Acinetobacter baumannii outer membrane protein liposome vaccine as well as preparation method and application thereof

Abstract

The invention discloses an acinetobacter baumannii outer membrane protein liposome vaccine as well as a preparation method and application thereof, and relates to the field of biological medicines. The acinetobacter baumannii outer membrane protein liposome vaccine is obtained by efficiently expressing the acinetobacter baumannii outer membrane protein through a genetic engineering means and combining the obtained multiple outer membrane proteins as antigens with a lipid mixture and an adjuvant, and the acinetobacter baumannii outer membrane protein liposome vaccine is proved to be capable of providing complete protection for mice challenged by lethal dose acinetobacter baumannii. Wherein the outer membrane protein loaded by the lipidosome realizes structural refolding, the protein immunogenicity is enhanced, and the lipidosome can reduce the toxicity of the outer membrane protein; omp38 + BauA + FimA may be an effective antigen combination for preventing acinetobacter baumannii infection; as a TLR2 agonist, Pam2CSK4 has a strong auxiliary effect on an acinetobacter baumannii liposome vaccine, can induce innate immune response activation, and provides a more comprehensive protection effect on a host.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an Acinetobacter baumannii outer membrane protein liposome vaccine and a preparation method and application thereof. Background Art

[0002] Acinetobacter baumannii (A.baumannii) is a Gram-negative bacterium that can cause serious infections in critically ill hospital patients, such as ventilator-associated pneumonia, wound infections, and bacteremia, and has become one of the main pathogens of nosocomial infections. Since A.baumannii is easily resistant to disinfectants and antibiotics, it poses a huge challenge to clinical anti-infection treatment and seriously threatens the life safety of patients. Vaccines are the most economical and effective means to control pathogen epidemic infections.

[0003] The vaccines for Acinetobacter baumannii currently under study are in various forms, including inactivated whole-bacter vaccines, polysaccharide vaccines, protein vaccines, DNA vaccines, subunit vaccines, outer membrane vesicle vaccines, and bacterial outer membrane protein complexes (OMCs). All of the above vaccines can have a certain protective effect on animals infected with Acinetobacter baumannii, but they are still in the experimental research stage, and no vaccine for Acinetobacter baumannii has entered clinical trials.

[0004] The bacterial outer membrane is a double-layer membrane structure containing a variety of integral membrane proteins, which have many important functions, including nutrient absorption, signal transduction, bacterial adhesion, and waste output. For pathogenic bacteria, outer membrane proteins also play an important role in evading host defense. Therefore, outer membrane proteins are considered to be the main protective antigens of Acinetobacter baumannii. The outer membrane protein complex contains rich antigens, and it has been proven to have certain protective and therapeutic effects as a preventive vaccine. However, because it is separated from the "membrane environment", the structure cannot be folded correctly, resulting in low immunogenicity and weak immune response. Moreover, there are too many types of antigens in the outer membrane protein complex, the effective antigen content is relatively low, and the endotoxin content is too high, which restricts the development of its vaccine.

[0005] In order to obtain a single-component outer membrane protein with high safety, clear composition and high purity, a prokaryotic expression vector was constructed by recombinant DNA technology to induce the expression of Acinetobacter baumannii outer membrane proteins in Escherichia coli, but most of these proteins still exist in the form of inclusion bodies. Due to the complex pathogenic mechanism of Acinetobacter baumannii and the variety of virulence proteins and pathogenic factors, one outer membrane protein may only provide limited protection. Taking the recombinant Staphylococcus aureus vaccine as an example, the vaccine uses a multivalent strategy, contains five effective antigens, and has now entered phase III clinical trials. Therefore, multi-component antigens may be an effective strategy for preparing Acinetobacter baumannii vaccines in the future.

[0006] The design and development of vaccines generally require adjuvants to assist antigens. Adjuvants may enhance immunogenicity and change the bias of immune responses, which is crucial to the safety and effectiveness of vaccines. Currently, most of the research and development of Acinetobacter baumannii vaccines focuses on outer membrane proteins, and none of them can solve the problem of misfolding in vitro. The screened antigens cannot provide complete protection, and no suitable vaccine adjuvants have been found.

[0007] In view of this, the present invention is proposed. Summary of the invention

[0008] The purpose of the present invention is to provide an Acinetobacter baumannii outer membrane protein liposome vaccine and a preparation method and application thereof. Experiments have shown that the Acinetobacter baumannii outer membrane protein liposome vaccine can provide complete protection for mice challenged with a lethal dose of Acinetobacter baumannii.

[0009] The present invention is achieved in that:

[0010] In the first aspect, the present invention provides an Acinetobacter baumannii outer membrane protein liposome vaccine, comprising liposomes formed by a lipid mixture and an Acinetobacter baumannii outer membrane protein loaded on the lipid mixture, wherein the Acinetobacter baumannii outer membrane protein is selected from at least one of Omp38, BauA, BamA, TamA, FimA and CaW.

[0011] In a second aspect, the present invention also provides a method for preparing an Acinetobacter baumannii outer membrane protein liposome vaccine, which comprises: using a surfactant to dissolve the Acinetobacter baumannii outer membrane protein and a lipid mixture respectively, and then using a dialysis method to prepare the outer membrane protein liposome.

[0012] In a third aspect, the present invention provides the use of the above-mentioned Acinetobacter baumannii outer membrane protein liposome vaccine or the Acinetobacter baumannii outer membrane protein liposome vaccine obtained by the above-mentioned preparation method in the preparation of products for preventing and / or treating diseases caused by Acinetobacter baumannii.

[0013] The present invention has the following beneficial effects:

[0014] The present invention efficiently expresses the outer membrane protein of Acinetobacter baumannii by means of genetic engineering, and combines the obtained multiple outer membrane proteins as antigens with lipid mixtures and adjuvants to obtain an Acinetobacter baumannii outer membrane protein liposome vaccine, which has been verified to provide complete protection to mice challenged with a lethal dose of Acinetobacter baumannii. Among them, the outer membrane protein loaded by liposomes achieves structural refolding, the immunogenicity of the protein is enhanced, and the liposomes may reduce the toxicity of the outer membrane protein; Omp38+BauA+FimA may be an effective antigen combination for preventing Acinetobacter baumannii infection; Pam2CSK4, as a TLR2 agonist, has a strong auxiliary effect on the Acinetobacter baumannii liposome vaccine, and may induce the activation of the innate immune response, providing a more comprehensive protection for the host. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0016] Figure 1 The nucleic acid electrophoresis after the plasmid digestion in Example 1;

[0017] Figure 2 The results of SDS-PAGE of Omp38, BauA, BamA, TamA, CaW and FimA after passing through a nickel column and undergoing an imidazole gradient elution in Example 1;

[0018] Figure 3 The SDS-PAGE test results of the five recombinant outer membrane proteoliposomes in Example 2;

[0019] Figure 4 The DLS results of five recombinant outer membrane proteoliposomes in Example 2;

[0020] Figure 5 The transmission electron micrographs of five recombinant outer membrane proteoliposomes in Example 2 are shown;

[0021] Figure 6 The results of the lethal challenge experiment of mice immunized with the combined outer membrane protein liposome vaccine by intramuscular injection in Example 4;

[0022] Figure 7 The results of the lethal challenge experiment of mice immunized with the combined outer membrane proteoliposome vaccine by intranasal drops in Example 4;

[0023] Figure 8 The SDS-PAGE test results of liposomes prepared with different OMC dosages in Experimental Example 1;

[0024] Fig. 9 The particle size distribution diagram of liposomes prepared with different OMC dosages in Experimental Example 1;

[0025] Fig.10 The results of SDS-PAGE detection of Mega-10 and OG dissolving OMC in Experimental Example 2;

[0026] Fig.11 The SDS-PAGE test results of Mega-10 liposomes and OG liposomes in Experimental Example 2;

[0027] Fig.12 The particle size distribution diagram of liposomes treated with different ultrasonic conditions in Experimental Example 2;

[0028] Fig.13 The SDS-PAGE test results (Mega-10) of liposomes prepared with different lipid mixture feed ratios in Experimental Example 3;

[0029] Fig.14 The SDS-PAGE test results (OG) of liposomes prepared with different lipid mixture feed ratios in Experimental Example 3;

[0030] Fig.15 The particle size distribution diagram (OG) of liposomes prepared with different lipid mixture feed ratios in Experimental Example 3;

[0031] Fig.16 This is the particle size distribution diagram of the liposomes after centrifugation at 1500-5500 rpm in Experimental Example 4;

[0032] Fig.17 The particle size distribution diagram of the liposomes after centrifugation at 5000, 8000, and 13000 rpm in Experimental Example 4;

[0033] Fig.18 The results of SDS-PAGE of liposomes after centrifugation at 5000, 8000, and 13000 rpm in Experimental Example 4;

[0034] Fig.19 This is the particle size distribution diagram of the liposomes in Experimental Example 5 after being treated with carbonate membranes with different pore sizes;

[0035] Fig. 20 The results of SDS-PAGE detection of liposomes after being treated with carbonate membranes of different pore sizes in Experimental Example 5;

[0036] Fig.21 The SDS-PAGE test results of liposomes after extruder and centrifugation in Experimental Example 6;

[0037] Fig. 22The particle size distribution diagram of the liposomes after extruder and centrifugation in Experimental Example 6;

[0038] Fig.23 It is the transmission electron microscope image of OMC-Lp in Experimental Example 6;

[0039] Fig.24 This is the serum IgG response level of mice after intramuscular injection of OMC in Experimental Example 7, **P<0.01;

[0040] Fig.25 This is the survival curve of mice after lethal challenge with OMC intramuscular injection or intranasal immunization in Experimental Example 7;

[0041] Fig.26 The bacterial colonization in the blood and lungs of mice immunized with OMC intramuscular injection after sublethal challenge in Experimental Example 7, where A-B: the bacterial colonization in the blood and lungs of mice in each group; *P<0.05, **P<0.01;

[0042] Fig. 27 The bacterial colonization in the blood and lungs of mice immunized with OMC intranasal drops in Experimental Example 7 after sublethal challenge, where A to B: the bacterial colonization in the blood and lungs of mice in each group;

[0043] Fig.28 It is the IgG response level of the serum of mice immunized with the recombinant proteoliposome vaccine in Experimental Example 8 (inactivated whole bacteria coating);

[0044] Fig.29 This is the survival status of mice immunized with the recombinant proteoliposome vaccine after lethal challenge in Experimental Example 8;

[0045] Fig.30 The serum IgG level (×1000) after mice were immunized with the combined outer membrane protein liposome vaccine in Experimental Example 8 (inactivated whole bacteria coating);

[0046] Fig.31 The results of the lethal challenge experiment of mice immunized with the combined outer membrane protein liposome vaccine in Experimental Example 8;

[0047] Fig.32 The serum specific IgG level of mice immunized with the combined outer membrane protein liposome vaccine in Experimental Example 8 after intramuscular injection;

[0048] Fig.33 The serum specific IgG level of mice after intranasal immunization with the combined outer membrane protein liposome vaccine in Experimental Example 8;

[0049] Fig.34 The serum FimA-specific IgG level of mice immunized with the combined outer membrane protein liposome vaccine in Experimental Example 8;

[0050] Fig.35The results of the lethal challenge experiment of mice immunized with the combined outer membrane protein liposome vaccine by intramuscular injection in Experimental Example 8;

[0051] Fig.36 These are the results of the lethal challenge experiment in mice immunized with the combined outer membrane protein liposome vaccine by nasal drops in Experimental Example 8. DETAILED DESCRIPTION

[0052] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0053] In view of the fact that the outer membrane protein structure of the current Acinetobacter baumannii vaccine cannot be correctly folded, resulting in low immunogenicity, the screened antigens cannot provide complete protection, and no suitable vaccine adjuvant has been screened, the present invention designs the following technical solutions to address the above defects:

[0054] Firstly, the present invention provides an Acinetobacter baumannii outer membrane protein liposome vaccine, which comprises outer membrane protein liposomes and an adjuvant, wherein the outer membrane protein liposomes are obtained by loading the outer membrane protein of Acinetobacter baumannii with a lipid mixture.

[0055] In the present invention, the role of the lipid mixture is to simulate the "membrane environment" and load the outer membrane protein in the lipid bilayer to improve the problem that the outer membrane protein structure cannot be correctly folded, so as to improve the immunogenicity. Regarding the composition of the lipid mixture, in some embodiments, the lipid mixture includes phospholipids and cholesterol, and the phospholipids can be egg phospholipids, cephalins, soybean lecithin and other synthetic phospholipids.

[0056] In some embodiments, the mass ratio of the Acinetobacter baumannii outer membrane protein to the lipid mixture is 1:1.8-2.2.

[0057] In some embodiments, the mass ratio of cholesterol to lecithin in the lipid mixture is 1:0.8-1.2.

[0058] In order to further improve the protective effect of the vaccine, the present invention screens the types of outer membrane protein antigens, and the outer membrane protein antigens obtained can be at least one selected from Omp38, BauA, BamA, TamA, FimA and CaW. Specifically, the six outer membrane proteins Omp38, BauA, BamA, TamA, FimA and CaW can be used as antigens to prepare vaccines, or any two, three, four, five or six of them can be selected as antigens to prepare vaccines.

[0059] In some embodiments, when the outer membrane protein Omp38 and BauA are combined as an outer membrane protein combination, the prepared vaccine has a better protective effect.

[0060] After further optimization of the technical solution of the present invention, it was found that when Omp38, BauA and FimA are used as antigen combinations, the prepared vaccine has the best protective effect. The feed ratio of Omp38, BauA and FimA can be set according to the commonly used antigen feed amount of vaccines, and the present invention is not limited to this. Conventionally, the antigen dosage per mouse is 20 micrograms. In some embodiments, the feed ratio of antigens Omp38, BauA and FimA is set to 1:1:1.

[0061] For the above outer membrane protein, the preparation method includes: connecting the gene fragments of Omp38, BauA, BamA, TamA, FimA and CaW into expression vectors respectively, transferring the obtained recombinant vector into host cells, culturing and inducing, and then isolating and purifying to obtain the recombinant protein.

[0062] In some embodiments, the expression vector includes pET-30a; the host bacteria include Escherichia coli BL21 (DE3). The expression vector and host bacteria are not limited to the above types, and those skilled in the art can select them according to experimental conditions and actual needs, as long as the recombinant protein can be obtained.

[0063] In some embodiments, the purification comprises subjecting the crude product to gel filtration chromatography; the filler in the gel filtration chromatography is Ni-NTA, and the eluent is a urea solution.

[0064] In some embodiments, the adjuvant is selected from at least one of TLR2 agonists such as Pam2CSK4, Pam3CSK4 and FSL-1, TLR4 agonists such as MPLA, GLA, TLR9 agonists such as CpG, Agatolimod, etc., and natural plant adjuvants such as QS-21 and β-glucan. When the antigen is a single outer membrane protein, the adjuvant is preferably MPLA and CpG, and more preferably, the adjuvant is CpG; when the antigen is a combination of multiple outer membrane proteins, the adjuvant is preferably QS-21 and Pam2CSK4; when the antigen is a combination of Omp38, BauA and FimA outer membrane proteins, the adjuvant is preferably Pam2CSK4.

[0065] In the present invention, the ratio of outer membrane proteoliposome to adjuvant can be set according to conventional proportions, and the present invention does not limit this.

[0066] The method for preparing the Acinetobacter baumannii outer membrane protein liposome vaccine using the above outer membrane protein, lipid mixture and adjuvant is as follows:

[0067] Surfactants are used to dissolve the outer membrane protein and lipid mixture of Acinetobacter baumannii respectively, and then the outer membrane protein liposomes are prepared by dialysis. Ultrasound, centrifugation, gradient extrusion and filtration can be used to reduce the particle size and improve the particle size distribution.

[0068] In some embodiments, the surfactant is selected from any one of decanoyl-N-methylglucamide (Mega-10) and octyl-β-D-pyranoglucoside (OG); in the present invention, when the surfactant is OG, the uniformity of the liposomes and the outer membrane protein encapsulation rate can be further improved.

[0069] In order to improve the uniformity of the prepared liposomes, the present invention also optimizes the feeding amount of the outer membrane protein. Experiments show that when the feeding amount is 0.8-1:2, the prepared outer membrane protein liposomes have better uniformity.

[0070] In order to further improve the encapsulation rate of the outer membrane protein, the present invention adjusts the feeding amount of the lipid mixture on the basis of the above ratio. Experiments show that when the surfactant is OG and the feeding amount of the outer membrane protein and the lipid mixture is 1:1-3, the prepared outer membrane protein liposomes have good uniformity and encapsulation rate; more preferably, when the feeding amount of the outer membrane protein and the lipid mixture is 1:1-1.2, the uniformity and encapsulation rate of the outer membrane protein liposomes are higher.

[0071] In order to improve the uniformity of liposome particle size, the outer membrane protein liposomes obtained are subjected to ultrasonic treatment after dialysis. In some embodiments, the ultrasound is probe ultrasound or water bath ultrasound. In contrast, probe ultrasound is more intense than water bath ultrasound and may be easier to obtain liposomes with uniform particle size.

[0072] Centrifugation can separate particles of different sizes by using their different sedimentation speeds under the action of centrifugal force. During the preparation of outer membrane proteoliposomes, by adjusting the centrifugal force, larger or smaller outer membrane proteoliposomes can be selectively removed, thereby making the particle size more uniform.

[0073] In some embodiments, the centrifugation speed is 1500-5500 rpm.

[0074] In some embodiments, the gradient extrusion is: using 800 nm, 400 nm, and 200 nm carbonate membranes to extrude the outer membrane proteoliposomes in sequence.

[0075] By optimizing the conditions of centrifugation and gradient extrusion, the invention can obtain more uniform outer membrane proteoliposomes with higher encapsulation rate. The centrifugation conditions are: 4000-5500rpm; the gradient extrusion is to extrude in sequence using 800nm, 400nm and 200nm carbonate membranes respectively.

[0076] The administration mode of the Acinetobacter baumannii outer membrane proteoliposome vaccine obtained by the above preparation method can be injection immunization or nasal immunization. Experiments have shown that the vaccine has achieved complete protection in both intramuscular injection and nasal immunization mice. Therefore, the vaccine of the present invention can be used to prepare products for preventing and / or treating diseases caused by Acinetobacter baumannii, and has good application prospects.

[0077] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0078] Example 1

[0079] This example is the expression and purification of recombinant proteins Omp38, BauA, BamA, TamA, FimA and CaW, and the specific steps are as follows:

[0080] 1. Construction of recombinant expression plasmids for candidate antigens BauA, BamA, TamA, CaW, and FimA

[0081] The protein sequences of BauA, BamA, TamA, CaW, and FimA were obtained from the UniProt database, which were derived from Omp38 UniProtKB / TrEMBL: Q6RYW5; BauA UniProtKB / TrEMBL: Q76HJ9; BamA UniProtKB / TrEMBL: A0A7S8WB78; TamA UniProtKB / TrEMBL: D0C629; FimA UniProtKB / TrEMBL: A0A427QVH0; CaW UniProtKB / TrEMBL: F0KGV8. Then, the sequences of BauA, BamA, TamA, CaW, and FimA were codon optimized (website: http: / / sg.idtdna.com / CodonOpt), and whole gene synthesis was performed. Then, the coding sequences were connected to the pET30a vector using the NdeI and XhoI restriction endonuclease sites, respectively, to obtain pET30a-AbBauA, pET30a-AbBamA, pET30a-AbTamA, pET30a-AbCaW, and pET30a-AbFimA plasmids.

[0082] 2. Plasmid Transformation

[0083] (1) Thaw BL21(DE3) competent cells on ice.

[0084] (2) Take 2 μL of each of the four plasmids mentioned above and add them to 100 μL of competent medium. Mix gently and let stand on ice for 30 min.

[0085] (3) After heat shock in a 42°C thermostatic metal bath for 90 seconds, the cells were placed on ice for 3 minutes.

[0086] (4) Add 500 μL of resistance-free LB medium to each tube and culture at 37°C and 220 rpm with shaking for 1 h.

[0087] (5) Centrifuge at 3000 rpm for 10 min at 4°C and resuspend the pellet in 100-200 μL LB medium.

[0088] (6) Spread the bacterial solution on a 50 μg / mL kanamycin-resistant LB plate and culture it at 37°C overnight to grow a single colony.

[0089] 3. Inducible expression of recombinant protein

[0090] (1) Use a 10 μL pipette tip to pick up a single clone on the LB plate, inoculate it into 30 mL LB liquid medium (50 μg / mL kanamycin resistance), and culture overnight at 37°C and 220 rpm.

[0091] (2) The bacterial solution was expanded to 3 L LB liquid medium (40 μg / mL kanamycin resistance) at a ratio of 1:100. The culture was continued at 37°C, 220 rpm for 3 h until the bacterial solution OD 600nm is 0.8.

[0092] (3) Add 3 mL of 1 M IPTG inducer and induce culture at 37°C and 220 rpm for 4 h.

[0093] (4) Centrifuge at 5000 rpm for 20 min and collect the bacterial precipitate.

[0094] (5) Add PBS at a ratio of 1:10 (w / v) to resuspend the cells.

[0095] (6) Bacterial destruction: Use homogenizer at 750 bar for 10 min.

[0096] (7) Centrifuge at 4°C and 5000 rpm for 10 min to obtain the supernatant. Centrifuge at 4°C and 12000 rpm for 30 min to obtain the precipitate, i.e., the inclusion bodies.

[0097] 4. Recombinant protein purification and concentration

[0098] (1) Dissolve the precipitate in 10-20 mL of 8 M urea.

[0099] (2) The Ni-NTA affinity chromatography column was washed three times with 20 mL of PBS and then equilibrated three times with 20 mL of 8 M urea.

[0100] (3) Add the inclusion body solution to the affinity chromatography column and let it stand for about 30 minutes.

[0101] (4) Elution: After the inclusion bodies are eluted, they are collected and then eluted with 15 mL each of 25 mM, 40 mM, 60 mM, 80 mM, 100 mM, 200 mM, 300 mM, 500 mM and 1 M imidazole solutions on the nickel column, and the eluate is collected.

[0102] (5) Take 10 μL of the eluate from each step and perform SDS-PAGE.

[0103] (6) Select an ultrafiltration tube with an appropriate pore size, add pure water to submerge the filter membrane, place it on ice to precool for 2 to 5 minutes, and then pour out the pure water.

[0104] (7) Pour the eluate with higher purity of the target band into the ultrafiltration tube in turn and centrifuge at 3000 rpm until it is concentrated to the required volume.

[0105] (8) Protein concentration was measured by BCA method.

[0106] The plasmid was double digested with Nde I and Xho I for identification. The results of nucleic acid electrophoresis after plasmid digestion were as follows: Figure 1 As shown, the molecular weight of the fragments was consistent with the expected value. Figure 1 Lane M (5 μL / well): DNA marker; Lane 1 (5 μL / well): pET30a-AbBauA; Lane 2 (5 μL / well): pET30a-AbFimA; Lane 3 (5 μL / well): pET30a-AbCaW; Lane 4 (5 μL / well): pET30a-AbBamA; Lane 5 (5 μL / well): pET30a-AbTamA.

[0107] The plasmids pET30a-AbBauA, pET30a-AbBamA, pET30a-AbTamA, pET30a-AbCaW, pET30a-AbFimA and other plasmids that were correctly identified by enzyme digestion were transferred into BL21 (DE3) competent cells. All proteins were expressed in the form of inclusion bodies using the induction conditions of 1mM IPTG, 37°C and 220rpm. Based on the previous work, the recombinant protein Omp38 was induced to express directly from the preserved glycerol bacteria. The protein molecular weight was calculated based on the length of the nucleic acid sequence. The molecular weight of the Omp38 protein is about 38kDa, the molecular weight of the BauA protein is about 79kDa, the molecular weight of the BamA protein is about 92kDa, the molecular weight of the TamA protein is about 96kDa, the molecular weight of the CaW protein is about 52kDa, and the molecular weight of the FimA protein is about 19kDa. The recombinant protein was purified by Ni-NTA affinity chromatography, and the eluate was subjected to SDS-PAGE. The results are as follows Figure 2 As shown, the molecular weight is consistent with the expected one.

[0108] Figure 2AF correspond to Omp38, BauA, BamA, TamA, CaW and FimA respectively after SDS-PAGE detection by nickel column and imidazole gradient elution, where lane M (5 μL / well) of 2A: pre-stained protein standard molecular weight; lane 1 (10 μL / well): bacterial solution after IPTG induction; lane 2 (10 μL / well): centrifugal precipitation after bacterial lysis; lane 3 (10 μL / well): supernatant of centrifuged inclusion bodies; lane 4 (10 μL / well): protein Lane 5 (10 μL / well): 25 mM imidazole eluent; Lane 6 (10 μL / well): 40 mM imidazole eluent; Lane 7 (10 μL / well): 60 mM imidazole eluent; Lane 8 (10 μL / well): 80 mM imidazole eluent; Lane 9 (10 μL / well): 100 mM imidazole eluent; Lane 10 (10 μL / well): 200 mM imidazole eluent; Lane 11 (10 μL / well): 300 mM imidazole eluent.

[0109] Lane M (5 μL / well) of 2B: pre-stained protein standard molecular weight; Lane 1 (10 μL / well): bacterial solution before IPTG induction; Lane 2 (10 μL / well): bacterial solution after IPTG induction; Lane 3 (10 μL / well): supernatant of centrifuged inclusion bodies; Lane 4 (10 μL / well): inclusion bodies; Lane 5 (10 μL / well): protein flow-through; Lane 6 (10 μL / well): 25 mM Lane 7 (10 μL / well): 40 mM imidazole eluent; Lane 8 (10 μL / well): 60 mM imidazole eluent; Lane 9 (10 μL / well): 80 mM imidazole eluent; Lane 10 (10 μL / well): 100 mM imidazole eluent; Lane 11 (10 μL / well): 200 mM imidazole eluent; Lane 12 (10 μL / well): 300 mM imidazole eluent.

[0110] Among them, lane M (5μL / well) of 2C: pre-stained protein standard molecular weight; lane 1 (10μL / well): protein flow-through; lane 2 (10μL / well): 25mM imidazole eluent; lane 3 (10μL / well): 40mM imidazole eluent; lane 4 (10μL / well): 60mM imidazole eluent; lane 5 (10μL / well): 80mM imidazole eluent; lane 6 (10μL / well): 100mM imidazole eluent; lane 7 (10μL / well): 200mM imidazole eluent; lane 8 (10μL / well): 300mM imidazole eluent.

[0111] Among them, lane M of 2D (5μL / well): pre-stained protein standard molecular weight; lane 1 (10μL / well): protein flow-through; lane 2 (10μL / well): 25mM imidazole eluent; lane 3 (10μL / well): 40mM imidazole eluent; lane 4 (10μL / well): 60mM imidazole eluent; lane 5 (10μL / well): 80mM imidazole eluent; lane 6 (10μL / well): 100mM imidazole eluent; lane 7 (10μL / well): 200mM imidazole eluent.

[0112] Lane M (5 μL / well) of 2E: prestained protein standard molecular weight; Lane 1 (10 μL / well): supernatant of lysed bacteria; Lane 2 (10 μL / well): supernatant of centrifuged inclusion bodies; Lane 3 (10 μL / well): protein flow-through; Lane 4 (10 μL / well): 25 mM imidazole eluent; Lane 5 (10 μL / well): 40 mM imidazole eluent; Lane 6 (10 μL / well): 60 Lane 7 (10 μL / well): 80 mM imidazole eluent; Lane 8 (10 μL / well): 100 mM imidazole eluent; Lane 9 (10 μL / well): 200 mM imidazole eluent; Lane 10 (10 μL / well): 300 mM imidazole eluent; Lane 11 (10 μL / well): 500 mM imidazole eluent; Lane 12 (10 μL / well): 1 M imidazole eluent.

[0113] Among them, lane M of 2F (5μL / well): pre-stained protein standard molecular weight; lane 1 (10μL / well): 25mM imidazole eluent; lane 2 (10μL / well): 40mM imidazole eluent; lane 3 (10μL / well): 60mM imidazole eluent; lane 4 (10μL / well): 80mM imidazole eluent; lane 5 (10μL / well): 100mM imidazole eluent; lane 6 (10μL / well): 200mM imidazole eluent; lane 7 (10μL / well): 300mM imidazole eluent; lane 8 (10μL / well): 500mM imidazole eluent; lane 9 (10μL / well): 1M imidazole eluent.

[0114] Example 2

[0115] This example is the preparation and characterization of recombinant outer membrane protein liposomes

[0116] The preparation method of each outer membrane protein liposome is as follows:

[0117] (1) 1 mg of outer membrane protein was added with 20% OG to a concentration of 1%. The outer membrane protein, cholesterol and phosphatidylcholine (dissolved in chloroform) were mixed at a mass ratio of 1:1:1 and incubated at room temperature for 30 min.

[0118] (2) The mixed solution was dialyzed against PBS at 4°C for 24 h, during which the solution was changed twice.

[0119] (3) In an ice bath, use a probe to sonicate the liposomes at 150 W for 10 min (run for 15 s and pause for 5 s).

[0120] (4) The liposomes were treated with 800 nm, 400 nm, and 200 nm carbonate membranes in sequence, and the liposomes were removed from the other side of the syringe after 21 squeezes.

[0121] (5) Centrifuge the liposomes at 4000 rpm for 10 min and collect the supernatant.

[0122] (6) Check the bands by SDS-PAGE.

[0123] SDS-PAGE results are as follows Figure 3 As shown, lane M (5 μL / well): pre-stained protein standard molecular weight; lane 1 (10 μL / well): Omp38-Lp; lane 2 (10 μL / well): BauA-Lp; lane 3 (10 μL / well): BamA-Lp; lane 4 (10 μL / well): TamA-Lp; lane 5 (10 μL / well): FimA-Lp, each protein band is clearly visible; DLS results and protein encapsulation efficiency are shown in Figure 4 ,As shown in Table 1, the particle size of each recombinant proteoliposome was about 200 nm, and the PDI was less than 0.3; Figure 5 Transmission electron micrographs of five recombinant proteins: A: Omp38-Lp; B: BauA-Lp; C: BamA-Lp; D: TamA-Lp; E: FimA-Lp. The liposomes are all regular spherical vesicles, which meet the requirements of animal immunization experiments.

[0124] Table 1 Characterization of five outer membrane proteoliposomes

[0125]

[0126]

[0127] Example 3

[0128] This embodiment is a method for preparing an outer membrane protein liposome vaccine of Acinetobacter baumannii, and the specific steps are as follows:

[0129] (1) using a surfactant to dissolve the outer membrane protein and the lipid mixture respectively, wherein the outer membrane protein is a composition of Omp38, BauA and FimA in a mass ratio of 1:1:1 obtained in Example 1, the mass ratio of the outer membrane protein to the lipid mixture is 1:1, the lipid mixture consists of lecithin and cholesterol in a mass ratio of 1:1, and the surfactant is OG;

[0130] (2) transferring the solution consisting of the outer membrane protein and lipid mixture into a dialysis bag with a molecular weight cutoff of 8-14 kD and dialyzing for 24-36 hours;

[0131] (3) After dialysis, the product was subjected to ultrasound, centrifugation and gradient extrusion. The ultrasound power was 150w for 10min and the centrifugal speed was 4000rpm. The product was extruded in sequence using 800nm, 400nm and 200nm carbonate membranes respectively. The vaccine dosage for a single mouse Pam2CSK4 was 7.5 micrograms and the dosage for a single mouse antigen was 20 micrograms.

[0132] Example 4

[0133] This example is an evaluation of the protective effect of the outer membrane protein liposome vaccine of Acinetobacter baumannii, as follows:

[0134] The Omp38-Lp, BauA-Lp and FimA-Lp obtained in Example 3 were combined with the Pam2CSK4 adjuvant to immunize mice by intramuscular injection and intranasal drip, and mice were challenged with a lethal dose of LAC-4 14 days after the last immunization. The results of the lethal dose challenge are shown in Figure 6-7 As shown, it was shown that the vaccine prepared in Example 3 achieved complete protection in both intramuscular injection and nasal drop immunization mice.

[0135] Experimental Example 1

[0136] This experiment is to compare the performance of liposomes obtained at different feed amounts of Acinetobacter baumannii outer membrane protein complex (OMC, Acinetobacter baumannii outer membrane protein complex obtained by extraction method). The other steps in the preparation method of the liposome are the same as those in Example 3. The mass ratio of OMC to lipid mixture is set as 1 mg: 2 mg, 0.5 mg: 2 mg and 0.25 mg: 2 mg. The SDS-PAGE results of the prepared liposomes are as follows: Figure 8 As shown, lane M (5 μL / well): pre-stained protein standard molecular weight; lane 1 (10 μL / well): 1 mg OMC-Lp (unfiltered); lane 2 (10 μL / well): 1 mg OMC-Lp (filtered); lane 3 (10 μL / well): 0.5 mg OMC-Lp (unfiltered); lane 4 (10 μL / well): 0.5 mg OMC-Lp (filtered); lane 5 (10 μL / well): 0.25 mg OMC-Lp (unfiltered); lane 6 (10 μL / well): 0.25 mg OMC-Lp (filtered).

[0137] Before filtration, the protein bands in the liposomes prepared at each feed amount were clear, and the overall content was proportional to the feed amount. However, after membrane filtration, no protein was found in the liposomes at all feed amounts, indicating that free OMC and large-size OMC-Lp accounted for a high proportion in the system.

[0138] PDI is an important reference indicator for describing the uniformity of particle distribution. The larger the value, the more uneven the particle size distribution. PDI < 0.3 is considered to be a system with relatively uniform particle size. Fig. 9 The DLS measurement results showed that before filtration, OMC-Lp was mostly one peak, and the particle distribution was disorderly and uneven. Filtration could improve the liposome particle size and PDI. The particle size and PDI of 1 mg OMC-Lp were better than those of 0.25 mg OMC-Lp and 0.5 mg OMC-Lp (Table 1).

[0139] Table 2 Characterization of liposomes prepared with different OMC dosages

[0140]

[0141] Based on the above results, 1 mg of protein was used for co-dialysis with 2 mg of lipid mixture to prepare liposomes.

[0142] Experimental Example 2

[0143] This experimental example is a screening experiment of surfactants and a comparison of ultrasonic methods. The other steps in the preparation method of liposomes are the same as those in Example 2.

[0144] Mega-10 and OG, two commonly used surfactants for dissolving outer membrane proteins, were selected. In order to improve the uniformity of liposomes and the encapsulation rate of outer membrane proteins, liposomes were prepared using Mega-10 and OG respectively. First, their solubilizing effects on OMC were compared. OMC was dissolved with Mega-10 and OG respectively, and the supernatant was taken for SDS-PAGE after centrifugation. The results are shown in the figure. Fig.10 As shown, lane M (5 μL / well): pre-stained protein standard molecular weight; lane 1 (10 μL / well): 1 mg / mL OMC; lane 2 (10 μL / well): Mega-10 OMC; lane 3 (10 μL / well): OG OMC.

[0145] Fig.10 The results showed that there was no significant difference between the two surfactants in the dissolution of OMC. Then, liposomes were prepared by dialysis, and the ratio of protein to lipid mixture was as described above.

[0146] In order to improve the uniformity of liposome particle size, the liposomes were ultrasonicated in a 37°C water bath for 1 h after dialysis and then filtered with a filter membrane. Figure 7 As shown, lane M (5 μL / well): pre-stained protein standard molecular weight; lane 1 (10 μL / well): 1 mg / mL OMC; lane 2 (10 μL / well): Mega-10 OMC-Lp; lane 3 (10 μL / well): OG OMC-Lp.

[0147] Fig.11 The SDS-PAGE results showed that the protein bands of Mega-10 liposomes and OG liposomes were not clear, indicating that the protein content in the liposomes was low and the protein encapsulation efficiency of the liposomes needed to be further improved. Fig.12 The DLS results showed that the particle size and PDI of OGBlank-Lp and OMC-Lp were better than those of Mega-10 Blank-Lp and OMC-Lp, indicating that water bath ultrasound can improve the uniformity of liposomes to a certain extent, but the improvement effect on Mega-10 liposomes is not good.

[0148] Probe ultrasound is more intense than waterbath ultrasound, and it may be easier to obtain liposomes with uniform particle size. When other conditions remain unchanged, probe ultrasound is used instead of waterbath ultrasound to treat liposomes. Compared with waterbath ultrasound, probe ultrasound helps to improve the uniformity of liposomes; the particle size of Mega-10 OMC-Lp in waterbath ultrasound is 563.7±274.0nm, and the PDI is 0.768±0.241; the particle size of probe ultrasound is 177.2±17.7nm, and the PDI is 0.461±0.095. The particle size of OG OMC-Lp in waterbath ultrasound is 247.4±9.2nm, and the PDI is 0.322±0.026; the particle size of probe ultrasound is 162.1±9.5nm, and the PDI is 0.305±0.040 (Table 3).

[0149] Table 3 Characterization of liposomes under different ultrasonic treatment conditions

[0150]

[0151]

[0152] The particle size and PDI of liposomes prepared based on OG were better than those of Mega-10, indicating that OG had a better effect on improving the particle size of liposomes, and probe ultrasound could effectively improve the uniformity of liposomes.

[0153] Experimental Example 3

[0154] This experiment is a performance comparison of liposomes obtained from lipid mixtures at different feed amounts. 1, 2, and 3 times the lipid mixture was mixed with OMC, respectively, and then dissolved with Mega-10 and OG, respectively. Liposomes were prepared by dialysis and treated with a liposome extruder (200 nm carbonate membrane) to further increase the OMC loading capacity in the liposomes.

[0155] The liposomes were prepared by Mega-10 dialysis method. The BCA method showed that the encapsulation efficiency of liposomes prepared by 1, 2, and 3 times lipid mixtures was 33.5%, 18.3%, and 33.0%, respectively. The SDS-PAGE results are shown in Fig.13As shown, wherein lane M (5 μL / well): pre-stained protein standard molecular weight; lane 1 (10 μL / well): 1-fold lipid mixture Mega10 OMC-Lp; lane 2 (10 μL / well): 1-fold lipid mixture Mega10 OMC-Lp (filtered); lane 3 (10 μL / well): 2-fold lipid mixture Mega10 OMC-Lp; lane 4 (10 μL / well): 2-fold lipid mixture Mega10 OMC-Lp (filtered); lane 5 (10 μL / well): 3-fold lipid mixture Mega10OMC-Lp; lane 6 (10 μL / well): 3-fold lipid mixture Mega10 OMC-Lp (filtered).

[0156] Fig.13 The SDS-PAGE results showed that after OMC was prepared into liposomes, the main protein bands changed, especially the protective antigen Omp38 protein disappeared, so the use of Mega-10 to prepare liposomes was stopped.

[0157] The liposomes were prepared by OG dialysis method, and the BCA method showed that the encapsulation efficiency of liposomes prepared by 1, 2, and 3 times lipid mixtures was 55.7%, 55.7%, and 40.5%, respectively. The SDS-PAGE results are shown in Fig.14 As shown, lane M (5 μL / well): pre-stained protein standard molecular weight; lane 1 (10 μL / well): 1-fold lipid mixture OG OMC-Lp; lane 2 (10 μL / well): 1-fold lipid mixture OG OMC-Lp (extruder treatment); lane 3 (10 μL / well): 1-fold lipid mixture OG OMC-Lp (filtered); lane 4 (10 μL / well): 2-fold lipid mixture OG OMC-Lp; lane 5 (10 μL / well): 2-fold lipid mixture OG OMC-Lp (filtered); lane 6 (10 μL / well): 3-fold lipid mixture OG OMC-Lp; lane 7 (10 μL / well): 3-fold lipid mixture OG OMC-Lp (extruder treatment); lane 8 (10 μL / well): 3-fold lipid mixture OG OMC-Lp (filtered).

[0158] Fig.14 The SDS-PAGE results showed that the main band was prominent and the bands were clear before extruder treatment. The bands could also be observed after extruder treatment and filtration. The concentrations of the main protein bands in the 1- and 2-fold lipid mixtures were similar and higher than those in the 3-fold lipid mixture group.

[0159] Fig.15The DLS measurement results showed that the particle size of OMC-Lp prepared with 1-fold lipid mixture was 505.7±10.5 nm, and the PDI was 0.320±0.041; the particle size of OMC-Lp prepared with 2-fold lipid mixture was 391.5±12.2 nm, and the PDI was 0.532±0.079; the particle size of OMC-Lp prepared with 3-fold lipid mixture was 356.7±10.2 nm, and the PDI was 0.433±0.022 (Table 3).

[0160] Table 4 Characterization of liposomes prepared with different lipid mixture feed ratios (OG)

[0161]

[0162] The above results showed that the liposomes prepared with 1-fold lipid mixture had the smallest PDI, and the 1-fold liposome mixture was used for subsequent experiments.

[0163] Experimental Example 4

[0164] This experiment is an experiment to improve the particle size distribution of liposomes by centrifugation. Differential centrifugation can separate particles of different sizes by using the difference in sedimentation speed under the action of centrifugal force. In the process of liposome preparation, by adjusting the centrifugal force, larger or smaller liposomes can be selectively removed, thereby obtaining liposomes with more uniform particle size.

[0165] This experiment used differential centrifugation to treat liposomes to investigate whether the uniformity of OMC liposomes could be improved. According to the preparation method of Example 2, dialyzed and ultrasonicated liposomes were obtained, and centrifuged at 1500 rpm, 2500 rpm, 3500 rpm, 4500 rpm, and 5500 rpm for 5 min, respectively, and the particle size distribution was measured. The results are as follows: Fig.16 shown.

[0166] The characterization data of liposomes before and after centrifugation are shown in Table 5:

[0167] Table 5 Characterization of liposomes after centrifugation at 1500-5500 rpm

[0168]

[0169] Compared with before centrifugation, the particle size of liposomes decreased from 1148.9±277.1nm to 294.2±15.2nm, and PDI decreased from 0.993±0.013 to 0.413±0.057 after centrifugation at 5500rpm, indicating that differential centrifugation has a certain effect on improving the uniformity of liposomes.

[0170] The centrifugal speed was further increased to 5000 rpm, 8000 rpm and 13000 rpm. At this time, the particle size of the liposomes was below 200 nm and the PDI was less than 0.3 ( Fig.17 , Table 6), but the SDS-PAGE results showed that the protein bands at each speed were no longer clear, indicating that OMC loss was serious under high speed ( Fig.18 ). Fig.13 Middle lane M (5 μL / well): pre-stained protein standard molecular weight; lane 1 (10 μL / well): 5000rpm OMC-Lp; lane 2 (10 μL / well): 8000rpm OMC-Lp; lane 3 (10 μL / well): 13000rpm OMC-Lp.

[0171] Table 6 Characterization of liposomes after centrifugation at 5000, 8000, and 13000 rpm

[0172]

[0173] Experimental Example 5

[0174] This experiment is an experiment to improve the particle size distribution of liposomes by gradient extrusion. Although high-speed centrifugation helps to improve the particle size distribution of liposomes, protein loss also increases with the increase of speed. Previous experiments have proved that the extruder has a certain effect on particle size improvement, so this experiment tried to use 800nm, 400nm, and 200nm carbonate membranes to treat liposomes in sequence.

[0175] Fig.19 The results in Table 7 show that as the pore size of the carbonate membrane decreases, the particle size and PDI of the liposomes decrease. However, even when the liposomes are extruded using a 200nm carbonate membrane, the particle size still reaches 494.7nm. After filtration with a 0.45μm filter membrane, obvious bands can still be observed, and the protein encapsulation efficiency is 45.04%. After filtration with a 0.22μm filter membrane, the bands disappear, and the protein loss is relatively large ( Fig. 20 ). Fig. 20 Middle lane M (5 μL / well): pre-stained protein standard molecular weight; Lane 1 (10 μL / well): 800 nm carbonate membrane OMC-Lp; Lane 2 (10 μL / well): 400 nm carbonate membrane OMC-Lp; Lane 3 (10 μL / well): 200 nm carbonate membrane OMC-Lp; Lane 4 (10 μL / well): 0.45 μm OMC-Lp; Lane 5 (10 μL / well): 0.22 μm OMC-Lp.

[0176] Table 7 Characterization of liposomes treated with carbonate membranes of different pore sizes

[0177]

[0178] Experimental Example 6

[0179] This experiment is an experiment to improve the particle size distribution of liposomes by combining gradient extrusion and differential centrifugation. In order to obtain liposomes with uniform particle size and high protein loading, we explored the combination of gradient extrusion and differential centrifugation to treat liposomes. After preparing liposomes by the dialysis method of Example 2, they were treated with 800nm, 400nm, and 200nm carbonate membranes in sequence. The SDS-PAGE results showed that the protein bands were intact ( Fig.21 ). Fig. 20 Lane M (5 μL / well): pre-stained protein standard molecular weight; Lane 1 (10 μL / well): OMC-Lp; Lane 2 (10 μL / well): 800nm ​​OMC-Lp; Lane 3 (10 μL / well): 400nm OMC-Lp; Lane 4 (10 μL / well): 200nm OMC-Lp.

[0180] DLS results are as follows Fig. 22 As shown in Table 7, after centrifugation at 2000 rpm, the particle size of OMC-Lp was 202.5±6.1 nm, PDI was 0.308±0.045, and the encapsulation efficiency was 71.6%; after centrifugation at 4000 rpm, the particle size of OMC-Lp was 188.3±4.1 nm, PDI was 0.278±0.048, and the protein encapsulation efficiency was 52.2%. Therefore, the centrifugation speed of 4000 rpm was used as one of the subsequent liposome treatment conditions.

[0181] Table 8 Characterization of liposomes after extruder and centrifugation

[0182]

[0183] Transmission electron microscopy was used to observe its morphology and structure ( Fig.23 ), presenting as regular vesicles. Satisfy the conditions of animal immunization experiments.

[0184] Experimental Example 7

[0185] This experiment is to evaluate the protective effect of OMC liposomes on Ab infection

[0186] OMC-Lp was combined with MPLA and CpG adjuvants to construct vaccines, and their protective effects against lethal doses of Acinetobacter baumannii were evaluated after immunization.

[0187] The Ab OMC liposome vaccine was immunized with mice by intramuscular injection and intranasal drops. OMC liposomes containing 20 μg protein were combined with MPLA or CpG adjuvants and immunized three times by intramuscular injection or intranasal drops at 0, 14, and 28 days. Ab challenge was performed 14 days after the last immunization to evaluate the protective effect of the vaccine against Ab infection.

[0188] (1) Serum IgG response

[0189] ELISA was used to detect the level of OMC-specific IgG in the serum of mice immunized with intramuscular injection. Fig.24 As shown, the OMC group, OMC-Lp group and OMC-Lp-MPLA group all produced significant specific IgG antibody responses, but there was no significant difference in IgG levels among the groups, indicating that liposome loading and the addition of adjuvants failed to significantly increase serum IgG levels.

[0190] (2) Evaluation of attack and protection effects

[0191] Mice in the intramuscular immunization group were challenged with a sublethal dose 14 days after the last immunization. OMC-Lp+CpG intramuscular injection only provided a survival rate of 37.5% ( Fig.25 ). Results of bacterial colonization experiment ( Fig.26 ) showed that the blood bacterial load of the OMC-Lp group and the OMC-Lp+MPLA group was significantly lower than that of the PBS group. No bacteria were found in the lungs of the OMC-Lp group.

[0192] Mice in the intranasal immunization group were challenged with a sublethal dose of the virus 14 days after the last immunization. Fig. 27 ) showed that the amount of bacterial colonization in the blood of the OMC-Lp+MPLA group showed a downward trend, and there was no statistical difference in the amount of bacterial colonization in the blood and lungs among the groups.

[0193] Experimental Example 8

[0194] Previous studies have shown that single outer membrane protein liposomes are more suitable for combination with CpG adjuvants, and combined outer membrane protein liposomes are more suitable for combination with QS-21 adjuvants and Pam2CSK4 adjuvants. Therefore, when screening adjuvants in this experiment, single outer membrane protein liposomes were directly combined with CpG adjuvants, and combined outer membrane protein liposomes were combined with QS-21 adjuvants and Pam2CSK4 adjuvants to test the protective effect against lethal doses of Acinetobacter baumannii.

[0195] 1. Evaluation of the protective effect of a single outer membrane protein liposome vaccine

[0196] 20 μg of protein quantified Omp38-Lp, BauA-Lp, BamA-Lp, and TamA-Lp were mixed with 20 μg of CpG and injected intramuscularly to immunize mice on days 0, 14, and 28, respectively. The mice were challenged with a lethal dose of the virus on day 42, and tail vein blood was collected from the mice before challenge to measure serum antibody titers.

[0197] (1) Serum IgG response

[0198] The 96-well plate was coated with inactivated LAC-4 bacteria. The ELISA results were as follows: Fig.28As shown, A: IgG response level of mouse serum diluted 100 times; B: IgG response level of mouse serum diluted 1000 times; C: IgG response level of mouse serum diluted 4000 times. The IgG level of mice in the recombinant protein group was significantly increased compared with the PBS group at a dilution factor of ×100 (P<0.01 or P<0.001 or P<0.0001), and the BauA-Lp+CpG group and the BamA-Lp+CpG group maintained a high IgG level at a dilution factor of ×1000 and ×4000, which was significantly different from the PBS group (P<0.05 or P<0.01).

[0199] (2) Evaluation of protection effect

[0200] like Fig.29 As shown in A, 14 days after the last immunization, mice were challenged with a lethal dose of LAC-4. The Blank-Lp+CpG group recovered the fastest, followed by the BauA-Lp+CpG group and the Omp38-Lp+CpG group. The TamA-Lp+CpG group had no record of death from the challenge, and the BamA-Lp+CpG group had the largest weight loss and the slowest recovery. Survival results after challenge ( Fig.29 B) showed that the survival of each group was poor, with the survival rate of Omp38-Lp+CpG group being 20%, and the survival rates of Blank-Lp+CpG group and BamA-Lp+CpG group being 10%.

[0201] 2. Evaluation of the protective effect of Omp38-Lp, BauA-Lp, BamA-Lp, and TamA-Lp recombinant proteoliposome vaccines

[0202] like Fig.29 As shown in A, Omp38-Lp and BauA-Lp alone caused little weight change in mice after challenge. The survival of mice was analyzed, and the survival rate of the Omp38-Lp group was 20%, and the death rates of the BauA-Lp group, BamA-Lp group and TamA-Lp group were faster in turn, so the recombinant protein combinations were determined as follows: Omp38-Lp group, Omp38-Lp+BauA-Lp group, Omp38-Lp+BauA-Lp+BamA-Lp group, Omp38-Lp+BauA-Lp+BamA-Lp group.

[0203] Combined outer membrane proteoliposomes (20 μg of each protein content) were combined with 20 μg of QS-21 and used to immunize mice on days 0, 14, and 28. Blood was collected from the tail vein of the mice after the three immunizations to measure serum IgG levels.

[0204] (1) Serum IgG response

[0205] The 96-well plate was coated with inactivated whole bacteria, and the IgG level test results were as follows Fig.30 As shown, there were statistical differences between each experimental group and the PBS group. As the number of recombinant proteins used for immunization increased, the IgG level tended to increase, and the IgG level in the Omp38-Lp+BauA-Lp+BamA-Lp+TamA-Lp group was the highest.

[0206] (2) Evaluation of protection effect

[0207] Omp38-Lp, BauA-Lp, BamA-Lp, TamA-Lp and QS-21 adjuvant were injected intramuscularly into mice, and a lethal dose of LAC-4 was used to challenge the virus 14 days after the last immunization. The results of the lethal dose challenge showed that ( Fig.31 ), the PBS group and Omp38-Lp+BauA-Lp+BamA-Lp+TamA-Lp group died first, and the Omp38-Lp group died the slowest. All groups of mice had poor resistance to LAC-4 infection. Based on the analysis of the mouse death rate, Omp38 and BauA were further studied as possible effective antigens.

[0208] The ELISA method detected that the levels of Omp38 and BauA specific antibodies in the three-component liposome vaccine were higher than those in the two-component liposome vaccine ( Fig.32 , Fig.33 ), the IgG antibody levels in the intramuscular injection group were higher than those in the nasal immunization group ( Fig.34 ). Fig.35 and 36 It shows that the Omp38-Lp+BauA-Lp+FimA-Lp group can completely protect mice from the effects of lethal doses of LAC-4 both by intramuscular injection and nasal immunization.

[0209] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An Acinetobacter baumannii outer membrane protein liposome vaccine, characterized in that: The invention comprises liposomes, wherein the liposomes are formed by a lipid mixture and an outer membrane protein of Acinetobacter baumannii loaded in the lipid mixture, wherein the outer membrane protein of Acinetobacter baumannii is selected from at least one of Omp38, BauA, BamA, TamA, FimA and CaW.

2. The Acinetobacter baumannii outer membrane protein liposome vaccine according to claim 1, characterized in that The outer membrane proteins in the Acinetobacter baumannii outer membrane protein liposome vaccine include Omp38 and BauA; Preferably, the outer membrane proteins in the Acinetobacter baumannii outer membrane protein liposome vaccine include Omp38, BauA and FimA.

3. The Acinetobacter baumannii outer membrane protein liposome vaccine according to claim 1, characterized in that The mass ratio of the Acinetobacter baumannii outer membrane protein to the lipid mixture is 1:1.8-2.2; Preferably, the lipid mixture comprises cholesterol and lecithin; Preferably, the mass ratio of cholesterol to lecithin in the lipid mixture is 1:0.8-1.

2.

4. The Acinetobacter baumannii outer membrane proteoliposome vaccine according to claim 1, characterized in that The Acinetobacter baumannii outer membrane proteoliposome vaccine also includes an adjuvant, which is selected from at least one of a TLR2 agonist, a TLR4 agonist, a TLR9 agonist and a natural plant adjuvant; Preferably, the TLR2 agonist comprises Pam2CSK 4、 Pam3CSK4 and FSL-1; Preferably, the TLR4 agonist includes MPLA and GLA; Preferably, the TLR9 agonist comprises CpG and Agatolimod; Preferably, the natural plant adjuvant comprises QS-21 and β-glucan; Preferably, the adjuvant is a TLR2 agonist; Preferably, the adjuvant is Pam2CSK4.

5. The Acinetobacter baumannii outer membrane proteoliposome vaccine according to claim 4, characterized in that The administration method of the Acinetobacter baumannii outer membrane protein liposome vaccine includes injection immunization and nasal drop immunization.

6. The method for preparing the Acinetobacter baumannii outer membrane proteoliposome vaccine according to any one of claims 1 to 5, characterized in that: include: Surfactants were used to dissolve the outer membrane protein and lipid mixture of Acinetobacter baumannii respectively, and then the outer membrane protein liposomes were prepared by dialysis method.

7. The preparation method according to claim 6, characterized in that: The surfactant is selected from any one of N-decanoyl-N-methylglucosamine and octyl-β-D-pyranoglucoside; Preferably, the surfactant is octyl-β-D-pyranoglucoside.

8. The preparation method according to claim 6, characterized in that: The preparation method further comprises subjecting the obtained outer membrane protein complex liposomes to ultrasound, centrifugation and gradient extrusion; Preferably, the ultrasound is probe ultrasound or water bath ultrasound; Preferably, the centrifugal speed is 4000-5500rpm; Preferably, the gradient extrusion is: using 800nm, 400nm, and 200nm carbonate membranes to extrude liposomes in sequence.

9. The preparation method according to claim 6, characterized in that: The preparation method of the outer membrane protein of Acinetobacter baumannii comprises: connecting the nucleotide sequence encoding Omp38, BauA, BamA, TamA, FimA and CaW antigens to an expression vector to obtain a recombinant expression vector, then transforming the vector into a host bacterium, culturing and inducing the recombinant bacterium to express the outer membrane protein, and then separating and purifying to obtain the recombinant protein; Preferably, the expression vector includes pET-30a; the host bacteria include: Escherichia coli BL21 (DE3); Preferably, the purification comprises subjecting the crude product to affinity chromatography; Preferably, the filler in the affinity chromatography is Ni-NTA, and the eluent is a urea solution.

10. Use of the Acinetobacter baumannii outer membrane proteosome vaccine according to any one of claims 1 to 5 or the Acinetobacter baumannii outer membrane proteosome vaccine obtained by the preparation method according to any one of claims 6 to 9 in preparing products for preventing and / or treating diseases caused by Acinetobacter baumannii.

Images