in water intake in mice after viral challenge;

[0035] Figure 9 This is a diagram showing the changes in the body posture of mice after challenge with the virus;

[0036] Figure 10 This shows the plating and culture of mouse lung tissue homogenate after challenge.

[0037] Figure 11 The bacterial load of Pseudomonas aeruginosa in mouse lung tissue after challenge;

[0038] Figure 12 This is a diagram showing the changes in mouse lung tissue after viral challenge.

[0039] Figure 13 Pathological section of mouse lung tissue after challenge (400x magnification);

[0040] Figure 14 To perform semi-quantitative analysis of lung injury scoring charts in each group of mice after viral challenge;

[0041] Figure 15 These are organ indices of mice after challenge with the virus; in the figure: a is the lung index; b is the liver index; c is the spleen index; d is the kidney index; e is the heart index;

[0042] Figure 16The figures show the secretion levels of mouse cytokines. In the figure: a represents the secretion level of IL-17A in mouse serum; b represents the secretion level of IL-4 in mouse serum; c represents the secretion level of IFN-γ in mouse serum; and d represents the secretion level of TNF-α in mouse serum.

[0043] Figure 17 This is a diagram showing the proportion of T lymphocyte subsets in the spleen of mice. Detailed Implementation

[0044] The specific embodiments of the present invention will be further described below with reference to the accompanying drawings. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0045] Example 1

[0046] A method for preparing a recombinant protein, comprising the following steps:

[0047] S1. The target gene PA1304 (nucleotide sequence as shown in SEQ ID NO:1) and expression plasmid pET22b(+) were double-digested with Xho I and EcoRI at 37℃.

[0048] S2. Recover the enzyme digestion product and ligate it with T4 DNA ligase at 16°C for 10 hours to obtain the ligation solution.

[0049] S3. The ligation solution was heat-shocked at 42℃ for 45s to transform *E. coli* DH5α competent cells, and plated onto LB agar plates containing ampicillin sodium. Positive colonies were selected for extraction of the recombinant plasmid, which was named pET22b(+)-PA1304. Figure 1 As shown; and the recombinant plasmid was verified by double enzyme digestion, the results are as follows. Figure 2 As shown, this indicates that the target gene has been successfully ligated into the expression vector;

[0050] S4. The positive clone recombinant plasmid was heat-shocked at 42℃ for 45s and introduced into Escherichia coli BL21 competent cells to obtain recombinant bacteria.

[0051] S5. Inoculate the recombinant bacteria into 100 mL of LB liquid medium containing ampicillin sodium at a final concentration of 100 μg / mL, and culture until OD. 600 The seed culture was obtained with a value of 0.5. The seed culture was inoculated into 2000 mL of LB liquid medium containing 100 μg / mL ampicillin sodium for fermentation. After 1 hour of culture, selenium disulfide was added to the medium at a concentration of 0.002 g / L, and the culture was continued until the OD value reached 0.5. 600The value was 0.4. IPTG was added at a concentration of 0.5 mmol / L to induce expression, and the cells were cultured at 30°C for 5 hours after induction.

[0052] S6. Centrifuge to collect bacterial cells, add PBS buffer to the bacterial cells at a weight ratio of 1:5, sonicate to disrupt the bacterial cells, and centrifuge to collect the supernatant.

[0053] S7. Filter the supernatant through a filter membrane with a pore size of 0.22 μm, and then purify the filtrate with a nickel column. First, elute with 30 mM imidazole elution buffer, and then elute the target protein with 100 mM imidazole elution buffer. Collect the target recombinant protein and sequence the amino acid sequence of the recombinant protein. The amino acid sequence is shown in SEQ ID NO:2.

[0054] S8. Dialyze the target protein in PBS dialysis buffer at 4°C for 24 hours using a dialysis membrane with a molecular cutoff of 20 kDa. Change the buffer every 8 hours to obtain a recombinant protein solution.

[0055] In this example, 8.6 mL of recombinant protein solution was obtained. The protein concentration was measured to be 7.83 mg / mL, and the calculated recombinant protein content was 67.34 mg.

[0056] Example 2

[0057] A method for preparing a recombinant protein, comprising the following steps:

[0058] S1. The target gene PA1304 (nucleotide sequence as shown in SEQ ID NO:1) and expression plasmid pET22b(+) were double-digested with Xho I and EcoRI at 37℃.

[0059] S2. Recover the enzyme digestion product and ligate it with T4 DNA ligase at 16°C for 10 hours to obtain the ligation solution.

[0060] S3. The ligation solution was heat-shocked at 42℃ for 45s to transform *E. coli* DH5α competent cells, and plated onto LB agar plates containing ampicillin sodium. Positive colonies were selected for extraction of the recombinant plasmid, which was named pET22b(+)-PA1304. Figure 1 As shown; and the recombinant plasmid was verified by double enzyme digestion, the results are as follows. Figure 2 As shown, this indicates that the target gene has been successfully ligated into the expression vector;

[0061] S4. The positive clone recombinant plasmid was heat-shocked at 42℃ for 45s and introduced into Escherichia coli BL21 competent cells to obtain recombinant bacteria.

[0062] S5. Inoculate the recombinant bacteria into 100 mL of LB liquid medium containing ampicillin sodium at a final concentration of 100 μg / mL, and culture until OD. 600 The seed culture was obtained when the OD value was 0.5. The seed culture was inoculated into 2000 mL of LB liquid medium containing 100 μg / mL ampicillin sodium. After culturing for 1 hour, selenium disulfide was added to the medium at a concentration of 0.008 g / L, and culturing continued until the OD value reached 0.5. 600 The value was 0.4. IPTG was added at a concentration of 0.5 mmol / L to induce expression, and the cells were cultured at 30°C for 5 hours after induction.

[0063] S6. Centrifuge to collect bacterial cells, add PBS buffer to the bacterial cells at a weight ratio of 1:5, sonicate to disrupt the bacterial cells, and centrifuge to collect the supernatant.

[0064] S7. Filter the supernatant through a filter membrane with a pore size of 0.22 μm, and then purify the filtrate with a nickel column. First, elute with 30 mM imidazole elution buffer, and then elute the target protein with 100 mM imidazole elution buffer. Collect the target recombinant protein and sequence the amino acid sequence of the recombinant protein. The amino acid sequence is shown in SEQ ID NO:2.

[0065] S8. Dialyze the target protein in PBS dialysis buffer at 4°C for 24 hours using a dialysis membrane with a molecular cutoff of 20 kDa. Change the buffer every 8 hours to obtain a recombinant protein solution.

[0066] In this example, 7.5 mL of recombinant protein solution was obtained. The protein concentration was measured to be 8.17 mg / mL, and the calculated recombinant protein content was 61.28 mg.

[0067] Example 3

[0068] A method for preparing a recombinant protein, comprising the following steps:

[0069] S1. The target gene PA1304 (nucleotide sequence as shown in SEQ ID NO:1) and expression plasmid pET22b(+) were double-digested with Xho I and EcoRI at 37℃.

[0070] S2. Recover the enzyme digestion product and ligate it with T4 DNA ligase at 16°C for 10 hours to obtain the ligation solution.

[0071] S3. The ligation solution was heat-shocked at 42℃ for 45s to transform *E. coli* DH5α competent cells, and plated onto LB agar plates containing ampicillin sodium. Positive colonies were selected for extraction of the recombinant plasmid, which was named pET22b(+)-PA1304. Figure 1 As shown; and the recombinant plasmid was verified by double enzyme digestion, the results are as follows. Figure 2 As shown, this indicates that the target gene has been successfully ligated into the expression vector;

[0072] S4. The positive clone recombinant plasmid was heat-shocked at 42℃ for 45s and introduced into Escherichia coli BL21 competent cells to obtain recombinant bacteria.

[0073] S5. Inoculate the recombinant bacteria into 100 mL of LB liquid medium containing ampicillin sodium at a final concentration of 100 μg / mL, and culture until OD. 600 The seed culture was obtained with a value of 0.5. The seed culture was inoculated into 2000 mL of LB liquid medium containing 100 μg / mL ampicillin sodium for fermentation. After 1 hour of culture, selenium disulfide was added to the medium at a concentration of 0.006 g / L, and the culture was continued until the OD value reached 0.5. 600 The value was 0.4. IPTG was added at a concentration of 0.5 mmol / L to induce expression, and the cells were cultured at 30°C for 5 hours after induction.

[0074] S6. Centrifuge to collect bacterial cells, add PBS buffer to the bacterial cells at a weight ratio of 1:5, sonicate to disrupt the bacterial cells, and centrifuge to collect the supernatant.

[0075] S7. Filter the supernatant through a filter membrane with a pore size of 0.22 μm, and then purify the filtrate with a nickel column. First, elute with 30 mM imidazole elution buffer, and then elute the target protein with 100 mM imidazole elution buffer. Collect the target recombinant protein and sequence the amino acid sequence of the recombinant protein. The amino acid sequence is shown in SEQ ID NO:2.

[0076] S8. Dialyze the target protein in PBS dialysis buffer at 4°C for 24 hours using a dialysis membrane with a molecular cutoff of 20 kDa. Change the buffer every 8 hours to obtain a recombinant protein solution.

[0077] In this example, 8.5 mL of recombinant protein solution was obtained. The protein concentration was measured to be 8.36 mg / mL, and the calculated recombinant protein content was 71.06 mg.

[0078] The purification effect of the supernatant obtained in step S6 and the recombinant protein solution obtained in step S8 was detected by SDS-PAGE. The results are as follows: Figure 3 As shown, the gel electrophoresis pattern revealed only a single 76kDa band in the purified protein, indicating that the PA1304 gene was successfully induced and purified.

[0079] Example 4

[0080] A vaccine against Pseudomonas aeruginosa comprises a recombinant protein with an amino acid sequence as shown in SEQ ID NO:2 and Freund's complete adjuvant. The recombinant protein is first adjusted to a recombinant protein solution with a recombinant protein concentration of 1 mg / mL using PBS buffer. Then, the recombinant protein solution is mixed with Freund's complete adjuvant at a volume ratio of 1:1 and ultrasonically mixed for 10 minutes to obtain the vaccine against Pseudomonas aeruginosa.

[0081] Example 5

[0082] A vaccine against Pseudomonas aeruginosa comprises a recombinant protein with an amino acid sequence as shown in SEQ ID NO:2 and Freund's incomplete adjuvant. The recombinant protein is first adjusted to a recombinant protein solution with a recombinant protein concentration of 1 mg / mL using PBS buffer. Then, the recombinant protein solution is mixed with Freund's incomplete adjuvant at a volume ratio of 1:1 and ultrasonically mixed for 15 minutes to obtain the vaccine against Pseudomonas aeruginosa.

[0083] Comparative Example

[0084] A method for preparing a recombinant protein, comprising the following steps:

[0085] S1. The target gene PA1304 (nucleotide sequence as shown in SEQ ID NO:1) and expression plasmid pET22b(+) were double-digested with Xho I and EcoRI at 37℃.

[0086] S2. Recover the enzyme digestion product and ligate it with T4 DNA ligase at 16°C for 10 hours to obtain the ligation solution.

[0087] S3. The ligation solution was heat-shocked at 42℃ for 45s to transform *E. coli* DH5α competent cells, and plated onto LB agar plates containing ampicillin sodium. Positive colonies were selected for extraction of the recombinant plasmid, which was named pET22b(+)-PA1304. Figure 1 As shown; and the recombinant plasmid was verified by double enzyme digestion, the results are as follows. Figure 2 As shown, this indicates that the target gene has been successfully ligated into the expression vector;

[0088] S4. The positive clone recombinant plasmid was heat-shocked at 42℃ for 45s and introduced into Escherichia coli BL21 competent cells to obtain recombinant bacteria.

[0089] S5. Inoculate the recombinant bacteria into 100 mL of LB liquid medium containing ampicillin sodium at a final concentration of 100 μg / mL, and culture until OD. 600 The seed culture was obtained with a value of 0.5. The seed culture was then inoculated into 2000 mL of LB liquid medium containing 100 μg / mL ampicillin sodium for fermentation until the OD value reached 0.5. 600The value was 0.4. IPTG was added at a concentration of 0.5 mmol / L to induce expression, and the cells were cultured at 30°C for 5 hours after induction.

[0090] S6. Centrifuge to collect bacterial cells, add PBS buffer to the bacterial cells at a weight ratio of 1:5, sonicate to disrupt the bacterial cells, and centrifuge to collect the supernatant.

[0091] S7. Filter the supernatant through a filter membrane with a pore size of 0.22 μm, and then purify the filtrate with a nickel column. First, elute with 30 mM imidazole elution buffer, and then elute the target protein with 100 mM imidazole elution buffer. Collect the target recombinant protein. The amino acid sequence of the recombinant protein is shown in SEQ ID NO:2.

[0092] S8. Dialyze the target protein in PBS dialysate at 4°C for 24 hours using a dialysis membrane with a molecular cutoff of 20 kDa. Change the medium every 8 hours to obtain a recombinant protein solution.

[0093] The comparative example yielded 7.0 mL of recombinant protein solution. The protein concentration was measured to be 6.53 mg / mL, and the calculated recombinant protein content was 45.71 mg.

[0094] SEQ ID NO:1

[0095]

[0096] SEQ ID NO:2

[0097] MPHPQPPIARRDAGSPYAWLEERDAPEVLDYLKAENAYLDSELADQAGLRETLFEEIRGRIRETDLSLPSPWGPWLYYQRTTAGDEYPRHYRCPRPADGSLAVDEGAEQLLLDPNQLADGGFLSLGAFSISPDQSLLAYSLDTSGDEIYRLFVKDLASGAVQALPFDDC DGSLTWANDNRTLFFGELDDTHRPHKLYRHRLGEAGAELVFEEGDGRFFLHCYRSSSERQLILLLNSKTTSEAWVLDADQPQGTFRCLAPREEGHEYYPDHGRLDGRGLWLIRSNQAGINFALYQAEESRPTREHWQLRVAHDPQRTLEDVSLNAEAVILGLRDGGLPVIE VQPQGLPAYRVQLPDAAYSLYVQDSLEFDSPCVRLRYEALNRPAQVRQLNLADGTQTVLKQTPVEGPFDADAYASRRIWASSGDGTQVPVSLVARKEVLDGIAAGRPAPLYLYGYGAYGHSLDPWFSHARLSLLERGFVFAIAHVRGGGELGEAWYRAGKLEHKQNSFDDF IACAEHLLAEGYCRPEGLAISGGSAGGLLIGAVLNQRPQLFGAAIAEVPFVDVLNSMLNPDLPLTVTEYDEWGNPQEPEVHARIAAYAPYENVRAQDYPHLLVVAGYNDSRVQYWEAAKWVAKLRATRTDANLLLLKTDLGAGHGGMSGRYQGLKDVALEYAFLLKVLGLV

[0098] Example 6

[0099] 1. Grouping and Immunization of Experimental Animals

[0100] Thirty-two clean-grade female BALB / c mice with similar physiological conditions, weight, and morphology were randomly divided into four groups of eight mice each: a blank control group (blank group), an adjuvant group, a vaccine group, and a recombinant protein group (PA group). The mice in each group were immunized as shown in Table 1.

[0101] Table 1. Grouping and Immunization of Experimental Animals

[0102]

[0103] Note: The recombinant protein content in the recombinant protein solution is 1 mg / mL.

[0104] Mice in each group were immunized via intramuscular injection in the thigh, with immunization occurring every 14 days. After each immunization, the mice were observed for 1-2 hours, and their condition, food intake, and weight were recorded every two days during the immunization period. Seven days after each immunization, blood was collected from the retro-orbital venous plexus, and the serum was stored at -80℃ for subsequent experiments.

[0105] 2. Antibody titer evaluation and validation of recombinant protein vaccines

[0106] 2.1 Antibody titer evaluation of recombinant protein vaccines

[0107] To detect the antibody titer level of the humoral immune response induced by recombinant protein, blood was collected from the retro-orbital venous plexus of mice in the vaccine group, adjuvant group, and blank control group (blank group) 7 days after each immunization. Serum was separated, and the titer of anti-recombinant protein vaccine IgG antibodies in the serum was detected by ELISA.

[0108] (1) Antibody coating: Take a clean, unopened 96-well ELISA plate and add the antigen solution diluted with coating buffer to each well according to the required number of wells. Add 100 μL (containing 50 ng of recombinant protein) to each well. After completing the antigen coating, incubate overnight at 4°C. When using, remove the coating solution, wash 2-3 times with PBST washing buffer, and then pat the ELISA plate dry on absorbent paper. Each wash should last 1-2 minutes.

[0109] (2) Blocking: Add 300 μL of 3% bovine serum albumin (BSA) to each well for blocking. After blocking at 37°C for 2 hours, wash each well. Add 300 μL of PBST washing solution to each well and wash 2-3 times with the washing solution. Pat dry on absorbent paper. Each wash should last 1-2 minutes.

[0110] (3) Serum dilution and sample addition: Start with a 1:500 dilution of serum and then perform serial dilutions of 2:1. Add the diluted serum to each well for antigen-antibody binding reaction. Incubate at 37°C for 1 hour, then wash each well with 300 μL of PBST washing buffer each time. Wash 2-3 times with the washing buffer and then pat the ELISA plate dry on absorbent paper. Each wash should last 1-2 minutes.

[0111] (4) Add secondary antibody solution: Add 100 μL of HRP-labeled goat anti-mouse IgG secondary antibody solution diluted at 1:5000, incubate at 37℃ for 1 h, pour out the reaction solution, add 300 μL of PBST washing solution quantitatively and wash 3 times or more, each time for 1 to 2 minutes, and pat dry on absorbent paper.

[0112] (5) Add substrate solution: Add 100 μL of TMB substrate colorimetric solution to each well in the dark and react at room temperature in the dark for 10 to 15 minutes.

[0113] (6) Termination of reaction: After the reaction is completed, add 50 μL of 2 mol / L H2SO4 to each well to terminate the reaction.

[0114] (7) OD value determination: The absorbance at 450 nm was measured using an ELISA reader. The results are shown in Table 2 and Figure 4 As shown.

[0115] Table 2. Antibody titers of recombinant protein vaccines

[0116]

[0117] 2.2 Purification and Validation of Recombinant Protein Polyclonal Antibody

[0118] To further verify the antibodies in the serum of immunized mice, we purified the antibodies in the serum of immunized mice using the following method:

[0119] (1) Pack Protein A packing into the chromatography column and equilibrate the packing with equilibration buffer of ≥ 5 times the packing volume.

[0120] (2) After the sample was filtered through a 0.22 μm filter membrane, the serum sample from the immunized mouse was loaded onto the column at a moderate flow rate.

[0121] (3) Clean the packing with a cleaning buffer of 5-10 times the packing volume.

[0122] (4) Use elution buffer with 3-5 times the volume of packing material for elution, and collect the eluent in separate tubes.

[0123] (5) Add 1 / 10 volume of neutralization buffer to the collected eluent.

[0124] (6) Packing material regeneration: Use 3 times the volume of equilibration buffer to clean the packing material, then use 2-3 times the packing material volume of 0.1-0.5M NaOH solution to flow through the packing material for 15-30 minutes to remove the antibodies and impurities bound to the packing material. Finally, use 5 times the packing material volume of sterile equilibration buffer to clean the packing material.

[0125] (7) The purified antibody and serum samples were subjected to SDS-PAGE protein electrophoresis under reducing conditions. The results are as follows: Figure 5 As shown.

[0126] IgG is the most important and persistent antibody type in the body, and a core marker of whether a vaccine can elicit long-lasting protective immunity. To evaluate the strength of the humoral immune response induced by recombinant protein vaccines, their immunogenicity, and the potential protective effect of the vaccines, this experiment used indirect ELISA to determine serum titers, as shown in Table 2. Figure 4 As shown, the measured titer reached over 1:128000; verification by SDS-PAGE under reducing conditions showed that the antibody cleaved into 50kD and 25kD heavy and light chains, and the purified antibody bands met expectations (e.g., Figure 5 (As shown in the figure). This indicates that the vaccine can effectively activate B cell differentiation and antibody secretion, inducing a strong humoral immune response, providing key immunological evidence for vaccine potency evaluation.

[0127] 3. Evaluation of the protective effect of recombinant protein active immunization in a mouse pneumonia model

[0128] Fifteen days after the last immunization, a challenge experiment was conducted. Mice in the blank control group (blank group), vaccine group, and recombinant protein group (PA group) were administered a nasal instillation of a concentration of 1×10⁻⁶. 9 CFU / mL 30μL / pseudomonas aeruginosa.

[0129] 3.1 Determination of mouse growth performance

[0130] For 7 days after the challenge, the survival, mental state, and food intake of the mice were observed daily. The daily weight, food intake, and water intake were recorded. The results are shown in Tables 3 to 5.

[0131] Table 3. Changes in mouse body weight

[0132]

[0133] From Table 3 and Figure 6 It can be seen that after challenge, the mice in the blank control group (blank group) experienced a continuous decrease in body weight, and their mental state and appetite were poor. The recombinant protein group (PA group) also showed a decrease in body weight, but the decrease was smaller than that in the blank control group (blank group). Both the blank control group (blank group) and the recombinant protein group (PA group) required 5-6 days to recover. The body weight of the vaccine group mice decreased steadily overall, with slight fluctuations after challenge, followed by rapid recovery and stabilization within 3-4 days. This indicates that vaccine immunization can effectively alleviate the damage and stress response caused by challenge, reduce the impact on the growth status of mice, and demonstrate that the vaccine has a good immunoprotective effect.

[0134] Table 4. Changes in food intake (g) in mice

[0135]

[0136] Table 5. Changes in water consumption in mice (g)

[0137]

[0138] From Tables 4 and 5 Figure 7 , 8 As shown, after challenge, the food and water intake of mice in the blank control group (blank group), recombinant protein group (PA group) and vaccine group all decreased to varying degrees. The blank control group (blank group) and recombinant protein group (PA group) showed larger decreases and longer recovery times, while the vaccine group showed smaller decreases and significantly faster recovery. This indicates that the vaccine can effectively alleviate the stress caused by challenge and improve the health and tolerance of mice.

[0139] One day after being challenged with Pseudomonas aeruginosa, mice exhibited decreased activity, huddling together, lethargy, rapid breathing, irregular breathing rhythm, and ruffled fur (as shown in the image). Figure 9 As shown), lose its luster (such as) Figure 9 Symptoms such as those shown in the image.

[0140] 3.2 Detection of bacterial load in lung tissue

[0141] Preparation of lung tissue homogenate: Three days after viral challenge, mice were euthanized by cervical dislocation, lung tissue was collected, weighed, and then 1 mL of PBS was added to homogenize it into a homogenate solution free of lumps. The homogenate was then serially diluted 10⁻⁶ times. 1 ~10 7 times.

[0142] Plate counting: Spread 100 μL of the diluted solution onto beef extract peptone agar plates and incubate at 37°C for 48 h. Figure 10 As shown, the CFU / g lung tissue ratio was calculated, as shown in Table 6 and... Figure 11 .

[0143] Table 6. Bacterial load 3 days after challenge

[0144]

[0145] From Table 6 and Figure 11 As shown, compared with the blank control group, the bacterial load in the vaccine group was significantly lower than that in the blank control group (P < 0.0005), indicating that a certain dose of recombinant protein vaccine can reduce the adhesion of Pseudomonas aeruginosa to the lungs of mice, thereby reducing the degree of lung infection.

[0146] 3.3 Pathological assessment of mouse lung tissue

[0147] Three days after viral challenge, three mice were randomly selected from each group, euthanized by cervical dislocation, and dissected. Lungs were removed from each mouse, and after morphological observation, a portion of the lung tissue was preserved by immersion in 4% paraformaldehyde solution. Histological sections were prepared, stained with hematoxylin and eosin, and then sealed with coverslips. The pathological characteristics of the mouse lung tissue were observed under a microscope at 400x magnification, and histopathological analysis was performed. The Szapiel scoring system was used for histopathological scoring: 0 points: no alveolitis; 1 point: mild alveolitis, with mononuclear cell infiltration visible locally and near the chest, covering less than 20% of the entire lung, and the alveolar structure was generally normal; 2 points: moderate alveolitis, with the lesion area accounting for 20%-50% of the entire lung; 3 points: severe pulmonary fibrosis, with the lesion area greater than 50% of the entire lung. The lung injury results for each group of mice are shown in Table 7. Figure 12 , 13 14.

[0148] An autopsy three days after the viral load revealed varying degrees of congestion and necrosis in the lungs (e.g., Figure 12 (As shown in the figure). Compared with the vaccine group, mice in the recombinant protein group (PA group) and the blank control group (blank group) showed obvious pneumonia symptoms, which indicates that the recombinant protein vaccine has a certain protective effect against pneumonia caused by Pseudomonas aeruginosa infection.

[0149] Table 7. Semi-quantitative analysis of lung injury scores in mice of each group

[0150]

[0151] Depend on Figure 13 HE staining of mouse lungs showed that the lung epithelium of the normal group mice was intact with no obvious damage; the blank control group mice showed obvious inflammatory cell infiltration, significantly increased smooth muscle thickness and area, and disordered surrounding tissue structure, indicating severe alveolitis; the adjuvant group mice showed reduced inflammatory cell infiltration, increased smooth muscle thickness and area, and reduced disordered surrounding tissue structure, indicating moderate alveolitis; the vaccine group mice showed only a small amount of inflammatory cell infiltration in some tissues without other obvious damage, indicating mild alveolitis. (Based on scoring) Figure 14 The results in Table 7 show that the lung tissue pathology score of the vaccine group was significantly lower than that of the control group (P<0.05), indicating that a certain dose of recombinant protein vaccine can alleviate lung damage caused by Pseudomonas aeruginosa in mice.

[0152] 3.4 Determination of organ indices in mice

[0153] Three days after the virus challenge, three mice from each group were randomly selected and euthanized by dislocation. The heart, liver, spleen, kidneys and lungs were removed and weighed. The organ index of each group was calculated. The results are shown in Table 8.

[0154] Cardiac index = Heart weight (mg) / Mouse weight (g) × 100%

[0155] Liver index = Liver weight (mg) / Mouse weight (g) × 100%

[0156] Spleen index = Spleen weight (mg) / Mouse weight (g) × 100%

[0157] Kidney index = Kidney weight (mg) / Mouse weight (g) × 100%

[0158] Lung tissue index = Lung tissue weight (mg) / Mouse weight (g) × 100%

[0159] Table 8. Mouse organ index

[0160] Organ index measurement is an important indicator for evaluating the protective effect of vaccines. After bacterial challenge, organs will increase in weight due to pathological damage such as inflammation, congestion, and edema. (See Table 8 and...) Figure 15 The results showed that, compared with the blank control group (blank group) and the recombinant protein group (PA group), the indices of organs such as liver, spleen, kidney, and heart in the vaccine group mice after challenge were not significantly different, which may indicate that the inflammatory response was not obvious. However, the lung index of the vaccine group mice was significantly reduced, and the difference was statistically significant (P<0.0005), indicating that immunization can alleviate lung damage caused by Pseudomonas aeruginosa challenge to a certain extent, reduce the degree of tissue edema and inflammatory response, and has a certain protective effect on the lungs of mice.

[0161] 4. Detection of serum IL-17A, IL-4, IFN-γ and TNF-α cytokine secretion levels

[0162] This experiment used magnetic microparticle chemiluminescence immunoassay to determine the secretion levels of IL-17A, IL-4, IFN-γ, and TNF-α cytokines in mice before and after bacterial challenge, using the Servicebio® Mouse MPCLIA kit.

[0163] The methods for detecting the secretion levels of IL-17A, IL-4, IFN-γ, and TNF-α cytokines are the same; taking IL-17A as an example, 15 μL of sample was added to a reaction vessel along with magnetic microparticles coated with anti-interleukin-17A monoclonal antibody and an anti-interleukin-17A monoclonal antibody-acridoid ester label. After incubation, interleukin-17A in the sample binds to the magnetic microparticles coated with anti-interleukin-17A monoclonal antibody, while another site of interleukin-17A in the sample binds to the anti-interleukin-17A monoclonal antibody-acridoid ester label, forming a "sandwich" structure complex. After the reaction, the magnetic field adsorbs the magnetic microparticles, and unbound substances are washed away. Pre-excitation and excitation solutions are added to the reaction vessel, and the luminescence value is measured. Based on the standard curve, the content of interleukin-17A in the sample can be calculated using a chemiluminescence analyzer. The results are shown in Table 9. Figure 16 .

[0164] Table 9. Serum secretion levels of IL-17A, IL-4, IFN-γ, and TNF-α cytokines

[0165]

[0166] The levels of IL-17A, IL-4, IFN-γ, and TNF-α cytokines in mouse serum before and after challenge were detected using magnetic microparticle chemiluminescence immunoassay to evaluate the level of cellular immune response induced by the vaccine. (See Table 9 and...) Figure 16 As shown, compared with the blank control group (blank group), the serum levels of IL-17A, IL-4, IFN-γ, and TNF-α in the vaccine group mice were all increased before and after challenge. Specifically, the IL-17A level increased significantly before and after challenge, indicating that the vaccine can induce Th17 cell activation and enhance mucosal immunity and local antibacterial infection ability. The IL-4 level increased significantly before and after challenge, indicating that the vaccine can activate the Th2 immune response and promote the production of related antibodies by humoral immunity. The IFN-γ level did not increase significantly before challenge, but increased significantly after challenge, indicating that bacterial infection can promote the vaccine to further induce the Th1 immune response and enhance the body's cellular immunity and anti-infection ability. The TNF-α level increased significantly after challenge, reflecting that the body's pro-inflammatory and immune regulatory pathways were further effectively activated, which is beneficial for enhancing phagocytic bactericidal and inflammation clearance capabilities. The above results indicate that the recombinant protein vaccine prepared in this invention can simultaneously activate Th1, Th2 and Th17 immune responses, effectively regulate the levels of cellular and humoral immunity, and provide important immunological evidence for the vaccine's effectiveness against Pseudomonas aeruginosa infection.

[0167] 5. Effects of recombinant protein on spleen T lymphocyte subsets in immunized mice

[0168] 5.1 Preparation of mouse spleen lymphocyte suspension

[0169] Mice were euthanized by cervical dislocation after challenge with the virus. After soaking in 75% ethanol for 1 min, the spleen was aseptically removed and placed in a sterile mortar. The spleen was ground, and 3.75 mL of PBS was added. The mixture was then filtered (through a 200-mesh sieve) to prepare a single-splenic cell suspension. The suspension was centrifuged, the supernatant was discarded, and 2 mL of erythrocyte lysis buffer was added. The suspension was gently mixed by pipetting, centrifuged again, washed three times with PBS, and centrifuged at 1500 r / min for 5 min. 2 mL of RPMI-1640 (complete culture medium) containing 10% fetal bovine serum was added, vortexed, and the cells were counted using trypan blue staining. Viable cells should be greater than 95%. The cell concentration was adjusted for later use.

[0170] 5.2 Effects of recombinant protein on spleen T lymphocyte subsets in immunized mice

[0171] (1) Lymphocyte extraction: Add 3 mL of lymphocyte separation medium to a 15 mL centrifuge tube. Slowly add the filtered mouse spleen cell suspension to the upper layer of the separation medium and centrifuge at 420×g for 30 min. Carefully aspirate the lymphocytes from the white membrane layer of the separation medium and place the lymphocytes in a new 15 mL centrifuge tube. Add 10 mL of PBS to the tube and wash once. Centrifuge at 200×g for 10 min and discard the supernatant.

[0172] (2) PBS washing: Add 1 mL of PBS to transfer the cells into a 1.5 mL EP tube and wash them again.

[0173] (3) Antibody incubation: Dilute the antibody according to the ratio, add 100 μL of antibody dilution solution to each tube, incubate the cells at 4℃ for 30 min, and collect the cell pellet by centrifugation.

[0174] (4) Washing cells with PBS: Add 1 mL of PBS, mix well, centrifuge at 2000 rpm for 5 min to collect cells. Add 200 μL of PBS to resuspend the cells, and mix the pellet by blowing and aspiration.

[0175] (5) Flow Cytometry Testing: Flow cytometry testing was performed using a FITC 488 to excite the FL1 channel and a PE 488 to excite the FL2 channel and an APC 638 to excite the FL4 channel. The results were analyzed using FlowJo software, as shown in Table 10. Figure 17 .

[0176] Table 10. Flow cytometry results of T lymphocyte subsets in mouse spleen

[0177]

[0178] Note: The same superscript letter indicates no significant difference between groups, while different letters indicate a significant difference between groups (P<0.05). For example, there is a significant difference between groups labeled a and b.

[0179] This study used flow cytometry to detect the proportion of T cell subsets in the spleen of mice in each group, as shown in Table 10 and... Figure 17 The results showed that there was no significant difference in the proportion of CD3⁺ T cells between the blank control group (blank group) and the adjuvant group, indicating that adjuvant stimulation alone could not effectively activate the body's T cell immune response. However, the proportion of CD3⁺ T cells in the vaccine group was significantly increased, and the difference was statistically significant, indicating that the vaccine of this invention can effectively promote T lymphocyte proliferation and activation, and enhance the body's overall cellular immune function. Compared with the blank control group (blank group) and the adjuvant group, the proportion of CD4⁺CD8⁻ helper T cells was significantly increased in the vaccine group, while the proportion of CD4⁻CD8⁺ cytotoxic T cells was significantly decreased. Simultaneously, the CD4 / CD8 ratio was significantly increased, indicating that the vaccine of this invention can effectively promote cytokine secretion, help B cells produce specific antibodies, and participate in the generation of immune memory cells, confirming that the vaccine can induce a specific immune response dominated by CD4⁺ T cells.

[0180] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and these variations still fall within the protection scope of the present invention.

Claims

1. A vaccine against Pseudomonas aeruginosa, characterized in that, Includes recombinant proteins, the amino acid sequence of which is shown in SEQ ID NO:

2.

2. The vaccine against Pseudomonas aeruginosa according to claim 1, characterized in that, It also includes adjuvants.

3. The vaccine against Pseudomonas aeruginosa according to claim 2, characterized in that, The adjuvant is either Freund's complete adjuvant or Freund's incomplete adjuvant.

4. A method for preparing a vaccine against Pseudomonas aeruginosa, characterized in that, Includes the following steps: S1. The target gene and expression plasmid pET22b(+) were double-digested with Xho I and EcoRI at 37℃. S2. Recover the enzyme digestion product and ligate it with T4 DNA ligase at 16°C for 10 hours to obtain the ligation solution. S3. The ligation solution was heat-shocked at 42℃ for 45s to transform E. coli DH5α competent cells, and positive clones of recombinant plasmids were screened. S4. The positive clone recombinant plasmid was heat-shocked at 42℃ for 45s and introduced into Escherichia coli BL21 competent cells to obtain recombinant bacteria. S5. Inoculate the recombinant bacteria into LB liquid medium containing ampicillin sodium and culture to obtain seed culture; The seed culture was then inoculated into LB liquid medium containing ampicillin sodium for fermentation. After 1 hour of culture, selenium disulfide was added to the medium at a rate of 0.002–0.008 g / L, and the culture was continued until the OD reached [value missing]. 600 The value was 0.

4. IPTG was added at a concentration of 0.5 mmol / L to induce expression, and the cells were cultured at 30°C for 5 hours after induction. S6. Centrifuge to collect bacterial cells, add PBS buffer to the bacterial cells at a weight ratio of 1:5, sonicate to disrupt the bacterial cells, and centrifuge to collect the supernatant. S7. Filter the supernatant through a filter membrane with a pore size of 0.22 μm, then purify the filtrate with a nickel column and collect the target protein; S8. Dialyze the target protein in PBS dialysate at 4°C for 24 hours using a dialysis membrane with a molecular cutoff of 20 kDa. Change the medium every 8 hours to obtain a prolyl oligopeptidase solution. S9. Mix the prolyl oligopeptidase solution with Freund's adjuvant and emulsify by ultrasonication to obtain the vaccine.

5. The method for preparing the vaccine against Pseudomonas aeruginosa according to claim 4, characterized in that, The target gene is the PA1304 gene.

6. The method for preparing the vaccine against Pseudomonas aeruginosa according to claim 5, characterized in that, The final concentration of ampicillin sodium described in step S5 is 100 μg / mL.

7. The method for preparing the vaccine against Pseudomonas aeruginosa according to claim 4, characterized in that, The process of mixing the recombinant protein solution with Freund's adjuvant involves first adjusting the recombinant protein with PBS buffer to a recombinant protein concentration of 1 mg / mL, and then mixing the recombinant protein solution with Freund's complete adjuvant at a volume ratio of 1:

1.

8. The method for preparing a Pseudomonas aeruginosa vaccine according to claim 4, characterized in that, The process of mixing the recombinant protein solution with Freund's adjuvant involves first adjusting the recombinant protein with PBS buffer to a recombinant protein solution with a recombinant protein content of 1 mg / mL, and then mixing the recombinant protein solution with Freund's incomplete adjuvant at a volume ratio of 1:

1.

9. The use of a recombinant protein in the preparation of a medicament for the prevention and / or treatment of Pseudomonas aeruginosa infection, characterized in that, The amino acid sequence of the recombinant protein is shown in SEQ ID NO:

2.

10. The use of the recombinant protein according to claim 9 in the preparation of a medicament for the prevention and / or treatment of Pseudomonas aeruginosa infection, characterized in that, The drug in question is a vaccine.

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