Acinetobacter baumannii A1S_3863 recombinant protein antigen, preparation method and application thereof

Abstract

The present application provides a recombinant protein antigen of Acinetobacter baumannii A1S_3863, 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_3863 of the present invention is shown in SEQ ID NO.1. The preparation process of the recombinant protein antigen of Acinetobacter baumannii A1S_3863 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_3863 has high purity and high yield, and is suitable for large-scale production. The vaccine prepared using the recombinant protein antigen of Acinetobacter baumannii A1S_3863 of the present application has no toxic side effects, can produce high-titer antibodies, has good immune protection effect against Acinetobacter baumannii infection, and is suitable for preparing specific antibodies.

Description

Technical Field

[0001] The present application belongs to the field of biomedicine technology, and in particular, relates to a recombinant protein antigen of Acinetobacter baumannii A1S_3863, 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.

[0004] However, the development of vaccines against Acinetobacter baumannii currently faces a series of challenges and difficulties. One of the main problems is the lack of effective antigen reserves, which restricts the progress of vaccine development. In addition, the diversity of Acinetobacter baumannii also poses a huge challenge to vaccine development. Therefore, the development of an effective and safe vaccine against Acinetobacter baumannii is one of the important issues that need to be urgently addressed in the current medical field. Summary of the invention

[0005] The purpose of the present application is to provide a recombinant protein antigen of Acinetobacter baumannii A1S_3863, 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.

[0006] In order to achieve the above application purpose, the technical solution adopted in this application is as follows:

[0007] In a first aspect, the present application provides a recombinant protein antigen of Acinetobacter baumannii A1S_3863, the amino acid sequence of the recombinant protein antigen of Acinetobacter baumannii A1S_3863 being shown as SEQ ID NO.1.

[0008] In a second aspect, the present application provides a gene encoding the recombinant protein antigen of Acinetobacter baumannii A1S_3863 described in the first aspect, and its nucleotide sequence is shown in SEQ ID NO.2.

[0009] In a third aspect, the present application provides a method for preparing the recombinant protein antigen of Acinetobacter baumannii A1S_3863 as described in the first aspect, comprising the following steps:

[0010] a) Plasmid construction, prokaryotic expression:

[0011] 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,

[0012] b) Collect by centrifugation:

[0013] 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;

[0014] c) Obtain A1S_3863 fusion protein containing GST tag:

[0015] 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 an A1S_3863 fusion protein containing a GST tag;

[0016] d) Extraction of target protein A1S_3863:

[0017] After the supernatant in step c) is completely bound to the GST-tagged protein purification resin, the A1S_3863 target protein and the GST tag are separated by enzyme cleavage and then collected;

[0018] e) Purification of A1S_3863 target protein:

[0019] The A1S_3863 target protein collected in step d) is further purified using an ion exchange column to obtain a high-purity A1S_3863 recombinant protein antigen.

[0020] According to a preferred embodiment, in step a), the vector is a pGEX-6P-2 plasmid, and the prokaryotic expression system is a competent Escherichia coli BL21 (DE3).

[0021] In a fourth aspect, the present application provides the use of the recombinant protein antigen of Acinetobacter baumannii A1S_3863 described in the first aspect in the preparation of a drug for treating or preventing Acinetobacter baumannii infection.

[0022] According to a preferred embodiment, the drug is an Acinetobacter baumannii vaccine.

[0023] In the fifth aspect, the present application provides an Acinetobacter baumannii A1S_3863-specific antibody prepared by immunizing an animal with the Acinetobacter baumannii A1S_3863 recombinant protein antigen described in the first aspect.

[0024] In a sixth aspect, the present application provides the use of the Acinetobacter baumannii A1S_3863-specific antibody described in the fifth aspect in the preparation of a drug for treating or preventing Acinetobacter baumannii infection.

[0025] In a seventh aspect, the present application provides a detection kit for Acinetobacter baumannii, comprising the recombinant protein antigen of Acinetobacter baumannii A1S_3863 described in the first aspect, and / or,

[0026] The fifth aspect is the Acinetobacter baumannii A1S_3863-specific antibody.

[0027] Based on the above technical solution, the recombinant protein antigen of Acinetobacter baumannii A1S_3863, the preparation method and the application thereof provided in the present application have at least the following beneficial technical effects:

[0028] The preparation process of the recombinant protein antigen of Acinetobacter baumannii A1S_3863 provided in the present application is simple, and the operation steps are easy to control and implement. The obtained recombinant protein of Acinetobacter baumannii A1S_3863 has high purity and high yield, and is suitable for large-scale production.

[0029] On the other hand, the vaccine prepared using the recombinant protein antigen of Acinetobacter baumannii A1S_3863 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

[0030] 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.

[0031] Figure 1 This is the result of enzyme digestion identification of the expression vector pGEX-6p-2-A1S_3863.

[0032] Figure 2 A graph showing the results of protein expression induced by recombinant engineering bacteria at 16°C.

[0033] 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 purification results.

[0034] Figure 4 This is the result of ion exchange column purification of A1S_3863 protein obtained by cleaving A1S_3863-GST fusion protein after induction of expression in recombinant engineering bacteria at 16°C. DETAILED DESCRIPTION

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] The A1S_3863 protein used in this application is a protein with unknown function in Acinetobacter baumannii. Through the principle of reverse vaccinology, bioinformatics technology is used to analyze potential proteins with high immunogenicity from the whole genome of Acinetobacter baumannii, including the A1S_3863 protein, which has a good immunogenic system and can induce the host to produce an immune response against bacteria. Analysis of the bacterial localization of the protein shows that it may be secreted outside the cell, and the secreted protein is usually a bacterial pathogenic toxin, etc., which participates in the signal of bacterial cell destruction, etc. Therefore, blocking A1S_3863 can interfere with these key processes and reduce bacterial infectivity. This application is expected to effectively prevent and control the infection of Acinetobacter baumannii by developing a vaccine for the A1S_3863 protein, providing a new means for clinical treatment.

[0042] The specific experimental process is as follows:

[0043] 1. Strains.

[0044] Acinetobacter baumannii strain ATCC17978 was purchased from the American ATCC strain library.

[0045] 2. Reagents.

[0046] 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; protein marker was a product of Yamei, and bacterial genome extraction kit, plasmid extraction kit, gel recovery kit, and ultrathin recovery kit were purchased from Tiangen; TMB colorimetric solution was a product of Tiangen; 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 A1S_3863 protein.

[0048] 1. According to the amino acid sequence of the A1S_3863 protein of Acinetobacter baumannii strain (GenBank: ABS90288.2), bioinformatics software was used for structural analysis to determine that the protein to be amplified was the A1S_3863 protein, whose amino acid sequence is shown in SEQ ID NO.1.

[0049] 2. According to the protein sequence of Acinetobacter baumannii A1S_3863 shown in SEQ ID NO.1, search for the DNA sequence expressing the recombinant protein, as shown in SEQ ID NO.2.

[0050] 3. According to the analysis results, the PCR method was used to amplify the gene fragment of A1S_3863 protein using the whole genome of Acinetobacter baumannii ATCC17978 as a template. The amplification steps are as follows:

[0051] 1) Design and synthesize the PCR primers for amplifying the target fragment encoding gene as follows, which are shown in SEQ ID NO.3 and SEQ ID NO.4 respectively:

[0052] Forward primer (SEQ ID NO.3) 5`-CGCGGATCCGGTGATGGCTACTATAATCAAAACTC-3` Reverse primer (SEQ ID NO.4) 5`-TTATGCGGCCGCTTAAAATGTAAATTTTTGCGCG-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 8 hours. The whole genome was extracted according to the bacterial genome extraction kit.

[0054] 3) The A1S_3863 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 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, 30 cycles, 72 °C full extension for 5 min to obtain PCR products.

[0058] 4) Recover the A1S_3863 PCR product using a gel recovery kit.

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

[0060] 1) The pGEX-6P-2 plasmid and A1S_3863 PCR product were digested with BamH I and Not I.

[0061] Among them, the 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 1h.

[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 vector to exogenous fragment of 1:2-10.

[0067] Among them, the connection 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 1h.

[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 minutes, heat shock in a 42℃ metal bath for 90 seconds, and quickly ice bath for 2 minutes. Add 600 μl LB blank medium, mix well, and place in a 37℃ shaker at 200rpm for 1 hour.

[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 an Amp-resistant LB plate. The plate was inverted and incubated in a 37°C incubator for 24 h.

[0072] 5) Screening and identification of pGEX-6P-2-A1S_3863 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] Among them, double enzyme digestion reaction system:

[0077] Components volume BamH 1μl Not I 1μl 10×CutSmart Buffer 2μl Recombinant 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 result of recombinant expression plasmid pGEX-6p-2-A1S_3863 after enzyme digestion. The target fragment produced after enzyme digestion is consistent with the theoretical value (1194 bp).

[0080] Example 2: Induced expression of A1S_3863 recombinant 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-A1S_3863 / BL21 (DE3) bacterial solution that was correctly identified by double enzyme digestion and add it to 10 mL of Amp-resistant LB medium, and culture it at 100 rpm and 37°C overnight. Take 2 mL of the overnight cultured bacterial solution and add it to 18 mL of Amp-resistant LB medium (the remaining bacterial solution was stored in a 4°C refrigerator for standby use), and culture it at 37°C for 2-3 h at 220 rpm. After secondary activation until OD600 is 0.6-1.0, add IPTG to a final concentration of 200 μM, and then place it on a shaker to induce expression at 16°C overnight.

[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, ultrasonic lyse for 5 min, centrifuge at 12000 rpm at 4℃ for 15 min, and collect the supernatant.

[0084] 2. 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 30 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.

[0085] 3. Protein expression detection: Take 10 μL of the treated supernatant samples and load them for SDS-PAGE electrophoresis. The voltage is 80 V for 30 min, then adjusted to 200 V. After 45 min of electrophoresis, take out the gel, place it in Coomassie Brilliant Blue staining solution for shaking staining, and then place it in decolorizing solution for shaking decolorization, and observe the results under the gel imaging system.

[0086] The results are as follows Figure 2As shown, lane "M": protein molecular weight standard (Marker); lane "-": protein band in the whole bacteria before induction of expression of recombinant engineering bacteria; lane "+": fusion protein obtained in the whole bacteria after induction of expression of recombinant engineering bacteria at 16°C; lane "s": fusion protein obtained in the supernatant after induction of expression of recombinant engineering bacteria at 16°C, indicated by the arrow; lane "p": protein sample in the precipitate after induction of expression of recombinant engineering bacteria at 16°C, it can be seen that the recombinant fusion protein band is between 50-75 KD, which is consistent with the target fusion protein size of 69 KD, and the recombinant fusion protein is mainly expressed in the supernatant of ultrasonic lysis in a soluble form. The gray value of the target band is calculated as the gray value of the entire lane, and the expression rate is 25.04%, with high expression efficiency.

[0087] The pGEX-6P-2-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-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 A1S_3863 recombinant protein antigen.

[0089] 1. Amplify the culture to obtain GST-A1S_3863 fusion protein.

[0090] Take the pGEX-6P-2-A1S_3863 / 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 on 100 mL LB ampicillin resistance medium and culture it at 37℃, 220 rpm overnight; add the activated 100 mL bacterial solution to 2 L LB medium containing Amp resistance for secondary activation, culture at 37℃ for 3-4 hours until OD600 is 0.8, add 1 mL IPTG (final concentration is 500 μM) and place it in a 16℃ shaker for induction for 12 hours, centrifuge at 4000rpm for 10 min to collect the bacteria, add 100 mL PBS to resuspend the bacteria, and use a high-pressure homogenizer to cycle 5-6 cycles. After breaking, use a floor-standing high-speed centrifuge at 12000 rpm for 30 min, collect the supernatant and 5 mL glutathione-agarose gel 4B binding; a large amount of A1S_3863 fusion protein containing GST tag was obtained.

[0091] The target protein and the GST tag were separated by enzyme digestion to obtain the A1S_3863 recombinant protein.

[0092] Specific:

[0093] 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, vertically rotate and digest overnight at 4°C, centrifuge and aspirate the supernatant, wash twice with 2 mL of PBS, take 10 μL of sample and denature it, load 10 μL for SDS-PAGE protein electrophoresis, observe the results under the phase system, and the molecular weight of the A1S_3863 protein obtained after digestion is about 44kD, which is consistent with the expected protein molecular weight. The electrophoresis results are shown in Figure 3 Lane "M": protein molecular weight standard (Marker); Lane 1: fusion protein obtained in the supernatant after induction expression of recombinant engineering bacteria at 16°C; Lane 2: beads spotting result after the fusion protein obtained in the supernatant binds to the beads; Lane 3: target protein after enzyme digestion of the bound beads; Lane 4: beads after enzyme digestion. The protein in lane 3 is the target A1S_3863 recombinant protein without the GST tag.

[0094] 3. Replace the buffer and store the target A1S_3863 recombinant protein in PBS (pH=6.0) buffer.

[0095] 4. Use an ion exchange column to further purify the A1S_3863 recombinant protein to obtain high-purity A1S_3863 recombinant protein.

[0096] Specific:

[0097] 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=6.0) solution; filter the sample stored in PBS (PH=6.0) 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_3863 protein purified after using an ion exchange column. After scanning with a gel scanner, the grayscale of the target band was analyzed to account for 96% of the protein grayscale of the entire lane, indicating that the protein purity is 96%. The high-purity recombinant protein obtained can be used for the preparation of subsequent immune preparations.

[0098] 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.

[0099] Example 4: Preparation of A1S_3863 recombinant protein subunit vaccine.

[0100] The A1S_3863 recombinant protein was adsorbed with different adjuvants for preliminary experiments and the adsorption effect was detected. It was found that Al(OH)3 had the best adsorption efficiency. Subsequently, Al(OH)3 was mainly used as an adjuvant component for adsorption to prepare subunit vaccines. The method is as follows: the aluminum hydroxide adjuvant was measured and adjusted to a concentration of 5 mg / ml with a pH 6.0 histidine diluent, and mixed thoroughly to obtain solution A; the A1S_3863 recombinant protein was diluted to 200 μg / mL with a pH 6.0 histidine diluent to obtain solution B; equal volumes of solution A and solution B were taken and rotated and suspended at 4°C for 2 hours to obtain the vaccine. Solution B of the adjuvant control preparation group was a histidine diluent without protein antigen, and adsorbed with solution A under the same conditions.

[0101] Example 5: Immunization of animals with A1S_3863 recombinant protein.

[0102] 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;

[0103] 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;

[0104] 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;

[0105] 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.

[0106] Example 6: Preparation of a detection kit for Acinetobacter baumannii A1S_3863-specific antibodies.

[0107] 1) Prepare the liquid.

[0108] 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;

[0109] ② Preparation of blocking solution: 1 g bovine serum albumin, dissolved in 100 mL antibody diluent (1:100);

[0110] 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;

[0111] 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;

[0112] ⑤ Chromogenic solution (TMB), a product of Tiangen Company;

[0113] ⑥ Preparation of stop solution (2 M H2SO4): Pour 22.2 mL of concentrated sulfuric acid into 177.8 mL of ddH2O.

[0114] 2) Preparation of A1S_3863 protein antibody detection kit.

[0115] ① Use coating solution to dilute the purified A1S_3863 recombinant protein to 4 μg / mL;

[0116] 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;

[0117] 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;

[0118] 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.

[0119] Example 7: Detection of specific antibodies after mice were immunized with A1S_3863 recombinant protein.

[0120] 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.

[0121] ① Dilute the serum 200 times and then dilute it 3 times;

[0122] 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;

[0123] ③ Dilute the HRP-labeled goat anti-mouse IgG antibody 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;

[0124] ④ Add 100 μl / well of substrate colorimetric solution (TMB) and react at room temperature in the dark for 5 min;

[0125] 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;

[0126] ⑥Result judgment: A 样品 ∕A 阴性 A value ≧2.1 was positive (the negative control was to make several groups of immune sera).

[0127] Table 1 Recombinant A1S_3863 protein immune serum antibody detection

[0128] Sample dilution A1S_3863 serum Adjuvant group serum 1:437400 0.438 0.044 1:145800 0.859 0.047 1:48600 1.27 0.046 1:16200 1.621 0.047 1:5400 1.909 0.051 1:1800 2.149 0.057 1:600 2.272 0.071 1:200 2.302 0.116

[0129] The results are shown in Table 1. The OD450 of the antibodies produced by mice immunized with A1S_3863 protein antigen was still 0.044 after being diluted 437400 times, and the antibody titer was higher than 1:437400. This shows that the A1S_3863 recombinant protein constructed by the present invention has strong immunogenicity and can stimulate mice to produce high-titer antigen-specific antibodies.

[0130] Example 8: Evaluation of the immune protective effect of A1S_3863 recombinant protein using bacteremia model.

[0131] 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.

[0132] Table 2 The protection results of mice challenged with Acinetobacter baumannii after immunization with recombinant A1S_3863 protein

[0133] Group Immune components Mouse (pcs) Number of dead mice mortality rate(%) Protection rate (%) A1S_3863 immunization group <![CDATA[A1S_3863 + Al(OH)3 Adjuvant]]> 10 4 40 56 Adjuvant control group <![CDATA[Histidine solution + Al(OH)3 adjuvant]]> 10 9 90 0

[0134] Table 2 shows the results of the animal immunization test. The mortality rate of the adjuvant control group was 90%, and the mortality rate of the A1S_3863 vaccine immunization group was 40%. 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 A1S_3863 protein vaccine immunization group was 56%.

[0135] Example 9: Preparation of A1S_3863-specific antibodies.

[0136] 1. Rabbit immunization: A1S_3863 recombinant protein was mixed with aluminum hydroxide adjuvant to immunize rabbits, 300 μg / rabbit, multiple intramuscular injection, and 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 1:145800.

[0137] 2. IgG purification: Dilute rabbit serum with PBS (1:1) 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, the concentration is 1.2 mg / ml.

[0138] Example 10: A1S_3863-specific antibody protects mice from challenge with Acinetobacter baumannii.

[0139] The bacteremia model of Example 8 was used, and rabbit IgG was injected 1 hour after infection with Acinetobacter baumannii.

[0140] 1. Randomly divide the mice into three groups, with 10 mice in each group:

[0141] - Experimental group: administered with A1S_3863 specific rabbit IgG.

[0142] -Control group A: administered with normal saline.

[0143] -Control group B: administered with normal rabbit IgG.

[0144] 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 experimental group mice were given 100 μg of A1S_3863-specific rabbit IgG by tail vein injection, the control group A mice were given an equal volume of normal saline, and the control group B mice were given 100 μg of normal rabbit IgG.

[0145] 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.

[0146] Table 3 Protective effect of A1S_3863 recombinant protein specific IgG on mouse bacteremia model

[0147] Group Immune components Mouse (pcs) Number of dead mice mortality rate(%) Protection rate (%) Experimental Group <![CDATA[A1S_3863 + Aluminum hydroxide adjuvant]]> 10 3 30 70 Control group A Normal saline 10 10 100 0 Control group B Normal rabbit IgG 10 8 80 20

[0148] The results are shown in Table 3. The experimental results show that the administration of purified A1S_3863-specific rabbit IgG significantly improved the resistance of mice to Acinetobacter baumannii infection, proving that the A1S_3863-specific antibody has a strong protective effect and can be used for the treatment of Acinetobacter baumannii infection.

[0149] The Acinetobacter baumannii A1S_3863 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:

[0150] (1) Efficient preparation: The method of the present invention can efficiently prepare high-purity and high-yield Acinetobacter baumannii A1S_3863 protein to meet the needs of large-scale vaccine preparation.

[0151] (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.

[0152] 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.

[0153] 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_3863 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 A1S_3863 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 A1S_3863 is shown in SEQ ID NO.

2.

4. A method for preparing Acinetobacter baumannii A1S_3863-specific antibodies by immunizing animals with recombinant protein antigens of Acinetobacter baumannii A1S_3863, characterized in that: The steps include: The recombinant protein antigen of Acinetobacter baumannii A1S_3863 was mixed with aluminum hydroxide adjuvant and used to immunize rabbits at 300 μg per rabbit by multiple intramuscular injections on the 1st, 14th and 21st days, respectively. 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 and obtain Acinetobacter baumannii A1S_3863-specific antibodies. The amino acid sequence of the recombinant protein antigen of Acinetobacter baumannii A1S_3863 is shown in SEQ ID NO.1.

Images