Use of a recombinant protein in the preparation of a Pseudomonas aeruginosa vaccine

Abstract

The present invention relates to the field of biomedicine, and specifically relates to the use of a recombinant protein in the preparation of a Pseudomonas aeruginosa vaccine, characterized in that the recombinant protein comprises the outer membrane carboxylate channel K6 protein of Pseudomonas aeruginosa, the Pseudomonas aeruginosa vaccine comprises 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 comprises 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. The Pseudomonas aeruginosa vaccine provided by the present invention exhibits better immune protection efficacy (the bacterial clearance rate can reach more than 99%) while having good safety, and has extremely high application value in the prevention and / or treatment of Pseudomonas aeruginosa infection (especially lung infection caused by Pseudomonas aeruginosa).

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and particularly to the use of a recombinant protein in the preparation of a Pseudomonas aeruginosa vaccine. Background Art

[0002] Pseudomonas aeruginosa (PA for short) is a clinically common opportunistic pathogen, which is characterized by a high infection rate, rapid mutation, strong pathogenicity and strong drug resistance. Although the isolation rate of Pseudomonas aeruginosa in the hospital environment has decreased in recent years, it is still an important cause of lower respiratory tract infections.

[0003] The clinical treatment of Pseudomonas aeruginosa infection mainly relies on antibiotics. However, due to its natural drug resistance and acquired drug resistance, there is still a lack of effective treatment and control strategies. Data from the China Surveillance for Bacterial Resistance (CHINET) show that in 2021, the isolation rate of Pseudomonas aeruginosa in general teaching hospitals ranked fourth among all isolated strains and third among isolated strains from respiratory specimens. In the past five years, the isolation rate of Pseudomonas aeruginosa has remained among the top four Gram-negative bacteria (10.6% - 13.3%). The pathogenic mechanism of Pseudomonas aeruginosa is complex and its drug resistance mechanism is diverse. Therefore, it is difficult for general antibiotic treatment to completely eliminate bacteria, and even more drug resistance may be generated.

[0004] Vaccines are an alternative strategy against Pseudomonas aeruginosa infection. Among them, immunization with outer membrane protein F or outer membrane protein I as an immunogen can trigger specific immune responses in animal models or clinical trials and is a potential vaccine candidate. The recombinant fusion protein vaccine based on outer membrane protein F and outer membrane protein I (also known as IC43) is the most effective candidate vaccine among the Pseudomonas aeruginosa vaccines entering phase III clinical trials, with better safety and immunogenicity. However, the results of the phase III clinical trial showed that there was no statistically significant difference in the infection rate between the patients vaccinated with this vaccine and those receiving the placebo. Although the prior art has tried to improve the immune protection of the vaccine based on outer membrane protein F and outer membrane protein I, it still faces certain problems in terms of safety and effectiveness. Summary of the Invention

[0005] In a first aspect, the present invention provides the use of a recombinant protein in the preparation of a Pseudomonas aeruginosa vaccine, characterized in that the recombinant protein comprises the outer membrane carboxylate channel K6 protein of Pseudomonas aeruginosa, the Pseudomonas aeruginosa vaccine comprises the recombinant protein and an outer membrane porin combination, and the recombinant protein is used to enhance the immunogenicity of the outer membrane porin combination; 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 is 1 - 3:3.

[0006] In some embodiments, the recombinant protein is obtained by expression from a recombinant expression vector, and the recombinant expression vector comprises the nucleotide sequence shown in SEQ ID NO:2.

[0007] In some embodiments, the mass ratio of the recombinant protein to the outer membrane porin combination is 1:2.

[0008] In some embodiments, the mass ratio of outer membrane porin F to outer membrane porin I is 1:1.

[0009] In some embodiments, the Pseudomonas aeruginosa vaccine further comprises an adjuvant.

[0010] In some embodiments, the adjuvant comprises MF59.

[0011] In some embodiments, the Pseudomonas aeruginosa vaccine is used for preventing and / or treating Pseudomonas aeruginosa infection.

[0012] In some embodiments, the Pseudomonas aeruginosa infection includes acute pneumonia caused by Pseudomonas aeruginosa.

[0013] In some embodiments, the Pseudomonas aeruginosa vaccine is used to reduce the colonization of Pseudomonas aeruginosa in the lungs of a subject.

[0014] In some embodiments, the amino acid sequence of the recombinant protein is as shown in SEQ ID NO:5.

[0015] In some embodiments, the bacterial clearance rate (Pseudomonas aeruginosa) of the Pseudomonas aeruginosa vaccine is at least 99%.

[0016] In a second aspect, the present invention provides a recombinant expression vector, characterized in that the recombinant expression vector comprises the nucleotide sequence shown in SEQ ID NO:2.

[0017] In some embodiments, the recombinant expression vector is a plasmid.

[0018] In some embodiments, the recombinant expression vector is a pET-28a(+) expression vector.

[0019] Third aspect, the present invention provides a Pseudomonas aeruginosa vaccine, characterized in that the Pseudomonas aeruginosa vaccine comprises a combination of a recombinant protein and outer membrane porins, 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 is 1 - 3:3.

[0020] In some embodiments, the recombinant protein is obtained by expression using a recombinant expression vector, and the recombinant expression vector comprises the nucleotide sequence shown in SEQ ID NO:2.

[0021] In some embodiments, the mass ratio of the recombinant protein to the outer membrane porin combination is 1:2.

[0022] In some embodiments, the mass ratio of outer membrane porin F to outer membrane porin I is 1:1.

[0023] In some embodiments, the Pseudomonas aeruginosa vaccine further comprises an adjuvant.

[0024] In some embodiments, the adjuvant comprises MF59.

[0025] In some embodiments, the amino acid sequence of the recombinant protein is as shown in SEQ ID NO:5.

[0026] Fourth aspect, the present invention provides the use of a recombinant protein in the preparation of a drug for treating and / or preventing bacterial infections, characterized in that the bacterial infections include infections caused by Pseudomonas aeruginosa and / or Acinetobacter baumannii, and the recombinant protein comprises the outer membrane carboxylate channel K6 protein of Pseudomonas aeruginosa.

[0027] In some embodiments, the recombinant protein further comprises a Pseudomonas aeruginosa heme uptake receptor.

[0028] In some embodiments, the bacterial infection is a Pseudomonas aeruginosa infection.

[0029] In some embodiments, the bacterial infection is an Acinetobacter baumannii infection.

[0030] In some embodiments, the bacterial infection is a hospital-acquired infection.

[0031] In some embodiments, the bacterial infection is a pulmonary infection.

[0032] In some embodiments, the bacterial infection is ventilator-associated pneumonia.

[0033] In some embodiments, the recombinant protein is obtained by recombinant expression using a recombinant expression vector comprising the nucleotide sequence shown in SEQ ID NO:2.

[0034] In some embodiments, the amino acid sequence of the recombinant protein is as shown in SEQ ID NO:5.

[0035] In some embodiments, the drug is in the form of a vaccine.

[0036] In some embodiments, the vaccine further comprises an adjuvant.

[0037] In some embodiments, the adjuvant comprises MF59.

[0038] 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 existing clinical bacterial resistance mechanisms and can effectively reduce infections caused by bacteria. It can not only be used for the treatment / prevention of Pseudomonas aeruginosa infections, but also has an immune protection effect against Acinetobacter baumannii infections, can significantly reduce bacterial colonization in the lungs of mice, has good immune protection efficacy, and is particularly suitable for the treatment and / or prevention of hospital-acquired infections (such as ventilator-associated pneumonia).

[0039] In a fifth aspect, the present invention provides the use of a recombinant protein in the preparation of 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.

[0040] In some embodiments, the recombinant protein is used in combination with an outer membrane porin combination.

[0041] In some embodiments, the outer membrane porin combination comprises outer membrane porin F and outer membrane porin I.

[0042] In some embodiments, the mass ratio of the recombinant protein to the outer membrane porin combination is 1 – 3:3.

[0043] In some embodiments, the recombinant protein is obtained by recombinant expression using a recombinant expression vector comprising the nucleotide sequence shown in SEQ ID NO:2.

[0044] In some embodiments, the mass ratio of the recombinant protein to the outer membrane porin combination is 1:2.

[0045] In some embodiments, the mass ratio of outer membrane porin F to outer membrane porin I is 1:1.

[0046] In some embodiments, the Pseudomonas aeruginosa vaccine further comprises an adjuvant.

[0047] In some embodiments, the adjuvant includes MF59.

[0048] In some embodiments, the amino acid sequence of the recombinant protein is as shown in SEQ ID NO:5.

[0049] Compared with the prior art, the beneficial effects of the present invention at least include the following aspects:

[0050] 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 it is found that the addition of the outer membrane carboxylate channel K6 protein of 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 porin combination is 1 / 3), which improves the original immune protection efficacy of outer membrane protein F and outer membrane protein I, increases the bacterial clearance rate of Pseudomonas aeruginosa from 90% to more than 99%, and is particularly suitable for clearing Pseudomonas aeruginosa colonized in the lungs of a subject 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 can not only improve the original immune protection efficacy of outer membrane porin F + outer membrane porin I, but also the safety of the Pseudomonas aeruginosa vaccine based on outer membrane protein F and outer membrane protein I that has been fully proven is not affected. Therefore, the Pseudomonas aeruginosa vaccine provided by the present invention exhibits better immune protection efficacy (the bacterial clearance rate can reach more than 99%) while having good safety, 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 technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the elements or parts are not necessarily drawn to actual scale. Obviously, the following-described drawings are some embodiments of the present invention, and those of ordinary skill in the art can obtain other drawings according to these drawings without creative efforts.

[0053] Figure 1 It is a diagram of the sequence identification result of the recombinant plasmid;

[0054] Figure 2Results diagram of the expression and purification of recombinant protein 1;

[0055] Figure 3 Results diagram for comparing the immunoprotective efficacy of different recombinant proteins;

[0056] Figure 4 Results diagram of the antibody titer of outer membrane carboxylate channel K6;

[0057] Figure 5 Results diagram for comparing the immunoprotective efficacy of different combinations of recombinant proteins;

[0058] Figure 6 Results diagram for comparing the immunoprotective efficacy of different combinations of recombinant proteins;

[0059] Figure 7 Results diagram of the safety of different combinations of recombinant proteins. Detailed implementation manners

[0060] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, 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 part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0061] As used herein, "and / or" includes any and all combinations of one or more of the listed related items.

[0062] As used herein, "a plurality" means two or more, i.e., it includes two, three, four, five, etc.

[0063] As used in this specification, the term "about" typically represents + / - 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.

[0064] In this specification, certain embodiments may be disclosed in a format that is within a certain range. It should be understood that this description of "within a certain range" is merely for convenience and brevity and should not be construed as a rigid limitation on the disclosed range. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and the individual numerical values within that range. For example, the description of the range 1 - 6 should be regarded as having specifically disclosed sub-ranges 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 the individual numbers within that range, such as 1, 2, 3, 4, 5, and 6. The above rules apply regardless of the breadth of the range.

[0065] Example 1

[0066] Synthesis of the nucleotide sequence of Pseudomonas aeruginosa outer membrane carboxylate channel K6 and construction of recombinant plasmid

[0067] The wild-type nucleotide sequence (SEQ ID NO:1) and the optimized nucleotide sequence (SEQ ID NO:2) of Pseudomonas aeruginosa outer membrane carboxylate channel K6 were gene-synthesized and respectively ligated to the pET-28a(+) expression vector to construct recombinant plasmids. The recombinant plasmids with correct plasmid sequences identified by sequencing reaction were subjected to subsequent expression and purification steps.

[0068] Expression of recombinant protein

[0069] Single colonies containing the recombinant plasmid were selected and inoculated into 3 mL of LB liquid medium (containing 50 μg / mL of Kan), and cultured overnight at 37°C with shaking at 220 pm. The next day, the overnight bacterial solution was taken to measure the OD630 value, calculate the concentration of the overnight bacterial solution, and inoculated into 3 mL of LB liquid medium (containing 50 μg / mL of Kan) to make the initial bacterial solution concentration 0.05 OD, and cultured at 37°C with shaking at 220 rpm until the logarithmic phase (about 0.4 - 0.8 OD). 1 mL of the bacterial solution was taken as the control group, and IPTG inducer (final concentration 0.1 mM) was added to the remaining bacterial solution, and cultured at 37°C with shaking for 4 h. 1 mL of the two groups of bacterial solutions was taken respectively, centrifuged at 12000×g for 2 min, and the bacterial cell pellet was resuspended with 100 μL / 1 OD of 1×loading buffer and lysed in a boiling water bath for 10 min, and 10 μL was taken for SDS-PAGE gel electrophoresis detection.

[0070] Purification of recombinant protein

[0071] Load Ni-NTA column material, rinse with deionized water to remove 20% ethanol, pass through the column with 3 volumes of 0.1 M EDTA-2Na, then rinse with deionized water, add 3 column volumes of NTA-0 buffer (pH = 8.0) to equilibrate the nickel column, add 5 column volumes of 0.1 M NiSO4 solution to chelate Ni2+, then add 3 column volumes of equilibration buffer (pH = 4.0) for washing, and finally wash with 3 column volumes of NTA-0 buffer (pH = 8.0) until the pH of the effluent is 8.0.

[0072] The supernatant protein solution is filtered through a 0.22 µm filter and reserved. The supernatant protein solution is loaded onto the column at a flow rate of 1 mL / min, and the column is washed with NTA-0 buffer (pH = 8.0) until the effluent contains no protein (the G250 detection solution does not change color). Elute with 20 mM, 60 mM, 200 mM, and 500 mM imidazole respectively, collect the eluate in fractions until the G250 detection solution does not change color, wash the column material with 3 column volumes of deionized water, and finally seal the column with 20% ethanol. Dialyze and concentrate the collected eluate, take 10 μL for SDS-PAGE gel electrophoresis detection, measure the protein concentration according to the steps in the BCA protein concentration assay kit instruction manual, and store in aliquots at -80 °C.

[0073] Figure 1 Show the sequence identification results of the recombinant plasmid, where M represents Marker, lanes 1 and 2 are both monoclonal clones of the recombinant plasmid containing the optimized nucleotide sequence of outer membrane carboxylate channel K6; lane 3 is a monoclonal clone of the recombinant plasmid containing the wild-type nucleotide sequence of outer membrane carboxylate channel K6. Although both the recombinant plasmid containing the wild-type nucleotide sequence of outer membrane carboxylate channel K6 and the recombinant plasmid containing the optimized nucleotide sequence of outer membrane carboxylate channel K6 can express outer membrane carboxylate channel K6 protein (the amino acid sequence is as shown in SEQ ID NO: 3), the recombinant plasmid containing the wild-type nucleotide sequence of outer membrane carboxylate channel K6 not only has a low expression level of the recombinant protein but also has low immunogenicity of the expressed recombinant protein. 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 outer membrane carboxylate channel K6 and the recombinant plasmid containing the optimized nucleotide sequence of outer membrane carboxylate channel K6. Therefore, the recombinant protein (abbreviated as recombinant protein 1) expressed by the recombinant plasmid containing the optimized nucleotide sequence of outer membrane carboxylate channel K6 is used for subsequent experiments in the present invention.

[0074] Expression and purification results of recombinant protein 1 (51 kDa), see Figure 2 (M represents Marker, Figure 2 The unit of the numbers on the right is kDa).

[0075]

[0076]

[0077] MSMTPIARAVAFAALGSSITVPTLAHAEFIKDSKASIELRNFYFNRDFRQEGASQSKAEEWAQGFLLRYESGYTEGTIGFGVDAIGLLGVKLDSSPDRSGTGLLKRDRETGRAQDDYGEAGITAKLRASKSTLKIGTLTPKLPVIMPNDSRLLPQTFQGGALNSMEIDGLTLDAGRLKKVNQRDSSDNEDMTITGGGKRNIVVRSGLTSDKFDFAGGSYKWTDNLSTSYHYGKLDNFYKQHYLGLVHTLPIADKQSLKSDIRWARSTDDGSSNVDNKALNAMFTYSLGYHAFGVGYQKMSGDTGFAYINGADPYLVNFIQIGDFANKDEKSWQARYDYNFAGVGIPGLTFMTRYVKGDNIDLLTTSGEGKEWERDMDIAYVFQSGPLKNLGVKWRNATMRTNYTNDYDENRLIVSYTLPLW (SEQ ID NO:3)

[0078] Example 2

[0079] Comparison of immunoprotective efficacy of different recombinant proteins

[0080] Using a method similar to that in Example 1, the following proteins of Pseudomonas aeruginosa were also expressed and purified respectively: outer membrane porin F, outer membrane porin I, outer membrane porin 86, outer membrane porin Q, and Pseudomonas aeruginosa heme uptake receptor.

[0081] On days 0, 14, and 28 respectively, 100 μL of recombinant protein was injected intramuscularly at two points in the thigh of C57BL / 6J mice for immunization (20 μg of recombinant protein / dose, resuspended in 50 μL of normal saline and added with 50 μL of adjuvant MF59). According to the different recombinant proteins selected, the experimental groups were divided into a model group, an outer membrane porin F group, an outer membrane porin I group, an outer membrane porin 86 group, an outer membrane porin Q group, a Pseudomonas aeruginosa heme receptor group, and an outer membrane carboxylate channel K6 group.

[0082] On the 14th day after the last immunization, the challenge protection efficacy was evaluated for each group. By intratracheal injection of a non-lethal dose of Pseudomonas aeruginosa PAO1 bacterial solution (2×10 7 CFU / mL), a mouse acute pneumonia model was established, and lung tissue was aseptically collected 24 h after infection for bacterial CFU counting.

[0083] The results are asFigure 3 As shown, the immunoprotective efficacy of outer membrane porin 86 and outer membrane porin Q is weak, and there is little difference in the Pseudomonas aeruginosa load compared with the model group; the immunoprotective efficacy of Pseudomonas aeruginosa heme uptake receptor is equivalent to that of outer membrane porin F and outer membrane porin I. The Pseudomonas aeruginosa load in the mice immunized with outer membrane carboxylate channel K6 decreased by about 1.8 logs (P = 0.0018), and more than 90% of the bacteria (Pseudomonas aeruginosa) could be cleared, which could significantly reduce the bacterial colonization in the lungs of mice, with good immunoprotective efficacy, superior to outer membrane porin F and outer membrane porin I.

[0084] Outer membrane carboxylate channel K6 can induce the production of high levels of specific antibodies

[0085] On days 0, 14, and 28 respectively, 100 μL of recombinant protein 1 was injected into the thigh muscles of C57BL / 6J mice at two points for immunization (20 μg of recombinant protein / dose, resuspended in 50 μL of normal saline and added with 50 μL of adjuvant MF59). Blood was collected from the orbital cavity 7 days after each immunization, and the serum was collected for ELISA detection of specific antibody levels. The results are as Figure 4 shown. The serum antibody titer of outer membrane carboxylate channel K6 protein could reach more than 10 4 after the last immunization, with good immunogenicity.

[0086] Predict the antigenic epitopes of outer membrane carboxylate channel K6 protein

[0087] Using the Protean software in DNAStar software, the amino acid sequence of outer membrane carboxylate channel K6 was analyzed for T cell and B cell antigenic epitopes: It was found that it contains 31 potential B cell antigenic 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 - 269, 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 antigenic 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).

[0088] The above experimental results show that the outer membrane carboxylate channel K6 is a protein antigen dominated by B-cell epitopes and also has T-cell epitopes. The outer membrane carboxylate channel K6 has good immunoprotective efficacy, even higher than that of outer membrane porin F and outer membrane porin I which have undergone clinical trials.

[0089] Example 3

[0090] Comparison of immunoprotective efficacy of different recombinant protein combinations

[0091] Considering that outer membrane porin F and outer membrane porin I have been well studied and have good immunogenicity and safety, therefore, this example focused on investigating whether the outer membrane carboxylate channel K6 can further improve the immunoprotective efficacy of outer membrane porin F and outer membrane porin I.

[0092] On days 0, 14, and **************, 100 μL of recombinant protein was injected into two points of the thigh muscle of C57BL / 6J mice for immunization (20 μg of recombinant protein / dose, resuspended in 50 μL of normal saline and added with 50 μL of adjuvant MF59). According to the different recombinant proteins selected, the experimental groups were divided into a model group, an outer membrane porin F + outer membrane porin I group (outer membrane porin F / I, 10 μg each), an outer membrane carboxylate channel K6 group (20 μg), and an outer membrane porin F + outer membrane porin I + outer membrane carboxylate channel K6 group (combination 1, 6.67 μg each).

[0093] The evaluation of the challenge protection efficacy was carried out on the 14th day after the last immunization in each group. By intratracheal injection of a non-lethal dose of Pseudomonas aeruginosa bacterial solution, an acute pneumonia model of mice was established, and the lung tissue was aseptically collected 24 h after infection for bacterial CFU counting.

[0094] The results are as Figure 5 shown. Compared with the model group, the Pseudomonas aeruginosa load in the outer membrane porin F + outer membrane porin I group decreased by about 1.5 logs, and the Pseudomonas aeruginosa load in the outer membrane carboxylate channel K6 group decreased by about 1.8 logs. While the Pseudomonas aeruginosa load in the mice of the outer membrane porin F + outer membrane porin I + outer membrane carboxylate channel K6 group further decreased, by about 2.5 logs, and more than 99% of the bacteria could be cleared. The above results indicate that the addition of the outer membrane carboxylate channel K6 can effectively enhance the immunoprotective efficacy of outer membrane porin F + outer membrane porin I.

[0095] Example 4

[0096] This example also tested the immunoprotective 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 Table 1 below and Figure 6 .

[0097] Table 1

[0098]

[0099] The above results show that the addition of outer membrane carboxylate channel K6 can enhance the original immunoprotective efficacy of outer membrane porin F + outer membrane porin I: when the mass of the added outer membrane carboxylate channel K6 is one-third of the total mass of outer membrane porin F + outer membrane porin I, the original immunoprotective efficacy of outer membrane porin F + outer membrane porin I can be enhanced, and when the mass of the added outer membrane carboxylate channel K6 is equal to the total mass of outer membrane porin F + outer membrane porin I, the immunoprotective efficacy of outer membrane porin F + outer membrane porin I will be further enhanced. However, the increase in the mass of pure outer membrane porin F or outer membrane porin I alone cannot enhance the immunoprotective efficacy of outer membrane porin F + outer membrane porin I.

[0100] In this example, the weight changes of the immunized mice were also recorded weekly, and the immunogenic responses of the mice after each immunization were observed. The weight records are as Figure 7 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 a stable weight gain during immunization, good mental state, and no adverse reactions at the inoculation site. That is, the addition of outer membrane carboxylate channel K6 does 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.

[0101] It should be noted that in this article, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements not only includes those elements, but also includes other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. Without further limitations, the element defined by the statement "including one..." does not exclude the existence of another identical element in the process, method, article or device including the element.

[0102] The embodiments of the present invention have been described above in conjunction with the accompanying drawings. However, the present invention is not limited to the above specific embodiments. The above specific embodiments are merely illustrative and not restrictive. Under the inspiration of the present invention, those of ordinary skill in the art can also make many forms without departing from the spirit and scope protected by the present invention and the claims. These all belong to the protection scope of the present invention.

Claims

1. Use of a recombinant protein in the preparation of a Pseudomonas aeruginosa vaccine, characterized in that, The recombinant protein is the outer membrane carboxylate channel K6 protein of Pseudomonas aeruginosa. The Pseudomonas aeruginosa vaccine comprises the recombinant protein and an outer membrane porin combination, and the recombinant protein is used to enhance the immunogenicity of the outer membrane porin combination; the outer membrane porin combination is composed of 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; the recombinant protein is obtained by expression through a recombinant expression vector, and the nucleotide sequence of the recombinant protein is as shown in SEQ ID NO:

2.

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

2.

3. The use according to claim 1, characterized in that, The Pseudomonas aeruginosa vaccine further comprises an adjuvant.

4. The use according to claim 3, characterized in that, The adjuvant comprises MF59.

5. The use according to claim 1, characterized in that, The Pseudomonas aeruginosa vaccine is used for preventing and / or treating Pseudomonas aeruginosa infection.

6. The use according to claim 5, characterized in that, The Pseudomonas aeruginosa infection includes acute pneumonia caused by Pseudomonas aeruginosa.

7. The use according to claim 6, characterized in that, The Pseudomonas aeruginosa vaccine is used for reducing the colonization of Pseudomonas aeruginosa in the lungs of a subject.

8. The use according to claim 1, characterized in that, The mass ratio of the outer membrane porin F to the outer membrane porin I is 1:

1.

9. The use according to claim 1, characterized in that, The bacterial clearance rate of the Pseudomonas aeruginosa vaccine is at least 99%.

10. A recombinant expression vector, characterized in that, The recombinant expression vector is used for expressing a recombinant protein, and the nucleotide sequence of the recombinant protein is as shown in SEQ ID NO:2.

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