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

Abstract

The present application provides a recombinant protein antigen of Acinetobacter baumannii fimbriae, 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 fimbriae of the present invention is shown in SEQ ID NO.1. The preparation process of the recombinant protein antigen of Acinetobacter baumannii fimbriae 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 fimbriae has a high purity and a high yield, which is suitable for large-scale production. The vaccine prepared using the recombinant protein antigen of Acinetobacter baumannii fimbriae 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 pili, a preparation method and an application thereof. Background Art

[0002] Acinetobacter baumannii is a common pathogen that has been widely recognized as one of the main pathogens of hospital-acquired infections. It mainly infects elderly and long-term hospitalized patients, especially those in intensive care units and other critically ill patients. Its infection can cause pneumonia, severe soft tissue or wound infection, catheter-related infection, urinary tract infection, bacteremia and secondary meningitis, among which pneumonia and bloodstream infection are the most common and serious. The increased drug resistance of Acinetobacter baumannii and its spread in medical environments make its infection treatment extremely difficult, and even lead to serious infection outbreaks in hospitals.

[0003] Currently, the treatment of Acinetobacter baumannii mainly relies on antibiotics. However, due to its high resistance, many conventional antibiotics have lost their therapeutic effect on the bacteria, which has intensified the demand for alternative treatment methods. Therefore, there is an urgent need to develop new methods for the prevention and treatment of Acinetobacter baumannii infection. Summary of the invention

[0004] The purpose of the present application is to provide a recombinant protein antigen of Acinetobacter baumannii fimbriae, 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 fimbriae, the amino acid sequence of the recombinant protein antigen of Acinetobacter baumannii fimbriae is 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 fimbriae 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 fimbriae 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) Obtain recombinant fimbriae 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 a HIS-tagged protein agarose high-speed purification resin to obtain a recombinant fimbriae protein containing a HIS tag;

[0015] d) Purification of recombinant fimbriae proteins:

[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; the collected fimbriae target protein is further purified using an ion exchange column to obtain a high-purity fimbriae recombinant protein antigen.

[0017] 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).

[0018] In a fourth aspect, the present application provides the use of the recombinant protein antigen of Acinetobacter baumannii fimbriae described in the first aspect in the preparation of a drug for treating or preventing Acinetobacter baumannii infection.

[0019] According to a preferred embodiment, the drug is an Acinetobacter baumannii vaccine.

[0020] In a fifth aspect, the present application provides an Acinetobacter baumannii fimbriae-specific antibody prepared by immunizing an animal with the Acinetobacter baumannii fimbriae recombinant protein antigen described in the first aspect.

[0021] In a sixth aspect, the present application provides the use of the Acinetobacter baumannii fimbriae-specific antibody described in the fifth aspect in the preparation of a drug for treating or preventing Acinetobacter baumannii infection.

[0022] In a seventh aspect, the present application provides a detection kit for Acinetobacter baumannii, comprising the Acinetobacter baumannii pilus recombinant protein antigen described in the first aspect, and / or,

[0023] The Acinetobacter baumannii fimbriae-specific antibody described in the fifth aspect.

[0024] Based on the above technical solution, the recombinant protein antigen of Acinetobacter baumannii fimbriae, preparation method and application thereof provided in the present application have at least the following beneficial technical effects:

[0025] 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 pilus recombinant protein antigen provided by the present application is simple, the operation steps are easy to control and implement, and the obtained Acinetobacter baumannii pilus recombinant protein has high purity and high yield, and is suitable for large-scale production.

[0026] On the other hand, the vaccine prepared using the recombinant protein antigen of Acinetobacter baumannii fimbriae 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

[0027] 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.

[0028] Figure 1 This is a diagram showing the results of plasmid restriction enzyme digestion identification of the recombinant engineering bacteria pET-28a-Pilin / BL21.

[0029] Figure 2 Results of inducing expression of recombinant protein by recombinant engineering bacteria pET-28a-Pilin / BL21.

[0030] Figure 3 This is a diagram showing the purification results of the recombinant protein obtained in the supernatant after the recombinant engineering bacteria were induced to express at 16°C.

[0031] Figure 4 This is the result of purifying the Pilin protein using an ion exchange column after inducing expression in the recombinant engineered bacteria at 16°C. DETAILED DESCRIPTION

[0032] 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.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] The terms "first" and "second" are used only for descriptive purposes to distinguish objects such as substances from each other, and should not be understood as indicating or implying relative importance or implicitly indicating the number of indicated technical features. For example, without departing from the scope of the embodiments of the present application, the first XX may also be referred to as the second XX, and similarly, the second XX may also be referred to as the first XX. Thus, features defined as "first" and "second" may explicitly or implicitly include one or more of the features.

[0039] The pili protein (Pilin) ​​used in this application is a protein located on the surface of Acinetobacter baumannii bacteria and is directly exposed to the host immune system. This makes it a potential immunogen that can induce the host to produce an immune response against bacteria. Pili protein (Pilin) ​​plays an important role in bacterial adhesion and biofilm formation. Biofilms can enhance bacterial drug resistance and infection persistence. Blocking Pilin can interfere with these key processes and reduce bacterial infectivity. Due to the surface exposure characteristics of pili protein (Pilin), it can be effectively recognized by the host immune system and produce a strong immune response to form specific antibodies. The immune response against Pilin can interfere with bacterial adhesion, destroy biofilm formation, reduce the virulence of pathogens, thereby reducing the infectivity and persistence of bacteria and improving the therapeutic effect. The recombinant protein of Acinetobacter baumannii pili in this application has good immunogenicity. After immunizing mice, it can effectively protect mice against infection with Acinetobacter baumannii, with a high protection rate and stable protection effect. The specific experimental process is as follows:

[0040] 1. Strains.

[0041] The Acinetobacter baumannii strain was purchased from ATCC with the strain number ATCC17978.

[0042] 2. Reagents.

[0043] HIS-tagged protein agarose high-speed purification resin (Ni-TED 6FF agarose gel) was a product of Sangon Biotechnology Co., Ltd.; PET-28a vector was purchased from Wuhan Jinkairui Co., Ltd.; nucleic acid (DNA) molecular weight standard (Marker) was purchased from Sangon Biotechnology Co., Ltd.; kanamycin and ampicillin were purchased from Sangon Biotechnology Co., Ltd.; ion exchange chromatography filler was purchased from Cytiva; and BCA protein determination kit was purchased from Yisheng Biotechnology Co., Ltd.

[0044] Example 1: Preparation of recombinant engineered bacteria of Acinetobacter baumannii Pilin protein.

[0045] 1. According to the amino acid sequence of the pilin protein of the Acinetobacter baumannii strain, the signal peptide, transmembrane structure, etc. of the sequence were analyzed using bioinformatics software to determine the pilin protein fragment that needs to be amplified. The pilin protein sequence that needs to be amplified is shown in SEQ ID NO.1.

[0046] 2. According to the Acinetobacter baumannii pilin protein sequence shown in SEQ ID NO.1 and the codons optimized in the Escherichia coli expression system, a DNA sequence for expressing the recombinant protein was obtained, as shown in SEQ ID NO.2.

[0047] 3. Synthesize DNA and insert the synthesized DNA into pET-28a plasmid to obtain recombinant plasmid pET-28a-pilin.

[0048] 4. Transformation: The synthesized recombinant plasmid was centrifuged at 12000 rpm for 1 min, 40 μL of deionized water was added to mix, 1 μL of plasmid solution was added to 20 μL of E. coli BL21 (DE3) competent cells, flicked to mix, and placed on ice for 30 min. Heat shock was then performed at 42°C for 90 s, and the cells were immediately placed on ice for 2 min. 0.5 mL of LB medium without antibiotics was added, and the cells were revived at 220 rpm / 37°C for 50 min. 50 μL of the solution was spread on a kanamycin-containing LB plate and cultured at 37°C overnight.

[0049] 5. Identification of recombinant engineered bacteria: pick a single clone, expand the culture, extract the plasmid and then identify it by enzyme digestion.

[0050] The results are as follows Figure 1 As shown, lane M: nucleic acid (DNA) molecular weight standard (Marker); lane 1: identification result of recombinant expression plasmid pET-28a-Pilin after enzyme digestion. After enzyme digestion, the target fragment is between 250bp-500bp, which is consistent with the theoretical size of 348bp.

[0051] Example 2: Recombinant expression of Acinetobacter baumannii pilin recombinant protein.

[0052] 1. Take 50μL of pET-28a-pilin / BL21 (DE3) bacterial solution that has been correctly identified by double enzyme digestion and add it to 10mL of kanamycin-resistant LB medium, and culture it at 100rpm and 37℃ overnight. Take 2mL of the overnight cultured bacterial solution and add it to 18mL of kanamycin-resistant LB medium (the remaining bacterial solution is stored in a 4℃ refrigerator for standby use), culture it at 37℃ for 2-3h, rotate at 220rpm, activate it for the second time until OD600 is 0.8-1.2, add IPTG to make the final concentration 200μM, and then place it on a shaker to induce expression at 16℃ overnight.

[0053] 2. Take out the induced bacterial solution, take 40 μl of the induced bacterial solution (+) to make SDS-PAGE samples, centrifuge at 13300 rpm for 1 min, discard the supernatant, add 1 ml of resuspension buffer (20 mM Tirs, 500 mM NaCl, pH8.0) and mix well, ultrasonically lyse for 5 min, centrifuge at 4°C and 13300 rpm for 1 min, collect the supernatant (s) and precipitate (p).

[0054] 3. Take 40 μl of the bacteria before and after induction and the supernatant (s) from the cell lysis in step 2 to prepare SDS-PAGE samples. Resuspend the precipitate after cell lysis and centrifugation with an equal volume of resuspension buffer, centrifuge at 4°C and 13,300 rpm for 1 min, discard the supernatant for washing, add an equal volume of resuspension buffer again to resuspend, and take 40 μl to prepare SDS-PAGE samples.

[0055] 4. Take the SDS-PAGE samples prepared in the above steps respectively and perform polyacrylamide gel electrophoresis experiment to observe the expression of the target protein.

[0056] Results Figure 2 , Figure 2 The results of induction expression of recombinant protein by recombinant engineering bacteria pET-28a-Pilin / BL21 are shown. Lane "M": protein molecular weight standard (Marker); Lane "-": protein band of recombinant engineering bacteria before induction expression; Lane "+": recombinant protein obtained in the whole bacteria after induction expression of recombinant engineering bacteria at 16°C. The proportion of target protein in all bacterial proteins was scanned, and the expression rate was calculated to be about 24.63%; Lane "S": recombinant protein obtained in the supernatant after induction expression of recombinant engineering bacteria at 16°C; Lane "P": recombinant protein obtained in the precipitate after induction expression of recombinant engineering bacteria at 16°C. The target protein is efficiently expressed mainly in the supernatant in a soluble form.

[0057] 5. The recombinant bacteria with expression were named pET-28a-pilin / BL21(DE3) and preserved.

[0058] The pET-28a-pilin / BL21 (DE3) recombinant engineering bacteria obtained by the present invention has the advantages of easy cultivation, easy 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 pET-28a-BL21 (DE3) recombinant engineering bacteria can maintain its original spatial conformation to the maximum extent while also having good solubility.

[0059] Example 3: Preparation of Pilin protein antigen.

[0060] 1. Scale up the culture to obtain the recombinant Pilin protein.

[0061] Take the pET-28a-Pilin / 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 100mL LB kanamycin resistance medium and culture it at 37℃, 220rpm overnight; add the activated 100mL bacterial solution to 2L LB medium containing kanamycin resistance for secondary activation, culture it at 37℃ for 3-4h until OD600 is 0.8, add 1mL IPTG (final concentration is 500uM) and place it in a 16℃ shaker for induction for 12h, then collect the bacteria by centrifugation at 4000rpm for 10min, add 100mL PBS to resuspend the bacteria, and use a high-pressure homogenizer to break it for 5-6 cycles. After breaking, use a floor-standing high-speed centrifuge to centrifuge it at 4℃ 12000rpm for 30min, collect the supernatant and combine it with the balanced 6mL Ni-TED 6FF agarose gel at 4℃ overnight to obtain a large amount of Pilin recombinant protein containing HIS tag.

[0062] 2. Extraction of Pilin recombinant protein.

[0063] After the binding is completed, the Ni-TED agarose gel is recovered after the supernatant is run through. The impurities are first washed with 100 mL of PBS, and then washed with 100 mL of 20 mM imidazole. Next, the target protein is eluted with 10 mL of 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 electrophoresis results are shown in Figure 3 Lane "M": protein molecular weight standard (Marker); Lane 1 shows the results of Ni-TED 6FF agarose gel specifically binding to the target protein in the supernatant and non-specific binding to the impurity protein; Lane 2 shows the results of beads washing impurity protein at 20mM imidazole concentration; Lanes 3-8 show the results of target protein elution at 30mM, 50mM, 100mM, 200mM, 300mM, and 500mM imidazole concentrations respectively; Lane 9 shows the results of Ni-TED 6FF agarose gel spotting after elution. The results show that the molecular weight of Pilin protein is about 12kD, which is consistent with the expected protein molecular weight, and Pilin protein is mainly eluted by 200mM imidazole.

[0064] 3. Collect the target protein eluted with 200 mM imidazole, replace the buffer, and store the target protein in PBS (pH = 7.0) buffer.

[0065] 4. Use ion exchange columns to further purify the Pilin recombinant protein to obtain high-purity Pilin protein.

[0066] 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 10mM NaCl and 20mM NaCl in turn to remove the impurities; elute the target protein using a linear method, and collect the effluent for electrophoresis detection, such as Figure 4 As shown, lane M: protein molecular weight standard (Marker); lane 1 represents the Pilin protein purified after using an ion exchange column. The proportion of the target protein band in the total protein was determined by grayscale scanning, and its purity was 96%.

[0067] 5. Determine the protein concentration according to the instructions of the BCA protein assay kit, and the final measured concentration is 3.1 mg / mL.

[0068] Example 4: Preparation of pilin recombinant protein subunit vaccine.

[0069] The recombinant pilin protein was adsorbed with different adjuvants for preliminary experiments and the adsorption effect was tested. 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 pilin 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.

[0070] Example 5: Immunization of mice with pilin recombinant protein.

[0071] 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;

[0072] 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;

[0073] 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;

[0074] 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.

[0075] Example 6: Preparation of a kit for detecting Acinetobacter baumannii pilin-specific antibodies.

[0076] 1) Prepare the liquid.

[0077] 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;

[0078] ② Preparation of blocking solution: 1 g bovine serum albumin, dissolved in 100 mL antibody diluent;

[0079] 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;

[0080] 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;

[0081] ⑤ Chromogenic solution (TMB), a product of Tiangen Company;

[0082] ⑥ Preparation of stop solution (2 M H2SO4): Pour 22.2 mL of concentrated sulfuric acid into 177.8 mL of ddH2O.

[0083] 2) Preparation of pilin protein antibody detection kit.

[0084] ① Dilute the purified pilin recombinant protein to 4 μg / mL using coating solution;

[0085] 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;

[0086] 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;

[0087] 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.

[0088] Example 7: Antibody detection after immunization with pilin recombinant protein.

[0089] 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.

[0090] ① Dilute the serum 2000 times and then dilute it 4 times;

[0091] 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;

[0092] 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;

[0093] ④ Add 100 μl / well of substrate colorimetric solution (TMB) and react at room temperature in the dark for 5 minutes;

[0094] ⑤ Add stop solution (2M H2SO4) and immediately place on a microplate reader to measure the OD value at a wavelength of 450nm;

[0095] ⑥Result judgment: A 样品 ∕A 阴性 A value ≥2.1 was considered positive (the negative control was mouse pre-immune serum).

[0096] Table 1 Antibody detection of pilin recombinant protein immune serum

[0097] Test sample dilution OD value 2000 1.411 8000 1.256 32000 0.867 128000 0.403 512000 0.155 2048000 0.076 8192000 0.056 32768000 0.053

[0098] The results are shown in Table 1. The OD450 value of the antibody produced by mice immunized with pilin protein antigen was 0.155 after dilution 512000 times, and the antibody titer reached 1:512000, indicating that the pilin recombinant protein constructed by the present invention can produce high-titer specific antibodies in immunized mice and has good immunogenicity.

[0099] Example 8: Evaluation of the immune protective effect of pilin recombinant protein using bacteremia model.

[0100] After the third immunization of mice, on the 14th day, a lethal dose of Acinetobacter baumannii was injected into the tail vein for challenge. 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.

[0101] Table 2 Protection results of recombinant protein immunization against bacteremia model in mice

[0102] Group Immune components Mouse (pcs) Number of dead mice mortality rate(%) Protection rate (%) Pilin immunization group <![CDATA[pilin + Al(OH)3 adjuvant]]> 10 0 0 100 Adjuvant control group <![CDATA[Histidine solution + Al(OH)3 adjuvant]]> 10 7 70 0

[0103] Table 2 shows the results of the animal immunization test. The mortality rate of the adjuvant control group was 70%, and the mortality rate of the pilin immunization group was 0%. According to the protection rate calculation formula: [Vaccine protection rate = (control group incidence rate - vaccination group incidence rate) / control group incidence rate * 100%], the vaccine protection rate of the pilin immunization group was 100%.

[0104] Example 9: The challenge protection of animals immunized with pilin recombinant protein was determined by immunizing mice using the pneumonia model.

[0105] The same immunization scheme as in Example 5, after the third immunization of mice, on the 7th day, endotracheal intubation was used to challenge with a lethal dose of Acinetobacter baumannii, and each C57BL / 6 mouse was injected with 5×10 6 CFU, and observed for 7 days. The survival rate of mice in each group was counted and the results are shown in Table 3.

[0106] Table 3 The protective effect of recombinant protein on the pneumonia model in mice

[0107] Group Immune components Mouse (pcs) Number of dead mice mortality rate(%) Protection rate (%) Pilin immunization group <![CDATA[pilin + Al(OH)3 adjuvant]]> 10 3 30 70 Adjuvant control group <![CDATA[Histidine solution + Al(OH)3 adjuvant]]> 10 10 100 0

[0108] Table 3 shows the results of the animal immunization test. The mortality rate of the adjuvant control group was 100%, and the mortality rate of the pilin 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 vaccine protection rate of the pilin immunization group was 70%.

[0109] Example 10: Preparation of pilin-specific antibodies.

[0110] The recombinant protein pilin was mixed with aluminum hydroxide adjuvant and used to immunize rabbits at 300 μg / rabbit. The rabbits were immunized three times at 0, 14 and 21 days respectively. Blood was collected at different time points to detect antibody titers. The rabbit serum was collected and purified using a protein A column to obtain IgG, which is a pilin-specific antibody with a titer of up to 1:243,000.

[0111] The Acinetobacter baumannii pilus recombinant protein antigen of the present 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:

[0112] (1) Efficient preparation: The method of the present invention can efficiently prepare high-purity and high-yield recombinant protein antigens of Acinetobacter baumannii fimbriae, meeting the needs of large-scale vaccine preparation.

[0113] (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.

[0114] 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.

[0115] 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 fimbriae 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 fimbriae 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 pilus is shown in SEQ ID NO.

2.

4. A method for preparing Acinetobacter baumannii fimbriae-specific antibodies by immunizing animals with recombinant protein antigens of Acinetobacter baumannii fimbriae, comprising the following steps: The recombinant protein antigen of Acinetobacter baumannii fimbriae was mixed with aluminum hydroxide adjuvant and used to immunize rabbits. The rabbits were immunized three times at 0, 14 and 21 days respectively. The rabbit serum was collected to obtain the specific antibody of Acinetobacter baumannii fimbriae. The amino acid sequence of the recombinant protein antigen of Acinetobacter baumannii fimbriae is shown in SEQ ID NO.1.

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