CN119080891A

CN119080891A - Acinetobacter baumannii TonB recombinant protein antigen, preparation method and application thereof

Info

Publication number: CN119080891A
Application number: CN202411594959.2A
Authority: CN
Inventors: 李彦; 石云; 谢雨; 王宁; 陈凯; 孙向成
Original assignee: West China Hospital of Sichuan University
Current assignee: West China Hospital of Sichuan University
Priority date: 2024-11-11
Filing date: 2024-11-11
Publication date: 2024-12-06
Anticipated expiration: 2044-11-11
Also published as: CN119080891B

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

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

Technical Field

The application belongs to the technical field of biological medicine, and particularly relates to a Acinetobacter baumannii TonB recombinant protein antigen, a preparation method and application thereof.

Background

Acinetobacter baumannii (Acinetobacter baumannii) is a common pathogenic bacterium and has been widely accepted as one of the major pathogens of hospital-acquired infections. The infection objects are mainly old and long-term hospitalized patients, especially intensive care units and other severe patients, and the infection can cause pneumonia, serious soft tissue or wound infection, intubation related infection, urinary tract infection, bacteremia, secondary meningitis and the like, wherein the pneumonia and blood flow infection are the most common and serious. The enhancement of acinetobacter baumannii resistance and the spread in medical environments makes its infection treatment exceptionally difficult, even leading to serious infection outbreaks in hospitals.

Currently, therapies against acinetobacter baumannii rely primarily on antibiotics, however, many conventional antibiotics have lost therapeutic efficacy against this bacterium due to their highly resistant nature, which exacerbates the need for alternative therapies. Therefore, there is an urgent need to develop new control methods for acinetobacter baumannii infection.

Disclosure of Invention

The application aims to provide an Acinetobacter baumannii TonB recombinant protein antigen, a preparation method and application thereof, and aims to solve the problem of poor treatment effect on Acinetobacter baumannii infection in the prior art.

In order to achieve the purposes of the application, the technical scheme adopted by the application is as follows:

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

In a second aspect, the application provides a gene for encoding the Acinetobacter baumannii TonB recombinant protein antigen in the first aspect, which is characterized in that the nucleotide sequence of the gene is shown as SEQ ID NO. 2.

In a third aspect, the application provides a preparation method of a TonB recombinant protein antigen of Acinetobacter baumannii, which is characterized by comprising the steps of a) constructing plasmids, connecting genes with nucleotide sequences shown as SEQ ID No.2 in expression vectors, obtaining constructed plasmids, transferring the plasmids into a prokaryotic expression system for induced expression, b) centrifugally collecting bacterial liquid after the induced expression, centrifuging, discarding supernatant, adding PBS buffer solution, uniformly mixing, performing ultrasonic lysis, centrifuging, collecting supernatant, c) obtaining TonB recombinant protein containing HIS tag, culturing the strains collected in step b) in LB medium containing kana resistance until OD600 is 0.8, adding IPTG inducer, centrifugally collecting thalli, crushing with a PBS (phosphate buffer solution), centrifuging, collecting supernatant and combining with HIS tag protein agarose high-speed resin, obtaining TonB recombinant protein containing HIS tag, extracting the protein with the target protein in step b protein, purifying by using a shaking table, purifying the target protein by using high-speed chromatography, and purifying the target protein, and further purifying the target protein by using a high-speed chromatography column chromatography, wherein the target protein is purified by using the target protein, and the target protein is purified by using the TonB recombinant protein after the target protein is subjected to purification.

According to a preferred embodiment, in step a), the expression vector is a pET-28a plasmid and the prokaryotic expression system is competent for E.coli BL21 (DE 3).

In a fourth aspect, the use of the acinetobacter baumanii TonB recombinant protein antigen of the first aspect in the preparation of a medicament for treating or preventing acinetobacter baumanii infection.

According to a preferred embodiment, the medicament is an acinetobacter baumannii vaccine.

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 of the first aspect.

In a sixth aspect, the application provides an application of the acinetobacter baumannii TonB specific antibody in the fifth aspect in preparing a medicament for treating or preventing acinetobacter baumannii infection.

In a seventh aspect, the application also provides an acinetobacter baumanii detection kit, which comprises the acinetobacter baumanii TonB recombinant protein antigen in the first aspect and/or the acinetobacter baumanii TonB specific antibody in the fifth aspect.

Based on the technical scheme, the Acinetobacter baumannii TonB recombinant protein antigen, the preparation method and the application thereof provided by the application have at least the following beneficial technical effects:

the Acinetobacter baumannii TonB recombinant protein antigen provided by the application provides a new antigen reserve for medicines for treating and preventing Acinetobacter baumannii infection, such as vaccines, and fills the gap in the current field. The preparation process of the Acinetobacter baumannii TonB recombinant protein antigen provided by the application is simple, the operation steps are easy to control and realize, and the obtained Acinetobacter baumannii TonB recombinant protein has higher purity and higher yield, and is suitable for large-scale production.

On the other hand, the vaccine prepared by utilizing the Acinetobacter baumannii TonB recombinant protein antigen disclosed by the application has no toxic or side effect, can generate high-titer antibodies, has a good immune protection effect on Acinetobacter baumannii infection, and is suitable for preparing specific antibodies.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are needed in the embodiments or the description of the prior art will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present application, and that other drawings can be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a diagram showing the results of the digestion and identification of the expression vector pET-28 a-Tonb.

FIG. 2 is a graph showing the results of induction of protein expression by TonB2 recombinant protein at 16 ℃.

FIG. 3 is an SDS-PAGE electrophoresis of purified TonB recombinant protein by Ni affinity chromatography.

FIG. 4 is a SDS-PAGE electrophoresis of further purified TonB recombinant protein using a desalting column.

Detailed Description

In order to make the technical problems, technical schemes and beneficial effects to be solved more clear, the application is further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the application.

In the present application, the term "and/or" describes an association relationship of an association object, which means that three relationships may exist, for example, a and/or B may mean that a exists alone, a and B exist together, and B exists alone. Wherein A, B may be singular or plural. The character "/" generally indicates that the context-dependent object is an "or" relationship.

In the present application, "at least one" means one or more, and "a plurality" means two or more. "at least one of" or the like means any combination of these items, including any combination of single item(s) or plural items(s). For example, "at least one (a), b, or c)", or "at least one (a, b, and c)", may each represent a, b, c, a-b (i.e., a and b), a-c, b-c, or a-b-c, wherein a, b, c may be single or multiple, respectively.

It should be understood that, in various embodiments of the present application, the sequence number of each process described above does not mean that the execution sequence of some or all of the steps may be executed in parallel or executed sequentially, and the execution sequence of each process should be determined by its functions and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

The terminology used in the embodiments of the application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. As used in this application and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

The weights of the relevant components mentioned in the description of the embodiments of the present application may refer not only to the specific contents of the components, but also to the proportional relationship between the weights of the components, so long as the contents of the relevant components in the description of the embodiments of the present application are scaled up or down within the scope of the disclosure of the embodiments of the present application. Specifically, the mass described in the specification of the embodiment of the application can be a mass unit which is known in the chemical industry field such as mu g, mg, g, kg.

The gram-negative bacteria need to obtain nutrients from the outside, wherein the nutrients comprise various basic substances such as iron, heme, vitamin B12, carbohydrate, various transition metal elements and the like. However, this process does not use ATP to provide energy for active transport, but relies on the TonB-ExbB-ExbD complex of the intima ExbB-ExbD and the periplasmic protein TonB to provide energy units for the dependent adventitial receptor (TBDTs) to enable the bacteria to actively transport the outside important nutrients into the cell. In addition, the TonB complex also has a sequence structure different in different bacteria, and TonB in gram-negative bacteria shows diversity, forming a family. In the research process aiming at Acinetobacter baumannii, the inventor utilizes a bioinformatics technology to predict an epitope, and discovers that a protein in the TonB family of the Acinetobacter baumannii (the amino acid sequence of the protein is shown as SEQ ID NO. 1) has rich antigenic determinants and has certain species specificity, so that the TonB protein can be used as a candidate vaccine of the Acinetobacter baumannii. Thus, the application constructs a related TonB gene recombination expression system, performs expression purification, obtains target protein, and immunizes animals. Experimental results show that the recombinant protein antigen induces a high-titer specific antibody, and the immunized mice can resist the attack of Acinetobacter baumannii, so that the survival rate is remarkably improved. The specific experimental process is as follows:

1. Strains.

Acinetobacter baumannii strain was purchased from ATCC and the strain number was ATCC17978.

2. And (3) a reagent.

HIS tag protein agarose high-speed purification resin (Ni-TED 6FF agarose gel) is a product of biological company, PET-28a carrier is purchased from Wuhan Jin Kairui company, and nucleic acid (DNA) molecular weight standard (Marker) is purchased from biological company. Kanamycin and ampicillin were purchased from Bio-workers, ion exchange chromatography packing was purchased from Cytiva, and BCA protein assay kit was purchased from the company Saint Bio-next.

Example 1 cloning of Acinetobacter baumannii TonB protein.

1. According to the amino acid sequence of Acinetobacter baumannii TonB protein, the amino acid sequence of TonB protein is shown as SEQ ID NO.1, the nucleotide sequence of the TonB protein is optimized according to the preference of E.coli codons, the nucleotide sequences are respectively shown as SEQ ID NO.2, DNA is synthesized and inserted into pET-28a plasmid, and recombinant plasmids pET-28a-Tonb are respectively obtained.

2. And (3) transformation.

The synthesized plasmid was centrifuged at 12000 rpm at 1 min, and 40. Mu.L of deionized water was added for mixing, and 20. Mu.L of E.coli BL21 (DE 3) competent was added to 1. Mu.L of the plasmid solution, and the mixture was gently flicked for mixing, and allowed to stand on ice for 30 min. 42. Heat shock 90 s at C, immediately standing on ice for 2 min, adding 0.5 mL of antibiotic-free LB medium, resuscitating at 220 rpm/37℃for 50 min, spreading 50 μl on LB plates of kanamycin, and culturing at 37℃overnight. Monoclonal is selected, amplified and cultured, and plasmids are extracted and then are subjected to enzyme digestion and identification by using Noc1 and Xho 1.

The identification result of pET-28a-Tonb is shown in figure 1, wherein lane M is a nucleic acid (DNA) molecular weight standard (Marker), lane 1 is an identification result of recombinant expression plasmid pET-28a-Tonb after enzyme digestion, and the target fragment after enzyme digestion is between 500 bp and 750 bp, which accords with the theoretical 596bp size.

Example 2 recombinant expression of Acinetobacter baumannii TonB recombinant proteins.

1. Selecting a monoclonal in 10 mL LB culture medium containing kanamycin resistance, culturing overnight at 220 rpm and 37 ℃, adding the overnight cultured bacterial liquid 2ml into 18 mL LB culture medium containing kanamycin resistance (the rest bacterial liquid is stored in a refrigerator at 4 ℃ for standby), culturing for 2-3 hours at 37 ℃, rotating at 220 rpm, activating for the second time until the OD600 is 0.6-0.8, preparing 40 mu l of pre-induction bacterial liquid (-) into an SDS-PAGE sample, adding IPTG into the rest bacterial liquid to make the final concentration of the bacterial liquid be 500 mu M, and then placing the bacterial liquid in a shaking table for 16 ℃ overnight induction expression.

2. Taking out the bacterial liquid after induction expression, taking 40 mu l of the bacterial liquid (+) after induction to prepare an SDS-PAGE sample, centrifuging 1min by 13300 rpm, discarding the supernatant, adding 1 ml heavy suspension buffer (20 mM Tirs,500 mM NaCl,pH 8.0) to mix uniformly, performing ultrasonic pyrolysis on 5min, centrifuging 1min by 13300 rpm at the temperature of 4 ℃, and collecting the supernatant.

3. Taking 40 μl of the bacterial-destroying supernatant(s) in step 2 to prepare an SDS-PAGE sample, re-suspending the pellet after bacterial-destroying centrifugation with an equal volume of re-suspension buffer, centrifuging at4 ℃ and 13300 rpm for 1 min, discarding the supernatant for washing, re-adding an equal volume of re-suspension buffer for re-suspension, and taking 40 μl to prepare the SDS-PAGE sample.

4. And respectively taking SDS-PAGE samples prepared in the steps, and carrying out polyacrylamide gel electrophoresis experiments to observe the expression condition of the target protein. The expressed recombinant engineering bacteria are named as pET-28a-Tonb/BL21 (DE 3) for seed preservation.

The expression result of the recombinant protein TonB is shown in figure 2, wherein a lane ' M ' is a protein molecular weight standard (Marker), a lane ' is a whole bacterial protein band of recombinant engineering bacteria before induced expression, a lane ' plus ' is the expression of the recombinant protein in the whole bacteria after the recombinant engineering bacteria are induced to express at 16 ℃, a lane's ' is the recombinant protein obtained in bacterial ultrasonic lysis supernatant after the recombinant engineering bacteria are induced to express at 16 ℃, and a lane ' p ' is a recombinant protein obtained in bacterial ultrasonic precipitation after the recombinant engineering bacteria are induced to express at 16 ℃. The recombinant protein band is 25-35 KD, the size of the recombinant protein is consistent with that of the target recombinant protein, and the recombinant protein is mainly expressed in a soluble form in the supernatant of bacterial ultrasonic lysis with high expression.

Therefore, the recombinant engineering bacteria obtained by the application have the advantages of easy culture, high expression efficiency, short purification period, safety, controllability and convenient operation, and are also beneficial to the expansion of the process of the mass production of the later-stage vaccine. Meanwhile, the protein expressed by the pET-28a-Tonb/BL21 (DE 3) recombinant engineering bacteria can keep the original spatial conformation to the maximum extent and has better solubility.

Example 3 purification of TonB recombinant protein.

1. And (5) culturing in an amplifying way to obtain the TonB recombinant protein.

Culturing pET-28a-TonB/BL21 (DE 3) strain stored in a refrigerator at-80 ℃ in 100ml LB culture medium containing kanamycin resistance at 37 ℃ and 220 rpm for overnight, adding activated 100ml bacterial liquid into 1L LB culture medium containing kanamycin resistance for secondary activation, culturing at 37 ℃ for 3-4 hours until OD600 is 0.8, adding 0.5 ml IPTG (with the final concentration of 500 mu M), placing in a 16 ℃ shaker for inducing 12 h, centrifuging 15min at 4000 rpm to collect bacterial cells, adding 80 ml heavy suspension buffer (1 xPBS, 10 mM imidazole and pH 7.4) to heavy suspension bacterial cells, circularly crushing for 3 cycles by using a high-pressure homogenizing instrument, using a floor type high-speed centrifuge 15000 rpm and centrifuging 30 min after crushing, and collecting supernatant to combine with 5ml Ni-NTA to obtain a large amount of TonB proteins containing His tags.

2. The TonB recombinant protein was purified using Ni affinity chromatography.

1) Collecting the mixture of target protein and Ni-NTA filler into a gravity column, collecting flow-through, washing the Ni-NTA filler with eluting buffer (1 xPBS,30 mM imidazole, pH 7.4) to obtain about 500 ml eluting volume, eluting the target protein with eluting buffer (1 xPBS,300 mM imidazole, pH 7.4) to obtain about 50ml eluting volume, and collecting 40 μl of the "flow-through", "eluting" and "eluting" solutions to obtain SDS-PAGE samples, and performing polyacrylamide gel electrophoresis experiments to observe purification.

2) The 10 μl SDS-PAGE sample is subjected to polyacrylamide gel protein electrophoresis, the result is observed under a phase system, the molecular weight of the TonB protein obtained after elution is 25-37 kDa, the molecular weight of the TonB protein is consistent with the expected molecular weight of the protein, the electrophoresis result is shown in FIG. 3, wherein a lane "M" represents a Marker, a lane 1 represents a flow-through, a lane 2-6 represents impurity washing, and a lane 7 represents elution of the target protein.

3. The TonB recombinant protein was further purified using a desalting column.

Taking out the desalting column, connecting and installing an NGC (natural gas chromatography) purifier, eluting the 5 column volumes of the desalting column with deionized water, fully balancing the column with filtered PBS (pH 7.4) solution, loading at uniform speed to allow protein molecules to fully enter a desalting filler medium, and collecting protein effluent for electrophoresis detection, as shown in FIG. 4, wherein lane 'M' represents Marker, lanes 1-2 represent TonB2 recombinant protein purified after the desalting column, and the concentration of the TonB2 recombinant protein is 95% by gray scale scanning.

4. The BCA method measures the protein concentration, and the TonB protein concentration is 1.92 mg/mL.

Example 4 preparation of recombinant protein subunit vaccine.

The TonB recombinant protein is adsorbed with different adjuvants such as aluminum hydroxide, aluminum phosphate, MF59, cpG and the like respectively for pre-experiment, the adsorption effect is detected, the adsorption efficiency of the aluminum hydroxide is found to be best, and the subsequent main Al (OH) ₃ is taken as an adjuvant component for adsorption to prepare subunit vaccine.

The method comprises the steps of weighing an aluminum hydroxide adjuvant, adjusting the concentration to 5 mg/mL by using histidine diluent with the pH of 6.0, fully and uniformly mixing to obtain a solution A, diluting the TonB recombinant protein to 200 mug/mL by using histidine diluent with the pH of 6.0 to obtain a solution B, and rotationally suspending and adsorbing the solution A and the solution B with equal volumes for 2 hours at the temperature of 4 ℃ to obtain the vaccine. The adjuvant control formulation group solution B is histidine diluent without protein antigen, and the same conditions are used for adsorption with the solution A.

Example 5 tonb recombinant protein animals were immunized.

1) For the first immunization, the subunit vaccine prepared in example 4 was intramuscular injected into the inner thigh of the mouse with an insulin needle, 200 μl of each C57BL/6 mouse was injected, 100 μl of each of the left and right thighs, and an adjuvant control group was set;

2) The second immunization is carried out on the 14 th day, the immune components are the same as the first immunization, the immune route is the same as the first immunization, and the immunization is intramuscular injected outside the thigh of the mice;

3) The third immunization is carried out on the 21 st day, the immune components are the same as the first immunization, the immunization route is the same as the first immunization, and the immunization is intramuscular injected into the inner side of the thigh of the mouse;

4) On day 7 after the third immunization, venous blood was collected from C57BL/6 mice, serum was centrifuged after clotting, and the IgG humoral response level of the mice after immunization was detected by ELISA.

Example 6 preparation of TonB-specific antibody detection kit.

1) A liquid is prepared.

① Preparing a coating liquid, namely weighing NaHCO ₃1.6 g,Na₂CO₃ 2.9.9 g, dissolving in 1L ddH ₂ O, and adjusting the pH to 9.6 by a pH meter;

② Preparing a sealing solution, namely dissolving 1 g bovine serum albumin in 100: 100mL antibody diluent (1:100);

③ Preparing antibody diluent, namely dissolving phosphate in 1L ddH ₂ O, adding 500 mu l of Tween20, and regulating the pH value to 7.4 by using a pH meter;

④ Preparing a washing solution, namely weighing 2.42 g Tris, dissolving in 1L ddH ₂ O, adding 500 mu l of Tween 20, and regulating the pH value to 7.4 by a PH meter;

⑤ Color development liquid (TMB) is a product of Tiangen corporation;

⑥ Preparation of stop solution (2M H ₂SO₄) 22.2: 22.2 mL concentrated sulfuric acid was poured into 177.8: 177.8 mL ddH ₂ O.

2) And (3) preparing a TonB recombinant protein antibody detection kit.

① Diluting the purified TonB recombinant protein to 4 mug/mL respectively by using coating liquid;

② Coating, namely adding recombinant protein diluent into an ELISA plate, and washing with a washing solution for 4 times after 100 μl/hole is subjected to 4 ℃ overnight;

③ Sealing, namely adding 100 μl/hole of sealing liquid into the ELISA plate, placing the ELISA plate at 37 ℃ for incubation for 2 hours, washing for 4 times;

④ ELISA coated plates, antibody diluent, enzyme-labeled secondary antibodies, substrate chromogenic solution (TMB) and stop solution are combined and packaged to complete the preparation of the kit and the preservation at 4 ℃.

Example 7 detection of specific antibodies after immunization with TonB recombinant proteins.

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

① Serial multiple dilution is carried out on serum to be detected;

② Taking a sealed ELISA plate, sequentially adding diluted serum and 100 mu l/hole, placing the ELISA plate in a 37 ℃ incubator for 2 hours, washing for 4 times, and air-drying;

③ Diluting HRP-labeled goat anti-mouse IgG antibody preservation solution according to a ratio of 1:10000 to prepare an antibody working solution;

④ Adding diluted antibody working solution, placing 100 μl/hole in a 37 ℃ incubator 45 min, washing for 4 times, and air drying;

⑤ Adding 100 μl/well of a substrate color development solution (TMB), and reacting at room temperature in the absence of light for 5 min;

⑥ Adding a stop solution (2M H ₂SO₄), and immediately placing on an enzyme-labeled instrument to measure the OD value at the wavelength of 450 nm;

⑦ The result shows that the A sample/A negative value is not less than 2.1 and positive (the negative control is the serum before the mouse immunization).

Table 1 TonB recombinant protein immune serum antibody detection

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

The results of the detection are shown in Table 1, and the results show that the antibody titer of the TonB protein immune serum reaches 1:512000. The TonB recombinant protein prepared by the invention has good immunogenicity, and can generate high-titer specific antibodies in immunized mice.

Example 8 immunoprotection effects of TonB recombinant proteins.

After the third immunization of mice, the mice were challenged with a lethal dose of Acinetobacter baumannii by tail vein injection on day 14, the amount of bacteria injected into each C57BL/6 mouse was 4X 10 ⁷ CFU, and the survival rate of each group of mice was counted after observation for 7 days, and the results are shown in Table 2.

TABLE 2 protection results of toxin challenge to mouse bacterial blood model after recombinant protein immunization

Table 2 shows the results of animal immunization experiments, the mortality of the adjuvant control group is 100%, the mortality of the TonB vaccine immunization group is 60%, and the ratio of vaccine protection = (incidence of control group-incidence of vaccinated group)/incidence of control group is 100%) is calculated according to the protection ratio calculation formula. The protection rate of the TonB protein vaccine immune group was 40%.

Example 9 toxicity protection of TonB recombinant protein immunized animals by immunization of mice using a model of pneumonia.

Following the third immunization of mice using the immunization protocol of example 5, the mice were challenged with a lethal dose of Acinetobacter baumannii by tracheal intubation on day 7, and the amount of bacteria injected into each C57BL/6 mouse was 5X 10 ⁶ CFU, and the survival rate of each group of mice was counted for 7 days, and the results are shown in Table 3.

TABLE 3 protection results of challenge to mice pneumonia model after recombinant protein immunization

Table 3 shows the results of animal immunization experiments, the mortality rate of the adjuvant control group is 100%, the mortality rate of the TonB vaccine immunization group is 50%, and the ratio is calculated according to the protection rate [ vaccine protection rate= (incidence of control group-incidence of vaccinated group)/incidence of control group ×100% ]. The protection rate of the TonB protein vaccine immune group was 50%.

Example 10 preparation of TonB-specific antibodies.

Mixing recombinant protein TonB with aluminum hydroxide adjuvant, immunizing a rabbit, 300 mug/rabbit, immunizing three times, taking blood at different time points to detect antibody titer, collecting rabbit serum, purifying by a protein A column to obtain IgG, and obtaining the TonB specific antibody.

The Acinetobacter baumannii TonB recombinant protein antigen provides an effective antigen reserve for the research and development of Acinetobacter baumannii vaccine, and fills the blank in the field at present. Has the following advantages:

(1) The preparation method is efficient, and the Acinetobacter baumannii TonB recombinant protein antigen with high purity and high yield can be efficiently prepared by the method, so that the requirement of large-scale vaccine preparation is met.

(2) The invention provides a new antigen storage scheme for developing the Acinetobacter baumannii vaccine, is hopeful to accelerate the development process of the Acinetobacter baumannii vaccine, improves the development efficiency and promotes the appearance of vaccine products.

The recombinant protein prepared by the application can be used for preparing related kits with other related reagents such as detection antibodies, color developing agents, terminators and the like.

The foregoing description of the preferred embodiments of the application is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the application.

Claims

1. 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.

2. A gene encoding the recombinant protein antigen of Acinetobacter baumannii TonB according to claim 1, characterized in that its nucleotide sequence is shown in SEQ ID NO.2.

3. A method for preparing the TonB recombinant protein antigen of Acinetobacter baumannii as claimed in claim 1, characterized in that the preparation method comprises the following steps: a) plasmid construction and prokaryotic expression: connecting the gene of the nucleotide sequence shown in SEQ ID NO.2 to an expression vector to obtain 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.

4 . The preparation method according to claim 3 , characterized in that, in step a), the expression vector is a pET-28a plasmid, and the prokaryotic expression system is a competent Escherichia coli BL21 (DE3).

5. Use of the recombinant protein antigen of Acinetobacter baumannii TonB according to claim 1 in the preparation of a medicament for treating or preventing Acinetobacter baumannii infection.

6. The use according to claim 5, characterized in that the drug is an Acinetobacter baumannii vaccine.

7. An Acinetobacter baumannii TonB-specific antibody prepared by immunizing an animal with the Acinetobacter baumannii TonB recombinant protein antigen according to claim 1.

8. Use of the Acinetobacter baumannii TonB-specific antibody according to claim 7 in the preparation of a medicament for treating or preventing Acinetobacter baumannii infection.

9. A detection kit for Acinetobacter baumannii, characterized in that it comprises the recombinant protein antigen of Acinetobacter baumannii TonB according to claim 1, and/or the specific antibody of Acinetobacter baumannii TonB according to claim 7.