Recombinant protein antigen of Acinetobacter baumannii A1S_1027, preparation method and application thereof

Abstract

The present application provides a recombinant protein antigen of Acinetobacter baumannii A1S_1027, 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 A1S_1027 of the present invention is shown in SEQ ID NO.1. The preparation process of the recombinant protein antigen of Acinetobacter baumannii A1S_1027 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 A1S_1027 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 A1S_1027 of the present application has no toxic side effects, can produce high-titer antibodies, has a good immune protection effect against Acinetobacter baumannii infection, is suitable for preparing specific antibodies, and is conducive to the research and development of Acinetobacter baumannii vaccines.

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 A1S_1027, a preparation method and an application thereof. Background Art

[0002] Acinetobacter baumannii is one of the most common and seriously drug-resistant bacteria in clinical practice. 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, intubation-related infection, urinary tract infection, bacteremia and secondary meningitis, among which pneumonia and bloodstream infection are the most common and serious. Its increased drug resistance and 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 options. Therefore, there is an urgent need to develop new methods for the prevention and treatment of Acinetobacter baumannii infections. Summary of the invention

[0004] The purpose of the present application is to provide a recombinant protein antigen of Acinetobacter baumannii A1S_1027, a preparation method and its application, 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 A1S_1027, the amino acid sequence of the recombinant protein antigen of Acinetobacter baumannii A1S_1027 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 A1S_1027 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 the recombinant protein antigen of Acinetobacter baumannii A1S_1027 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 A1S_1027 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 A1S_1027 recombinant protein containing HIS tag;

[0015] d) Extraction of target protein A1S_1027:

[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 A1S_1027 target protein:

[0018] The A1S_1027 target protein collected in step d) is further purified using an ion exchange column to obtain a high-purity A1S_1027 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 A1S_1027 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 a fifth aspect, the present application provides an Acinetobacter baumannii A1S_1027-specific antibody prepared by immunizing an animal with the Acinetobacter baumannii A1S_1027 recombinant protein antigen described in the first aspect.

[0023] In a sixth aspect, the present application provides the use of the Acinetobacter baumannii A1S_1027-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 A1S_1027 described in the first aspect, and / or,

[0025] The fifth aspect is the Acinetobacter baumannii A1S_1027-specific antibody.

[0026] Based on the above technical solution, the recombinant protein antigen of Acinetobacter baumannii A1S_1027, 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 preparation process of the Acinetobacter baumannii A1S_1027 recombinant protein antigen provided by the present application is simple, the operation steps are easy to control and implement, and the obtained Acinetobacter baumannii A1S_1027 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 A1S_1027 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-A1S_1027.

[0031] Figure 2 A graph showing the results of inducing protein expression of A1S_1027 recombinant protein at 16°C.

[0032] Figure 3 This is a diagram showing 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 a diagram showing the results of recombinant protein A1S_1027 purified by affinity chromatography after purification using an ion exchange column. 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 hypothetical protein A1S_1027 used in the present invention is one of the target proteins that can be used as candidate vaccine antigens, which was screened from the genome sequence of Acinetobacter baumannii using reverse vaccinology and bioinformatics methods. The hypothetical protein A1S_1027 is functionally annotated as a potential signal peptide "putative signal peptide", and the gene encoding the hypothetical protein A1S_1027 expresses a sequence of 369 amino acids with a molecular weight of about 41.19 kDa. The inventor 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 to select the 26th-369th amino acids of the protein for expression. It has been verified by the present invention 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 recombinant protein has good immunogenicity and can effectively protect mice against infection with Acinetobacter baumannii after immunization, with a high protection rate and stable protection effect. The specific experimental process is as follows:

[0041] 1. Strains.

[0042] The Acinetobacter baumannii strain was purchased from ATCC, USA, with the strain number ATCC17978.

[0043] 2. Reagents.

[0044] HIS tag 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.

[0045] Example 1: Construction of the recombinant engineering bacteria pET-28a-A1S_1027 / BL21 (DE3) expressing the A1S_1027 protein.

[0046] 1. According to the amino acid sequence of the A1S_1027 protein of Acinetobacter baumannii strain (GenBank: ABO11459.2), the bioinformatics software was used for structural analysis to determine that the protein to be expressed was the amino acid sequence of positions 26-369 of the A1S_1027 protein, and the sequence was shown in SEQ ID NO.1.

[0047] 2. Based on the A1S_1027 protein sequence shown in SEQ ID NO.1 and the codons optimized in the E. coli expression system, a DNA sequence expressing the recombinant protein was obtained, and the restriction site sequence of the connection vector was taken into consideration to synthesize the DNA shown in SEQ ID NO.2.

[0048] 3. Ligate the synthesized DNA into the pET-28a plasmid to obtain the recombinant plasmid pET-28a-A1S_1027.

[0049] 4. Transformation: Centrifuge the recombinant plasmid pET-28a-A1S_1027 at 12000 rpm for 1 min, add 40 μL of deionized water and mix well, take 1 μL of the plasmid solution and add 20 μL of E. coli BL21 (DE3) competent cells, flick to mix well, and place on ice for 30 min. Heat shock at 42 ℃ for 90 s, immediately place 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 containing kanamycin, and culture at 37 ℃ overnight.

[0050] 5. 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 Lane M: nucleic acid (DNA) molecular weight standard (Marker); Lane 1: identification results of the recombinant expression plasmid pET-28a-A1S_1027 after enzyme digestion. The target fragment after enzyme digestion is between 1000 bp and 1500 bp, which is consistent with the theoretical size of 1038 bp.

[0052] Example 2: Induced expression of Acinetobacter baumannii A1S_1027 protein and identification of the expression form.

[0053] 1. Induce expression of target protein.

[0054] 1) Take 50 μL of pET-28a-A1S_1027 / BL21 (DE3) bacterial suspension 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 1 mL of the overnight cultured bacterial suspension and add it to 19 mL of kanamycin-resistant LB medium, and culture it at 37 ℃ for 2-3 h at 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 on 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, centrifuge at 12,000 rpm for 15 min at 4 °C, and collect the supernatant.

[0056] 3) Preparation of SDS-PAGE electrophoresis gel: Pour 5% concentrated gel into the gel plate, add distilled water to flatten the gel, leave it at room temperature for 40 min to solidify, drain the upper layer of distilled water, pour in 10% separation gel, immediately insert the comb, leave it at room temperature for 40 min 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 100 V for 20 min, then adjusted to 200 V for 40 min, the gel was taken out, 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 2 As shown, lane "M" is a protein molecular weight standard (Marker); lane "-": whole-cell protein bands of the recombinant engineering bacteria before induction expression; lane "+": expression of the recombinant protein in the whole cell after induction expression of the recombinant engineering bacteria at 16°C; lane "s": recombinant protein obtained in the supernatant of bacterial ultrasonic lysis after induction expression of the recombinant engineering bacteria at 16°C ( Figure 2 Indicated by the arrow); Lane "p": Samples precipitated after induction expression of recombinant engineering bacteria at 16°C, and recombinant proteins obtained in bacterial ultrasonic precipitation. It can be seen that the recombinant protein band is between 37-50 KD, which is consistent with the size of the target recombinant protein of 39 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 38.18%, and the expression level is high.

[0059] The pET-28a-A1S_1027 / BL21 (DE3) recombinant engineering bacteria obtained by the present invention has the advantages of easy cultivation, high efficiency of recombinant protein expression, short purification cycle, safety and controllability, and convenient operation, and is also conducive to the process expansion of large-scale production of vaccines in the later stage. At the same time, the protein expressed by the pET-28a-A1S_1027 / BL21 (DE3) recombinant engineering bacteria exists in a soluble form, which can maximize the maintenance of its original spatial conformation and immune activity.

[0060] Example 3: Preparation of A1S_1027 recombinant protein antigen.

[0061] 1. Scale up the culture to obtain the recombinant protein A1S_1027.

[0062] Take the pET-28a-A1S_1027 / 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 50 mL LB kanamycin resistance medium and culture it at 37 ℃, 220 rpm overnight; add the activated 50 mL bacterial solution to 1 L LB medium containing kanamycin resistance for secondary activation, culture at 37 ℃ for 3-4 hours until OD600 is 0.8, add 0.2 mL IPTG (final concentration is 200 μM) and place it in a 16 ℃ shaker for induction for 12 hours, centrifuge at 4000 rpm for 10 min to collect the bacteria, add 80 mL PBS to resuspend the bacteria, and use a high-pressure homogenizer to cycle and break it for 5-6 cycles. After breaking, use a floor-standing high-speed centrifuge at 4 ℃ 12000 rpm for 30 min, collect the supernatant and balance it with 5 mL Ni-TED 6FF agarose gel at 4 ℃ Overnight binding allowed the A1S_1027 recombinant protein containing the HIS tag to bind to the agarose gel.

[0063] 2. Purification of A1S_1027 recombinant protein.

[0064] After the binding is completed, the Ni-TED 6FF agarose gel is recovered after the supernatant is run through. It is first washed with 100 mL of PBS to remove impurities, and then washed with 100 mL of 10 mM imidazole. Next, the target protein is eluted with 10 mL of 20 mM, 30 mM, 50 mM, 100 mM, 200 mM, 300 mM, and 500 mM imidazole, respectively. Finally, the agarose gel is resuspended with PBS, and 10 μL of the above samples are taken for denaturation treatment and SDS-PAGE protein electrophoresis. The results are observed under the gel phase system. The electrophoresis results show that the molecular weight of the A1S_1027 protein is about 39 kD, which is consistent with the expected protein molecular weight, and the A1S_1027 protein is mainly eluted by 30 mM, 50 mM, and 100 mM imidazole. The electrophoresis results are shown in Figure 3 Lane 1 represents the protein bound to Ni-TED 6FF agarose gel in the supernatant of lysed bacteria; Lanes 2-8 represent the elution results of the target protein at 20 mM, 30 mM, 50 mM, 100 mM, 200 mM, 300 mM, and 500 mM imidazole concentrations, respectively; Lane 9 represents the Ni-TED 6FF agarose gel spotting results after the elution.

[0065] 3. Collect the target protein eluted with 30 mM, 50 mM, and 100 mM imidazole, replace the buffer, and store the target protein in PBS (pH=7.0) buffer.

[0066] 4. Use an ion exchange column to further purify the A1S_1027 recombinant protein to obtain high-purity A1S_1027 recombinant protein. Specifically:

[0067] 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=7.0) solution; filter the sample stored in PBS (PH=7.0) buffer, and slowly load the sample to allow the protein to fully bind; rinse the impurities on the exchange column with 10 mM NaCl and 20 mM 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 4 As shown, lane 1 represents the A1S_1027 protein purified by ion exchange column, and the gray value of the target band and the gray value of the entire lane are scanned, and the protein purity is calculated to be 96%.

[0068] 5. Determine the protein concentration according to the instructions of the BCA protein assay kit, and the final measured concentration is 2.2 mg / mL.

[0069] Example 4: Preparation of A1S_1027 recombinant protein immune preparation.

[0070] A1S_1027 recombinant protein and Al(OH)3 were used as adjuvant components for adsorption to prepare subunit vaccine.

[0071] The method is as follows: the aluminum hydroxide adjuvant is measured and adjusted to 5 mg / ml with histidine diluent at pH 6.0, and mixed thoroughly to obtain solution A; the A1S_1027 recombinant protein is diluted to 200 μg / mL with histidine diluent at pH 6.0 to obtain solution B; equal volumes of solution A and solution B are taken and rotated and suspended for adsorption for 2 hours at 4°C to obtain the vaccine. Solution B of the adjuvant control preparation group is a histidine diluent without protein antigen, and adsorption is carried out with solution A under the same conditions.

[0072] Example 5: Immunization of animals with A1S_1027 recombinant protein.

[0073] 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;

[0074] 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;

[0075] 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;

[0076] 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.

[0077] Example 6: Preparation of a detection kit for Acinetobacter baumannii A1S_1027-specific antibodies.

[0078] 1) Prepare the liquid.

[0079] 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;

[0080] ② Preparation of blocking solution: 1 g bovine serum albumin, dissolved in 100 mL antibody diluent;

[0081] 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;

[0082] 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;

[0083] ⑤ Chromogenic solution (TMB), a product of Tiangen Company;

[0084] ⑥ Preparation of stop solution (2 M H2SO4): Pour 22.2 mL of concentrated sulfuric acid into 177.8 mL of ddH2O.

[0085] 2) Preparation of A1S_1027 protein antibody detection kit.

[0086] ① Use coating solution to dilute the purified A1S_1027 recombinant protein to 4μg / mL;

[0087] 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;

[0088] 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

[0089] 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.

[0090] Example 7: Antibody detection after immunization with A1S_1027 recombinant protein.

[0091] 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.

[0092] ① After diluting the serum 1000 times, perform a 5-fold dilution;

[0093] 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;

[0094] 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;

[0095] ④ Add 100 μl / well of substrate colorimetric solution (TMB) and react at room temperature in the dark for 5 minutes;

[0096] ⑤ Add stop solution (2 M H2SO4) and immediately place on a microplate reader to measure the OD value at a wavelength of 450nm;

[0097] ⑥ Result judgment: A sample / A negative value ≧2.1 is positive (negative control is mouse pre-immune serum).

[0098] Table 1 Recombinant A1S_1027 protein immune serum antibody detection

[0099] Serum dilution Adjuvant group serum A1S_1027 immune group serum 1000 0.044 2.619 5000 0.044 1.389 25000 0.044 0.538 125000 0.041 0.174

[0100] The results are shown in Table 1. The OD450 of the antibodies produced by mice immunized with A1S_1027 protein antigen was still as high as 0.174 after being diluted 125,000 times, and the antibody titer was higher than 1:125,000. This indicates that the A1S_1027 recombinant protein constructed by the present invention can produce high-titer specific antibodies in immunized mice and has good immunogenicity.

[0101] Example 8: Pneumonia model to evaluate the immune protection effect of A1S_1027 recombinant protein.

[0102] The same immunization scheme as in Example 5, after the third immunization of mice, on the 14th day, the tail vein was injected with a lethal dose of Acinetobacter baumannii for challenge, and 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.

[0103] Table 2 Protection results of recombinant protein immunization against bacteremia model in mice

[0104] Group Immune components Mouse (pcs) Number of dead mice mortality rate(%) Protection rate (%) A1S_1027 immune group <![CDATA[A1S_1027 + Aluminum hydroxide adjuvant]]> 10 3 30 62.5 Adjuvant control group <![CDATA[Histidine solution + Al(OH)3 adjuvant]]> 10 8 80 0

[0105] Table 2 shows the results of the animal immunization test. The mortality rate of the adjuvant control group was 80%, and the mortality rate of the A1S_1027 vaccine immunization group was 30%. 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 A1S_1027 protein vaccine immunization group was 62.5%.

[0106] Example 9: Preparation of A1S_1027-specific antibodies.

[0107] 1. Rabbit immunization: A1S_1027 recombinant protein was mixed with aluminum hydroxide adjuvant to immunize rabbits, 300 μg / rabbit, multiple intramuscular injections, and boosted every two weeks for a total of three immunizations. Two weeks after the last immunization, rabbit blood was collected and placed in a serum separation tube. After standing at room temperature for 2 hours, the blood was centrifuged at 4 °C (3000 rpm, 10 min) to obtain serum. Referring to the method of Example 7, the titer of rabbit antibodies was detected using an ELISA kit, and the antibody titer was as high as 1:81000.

[0108] 2. IgG purification: Dilute rabbit serum with PBS (1:1 volume ratio) to make it suitable for affinity chromatography, and load the diluted serum onto a well-equilibrated protein A / G affinity chromatography column. Wash the column with PBS to remove nonspecifically bound proteins. Elute IgG with elution buffer (0.1 M citric acid, pH 3.0). Collect the eluate and immediately add 1 / 10 volume of neutralization buffer. Determine the IgG concentration by BCA method, which is 2.13 mg / ml.

[0109] Example 10: A1S_1027-specific antibody protects mice from challenge with Acinetobacter baumannii.

[0110] The bacteremia model of Example 8 was used, and rabbit IgG was injected 1 hour after infection with Acinetobacter baumannii.

[0111] 1. Randomly divide the mice into three groups, with 10 mice in each group:

[0112] - Experimental group: administered with A1S_1027-specific rabbit IgG.

[0113] -Control group A: administered with normal saline.

[0114] -Control group B: administered with normal rabbit IgG.

[0115] 2. Mouse bacteremia model: The bacteremia model was established by infecting mice according to the method of Example 8. One hour after infection with Acinetobacter baumannii, the mice in the experimental group were given 100 μg of A1S_1027-specific rabbit IgG by tail vein injection, the mice in the control group A were given an equal volume of normal saline, and the mice in the control group B were given 100 μg of normal rabbit IgG.

[0116] 3. Observe the survival of mice: Observe the survival of mice daily and record clinical symptoms, including mental state, activity level, weight changes, etc. Record the survival of mice 7 days after the challenge and calculate the survival rate.

[0117] Table 3 Protective effect of A1S_1027 recombinant protein specific IgG on mouse bacteremia model

[0118] Group Immune components Mouse (pcs) Number of dead mice mortality rate(%) Protection rate (%) Experimental Group <![CDATA[A1S_1027 + Aluminum hydroxide adjuvant]]> 10 5 50 50 Control group A Normal saline 10 10 100 0 Control group B Normal rabbit IgG 10 99 90 10

[0119] The results are shown in Table 3. The experimental results show that the administration of purified A1S_1027-specific rabbit IgG significantly improved the resistance of mice to Acinetobacter baumannii infection, proving that the A1S_1027-specific antibody has a strong protective effect and can be used for the treatment of Acinetobacter baumannii infection.

[0120] The Acinetobacter baumannii A1S_1027 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:

[0121] (1) Efficient preparation: The method of the present invention can efficiently prepare high-purity and high-yield recombinant protein antigen of Acinetobacter baumannii A1S_1027, meeting the needs of large-scale vaccine preparation.

[0122] (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.

[0123] 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.

[0124] 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 A1S_1027 in the preparation of a drug for treating or preventing infection with Acinetobacter baumannii, characterized in that: The amino acid sequence of the recombinant protein antigen of Acinetobacter baumannii A1S_1027 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 encoding the recombinant protein antigen gene of Acinetobacter baumannii A1S_1027 is shown in positions 7-1038 of SEQ ID NO.

2.

4. A method for preparing Acinetobacter baumannii A1S_1027-specific antibodies by immunizing animals with recombinant protein antigens of Acinetobacter baumannii A1S_1027, characterized in that: The steps include: The recombinant protein of Acinetobacter baumannii A1S_1027 was mixed with aluminum hydroxide adjuvant and used to immunize rabbits at 300 μg per rabbit by multi-point intramuscular injection. The immunization was boosted every two weeks for a total of three times. Two weeks after the last immunization, the rabbit blood was collected and placed in a serum separation tube. After standing at room temperature for 2 hours, the blood was centrifuged at 4°C to obtain serum. The specific antibody of Acinetobacter baumannii A1S_1027 was obtained, wherein the amino acid sequence of the recombinant protein antigen of Acinetobacter baumannii A1S_1027 is shown in SEQ ID NO.1.

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