CN113214364B

CN113214364B - Excavation and verification of multiple-drug-resistant acinetobacter baumannii recognition element

Info

Publication number: CN113214364B
Application number: CN202010069798.0A
Authority: CN
Inventors: 黄鹤; 徐京芝; 康广博; 李晓波
Original assignee: Tianjin University
Current assignee: Tianjin University
Priority date: 2020-01-21
Filing date: 2020-01-21
Publication date: 2022-11-22
Anticipated expiration: 2040-01-21
Also published as: CN113214364A

Abstract

The invention relates to the technical field of bioengineering, in particular to acinetobacter baumannii phage fiber protein, a coding gene thereof, a recombinant expression vector, a preparation method and application thereof. The acinetobacter baumannii phage fiber protein provided by the invention has an amino acid sequence shown as SEQ ID No.1 or 2 or a sequence with at least 80% of sequence homology with the amino acid sequence, has stronger specificity in the aspect of identifying and detecting acinetobacter baumannii, and can be used for preparing a reagent for identifying acinetobacter baumannii. The method for preparing acinetobacter baumannii bacteriophage tail fiber protein can realize large-scale expression and preparation of the acinetobacter baumannii bacteriophage tail fiber protein, and has the advantages of simple separation and purification and low cost.

Description

Excavation and verification of multiple-drug-resistant acinetobacter baumannii recognition element

Technical Field

The invention relates to the technical field of bioengineering, in particular to acinetobacter baumannii phage fiber protein, a coding gene thereof, a recombinant expression vector, a preparation method and application thereof.

Background

Acinetobacter baumannii is a non-fermented gram-negative bacterium widely existing in water, soil, sewage and many medical environments, is a common conditional pathogen, and can cause serious infection of hospital skin, urinary system and the like. With the long-term use of a large number of broad-spectrum antibacterial drugs, particularly the abuse of antibiotics, the problem of antibiotic resistance has been exacerbated year by year. In many countries, acinetobacter baumannii has become a multi-drug resistant and pan-drug resistant strain, and is also a major pathogenic bacterium causing nosocomial infections in patients. Pathogen-specific detection is critical to the treatment of acinetobacter baumannii threats. Therefore, how to detect pathogens has become a focus of clinical attention; we must find alternative methods to detect and treat these bacteria. Conventional clinical standard diagnostic methods invariably require time-consuming bacterial cultures and laborious DNA extraction and the like. To solve this problem, many other efforts are required to develop a rapid and simple bacterial detection method. Researchers have begun to explore new approaches in which bacteriophages and their bacteriophage fiber proteins are considered useful diagnostic tools.

A bacteriophage is a virus that infects microorganisms such as bacteria, and like other viruses, a bacteriophage is an obligate parasite that must rely on host cells to achieve its own survival and reproduction. When the phage infects a host bacterium, the phage injects genetic material such as DNA into the host bacterium, and performs self-replication by using DNA replication of the host bacterium and a protein expression system. Upon infection, bacteriophages utilize adhesion structures to bind to their bacterial host. The specificity of a phage is defined by its own set of proteins that are important for binding to the target cell. Phage-bacteria systems have evolved in nature for a considerable time, so that phages recognize their host bacteria in a highly specific manner and have a high level of binding affinity.

Recently, although the specificity of intact phage for detecting bacteria has been reported to be high, its inherent strong solubility in host bacteria has prevented downstream recognition and detection, and fiber protein has been considered to be a more suitable biotin responsible for the specific initial recognition of host bacteria. Thus, fiber proteins may be used as potential biorecognition elements for detecting bacteria. Through gene engineering technology, large amount of artificial phage fiber protein may be synthesized. Therefore, the bacteriophage and the fiber protein thereof have great application prospect. However, the research on the Acinetobacter baumannii fiber protein is less at present, and whether the fiber protein has the recognition effect on the Acinetobacter baumannii or not is still lack of related data.

Disclosure of Invention

In view of the above, the technical problem to be solved by the present invention is to provide acinetobacter baumannii phage fiber protein, its encoding gene, recombinant expression vector, and preparation method and application thereof. The fiber protein has good application prospect in the aspect of specific detection of acinetobacter baumannii.

The invention provides acinetobacter baumannii phage fiber protein which has an amino acid sequence shown as SEQ ID NO.1 or a sequence with at least 80 percent of sequence homology with the amino acid sequence;

or a sequence having the amino acid sequence shown as SEQ ID No.2 or having at least 80% sequence homology therewith.

In some embodiments, the fiber protein of the Acinetobacter baumannii phage provided by the present invention has an amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO. 2.

Wherein, the fiber protein shown in SEQ ID NO.1 consists of 202 amino acid residues; has a molecular weight of 22kDa, and the fiber protein shown in SEQ ID NO; the molecular weight is 76kDa.

The acinetobacter baumannii phage fiber protein is an identifying element of acinetobacter baumannii, particularly multiple drug-resistant acinetobacter baumannii, and has good specificity and anti-interference capability.

The invention also provides a polynucleotide for coding the acinetobacter baumannii phage fiber protein.

In the present invention, the "polynucleotide" refers to a biological macromolecular compound formed by polymerizing a plurality of nucleotides, which can be ribonucleic acid or deoxyribonucleic acid and their modifications, including double-stranded or single-stranded DNA, cDNA, RNA, mRNA, etc., and can be circular or linear, or can be a part of a circular vector or a fragment in a genome.

In some embodiments, the polynucleotide encoding acinetobacter baumannii phage fiber protein is DNA. The sequence of the polynucleotide is shown in SEQ ID NO.3 or SEQ ID NO. 4.

Wherein, SEQ ID NO.3 encodes the fiber protein shown in SEQ ID NO. 1; the fiber protein shown in SEQ ID NO.2 is encoded by SEQ ID NO. 4.

The invention also provides a nucleic acid vector comprising the polynucleotide.

The nucleic acid vector consists of a backbone vector and a polynucleotide. In some embodiments, the backbone vector of the nucleic acid vector is pET-32a, pET-32a-gp52, or pET-32a-gp53.

The invention also provides a recombinant host, which is transformed with the nucleic acid vector.

The preparation method of the recombinant host comprises the following steps: the recombinant expression vector is transformed into a host. In the recombinant host, the host is escherichia coli. In some embodiments, the host is E.coli Trans B.

The preparation method of the acinetobacter baumannii bacteriophage fiber protein comprises the following steps: culturing the recombinant host, and inducing the expression of the acinetobacter baumannii bacteriophage fiber protein.

The invention also provides an antibody of the acinetobacter baumannii phage fiber protein.

The term "antibody" as used herein includes any isotype antibody or immunoglobulin or antibody fragment that retains specific binding to an antigen, including but not limited to Fab, fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single chain antibodies, and fusion proteins comprising an antigen-binding portion of an antibody and a non-antibody protein. The antibody may be labeled and detected, for example, by a radioisotope, an enzyme capable of producing a detectable substance, a fluorescent protein, biotin, or the like. The antibodies can also be bound to a solid support, including but not limited to polystyrene plates or beads, and the like.

The acinetobacter baumannii phage fiber protein or the antibody of the invention can be applied to the preparation of the reagent of acinetobacter baumannii.

Research shows that the process of phage specificity recognition of host bacteria is mediated by fiber protein on fiber, which utilizes C-terminal binding domain to specifically recognize lipopolysaccharide receptor on host bacteria cell surface, and has not shown bacteriolytic activity. Therefore, the fiber protein is expressed by utilizing the genetic engineering technology and has important significance in the detection of pathogenic bacteria by taking the fiber protein as a specific recognition molecule. Therefore, on the one hand, the fiber protein of the acinetobacter baumannii phage can be used as a detection reagent of bacteria based on the specificity recognition function of the acinetobacter baumannii phage, and on the other hand, the fiber protein gene can be introduced into other phage based on the specificity recognition function of the protein, so that the phage can recognize both the original host and the acinetobacter baumannii.

The invention also provides a reagent for detecting the acinetobacter baumannii, which comprises at least one of the following I) to II):

i) The acinetobacter baumannii phage fiber protein is prepared by the method;

II), chemically or biologically labeled, fiber protein of the Acinetobacter baumannii phage of the present invention;

III) conjugate prepared by coupling the Acinetobacter baumannii phage fiber protein with a solid medium or a semisolid medium;

IV) an antibody of the invention;

v), the antibody being chemically or biologically labeled;

VI), the antibody of the invention and a solid medium or a semisolid medium.

The solid phase medium or semi-solid medium refers to any support to which the monoclonal antibody or labeled monoclonal antibody of the present invention can be attached, including but not limited to nitrocellulose membrane, polyvinylidene fluoride (PVDF) membrane, iPDMS chip, microwell plate, polystyrene plate, microparticle, microcarrier, gel, etc.

The method for detecting the acinetobacter baumannii is a method for detecting the acinetobacter baumannii by immunology. The immunological detection is a technology for researching qualitative, quantitative or positioning of intracellular antigens (polypeptides and proteins) or antibodies by applying the principle of immunological basic principle, namely antigen-antibody reaction, namely the principle of specific combination of antigen and antibody, and developing color development agents (fluorescein, enzyme, metal ions and isotopes) for marking the antibodies through chemical reaction, wherein the technology comprises but is not limited to enzyme-linked immunoassay (indirect, direct or double antibody sandwich method), immunofluorescence, radioimmunoassay, immunoblot, immunohistochemistry, co-immunoprecipitation, chromatin co-precipitation and the like.

In some embodiments, the detection of acinetobacter baumannii is performed by bioluminescence. The reagent for detecting acinetobacter baumannii comprises: acinetobacter baumannii phage fiber protein-bound magnetic beads.

The preparation method of the Acinetobacter baumannii phage fiber protein combined magnetic bead comprises the following steps: mixing the activated magnetic beads with the fiber protein, incubating and sealing to obtain the fiber protein.

The invention also provides a method for detecting the acinetobacter baumannii, which uses the reagent to detect.

The bioluminescence detection method of acinetobacter baumannii comprises the following steps: mixing a sample to be detected with the magnetic beads combined with the fiber protein, incubating, separating the magnetic beads, cracking with ATP lysate, reacting with ATP bioluminescence solution, and judging the concentration of the acinetobacter baumannii according to a luminescent signal.

The acinetobacter baumannii phage fiber protein provided by the invention has an amino acid sequence shown as SEQ ID No.1 or 2 or a sequence with at least 80% of sequence homology with the amino acid sequence, has stronger specificity in the aspect of identifying and detecting acinetobacter baumannii, and can be used for preparing a reagent for identifying acinetobacter baumannii. The preparation method of acinetobacter baumannii phage fiber protein provided by the invention can realize large-scale expression and preparation of acinetobacter baumannii phage fiber protein, and has the advantages of simple separation and purification and low cost.

Drawings

The invention is further illustrated by the following examples in conjunction with the accompanying drawings:

FIG. 1 shows plaque morphology;

FIG. 2 shows phage morphology;

FIG. 3 is the electrophoresis result of the phage AbTJ genome;

FIG. 4 shows SDS-PAGE electrophoretic analysis;

FIG. 5 shows a working curve of bioluminescence intensity versus Acinetobacter baumannii concentration.

Detailed Description

The invention provides acinetobacter baumannii phage fiber protein, a coding gene, a recombinant expression vector, a preparation method and application thereof. Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The instruments adopted by the invention are all common commercial products and can be purchased in the market.

1. Bacterial strains and plasmids

Host: acinetobacter baumannii MDR-TJ is obtained from separation, purification and preservation in the laboratory.

A separation method of acinetobacter baumannii phage AbTJ is disclosed in the literature' Yujindan, separation and identification of acinetobacter baumannii phage and research on biological characteristics [ D ]. Jilin university, 2013 ] "

Escherichia coli competence Trans B purchased from Beijing Tiangen Biochemical technology Ltd.

Expression vector pET-32a was purchased from Novengen.

2. Culture medium and reagent

LB medium, OXOID brand, 10g of trypsin, 5g of yeast extract, 10g of sodium chloride, and supplementing water to IL. And (3) adding 15g of agar powder into the LB solid culture medium on the basis of the LB culture medium. Autoclaving at 121 deg.C for 20min. LB semisolid culture Medium: 7.5g of agar powder is added on the basis of LB culture medium. Autoclaving at 121 deg.C for 20min.

PBS buffer: 8.18g of sodium chloride, 0.2g of potassium chloride, 1.42g of disodium hydrogen phosphate and 0.24g of potassium dihydrogen phosphate, water is supplemented to 1L, and the pH is adjusted to 7.4.

DNA Marker available from Novengen.

The invention provides an amino acid sequence of acinetobacter baumannii bacteriophage fiber protein, which is shown as Seq ID NO.1 or SEQ ID NO.2; the nucleotide sequence for coding the fiber protein is shown as SEQ ID NO.3 and SEQ ID NO.4, the nucleotide sequence of the acinetobacter baumannii phage fiber protein and a vector pET-32a are recombined to form a recombinant expression vector, the recombinant expression vector is transformed into Trans B to form recombinant engineering bacteria, and then the fiber protein is expressed and purified. The acinetobacter baumannii phage fiber protein provided by the invention has a remarkable effect in the aspect of specific identification and detection of acinetobacter baumannii, and can be used for preparing a reagent for detecting the acinetobacter baumannii, such as a kit for preparing clinical detection of the acinetobacter baumannii. The preparation method of acinetobacter baumannii phage fiber protein provided by the invention can realize large-scale expression and preparation of acinetobacter baumannii phage fiber protein, and has the advantages of simple separation and purification and low cost.

The invention is further illustrated by the following examples:

example 1 isolation and purification of Acinetobacter baumannii phage

Taking 1L of sewage before hospital treatment, adding CaCl ₂ Centrifuging at 8000rpm for 5min at 4 deg.C until lmmol/L, removing precipitate particles in sewage, collecting 250mL of treated sewage supernatant, placing into a triangular flask, adding 50mL of LB culture medium and 1mL of Acinetobacter baumannii (in several growth phases), shaking at 37 deg.C, and culturing overnight. 10000rpm, centrifuging for 5min, taking supernatant, filtering with 0.22 μm filter membrane to remove mixed bacteria, and obtaining phage stock solution. The obtained phage stock solution was diluted by a gradient of 10 times (10- ¹ -10- ⁸ ) Taking 8 centrifugal tubes, adding 10mL of treated supernatant into each centrifugal tube, then respectively adding 10mL of Acinetobacter baumannii bacterial liquid, mixing and standing for 15min, adding a semisolid culture medium melted at about 55 ℃, quickly mixing uniformly, then uniformly paving on a solid nutrient agar culture medium, cooling for 15min, inverting at 37 ℃, culturing overnight for 16-18 h, and observing the growth condition of plaques. Single plaques on double-layer agar plates are picked and inoculated into corresponding host bacterial suspensions, and the amplified phage are cultured by shaking at 37 ℃. Obtaining purer bacteriophage after 3-5 times of repeated purification, diluting the bacteriophage supernatant obtained by final purification by 10 times,taking 1ml of diluted supernatant to react with the host bacterial liquid with the same volume to prepare plaques, and observing the plaque morphology as shown in figure 1.

Example 2 preparation of phage particles

(1) Inoculating host bacteria (capable of forming plaques in double-layer agar medium) into 100mL LB liquid medium, and performing shake culture in a shaker at 37 ℃ until logarithmic phase;

(2) Inoculating single plaque, continuously performing oscillation culture until the mixed solution is clear, centrifuging the mixed solution for 10min at 5000g, and removing precipitate;

(3) Adding DNase I and RNaseA into the supernatant until the final concentration is 1 mu g/mL, and carrying out warm bath at 37 ℃ for 30min;

(4) Adding chloroform to a final concentration of 1% (v/v), and performing warm bath at 37 deg.C for 30min;

(5) Adding NaCl according to the proportion of 5.84g/100mL, fully dissolving, then carrying out ice-bath for 1h, centrifuging for 10min at 10000g, removing bacterial debris, and collecting supernatant;

(6) Adding solid PEG 8000 to final concentration of 10% (w/v), shaking to dissolve, standing overnight at 4 deg.C to completely precipitate phage particles, centrifuging at 4 deg.C for 10min at 12 000g, and discarding supernatant;

(7) Adding TM liquid into every 100mL of original bacterial liquid in a proportion of 2mL to obtain suspension precipitate;

(8) Adding equal volume of chloroform, shaking for 30s, centrifuging at 5000g for 10min, and collecting the upper aqueous phase;

(9) Then extracting with chloroform of the same volume for 1 time to obtain phage particles.

Example 3 Electron microscopy identification of phages

Dropping 30 μ L of phage particle suspension on copper mesh, precipitating for 15min, carefully sucking off the excess liquid from the side with filter paper, adding 2% phosphotungstic acid (PTA, pH7.0) onto the copper mesh, dyeing for 20min, and observing the phage morphology with TEM with acceleration voltage of 80kV after all samples are processed. The electron micrograph shows that it belongs to the family of the brachycoviridae (Podoviridae), the head diameter being about 50nm and the tail length being about 15nm. The phage morphology is shown in FIG. 2.

Example 4 extraction of phage genome

Referring to the second edition of molecular cloning, experimental guidelines for phage DNA extraction, the procedure was as follows:

(1) Adding DNase I into the phage particles subjected to primary purification to the final concentration of 5 mu g/mL and RNaseA to 1 mu g/mL, and incubating for 1h at 37 ℃;

(2) Adding EDTA (pH8.0) to a final concentration of 20mmol/L, adding proteinase K to a final concentration of 50. Mu.g/mL, adding 10% SDS to a final concentration of 0.5%, and incubating at 56 ℃ for 1h;

(3) Mixing uniformly with equal volume of phenol, chloroform, isoamyl alcohol (25;

(4) Step (3) is repeated 3 times

(5) Adding equal volume of chloroform, mixing uniformly at 12000rpm for 10min, and collecting supernatant;

(6) Adding precooled 95% ethanol with twice volume, mixing uniformly, 12000rpm,10min, and precipitating DNA;

(7) Washing the precipitate with 75% ethanol twice, centrifuging, removing ethanol, air drying, dissolving in 50 μ L double distilled water, and storing at-20 deg.C; electrophoresis is shown in FIG. 3.

Example 5 phage genomic DNA sequencing and annotation

Phage DNA sequencing (Wuhan Bennacidae technical services Co., ltd., using Illumina NovaSeq for sequencing).

As a result of sequencing, the nucleotide sequence of the phage AbTJ is a circular genome containing 42670bp, has a GC content of 39.32%, and contains 62 putative functional genes.

Open Reading Frames (ORFs) of the phage genome were analyzed using ORF finder software, the predicted genes were determined by Genemark, the predicted genes were compared to known gene and protein sequences using BLAST, and tRNAscan-SE Search Server performed a Search for tRNA coding sequences in the phage genome.

Functional proteins have 13 distinct functional genes, which can be classified into 4 types: DNA binding proteins, DNA assembly proteins, phage structural proteins, proteins associated with phage metabolism, the remainder are putative functional proteins.

The nucleotide sequences of ORF52 and ORF53 of phage AbTJ are shown in SEQ ID NO: 3. SEQ ID NO:4, the coded protein consists of 202 and 699 amino acids; the Blastp result shows that the proteins coded by ORF52 and ORF53 have high similarity with the fiber protein coded by phage Ab105-1phi (No. KT588074.1).

EXAMPLE 6 cloning, expression and purification of the Gene of the phage fiber protein

Artificially synthesizing gp52/gp53 gene segments of the phage, introducing XbaI and Xho I enzyme cutting sites at the upstream and the downstream of the phage respectively, and adding a ribosome binding site at the upstream and the downstream of the phage. The total length of the artificially synthesized gene fragment is 657bp and 2148bp, and the nucleotide sequence is shown as SEQ ID NO:5 and SEQ ID NO:6, 202 and 699 amino acids are coded.

The artificially synthesized gp52 and gp53 gene fragments obtained in example 6 were ligated between Xba1 and Xho1 restriction sites of pET32a vector to obtain pET32 a-gp52/pET 32a-gp53 recombinant vector; the obtained recombinant vector was transformed into E.coli Trans B competent cells, and spread on LB solid culture plate containing 10mg/ml kanamycin, and cultured at 37 ℃ for 16 hours; inoculating the positive clone strain into LB liquid culture medium containing 10mg/ml kanamycin, and performing shake culture at 37 ℃ and 200rpm overnight; the overnight cultured positive clone strain was inoculated into 500ml of LB liquid medium (containing 10mg/ml kanamycin) at a dilution ratio of 1:100, shake-cultured at 37 ℃ at 200rpm until OD600 reached 0.6, added with 1mM final concentration of isopropylthio-. Beta. -D-galactoside (IPTG), and subjected to inducible expression of phage fiber protein at 18 ℃ for 16 hours.

The cells obtained by the induction expression in example 6 were collected by centrifugation at 12000rpm at 4 ℃ and washed three times with PBS, and then the cells were resuspended in PBS buffer. Carrying out crushing and cracking on the thalli on ice by using an ultrasonic crushing method (power is 80%,5s/5s pulse circulation), carrying out high-speed centrifugation at 12000rpm and 4 ℃, and collecting supernatant; purifying the filtered supernatant with HIS affinity chromatography nickel column (GEHealthCare, sweden) to obtain fiber protein with amino acid sequence shown in SEQ ID NO:1 and SEQ ID NO:2, SDS-PAGE electrophoresis analysis is carried out, and the electrophoresis detection result is shown in FIG. 4, the left image is the SDS-PAGE electrophoresis image of gp52, the right image is the SDS-PAGE electrophoresis image of gp53, and the sizes of the SDS-PAGE electrophoresis images are consistent with the expected molecular weight (22 kD/76 kD).

Example 7 detection of Acinetobacter baumannii by bioluminescence method based on recognition of Acinetobacter baumannii phage fiber protein

1. Preparation of detection reagent

(1) Phage fiber protein binding beads: 1mL of magnetic beads are taken and washed for 2 times by 1mL of PBST; 1mL of activation buffer and 2mL of 0.25mg mL were added ^-1 The fiber protein solution, room temperature reaction for 6 hours, followed by PBST washing 2 times, adding 1mL of the blocking agent, room temperature reaction for 60min, PBST washing 2 times, 1.0mL 10mM PBS buffer heavy suspension, 4 degrees C storage.

(2) Acinetobacter baumannii suspension standard: taking single colony of Acinetobacter baumannii to inoculate in LB culture solution, shaking and culturing for 4 hours at 37 ℃, resuspending the bacteria by PBS buffer solution and adjusting the concentration of the bacteria to 1.0 multiplied by 10 ⁶ CFU/mL。

(3) ATP bioluminescence assay kit, purchased from Hao Biotechnology Limited, hao, japan.

2. And (3) detection:

diluting an Acinetobacter baumannii suspension standard substance with a buffer solution to prepare Acinetobacter baumannii working bacteria liquid, adding 25 mu L of magnetic bead suspension of fiber protein (taking the buffer solution as a solvent) into 100 mu L of the Acinetobacter baumannii working bacteria liquid, incubating for 1h at room temperature, carrying out magnetic separation to obtain an Acinetobacter baumannii-fiber protein-magnetic bead compound, washing for 3 times by using a washing solution, adding 100 mu L of ATP lysate, continuing reacting for 2min until the Acinetobacter baumannii is lysed, taking 50 mu L of lysate, injecting 50 mu L of ATP bioluminescent solution through a chemiluminescence detector to react with the ATP bioluminescent solution, recording a bioluminescent signal, drawing a graph by using bioluminescent intensity to the concentration of the Acinetobacter baumannii, and drawing a working curve.

3. As a result, the

In the detection method 2, the concentrations of the Acinetobacter baumannii working bacteria liquid are respectively 1.5 multiplied by 10 ² 、1.5×10 ³ 、1.5×10 ⁴ 、1.5×10 ⁵ 、1.5×10 ⁶ 、1.5×10 ⁷ And 1.5X 10 ⁸ CFU/mL, the bioluminescence intensity plotted against Acinetobacter baumannii concentration, the obtained working curve is shown in FIG. 5, the bioluminescence intensity is continuously increased along with the increase of the Acinetobacter baumannii concentration, and the bioluminescence intensity is 1.5 multiplied by 10 ³ -1.5×10 ⁷ In the CFU/mL rangeExhibit a good linear relationship, respectively R ² =0.9963 and R ² =0.9969, which indicates that acinetobacter baumannii can be sensitively detected by using fiber proteins gp52 and gp53 as acinetobacter baumannii molecular recognition reagents.

In the step (1), 10% glucose injection, excrement, urine, sputum and domestic sewage of healthy volunteers are respectively selected as biological samples, after the biological samples are respectively treated, acinetobacter baumannii with known concentration is added into the biological samples as samples to be detected, and the detection is carried out according to the method. The results are shown in tables 1 and 2, and the recovery rate of acinetobacter baumannii in the sample to be tested is more than 50%, and the RSD value is less than 7.6%, which shows that the acinetobacter baumannii can be accurately detected by using the fiber proteins gp52 and gp53 as the molecular recognition reagent of acinetobacter baumannii.

Table 1.Gp52-MBs detected anti-interference effect of acinetobacter baumannii (n = 4).

Table 2.Gp53-MBs detect anti-interference effect of Acinetobacter baumannii (n = 4).

The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.

Sequence listing

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Glu Leu Ala Asn Pro His Ile Phe Val Ser Gly Ile Phe Ser Gly Thr

625 630 635 640

Gly Leu Gly Gly Gly Lys Ala Ile Leu Pro Pro Thr Leu Gln Ala Ile

645 650 655

Glu Ala Tyr Ser Gly Ser Trp Ser Ser Phe Lys Val Ala Ala Gly Val

660 665 670

Lys Leu Phe Phe Ile Asp Leu Thr Gly Ala Leu Val Asp Pro Met Arg

675 680 685

Phe Ser Val Ser Phe Thr Leu Glu Ser Gly Ile

690 695

<210> 3

<211> 609

<212> DNA

<400> 3

atggataagt tagaagaatt tagcctgaac gggccaaaaa ataccgatgg actaacttta 60

ttaagtggct ttccatcaaa tcagaaaccc gctcgtcaat ggtttaactg gttatttaat 120

tcaataacca aaaagattaa tgaaatcatt gatggcaagc tggatgcagg tgctaatgct 180

gtttctgcag caaagttaga aaagggacga aagataaatt tcactggtgt aatccagggt 240

agtggcacat ttgatggctc taaagatatt actatcgata cggtcgacgg tggaaccata 300

ggtgaaaaag ctatcgctat tattcgcctc aacggctcaa cgttcgattt agtcaaaagc 360

cgtggctttg cttcagttga aaataaaggt ggcggccaaa tcgaatttac tttgagtgag 420

gatgcacctg atacagatta tggcatcgta tgtactggaa caagcaatcg agctgatgca 480

gttagcttgc aagagcgtga agactttgcc cggacaaaca ccaaattccg tctgatggga 540

gcatttggtg gtgataatac acaaggtgct tatacccctc aaatatgcac tgtagtcgtc 600

tattactaa 609

<210> 4

<211> 2100

<212> DNA

<400> 4

atgaccgcct tctggcggtt ttttgttgca tggagatcca aaaagatggc aacaaactgg 60

aatgcggttt taaacaacac aaataacttc aatgatgtat tggcaatttt aaaaaaactg 120

ctagcactca tgggcgattt atctactctt gattcatcag aggttctttt acgtattgat 180

gagattatta actcatcggt tgctgatttt aatgaaaaac aaattcaggc ttttaaggat 240

ctcaaagaag caattgaagt tgctagtgca gccggagcag gcgaaaacgg gtggatagat 300

acactggtac ttactttaac tggagaaaat ctcagggaat ttaataaaaa aactatcagt 360

actttagatt gtattgatga tttagctact acattgccat ggccaggccg caccgtaaat 420

gtacgatctg tgataaaaga taaacattta gggggaggaa cttttgtatt tagtgctgat 480

agttcaaaag ttccagacgg ttatattgtt gttgctgcaa atggcgggaa ttgggtgaaa 540

atcacagttg cttttccaac aattgatgat tttggcggtc tgggtgacga cccaaattat 600

gacgatgcgg acgcatttat tcgatgtgcg ttgagtccat acacaggttc gaatatttat 660

cttgctaaca gacaagttga atatcgcatc aataaacaag ttgattgcaa gggtaaaggg 720

attgttggag gtggatttag tagacaaaat gccactgcgt atgcaatgaa ctctctaaag 780

gtaagaccag gtgattattc aaattcaaat acccttctta ataatgtggc atttattaac 840

gtgggcgccg aagtaagaga tttgcaatta gtaagtgagg gtgtttcaga aaatatttcg 900

ggtttaaaag ttgatggtta taacttcaca ctttcaaatg caaatatttc tgggttttac 960

aatcaagtat atttatcgaa tgcaacggtc tcgttccgtg tccaaaattt gatgtcaatt 1020

agtgcatcaa atgcagggtt ttatattgct gatgttgatt ctaaacaaag tactactgca 1080

tattttgaca actgctcatg gcaatggggt aaataccctg tgctgtttgc taaagaagct 1140

tatcagtgcg tctttaataa tattattctt gaatatatgc agtacggttt aacagcgggt 1200

atctggtcga attgctcatt caatgcaatt tgggcagagc aaacaagaga tggggtagct 1260

cgtgactggc ttgtaaatac ttcttatcaa caaacattta attgcatcac taataatcta 1320

tacattagaa cgccttggct aaatcgagct gacacaaccg ctttggctgt atcagataat 1380

attggggggg ttgtaattga taaaagtcga attactttaa gcggtgcgac gggtgcgaaa 1440

atacaacttt ctccatctgg tttagcaaca ctttttgcta attggtatgg tggtactaat 1500

cgaagattat taattactac tcaaccgact gcggcagatt ctgggtataa aacaccaatc 1560

cacatcaatg caccgaatag cgaattgtat tttggaaatc aggatgaaac atcagttgca 1620

agtgtagtat ttaagcgagt aattggcgct actgctacaa atacacctta tattgcgtca 1680

gactcgtgga ctaaaaaaat tcgaaagtgg aatacttaca atcacgaagt atcaaaagtc 1740

gggcgtttta ttgcaccgat gatgctgact tatgatgtca actttactac ccaacaaaat 1800

aacgcaggct ggtctatctc aaaagagtct acgggagtct atagattgca acgagatgcg 1860

ggcgtaacaa ctgaattagc aaatccgcac atttttgttt ctggcatttt ttctggaacg 1920

ggattaggtg gtggaaaggc tatattgcca ccgacgctac aagcaattga agcatacagt 1980

gggagttggt cgtctttcaa agttgcagct ggagtcaagt tgttttttat agatttgaca 2040

ggtgcgttag ttgatccaat gcgcttctca gtatcattca cactagaatc aggaatttaa 2100

<210> 5

<211> 657

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

tctagaaata attttgttta actttaagaa ggagatatac atatggataa gttagaagaa 60

tttagcctga acgggccaaa aaataccgat ggactaactt tattaagtgg ctttccatca 120

aatcagaaac ccgctcgtca atggtttaac tggttattta attcaataac caaaaagatt 180

aatgaaatca ttgatggcaa gctggatgca ggtgctaatg ctgtttctgc agcaaagtta 240

gaaaagggac gaaagataaa tttcactggt gtaatccagg gtagtggcac atttgatggc 300

tctaaagata ttactatcga tacggtcgac ggtggaacca taggtgaaaa agctatcgct 360

attattcgcc tcaacggctc aacgttcgat ttagtcaaaa gccgtggctt tgcttcagtt 420

gaaaataaag gtggcggcca aatcgaattt actttgagtg aggatgcacc tgatacagat 480

tatggcatcg tatgtactgg aacaagcaat cgagctgatg cagttagctt gcaagagcgt 540

gaagactttg cccggacaaa caccaaattc cgtctgatgg gagcatttgg tggtgataat 600

acacaaggtg cttatacccc tcaaatatgc actgtagtcg tctattacta actcgag 657

<210> 6

<211> 2148

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

tctagaaata attttgttta actttaagaa ggagatatac atatgaccgc cttctggcgg 60

ttttttgttg catggagatc caaaaagatg gcaacaaact ggaatgcggt tttaaacaac 120

acaaataact tcaatgatgt attggcaatt ttaaaaaaac tgctagcact catgggcgat 180

ttatctactc ttgattcatc agaggttctt ttacgtattg atgagattat taactcatcg 240

gttgctgatt ttaatgaaaa acaaattcag gcttttaagg atctcaaaga agcaattgaa 300

gttgctagtg cagccggagc aggcgaaaac gggtggatag atacactggt acttacttta 360

actggagaaa atctcaggga atttaataaa aaaactatca gtactttaga ttgtattgat 420

gatttagcta ctacattgcc atggccaggc cgcaccgtaa atgtacgatc tgtgataaaa 480

gataaacatt tagggggagg aacttttgta tttagtgctg atagttcaaa agttccagac 540

ggttatattg ttgttgctgc aaatggcggg aattgggtga aaatcacagt tgcttttcca 600

acaattgatg attttggcgg tctgggtgac gacccaaatt atgacgatgc ggacgcattt 660

attcgatgtg cgttgagtcc atacacaggt tcgaatattt atcttgctaa cagacaagtt 720

gaatatcgca tcaataaaca agttgattgc aagggtaaag ggattgttgg aggtggattt 780

agtagacaaa atgccactgc gtatgcaatg aactctctaa aggtaagacc aggtgattat 840

tcaaattcaa atacccttct taataatgtg gcatttatta acgtgggcgc cgaagtaaga 900

gatttgcaat tagtaagtga gggtgtttca gaaaatattt cgggtttaaa agttgatggt 960

tataacttca cactttcaaa tgcaaatatt tctgggtttt acaatcaagt atatttatcg 1020

aatgcaacgg tctcgttccg tgtccaaaat ttgatgtcaa ttagtgcatc aaatgcaggg 1080

ttttatattg ctgatgttga ttctaaacaa agtactactg catattttga caactgctca 1140

tggcaatggg gtaaataccc tgtgctgttt gctaaagaag cttatcagtg cgtctttaat 1200

aatattattc ttgaatatat gcagtacggt ttaacagcgg gtatctggtc gaattgctca 1260

ttcaatgcaa tttgggcaga gcaaacaaga gatggggtag ctcgtgactg gcttgtaaat 1320

acttcttatc aacaaacatt taattgcatc actaataatc tatacattag aacgccttgg 1380

ctaaatcgag ctgacacaac cgctttggct gtatcagata atattggggg ggttgtaatt 1440

gataaaagtc gaattacttt aagcggtgcg acgggtgcga aaatacaact ttctccatct 1500

ggtttagcaa cactttttgc taattggtat ggtggtacta atcgaagatt attaattact 1560

actcaaccga ctgcggcaga ttctgggtat aaaacaccaa tccacatcaa tgcaccgaat 1620

agcgaattgt attttggaaa tcaggatgaa acatcagttg caagtgtagt atttaagcga 1680

gtaattggcg ctactgctac aaatacacct tatattgcgt cagactcgtg gactaaaaaa 1740

attcgaaagt ggaatactta caatcacgaa gtatcaaaag tcgggcgttt tattgcaccg 1800

atgatgctga cttatgatgt caactttact acccaacaaa ataacgcagg ctggtctatc 1860

tcaaaagagt ctacgggagt ctatagattg caacgagatg cgggcgtaac aactgaatta 1920

gcaaatccgc acatttttgt ttctggcatt ttttctggaa cgggattagg tggtggaaag 1980

gctatattgc caccgacgct acaagcaatt gaagcataca gtgggagttg gtcgtctttc 2040

aaagttgcag ctggagtcaa gttgtttttt atagatttga caggtgcgtt agttgatcca 2100

atgcgcttct cagtatcatt cacactagaa tcaggaattt aactcgag 2148

Claims

1. The application of acinetobacter baumannii bacteriophage fiber protein or an antibody thereof in preparing a reagent for detecting acinetobacter baumannii; the amino acid sequence of the acinetobacter baumannii phage fiber protein is shown in SEQ ID NO.1 or SEQ ID NO. 2.

2. A method for detecting acinetobacter baumannii for non-diagnostic purposes, which uses a reagent for detecting the acinetobacter baumannii for detection; the reagent for detecting the acinetobacter baumannii comprises at least one of the following I) to V):

i) The acinetobacter baumannii bacteriophage fiber protein is chemically or biologically marked;

II) conjugate prepared by coupling acinetobacter baumannii bacteriophage fiber protein with solid medium or semisolid medium;

III), an antibody to Acinetobacter baumannii phage fiber protein;

IV), chemically or biologically labeled acinetobacter baumannii phage fiber protein antibodies;

v) and a conjugate prepared by coupling the acinetobacter baumannii phage fiber protein antibody with a solid medium or a semisolid medium, wherein the amino acid sequence of the acinetobacter baumannii phage fiber protein is shown as SEQ ID NO.1 or SEQ ID NO. 2.