Recombinant protein antigen of Acinetobacter baumannii BA71_03257, preparation method and application thereof

Abstract

The present application provides a recombinant protein antigen of Acinetobacter baumannii BA71_03257, 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_03257 of the present invention is shown in SEQ ID NO.1. The preparation process of the recombinant protein antigen of Acinetobacter baumannii BA71_03257 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 BA71_03257 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 BA71_03257 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_03257, a preparation method and an application thereof. Background Art

[0002] Acinetobacter baumannii infection is a serious hospital-acquired infection in clinical practice, posing a serious threat to public health and clinical treatment. Acinetobacter baumannii is extremely resistant to conventional antibiotics, especially carbapenem antibiotics, which makes its infection treatment extremely difficult. In addition, Acinetobacter baumannii is highly contagious in hospital environments, especially for patients with weak immune systems, such as critically ill patients and immunosuppressed patients, whose infection risk is more prominent. Due to the increase in multidrug-resistant strains and high morbidity and mortality rates, Acinetobacter baumannii has been included in the World Health Organization (WHO)'s list of key research areas for drug-resistant bacteria and antimicrobial resistance.

[0003] Acinetobacter baumannii infection poses a serious threat to patients' health and may lead to a variety of serious complications, such as lung infection, sepsis, meningitis, etc., and even endanger life. Under the current circumstances, there is no effective vaccine specifically for Acinetobacter baumannii on the market, which has led to a series of challenges in the prevention and control of Acinetobacter baumannii infection. Therefore, it is particularly urgent and necessary to develop a vaccine that can prevent and control 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_03257, 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_03257, the amino acid sequence of the recombinant protein antigen of Acinetobacter baumannii BA71_03257 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_03257 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_03257 as described in the first aspect, comprising the following steps:

[0009] a) Plasmid construction, prokaryotic expression:

[0010] The nucleotide sequence shown in SEQ ID NO.2 is cloned into a vector, amplified by PCR to obtain a PCR product, and the obtained PCR product is connected to a vector to construct a recombinant expression vector; the constructed recombinant expression vector is introduced into a prokaryotic expression system to induce 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) Obtain BA71_03257 fusion protein containing GST tag:

[0014] The strain collected in step b) is cultured in LB medium containing ampicillin 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 a GST-tagged protein purification resin to obtain a BA71_03257 fusion protein containing a GST tag;

[0015] d) Extraction of BA71_03257 target protein:

[0016] After the supernatant in step c) is completely bound to the GST-tagged protein purification resin, the BA71_03257 target protein and the GST tag are separated by enzyme cleavage and then collected;

[0017] e) Purification of BA71_03257 target protein:

[0018] The BA71_03257 target protein collected in step d) is further purified using an ion exchange column to obtain a high-purity BA71_03257 recombinant protein antigen.

[0019] According to a preferred embodiment, in step a), the expression vector is a pGEX-6P-2 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_03257 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_03257-specific antibody prepared by immunizing an animal with the Acinetobacter baumannii BA71_03257 recombinant protein antigen described in the first aspect.

[0023] In a sixth aspect, the present application provides the use of the Acinetobacter baumannii BA71_03257-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_03257 described in the first aspect, and / or,

[0025] The fifth aspect is the Acinetobacter baumannii BA71_03257-specific antibody.

[0026] Based on the above technical solution, the recombinant protein antigen of Acinetobacter baumannii BA71_03257, 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 BA71_03257 recombinant protein antigen provided by the present application is simple, the operation steps are easy to control and implement, and the obtained Acinetobacter baumannii BA71_03257 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_03257 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 recombinant expression plasmid pGEX-6p-2-BA71_03257.

[0031] Figure 2 A graph showing the results of protein expression induced by recombinant engineering bacteria at 16°C.

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

[0033] Figure 4 This is the result of ion exchange column purification of BA71_03257 protein obtained by cleavage of BA71_03257-GST fusion protein after induction of expression in recombinant engineering 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 in conjunction with the embodiments below. 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 BA71_03257 protein used in this application is a hypothetical protein of Acinetobacter baumannii. Through gene annotation, it may be a signal peptide. Analysis of the bacteria of its protein shows that the protein may be a secreted protein. Secreted proteins usually play an important role in the interaction between bacteria and host cells. Many bacterial toxins exist in the form of secreted proteins. Bacteria act on host cells through secreted proteins, destroying host cells to promote the survival of bacteria. This application uses BA71_03257 protein as a target to develop a vaccine, which is expected to effectively prevent and control infections of Acinetobacter baumannii and provide new means for clinical treatment. The specific experimental process is as follows:

[0041] 1. Strains.

[0042] Acinetobacter baumannii strain ATCC17978 was purchased from the American ATCC strain library.

[0043] 2. Reagents.

[0044] Plasmid pGEX-6p-2 (purchased from GE), Escherichia coli strain BL21 (DE3) (purchased from TOLOBIO); 2× High Fidelity PCR Master Mix, DNA Marker, and DNA Ligation Mix were purchased from Sangon Biotechnology Co., Ltd.; restriction endonucleases BamH I and Not I were purchased from NEB;

[0045] Protein marker was a product of Yamei Company, bacterial genome extraction kit, plasmid extraction kit, gel recovery kit, and ultrathin recovery kit were purchased from Tiangen Company; TMB colorimetric solution was a product of Tiangen Company;

[0046] GST Bestarose 4FF and protein A / G affinity chromatography columns were products of Bergeron; ion exchange chromatography fillers were products of Cytiva; and Al(OH)3 was a product of Thermo.

[0047] Example 1: Preparation of recombinant engineered bacteria of Acinetobacter baumannii BA71_03257 protein.

[0048] 1. According to the amino acid sequence of the protein of Acinetobacter baumannii strain BA71_03257 (GenBank: EZF13770.1), the bioinformatics software was used for structural analysis to determine that the protein to be amplified was the amino acid sequence of positions 21-143 of the BA71_03257 protein, as shown in SEQ ID NO.1.

[0049] 2. Based on the amino acid sequence shown in SEQ ID NO.1, search for the DNA sequence expressing the recombinant protein, as shown in SEQ ID NO.2.

[0050] 3. The PCR method was used to amplify the gene fragment of BA71_03257 protein using the whole genome of Acinetobacter baumannii ATCC17978 as a template. The amplification steps were as follows:

[0051] 1) Design and synthesize PCR primers. The sequences of PCR primers are as follows: SEQ ID NO.3 and SEQ ID NO.4:

[0052] Forward primer (SEQ ID NO.3) 5`-CGCGGATCCACTACCGCACCTCTTTCTTCTTATC-3` Reverse primer (SEQ ID NO.4) 5`-TTATGCGGCCGCTTAATCACACTTTGCTAAGTTGTCTTTA-3`

[0053] 2) The Acinetobacter baumannii ATCC17978 strain was taken out of the -80 ℃ freezer and revived by the three-line method on TSA solid medium, cultured at 37 ℃ overnight, and then a single colony was picked and inoculated into TSB liquid medium and cultured for 5 hours. The whole genome was extracted according to the bacterial genome extraction kit.

[0054] 3) The BA71_03257 protein gene fragment was amplified by PCR using the whole genome DNA of Acinetobacter baumannii ATCC17978 as a template.

[0055] PCR system:

[0056] Components volume Template (200 ng / μl) 2μl Forward primer (1 μM) 1μl Reverse primer (1 μM) 1μl 2×High Fidelity PCR Master Mix 20μl Sterile double distilled water 16μl Total volume (make up with sterile deionized water) 40μl

[0057] PCR amplification reaction conditions: 95 °C pre-denaturation for 30 s, 95 °C denaturation for 15 s, 58 °C annealing for 15 s, 72 °C extension for 1 min, 35 cycles, and 72 °C full extension for 5 min to obtain PCR products.

[0058] 4) Use a gel recovery kit to recover the BA71_03257 target gene PCR product.

[0059] 3. Identification and cloning of PCR products, the steps are as follows:

[0060] 1) The pGEX-6P-2 plasmid and BA71_03257 PCR product were digested with BamH I and Not I.

[0061] Enzyme digestion reaction system:

[0062] Components volume BamH 2μl Not I 2μl 10×K CutSmart Buffer 4μl Plasmid or PCR product 1 μg Total volume (make up with sterile deionized water) 40μl

[0063] Digest at 37℃ for 1 h.

[0064] 2) Use a gel recovery kit to recover the pGEX-6P-2 plasmid and the PCR product digested with BamH I and Not I.

[0065] 3) Connect and transform.

[0066] The nucleic acid concentration of the target gene digestion product was determined by ultraviolet spectrophotometer, and the following ligation reaction system was designed based on the general molar ratio of plasmid to exogenous fragment of 1:2-10.

[0067] Ligation reaction system:

[0068] Components volume T4 DNA Ligase 0.5μl Target gene digestion recovery product 4.3μl PGEX-6P-2 digestion recovery product 0.7μl 10×ligation buffer 1μl Sterile deionized water 3.5μl Total volume 10μl

[0069] Mix well and connect at 16℃ for 1 h.

[0070] 4) Take 3 tubes of E. coli BL21 (DE3) competent cells from the -80 ℃ refrigerator, add pGEX-6P-2 plasmid to the first tube as a positive control, add DNA ligation product to the second tube, and do not add exogenous DNA to the third tube as a negative control. Ice bath for 30 min, heat shock in a 42 ℃ metal bath for 90 s, and quickly ice bath for 2 min. Add 600 μl LB blank medium, mix well, and place in a 37 ℃ shaker at 200 rpm for 1 h.

[0071] Each tube was centrifuged at 5000 rpm for 5 min at room temperature, 400 μl of supernatant was discarded, the bacteria were resuspended, and 100 μl was spread on Amp-resistant LB plates. The plates were inverted and cultured in a 37 ℃ incubator for 24 h.

[0072] 5) Screening and identification of pGEX-6P-2-BA71_03257 positive recombinant plasmid.

[0073] ① No colonies appeared on the negative control plate; the positive control plate was full of colonies, indicating that the competent cells were made correctly and the results were reliable. Pick a single colony that grew well on the ligation product transformation plate, inoculate it in Amp-resistant LB medium, and culture it at 37°C overnight with shaking;

[0074] ②Plasmid extraction: refer to the instructions of the plasmid extraction kit;

[0075] ③ Double digestion of plasmid DNA with BamH I and Not I;

[0076] Double enzyme digestion reaction system:

[0077] Components volume BamH 1μl Not I 1μl 10×CutSmart Buffer 2μl Recombinant pGEX-6P-2-BA71_03257 plasmid 1 μg Total volume (make up with sterile deionized water) 20μl

[0078] Enzyme digestion at 37 °C for 1 h;

[0079] ④1% agarose gel electrophoresis to detect the double enzyme digestion results. Figure 1 As shown, lane M: nucleic acid (DNA) molecular weight standard (Marker); lane 1: identification results of the recombinant expression plasmid pGEX-6p-2-BA71_03257 after enzyme digestion. The target protein DNA fragment produced after enzyme digestion is approximately 250-500 bp, which is consistent with its theoretical size of 369 bp.

[0080] Example 2: Induced expression of recombinant BA71_03257 protein in Escherichia coli and identification of the expression form.

[0081] 1. Induce expression of target protein.

[0082] 1) Take 50 μL of pGEX-6P-2-BA71_03257 / BL21 (DE3) recombinant engineering bacteria identified by double enzyme digestion and add it to 10 mL of Amp-resistant LB medium, and culture it at 100 rpm and 37 ℃ overnight. Take 2 mL of the overnight cultured bacteria and add it to 18 mL of Amp-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.

[0083] 2) Take out the bacterial solution after induced expression, centrifuge at 4000 rpm for 2 min, discard the supernatant, add 1 mL PBS buffer and mix well, ultrasonically lyse for 5 min, and then centrifuge at 12000 rpm at 4 ℃ for 15 min, and collect the supernatant and precipitate samples of the bacterial lysate respectively.

[0084] 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.

[0085] 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.

[0086] The results are as follows Figure 2As shown, lane "M" is a protein molecular weight standard (Marker); lane "-": whole bacterial protein band of recombinant engineering bacteria before induction expression; lane "+": expression of recombinant fusion protein in whole bacteria after induction expression of recombinant engineering bacteria at 16°C; lane "s": recombinant fusion protein with GST tag obtained in the supernatant of bacterial ultrasonic lysis after induction expression of recombinant engineering bacteria at 16°C (indicated by arrow); lane "p": protein in the sample precipitated after induction expression of recombinant engineering bacteria at 16°C. It can be seen that the recombinant fusion protein band is between 37-50 KD, which is consistent with the size of the target fusion protein of 39 KD, and the recombinant fusion protein is mainly expressed in the supernatant of bacterial ultrasonic lysis in a soluble form. The gray value of the scanned target band accounts for the gray value of the entire lane, and the expression rate of the recombinant protein is about 16.71%.

[0087] The pGEX-6P-2-BA71_03257 / BL21 (DE3) recombinant engineering bacteria obtained by the present invention has the advantages of easy cultivation, high protein expression, short purification cycle, safety and controllability, and convenient operation, and is also conducive to the process expansion of large-scale vaccine production in the later stage. At the same time, the protein expressed by the pGEX-6P-2-BA71_03257 / BL21 (DE3) recombinant engineering bacteria can maintain its original spatial conformation to the maximum extent while also having good solubility.

[0088] Example 3: Preparation of BA71_03257 protein antigen.

[0089] 1. Amplify the culture to obtain the recombinant fusion protein BA71_03257 with GST tag.

[0090] Take the pGEX-6P-2-BA71_03257 / BL21 (DE3) strain stored in a -80℃ refrigerator and inoculate it on an LB ampicillin resistance plate and culture it at 37℃ overnight; pick a single colony and inoculate it in 100 mL LB ampicillin resistance medium and culture it at 37℃, 220 rpm overnight; add 100 mL of activated bacterial solution to 2 L of LB medium containing Amp resistance for secondary activation, culture at 37℃ for 3-4 hours until OD600 is 0.8, add 0.4 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 100 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 4℃ 12000 rpm for 30 min, collect the supernatant and 5 mL glutathione-agarose gel 4B binding; a large amount of BA71_03257 fusion protein containing GST tag was obtained.

[0091] 2. Use the enzyme digestion method to separate the target protein and the GST tag to obtain the BA71_03257 recombinant protein.

[0092] Add 5 mL of PBS and 150 μL PreScission protease (PP enzyme) to about 5 mL of glutathione-agarose gel 4B bound to the target protein to remove the GST tag, vertically rotate the enzyme digestion at 4 °C overnight, centrifuge and absorb the supernatant, wash twice with 2 mL PBS, take 10 μL of the sample for denaturation treatment, load 10 μL for SDS-PAGE protein electrophoresis, observe the results under the phase system, the molecular weight of the BA71_03257 protein obtained after enzyme digestion is about 14 kD, which is consistent with the expected protein molecular weight. The electrophoresis results are shown in Figure 3 Lane "M" is a protein molecular weight standard (Marker); Lane 1: Fusion protein obtained in the supernatant after induction expression of the recombinant engineered bacteria at 16°C; Lane 2: Beads spotting results after the fusion protein obtained in the supernatant binds to the beads, showing the GST fusion protein bound to the beads; Lane 3: Target protein BA71_03257 after PP enzyme digestion of the fusion protein bound to the beads; Lane 4: The protein remaining on the beads after digestion is the incompletely removed fusion protein and PP enzyme.

[0093] 3. Replace the buffer and store the target protein BA71_03257 in PBS (pH=8.0) buffer.

[0094] 4. Use an ion exchange column to further purify the BA71_03257 recombinant protein to obtain high-purity BA71_03257 recombinant protein. Specifically:

[0095] Connect and install the ion exchange column, 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 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 BA71_03257 protein purified by ion exchange column. After scanning, the gray value of the target band accounts for the gray value of the entire lane, and the purity of the target protein is calculated to be 92%.

[0096] 5. Determine the protein concentration according to the instructions of the BCA protein assay kit, and the final measured concentration is 1.6 mg / mL.

[0097] Example 4: Preparation of BA71_03257 recombinant protein subunit vaccine.

[0098] The recombinant BA71_03257 protein was adsorbed with Al(OH)3 to prepare a subunit vaccine. The method was as follows: the aluminum hydroxide adjuvant was measured and adjusted to a concentration of 5 mg / ml with a histidine diluent at pH 6.0, and the solution A was obtained; the BA71_03257 recombinant protein was diluted to 200 μg / mL with a histidine diluent at pH 6.0 to obtain a solution B; equal volumes of solution A and solution B were taken and rotated and suspended at 4°C for 2 hours to obtain the vaccine. Solution B of the adjuvant control preparation group was a histidine diluent without protein antigen, and was adsorbed with solution A under the same conditions.

[0099] Example 5: Immunization of animals with BA71_03257 recombinant protein vaccine preparation.

[0100] 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;

[0101] 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;

[0102] 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;

[0103] 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.

[0104] Example 6: Preparation of a detection kit for specific antibodies against Acinetobacter baumannii BA71_03257.

[0105] 1) Prepare the liquid.

[0106] 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;

[0107] ② Preparation of blocking solution: 1 g bovine serum albumin, dissolved in 100 mL antibody diluent;

[0108] ③ Preparation of antibody diluent: dissolve phosphate in 1 L ddH2O, add 500 μl Tween 20, and adjust the pH to 7.4 using a pH meter;

[0109] 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;

[0110] ⑤ Color developing solution (TMB), a product of Tiangen Company;

[0111] ⑥ Preparation of stop solution (2 M H2SO4): Pour 22.2 mL of concentrated sulfuric acid into 177.8 mL of ddH2O.

[0112] 2) Preparation of BA71_03257 protein antibody detection kit.

[0113] ① Dilute the purified BA71_03257 recombinant protein to 4 μg / mL using coating solution;

[0114] 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;

[0115] 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;

[0116] 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.

[0117] Example 7: Detection of specific antibodies after mice were immunized with BA71_03257 recombinant protein.

[0118] 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.

[0119] ① After diluting the serum 1000 times, perform a 5-fold dilution;

[0120] 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;

[0121] ③ Dilute the HRP-labeled goat anti-mouse IgG antibody storage solution at 1:10000 to prepare the antibody working solution; add 100 μl / well of the diluted antibody working solution, place in a 37°C incubator for 45 min, wash 4 times, and control to dryness;

[0122] ④ Add 100 μl / well of substrate colorimetric solution (TMB) and react at room temperature in the dark for 5 min;

[0123] ⑤ Add stop solution (2 M H2SO4) and immediately place on a microplate reader to measure the OD value at a wavelength of 450 nm;

[0124] ⑥Result judgment: A 样品 ∕A 阴性值 ≧2.1 is positive (negative control is to make several groups of immune sera).

[0125] Table 1 Recombinant BA71_03257 protein immune serum antibody detection

[0126] Serum dilution Adjuvant control BA71_03257 serum 1000 0.049 1.791 5000 0.037 0.934 25000 0.035 0.307 125000 0.036 0.101

[0127] The results are shown in Table 1. The OD450 of the antibodies produced by mice immunized with BA71_03257 protein antigen was still as high as 0.101 after dilution 125,000 times, and the antibody titer was higher than 1: 125,000. This shows that the BA71_03257 recombinant protein constructed by the present invention has strong immunogenicity and can stimulate mice to produce high-titer antigen-specific antibodies.

[0128] Example 8: Evaluation of the immune protection effect of BA71_03257 recombinant protein using bacteremia model.

[0129] Culture Acinetobacter baumannii until the logarithmic growth phase, collect the bacteria and adjust the concentration to 1.5 × 10 8 CFU / mL. After the third immunization of mice, 200 μL of Acinetobacter baumannii was injected into the tail vein on the 14th day for challenge. The amount of bacterial liquid injected into each C57BL / 6 mouse was 3×10 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.

[0130] Table 2 The protection results of mice challenged with Acinetobacter baumannii after immunization with recombinant BA71_03257 protein

[0131] Group Immune components Mouse (pcs) Number of dead mice mortality rate(%) Protection rate (%) BA71_03257 immunization group <![CDATA[BA71_03257 + Al(OH)3 Adjuvant]]> 10 6 60 40 Adjuvant control group <![CDATA[Histidine solution + Al(OH)3 adjuvant]]> 10 10 100 0

[0132] 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_03257 vaccine immunization group was 60%. According to the vaccine 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_03257 protein vaccine immunization group was 40%.

[0133] Example 9: Preparation of BA71_03257-specific antibodies.

[0134] Rabbit immunization: BA71_03257 recombinant protein was mixed with aluminum hydroxide adjuvant, and the preparation method was the same as in Example 4. Rabbits were immunized with 300 μg / rabbit, and multiple intramuscular injections were used for immunization. The immunization procedure was the same as in Example 5. 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 in Example 7, the titer of rabbit antibodies was detected using an ELISA kit, and the antibody titer was as high as 1:243000.

[0135] The Acinetobacter baumannii BA71_03257 recombinant protein antigen of the present invention provides an effective antigen reserve for the development of Acinetobacter baumannii vaccines, filling the current gap in this field. It has the following advantages:

[0136] (1) Efficient preparation: The method of the present invention can efficiently prepare high-purity and high-yield Acinetobacter baumannii BA71_03257 protein to meet the needs of large-scale vaccine preparation.

[0137] (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.

[0138] 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.

[0139] 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_03257 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_03257 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_03257 is shown in SEQ ID NO.

2.

4. A method for preparing Acinetobacter baumannii BA71_03257-specific antibodies by immunizing animals with recombinant protein antigens of Acinetobacter baumannii BA71_03257, characterized in that: The steps include: The recombinant protein antigen of Acinetobacter baumannii BA71_03257 was mixed with aluminum hydroxide adjuvant to immunize rabbits, 300 μg / rabbit, and immunized once on the 0th day, the 14th day and the 21st day respectively. Two weeks after the last immunization, the blood of the rabbits 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, and specific antibodies against Acinetobacter baumannii BA71_03257 were obtained, wherein the amino acid sequence of the recombinant protein antigen of Acinetobacter baumannii BA71_03257 is shown in SEQ ID NO.1.

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