Application of acinetobacter baumannii outer membrane protein OmpA in aspect of cross protection

Abstract

The invention discloses an application of an outer membrane protein OmpA of acinetobacter baumannii in the aspect of cross protection. Particularly, the outer membrane protein OmpA of the acinetobacter baumannii can play a role in preventing or treating infection of drug-resistant bacteria of the acinetobacter baumannii and other bacteria. The acinetobacter baumannii outer membrane protein OmpA disclosed by the invention has a cross protection effect, can play a role in preventing or treating infection of drug-resistant bacteria of the acinetobacter baumannii and other bacteria, and can play a better treatment role in respiratory tract infection diseases. The application of the acinetobacter baumannii outer membrane protein OmpA is wider.

Description

Technical Field

[0001] The invention relates to the technical field of molecular biology, and in particular to the application of outer membrane protein OmpA of Acinetobacter baumannii in cross protection. Background Art

[0002] Among the Omps of Acinetobacter baumannii, OmpA is the most intensively studied virulence factor. OmpA plays a key role in regulating the adhesion, invasiveness and biofilm formation of Acinetobacter baumannii and the host immune response. Excessive production of OmpA is an independent risk factor for mortality from nosocomial pneumonia and bacteremia caused by Acinetobacter baumannii. In addition, the OmpA expression level measured by qRT-PCR can be used as a rapid diagnostic indicator for antibiotic-resistant Acinetobacter baumannii, and the results are highly consistent with the traditional MIC analysis results.

[0003] With the resolution of the native structure of OmpA, researchers found that the amino acid sequences of Acinetobacter baumannii OmpA (AbOmpA) from multiple clinical isolates were highly conserved (>89%), while they were not homologous to the human proteome. By comparing various OmpA-like proteins, two conserved amino acids, Arg286 and Asp271, in the C-terminal domain of OmpA were identified, which non-covalently bind to diaminopimelic acid (DAP), a component of peptidoglycan (PNG). This interaction suggests that OmpA plays a key role in maintaining bacterial integrity. In general, in GNB species, OmpA stabilizes its structure by self-dimerization, thereby preventing the cleavage of the curved connection between the β-barrel structure and the periplasm.

[0004] Acinetobacter baumannii OmpA has the following advantages: it is essential for Acinetobacter baumannii to adhere to and invade epithelial cells; the penetration of small molecule antibiotics through Acinetobacter baumannii OmpA is of great significance for bacterial resistance; Acinetobacter baumannii OmpA has strong immunogenicity and immune protection as a vaccine antigen; Acinetobacter baumannii OmpA may have anti-infection and anti-cancer activities by regulating the activity of immune cells. Studying the use of Acinetobacter baumannii OmpA has far-reaching significance. Summary of the invention

[0005] The purpose of the present invention is to provide an application of the outer membrane protein OmpA of Acinetobacter baumannii in cross protection, which can prevent or treat infections caused by drug-resistant bacteria of Baumannii and other bacteria.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] The invention provides an application of the Acinetobacter baumannii outer membrane protein OmpA in short-term immunity. The Acinetobacter baumannii outer membrane protein OmpA can prevent or treat infections caused by drug-resistant bacteria of Acinetobacter baumannii and other bacteria.

[0008] Furthermore, the amino acid sequence of the Acinetobacter baumannii outer membrane protein OmpA is shown in SEQ ID NO:1.

[0009] Furthermore, the nucleotide sequence of the Acinetobacter baumannii outer membrane protein OmpA is shown in SEQ ID NO:2.

[0010] Furthermore, the other bacteria include strains that can cause respiratory tract infections.

[0011] Furthermore, the strains capable of causing respiratory tract infection include Gram-positive cocci, Gram-negative bacilli, and Gram-negative cocci.

[0012] Furthermore, the Gram-positive cocci include hemolytic streptococci, viridans streptococci, Streptococcus pneumoniae, and Staphylococcus aureus; the Gram-negative bacilli include Klebsiella pneumoniae, Escherichia coli, and Pseudomonas aeruginosa; the Gram-negative cocci include Moraxella catarrhalis, Haemophilus influenzae, and Legionella pneumophila.

[0013] Furthermore, in the validation test of short-term immunization, the outer membrane protein OmpA of Acinetobacter baumannii was mixed with the adjuvant in a 1:1 ratio.

[0014] Based on the above technical solution, the embodiments of the present invention can at least produce the following technical effects:

[0015] The outer membrane protein OmpA of Acinetobacter baumannii has a cross-protective effect, can prevent or treat infections caused by drug-resistant bacteria and other bacteria, and can play a better therapeutic role in respiratory tract infections, making the application of the outer membrane protein OmpA of Acinetobacter baumannii more extensive. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the technical solutions in the present invention or the prior art, 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 invention. For ordinary technicians in this field, other drawings can be obtained based on the structures shown in these drawings without paying creative work.

[0017] Figure 1 This is a diagram showing the immune effect of the outer membrane protein OmpA of Acinetobacter baumannii according to an embodiment of the present invention. DETAILED DESCRIPTION

[0018] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0019] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0020] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0021] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0022] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0023] SEQ ID NO: 1:

[0024] MANAGVTVTPLLLGYTFQDSQHNNGGKDGNLTNGPELQDDLFVGAALGIELTPWLG

[0025] FAEYNQ

[0026] VKGDVDGASAGAEYKQKQINGNFYVTSDLITKNYDSKIKPYVLLGAGHYKYDFDGV

[0027] NRGTRGTS

[0028] EEGTLGNAGVGAFWRLNDALSLRTEARATYNADEEFWNYTALAGLNVVLGGHLKP

[0029] AAPVVEVA

[0030] PVEPTPVTPQPQELTEDLNMELRVFFDTNKSNIKDQYKPEIAKVAEKLSEYPNATARIE

[0031] GHTDNT

[0032] GPRKLNERLSLARANSVKSALVNEYNVDASRLSTQGFAWDQPIADNKTKEGRAMNR

[0033] RVFATITG

[0034] SRTVVVQPGQEAAAPAAAQ

[0035] SEQ ID NO:2

[0036] ATGGCTAATGCTGGCGTAACAGTTACTCCATTATTGCTTGGTTACACTTTCCAAGAC

[0037] AGCCAAC

[0038] ACAACAATGGCGGTAAAGATGGTAACTTAACTAACGGTCCTGAGTTACAAGACGA

[0039] TTTATTCG

[0040] TTGGCGCAGCTCTTGGTATCGAGTTAACTCCATGGTTAGGTTTCGAAGCTGAATATA

[0041] ACCAAGT

[0042] TAAAGGCGACGTAGACGGCGCTTCTGCTGGTGCTGAATATAAACAAAAACAAATC

[0043] AACGGTA

[0044] ACTTCTATGTTACTTCTGATTTAATTACTAAAAACTACGACAGCAAAATCAAGCCGT

[0045] ACGTATT

[0046] ATTAGGTGCTGGTCACTATAAATACGACTTTGATGGCGTAAACCGTGGTACACGTG

[0047] GTACTTCT

[0048] GAAGAAGGTACTTTAGGTAACGCTGGTGTTGGTGCTTTTCTGGCGCTTAAACGACG

[0049] CTTTATCTC

[0050] TTCGTACTGAAGCTCGTGCTACTTATAATGCTGATGAAGAGTTCTGGAACTATACAG

[0051] CTCTTGC

[0052] TGGCTTAAACGTAGTTCTTGGTGGTCACTTGAAGCCTGCTGCTCCTGTAGTAGAAG

[0053] TTGCTCCA

[0054] GTTGAACCAACTCCAGTTACTCCACAACCACAAGAGTTAACTGAAGACCTTAAC

[0055] TGGAACTT

[0056] CGTGTGTTCTTTGATACTAACAAATCAAACATCAAAGACCAATACAAGCCAGAAAT

[0057] TGCTAAA

[0058] GTTGCTGAAAAATTATCTGAATACCCTAACGCTACTGCACGTATCGAAGGTCACAC

[0059] AGATAAC

[0060] ACTGGTCCACGTAAGTTGAACGAACGTTTATCTTTAGCTCGTGCTAACTCTGTTAA

[0061] ATCAGCTCT

[0062] TGTAAACGAATACAACGTTGATGCTTCTCGTTTGTCTACTCAAGGTTTCGCTTGGGA

[0063] TCAACCG

[0064] ATTGCTGACAACAAAACTAAAAGGTCGTGCTATGAACCGTCGTGTATTCGCGA

[0065] CAATCACT

[0066] GGTAGCCGTACTGTAGTAGTTCAACCTGGTCAAGAAGCGGCAGCTCCTGCAGCAGCTCAATAA

[0067] Example

[0068] Comparison of the effects of virus attack at different times after short-term immunization with OmpA

[0069] 1. Experimental Preparation

[0070] 1.1 Experimental samples

[0071] The purified OmpA protein sample with a purity of 98% was diluted to a concentration of 200 ug / ml and mixed with an aluminum adjuvant (aluminum hydroxide, concentration 2000 ug / ml) in a 1:1 ratio.

[0072] 1.2 Animal experiments

[0073] Strain: BALB / c mice (SPF grade)

[0074] Gender: Female

[0075] Age: 6-8 years old (about 20±1g)

[0076] Rearing conditions: temperature: 25±2℃, relative humidity: 40-60%, light: 12-hour light-dark cycle, drinking water: filtered water, free drinking, feed: complete nutritional granular feed for mice.

[0077] 2. Experimental steps

[0078] 2.1 Bacterial culture is shown in Table 1

[0079] The standard strain of Acinetobacter baumannii ATCC17978, the clinical drug-resistant strain of Acinetobacter baumannii XH733, and the standard strain of Klebsiella pneumoniae ATCC700721 ​​were streaked on LB solid medium, cultured overnight at 37°C, and single clones were picked and inoculated into 5 mL LB liquid medium, and cultured at 37°C in a shaker at 220 rpm / min for 12 h. 50 μL of the bacterial solution was transferred to 5 mL LB liquid medium and cultured at 37°C in a shaker at 220 rpm / min until OD 600 About 2.0.

[0080] Take 1mL of bacterial solution into a sterile test tube, add 9mL of sterile saline to make a bacterial suspension with a dilution gradient of 10-1. Dilute the bacterial suspension according to Table 1, and finally select the bacterial suspensions numbered 5, 6, and 7 as the bacterial density counting gradient. Make three replicate wells.

[0081] Prepare an LB solid culture dish, take 10uL of the above bacterial suspension, and dot it onto the LB solid culture medium. Slowly tilt the culture dish to let it slide down. Incubate at 37℃ for 24h and then count.

[0082] According to the live bacteria count result, count the live bacteria content of the bacterial suspension before. Get the OD value of the current bacterial suspension. 600 = 2.0. This experiment was performed three times and the average value was obtained.

[0083] Table 1

[0084]

[0085] 2.2 Confirmation of LD90 of the challenge bacteria

[0086] The mice were randomly divided into 5 groups, with 10 BABL / c mice in each group. The mice were intraperitoneally injected with 200 μl of bacterial suspension of different concentrations, and the LD 70-90 .

[0087] 2.3 Immunity

[0088] The mice were randomly divided into groups, with 10 mice in each group. The specific groups are shown in Table 2. 100ul of immune sample, i.e., 10ug of OmpA protein sample per mouse, was injected subcutaneously into the corresponding experimental groups, and the same volume of normal saline was injected into the control group.

[0089] Table 2

[0090] Group Immunization process way Control group 1 Acinetobacter baumannii standard strain ATCC17978 SC Control group 2 Clinical drug-resistant strain XH733 of Acinetobacter baumannii SC Control group 3 Klebsiella pneumoniae standard strain ATCC700721 SC Experimental Group 1 Acinetobacter baumannii standard strain ATCC17978 SC Experimental Group 2 Clinical drug-resistant strain XH733 of Acinetobacter baumannii SC Experimental Group 3 Klebsiella pneumoniae standard strain ATCC700721 SC

[0091] 2.4 Intraperitoneal challenge with Acinetobacter baumannii

[0092] Bacterial culture: Take a single clone of the standard strain of Acinetobacter baumannii ATCC17978, the clinical drug-resistant strain of Acinetobacter baumannii XH733, and the standard strain of Klebsiella pneumoniae ATCC700721 ​​and inoculate it into 50 mL LB liquid culture medium, and culture it at 37°C in a shaker at 220 rpm / min for 12 hours. Take 500 μL of the bacterial solution and transfer it to 50 mL LB liquid culture medium, and culture it at 37°C in a shaker at 220 rpm / min until the OD 600 About 2.0. Dilute the bacterial solution with sterile saline to LD 90 The concentration at that time.

[0093] Mouse challenge: On the 7th day after the first immunization, 200 μl of bacterial suspension was injected into the peritoneal cavity of mice at a concentration of LD 90 Concentration of corresponding bacterial suspension.

[0094] The mortality of mice was recorded every 8 hours after bacterial challenge for 7 days. After the observation period, the surviving animals were euthanized by CO2 inhalation.

[0095] 3. Experimental results

[0096] The immune effect of the extracellular protein OmpA of Acinetobacter baumannii Figure 1 Shown

[0097] Table 3

[0098] Group Immunization process Protection rate% Control group 1 Acinetobacter baumannii standard strain ATCC17978 20 Control group 2 Clinical drug-resistant strain XH733 of Acinetobacter baumannii 40 Control group 3 Klebsiella pneumoniae standard strain ATCC700721 20 Experimental Group 1 Acinetobacter baumannii standard strain ATCC17978 80 Experimental Group 2 Clinical drug-resistant strain XH733 of Acinetobacter baumannii 100 Experimental Group 3 Klebsiella pneumoniae standard strain ATCC700721 100

[0099] Finally, it should be noted that:

[0100] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions described in the above embodiments may still be modified, or some or all of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of the outer membrane protein OmpA of Acinetobacter baumannii in cross protection, characterized in that: The outer membrane protein OmpA of Acinetobacter baumannii can prevent or treat infections caused by drug-resistant bacteria and other bacteria.

2. The use of the Acinetobacter baumannii outer membrane protein OmpA in cross protection according to claim 1, characterized in that: The amino acid sequence of the outer membrane protein OmpA of Acinetobacter baumannii is shown in SEQ ID NO:

1.

3. The use of the Acinetobacter baumannii outer membrane protein OmpA in cross protection according to claim 1, characterized in that: The nucleotide sequence of the Acinetobacter baumannii outer membrane protein OmpA is shown in SEQ ID NO:

2.

4. The use of the Acinetobacter baumannii outer membrane protein OmpA in cross protection according to claim 1, characterized in that: The other bacteria include strains that can cause respiratory tract infections.

5. The use of the Acinetobacter baumannii outer membrane protein OmpA in cross protection according to claim 4, characterized in that: The bacterial strains capable of causing respiratory tract infection include Gram-positive cocci, Gram-negative bacilli, and Gram-negative cocci.

6. The use of the Acinetobacter baumannii outer membrane protein OmpA in cross protection according to claim 5, characterized in that: The Gram-positive cocci include hemolytic streptococci, viridans streptococci, Streptococcus pneumoniae, and Staphylococcus aureus; the Gram-negative bacilli include Klebsiella pneumoniae, Escherichia coli, and Pseudomonas aeruginosa; the Gram-negative cocci include Moraxella catarrhalis, Haemophilus influenzae, and Legionella pneumophila.

7. The use of the Acinetobacter baumannii outer membrane protein OmpA in cross protection according to claim 1, characterized in that: In the validation test of short-term immunization, the outer membrane protein OmpA of Acinetobacter baumannii was mixed with adjuvant in a 1:1 ratio.

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