Soluble acinetobacter baumannii recombinant NucAb protein as well as expression and purification method and application thereof

Abstract

The invention discloses a soluble acinetobacter baumannii recombinant NucAb protein as well as an expression and purification method and application thereof, and relates to the technical field of gene engineering. The recombinant NucAb protein is obtained by expressing a recombinant vector in host bacteria, wherein the recombinant vector is constructed by introducing a NucAb gene with an amino acid sequence as shown in SEQ ID NO: 1 into an expression vector; the expression and purification method comprises the steps of recombinant plasmid construction, induced expression, nickel column affinity chromatography, ion exchange chromatography and the like. By optimizing an expression system and a two-step chromatographic purification process, efficient soluble expression and high-purity separation of the NucAb protein are achieved, the purity is higher than 90%, and the recovery rate reaches 85% or above. The protein has the capability of stimulating high-efficiency specific antibody reaction, the antibody titer reaches 1: 25600, and a technical support is provided for developing vaccines and therapeutic drugs for resisting acinetobacter baumannii infection.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of genetic engineering, in particular to a soluble Acinetobacter baumannii recombinant NucAb protein, an expression and purification method thereof and application. BACKGROUND

[0002] Acinetobacter baumannii is an important opportunistic pathogen, which mainly causes catheter-related pneumonia, traumatic infection, urogenital system infection and catheter-related septicemia, etc. Due to the increasingly serious drug resistance of the strain, especially the multiple drug resistance characteristics of the hospital-acquired infection strain, the therapeutic effect of conventional antimicrobial drugs is significantly reduced. Due to the abuse of antibiotics, "multiple drug-resistant Acinetobacter baumannii" (MDR-AB) and "pan-drug-resistant Acinetobacter baumannii" (XDR-AB) have appeared, which poses a great challenge to clinical treatment. Under this background, vaccination is considered as a potential alternative strategy to prevent and control Acinetobacter baumannii infection, but there is no specific vaccine approved for marketing in the world at present.

[0003] In the past decade, the development of Acinetobacter baumannii vaccine mainly focuses on the recombinant antigen technology route, but there is no Acinetobacter baumannii vaccine approved for marketing in the world at present. Animal model experiments show that the vaccine based on a single recombinant protein can provide partial protection, but the cross-protection efficiency of heterogeneous strains is limited, which indicates that the existing vaccine design strategy still needs to be optimized.

[0004] Acinetobacter baumannii outer membrane nuclease (NucAb) is a protein in the outer membrane of Acinetobacter baumannii, which has nuclease activity. Both gram-negative bacteria and gram-positive bacteria can produce outer membrane nuclease (NucAb). Compared with other candidate antigens, NucAb protein exhibits excellent vaccine candidate characteristics: ① located in the bacterial outer membrane, which is beneficial for the recognition of the immune system; ② with high adhesion probability, involved in the pathogenic process; ③ no homology with human proteins, which can reduce the risk of autoimmunity; ④ carrying abundant B cell and T cell epitopes. These characteristics suggest that NucAb protein can be used as an innovative target for developing broad-spectrum Acinetobacter baumannii vaccine. However, how to efficiently express and purify the protein through genetic engineering technology while ensuring its immunogenicity and functionality is still a technical problem to be solved.

[0005] NucAb proteins have amino acid sequences rich in hydrophobic domains (such as the HNH nuclease domain), which easily form inclusion bodies in prokaryotic expression systems such as E. coli (requiring complex steps such as urea dissolution and refolding), resulting in extremely low soluble expression rates (typically <10%). A search revealed reports on soluble protein expression, such as patent application CN115991745A, which discloses a recombinant antigen protein of Helicobacter pylori, TatB, its preparation method, and applications. This involves the soluble expression of Helicobacter pylori TatB protein, but it is a membrane transport protein (molecular weight 16.8 kDa), significantly different from NucAb in molecular weight, structure (distribution of hydrophobic regions, isoelectric point), and function (nuclease activity), thus failing to provide technical guidance for soluble NucAb expression. Currently, no soluble expression methods for NucAb proteins have been found. The protein mostly exists in inclusion body form (requiring dissolution in 8M urea followed by purification), with purification yields of only 5-10 mg / L, severely limiting its application as a vaccine antigen. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method and application for expressing and purifying soluble Acinetobacter baumannii recombinant NucAb protein, which can obtain high-purity target protein through a simple process.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] A soluble Acinetobacter baumannii recombinant NucAb protein was obtained by expressing a recombinant vector constructed by introducing the NucAb gene with the amino acid sequence shown in SEQ ID NO:1 into an expression vector in a host bacterium.

[0009] Furthermore, the expression vector is plasmid pET28a(+), and the host bacterium is E. coli Rosetta(DE3).

[0010] Furthermore, the recombinant NucAb protein has a molecular weight of 44 kDa.

[0011] Furthermore, the present invention provides a method for expressing and purifying the above-mentioned soluble Acinetobacter baumannii recombinant NucAb protein, comprising the following steps:

[0012] S1, Constructing a prokaryotic expression plasmid: The nucleotide sequence corresponding to the target gene with the amino acid sequence shown in SEQ ID NO:1 is ligated into the expression vector plasmid pET28a(+) to obtain the recombinant plasmid pET28a(+)-NucAb. The recombinant plasmid is then transformed into the host bacterium E. coli Rosetta(DE3) to induce the expression of the exogenous protein, resulting in recombinant Escherichia coli.

[0013] S2, High-pressure sterilization and centrifugation: Collect the recombinant Escherichia coli cells from step S1, mix and suspend them in a equilibration solution, pre-cool at 4°C, and then perform high-pressure sterilization at 700-800 bar. Collect the supernatant. The equilibration solution includes 50 mM Hepes, 100 mM KCl, 10% glycerol, and has a pH of 7.5.

[0014] S3, Ni-NTA chromatography column purification: The supernatant from step S2 is passed through a Ni-NTA affinity chromatography column and eluted sequentially with eluents containing 20-500 mM imidazole. The eluent containing 100 mM imidazole is collected and dialyzed overnight at 4°C. The pH of the eluent is 7.5, and it also includes 50 mM Hepes, 100 mM KCl, and 10% glycerol. The dialysate includes 50 mM Hepes, 150 mM KCl, 1 mM DTT, and 10% glycerol, and has a pH of 7.5.

[0015] S4, Anion exchange column purification: The protein dialyzed overnight in step S3 is purified by anion exchange column using linear elution with solutions A and B to obtain the purified protein. Solution A consists of 50 mM Hepes, 150 mM KCl, 1 mM DTT, and 10% glycerol, with a pH of 7.5. Solution B consists of 50 mM Hepes, 500 mM KCl, 1 mM DTT, and 10% glycerol, with a pH of 7.5. The packing material used for the anion exchange column purification is Capto. TM Q.

[0016] Furthermore, in step S1, the target gene is ligated to the expression vector pET28a(+) via the restriction sites of BamHI and XhoI.

[0017] Furthermore, in step S2, the bacterial cells and equilibration solution are mixed and suspended at a volume ratio of 1:10; after high-pressure sterilization, the mixture is centrifuged at 4°C and 12,000 rpm for 15 minutes.

[0018] Furthermore, in step S3, the Ni-NTA affinity chromatography column is packed with HisSep Ni-NTA Agarose Resin; the amount of packing material used is 100 mL per 100 g of ruptured bacterial cells wet weight.

[0019] Furthermore, in step S4, the amount of packing material used is 100 mL per 100 g of broken bacterial cells wet weight.

[0020] Furthermore, the present invention provides the use of the above-mentioned soluble Acinetobacter baumannii recombinant NucAb protein in the preparation of an anti-Acinetobacter baumannii vaccine composition, wherein the vaccine composition further comprises a pharmaceutically acceptable adjuvant, wherein the concentration of NucAb protein is ≥1 mg / mL.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] (1) This invention utilizes the pET28a(+) vector and the E. coli Rosetta(DE3) host system to construct recombinant NucAb protein. Combined with high-pressure sterilization, Ni-NTA affinity chromatography, and anion exchange chromatography, it achieves efficient soluble expression and high-purity (>90%) preparation of the target protein. This process is simple to operate, has good reproducibility, and achieves a recovery rate of over 85%. Through synergistic design, this invention significantly improves the soluble expression and purification efficiency of NucAb, solving the problem of large-scale production of outer membrane proteins.

[0023] (2) The recombinant NucAb protein of this invention, as a candidate vaccine antigen for Acinetobacter baumannii, can effectively stimulate the body to produce high-titer specific antibodies (titer up to 1:25600), and by specifically inhibiting the nuclease activity mediated by its HNH domain, it can effectively block the immune escape mechanism of pathogens, thereby enhancing the clearance efficiency of phagocytes against bacteria. Animal experiments have confirmed that this protein has a significant immunoprotective effect against Acinetobacter baumannii infection, providing a reliable candidate target for the development of broad-spectrum vaccines.

[0024] (3) This invention is the first to combine the conserved functional epitopes of the NucAb protein with vaccine design. Its sequence has no homology with human proteins, which can reduce the risk of autoimmunity, while carrying abundant B / T cell epitopes. In addition, this protein is located on the bacterial outer membrane, making it easily recognized by the immune system, providing a new solution for the clinical prevention and control of multidrug-resistant Acinetobacter baumannii. Attached Figure Description

[0025] Figure 1 The results of Ni-NTA purification and identification for pET28a(+)-NucAb protein expression:

[0026] The lanes in the diagram represent: Lane M: Protein molecular weight standard; Lane 1: Complete lysate; Lane 2: Supernatant; Lane 3: Precipitate; Lane 4: Breakthrough buffer; Lane 5: Elution buffer containing 20 mM imidazole; Lane 6: Elution buffer containing 50 mM imidazole; Lane 7: Elution buffer containing 100 mM imidazole; Lane 8: Elution buffer containing 200 mM imidazole; Lane 9: Elution buffer containing 300 mM imidazole; Lane 10: Elution buffer containing 500 mM imidazole.

[0027] Figure 2 Purification results of recombinant protein with E. coli BL21(DE3) as host bacterium:

[0028] The lanes in the diagram represent: Lane M: Protein molecular weight standard; Lane 1: Complete lysate; Lane 2: Supernatant; Lane 3: Precipitate; Lane 4: Breakthrough buffer; Lane 5: Elution buffer containing 20 mM imidazole; Lane 6: Elution buffer containing 50 mM imidazole; Lane 7: Elution buffer containing 100 mM imidazole; Lane 8: Elution buffer containing 200 mM imidazole; Lane 9: Elution buffer containing 300 mM imidazole; Lane 10: Elution buffer containing 500 mM imidazole.

[0029] Figure 3 Results of ion column purification of pET28a(+)-NucAb recombinant protein:

[0030] The lanes in the figure are as follows: Lane M: Protein molecular weight standard; Lane 1: Before loading; Lane 2: Breakthrough buffer; Lane 3: 0-20% B elution buffer 1; Lane 4: 0-20% B elution buffer 2; Lane 5: 20-40% B elution buffer 1; Lane 6: 20-40% B elution buffer 2; Lane 7: 20-40% B elution buffer 3; Lane 8: 500mM KCl elution buffer.

[0031] Figure 4 The antibody titer after NucAb protein immunization. Detailed Implementation

[0032] The present invention will be further described below with reference to the accompanying drawings and embodiments. The embodiments of the present invention include, but are not limited to, the following embodiments.

[0033] This embodiment provides a soluble recombinant NucAb protein from Acinetobacter baumannii and its expression and purification method. The NucAb protein is an outer membrane nuclease of Acinetobacter baumannii. Using the pET28a(+) plasmid as a vector, the plasmid was amplified in *E. coli* Rosetta (DE3), and the solubility of the recombinant protein was analyzed. Then, the optimal expression conditions for the recombinant protein were screened, achieving soluble expression of the target protein. The purification process in this embodiment is simple, reproducible, and yields a high-purity target protein. Furthermore, this recombinant protein can stimulate the body to produce specific antibodies, providing highly effective immunoprotection against Acinetobacter baumannii infection, and holds promise as a subunit vaccine antigen for Acinetobacter baumannii.

[0034] In this embodiment, the NucAb gene was cloned and expressed, constructed into the pET28a(+)-NucAb plasmid, transformed into the host bacterium E.coli Rosetta(DE3), and induced for recombinant expression overnight at 16°C with 0.5 mM IPTG. The prepared antigen was then purified by high-pressure centrifugation after sterilization.

[0035] The experimental strains, plasmids, mice, reagents and instruments used in this embodiment were all purchased from the market. Among them, pET28a(+) plasmid and E. coli TOP10 were purchased from Wuhan Jinkairui Biotechnology Co., Ltd., E. coli Rosetta(DE3) was purchased from Beijing TransGen Biotechnology Co., Ltd., and BALB / c mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The sources of the main reagents are shown in Table 1.

[0036] Table 1. Sources of main reagents

[0037]

[0038]

[0039] The construction, expression, and purification methods of soluble Acinetobacter baumannii recombinant NucAb protein provided in this embodiment are as follows:

[0040] I. Cloning and Construction of the NucAb Gene

[0041] 1) NCBI sequence alignment revealed that the NucAb amino acid sequences of different strains of Acinetobacter baumannii are highly conserved. Based on the NucAb amino acid sequence, the target gene fragment was obtained, as shown in SEQ ID NO:1. In this invention, the NucAb amino acid sequence was modified by adding two amino acids, GS (glycine-serine), to its N-terminus and two amino acids, LE (leucine-glutamic acid), to its C-terminus. The GS added to the N-terminus is not a residue from enzyme digestion but a flexible linker peptide actively introduced through molecular design: the small side chain of glycine (G) reduces steric hindrance; the polar side chain of serine (S) increases local hydrophilicity. Together, they reduce the hydrophobicity of the N-terminus and decrease inclusion body formation. The LE added to the C-terminus is a charge-balancing peptide: the hydrophobic side chain of leucine (L) enhances the structural stability of the C-terminus, and the negative charge of glutamic acid (E) balances the local positive charge, reducing protein aggregation.

[0042] SEQ ID NO:1 (Amino acid sequence of the NucAb gene):

[0043] GSMQLSIFEHYKQLLKNAKKYKIPPREKTFFDTAIRNHYENPTTELLEFFLNPTESHDLGDLFWKGFCDVLQQEASLSKLDLGNIVKLEREYATHQGNRIDLWIETDTCFILLEAKIYHHQNNPFQDYIQFAQSKNQSKNKQIVGVILSIAGKSEKKGWLGLSYQQIVNSIRPYLAEQMLANPM NKWNLFAREFLLHLDSYYRIKNLDMNRVQFILDHYKEIEELQRLRTSTISEVVDSLSQQLNEMIDGYESENKYESWGGIRFYNKAWGNKSNTTLLIKQEDGQTVIKVITYILNLSLELEEEAFSILGTQTDSRYLDQKVENYRRGTERWLCIYWRSPENNLTAITDLLFEKVKLLDTIERTLKLE

[0044] 2) The target gene was synthesized in its entirety and ligated into the expression vector pET28a(+) (with a His tag) via BamHI and XhoI restriction sites. The plasmid sequencing results were identical to the target gene sequence, and analysis confirmed the absence of amino acid mutations. The recombinant plasmid was extracted from the recombinant pET28a(+) / NucAb / E. coli TOP10 strain.

[0045] 3) The recombinant plasmid was transformed into the expression strain E. coli Rosetta(DE3) to obtain recombinant E. coli pET28a(+) / NucAb / E. coli Rosetta(DE3) that can express NucAb.

[0046] II. Induced Expression of NucAb Protein

[0047] 1) Activation of bacteria: Take the candidate antigen protein expression bacteria pET28a(+) / NucAb / E.coliRosetta(DE3) stored at -80℃ and activate them by inoculation at a ratio of 1:1000, that is, add 10μL of glycerol bacterial solution to 10mL of LB medium containing Kana and Chl resistance, and incubate overnight at 37℃ and 220rpm for 16h.

[0048] 2) Secondary activation induction: Perform secondary activation by inoculating the bacteria at a 1:100 ratio. Take 200 μL of overnight bacterial culture and add it to 20 mL of LB medium containing Kanamycin and Chl resistant bacteria. Incubate at 37°C and 220 rpm for 5-6 hours. Activate to OD. 600When the concentration is 0.6, add IPTG to a final concentration of 0.5 mM, and incubate overnight at 16°C and 150 rpm on a shaker. (Preparation of IPTG solution: Dissolve 2.38 g of IPTG in 10 mL of Grade I water, and filter through a 0.22 μm sterile filter after complete dissolution).

[0049] 3) After inducing expression, remove the bacterial culture, centrifuge at 4℃ and 6000 rpm for 15 min, discard the supernatant, add 1-2 mL of equilibration buffer to resuspend and mix well, sonicate for 15 min to break down the bacteria, and collect the lysed culture. Centrifuge at 4℃ and 12000 rpm for 15 min to separate the supernatant and precipitate.

[0050] 4) Processing the supernatant and precipitate: Take 20 μL of supernatant and add 5 μL of 5× protein loading buffer, boil for 10 min, and then centrifuge at 14000 rpm for 3 min; add 30 μL of equilibration buffer to the precipitate to resuspend the bacterial cells, take 20 μL of the resuspended bacterial solution and add 5 μL of 5× protein loading buffer, boil for 10 min, and then centrifuge at 14000 rpm for 3 min.

[0051] 5) Pour 12.5% ​​separating gel into the gel casting plate, add distilled water to flatten the gel, and let it solidify at room temperature for 30 minutes. Pour off the top layer of distilled water, then pour in the stacking gel, immediately insert the comb, and let it solidify at room temperature for 30 minutes. Take 10 μL of the treated supernatant and precipitate respectively, and perform SDS-PAGE electrophoresis. Initially, electrophoresis at 80V for 30 minutes, then adjust to 180V and electrophoresis for 1-2 hours. After electrophoresis, remove the gel, place it in staining solution, shake to stain, then place it in water, shake to destain, and observe the results under an imaging system. The results are as follows: Figure 1 As shown, NucAb is a soluble protein.

[0052] Next, the following control experiment was conducted: E. coli BL21(DE3) was selected as the host bacterium for protein expression and purification. The purification results are as follows: Figure 2 As shown in the figure, when using E. coli BL21(DE3) as the host bacterium, the NucAb protein is almost entirely contained in the precipitate and expressed as inclusion bodies. This indicates that only when using E. coli Rosetta(DE3) as the host bacterium can the NucAb protein achieve soluble expression, and conventional vectors / host bacteria cannot solve the problem of soluble NucAb expression.

[0053] III. Preparation of NucAb Antigen

[0054] 1) Scale-up culture to obtain protein: Take 20 μL of pET28a(+) / NucAb / E. coli Rosetta(DE3) bacterial culture stored at 4℃ and add it to 20 mL of LB medium containing Kana and Chl resistance. Incubate overnight at 37℃ and 200 rpm for primary activation. Take 20 mL of the primary activated bacterial culture and add it to 2000 mL of LB medium containing Kana and Chl resistance for secondary activation. Incubate at 37℃ for 5-6 h. Add 0.5 mM IPTG and incubate overnight at 16℃ and 150 rpm for induction. Centrifuge at 4℃ and 6000 rpm for 15 min to collect the bacterial cells. Resuspend the bacterial cells in 50 mL of equilibration buffer. Perform high-pressure lysis of the bacterial culture (700-800 bar). Centrifuge and collect the supernatant.

[0055] 2) Protein binding to packing material: After lysis, the solution was centrifuged at 12,000 rpm for 15 min at 4°C. The supernatant was collected and purified using a Ni column. In this example, the Ni column was a Ni-NTA affinity chromatography column, and the packing material was HisSep Ni-NTA Agarose Resin. The packing material volume was 100 mL per 100 g of lysed bacterial wet weight. Impurities were washed with 5 column volumes of elution buffer containing 20 mM imidazole and 5 column volumes of elution buffer containing 50 mM imidazole. Samples were then collected. The collected samples were eluted sequentially with 5 column volumes of elution buffer containing 100 mM, 200 mM, 300 mM, 400 mM, and 500 mM imidazole to remove NucAb proteins. Samples were then subjected to SDS-PAGE electrophoresis. Collect the eluent containing 100 mM imidazole and dialyze overnight at 4°C (dialysis buffer: 50 mM Hepes, 150 mM KCl, 1 mM DTT, 10% glycerol, pH 7.5). The eluent contains different concentrations of imidazole, as well as 50 mM Hepes, 100 mM KCl, 10% glycerol, and has a pH of 7.5.

[0056] 3) Using Capto TM Q-ion column purification; Capto was rinsed with pure water and equilibration buffer. TM The Q ion column was run three times, and the protein solution dialyzed overnight in step 2) was added. Linear elution was performed using solution A (50 mM Hepes, 150 mM KCl, 1 mM DTT, 10% glycerol, pH 7.5) and solution B (50 mM Hepes, 500 mM KCl, 1 mM DTT, 10% glycerol, pH 7.5). Approximately two column volumes were collected for each gradient (the exact amount depends on the UV absorption peak of the chromatography instrument). The samples from each gradient were labeled and analyzed by electrophoresis (results are shown in the figure). Figure 3 As shown in the figure, the final NucAb protein with a molecular weight of 44 kDa and high purity was obtained.

[0057] IV. Antibody titer assessment after NucAb protein immunization

[0058] BALB / c mice aged 6-8 weeks were used, with 5 mice in each group, divided into two groups: an experimental group and a control group. All mice were housed in an SPF-grade environment with free access to food and water and underwent acclimatization for 1 week. Mice in the experimental group were injected with NucAb antigen and adjuvant (French complete adjuvant / French incomplete adjuvant), while mice in the control group were injected with adjuvant + PBS buffer.

[0059] NucAb antigen is recombinantly expressed and purified Acinetobacter baumannii NucAb protein at a concentration ≥1 mg / mL and an endotoxin content <0.1 EU / μg. For primary immunization, on day 0, each mouse was intraperitoneally injected with a 100 μL mixture containing 20 μg NucAb antigen and an equal volume of Freund's complete adjuvant (CFA). Booster immunizations were performed on days 14 and 28, each time with the same dose of NucAb antigen and Freund's incomplete adjuvant (IFA). The control group was injected with an equal volume of adjuvant + PBS buffer. Blood was collected from the tail vein, approximately 100 μL of whole blood each time. The blood was allowed to stand for 30 minutes, then centrifuged at 4°C (3000 rpm, 10 minutes), and the serum was separated and stored at -80°C for later use.

[0060] Antibody titers were evaluated using the ELISA method.

[0061] Coating antigen: NucAb protein (2 μg / mL, pH 9.6 carbonate buffer, 4°C overnight).

[0062] Sealing: After washing the plate, seal with 3% BSA, 200 μL / well, at 37°C for 1 hour.

[0063] Serum dilution gradient: Serum from immunized mice was serially diluted starting at 1:100. Simultaneously, serum from control mice was used as a negative control, and PBST was used as a blank control. (Add 100 μL of PBST to all wells of the ELISA plate except for rows 1 and 12. Add 200 μL of diluted immunized mouse serum to the wells in row 1, then transfer 100 μL from row 1 to row 2 and mix 10 times using a multi-channel pipette. Repeat this process, performing 2-fold serial dilutions until row 11. Finally, discard the 100 μL solution in row 11.) Incubate at 37°C for 1 hour.

[0064] Secondary antibody addition: After washing the plate, add 100 μL of HRP-labeled goat anti-mouse IgG secondary antibody and incubate at 37°C for 45 minutes at a dilution ratio of 1:10000.

[0065] Color development and termination: Wash the plate again, add 100 μL of TMB substrate, and incubate at 37°C in the dark for 10 minutes. After the color development is complete, terminate the reaction with 50 μL of stop solution.

[0066] Titer determination: After the reaction was terminated, the OD value of each well was measured using a microplate reader within 15 minutes, and the optical density at 450 nm was recorded. The antibody titer was determined by the highest dilution with an OD value ≥ 2.1 times that of the negative control. This process allows for a systematic assessment of the antibody response strength of immunized mice to NucAb protein (results are shown in Figure 1). Figure 4 (As shown in the figure). It can be seen that the mice immunized with NucAb antigen all showed good antibody humoral response levels, the antigen immunization effect was in line with the experimental expectations, and the antibody titer reached 1:25600.

[0067] NucAb protein, a key virulence factor in Acinetobacter baumannii, possesses nuclease activity and helps bacteria evade the host's innate immune defenses by degrading neutrophil extracellular traps (NETs). The HNH nuclease domain of NucAb is its catalytic active center; this region is highly conserved across different strains of Acinetobacter baumannii. Antibodies targeting this region can achieve "functional neutralization," effectively blocking its nuclease activity, thereby restoring the NETs' trapping effect on bacteria, enhancing the clearance efficiency of phagocytes, and reducing bacterial dissemination within the host. This strategy based on the functional neutralization of a key virulence factor is less likely to induce bacterial resistance compared to traditional bactericidal or bacteriostatic mechanisms. Furthermore, the localization of NucAb protein on the bacterial outer membrane makes it more easily recognized by the immune system, further enhancing its potential as a vaccine antigen.

[0068] Experiments have demonstrated that the recombinant NucAb protein can be constructed using the pET28a(+) vector and the E. coli Rosetta(DE3) host system. By combining high-pressure sterilization, Ni-NTA affinity chromatography, and anion exchange chromatography, the limitations of traditional single-column purification are overcome, resulting in a NucAb protein purity >90% and a target protein recovery rate of over 85%, demonstrating significant advantages for large-scale production.

[0069] The above embodiments are merely one of the preferred embodiments of the present invention and should not be used to limit the scope of protection of the present invention. Any modifications or refinements made to the main design concept and spirit of the present invention that are not of substantial significance, but solve the same technical problem as the present invention, should be included within the scope of protection of the present invention.

Claims

1. A soluble Acinetobacter baumannii recombinant NucAb protein, characterized in that, The recombinant vector, constructed by introducing the NucAb gene (as shown in SEQ ID NO:1) into an expression vector, was expressed in a host bacterium.

2. The soluble Acinetobacter baumannii recombinant NucAb protein according to claim 1, characterized in that, The expression vector is plasmid pET28a(+), and the host bacterium is E. coli Rosetta(DE3).

3. The soluble Acinetobacter baumannii recombinant NucAb protein according to claim 2, characterized in that, The recombinant NucAb protein has a molecular weight of 44 kDa.

4. The method for expression and purification of soluble Acinetobacter baumannii recombinant NucAb protein as described in claim 3, characterized in that, Includes the following steps: S1, Constructing a prokaryotic expression plasmid: The nucleotide sequence corresponding to the target gene with the amino acid sequence shown in SEQ ID NO:1 is ligated into the expression vector plasmid pET28a(+) to obtain the recombinant plasmid pET28a(+)-NucAb. The recombinant plasmid is then transformed into the host bacterium E. coli Rosetta(DE3) to induce the expression of the exogenous protein, resulting in recombinant Escherichia coli. S2, High-pressure sterilization and centrifugation: Collect the recombinant Escherichia coli cells from step S1, suspend them in a equilibration solution, pre-cool at 4°C, and then sterilize them under high pressure at 700-800 bar. Collect the supernatant. The equilibration solution includes 50 mM Hepes, 100 mM KCl, and 10% glycerol, and the pH is 7.

5. S3, Ni-NTA chromatography column purification: The supernatant from step S2 is passed through a Ni-NTA affinity chromatography column and eluted sequentially with eluents containing 20-500 mM imidazole. The eluent containing 100 mM imidazole is collected and dialyzed overnight at 4°C. The pH of the eluent is 7.5, and it also includes 50 mM Hepes, 100 mM KCl, and 10% glycerol. The dialysate includes 50 mM Hepes, 150 mM KCl, 1 mM DTT, and 10% glycerol, and has a pH of 7.

5. S4, Anion exchange column purification: The protein dialyzed overnight in step S3 is purified by anion exchange column using linear elution with solutions A and B to obtain the purified protein. Solution A consists of 50 mM Hepes, 150 mM KCl, 1 mM DTT, and 10% glycerol, with a pH of 7.

5. Solution B consists of 50 mM Hepes, 500 mM KCl, 1 mM DTT, and 10% glycerol, with a pH of 7.

5. The packing material used for the anion exchange column purification is Capto. TM Q.

5. The method for expression and purification of recombinant NucAb protein from soluble Acinetobacter baumannii according to claim 4, characterized in that, In step S1, the target gene is ligated to the expression vector pET28a(+) via the restriction sites of BamHI and XhoI.

6. The method for expression and purification of recombinant NucAb protein from soluble Acinetobacter baumannii according to claim 5, characterized in that, In step S2, the bacterial cells and equilibration solution are mixed and suspended at a volume ratio of 1:10; after high-pressure sterilization, the mixture is centrifuged at 4°C and 12,000 rpm for 15 min.

7. The method for expression and purification of recombinant NucAb protein from soluble Acinetobacter baumannii according to claim 6, characterized in that, In step S3, the Ni-NTA affinity chromatography column is packed with HisSep Ni-NTA Agarose Resin; the packing volume is 100 mL per 100 g of ruptured bacterial cells wet weight.

8. The method for expression and purification of recombinant NucAb protein from soluble Acinetobacter baumannii according to claim 7, characterized in that, In step S4, the amount of packing material used is 100mL for every 100g of broken bacterial cells in wet weight.

9. The use of the soluble Acinetobacter baumannii recombinant NucAb protein according to any one of claims 1 to 3 in the preparation of an anti-Acinetobacter baumannii vaccine composition, characterized in that, The vaccine composition also includes a pharmaceutically acceptable adjuvant, wherein the concentration of NucAb protein is ≥1 mg / mL.

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