Application of acinetobacter baumannii outer membrane protein OmpA in short-term immunity

Abstract

The invention discloses application of an outer membrane protein OmpA of acinetobacter baumannii in short-term immunity, and particularly, the outer membrane protein OmpA of the acinetobacter baumannii can play an emergency prevention and treatment role on acinetobacter baumannii infection. After the acinetobacter baumannii outer membrane protein OmpA is injected, the shortest time of short-term immunization is 3 days. The acinetobacter baumannii can be emergently prevented and treated. 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 application of outer membrane protein OmpA of Acinetobacter baumannii in short-term immunity. 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 short-term immunity, which can play an emergency preventive and therapeutic role on Acinetobacter baumannii infection, thereby bringing more convenience to the masses.

[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 play an emergency preventive and therapeutic role on Acinetobacter baumannii infection.

[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 short-term immunization lasts for at least 3 days.

[0011] 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.

[0012] Furthermore, the onset time of the short-term immunity is from the 7th day (protection rate 100%) to the 14th day (protection rate 50%).

[0013] Furthermore, after the effect of the short-term immunity drops below 50%, a 100% protection rate can be regained by repeating the immunization with the outer membrane protein OmpA.

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

[0015] The short-term immunity of Acinetobacter baumannii outer membrane protein OmpA after injection is as short as 3 days. It can play an emergency prevention and treatment role in Acinetobacter baumannii infection, making the application of Acinetobacter baumannii outer membrane protein OmpA 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 those skilled in the art. 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

[0076] Feeding conditions: Temperature: 25±2℃, Relative humidity: 40-60%, Lighting: 12-hour light-dark cycle, Drinking water: Filtered water, free to drink, Feed: Complete nutrient pellet feed for mice

[0077] 2. Experimental steps

[0078] 2.1 Bacterial culture is shown in Table 1

[0079] Take the glycerol bacteria of Acinetobacter baumannii ATCC17978 strain and streak them on LB solid medium. After culturing overnight at 37°C, pick a single clone and inoculate it into 5mL LB liquid medium and culture it in a shaker at 37°C at 220rpm / min for 12h. Take 50μL of the bacterial solution and transfer it to 5mL LB liquid medium and culture it in a shaker at 37°C at 220rpm / min until the OD600 is 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 the table below, 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, and get the bacteria count in the current bacterial suspension when OD600 = 2.0. This experiment was done 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]

[0091]

[0092] 2.4 Intraperitoneal challenge with Acinetobacter baumannii

[0093] Bacterial culture: Take a single clone of Acinetobacter baumannii ATCC17978 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 h. Take 500 μL of bacterial solution and transfer it into 50 mL LB liquid culture medium, and culture it at 37°C in a shaker at 220 rpm / min until OD 600 About 2.0. Dilute the bacterial solution with sterile saline to LD 90 The concentration at that time.

[0094] Mouse challenge: 200 μl of bacterial suspension was injected into the mouse peritoneal cavity at a concentration of LD 90 concentration of ATCC17978.

[0095] 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.

[0096] 3. Experimental results

[0097] The immune effect of the extracellular protein OmpA of Acinetobacter baumannii Figure 1 Shown

[0098] Table 3

[0099] Group Immunization process Protection rate% Control group 1 Normal saline 0 Experimental Group 1 OmpA (0 / 0 / 3d immune, 7d challenge) 100 Experimental Group 2 OmpA (0 / 0 / 7d immune, 11d challenge) 100 Experimental Group 3 OmpA (0 / 0 / 7d immune, 14d challenge) 60 Experimental Group 4 OmpA (0 / 0 / 7d immune, 17d challenge) 50 Experimental Group 5 OmpA (0 / 0 / 7d immune, 21d challenge) 30 Experimental Group 6 OmpA (0 / 0 / 7d immune, 24d challenge) 20 Experimental Group 7 OmpA (0 / 0 / 7 / 21d immune, 21d attack) 100

[0100] Finally, it should be noted that:

[0101] 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 Acinetobacter baumannii outer membrane protein OmpA in short-term immunity, It is characterized in that The outer membrane protein OmpA of Acinetobacter baumannii can play an emergency preventive and therapeutic role in Acinetobacter baumannii infection.

2. The use of the outer membrane protein OmpA of Acinetobacter baumannii according to claim 1 in short-term immunity, It is 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 outer membrane protein OmpA of Acinetobacter baumannii according to claim 1 in short-term immunity, It is 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 outer membrane protein OmpA of Acinetobacter baumannii according to claim 1 in short-term immunity, It is characterized in that The short-term immunization lasts for at least 3 days.

5. The use of the outer membrane protein OmpA of Acinetobacter baumannii according to claim 1 in short-term immunity, It is 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.

6. Use of the outer membrane protein OmpA of Acinetobacter baumannii according to claim 5 in short-term immunity, It is characterized in that The short-term immunity starts to take effect on the 7th day, with a protection rate of 100%; until the 14th day, the protection rate is 50%.

7. Use of the outer membrane protein OmpA of Acinetobacter baumannii according to claim 5 in short-term immunity, It is characterized in that After the effect of the short-term immunity drops below 50%, a 100% protection rate can be regained by repeating the immunization with the outer membrane protein OmpA.

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