Acinetobacter baumannii TonB recombinant protein antigen, preparation method and application thereof

Abstract

The present application provides a recombinant protein antigen of Acinetobacter baumannii TonB, a preparation method and its application, and relates to the field of biomedical technology. The amino acid sequence of the recombinant protein antigen of Acinetobacter baumannii TonB of the present invention is shown in SEQ ID NO.1. The preparation process of the recombinant protein antigen of Acinetobacter baumannii TonB provided in the present application is simple, the operation steps are easy to control and easy to implement, and the obtained recombinant protein of Acinetobacter baumannii TonB has high purity and high yield, and is suitable for large-scale production. The vaccine prepared using the recombinant protein antigen of Acinetobacter baumannii TonB of the present application has no toxic side effects, can produce high-titer antibodies, has good immune protection effect against Acinetobacter baumannii infection, and is suitable for preparing specific antibodies.

Description

Technical Field

[0001] The present application belongs to the field of biomedicine technology, and in particular relates to a recombinant protein antigen of Acinetobacter baumannii TonB, a preparation method and application thereof. Background Art

[0002] Acinetobacter baumannii is a common pathogen that has been widely recognized as one of the main pathogens of hospital-acquired infections. It mainly infects elderly and long-term hospitalized patients, especially those in intensive care units and other critically ill patients. Its infection can cause pneumonia, severe soft tissue or wound infection, catheter-related infection, urinary tract infection, bacteremia and secondary meningitis, among which pneumonia and bloodstream infection are the most common and serious. The increased drug resistance of Acinetobacter baumannii and its spread in medical environments make its infection treatment extremely difficult, and even lead to serious infection outbreaks in hospitals.

[0003] Currently, the treatment of Acinetobacter baumannii mainly relies on antibiotics. However, due to its high resistance, many conventional antibiotics have lost their therapeutic effect on the bacteria, which has intensified the demand for alternative treatment methods. Therefore, there is an urgent need to develop new methods for the prevention and treatment of Acinetobacter baumannii infection. Summary of the invention

[0004] The purpose of the present application is to provide a recombinant protein antigen of Acinetobacter baumannii TonB, a preparation method and application thereof, aiming to solve the problem of poor therapeutic effect against Acinetobacter baumannii infection in the prior art.

[0005] In order to achieve the above application purpose, the technical solution adopted in this application is as follows:

[0006] In a first aspect, the present application provides a recombinant protein antigen of Acinetobacter baumannii TonB, characterized in that the amino acid sequence of the recombinant protein antigen of Acinetobacter baumannii TonB is shown in SEQ ID NO.1.

[0007] In a second aspect, the present application provides a gene encoding the recombinant protein antigen of Acinetobacter baumannii TonB described in the first aspect, characterized in that its nucleotide sequence is shown in SEQ ID NO.2.

[0008] In a third aspect, the present application provides a method for preparing a recombinant protein antigen of Acinetobacter baumannii TonB as described in the first aspect, the preparation method comprising the following steps: a) plasmid construction, prokaryotic expression: connecting the gene of the nucleotide sequence shown in SEQ ID NO.2 to an expression vector, obtaining a constructed plasmid and transferring it into a prokaryotic expression system for induced expression; b) centrifugal collection: collecting the bacterial solution after induced expression and centrifuging it, discarding the supernatant, adding PBS buffer to mix, using ultrasonic lysis, and then centrifuging it to collect the supernatant; c) obtaining the TonB recombinant protein containing a HIS tag: culturing the strain collected in step b) in an LB medium containing kanamycin resistance until OD600 is 0.8, adding IPTG inducer and placing it in a shaker for induction, and then centrifuging to collect the bacteria , add PBS to resuspend the bacteria, break them with a high-pressure homogenizer, centrifuge after breaking, collect the supernatant and combine it with the HIS-tagged protein agarose high-speed purification resin to obtain the TonB recombinant protein containing the HIS tag; d) Extract the TonB target protein: after the supernatant in step c) is combined with the HIS-tagged protein agarose high-speed purification resin, use Ni affinity chromatography to elute the target protein and collect it; e) Purify the TonB target protein: use gel filtration chromatography or desalting column to further purify the TonB recombinant protein to obtain a high-purity TonB recombinant protein antigen.

[0009] According to a preferred embodiment, in step a), the expression vector is a pET-28a plasmid, and the prokaryotic expression system is a competent Escherichia coli BL21 (DE3).

[0010] In a fourth aspect, use of the recombinant protein antigen of Acinetobacter baumannii TonB described in the first aspect in the preparation of a drug for treating or preventing Acinetobacter baumannii infection.

[0011] According to a preferred embodiment, the drug is an Acinetobacter baumannii vaccine.

[0012] In a fifth aspect, the present application provides an Acinetobacter baumannii TonB-specific antibody prepared by immunizing an animal with the Acinetobacter baumannii TonB recombinant protein antigen described in the first aspect.

[0013] In a sixth aspect, the present application provides use of the Acinetobacter baumannii TonB-specific antibody described in the fifth aspect in the preparation of a drug for treating or preventing Acinetobacter baumannii infection.

[0014] In a seventh aspect, the present application further provides an Acinetobacter baumannii detection kit, comprising the Acinetobacter baumannii TonB recombinant protein antigen described in the first aspect, and / or the Acinetobacter baumannii TonB specific antibody described in the fifth aspect.

[0015] Based on the above technical solution, the recombinant protein antigen of Acinetobacter baumannii TonB, preparation method and application thereof provided in the present application have at least the following beneficial technical effects:

[0016] The present application provides a new antigen reserve for drugs such as vaccines for treating and preventing Acinetobacter baumannii infection, filling the current gap in this field. The preparation process of the Acinetobacter baumannii TonB recombinant protein antigen provided by the present application is simple, the operation steps are easy to control and implement, and the obtained Acinetobacter baumannii TonB recombinant protein has high purity and high yield, and is suitable for large-scale production.

[0017] On the other hand, the vaccine prepared using the recombinant protein antigen of Acinetobacter baumannii TonB of the present application has no toxic side effects, can produce high-titer antibodies, has good immune protection effect against Acinetobacter baumannii infection, and is suitable for preparing specific antibodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0019] Figure 1 This is the result of enzyme digestion identification of the expression vector pET-28a-Tonb.

[0020] Figure 2 This is a graph showing the results of inducing protein expression of TonB2 recombinant protein at 16°C.

[0021] Figure 3 This is the SDS-PAGE electrophoresis of TonB recombinant protein purified by Ni affinity chromatography.

[0022] Figure 4 The figure is the SDS-PAGE electrophoresis of further purification of TonB recombinant protein using a desalting column. DETAILED DESCRIPTION

[0023] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present application more clearly understood, the present application is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0024] In this application, the term "and / or" describes the association relationship of associated objects, indicating that there may be three relationships. For example, A and / or B can mean: A exists alone, A and B exist at the same time, and B exists alone. A and B can be singular or plural. The character " / " generally indicates that the associated objects are in an "or" relationship.

[0025] In this application, "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single or plural items. For example, "at least one of a, b, or c", or "at least one of a, b, and c" can all mean: a, b, c, ab (i.e. a and b), ac, bc, or abc, where a, b, and c can be single or multiple.

[0026] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean the order of execution, some or all of the steps can be executed in parallel or sequentially, and the execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0027] The terms used in the embodiments of the present application are only for the purpose of describing specific embodiments, and are not intended to limit the present application. The singular forms "a", "said" and "the" used in the embodiments of the present application and the appended claims are also intended to include plural forms, unless the context clearly indicates other meanings.

[0028] The weight of the relevant components mentioned in the embodiment description of the present application can not only refer to the specific content of each component, but also indicate the proportional relationship between the weights of the components. Therefore, as long as the content of the relevant components is proportionally enlarged or reduced according to the embodiment description of the present application, it is within the scope disclosed in the embodiment description of the present application. Specifically, the mass described in the embodiment description of the present application can be a mass unit known in the chemical industry such as µg, mg, g, kg, etc.

[0029] Gram-negative bacteria need to obtain nutrients from the outside world for survival, including: iron, heme, vitamin B12, carbohydrates and various transition metal elements and other basic substances. However, this process cannot use ATP to provide energy to achieve active transport, but relies on the TonB-ExbB-ExbD complex composed of the inner membrane ExbB-ExbD and the periplasmic protein TonB to provide energy units for the dependent outer membrane receptors (TBDTs), so that bacteria can actively transport important nutrients from the outside into the cell. In addition, the TonB complex also has different sequence structures in different bacteria, and TonB in Gram-negative bacteria shows diversity, forming a family. In the process of studying Acinetobacter baumannii, the applicant used bioinformatics technology to predict antigenic epitopes and found that a protein in the TonB family of the bacterium (whose amino acid sequence is shown in SEQ ID NO.1) has a relatively rich antigenic determinant cluster, and the protein sequence has certain species specificity. Therefore, this TonB protein can be used as a candidate vaccine for Acinetobacter baumannii. Therefore, the present application constructed a relevant TonB gene recombinant expression system, performed expression purification, obtained the target protein, and immunized animals. The experimental results showed that this recombinant protein antigen induced high-titer specific antibodies, and enabled the immunized mice to resist the attack of Acinetobacter baumannii, significantly improving the survival rate. The specific experimental process is as follows:

[0030] 1. Strains.

[0031] The Acinetobacter baumannii strain was purchased from ATCC with the strain number: ATCC17978.

[0032] 2. Reagents.

[0033] HIS tag protein agarose high-speed purification resin (Ni-TED 6FF agarose gel) was purchased from Sangon Biotechnology Co., Ltd.; PET-28a vector was purchased from Wuhan Jinkairui Co., Ltd.; nucleic acid (DNA) molecular weight standard (Marker) was purchased from Sangon Biotechnology Co., Ltd.; kanamycin and ampicillin were purchased from Sangon Biotechnology Co., Ltd.; ion exchange chromatography filler was purchased from Cytiva; BCA protein determination kit was purchased from Yisheng Biotechnology Co., Ltd.

[0034] Example 1: Cloning of Acinetobacter baumannii TonB protein.

[0035] 1. According to the amino acid sequence of TonB protein of Acinetobacter baumannii, the amino acid sequence of TonB protein is shown in SEQ ID NO.1, and its nucleotide sequence is optimized according to the codon preference of Escherichia coli, and the nucleotide sequence is shown in SEQ ID NO.2, respectively. DNA is synthesized and inserted into pET-28a plasmid to obtain recombinant plasmid pET-28a-Tonb.

[0036] 2. Conversion.

[0037] The synthesized plasmid was centrifuged at 12000 rpm for 1 min, 40 μL of deionized water was added to mix, 1 μL of plasmid solution was added to 20 μL of E. coli BL21 (DE3) competent cells, flicked to mix, and placed on ice for 30 min. Heat shock was performed at 42 °C for 90 s, and the cells were immediately placed on ice for 2 min. 0.5 mL of LB medium without antibiotics was added, and the cells were revived at 220 rpm / 37 °C for 50 min. 50 μL of the culture was spread on a kanamycin LB plate and cultured at 37 °C overnight. Single clones were picked, expanded, and the plasmids were extracted and identified by Noc1 and Xho1 digestion.

[0038] The identification results of pET-28a-Tonb are as follows Figure 1 Lane M: nucleic acid (DNA) molecular weight standard (Marker); Lane 1: identification results of the recombinant expression plasmid pET-28a-Tonb after enzyme digestion. The target fragment after enzyme digestion is between 500 bp and 750 bp, which is consistent with the theoretical size of 596 bp.

[0039] Example 2: Recombinant expression of Acinetobacter baumannii TonB recombinant protein.

[0040] 1. Pick a single clone and place it in 10 mL of LB medium containing kanamycin resistance, culture it at 220 rpm and 37℃ overnight, then take 2 ml of the overnight cultured bacterial solution and add it to 18 mL of LB medium containing kanamycin resistance (the remaining bacterial solution is stored in a 4℃ refrigerator for standby use), culture it at 37℃ for 2~3 h, rotate at 220 rpm, and activate it for the second time until the OD600 is 0.6~0.8, take 40 μl of the pre-induction bacterial solution (-) to prepare SDS-PAGE samples, add IPTG to the remaining bacterial solution to make its final concentration 500 μM, and then place it in a shaker at 16℃ overnight to induce expression.

[0041] 2. Take out the induced bacterial solution, take 40 μl of the induced bacterial solution (+) to make SDS-PAGE samples, centrifuge at 13300 rpm for 1 min, discard the supernatant, add 1 ml of resuspension buffer (20 mM Tirs, 500 mM NaCl, pH 8.0), mix well, ultrasonically lyse for 5 min, centrifuge at 4 ℃ and 13300 rpm for 1 min, and collect the supernatant.

[0042] 3. Take 40 μl of the supernatant (s) from step 2 to prepare the SDS-PAGE sample. Resuspend the precipitate after centrifugation with an equal volume of resuspension buffer, centrifuge at 4°C, 13300 rpm for 1 min, discard the supernatant for washing, add an equal volume of resuspension buffer again to resuspend, and take 40 μl to prepare the SDS-PAGE sample.

[0043] 4. Take the SDS-PAGE samples prepared in the above steps and perform polyacrylamide gel electrophoresis to observe the expression of the target protein. The recombinant engineering bacteria with expression are named pET-28a-Tonb / BL21 (DE3) for seed preservation.

[0044] The expression results of the recombinant protein TonB are shown in Figure 2 As shown, lane "M" is a protein molecular weight standard (Marker); lane "-" is the whole bacterial protein band of the recombinant engineering bacteria before induction expression; lane "+" is the expression of the recombinant protein in the whole bacteria after the recombinant engineering bacteria were induced to express at 16°C; lane "s" is the recombinant protein obtained in the supernatant of bacterial ultrasonic lysis after the recombinant engineering bacteria were induced to express at 16°C; lane "p" is the recombinant protein obtained in the bacterial ultrasonic precipitation of the sample precipitated after the recombinant engineering bacteria were induced to express at 16°C. It can be seen that the recombinant protein band is between 25-35 KD, which is consistent with the size of the target recombinant protein, and the recombinant protein is mainly expressed in the supernatant of bacterial ultrasonic lysis in a soluble form with high expression.

[0045] It can be seen that the recombinant engineering bacteria obtained in this application have the advantages of easy cultivation, high expression efficiency, short purification cycle, safety and controllability, and convenient operation, and are also conducive to the process expansion of large-scale vaccine production in the later stage. At the same time, the protein expressed by the pET-28a-Tonb / BL21 (DE3) recombinant engineering bacteria can maintain its original spatial conformation to the maximum extent while also having good solubility.

[0046] Example 3: Purification of TonB recombinant protein.

[0047] 1. Amplify the culture to obtain TonB recombinant protein.

[0048] The pET-28a-TonB / BL21 (DE3) strain stored in a -80 ℃ refrigerator was placed in 100 ml of LB medium containing kanamycin resistance and cultured overnight at 37 ℃ and 220 rpm. The activated 100 ml bacterial solution was added to 1 L of LB medium containing kanamycin resistance for secondary activation, and cultured at 37 ℃ for 3-4 h until OD600 reached 0.8. 0.5 ml IPTG (final concentration of 500 μM) was added and induced in a 16 ℃ shaker for 12 h. The cells were collected by centrifugation at 4000 rpm for 15 min, and then 80 ml of resuspension buffer (1xPBS, 10 mM imidazole, pH 7.4) was added to resuspend the cells. The cells were broken by a high-pressure homogenizer for 3 cycles. After breaking, a floor-standing high-speed centrifuge was used to centrifuge at 15000 rpm for 30 min. The supernatant was collected and combined with 5 ml Ni-NTA to obtain a large amount of TonB target protein containing a His tag.

[0049] 2. Purify TonB recombinant protein using Ni affinity chromatography.

[0050] 1) Collect the mixture of target protein and Ni-NTA filler into the gravity column, and collect the flow-through; then use wash buffer (1xPBS, 30 mM imidazole, pH 7.4) to rinse the Ni-NTA filler, and the wash volume is about 500 ml; finally, use elution buffer (1xPBS, 300 mM imidazole, pH 7.4) to elute the target protein, and the elution volume is about 50 ml. Then take 40 μl of the liquid in the "flow-through", "wash" and "elution" respectively to prepare SDS-PAGE samples, and perform polyacrylamide gel electrophoresis experiments to observe the purification status.

[0051] 2) Load 10 μl of SDS-PAGE sample for polyacrylamide gel protein electrophoresis and observe the results under the phase system. After elution, the molecular weight of the TonB protein obtained is between 25-37 kDa, which is consistent with the expected protein molecular weight. The electrophoresis results are shown in Figure 3 , where lane "M" represents the Marker, lane 1 represents the flow-through, lanes 2-6 represent the wash, and lane 7 represents the elution of the target protein.

[0052] 3. Use a desalting column to further purify the TonB recombinant protein.

[0053] Remove the desalting column and connect it to the NGC purifier. Rinse the desalting column with deionized water for 5 column volumes, and then fully balance the column with filtered PBS (pH 7.4) solution. Load the sample at a uniform rate to allow the protein molecules to fully enter the desalting filler medium, and collect the protein effluent for electrophoresis detection. Figure 4As shown, lane "M" represents Marker, lanes 1-2 represent TonB2 recombinant protein purified using a desalting column, and its concentration is 95% as determined by grayscale scanning.

[0054] 4. The protein concentration was determined by BCA method. The concentration of TonB protein was 1.92 mg / mL.

[0055] Example 4: Preparation of recombinant protein subunit vaccine.

[0056] The TonB recombinant protein was adsorbed with different adjuvants such as aluminum hydroxide, aluminum phosphate, MF59, CpG, etc. for preliminary experiments and the adsorption effect was tested. It was found that aluminum hydroxide had the best adsorption efficiency. Subsequently, Al(OH)3 was mainly used as the adjuvant component for adsorption to prepare subunit vaccines.

[0057] The method is as follows: Aluminum hydroxide adjuvant was measured and adjusted to 5 mg / ml with histidine diluent at pH 6.0, and mixed thoroughly to obtain solution A; TonB recombinant protein was diluted to 200 μg / mL with histidine diluent at pH 6.0 to obtain solution B; equal volumes of solution A and solution B were taken and rotated and suspended at 4 °C for 2 hours to obtain the vaccine. Solution B of the adjuvant control preparation group was histidine diluent without protein antigen, and adsorbed with solution A under the same conditions.

[0058] Example 5: Immunization of animals with recombinant TonB protein.

[0059] 1) For the first immunization, the subunit vaccine prepared in Example 4 was injected intramuscularly into the inner thigh of mice using an insulin needle. The injection volume for each C57BL / 6 mouse was 200 μL, 100 μL each into the left and right thighs, and an adjuvant control group was set up;

[0060] 2) On the 14th day, the second immunization was performed with the same immune components and injection volume as the first immunization, and the immunization route was the same as the first immunization. This immunization was injected intramuscularly into the outer thigh of the mouse;

[0061] 3) The third immunization was carried out on the 21st day. The immunization components were the same as above, the injection volume was the same as the first immunization, and the immunization route was the same as above. This immunization was injected intramuscularly into the inner thigh of the mouse;

[0062] 4) On the 7th day after the third immunization, venous blood was collected from C57BL / 6 mice, and serum was separated by centrifugation after coagulation. The IgG humoral response level of mice after immunization was detected by ELISA.

[0063] Example 6: Preparation of TonB-specific antibody detection kit.

[0064] 1) Prepare the liquid.

[0065] ① Preparation of coating solution: weigh 1.6 g NaHCO3 and 2.9 g Na2CO3, dissolve in 1 L ddH2O, and adjust the pH to 9.6 using a pH meter;

[0066] ② Preparation of blocking solution: 1 g bovine serum albumin, dissolved in 100 mL antibody diluent (1:100);

[0067] ③ Preparation of antibody diluent: dissolve phosphate in 1 L ddH2O, add 500 μl Tween 20, and adjust the pH to 7.4 using a pH meter;

[0068] ④ Preparation of washing solution: weigh 2.42 g Tris and dissolve it in 1 L ddH2O, then add 500 μl Tween 20, and adjust the pH to 7.4 using a pH meter;

[0069] ⑤ Chromogenic solution (TMB), a product of Tiangen Company;

[0070] ⑥ Preparation of stop solution (2 M H2SO4): Pour 22.2 mL of concentrated sulfuric acid into 177.8 mL of ddH2O.

[0071] 2) Preparation of TonB recombinant protein antibody detection kit.

[0072] ① Dilute the purified TonB recombinant protein to 4 μg / mL using coating solution;

[0073] ② Coating: Add the recombinant protein dilution to the ELISA plate, 100 μl / well, incubate at 4°C overnight, and then wash 4 times with washing solution;

[0074] ③ Blocking: Add 100 μl / well of blocking solution to the ELISA plate, incubate at 37°C for 2 hours, wash 4 times; dry the kit;

[0075] ④ Package the ELISA coated plate and antibody diluent, enzyme-labeled secondary antibody, substrate colorimetric solution (TMB), and stop solution to complete the preparation of the kit and store it at 4°C.

[0076] Example 7: Detection of specific antibodies after immunization with TonB recombinant protein.

[0077] On the 7th day after the third immunization, blood was collected from C57Bl / 6 mice, and the IgG humoral response level of the mice after immunization was detected by ELISA.

[0078] ① Perform serial dilution of the serum to be tested;

[0079] ② Take the blocked ELISA plate, add diluted serum in sequence, 100 μl / well, place in a 37°C incubator for 2 hours, wash 4 times, and air dry;

[0080] ③ Dilute the HRP-labeled goat anti-mouse IgG antibody storage solution at 1:10000 to prepare the antibody working solution;

[0081] ④ Add diluted antibody working solution, 100 μl / well, place in a 37°C incubator for 45 min, wash 4 times, and air dry;

[0082] ⑤ Add 100 μl / well of substrate colorimetric solution (TMB) and react at room temperature in the dark for 5 min;

[0083] ⑥ Add stop solution (2M H2SO4) and immediately place on a microplate reader to measure the OD value at a wavelength of 450 nm;

[0084] ⑦ Result judgment: A sample / A negative value ≧2.1 is positive (negative control is mouse pre-immune serum).

[0085] Table 1 TonB recombinant protein immune serum antibody detection

[0086] Serum dilution TonB (OD value) 2000 2.38 8000 1.521 32000 0.687 128000 0.265 512000 0.111 2048000 0.064 8192000 0.052 32768000 0.048

[0087] The test results are shown in Table 1, which show that the antibody titer of the TonB protein immune serum reaches 1:512000, indicating that the TonB recombinant protein prepared by the present invention has good immunogenicity and can produce high-titer specific antibodies in the immune mice.

[0088] Example 8: Immune protection effect of TonB recombinant protein.

[0089] After the third immunization of mice, on the 14th day, a lethal dose of Acinetobacter baumannii was injected into the tail vein for challenge. The amount of bacterial solution injected into each C57BL / 6 mouse was 4×10 7 CFU, and observed for 7 days. The survival rate of mice in each group was counted and the results are shown in Table 2.

[0090] Table 2 Protection results of recombinant protein immunization against bacteremia in mice

[0091]

[0092] Table 2 shows the results of the animal immunization test. The mortality rate of the adjuvant control group was 100%, and the mortality rate of the TonB vaccine immunization group was 60%. According to the protection rate calculation formula: [Vaccine protection rate = (control group incidence rate - vaccination group incidence rate) / control group incidence rate * 100%), the protection rate of the TonB protein vaccine immunization group was 40%.

[0093] Example 9: The challenge protection of animals immunized with TonB recombinant protein was determined by immunizing mice using the pneumonia model.

[0094] The same immunization scheme as in Example 5, after the third immunization of mice, on the 7th day, endotracheal intubation was used to challenge with a lethal dose of Acinetobacter baumannii, and each C57BL / 6 mouse was injected with 5×10 6 CFU, and observed for 7 days. The survival rate of mice in each group was counted and the results are shown in Table 3.

[0095] Table 3 The protective effect of recombinant protein on the pneumonia model in mice

[0096]

[0097] Table 3 shows the results of the animal immunization test. The mortality rate of the adjuvant control group was 100%, and the mortality rate of the TonB vaccine immunization group was 50%. According to the protection rate calculation formula: [Vaccine protection rate = (control group incidence rate - vaccination group incidence rate) / control group incidence rate * 100%). The protection rate of the TonB protein vaccine immunization group was 50%.

[0098] Example 10: Preparation of TonB-specific antibodies.

[0099] The recombinant protein TonB was mixed with aluminum hydroxide adjuvant and used to immunize rabbits at 300 μg / rabbit for three times. Blood was collected at different time points to detect antibody titers. Rabbit serum was collected and purified using a protein A column to obtain IgG, thereby obtaining TonB-specific antibodies.

[0100] The Acinetobacter baumannii TonB recombinant protein antigen of this application provides an effective antigen reserve for the development of Acinetobacter baumannii vaccines, filling the current gap in this field. It has the following advantages:

[0101] (1) Efficient preparation: The method of the present invention can efficiently prepare high-purity and high-yield recombinant protein antigen of Acinetobacter baumannii TonB, meeting the needs of large-scale vaccine preparation.

[0102] (2) Improving vaccine research and development efficiency: The present invention provides a new antigen reserve solution for the development of Acinetobacter baumannii vaccines, which is expected to accelerate the development of Acinetobacter baumannii vaccines, improve research and development efficiency, and promote the launch of vaccine products.

[0103] The recombinant protein prepared in the present application can be used with other relevant reagents, such as detection antibodies, colorimetric agents, terminators, etc., to prepare relevant kits.

[0104] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present application should be included in the protection scope of the present application.

Claims

1. Use of a recombinant protein antigen of Acinetobacter baumannii TonB in the preparation of a drug for treating or preventing infection by Acinetobacter baumannii, characterized in that: The amino acid sequence of the Acinetobacter baumannii TonB recombinant protein antigen is shown in SEQ ID NO.1, the drug is an Acinetobacter baumannii vaccine, and the nucleotide sequence of the gene encoding the Acinetobacter baumannii TonB recombinant protein antigen is shown in positions 3-590 of SEQ ID NO.2.

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