Recombinant protein antigen of Acinetobacter baumannii BA71_01558, preparation method and application thereof

Abstract

The present application provides a recombinant protein antigen of Acinetobacter baumannii BA71_01558, 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 BA71_01558 of the present invention is shown in SEQ ID NO.1. The preparation process of the recombinant protein antigen of Acinetobacter baumannii BA71_01558 provided by the present application is simple, the operation steps are easy to control and easy to implement, and the obtained recombinant protein of Acinetobacter baumannii BA711_01558 has a high purity and a high yield, and is suitable for large-scale production. The vaccine prepared using the recombinant protein antigen of Acinetobacter baumannii BA711_01558 of the present application has no toxic side effects, can produce high-titer antibodies, has a 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 BA71_01558, a preparation method and an 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 BA71_01558, a preparation method and application thereof, aiming to solve the problem of poor treatment and prevention effects of 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 BA71_01558, the amino acid sequence of the recombinant protein antigen of Acinetobacter baumannii BA71_01558 being 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 BA71_01558 described in the first aspect, and 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 BA71_01558 as described in the first aspect, the preparation method comprising the following steps:

[0009] a) Plasmid construction, prokaryotic expression:

[0010] Connect the gene of the nucleotide sequence shown in SEQ ID NO.2 to the expression vector to obtain the constructed plasmid and transfer it into the prokaryotic expression system for induced expression;

[0011] b) Collect by centrifugation:

[0012] The bacterial solution after induced expression was collected and centrifuged, the supernatant was discarded, PBS buffer was added and mixed, and then ultrasonic lysis was performed, and then centrifuged again to collect the supernatant;

[0013] c) Obtaining BA71_01558 recombinant protein containing HIS tag:

[0014] The strain collected in step b) is cultured in LB medium containing kanamycin resistance until OD600 is 0.8, IPTG inducer is added and the cells are placed in a shaker for induction, and then the cells are collected by centrifugation, PBS is added to resuspend the cells, and the cells are broken by a high-pressure homogenizer. After breaking, the cells are centrifuged, and the supernatant is collected and combined with HIS-tagged protein agarose high-speed purification resin to obtain BA71_01558 recombinant protein containing HIS tag;

[0015] d) Extraction of BA71_01558 target protein:

[0016] After the supernatant in step c) is bound to the HIS-tagged protein agarose high-speed purification resin, the impurities are washed with PBS buffer, and then washed with low-mass molar concentration imidazole, and then the target protein is eluted with high-mass molar concentration imidazole and collected and the buffer is replaced;

[0017] e) Purification of BA71_01558 target protein:

[0018] The BA71_01558 target protein collected in step d) is further purified using an ion exchange column to obtain a high-purity BA71_01558 recombinant protein antigen.

[0019] 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).

[0020] In a fourth aspect, the present application provides the use of the recombinant protein antigen of Acinetobacter baumannii BA71_01558 described in the first aspect in the preparation of a drug for treating or preventing Acinetobacter baumannii infection.

[0021] According to a preferred embodiment, the drug is an Acinetobacter baumannii vaccine.

[0022] In the fifth aspect, the present application provides an Acinetobacter baumannii BA71_01558-specific antibody prepared by immunizing an animal with the Acinetobacter baumannii BA71_01558 recombinant protein antigen described in the first aspect.

[0023] In a sixth aspect, the present application provides the use of the Acinetobacter baumannii BA71_01558-specific antibody described in the fifth aspect in the preparation of a drug for treating or preventing Acinetobacter baumannii infection.

[0024] In a seventh aspect, the present application provides a detection kit for Acinetobacter baumannii, comprising the recombinant protein antigen of Acinetobacter baumannii BA71_01558 described in the first aspect, and / or,

[0025] The fifth aspect is the Acinetobacter baumannii BA71_01558-specific antibody.

[0026] Based on the above technical solution, the recombinant protein antigen of Acinetobacter baumannii BA71_01558, the preparation method and the application thereof provided in the present application have at least the following beneficial technical effects:

[0027] 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 present application provides an Acinetobacter baumannii BA71_01558 recombinant protein antigen with high efficiency, simple preparation process, easy control and easy implementation of the operation steps, and the obtained Acinetobacter baumannii BA71_01558 recombinant protein has high purity and high yield, and is suitable for large-scale production.

[0028] On the other hand, the vaccine prepared using the recombinant protein antigen of Acinetobacter baumannii BA71_01558 of the present application provided in the embodiments 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

[0029] 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.

[0030] Figure 1 This is the result of enzyme digestion identification of the expression vector pET-28a-BA71_01558.

[0031] Figure 2 The figure shows the results of protein expression induction at 16°C.

[0032] Figure 3 This is a diagram of the recombinant protein obtained from the supernatant of the recombinant engineered bacteria after induction of expression at 16°C and its purification results.

[0033] Figure 4 This is the result of purifying the BA71_01558 protein using an ion exchange column after inducing expression in the recombinant engineered bacteria at 16°C. DETAILED DESCRIPTION

[0034] 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.

[0035] 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.

[0036] In this application, "at least one" means one or more, and "plurality" 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 items 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] The terms "first" and "second" are used only for descriptive purposes to distinguish objects such as substances from each other, and should not be understood as indicating or implying relative importance or implicitly indicating the number of indicated technical features. For example, without departing from the scope of the embodiments of the present application, the first XX may also be referred to as the second XX, and similarly, the second XX may also be referred to as the first XX. Thus, features defined as "first" and "second" may explicitly or implicitly include one or more of the features.

[0041] The hypothetical protein BA71_01558 in this application is one of the target proteins that can be used as candidate antigens for vaccines, which was screened from the genome sequence of Acinetobacter baumannii by the inventor using reverse vaccinology. The hypothetical protein BA71_01558 is a SIMPL domain-containing protein. The gene encoding the hypothetical protein BA71_01558 expresses a sequence of 233 amino acids with a molecular weight of about 26 kDa. The inventor of this application initially attempted to express the full-length protein of the protein. After the recombinant plasmid encoding the protein was successfully constructed, the expression level was low and it mainly existed in the form of inclusion bodies. The properties of the protein were then analyzed by bioinformatics, and the characteristics of its transmembrane hydrophobic structure and signal peptide were combined. The 23rd-233rd amino acids of the protein were selected for expression. The inventor's experiments verified that the recombinant protein can be efficiently expressed and expressed in a soluble form, which is more conducive to maintaining the natural structure and activity of the protein, and provides a basis for the subsequent large-scale production of the protein. The Acinetobacter baumannii BA71_01558 recombinant protein of the present application has good immunogenicity, and can effectively protect mice against infection by Acinetobacter baumannii after immunization, and has a high protection rate and a stable protection effect. The specific experimental process is as follows.

[0042] 1. Strains.

[0043] The Acinetobacter baumannii strain was purchased from ATCC with the strain number ATCC17978.

[0044] 2. Reagents.

[0045] HIS-tagged protein agarose high-speed purification resin (Ni-TED 6FF agarose gel) was a product of 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; and BCA protein determination kit was purchased from Yisheng Biotechnology Co., Ltd.

[0046] Example 1: Construction of recombinant expression engineering bacteria of Acinetobacter baumannii BA71_01558 protein.

[0047] 1. According to the amino acid sequence of the protein BA71_01558 of the Acinetobacter baumannii strain (GenBank: EZF15418.1), the signal peptide and transmembrane domain of the protein were structurally analyzed using bioinformatics software, thereby determining that the protein to be amplified is the amino acid sequence of positions 23-233 of the BA71_01558 protein. The BA71_01558 protein sequence to be amplified is shown in SEQ ID NO.1.

[0048] 2. Based on the BA71_01558 protein sequence shown in SEQ ID NO.1 and the optimized codons of the E. coli expression system, the DNA sequence expressing the recombinant protein was obtained, and considering the restriction site sequence of the connection vector, a biological company was commissioned to synthesize DNA, and the DNA sequence was shown in SEQ ID NO.2. The synthesized DNA was linked to the pET-28a plasmid to obtain the recombinant plasmid pET-28a-BA71_01558.

[0049] 3. Transformation: Centrifuge the synthesized recombinant plasmid pET-28a-BA71_01558 at 12000 rpm for 1 min, add 40 μL of deionized water to mix, take 1 μL of plasmid solution and add 20 μL of E. coli BL21 (DE3) competent cells, flick to mix, and let stand on ice for 30 min. Then heat shock at 42 ℃ for 90 s, immediately let stand on ice for 2 min, add 0.5 mL of LB medium without antibiotics, resuscitate at 220 rpm / 37℃ for 50 min, take 50 μL and spread on LB plate with kanamycin, and culture at 37 ℃ overnight.

[0050] 4. Identification of recombinant engineered bacteria: pick a single clone, expand the culture, extract the plasmid and then identify it by enzyme digestion.

[0051] The results are as follows Figure 1 As shown, lane M: nucleic acid (DNA) molecular weight standard (Marker); lane 1: identification result of recombinant expression plasmid pET-28a-BA71_01558 after enzyme digestion. It can be seen that the target fragment after enzyme digestion is consistent with the theoretical 639bp.

[0052] Example 2: Induced expression, purification and identification of the expression form of the Acinetobacter baumannii BA71_01558 protein in the prokaryotic expression system - Escherichia coli.

[0053] 1. Induce expression of target protein.

[0054] 1) Take 100 μL of pET-28a-BA71_01558 / BL21 (DE3) bacterial solution that was correctly identified by double enzyme digestion and add it to 10 mL of kanamycin-resistant LB medium, and culture it at 100 rpm and 37℃ overnight. Take 2 mL of the overnight cultured bacterial solution and add it to 18 mL of kanamycin-resistant LB medium (the remaining bacterial solution was stored in a 4℃ refrigerator for standby use), culture it at 37℃ for 2-3h and 220 rpm. After secondary activation until OD600 is 0.8-1.2, add IPTG to a final concentration of 200 μM, and then place it in a shaker at 16℃ overnight to induce expression.

[0055] 2) Take out the induced bacterial solution, centrifuge at 10,000 rpm for 1 min, discard the supernatant, add 1 mL PBS buffer and mix well, ultrasonically lyse for 5 min, and then centrifuge at 12,000 rpm for 15 min at 4°C, collect the supernatant and precipitate samples.

[0056] 3) Preparation of SDS-PAGE electrophoresis gel: Pour 5% concentrated gel into the gel plate, add distilled water to flatten the gel, and leave it at room temperature for 30 minutes to solidify. Pour out the distilled water on the upper layer, and then pour in 10% separation gel. Immediately insert the comb and leave it at room temperature for 40 minutes to solidify for later use.

[0057] 4) Protein expression detection: 10 μL of the treated supernatant samples were loaded for SDS-PAGE electrophoresis. The voltage was first 80 V for 30 min, then adjusted to 200 V. After 45 min of electrophoresis, the gel was taken out and placed in Coomassie Brilliant Blue staining solution for shaking staining, and then placed in destaining solution for shaking destaining, and the results were observed under the gel imaging system.

[0058] The results are as follows Figure 2As 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 (indicated by the arrow); 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 around 25 KD, which is consistent with the size of the target recombinant protein of 24 KD, and the recombinant protein is mainly expressed in the supernatant of bacterial ultrasonic lysis in a soluble form. The expression rate of the target protein band (the ratio of the gray value of the target band to the gray value of the entire band) is 17.30% through analysis, and the expression amount is high.

[0059] It can be seen that the pET-28a-BA71_01558 / BL21 (DE3) 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-BA71_01558 / BL21 (DE3) recombinant engineering bacteria can maintain its original spatial conformation to the maximum extent while also having good solubility.

[0060] Example 3: Preparation of BA71_01558 recombinant protein antigen.

[0061] 1. Scale up the culture to obtain the recombinant protein HIS-BA71_01558.

[0062] Take the pET-28a-BA71_01558 / BL21 (DE3) strain stored in a -80 ℃ refrigerator and inoculate it on an LB kanamycin resistance plate and culture it at 37 ℃ overnight; pick a single colony and inoculate it in 100 mL LB kanamycin resistance medium and culture it at 37 ℃ and 220 rpm overnight; add the activated 100 mL bacterial solution to 2 L LB medium containing kanamycin resistance for secondary activation, culture it at 37 ℃ for 3-4 h until OD600 is 0.8, add 1 mL IPTG inducer (final concentration is 500 μM) and place it in a 16 ℃ shaker for induction for 12 h, collect the bacteria by centrifugation at 4000 rpm for 10 min, add 80 ml PBS to resuspend the bacteria, and use a high-pressure homogenizer to cycle and break it for 5 cycles. After breaking, use a floor-standing high-speed centrifuge at 12000 rpm for 30 min, collect the supernatant and balance it with 5 mL Ni-TED 6FF agarose gel 4 ℃ overnight to obtain a large amount of BA71_01558 recombinant protein containing HIS tag.

[0063] 2. Extraction of HIS-BA71_01558.

[0064] After the binding is completed, the Ni-TED agarose gel is recovered after the supernatant is passed through. The impurities are first washed with 100 mL of PBS, and then washed with 100 mL of 20 mM, 50 mL of 30 mM, and 50 mL of 50 mM imidazole respectively. Next, the target protein is eluted with 10 mL of 100 mM, 200 mM, 300 mM, and 500 mM imidazole respectively. Finally, the agarose gel is resuspended with PBS, and 10 μL of the sample is taken for denaturation treatment and SDS-PAGE protein electrophoresis, and the results are observed under the phase system.

[0065] The electrophoresis results are shown in Figure 3 Lane "M" is a protein molecular weight standard (Marker), lane 1 represents the protein bound to the supernatant of Ni-TED 6FF agarose gel and lysed bacteria; lanes 2-4 represent the protein results of beads washed with 20 mM, 30 mM, and 50 mM imidazole; lanes 5-8 represent the target protein eluted at 100 mM, 200 mM, 300 mM, and 500 mM imidazole concentrations; lane 9 represents the Ni-TED agarose gel spotting results after elution. The electrophoresis results show that the molecular weight of the BA71_01558 recombinant protein is about 24kD, which is consistent with the expected protein molecular weight, and the HIS-BA71_01558 protein is mainly eluted by 100 mM, 200 mM, and 300 mM imidazole.

[0066] 3. Collect the target protein eluted with 100mM, 200mM, and 300mM imidazole, replace the buffer, and store the target protein in PBS (pH=8.0) buffer.

[0067] 4. Use an ion exchange column to further purify the BA71_01558 recombinant protein to obtain high-purity BA71_01558 recombinant protein. Specifically:

[0068] Take out the ion exchange column, connect and install it, rinse the exchange column with deionized water for 5 column volumes, and fully balance the column with filtered PBS (pH=8.0) solution; filter the sample stored in PBS (PH=8.0) buffer, and slowly load the sample to allow the protein to fully bind; rinse the impurities on the exchange column with 10mM NaCl and 20mM NaCl in turn to remove the impurities; elute the target protein using a linear method, and collect the effluent for electrophoresis detection, such as Figure 4As shown, lane M: protein molecular weight standard (Marker); lane 1 represents the BA71_01558 recombinant protein purified after using an ion exchange column. After scanning, the gray value of the target protein accounts for the gray value of the entire lane, and the protein purity is calculated to be 92%.

[0069] 5. Determine the protein concentration according to the instructions of the BCA protein assay kit, and the final assay concentration is 1.5 mg / mL.

[0070] Example 4: Preparation of BA71_01558 recombinant protein immune preparation.

[0071] The BA71_01558 recombinant protein and Al(OH)3 were used as adjuvant components for adsorption to prepare subunit vaccine.

[0072] The method is as follows: the aluminum hydroxide adjuvant is measured and adjusted to a concentration of 5 mg / ml with a histidine diluent of pH 6.0, and mixed thoroughly to obtain solution A; the BA71_01558 recombinant protein is diluted to 200 μg / mL with a histidine diluent of pH 6.0 to obtain solution B; equal volumes of solution A and solution B are taken and rotated and suspended for adsorption at 4°C for 2 hours to obtain a subunit vaccine. Solution B of the adjuvant control preparation group is a histidine diluent without protein antigen, and adsorption is performed with solution A under the same conditions.

[0073] Example 5: Immunization of mice with BA71_01558 recombinant protein.

[0074] The experimental mice were purchased from Hunan Lesk Jingda Experiment Co., Ltd. They were SPF-grade female healthy mice weighing about 18-20 g.

[0075] 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;

[0076] 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;

[0077] 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;

[0078] 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.

[0079] Example 6: Preparation of a specific antibody detection kit for Acinetobacter baumannii BA71_01558.

[0080] 1) Prepare the liquid.

[0081] 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;

[0082] ② Preparation of blocking solution: 1 g bovine serum albumin, dissolved in 100 mL antibody diluent;

[0083] ③ Preparation of antibody diluent: Dissolve 0.1M phosphate (10X PBS) in 1 L ddH2O, add 500 μl Tween 20, and adjust the pH to 7.4 using a pH meter;

[0084] 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;

[0085] ⑤ Chromogenic solution (TMB), a product of Tiangen Company;

[0086] ⑥ Preparation of stop solution (2 M H2SO4): Pour 22.2 mL of concentrated sulfuric acid into 177.8 mL of ddH2O.

[0087] 2) Preparation of BA71_01558 protein antibody detection kit.

[0088] ① Dilute the purified BA71_01558 recombinant protein to 4 μg / mL using coating solution;

[0089] 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;

[0090] 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;

[0091] ④ Combine 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 at 4°C.

[0092] Example 7: Antibody detection after immunization with BA71_01558 recombinant protein.

[0093] On the 7th day after the third immunization, blood was collected from C57Bl / 6 mice, and the IgG humoral response level of mice after immunization was detected by ELISA.

[0094] ① After diluting the serum 1000 times, perform a 5-fold dilution;

[0095] 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 control dryness;

[0096] ③ Dilute the HRP-labeled goat anti-mouse IgG antibody storage solution at 1:10000 to prepare the antibody working solution; add the diluted antibody working solution, 100 μl / well, place in a 37°C incubator for 45 minutes, wash 4 times, and control dryness;

[0097] ④ Add 100 μl / well of substrate colorimetric solution (TMB) and react at room temperature in the dark for 5 minutes;

[0098] ⑤ Add stop solution (2M H2SO4) and immediately place on a microplate reader to measure the OD value at a wavelength of 450nm;

[0099] ⑥ Result judgment: A sample / A negative value ≥ 2.1 is positive (negative control is mouse pre-immune serum).

[0100] Table 1 Recombinant BA71_01558 protein immune serum antibody detection

[0101] Serum dilution Adjuvant group serum BA71_01558 immune group serum 1000 0.053 2.552 5000 0.038 2.437 25000 0.04 1.694 125000 0.045 0.788

[0102] The results are shown in Table 1. The OD450 of the antibodies produced by mice immunized with BA71_01558 protein antigen was still as high as 0.788 after dilution 125,000 times, and the antibody titer was higher than 1:125,000. This indicates that the BA71_01558 recombinant protein constructed by the present invention can produce high-titer specific antibodies in immunized mice and has good immunogenicity.

[0103] Example 8: Pneumonia model was used to evaluate the immune protection effect of BA71_01558 recombinant protein.

[0104] 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 20 μl of bacterial solution, with a bacterial amount of 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 2.

[0105] Table 2 The protective effect of recombinant protein on the pneumonia model in mice

[0106] Group Immune components Mouse (pcs) Number of dead mice mortality rate(%) Protection rate (%) BA71_01558 immunization group <![CDATA[BA71_01558 + Al(OH)3 Adjuvant]]> 10 7 70 30 Adjuvant control group <![CDATA[Histidine solution + Al(OH)3 adjuvant]]> 10 10 100 0

[0107] 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 BA71_01558 immunization group was 70%. 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 BA71_01558 immunization group was 30%.

[0108] Example 9: Preparation of BA71_01558-specific antibodies.

[0109] The BA71_01558 recombinant protein was mixed with aluminum hydroxide adjuvant and used to immunize rabbits at 300 μg / rabbit. The immunization procedure was the same as in Example 5. The rabbits were immunized three times. Blood was collected at different time points to detect the antibody titer. Rabbit serum was collected and the antibody titer was as high as 1:243000. The IgG in the serum was purified by protein A column, which was the BA71_01558 specific antibody.

[0110] Table 3 Recombinant BA71_01558 protein rabbit immune serum antibody detection

[0111] Serum dilution BA71_01558 immune group serum 1000 1.4153 3000 1.0393 9000 0.9436 27000 0.504 81000 0.37 243000 0.26 729000 0.075 2187000 0.062

[0112] The Acinetobacter baumannii BA71_01558 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:

[0113] (1) Efficient preparation: The method of the present invention can efficiently prepare high-purity and high-yield recombinant protein antigens of Acinetobacter baumannii BA71_01558, meeting the needs of large-scale vaccine preparation.

[0114] (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.

[0115] 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.

[0116] 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 BA71_01558 in the preparation of a drug for treating or preventing infection by Acinetobacter baumannii, characterized in that: The amino acid sequence of the recombinant protein antigen of Acinetobacter baumannii BA71_01558 is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that The drug is an Acinetobacter baumannii vaccine.

3. The use according to claim 1, characterized in that: The nucleotide sequence of the gene encoding the recombinant protein antigen of Acinetobacter baumannii BA71_01558 is shown in positions 7-639 of SEQ ID NO.

2.

4. A method for preparing Acinetobacter baumannii BA71_01558-specific antibodies by immunizing animals with the recombinant protein antigen of Acinetobacter baumannii BA71_01558 described in claim 1, characterized in that: The steps include: The recombinant protein antigen of Acinetobacter baumannii BA71_01558 was mixed with aluminum hydroxide adjuvant and used to immunize rabbits, 300 μg / rabbit, and the rabbits were immunized three times on the 1st day, the 14th day and the 21st day respectively. On the 7th day after the third immunization, the venous blood of the mice was collected, and the serum was separated by centrifugation after coagulation. The IgG in the serum was purified by protein A column to obtain BA71_01558-specific antibodies, wherein the amino acid sequence of the recombinant protein antigen of Acinetobacter baumannii BA71_01558 is shown in SEQ ID NO.1.

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