Application of recombinant protein in preparation of pseudomonas aeruginosa vaccine

Abstract

The invention relates to the field of biological medicine, in particular to application of a recombinant protein in preparation of a pseudomonas aeruginosa vaccine, which is characterized in that the recombinant protein comprises an outer membrane carboxylate channel K6 protein of pseudomonas aeruginosa, and the pseudomonas aeruginosa vaccine comprises the recombinant protein and an outer membrane pore protein combination, the recombinant protein is used for improving the immune efficacy of the outer membrane pore protein combination; the outer membrane pore protein combination comprises outer membrane pore protein F and outer membrane pore protein I; wherein the mass ratio of the recombinant protein to the outer membrane pore protein combination is (1-3): 3. The pseudomonas aeruginosa vaccine provided by the invention shows better immune protection efficacy (the bacterial clearance rate can reach 99% or above), is good in safety, and has extremely high application value in prevention and / or treatment of pseudomonas aeruginosa infection (especially pulmonary infection caused by pseudomonas aeruginosa).

Description

[0001] This application is a divisional application based on the Chinese invention patent application with application number 202411867477.X, application date December 18, 2024, and invention name “Use of a recombinant protein in the preparation of Pseudomonas aeruginosa vaccine”. Technical Field

[0002] The present invention relates to the field of biomedicine, and in particular to use of a recombinant protein in preparing a Pseudomonas aeruginosa vaccine. Background Art

[0003] Pseudomonas aeruginosa (PA) is a common opportunistic pathogen in clinical practice, characterized by high infection rates, rapid mutation, strong pathogenicity, and strong drug resistance. Although the isolation rate of P. aeruginosa in hospital settings has decreased in recent years, it remains a major cause of lower respiratory tract infections.

[0004] Pseudomonas aeruginosa infections are primarily treated clinically with antibiotics, but due to both natural and acquired resistance, effective treatment and control strategies are still lacking. Data from the China Surveillance for Bacterial Resistance (CHINET) indicate that in 2021, the isolation rate of P. aeruginosa in comprehensive teaching hospitals ranked fourth among all strains isolated and third among strains isolated from respiratory specimens. Over the past five years, the isolation rate of P. aeruginosa has consistently ranked among the top four Gram-negative bacteria (10.6%-13.3%). P. aeruginosa has complex pathogenic mechanisms and diverse resistance mechanisms. Therefore, complete bacterial eradication with conventional antibiotics is difficult, and may even lead to increased resistance.

[0005] Vaccines are an alternative strategy for combating Pseudomonas aeruginosa infection. Immunization with outer membrane protein F or outer membrane protein I as an immunogen can elicit specific immune responses in animal models or clinical trials, making them potential vaccine candidates. A recombinant fusion protein vaccine based on outer membrane protein F and outer membrane protein I (also known as IC43) is the most effective candidate for Pseudomonas aeruginosa vaccines entering Phase III clinical trials, offering improved safety and immunogenicity. However, the results of Phase III clinical trials showed that the infection rate among patients receiving this vaccine was not statistically significantly different from that of patients receiving a placebo. Although existing technologies attempt to enhance the immune protection of vaccines based on outer membrane protein F and outer membrane protein I, they still face certain challenges in terms of safety and efficacy. Summary of the Invention

[0006] In the first aspect, the present invention provides a use of a recombinant protein in preparing a Pseudomonas aeruginosa vaccine, characterized in that the recombinant protein includes the outer membrane carboxylate channel K6 protein of Pseudomonas aeruginosa, the Pseudomonas aeruginosa vaccine includes the recombinant protein and an outer membrane porin combination, and the recombinant protein is used to enhance the immune efficacy of the outer membrane porin combination; the outer membrane porin combination includes outer membrane porin F and outer membrane porin I; wherein the mass ratio of the recombinant protein to the outer membrane porin combination is 1-3:3.

[0007] In some embodiments, the recombinant protein is expressed by a recombinant expression vector, and the recombinant expression vector comprises the nucleotide sequence shown in SEQ ID NO:2.

[0008] In some embodiments, the mass ratio of the recombinant protein to the outer membrane porin is 1:2.

[0009] In some embodiments, the mass ratio of the outer membrane porin F to the outer membrane porin I is 1:1.

[0010] In some embodiments, the Pseudomonas aeruginosa vaccine further comprises an adjuvant.

[0011] In some embodiments, the adjuvant comprises MF59.

[0012] In some embodiments, the Pseudomonas aeruginosa vaccine is used to prevent and / or treat Pseudomonas aeruginosa infection.

[0013] In some embodiments, the Pseudomonas aeruginosa infection comprises acute pneumonia caused by Pseudomonas aeruginosa.

[0014] In some embodiments, the Pseudomonas aeruginosa vaccine is used to reduce colonization of the lungs of a subject by the Pseudomonas aeruginosa.

[0015] In some embodiments, the amino acid sequence of the recombinant protein is shown in SEQ ID NO:3.

[0016] In some embodiments, the Pseudomonas aeruginosa vaccine has a bacteria (Pseudomonas aeruginosa) clearance rate of at least 99%.

[0017] In a second aspect, the present invention provides a recombinant expression vector, characterized in that the recombinant expression vector comprises a nucleotide sequence as shown in SEQ ID NO: 2.

[0018] In some embodiments, the recombinant expression vector is a plasmid.

[0019] In some embodiments, the recombinant expression vector is a pET-28a(+) expression vector.

[0020] In a third aspect, the present invention provides a Pseudomonas aeruginosa vaccine, characterized in that the Pseudomonas aeruginosa vaccine comprises a recombinant protein and an outer membrane porin combination, wherein the recombinant protein comprises the outer membrane carboxylate channel K6 protein of Pseudomonas aeruginosa, and the outer membrane porin combination comprises outer membrane porin F and outer membrane porin I; wherein the mass ratio of the recombinant protein to the outer membrane porin combination comprises 1-3:3.

[0021] In some embodiments, the recombinant protein is expressed by a recombinant expression vector, and the recombinant expression vector comprises the nucleotide sequence shown in SEQ ID NO:2.

[0022] In some embodiments, the mass ratio of the recombinant protein to the outer membrane porin is 1:2.

[0023] In some embodiments, the mass ratio of the outer membrane porin F to the outer membrane porin I is 1:1.

[0024] In some embodiments, the Pseudomonas aeruginosa vaccine further comprises an adjuvant.

[0025] In some embodiments, the adjuvant comprises MF59.

[0026] In some embodiments, the amino acid sequence of the recombinant protein is shown in SEQ ID NO:3.

[0027] In a fourth aspect, the present invention provides a use of a recombinant protein in the preparation of a medicament for treating and / or preventing bacterial infection, characterized in that the bacterial infection includes infection caused by Pseudomonas aeruginosa and / or Acinetobacter baumannii, and the recombinant protein includes the outer membrane carboxylate channel K6 protein of Pseudomonas aeruginosa.

[0028] In some embodiments, the recombinant protein further comprises a Pseudomonas aeruginosa heme uptake receptor.

[0029] In some embodiments, the bacterial infection is a Pseudomonas aeruginosa infection.

[0030] In some embodiments, the bacterial infection is an Acinetobacter baumannii infection.

[0031] In some embodiments, the bacterial infection is a hospital-acquired infection.

[0032] In some embodiments, the bacterial infection is a lung infection.

[0033] In some embodiments, the bacterial infection is ventilator-associated pneumonia.

[0034] In some embodiments, the recombinant protein is expressed by a recombinant expression vector, and the recombinant expression vector comprises the nucleotide sequence shown in SEQ ID NO:2.

[0035] In some embodiments, the amino acid sequence of the recombinant protein is shown in SEQ ID NO: 3.

[0036] In some embodiments, the medicament is in the form of a vaccine.

[0037] In some embodiments, the vaccine further comprises an adjuvant.

[0038] In some embodiments, the adjuvant comprises MF59.

[0039] It has been verified that the recombinant protein provided by the present invention (i.e., the outer membrane carboxylate channel K6 protein of Pseudomonas aeruginosa) is not affected by the existing bacterial resistance mechanisms in clinical practice, can effectively reduce infections caused by bacteria, and can not only be used to treat / prevent Pseudomonas aeruginosa infections, but also has an immune protective effect against Acinetobacter baumannii infections. It can significantly reduce bacterial colonization in the lungs of mice, has good immune protective efficacy, and is particularly suitable for the treatment and / or prevention of hospital-acquired infections (such as mechanical ventilation-associated pneumonia).

[0040] In a fifth aspect, the present invention provides a use of a recombinant protein in preparing a Pseudomonas aeruginosa vaccine, characterized in that the recombinant protein comprises the outer membrane carboxylate channel K6 protein of Pseudomonas aeruginosa and / or the heme uptake receptor of Pseudomonas aeruginosa.

[0041] In some embodiments, the recombinant protein is used in combination with an outer membrane porin.

[0042] In some embodiments, the outer membrane porin combination includes outer membrane porin F and outer membrane porin I.

[0043] In some embodiments, the mass ratio of the recombinant protein to the outer membrane porin is 1-3:3.

[0044] In some embodiments, the recombinant protein is expressed by a recombinant expression vector, and the recombinant expression vector comprises the nucleotide sequence shown in SEQ ID NO:2.

[0045] In some embodiments, the mass ratio of the recombinant protein to the outer membrane porin is 1:2.

[0046] In some embodiments, the mass ratio of the outer membrane porin F to the outer membrane porin I is 1:1.

[0047] In some embodiments, the Pseudomonas aeruginosa vaccine further comprises an adjuvant.

[0048] In some embodiments, the adjuvant comprises MF59.

[0049] In some embodiments, the amino acid sequence of the recombinant protein is shown in SEQ ID NO: 3.

[0050] Compared with the prior art, the beneficial effects of the present invention include at least the following aspects: In order to further improve the immune protection efficacy of the Pseudomonas aeruginosa vaccine based on outer membrane protein F and outer membrane protein I, the present invention creatively combines the outer membrane carboxylate channel K6 protein of Pseudomonas aeruginosa with outer membrane protein F and outer membrane protein I, and finds that the addition of the outer membrane carboxylate channel K6 protein of Pseudomonas aeruginosa in the technical solution of the present invention can produce a synergistic effect with outer membrane protein F and outer membrane protein I (for example, when the mass ratio of the outer membrane carboxylate channel K6 protein of Pseudomonas aeruginosa to the outer membrane pore protein combination is 1 / 3), thereby improving the original immune protection efficacy of outer membrane protein F and outer membrane protein I, and increasing the bacterial clearance rate of Pseudomonas aeruginosa from 90% to more than 99%. It is particularly suitable for clearing Pseudomonas aeruginosa colonized in the lungs of subjects, so as to enhance the immune protection efficacy of the Pseudomonas aeruginosa vaccine based on outer membrane protein F and outer membrane protein I.

[0051] In addition, it should be emphasized that the addition of the outer membrane carboxylate channel K6 protein not only enhances the original immune protection efficacy of outer membrane porin F + outer membrane porin I, but also does not affect the safety of the well-proven Pseudomonas aeruginosa vaccine based on outer membrane protein F and outer membrane protein I. Therefore, the Pseudomonas aeruginosa vaccine provided by the present invention exhibits better immune protection efficacy (bacterial clearance rate can reach more than 99%) while being safe, and has extremely high application value in preventing and / or treating Pseudomonas aeruginosa infections, especially lung infections caused by Pseudomonas aeruginosa (such as acute pneumonia). BRIEF DESCRIPTION OF THE DRAWINGS

[0052] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following is a brief introduction to the drawings required for the embodiments or the description of the prior art. In all drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the various elements or parts are not necessarily drawn according to the actual scale. Obviously, the drawings described below are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings without inventive work.

[0053] Figure 1 Figure 1 is the sequence identification result of the recombinant plasmid; Figure 2 This is the result diagram of expression and purification of recombinant protein 1; Figure 3 This is a graph comparing the immune protection efficacy of different recombinant proteins; Figure 4 This is the antibody gradient result diagram of the outer membrane carboxylate channel K6; Figure 5 This figure shows the comparison of the immune protection efficacy of different recombinant protein combinations; Figure 6 This figure shows the comparison of the immune protection efficacy of different recombinant protein combinations; Figure 7 Figure 2 shows the safety results of different recombinant protein combinations. DETAILED DESCRIPTION

[0054] To make the purpose, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0055] As used herein, "and / or" includes any and all combinations of one or more of the associated listed items.

[0056] Herein, "plurality" means two or more than two, ie, it includes two, three, four, five, etc.

[0057] As used in this specification, the term "about" typically means + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically + / - 2% of the stated value, even more typically + / - 1% of the stated value, and even more typically + / - 0.5% of the stated value.

[0058] In this specification, certain embodiments may be disclosed in a format that is within a range. It should be understood that this description of "within a range" is merely for convenience and brevity and should not be interpreted as a rigid limitation on the disclosed range. Therefore, the description of a range should be considered to have specifically disclosed all possible subranges and individual numerical values ​​within this range. For example, the description of a range of 1-6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within this range, such as 1, 2, 3, 4, 5, and 6. Regardless of the breadth of the range, the above rules apply.

[0059] Example 1 Nucleotide Sequence Synthesis and Recombinant Plasmid Construction of the Outer Membrane Carboxylate Channel K6 of Pseudomonas aeruginosa The wild-type nucleotide sequence (SEQ ID NO: 1) and the optimized nucleotide sequence (SEQ ID NO: 2) of the Pseudomonas aeruginosa outer membrane carboxylate channel K6 were synthesized and ligated into the pET-28a(+) expression vector to construct recombinant plasmids. Sequencing confirmed the correct recombinant plasmid sequence for subsequent expression and purification steps.

[0060] Expression of recombinant proteins Select a single colony containing the recombinant plasmid and incubate it in 3 mL of LB liquid medium (containing 50 μg / mL Kan) with shaking at 37°C, 220 rpm, and culture overnight with shaking. The next day, measure the OD630 value of the overnight culture suspension, calculate the concentration, and inoculate it into 3 mL of LB liquid medium (containing 50 μg / mL Kan) to a starting concentration of 0.05 OD. Culture at 37°C, 220 rpm, and shake until the culture reaches logarithmic phase (approximately 0.4-0.8 OD). Prepare a 1 mL control culture. Add IPTG (final concentration 0.1 mM) to the remaining culture and culture at 37°C with shaking for 4 h. Take 1 mL of each culture suspension and centrifuge at 12,000 × g for 2 min. Resuspend the pellet in 100 μL of 1× loading buffer per OD 100 and lyse in a boiling water bath for 10 min. A 10 μL sample is analyzed by SDS-PAGE gel electrophoresis.

[0061] Purification of recombinant proteins Fill the Ni-NTA column, rinse with deionized water to remove 20% ethanol, pass 3 times the volume of 0.1 M EDTA-2Na through the column, rinse with deionized water, add 3 times the column volume of NTA-0 buffer (pH = 8.0) to equilibrate the nickel column, add 5 times the column volume of 0.1 M NiSO4 solution to chelate Ni2+, then add 3 times the column volume of equilibration buffer (pH = 4.0) to wash, and finally wash with 3 times the column volume of NTA-0 buffer (pH = 8.0) until the pH of the effluent is 8.0.

[0062] The supernatant protein solution was filtered through a 0.22 µm filter and set aside. The supernatant protein solution was loaded onto the column at a flow rate of 1 mL / min and washed with NTA-0 buffer (pH 8.0) until the flow-through was protein-free (G250 assay solution did not change color). Elution was performed with 20 mM, 60 mM, 200 mM, and 500 mM imidazole, respectively. The eluate was collected in fractions until the G250 assay solution did not change color. The column was washed with 3 column volumes of deionized water and finally sealed with 20% ethanol. The collected eluate was dialyzed and concentrated, and 10 μL was aliquoted for SDS-PAGE gel electrophoresis. Protein concentration was determined according to the BCA protein concentration assay kit instructions. Aliquots were stored frozen at -80°C.

[0063] Figure 1 The sequence identification results of the recombinant plasmids are shown, where M represents Marker. Lanes 1 and 2 are both single clones of recombinant plasmids containing the optimized nucleotide sequence of the outer membrane carboxylate channel K6; Lane 3 is a single clone of the recombinant plasmid containing the wild-type nucleotide sequence of the outer membrane carboxylate channel K6. Although both the recombinant plasmid containing the wild-type nucleotide sequence of the outer membrane carboxylate channel K6 and the recombinant plasmid containing the optimized nucleotide sequence of the outer membrane carboxylate channel K6 can express the outer membrane carboxylate channel K6 protein (amino acid sequence shown in SEQ ID NO: 3), the recombinant plasmid containing the wild-type nucleotide sequence of the outer membrane carboxylate channel K6 not only has a low expression level of the recombinant protein, but also has low immunogenicity. This may be due to the different spatial structures of the recombinant proteins expressed by the recombinant plasmid containing the wild-type nucleotide sequence of the outer membrane carboxylate channel K6 and the recombinant plasmid containing the optimized nucleotide sequence of the outer membrane carboxylate channel K6. Therefore, the present invention uses the recombinant protein expressed by the recombinant plasmid containing the optimized nucleotide sequence of the outer membrane carboxylate channel K6 (referred to as recombinant protein 1) for subsequent experiments.

[0064] Expression and purification results of recombinant protein 1 (51 kDa) are shown in Figure 2 (M stands for Marker, Figure 2 The unit of the number on the right is kDa).

[0065] MSMTPIARAVAFAALGSSITVPTLAHAEFIKDSKASIELRNFYFNRDFRQEGASQSKAEEWAQGFLLRYESGYTEGTIGFGVDAIGLGVKLDSSPDRSGTGLLKR DRETGRAQDDYGEAGITAKLRASKSTLKIGTLTPKLPVIMPNDSRLLPQTFQGGALNSMEIDGLTLDAGRLKKVNQRDSSDNEDMTITGGGKRNIVVRSGLTSDKF DFAGGSYKWTDNLSTSYHYGKLDNFYKQHYLGLVHTLPIADKQSLKSDIRWARSTDDGSSNVDNKALNAMFTYSLGYHAFGVGYQKMSGDTGFAYINGADPYLVNF IQIGDFANKDEKSWQARYDYNFAGVGIPGLTFMTRYVKGDNIDLLTTSGEGKEWERDMDIAYVFQSGPLKNLGVKWRNATMRTNYTNDYDENRLIVSYTLPLW (SEQ ID NO:3) Example 2 Comparison of immune protection efficacy of different recombinant proteins Using a similar method to Example 1, the following proteins of Pseudomonas aeruginosa were expressed and purified: outer membrane porin F, outer membrane porin I, outer membrane porin 86, outer membrane porin Q and Pseudomonas aeruginosa heme uptake receptor.

[0066] C57BL / 6J mice were immunized by two injections of 100 μL of recombinant protein (20 μg recombinant protein / dose, resuspended in 50 μL saline and supplemented with 50 μL of MF59 adjuvant) into the thigh muscles on days 0, 14, and 28. The experimental groups were divided into the model group, the outer membrane porin F group, the outer membrane porin I group, the outer membrane porin 86 group, the outer membrane porin Q group, the Pseudomonas aeruginosa heme receptor group, and the outer membrane carboxylate channel K6 group, depending on the recombinant protein used.

[0067] The protective efficacy of each group was evaluated on the 14th day after the last immunization. A non-lethal dose of Pseudomonas aeruginosa PAO1 solution (2×10 7 CFU / mL) was used to establish an acute pneumonia model in mice, and lung tissues were obtained aseptically 24 h after infection for bacterial CFU count.

[0068] The results are as follows Figure 3As shown, the immune protective efficacy of outer membrane porin 86 and outer membrane porin Q was weak, with the P. aeruginosa load not much different from that of the model group; the immune protective efficacy of the P. aeruginosa heme uptake receptor was comparable to that of outer membrane porin F and outer membrane porin I. The P. aeruginosa load of mice immunized with outer membrane carboxylate channel K6 decreased by approximately 1.8 log (P=0.0018), and was able to eliminate more than 90% of bacteria (P. aeruginosa), significantly reducing bacterial colonization in the mouse lungs, demonstrating good immune protective efficacy that was superior to that of outer membrane porin F and outer membrane porin I.

[0069] Outer membrane carboxylate channel K6 can induce high levels of specific antibodies On days 0, 14, and 28, C57BL / 6J mice were immunized by injecting 100 μL of recombinant protein 1 into two points of the thigh muscle (20 μg of recombinant protein / dose, resuspended in 50 μL of saline and supplemented with 50 μL of adjuvant MF59). Blood was collected from the orbits 7 days after each immunization, and serum was collected for ELISA testing of specific antibody levels. The results are shown in Table 1. Figure 4 As shown. The outer membrane carboxylate channel K6 protein can reach 10 after the last immunization 4 The serum antibody titers above these levels have good immunogenicity.

[0070] Prediction of antigenic epitopes of outer membrane carboxylate channel K6 protein The amino acid sequence of the outer membrane carboxylate channel K6 was analyzed for T cell and B cell antigen epitopes using the Protean software in DNAStar software: the analysis found that it contains 31 potential B cell antigen epitopes (11-14, 23-27, 33-35, 51-58, 75-77, 79-87, 90-115, 117-120, 121-123, 145-148, 169-171, 173-187, 189-193, 195-198, 202-204, 215-218, 224-228, 245-250, 254-265, 267-271, 69, 280-294, 315-320, 328-335, 338-343, 358-361, 371-373, 378-381, 392-395, 397-403, 411-413, 415-418), and 15 potential T cell antigen epitopes (5-8, 10-13, 53-61, 112-117, 155-157, 164-166, 175-179, 219-222, 224-226, 231-241, 244-248, 267-271, 308-310, 317-322, 368-372).

[0071] These experimental results demonstrate that the outer membrane carboxylate channel K6 is a protein antigen with a predominantly B-cell epitope, but also contains T-cell epitopes. It demonstrates excellent immune protection, even surpassing that of the clinically tested outer membrane porin F and porin I.

[0072] Example 3 Comparison of immune protection efficacy of different recombinant protein combinations Considering that outer membrane porin F and outer membrane porin I have been fully studied and have good immunogenicity and safety, this example focuses on whether the outer membrane carboxylate channel K6 can further improve the immune protection efficacy of outer membrane porin F and outer membrane porin I.

[0073] C57BL / 6J mice were immunized with 100 μL of recombinant protein (20 μg recombinant protein / dose, resuspended in 50 μL saline and supplemented with 50 μL of MF59 adjuvant) injected into the thigh muscles at two points on days 0, 14, and 28. Depending on the recombinant protein used, the experimental groups were divided into a model group, a Porin F + Porin I group (Porin F / I, 10 μg each), a Porin K6 group (20 μg), and a Porin F + Porin I + Porin K6 group (Combination 1, 6.67 μg each).

[0074] Protective efficacy against infection was evaluated in all groups 14 days after the last immunization. An acute pneumonia model was established in mice by intratracheal injection of a nonlethal dose of Pseudomonas aeruginosa, and lung tissue was aseptically harvested 24 hours after infection for bacterial CFU counts.

[0075] The results are as follows Figure 5 As shown, compared with the model group, the Pseudomonas aeruginosa load in the Outer Membrane Porin F + Outer Membrane Porin I group decreased by approximately 1.5 logs, and the Pseudomonas aeruginosa load in the Outer Membrane Porin F + Outer Membrane Porin I group decreased by approximately 1.8 logs. The Pseudomonas aeruginosa load in mice in the Outer Membrane Porin F + Outer Membrane Porin I + Outer Membrane Porin K6 group decreased further, by approximately 2.5 logs, and was able to eliminate more than 99% of the bacteria. These results indicate that the addition of the Outer Membrane Porin F + Outer Membrane Porin I group can effectively enhance the immune protection of mice.

[0076] Example 4 This example also tested the immune protection efficacy of different mass ratios of outer membrane porin F, outer membrane porin I and outer membrane carboxylate channel K6. The results are shown in Tables 1 and Figure 6 .

[0077] Table 1 The above results show that the addition of outer membrane carboxylate channel K6 can enhance the original immune protection efficacy of outer membrane porin F + outer membrane porin I: when the mass of outer membrane carboxylate channel K6 added is one-third of the total mass of outer membrane porin F + outer membrane porin I, the original immune protection efficacy of outer membrane porin F + outer membrane porin I can be enhanced. When the mass of outer membrane carboxylate channel K6 added is equal to the total mass of outer membrane porin F + outer membrane porin I, the immune protection efficacy of outer membrane porin F + outer membrane porin I is further enhanced. However, simply increasing the mass of outer membrane porin F or outer membrane porin I cannot enhance the immune protection efficacy of outer membrane porin F + outer membrane porin I.

[0078] This example also records the weight changes of mice after immunization every week, and observes the immunogenic response of mice after each immunization. Figure 7 As shown, it was found that the mice in the outer membrane porin F + outer membrane porin I + outer membrane carboxylate channel K6 group (combination 1) had stable weight gain during the immunization period, were in good mental state, and no adverse reactions at the injection site occurred. That is, the addition of outer membrane carboxylate channel K6 did not reduce the original safety of outer membrane porin F + outer membrane porin I, and it is suitable for combination with outer membrane porin F + outer membrane porin I as a Pseudomonas aeruginosa vaccine.

[0079] It should be noted that, in this document, the terms "comprises," "includes," or any other variations thereof are intended to encompass non-exclusive inclusion, such that a process, method, article, or apparatus comprising a series of elements includes not only those elements but also other elements not explicitly listed, or elements inherent to such process, method, article, or apparatus. In the absence of further limitations, an element defined by the phrase "comprising a ..." does not exclude the presence of other identical elements in the process, method, article, or apparatus comprising the element.

[0080] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation methods. The above-mentioned specific implementation methods are merely illustrative and not restrictive. Under the guidance of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the present invention and the claims, all of which are protected by the present invention.

Claims

1. A Pseudomonas aeruginosa vaccine, characterized in that: The Pseudomonas aeruginosa vaccine includes a recombinant protein and an outer membrane porin combination, wherein the recombinant protein includes the outer membrane carboxylate channel K6 protein of Pseudomonas aeruginosa, and the outer membrane porin combination includes outer membrane porin F and outer membrane porin I; wherein the mass ratio of the recombinant protein to the outer membrane porin combination includes 1-3:3; the recombinant protein is expressed by a recombinant expression vector, and the recombinant expression vector includes the nucleotide sequence shown in SEQ ID NO:

2.

2. The vaccine according to claim 1, wherein The mass ratio of the recombinant protein to the outer membrane porin is 1:

2.

3. The vaccine according to claim 1, wherein The Pseudomonas aeruginosa vaccine further includes an adjuvant.

4. The vaccine according to claim 3, wherein The adjuvant includes MF59.

5. The vaccine according to claim 1, wherein The mass ratio of the outer membrane porin F to the outer membrane porin I is 1:

1.

6. Use of a recombinant protein in the preparation of a Pseudomonas aeruginosa vaccine, characterized in that: The recombinant protein includes the outer membrane carboxylate channel K6 protein of Pseudomonas aeruginosa. The recombinant protein is expressed by a recombinant expression vector. The recombinant expression vector includes the nucleotide sequence shown in SEQ ID NO:

2.

7. The use according to claim 6, characterized in that The recombinant protein also includes a Pseudomonas aeruginosa heme uptake receptor.

8. The use according to claim 6, characterized in that The recombinant protein is used in combination with an outer membrane porin combination; the outer membrane porin combination includes outer membrane porin F and outer membrane porin I.

9. Use of a recombinant protein in the preparation of a medicament for treating and / or preventing bacterial infection, characterized in that: The bacterial infection includes infection caused by Pseudomonas aeruginosa and / or Acinetobacter baumannii, and the recombinant protein includes the outer membrane carboxylate channel K6 protein of Pseudomonas aeruginosa; the recombinant protein is expressed by a recombinant expression vector, and the recombinant expression vector includes the nucleotide sequence shown in SEQ ID NO:

2.

10. The use according to claim 9, characterized in that The bacterial infection is mechanical ventilation-associated pneumonia.

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