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

Technical Field

[0001] 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

[0002] Pseudomonas aeruginosa (PA) is a common clinical conditional pathogen with the characteristics of high infection rate, rapid mutation, strong pathogenicity and strong drug resistance. Although the isolation rate of Pseudomonas aeruginosa in hospital environment has decreased in recent years, it is still an important cause of lower respiratory tract infection.

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

[0004] Vaccines are an alternative strategy to combat Pseudomonas aeruginosa infection. Among them, immunization with outer membrane protein F or outer membrane protein I as an immunogen can induce 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 for Pseudomonas aeruginosa vaccines that has entered Phase III clinical trials, with better safety and immunogenicity. However, the results of Phase III clinical trials showed that the infection rate of patients vaccinated with the 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 problems in terms of safety and effectiveness. Summary of the invention

[0005] In the first aspect, the present invention provides a 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 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 includes 1-3:3.

[0006] In some embodiments, the recombinant protein is expressed by a recombinant expression vector, and the recombinant expression vector comprises a nucleotide sequence as 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 the outer membrane porin F to the 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 to prevent and / or treat Pseudomonas aeruginosa infection.

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

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

[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 Pseudomonas aeruginosa vaccine has a bacteria (Pseudomonas aeruginosa) clearance rate of at least 99%.

[0016] 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.

[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] 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 an 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.

[0020] In some embodiments, the recombinant protein is expressed by a recombinant expression vector, and the recombinant expression vector comprises a nucleotide sequence as 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 the outer membrane porin F to the 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] 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.

[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 lung infection.

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

[0033] In some embodiments, the recombinant protein is expressed by a recombinant expression vector, and the recombinant expression vector comprises a nucleotide sequence as 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 medicament 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 the existing bacterial resistance mechanism in clinical practice, can effectively reduce infections caused by bacteria, can not only be used to treat / prevent Pseudomonas aeruginosa infections, but also has an immunoprotective effect on Acinetobacter baumannii infections, can significantly reduce bacterial colonization in the lungs of mice, has good immunoprotective efficacy, and is particularly suitable for the treatment and / or prevention of hospital-acquired infections (e.g., mechanical ventilation-associated pneumonia).

[0039] In a fifth aspect, the present invention provides a use of a recombinant protein in the preparation of a Pseudomonas aeruginosa vaccine, characterized in that the recombinant protein comprises an outer membrane carboxylate channel K6 protein of Pseudomonas aeruginosa and / or a heme uptake receptor of Pseudomonas aeruginosa.

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

[0041] In some embodiments, the outer membrane porin combination includes 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 comprises 1-3:3.

[0043] In some embodiments, the recombinant protein is expressed by a recombinant expression vector, and the recombinant expression vector comprises a nucleotide sequence as 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 the outer membrane porin F to the 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 comprises 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 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.

[0050] In addition, it should be emphasized that the addition of the outer membrane carboxylate channel K6 protein can not only enhance the original immune protection efficacy of the outer membrane porin F + outer membrane porin I, but also the safety of the Pseudomonas aeruginosa vaccine based on the outer membrane protein F and outer membrane protein I, which has been fully confirmed, 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%), and has good safety, and has extremely high application value in preventing and / or treating Pseudomonas aeruginosa infection (especially lung infection caused by Pseudomonas aeruginosa (such as acute pneumonia)). BRIEF DESCRIPTION OF THE DRAWINGS

[0051] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, each element or part is 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 paying creative labor.

[0052] Figure 1 Figure 1 is the sequence identification result of the recombinant plasmid; Figure 2 This is a diagram showing the expression and purification results 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 is a graph comparing the immune protection efficacy of different recombinant protein combinations; Figure 6 This is a graph comparing the immune protection efficacy of different recombinant protein combinations; Figure 7 The figure shows the safety results of different recombinant protein combinations. DETAILED DESCRIPTION

[0053] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are 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 creative work are within the scope of protection of the present invention.

[0054] Herein "and / or" includes any and all combinations of one or more of the associated listed items.

[0055] Herein, "plurality" means two or more than two, ie, it includes two, three, four, five, etc.

[0056] 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.

[0057] In this specification, some embodiments may be disclosed in a format of being in a range. It should be understood that this description of "being in a range" is only for convenience and brevity, and should not be interpreted 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 independent numerical values ​​within this range. For example, the description of the range 1-6 should be considered to have 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 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.

[0058] Embodiment 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 outer membrane carboxylate channel K6 of Pseudomonas aeruginosa were synthesized and connected to the pET-28a(+) expression vector to construct a recombinant plasmid. The recombinant plasmid with the correct plasmid sequence identified by sequencing reaction was subjected to subsequent expression and purification steps.

[0059] Expression of recombinant proteins Select a single clone containing the recombinant plasmid and place it in 3 mL LB liquid medium (containing Kan 50 μg / mL), shake and culture it at 37℃ 220pm overnight. The next day, measure the OD630 value of the overnight bacterial solution, calculate the concentration of the overnight bacterial solution, and inoculate it into 3 mL LB liquid medium (containing Kan 50 μg / mL) to make the initial bacterial solution concentration 0.05 OD, and shake and culture it at 37℃ 220 rpm to the logarithmic phase (about 0.4-0.8OD). Take 1 mL of bacterial solution as the control group, add IPTG inducer (final concentration 0.1 mM) to the remaining bacterial solution, shake and culture at 37℃ for 4 h, take 1 mL of the bacterial solution from each group, centrifuge at 12000×g for 2 min, resuspend the bacterial pellet with 100 μL / 1 OD 1× loading buffer, lyse it in a boiling water bath for 10 min, and take 10 μL for SDS-PAGE gel electrophoresis.

[0060] Purification of recombinant proteins Fill the Ni-NTA column, rinse with deionized water to remove 20% ethanol, pass 3 volumes of 0.1 M EDTA-2Na through the column, 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+, add 3 column volumes of equilibration buffer (pH=4.0) to wash, and finally wash with 3 column volumes of NTA-0 buffer (pH=8.0) until the pH of the effluent is 8.0.

[0061] The supernatant protein solution was filtered through a 0.22 µm filter and then used. The supernatant protein solution was loaded at a flow rate of 1 mL / min, and the column was washed with NTA-0 buffer (pH = 8.0) until the effluent contained no protein (the G250 detection solution did not change color). The column was eluted with 20 mM, 60 mM, 200 mM and 500 mM imidazole, and the eluate was collected in sections until the G250 detection solution did not change color. The column material was washed with 3 column volumes of deionized water, and finally the column was sealed with 20% ethanol. The collected eluate was dialyzed and concentrated, and 10 μL was taken for SDS-PAGE gel electrophoresis detection. The protein concentration was determined according to the instructions of the BCA protein concentration determination kit, and the aliquot was frozen at -80°C.

[0062] Figure 1 The sequence identification results of the recombinant plasmid are shown, where M represents Marker, and lanes 1 and 2 are monoclones of recombinant plasmids containing the optimized nucleotide sequence of the outer membrane carboxylate channel K6; lane 3 is a monoclone of the recombinant plasmid containing the wild-type nucleotide sequence of the outer membrane carboxylate channel K6. Although 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 both express the outer membrane carboxylate channel K6 protein (amino acid sequence as 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 of the expressed recombinant protein, which 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.

[0063] Expression and purification results of recombinant protein 1 (51 kDa), see Figure 2 (M stands for Marker, Figure 2 The numbers on the right are in kDa).

[0064] MSMTPIARAVAFAALGSSITVPTLAHAEFIKDSKASIELRNFYFNRDFRQEGASQSKAEEWAQGFLLRYESGYTEGTIGFGVDAIGLGVKLDSSPDRSGTGLLKR DRETGRAQDDYGEAGITAKLRASKSTLKIGTLTPKLPVIMPNDSRLLPQTFQGGALNSMEIDGLTLDAGRLKKVNQRDSSDNEDMTITGGGKRNIVVRSGLTSDKF DFAGGSYKWTDNLSTSYHYGKLDNFYKQHYLGLVHTLPIADKQSLKSDIRWARSTDDGSSNVDNKALNAMFTYSLGYHAFGVGYQKMSGDTGFAYINGADPYLVNF IQIGDFANKDEKSWQARYDYNFAGVGIPGLTFMTRYVKGDNIDLLTTSGEGKEWERDMDIAYVFQSGPLKNLGVKWRNATMRTNYTNDYDENRLIVSYTLPLW (SEQ ID NO:3) Embodiment 2 Comparison of immune protection efficacy of different recombinant proteins Using a similar method to that of 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.

[0065] On days 0, 14, and 28, 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 saline and added with 50 μL of adjuvant MF59). According to the different recombinant proteins selected, the experimental groups were divided into model group, outer membrane porin F group, outer membrane porin I group, outer membrane porin 86 group, outer membrane porin Q group, Pseudomonas aeruginosa heme receptor group, and outer membrane carboxylate channel K6 group.

[0066] 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.

[0067] The results are as follows Figure 3As shown, the immune protection efficacy of outer membrane porin 86 and outer membrane porin Q was weak, and the Pseudomonas aeruginosa load was not much different from that of the model group; the immune protection efficacy of the Pseudomonas aeruginosa heme uptake receptor was comparable to that of outer membrane porin F and outer membrane porin I. The Pseudomonas aeruginosa load of mice immunized with outer membrane carboxylate channel K6 decreased by about 1.8 log (P=0.0018), and more than 90% of bacteria (Pseudomonas aeruginosa) were eliminated, which could significantly reduce bacterial colonization in the lungs of mice, and had good immune protection efficacy, which was better than that of outer membrane porin F and outer membrane porin I.

[0068] Outer membrane carboxylate channel K6 can induce high levels of specific antibodies On days 0, 14, and 28, 100 μL of recombinant protein 1 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 saline and added with 50 μL of adjuvant MF59). Orbital blood was collected 7 days after each immunization, and serum was collected for ELISA detection of specific antibody levels. The results are shown in Figure 4 As shown. The outer membrane carboxylate channel K6 protein can reach 10 after the last immunization 4 Serum antibody titers above the above level have good immunogenicity.

[0069] 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 contained 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, 267-2 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).

[0070] The above experimental results show that the outer membrane carboxylate channel K6 is a protein antigen with a dominant B cell antigen epitope and also has a T cell antigen epitope. The outer membrane carboxylate channel K6 has a good immune protection effect, even higher than the outer membrane porin F and outer membrane porin I that have been clinically tested.

[0071] Embodiment 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.

[0072] On days 0, 14, and 28, 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 saline and added with 50 μL of adjuvant MF59). According to the different recombinant proteins selected, the experimental groups were divided into model group, outer membrane porin F+outer membrane porin I group (outer membrane porin F / I, 10 μg each), outer membrane carboxylate channel K6 group (20 μg) and outer membrane porin F+outer membrane porin I+outer membrane carboxylate channel K6 group (combination 1, 6.67 μg each).

[0073] The protective efficacy of challenge was evaluated in each group on the 14th day after the last immunization. The mouse acute pneumonia model was established by tracheal injection of a non-lethal dose of Pseudomonas aeruginosa liquid, and the lung tissue was aseptically obtained 24 hours after infection for bacterial CFU count.

[0074] 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 about 1.5 log, and the Pseudomonas aeruginosa load in the outer membrane carboxylate channel K6 group decreased by about 1.8 log. The Pseudomonas aeruginosa load in the outer membrane porin F + outer membrane porin I + outer membrane carboxylate channel K6 group of mice further decreased by about 2.5 log, and more than 99% of the bacteria were eliminated. The above results indicate that the addition of outer membrane carboxylate channel K6 can effectively enhance the immune protection efficacy of outer membrane porin F + outer membrane porin I.

[0075] Embodiment 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 .

[0076] 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 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 immune protection 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 immune protection efficacy of outer membrane porin F + outer membrane porin I will be further enhanced. However, the increase in the mass of outer membrane porin F or outer membrane porin I alone cannot enhance the immune protection efficacy of outer membrane porin F + outer membrane porin I.

[0077] 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 a stable weight gain during the immunization period, were in good mental state, and no adverse reactions occurred at the injection 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 is suitable for use in combination with outer membrane porin F + outer membrane porin I as a Pseudomonas aeruginosa vaccine.

[0078] It should be noted that, in this article, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the sentence "comprises a ..." does not exclude the existence of other identical elements in the process, method, article or device including the element.

[0079] 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 enlightenment of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the purpose of the present invention and the claims, which all fall within the protection 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 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 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 IDNO:

2.

2. The use according to claim 1, characterized in that 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 also includes an adjuvant.

4. The use according to claim 3, characterized in that The adjuvant includes 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 to reduce the colonization of the 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 elimination rate of the Pseudomonas aeruginosa vaccine is at least 99%.

10. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the nucleotide sequence shown in SEQ ID NO:2.

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