CN110713524A - High-sensitivity acinetobacter baumannii antigen Elisa determination kit - Google Patents

High-sensitivity acinetobacter baumannii antigen Elisa determination kit Download PDF

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CN110713524A
CN110713524A CN201911052219.5A CN201911052219A CN110713524A CN 110713524 A CN110713524 A CN 110713524A CN 201911052219 A CN201911052219 A CN 201911052219A CN 110713524 A CN110713524 A CN 110713524A
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杨波
胡征
王毅
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Abstract

The invention relates to a high-sensitivity acinetobacter baumannii antigen Elisa determination kit and a preparation method thereof. The kit comprises: the kit comprises an enzyme label plate coated with polyclonal antibodies against Acinetobacter baumannii Fhue and Pilf protein, polyclonal antibodies against Acinetobacter baumannii OmpA and ponA protein, positive control and negative control of Acinetobacter baumannii, a washing solution, an enzyme-labeled secondary antibody, an enzyme chromogenic substrate and a stop solution. The kit provided by the invention is used for carrying out combined detection on four specific surface proteins of acinetobacter baumannii, greatly improves the sensitivity, specificity and repeatability of acinetobacter baumannii detection, and can be used for non-diagnostic detection and research work on acinetobacter baumannii.

Description

High-sensitivity acinetobacter baumannii antigen Elisa determination kit
Technical Field
The invention belongs to the field of biotechnology and infectious disease diagnosis research, relates to an Elisa detection kit, and particularly relates to a high-sensitivity Acinetobacter baumannii antigen Elisa detection kit and a preparation method thereof.
Background
Acinetobacter baumannii (Ab) is a non-fermented gram-negative bacillus, widely exists in the nature and belongs to conditional pathogenic bacteria. The bacterium is an important pathogenic bacterium of hospital infection, mainly causes respiratory tract infection, and also can cause bacteremia, urinary system infection, secondary meningitis, operation site infection, ventilator-associated pneumonia and the like. The resistance rate to commonly used antibiotics tends to increase year by year and is of serious concern to clinicians and microbiologists. Domestic data indicate that a. baumann ii accounts for approximately 70% or more of clinically isolated acinetobacter. The drug resistance rate of baumann ii to the third and fourth generation cephalosporins reaches 63.0-89.9%, and the drug resistance rate of the bacteria to the four aminoglycosides (amikacin, gentamicin, netilmicin, tobramycin) and ciprofloxacin reaches 96.3%. The vast majority of strains currently in China remain sensitive to imipenem, meropenem, cefperazone/sulbactam and polymyxin B, but have poor effects in the treatment of respiratory tract infections. In view of the recent trend toward further increase in the drug resistance of acinetobacter baumannii, this should be highly appreciated by clinicians and the microbiology. The clinician should pay attention to the acquired acinetobacter baumannii infection, and closely cooperate with the clinical microorganism laboratory to enhance the monitoring of the infection and effectively prevent and control the infection.
The existing method for detecting the pathogen in the respiratory tract mainly adopts the traditional method, namely a separation identification method, the method needs long time, generally takes 2-3 days, and the requirement of quick identification is difficult to meet; the PCR technology developed in recent years is a quick, sensitive and specific technology, but at present, the technology still depends on the previous enrichment step of the traditional method, and PCR inhibitors are often contained in the enrichment liquid, so that the amplification effect is influenced. Meanwhile, the technology also needs professional detection equipment, and is not suitable for bedside detection. In addition, false positives are often caused by the sensitivity of this technique. Antibody-based immunological detection has become an indispensable important technical means for the detection of human pathogenic microorganisms. Various specific immunoassay techniques, such as Radioimmunoassay (RIA), Enzyme Immunoassay (EIA), Fluorescence Immunoassay (FIA), Chemiluminescence Immunoassay (CIA), immunoprecipitation, immunoagglutination, ELISA detection kit, immune colloidal gold test strip, immune latex detection reagent, and the like, have been developed. Among them, ELISA and other immunological detection techniques based on antibody have become an indispensable important means for detecting pathogenic microorganisms based on its characteristics of simplicity, rapidness, sensitivity, accuracy and practicality. Therefore, research and development of antibodies against pathogenic microorganisms with proprietary intellectual property rights are the basis for development of ELISA detection methods with proprietary intellectual property rights.
The choice of antigenic component is critical to the production of specific antibodies. The Acinetobacter baumannii fhuE receptor, OmpA, PilF and PonA proteins are important molecules positioned on the cell surface, the research selects surface proteins with interspecific specificity, such as Fhue receptor, OmpA, PilF and PonA, as alternative antigens, and simultaneously prepares a polyclonal antibody with good specificity through technical means of gene optimization, fusion expression and the like, and applies the polyclonal antibody to the preparation of an Acinetobacter baumannii EIisa detection kit.
Disclosure of Invention
In order to solve the technical problems in the background art, the invention provides the Acinetobacter baumannii antigen Elisa assay kit with high sensitivity and a preparation method, wherein the assay kit can improve the sensitivity, specificity and repeatability of Acinetobacter baumannii detection, is simple to operate, is low in cost, and can rapidly and quickly detect the Acinetobacter baumannii.
In order to achieve the purpose, the invention adopts the following technical scheme:
a high-sensitivity acinetobacter baumannii antigen Elisa assay kit is characterized in that: the high-sensitivity acinetobacter baumannii antigen Elisa determination kit comprises an enzyme label plate coated with a polyclonal antibody of anti-acinetobacter baumannii surface protein (Fhue and Pilf), an anti-acinetobacter baumannii surface protein (OmpA and ponA) polyclonal antibody, acinetobacter baumannii positive control, negative control, washing liquid, enzyme-labeled secondary antibody, enzyme chromogenic substrate and termination liquid; the acinetobacter baumannii positive control is inactivated acinetobacter baumannii bacterial liquid; the negative control is inactivated escherichia coli liquid; the washing solution comprises the following components in percentage by weight: 1.4g/L of disodium hydrogen phosphate, 0.2g/L of sodium dihydrogen phosphate, 8.5g/L of sodium chloride and 200.5mL/L of Tween, wherein the pH value of the washing solution is 7.4; the enzyme-labeled secondary antibody is goat anti-rabbit IgG labeled by horseradish peroxidase; the enzyme chromogenic substrate is TMB chromogenic solution; the stop solution is a 1M HCL solution.
A preparation method of a high-sensitivity Acinetobacter baumannii antigen Elisa assay kit is characterized by comprising the following steps: the high-sensitivity acinetobacter baumannii antigen Elisa determination kit comprises an enzyme label plate coated with a polyclonal antibody of anti-acinetobacter baumannii surface protein (Fhue and Pilf), an anti-acinetobacter baumannii surface protein (OmpA and ponA) polyclonal antibody, acinetobacter baumannii positive control, negative control, washing liquid, enzyme-labeled secondary antibody, enzyme chromogenic substrate and termination liquid; the acinetobacter baumannii positive control is inactivated acinetobacter baumannii bacterial liquid; the negative control is inactivated escherichia coli liquid; the washing solution comprises the following components in percentage by weight: 1.4g/L of disodium hydrogen phosphate, 0.2g/L of sodium dihydrogen phosphate, 8.5g/L of sodium chloride and 200.5mL/L of Tween, wherein the pH value of the washing solution is 7.4; the enzyme-labeled secondary antibody is goat anti-rabbit IgG labeled by horseradish peroxidase; the enzyme chromogenic substrate is TMB chromogenic solution; the stop solution is 1M HCL solution;
the preparation method of the ELISA plate for coating the polyclonal antibody of the anti-Acinetobacter baumannii surface protein (Fhue and Pilf) comprises the following steps:
1) preparation of polyclonal antibodies against acinetobacter baumannii surface protein (Fhue and Pilf):
1.1) respectively obtaining peptide segments with most abundant antigenic epitopes in the extracellular domains of the Acinetobacter baumannii surface protein Fhue and the surface protein Pilf, finding out the gene coding sequence of the peptide segments, optimizing the gene coding sequence of the peptide segments, and connecting the optimized gene coding sequence of the peptide segments by using the coding sequence of flexible connecting peptide to form a fusion gene; the Acinetobacter baumannii surface protein Fhue and the surface protein Pilf have access numbers of KMV27515 and AJF80497 in an NCBI protein database respectively; the sequence of the flexible connecting peptide is ggsggsggsggs; simultaneously, enzyme cutting site NdeI is introduced into the 5 'end of the fusion gene, and termination signal TAA and enzyme cutting site BamHI are introduced into the 3' end of the fusion gene, and then a complete gene sequence is chemically synthesized and is marked as FhuPil;
the complete sequence of the gene of the FhuPil is as follows:
CATATGTTCGATGGCAACTACCTGGACCCGGTAGAAGGTAACTCTACTGAAGTTGGTCTCAAATCCGCTTGGTTTGACGGCCGTCTGAACGGTACCCTGGCTCTGTACCACATCAAACAGGACAACCTGGCACAGGAAGCGGGCCAAGTTACCCGTAACGGTGTTAAAGAGACTTACTACCGTGCAGCGAAAGGCGCTACATCCGAAGGTTTTGAAGTTGAAGTATCAGGTCAGATCACTCCAGATTGGAACATCACCGCAGGTTACTCTCAATTTTCTGCTAAGGACGCGAACGATGCGGACGTAAACACTCAGCTTCCGCGTAAAATGATCCAGAGCTTCACTACCTATAAACTGCCTGGTAAACTGGAAAACATCACAGTTGGCGGTGGCGTAAACTGGCAGTCTTCTACCTACGTGAACGCAAAAAACCCGAAAAAAGTAATCGAAAAAGTTGAGCAGGGTGACTACGCTCTGGTAAACCTGATGGCGCGTTACCAGATCACTAAGGACTTTTCTGCACAGCTTAACATTAACAACGTTTTCGGTGGTTCTGGTGGTTCTGGTGGTTCTGGTGGTTCTGCGGTTAAGGTACGCACTCAACTGGCGGCTGAATATATCCGTTCAGGTGATCTGGACTCCGCGAAACGCTCCCTGGACCAGGCCCTGAGCGTTGACTCTCGTGACGCGACAGCAAACATGATGATGGGCATCCTGCTGCAGCAGGAGGGCTCTAAATCTAACCTGGAGAAAGCGGAGCACTACTTCAAACGTGCTATCAGTTCTGAACCGGATAACGCTCAGGCGCGCAACAACTATGGTACCTATCTGTACCAGATGGAACGTTATAACGACGCGATTGAACAGTTTCGTATCGCAGGTGCGACCCTGGGTTATGATCAGCGTTATCAGGCGCTGGAAAACCTGGGCCGCATCTACCTGAAGCTGGGTAACATCGCCAGCGCTGAAAAAACTTTCAAGCAGGCACTGCTGGCGAACCGTGACTCCTACATCTCTATGCTGGAGCTGGCTGAAATCTTTTACCTGCAGCAGTAAGGATCC；
the protein sequence encoded by the FhuPil gene is as follows:
MFDGNYLDPVEGNSTEVGLKSAWFDGRLNGTLALYHIKQDNLAQEAGQVTRNGVKETYYRAAKGATSEGFEVEVSGQITPDWNITAGYSQFSAKDANDADVNTQLPRKMIQSFTTYKLPGKLENITVGGGVNWQSSTYVNAKNPKKVIEKVEQGDYALVNLMARYQITKDFSAQLNINNVFGGSGGSGGSGGSAVKVRTQLAAEYIRSGDLDSAKRSLDQALSVDSRDATANMMMGILLQQEGSKSNLEKAEHYFKRAISSEPDNAQARNNYGTYLYQMERYNDAIEQFRIAGATLGYDQRYQALENLGRIYLKLGNIASAEKTFKQALLANRDSYISMLELAEIFYLQQ；
the protein sequence coded by the FhuPil gene is 509-688aa of the surface protein Fhue of acinetobacter baumannii and 28-184aa of the surface protein Pilf; the two protein sequences are connected by flexible connecting peptide ggsggsggs;
1.2) cloning the complete sequence of the FhuPil gene into a prokaryotic expression vector pET-28a (+) by a conventional method, transferring the FhuPil gene into E.coli BL21(DE3) bacteria, inducing recombinant escherichia coli expression by IPTG, and using Ni2+Purifying the recombinant His-FhuPil protein by affinity chromatography; the recombinant protein is used as an immune antigen, is mixed with Freund's adjuvant and then repeatedly and artificially immunizes healthy New Zealand white rabbits,performing titer determination by blood drawing, separating and purifying high-titer recombinant protein antibodies, and finally obtaining polyclonal antibodies against acinetobacter baumannii surface proteins (Fhue and Pilf);
2) coating of anti-acinetobacter baumannii surface protein (Fhue and Pilf) polyclonal antibody:
diluting the polyclonal antibody against Acinetobacter baumannii surface protein (Fhue and Pilf) prepared in the step 1) to the concentration of 10 mu g/mL by using PBS buffer solution, coating a 96-hole EIA high-efficiency binding enzyme standard plate according to the amount of 100 mu L/hole, and carrying out 2 hours at 37 ℃; taking out, washing the plate for three times by using 250 mu L of washing liquid, and spin-drying; using a washing solution containing 1% BSA as a blocking solution, adding an enzyme label plate according to the amount of 250 mu L/hole, and blocking for 1 hour at 37 ℃; taking out, washing the plate with 250 μ L of washing solution for 3 times, each time for one minute, spin-drying, and storing in sealed condition;
wherein the PBS buffer solution comprises the following components in percentage by weight: 1.4g/L of disodium hydrogen phosphate, 0.2g/L of sodium dihydrogen phosphate and 8.5g/L of sodium chloride; the pH of the PBS buffer was 7.4;
the washing solution comprises the following components in percentage by weight: 1.4g/L of disodium hydrogen phosphate, 0.2g/L of sodium dihydrogen phosphate, 8.5g/L of sodium chloride and 200.5mL/L of Tween, wherein the pH value of the washing solution is 7.4;
the blocking solution was an aqueous solution of a washing solution containing 1% BSA, and the pH of the blocking solution was 7.4.
The preparation method of the anti-acinetobacter baumannii surface protein (OmpA and ponA) polyclonal antibody comprises the following steps:
1) respectively obtaining peptide segments with the most abundant antigenic epitopes in the surface protein OmpA and the surface protein ponA extracellular domain of the acinetobacter baumannii, finding out the gene coding sequence of the peptide segment, optimizing the gene coding sequence of the peptide segment, and connecting the optimized gene coding sequence of the peptide segment by using the coding sequence of rigid connecting peptide to form a fusion gene; the Acinetobacter baumannii surface protein OmpA and the surface protein ponA have access numbers in an NCBI protein database of AJF83030 and ADX05080 respectively; the sequence of the rigid linker peptide is eaaakaaaak; simultaneously, enzyme cutting site NdeI is introduced into the 5 'end of the fusion gene, and termination signal TAA and enzyme cutting site BamHI are introduced into the 3' end of the fusion gene, and then a complete gene sequence is chemically synthesized and is marked as OmpPon;
the complete gene sequence of OmpPon is as follows:
CATATGCTGGGTTACACCTTCCAGGATACTCAGCACAACAACGGCGGTAAAGACGGTGAACTGACCAACGGTCCGGAACTGCAGGACGACCTGTTCGTTGGTGCTGCGCTGGGTATCGAACTGACTCCGTGGCTGGGTTTCGAAGCCGAGTATAACCAGGTTAAGGGTGACGTGGATGGCCTGGCAGCGGGTGCTGAATACAAACAGAAACAGATCAACGGTAACTTCTACGTTACCAGCGACCTGATTACCAAGAACTATGACTCTAAAATCAAACCGTATGTTCTGCTGGGTGCGGGCCACTACAAATACGAAATCCCGGACCTTTCCTATCACAACGACGAGGAAGGCACTCTGGGTAACGCGGGTGTTGGTGCTTTCTGGCGTCTGAACGACGCTCTGTCTCTGCGTACCGAAGCTCGTGGTACCTATAACGAAGCTGCTGCTGCTAAAGAAGCTGCTGCTGCTAAAGGCTCTATCGAAGCTATCGTAGGTGGTTACAACTTCTACCAGTCCAAGTTTAACCGTGCGCTCCAGGGCTGGCGCCAGCCGGGCTCTACCATTAAACCTTTCCTGTACGCTCTGGCTCTGGAACGTGGCATGACCCCGTACAGCATGGTAAACGATTCTCCGATCACTATTGGTAAATGGACCCCAAAAAATTCTGACGGCCGTTACCTGGGTATGATCCCGCTGCGTCGCGCTCTGTACCTGTCCCGTAACACTGTATCCGTTCGTCTGCTGCAGACTGTTGGCATCGAACGTACCCGCCAACTGTTTATGGATTTCGGTCTGCAGGAAGACCAGATTCCACGTAACTACACTATCGCTCTGGGCACTCCGCAGGTACTGCCGATCCAGATGGCTACCGGCTACGCTACTTTCGCTAATGGCGGCTACCGTGTTCAGCCACATTTCATCCAGCGTATCGAAGACGCGTATGGTAAAGTAATTTACGAAGCTAAACCGGAATATAAGGATCC；
the protein sequence encoded by the OmpPon gene is:
MLGYTFQDTQHNNGGKDGELTNGPELQDDLFVGAALGIELTPWLGFEAEYNQVKGDVDGLAAGAEYKQKQINGNFYVTSDLITKNYDSKIKPYVLLGAGHYKYEIPDLSYHNDEEGTLGNAGVGAFWRLNDALSLRTEARGTYNEAAAAKEAAAAKGSIEAIVGGYNFYQSKFNRALQGWRQPGSTIKPFLYALALERGMTPYSMVNDSPITIGKWTPKNSDGRYLGMIPLRRALYLSRNTVSVRLLQTVGIERTRQLFMDFGLQEDQIPRNYTIALGTPQVLPIQMATGYATFANGGYRVQPHFIQRIEDAYGKVIYEAKPEY；
the protein sequence coded by the OmpPon gene is 31-173aa of the Acinetobacter baumannii surface protein OmpA and 406-572aa of the surface protein ponA; the middle of the two protein sequences is connected by rigid connecting peptide eaaakaaaak;
2) cloning OmpPon gene complete sequence into prokaryotic expression vector pET-28a (+) according to conventional method, transferring into E.coli BL21(DE3) bacteria, inducing recombinant Escherichia coli expression with IPTG, and using Ni2+Purifying the recombinant His-OmpPon protein by affinity chromatography; the recombinant protein is used as an immune antigenMixing the antibody with Freund's adjuvant, repeatedly and artificially immunizing healthy New Zealand white rabbits, performing titer determination by blood drawing, separating high-titer recombinant protein antibodies and purifying to finally obtain anti-Acinetobacter baumannii OmpA and ponA protein polyclonal antibodies, and diluting with a confining liquid to a final concentration of 20 mug/mL;
the sealing liquid comprises the following components in percentage by weight: 1.4g/L of disodium hydrogen phosphate, 0.2g/L of sodium dihydrogen phosphate, 8.5g/L of sodium chloride and 10g/L of bovine serum albumin, wherein the pH value of the sealing liquid is 7.4.
Compared with the prior art, the invention has the following advantages:
(1) the invention adopts structural analysis, gene optimization and other modes to construct two brand new fusion genes, and successfully obtains soluble recombinant Fhue/Pilf fusion protein and OmpA/ponA fusion protein for the first time through soluble over-expression. The two fusion proteins have high expression amount, low preparation cost, good protein solubility, strong antigenicity, high antibody titer and low cost.
(2) The antibody prepared by utilizing the four protein exposed regions on the surface of the acinetobacter baumannii for the first time has high titer, more antigen binding sites, strong capture capacity, no site competition problem and high detection sensitivity of the kit. The detection sensitivity of the kit to the Acinetobacter baumannii standard strain ATCC19606 reaches 1 multiplied by 103CFU/mL is obviously higher than that of the traditional microorganism detection method, and has the advantages of rapidness, high efficiency and the like.
(3) The Elisa kit has good specificity, and results of specificity experiments carried out by 6 Acinetobacter baumannii strains and 17 non-Acinetobacter baumannii standard strains (containing most common respiratory pathogens) show that the test strip has good specificity and stability, can detect all tested Acinetobacter baumannii, has no cross reaction with all non-Acinetobacter baumannii standard strains, and is very suitable for clinical non-diagnostic application.
Detailed Description
The methods used in the following examples are conventional methods unless otherwise specified.
Example 1
Preparation of polyclonal antibodies against acinetobacter baumannii Fhue and Pilf proteins:
1.1) cloning of Acinetobacter baumannii FhuPil fusion Gene
The method comprises the steps of obtaining peptide segments with most abundant antigenic epitopes in extracellular domains of Acinetobacter baumannii surface proteins Fhue and Pilf (wherein accession numbers in an NCBI protein database are KMV27515 and AJF80497 respectively), finding out gene coding sequences of the peptide segments, optimizing the gene coding sequences of the peptide segments, and connecting the two sequences by using a coding sequence of flexible connecting peptide (ggsgggsgggs) to form a fusion gene. Meanwhile, enzyme cutting site NdeI is introduced into the 5 'end of the fusion gene, and termination signal TAA and enzyme cutting site BamHI are introduced into the 3' end of the fusion gene, and then a complete gene sequence is chemically synthesized and is marked as FhuPil. The complete gene sequence and the coded amino acid sequence are shown in a sequence table. Specifically, the protein sequence encoded by the FhuPil gene is 509-688aa of the surface protein Fhue of acinetobacter baumannii and 28-184aa of the surface protein Pilf, and the two protein sequences are connected by flexible connecting peptide (ggsggsggs). The gene sequence is delivered to Nanjing Jinslei Biotech, Inc. for complete gene chemical synthesis, and the artificially synthesized gene fragment is connected to vector pUC57 when delivered. The vector pUC57 containing the artificially synthesized DNA fragment was digested with NdeI and BamHI, and the desired fragment was recovered by a conventional method and used. And simultaneously carrying out double enzyme digestion on the vector pET-28a (+) by NdeI and BamHI, connecting the FhuPil gene obtained after double enzyme digestion into the pET-28a (+) vector according to a conventional molecular biology method, and transforming Escherichia coli TOP10 to construct the pET-FhuPil expression vector. The construction of the expression vector is verified to be correct by enzyme digestion and sequence determination. The vector expresses recombinant FhuPil fusion protein.
1.2) expression and purification of Acinetobacter baumannii FhuPil fusion protein
Culturing the positive clone bacteria, extracting plasmid, transferring into competent E.coliBL21(DE3) according to conventional technique, coating the bacteria liquid on LB plate containing 50 ug/mL kanamycin, and screening expression strain according to conventional method. Individual colonies transformed with pET-FhuPil having the ability to express a foreign protein were picked and inoculated into 100mL of LB medium and cultured overnight at 37 ℃. After taking out the bacterial liquid, the bacterial liquid is prepared according to the following steps of 1: 100 is inoculated in 100mL of LB medium containing 50. mu.g/mL kanamycin, cultured at 30 ℃ until OD600 is 0.6, added with 1mol/L IPTG to a final concentration of 0.5mmol/L, and cultured with shaking at 18 ℃ to induce expression of the fusion protein. After 12h of induction, the thalli are collected by centrifugation for 10min at 8000 r/min. The resulting mycelia were inoculated with 50mM BufferA (50mM Na)3PO40.5M NaCl; pH7.4) was washed 3 times and 50mL of loading buffer (50mM Na)3PO40.5M NaCl; 5mM imidazole, pH7.4) followed by resuspension, sonication, operating under the following conditions: the power is 50W, the working time is 2s, the interval time is 3s, the alarm temperature is 60 ℃, and the total time is 30 min. After the ultrasonic treatment is finished, the mixture is centrifuged at 12000g for 15min, and then the precipitate and the supernatant are respectively collected for electrophoresis detection. The recombinant FhuPil fusion protein was found to be present in the bacterial cells in solubilized form. Thin-layer scanning showed that the recombinant protein accounted for more than 30% of the total bacterial protein. The wild type FhuPil gene which is not subjected to sequence optimization is expressed in the same way, the expression product only accounts for about 4% of the total protein of the thallus, the expression amount is weak, and the gene optimization effect is good. The sonicated supernatant obtained above was filtered through a 0.45 μm filter and purified by His Trap affinity columns (product of GEHealthcare Co.) according to the method described in the specification. The specific method comprises the following steps:
1.2.1) connecting a chromatography system, wherein the system comprises a sample inlet pipe, a peristaltic pump (Shanghai Huxi analytical instrument factory, model DHL-A), a chromatography column (GE healthcare product, product name His trade affinity columns) and an ultraviolet detector (Shanghai Huxi analytical instrument factory, model HD1), the column volume is 2ml, and the ultraviolet detector is preheated for about 30min until the reading is stable;
1.2.2) proofreading T%: adjusting a brightness knob to display 100%;
1.2.3) rotational sensitivity to the appropriate position, typically 0.2A;
1.2.4) equilibrating the chromatography system with the above buffer until the reading is stable and then rotating "zero" to show "000";
1.2.5) applying protein sample, controlling the flow rate within 5ml/min, and collecting penetration liquid;
1.2.6) washing away unbound protein with loading buffer, recording the reading during the process until the reading does not change any more, and collecting the eluate;
1.2.7) eluting with BufferA +10mM imidazole, and collecting the elution peak;
1.2.8) eluting with BufferA +20mM imidazole, and collecting the elution peak;
1.2.9) eluting with BufferA +40mM imidazole, and collecting an elution peak;
1.2.10) eluting with BufferA +100mM imidazole, and collecting the elution peak;
1.2.11) eluting with BufferA +150mM imidazole, and collecting the elution peak;
1.2.12) taking 100ul of each elution peak sample to carry out SDS-PAGE electrophoresis;
1.2.13) was eluted at 40mM imidazole, and the target protein was found to have a purity of 90% or more, and was adjusted to 0.2mg/mL for use after measuring the protein concentration with the bradford kit. So as to prepare the Acinetobacter baumannii FhuPil fusion protein.
1.3) preparation of polyclonal antibody against Acinetobacter baumannii Fhue and Pilf proteins
1.3.1) mixing the acinetobacter baumannii FhuPil fusion protein prepared in the step (1.2) with Freund's complete adjuvant, emulsifying to serve as immunogen to immunize 2 male New Zealand rabbits, wherein the total amount of subcutaneous injection for each rabbit is 2ml, and the total amount of antigen is 2 mg/rabbit. And then, immunizing once every two weeks by using emulsion formed by the FhuPil fusion protein and Freund's incomplete adjuvant, wherein the immunization is carried out for 5 times totally, and the dosage of the antigen is the same as that of the primary immunization. Large amount of blood is taken 3-5 days after five-immunization, placed at 37 ℃ for 1 hour, then placed in a refrigerator at 4 ℃ overnight, and serum is taken every other day.
1.3.2) determination of the potency of the polyclonal antibody
The FhuPil fusion protein is used as a coating antigen, the coating concentration is 5 mu g/ml, each hole is coated with 100 mu l, and the level of serum antibody is detected by an indirect ELISA method. The serum dilution times of the experimental groups are as follows: 1: 200. 1: 400. 1: 800. 1: 1600. 1: 3200. 1: 6400. 1: 12800. 1: 25600. 1: 51200. 1: 102400, 1: 204800;
the ELISA plate is coated with bovine serum albumin as a negative control, and an enzyme-linked detector is used for measuring OD450, so that the positive result is obtained when the P/N value is more than 2.1. The results showed that the serum antibody titers of 2 rabbits all reached 1: 102400, it is indicated that the immune effect is better.
1.3.3) extraction of polyclonal antibodies
The antibodies were purified using a GE-HiTrap Protein A HP pre-packed column as described, in the following manner:
1.3.3.1) 5mL of antiserum was taken, 0.5mL of 1M Tris (pH8.0) was added to adjust to pH8.0, and 20,000 g was centrifuged for 20min to remove the precipitate.
1.3.3.2) was applied to the column, and then washed with 10 column volumes of buffer A (100mM Tris-Cl, pH8.0) and then with 10 column volumes of buffer B (10mM Tris-Cl, pH 8.0).
1.3.3.3) eluted IgG with approximately three column volumes of IgG elution buffer (100mM glycine, pH 3.0). (0.1 mL IgG-neutralizing buffer (1M Tris-Cl, pH8.0) was preloaded into the collection tube, 0.9mL of eluent was added to each tube)
1.3.3.4) the eluate was dialyzed against 50 volumes of Tris (10mM Tris-Cl, pH 8.0).
1.3.3.5) ultrafiltering and concentrating, adjusting the concentration to 5mg/ml, and storing at-70 ℃ for later use. So as to prepare the polyclonal antibody against the Acinetobacter baumannii Fhue and Pilf proteins.
Example 2
Preparation of polyclonal antibodies against Acinetobacter baumannii OmpA and ponA proteins:
1.1) cloning of Acinetobacter baumannii OmpPon fusion Gene
The method comprises the steps of obtaining peptide segments with the most abundant antigenic epitopes in extracellular domains of acinetobacter baumannii surface proteins OmpA and ponA (the accession numbers in an NCBI protein database are AJF83030 and ADX05080 respectively), finding out gene coding sequences of the peptide segments, optimizing the gene coding sequences of the peptide segments, and connecting the two sequences by using coding sequences of rigid connecting peptide (eaaakaaaak) to form a fusion gene. Meanwhile, after introducing a restriction enzyme site NdeI at the 5 'end and a termination signal TAA and a restriction enzyme site BamHI at the 3' end of the fusion gene, a complete gene sequence is chemically synthesized and is marked as OmpPon. The complete gene sequence and the coded amino acid sequence are shown in a sequence table. Specifically, the protein sequence encoded by the OmpPon gene is 31-173aa of the Acinetobacter baumannii surface protein OmpA and 406-572aa of the surface protein ponA, and the two protein sequences are connected by a rigid connecting peptide (eaaakaaaak). The gene sequence is delivered to Nanjing Jinslei Biotech, Inc. for complete gene chemical synthesis, and the artificially synthesized gene fragment is connected to vector pUC57 when delivered. The vector pUC57 containing the artificially synthesized DNA fragment was digested with NdeI and BamHI, and the desired fragment was recovered by a conventional method and used. And simultaneously carrying out double enzyme digestion on the vector pET-28a (+) by NdeI and BamHI, connecting the OmpPon gene obtained after double enzyme digestion into the pET-28a (+) vector according to a conventional molecular biology method, and transforming Escherichia coli TOP10 to construct a pET-OmpPon expression vector. The construction of the expression vector is verified to be correct by enzyme digestion and sequence determination. The vector expresses recombinant OmpPon fusion protein.
1.2) expression and purification of Acinetobacter baumannii OmpPon fusion protein
Culturing the positive clone bacteria, extracting plasmid, transferring into competent E.coliBL21(DE3) according to conventional technique, coating the bacteria liquid on LB plate containing 50 ug/mL kanamycin, and screening expression strain according to conventional method. Individual colonies transformed with pET-OmpPon having the ability to express a foreign protein were picked and inoculated into 100mL of LB medium and cultured overnight at 37 ℃. After taking out the bacterial liquid, the bacterial liquid is prepared according to the following steps of 1: 100 was inoculated into 100mL of LB medium containing 50. mu.g/mL of kanamycin, and when the cells were cultured at 30 ℃ until OD600 became 0.6, 1mol/L of IPTG was added to a final concentration of 1mmol/L, and the cells were cultured with shaking at 37 ℃ to induce expression of the fusion protein. After inducing for 6h, centrifuging at 8000r/min for 10min, and collecting thallus. The resulting mycelia were inoculated with 50mM BufferA (50mM Na)3PO40.5M NaCl; pH7.4) was washed 3 times and 50mL of loading buffer (50mM Na)3PO40.5M NaCl; 5mM imidazole, pH7.4) followed by resuspension, sonication, operating under the following conditions: the power is 50W, the working time is 2s, the interval time is 3s, the alarm temperature is 60 ℃, and the total time is 30 min. After the ultrasonic treatment is finished, the mixture is centrifuged at 12000g for 15min, and then the precipitate and the supernatant are respectively collected for electrophoresis detection. The recombinant OmpPon fusion protein was found to exist in the cell in a partially solubilized form (the other part was found to exist in the form of inclusion bodies). Thin-layer scanning showed that the recombinant protein accounted for more than 30% of the total bacterial protein. While the wild-type OmpPon gene which was not sequence-optimized was as aboveThe expression is carried out in the same way, and no expression product is found, which indicates that the gene optimization effect is good. The sonicated supernatant obtained above was filtered through a 0.45 μm filter and purified by His Trap affinity columns (Gehethecare corporation) according to the following method:
1.2.1) connecting a chromatography system, wherein the system comprises a sample inlet pipe, a peristaltic pump (Shanghai Huxi analytical instrument factory, model DHL-A), a chromatography column (GE healthcare product, product name His trade affinity columns) and an ultraviolet detector (Shanghai Huxi analytical instrument factory, model HD1), the column volume is 2ml, and the ultraviolet detector is preheated for about 30min until the reading is stable;
1.2.2) proofreading T%: adjusting a brightness knob to display 100%;
1.2.3) rotational sensitivity to the appropriate position, typically 0.2A;
1.2.4) equilibrating the chromatography system with the above buffer until the reading is stable and then rotating "zero" to show "000";
1.2.5) applying protein sample, controlling the flow rate within 5ml/min, and collecting penetration liquid;
1.2.6) washing away unbound protein with loading buffer, recording the reading during the process until the reading does not change any more, and collecting the eluate;
1.2.7) eluting with BufferA +10mM imidazole, and collecting the elution peak;
1.2.8) eluting with BufferA +20mM imidazole, and collecting the elution peak;
1.2.9) eluting with BufferA +40mM imidazole, and collecting an elution peak;
1.2.10) eluting with BufferA +100mM imidazole, and collecting the elution peak;
1.2.11) eluting with BufferA +150mM imidazole, and collecting the elution peak;
1.2.12) taking 100ul of each elution peak sample to carry out SDS-PAGE electrophoresis;
1.2.13) was eluted at 40mM imidazole, and the target protein was found to have a purity of 90% or more, and was adjusted to 0.2mg/mL for use after measuring the protein concentration with the bradford kit. Thus obtaining the Acinetobacter baumannii OmpPon fusion protein.
1.3) preparation of polyclonal antibody against Acinetobacter baumannii OmpA and ponA proteins
1.3.1) mixing the Acinetobacter baumannii OmpPon fusion protein prepared in the step (1.2) with Freund's complete adjuvant, emulsifying to serve as immunogen to immunize 2 male New Zealand rabbits, wherein the total amount of subcutaneous injection of each rabbit is 2ml, and the total amount of antigen is 2 mg/rabbit. And then, the emulsion formed by the OmpPon fusion protein and Freund's incomplete adjuvant is used for immunization once every two weeks for 5 times, and the dosage of the antigen is the same as that of the primary immunization. Large amount of blood is taken 3-5 days after five-immunization, placed at 37 ℃ for 1 hour, then placed in a refrigerator at 4 ℃ overnight, and serum is taken every other day.
1.3.2) determination of the potency of the polyclonal antibody
OmpPon fusion protein is used as a coating antigen, the coating concentration is 5 mu g/ml, each well is coated with 100 mu l, and the level of serum antibody is detected by an indirect ELISA method. The serum dilution times of the experimental groups are as follows: 1: 200. 1: 400. 1: 800. 1: 1600. 1: 3200. 1: 6400. 1: 12800. 1: 25600. 1: 51200. 1: 102400, 1: 204800;
the ELISA plate is coated with bovine serum albumin as a negative control, and an enzyme-linked detector is used for measuring OD450, so that the positive result is obtained when the P/N value is more than 2.1. The results showed that the serum antibody titers of 2 rabbits all reached 1: 102400, it is indicated that the immune effect is better.
1.3.3) extraction of polyclonal antibodies
The antibodies were purified using a GE-HiTrap Protein A HP pre-packed column as described, in the following manner:
1.3.3.1) 5mL of antiserum was taken, 0.5mL of 1M Tris (pH8.0) was added to adjust to pH8.0, and 20,000 g was centrifuged for 20min to remove the precipitate.
1.3.3.2) was applied to the column, and then washed with 10 column volumes of buffer A (100mM Tris-Cl, pH8.0) and then with 10 column volumes of buffer B (10mM Tris-Cl, pH 8.0).
1.3.3.3) eluted IgG with approximately three column volumes of IgG elution buffer (100mM glycine, pH 3.0). (0.1 mL IgG-neutralizing buffer (1M Tris-Cl, pH8.0) was preloaded into the collection tube, 0.9mL of eluent was added to each tube)
1.3.3.4) the eluate was dialyzed against 50 volumes of Tris (10mM Tris-Cl, pH 8.0).
1.3.3.5) ultrafiltering and concentrating, adjusting the concentration to 5mg/ml, and storing at-70 ℃ for later use. Thus, polyclonal antibodies against OmpA and ponA proteins of acinetobacter baumannii are prepared.
Example 3
High-sensitivity Acinetobacter baumannii antigen Elisa determination kit composition
An ELISA plate coated with anti-Acinetobacter baumannii Fhue and Pilf protein polyclonal antibody, anti-Acinetobacter baumannii OmpA and ponA protein polyclonal antibody, Acinetobacter baumannii positive control, negative control, washing liquid, enzyme-labeled secondary antibody, enzyme chromogenic substrate and stop solution jointly form the high-sensitivity Acinetobacter baumannii antigen Elisa determination kit.
(1) ELISA plate coated with polyclonal antibody against Acinetobacter baumannii Fhue and Pilf protein
The polyclonal antibody against Acinetobacter baumannii Fhue and Pilf protein (prepared in example 1) was diluted to a concentration of 10. mu.g/mL with PBS buffer, and a 96-well EIA high-efficiency binding ELISA plate (model: corning costar2592) was coated at 100. mu.L/well and coated at 37 ℃ for 2 hours. After being taken out, the plate is washed three times by 250 mu L of washing liquid and is dried. Using a washing solution containing 1% BSA as a blocking solution, 250. mu.L/well of the blocking solution was applied to an ELISA plate, and the plate was blocked at 37 ℃ for 1 hour. Taking out, washing the plate with 250 μ L of washing solution for 3 times, one minute each time, spin-drying, sealing, and storing.
Wherein, the PBS buffer solution formula: 1.4g of disodium hydrogen phosphate, 0.2g of sodium dihydrogen phosphate, 8.5g of sodium chloride and 1000mL of deionized water with the pH value of 7.4; washing liquid: PBS aqueous solution containing 0.01% Tween-20, pH 7.4; sealing liquid: aqueous washing containing 1% BSA, pH 7.4.
(2) Polyclonal antibody against OmpA and ponA proteins of acinetobacter baumannii
The polyclonal antibodies against Acinetobacter baumannii OmpA and ponA proteins as described in example 2 were prepared into a 20. mu.g/mL solution using the above blocking solution, and packaged in an amount of 5mL per kit.
(3) Enzyme-labeled secondary antibody
The enzyme-labeled secondary antibody is horseradish peroxidase-labeled goat anti-rabbit IgG which is purchased from Beijing Ceh Biotechnology Co., Ltd in the example, is product number 030005-G, has a concentration of 1mg/mL, and is diluted 3000 times by PBS buffer solution when in use. The mixture was packaged in an amount of 0.1mL per kit.
(4) Enzyme chromogenic substrate
The enzyme chromogenic substrate preparation process is as follows:
solution A: (configuration amount 1L)
1. 3.14g of citric acid (containing 1 molecule of crystal water and having a molecular weight of 210.14g) and 11.56g of sodium acetate (containing 3 molecules of crystal water and having a molecular weight of 136.0) were weighed and dissolved in 970mL of double distilled water to prepare an aqueous solution of sodium acetate having a pH of 5.0.
2. Weighing 0.08g of phenacetin, adding 30mL of double distilled water, heating to 100 ℃, and adding the mixture into the solution in the first step after completely dissolving.
3. Then 0.5g of carbamide peroxide is added and mixed evenly.
And B, liquid B: (configuration amount 1L)
Adding 500mL of methanol into a 2L beaker, adding 1.27g of 3,3,5, 5-tetramethylbenzidine TMB (SIGMA), heating at 60 ℃ to dissolve, and adding 500mL of glycerol.
A, B liquid 1: 1, mixing to prepare an enzyme chromogenic substrate. The solution A and the solution B were packaged in an amount of 5mL each per kit.
(5) Positive and negative controls
The positive control is formaldehyde-inactivated acinetobacter baumannii (ATCC19606) prepared as follows: taking Acinetobacter baumannii bacterial liquid cultured by a chocolate liquid culture medium, counting plates, centrifuging and collecting thalli, resuspending the thalli by using normal saline and adjusting the concentration of the thalli to 1 x 109CFU/mL. 5mL of the bacterial solution was added with analytically pure formaldehyde to a final concentration of 1% and inactivated overnight at 4 ℃. And centrifuging 12000g for 10 minutes, then removing the supernatant, adding 2mL of PBS buffer solution into the precipitate, and resuspending to obtain the positive control.
Negative control: the negative control was formaldehyde-inactivated escherichia coli (ATCC 25922) prepared as follows: taking Escherichia coli cultured in LB liquid culture medium, counting, centrifuging, collecting thallus, resuspending thallus with normal saline, and adjusting thallus concentration to 1 × 109CFU/mL. Adding analytically pure formaldehyde into 5mL of bacterial liquid until the final concentration is 1%, and inactivating at 4 DEG COvernight. Centrifuging 12000g for 10 minutes, discarding the supernatant, adding 2mL PBS buffer solution into the precipitate, and resuspending to obtain the negative control.
(6) Cleaning solution
The specific preparation method of the PBST solution is as described in (1). Packaging the obtained product in an amount of 200mL per kit.
(7) Stopping liquid
1M HCl solution prepared with double distilled water. Packaging the obtained product in an amount of 10mL per kit.
Example 4
Use method of high-sensitivity acinetobacter baumannii antigen Elisa determination kit
1) Treatment of samples to be examined
A pharyngeal swab of a subject is obtained by a conventional method, and the pharyngeal swab is inserted into a soft plastic tube containing 500. mu.L of a washing solution (PBST), and the tube wall of the plastic tube is pressed to sufficiently dissolve a sample on the swab.
2) Adding control and sample to be tested
Adding 100 mu L of sample to be detected into corresponding enzyme labeled holes, setting 1 hole of positive control (100 mu L/hole) and 3 holes of negative control (100 mu L/hole), incubating for 1 hour at 37 ℃, washing the plate for 3 times by using 250 mu L of washing liquid, and drying by spinning.
3) Adding polyclonal antibodies against OmpA and ponA proteins of acinetobacter baumannii
Adding the work solution of anti-Acinetobacter baumannii OmpA and ponA protein polyclonal antibody in the kit, incubating at the temperature of 37 ℃ for 1 hour at a concentration of 50 mu L/hole, washing the plate for 3 times by using 250 mu L of PBST washing solution, and drying by spinning.
4) Adding enzyme-labeled secondary antibody
The enzyme-labeled secondary antibody in the kit described in example 3 was diluted 1:3000 with PBS buffer to prepare a working solution, and corresponding enzyme-labeled wells were added at 50. mu.L/well, incubated at 37 ℃ for 1 hour, and then washed 3 times with 250. mu.L of PBST washing solution, and spun-dried.
5) Adding enzyme chromogenic substrate
Add freshly prepared enzyme chromogenic substrate in the kit, 50. mu.L/well, develop for 15 minutes.
6) Adding stop solution
The reaction was stopped by adding 50. mu.L of stop solution to each well.
7) Measuring OD450nm value
The microplate was placed in a microplate reader to determine the OD450nm value.
8) Determination of results
Respectively reading the OD450nm values of the 3-hole negative quality control sample and the 1-hole positive quality control sample; the sum of the average value of the OD450nm readings of the 3-well negative quality control sample and the 3-fold standard deviation is the CUT-OFF value; if the detected OD450nm value of the human pharynx swab sample is larger than the CUT-OFF value, the acinetobacter baumannii antigen in the clinical pharynx swab is judged to be positive, otherwise, the acinetobacter baumannii antigen in the human pharynx swab sample is judged to be negative; if the OD450nm value of the positive quality control sample is less than the CUT-OFF value, the kit is invalid.
Example 5
Specificity and sensitivity determination of high-sensitivity acinetobacter baumannii antigen Elisa determination kit
1) Specific assay
In order to verify the specificity of the high-sensitivity acinetobacter baumannii antigen Elisa assay kit, the kit composition and method are 2 × 10 for concentration according to the embodiment 3 and the embodiment 45CFU/mL of 6 Acinetobacter baumannii strains and 17 non-Acinetobacter baumannii standard strains were tested and shown in Table 1. The result shows that the detection results of the kit are positive for all 6 Acinetobacter baumannii strains, and the detection results of the kit are negative for other 17 respiratory common pathogenic microorganisms. The kit showed good specificity.
TABLE 1
Figure BDA0002255599460000141
At the same time, the concentration is 2 multiplied by 105120 Acinetobacter baumannii clinical isolates of CFU/mL are detected by the kit, the results are positive, and the kit is shown to be used for clinical diagnosisHigh assay coverage of isolated acinetobacter baumannii.
2) Sensitivity assay
Inoculating Acinetobacter baumannii ATCC19606 strain in sheep blood chocolate culture medium, culturing at 35 deg.C for 24 hr, diluting with normal saline 10 times gradient, and counting to obtain 10-fold thallus concentration8-102CFU/mL of the bacterial solution, 100. mu.L of the bacterial solution was dropped on the microplate, and detection was performed according to the kit composition and method described in examples 3 and 4. The result shows that the detection sensitivity of the kit is 103CFU/mL。
Sequence listing
<110> Hubei university of industry
<120> high-sensitivity acinetobacter baumannii antigen Elisa determination kit
<141>2019-10-31
<160>4
<170>SIPOSequenceListing 1.0
<210>1
<211>1062
<212>DNA
<213> complete sequence of the Gene of FhuPil (FhuPil)
<400>1
catatgttcg atggcaacta cctggacccg gtagaaggta actctactga agttggtctc 60
aaatccgctt ggtttgacgg ccgtctgaac ggtaccctgg ctctgtacca catcaaacag 120
gacaacctgg cacaggaagc gggccaagtt acccgtaacg gtgttaaaga gacttactac 180
cgtgcagcga aaggcgctac atccgaaggt tttgaagttg aagtatcagg tcagatcact 240
ccagattgga acatcaccgc aggttactct caattttctg ctaaggacgc gaacgatgcg 300
gacgtaaaca ctcagcttcc gcgtaaaatg atccagagct tcactaccta taaactgcct 360
ggtaaactgg aaaacatcac agttggcggt ggcgtaaact ggcagtcttc tacctacgtg 420
aacgcaaaaa acccgaaaaa agtaatcgaa aaagttgagc agggtgacta cgctctggta 480
aacctgatgg cgcgttacca gatcactaag gacttttctg cacagcttaa cattaacaac 540
gttttcggtg gttctggtgg ttctggtggt tctggtggtt ctgcggttaa ggtacgcact 600
caactggcgg ctgaatatat ccgttcaggt gatctggact ccgcgaaacg ctccctggac 660
caggccctga gcgttgactc tcgtgacgcg acagcaaaca tgatgatggg catcctgctg 720
cagcaggagg gctctaaatc taacctggag aaagcggagc actacttcaa acgtgctatc 780
agttctgaac cggataacgc tcaggcgcgc aacaactatg gtacctatct gtaccagatg 840
gaacgttata acgacgcgat tgaacagttt cgtatcgcag gtgcgaccct gggttatgat 900
cagcgttatc aggcgctgga aaacctgggc cgcatctacc tgaagctggg taacatcgcc 960
agcgctgaaa aaactttcaa gcaggcactg ctggcgaacc gtgactccta catctctatg 1020
ctggagctgg ctgaaatctt ttacctgcag cagtaaggat cc 1062
<210>2
<211>350
<212>PRT
<213> protein sequence of FhuPil (FhuPil)
<400>2
Met Phe Asp Gly Asn Tyr Leu Asp Pro Val Glu Gly Asn Ser Thr Glu
1 5 10 15
Val Gly Leu Lys Ser Ala Trp Phe Asp Gly Arg Leu Asn Gly Thr Leu
20 25 30
Ala Leu Tyr His Ile Lys Gln Asp Asn Leu Ala Gln Glu Ala Gly Gln
35 40 45
Val Thr Arg Asn Gly Val Lys Glu Thr Tyr Tyr Arg Ala Ala Lys Gly
50 55 60
Ala Thr Ser Glu Gly Phe Glu Val Glu Val Ser Gly Gln Ile Thr Pro
65 70 75 80
Asp Trp Asn Ile Thr Ala Gly Tyr Ser Gln Phe Ser Ala Lys Asp Ala
85 90 95
Asn Asp Ala Asp Val Asn Thr Gln Leu Pro Arg Lys Met Ile Gln Ser
100 105 110
Phe Thr Thr Tyr Lys Leu Pro Gly Lys Leu Glu Asn Ile Thr Val Gly
115 120 125
Gly Gly Val Asn Trp Gln Ser Ser Thr Tyr Val Asn Ala Lys Asn Pro
130 135 140
Lys Lys Val Ile Glu Lys Val Glu Gln Gly Asp Tyr Ala Leu Val Asn
145 150 155 160
Leu Met Ala Arg Tyr Gln Ile Thr Lys Asp Phe Ser Ala Gln Leu Asn
165 170 175
Ile Asn Asn Val Phe Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly
180 185 190
Ser Ala Val Lys Val Arg Thr Gln Leu Ala Ala Glu Tyr Ile Arg Ser
195 200 205
Gly Asp Leu Asp Ser Ala Lys Arg Ser Leu Asp Gln Ala Leu Ser Val
210 215 220
Asp Ser Arg Asp Ala Thr Ala Asn Met Met Met Gly Ile Leu Leu Gln
225 230 235 240
Gln Glu Gly Ser Lys Ser Asn Leu Glu Lys Ala Glu His Tyr Phe Lys
245 250 255
Arg Ala Ile Ser Ser Glu Pro Asp Asn Ala Gln Ala Arg Asn Asn Tyr
260 265 270
Gly Thr Tyr Leu Tyr Gln Met Glu Arg Tyr Asn Asp Ala Ile Glu Gln
275 280 285
Phe Arg Ile Ala Gly Ala Thr Leu Gly Tyr Asp Gln Arg Tyr Gln Ala
290 295 300
Leu Glu Asn Leu Gly Arg Ile Tyr Leu Lys Leu Gly Asn Ile Ala Ser
305 310 315 320
Ala Glu Lys Thr Phe Lys Gln Ala Leu Leu Ala Asn Arg Asp Ser Tyr
325 330 335
Ile Ser Met Leu Glu Leu Ala Glu Ile Phe Tyr Leu Gln Gln
340 345 350
<210>3
<211>983
<212>DNA
<213> complete Gene sequence of OmpPon (OmpPon)
<400>3
catatgctgg gttacacctt ccaggatact cagcacaaca acggcggtaa agacggtgaa 60
ctgaccaacg gtccggaact gcaggacgac ctgttcgttg gtgctgcgct gggtatcgaa 120
ctgactccgt ggctgggttt cgaagccgag tataaccagg ttaagggtga cgtggatggc 180
ctggcagcgg gtgctgaata caaacagaaa cagatcaacg gtaacttcta cgttaccagc 240
gacctgatta ccaagaacta tgactctaaa atcaaaccgt atgttctgct gggtgcgggc 300
cactacaaat acgaaatccc ggacctttcc tatcacaacg acgaggaagg cactctgggt 360
aacgcgggtg ttggtgcttt ctggcgtctg aacgacgctctgtctctgcg taccgaagct 420
cgtggtacct ataacgaagc tgctgctgct aaagaagctg ctgctgctaa aggctctatc 480
gaagctatcg taggtggtta caacttctac cagtccaagt ttaaccgtgc gctccagggc 540
tggcgccagc cgggctctac cattaaacct ttcctgtacg ctctggctct ggaacgtggc 600
atgaccccgt acagcatggt aaacgattct ccgatcacta ttggtaaatg gaccccaaaa 660
aattctgacg gccgttacct gggtatgatc ccgctgcgtc gcgctctgta cctgtcccgt 720
aacactgtat ccgttcgtct gctgcagact gttggcatcg aacgtacccg ccaactgttt 780
atggatttcg gtctgcagga agaccagatt ccacgtaact acactatcgc tctgggcact 840
ccgcaggtac tgccgatcca gatggctacc ggctacgcta ctttcgctaa tggcggctac 900
cgtgttcagc cacatttcat ccagcgtatc gaagacgcgt atggtaaagt aatttacgaa 960
gctaaaccgg aatataagga tcc 983
<210>4
<211>324
<212>PRT
<213> protein sequence of OmpPon (OmpPon)
<400>4
Met Leu Gly Tyr Thr Phe Gln Asp Thr Gln His Asn Asn Gly Gly Lys
1 5 10 15
Asp Gly Glu Leu Thr Asn Gly Pro Glu Leu Gln Asp Asp Leu Phe Val
20 25 30
Gly Ala Ala Leu Gly Ile Glu Leu Thr Pro Trp Leu Gly Phe Glu Ala
35 40 45
Glu Tyr Asn Gln Val Lys Gly Asp Val Asp Gly Leu Ala Ala Gly Ala
50 55 60
Glu Tyr Lys Gln Lys Gln Ile Asn Gly Asn Phe Tyr Val Thr Ser Asp
65 70 75 80
Leu Ile Thr Lys Asn Tyr Asp Ser Lys Ile Lys Pro Tyr Val Leu Leu
85 90 95
Gly Ala Gly His Tyr Lys Tyr Glu Ile Pro Asp Leu Ser Tyr His Asn
100 105 110
Asp Glu Glu Gly Thr Leu Gly Asn Ala Gly Val Gly Ala Phe Trp Arg
115 120 125
Leu Asn Asp Ala Leu Ser Leu Arg Thr Glu Ala Arg Gly Thr Tyr Asn
130 135 140
Glu Ala Ala Ala Ala Lys Glu Ala Ala Ala Ala Lys Gly Ser Ile Glu
145 150 155 160
Ala Ile Val Gly Gly Tyr Asn Phe Tyr Gln Ser Lys Phe Asn Arg Ala
165 170 175
Leu Gln Gly Trp Arg Gln Pro Gly Ser Thr Ile Lys Pro Phe Leu Tyr
180 185 190
Ala Leu Ala Leu Glu Arg Gly Met Thr Pro Tyr Ser Met Val Asn Asp
195 200 205
Ser Pro Ile Thr Ile Gly Lys Trp Thr Pro Lys Asn Ser Asp Gly Arg
210 215 220
Tyr Leu Gly Met Ile Pro Leu Arg Arg Ala Leu Tyr Leu Ser Arg Asn
225 230 235 240
Thr Val Ser Val Arg Leu Leu Gln Thr Val Gly Ile Glu Arg Thr Arg
245 250 255
Gln Leu Phe Met Asp Phe Gly Leu Gln Glu Asp Gln Ile Pro Arg Asn
260 265 270
Tyr Thr Ile Ala Leu Gly Thr Pro Gln Val Leu Pro Ile Gln Met Ala
275 280 285
Thr Gly Tyr Ala Thr Phe Ala Asn Gly Gly Tyr Arg Val Gln Pro His
290 295 300
Phe Ile Gln Arg Ile Glu Asp Ala Tyr Gly Lys Val Ile Tyr Glu Ala
305 310 315 320
Lys Pro Glu Tyr

Claims (8)

1. An acinetobacter baumannii surface protein (Fhue and Pilf), characterized in that: the protein sequence of the acinetobacter baumannii surface protein (Fhue and Pilf) is as follows:
MFDGNYLDPVEGNSTEVGLKSAWFDGRLNGTLALYHIKQDNLAQEAGQVTRNGVKETYYRAAKGATSEGFEVEVSGQITPDWNITAGYSQFSAKDANDADVNTQLPRKMIQSFTTYKLPGKLENITVGGGVNWQSSTYVNAKNPKKVIEKVEQGDYALVNLMARYQITKDFSAQLNINNVFGGSGGSGGSGGSAVKVRTQLAAEYIRSGDLDSAKRSLDQALSVDSRDATANMMMGILLQQEGSKSNLEKAEHYFKRAISSEPDNAQARNNYGTYLYQMERYNDAIEQFRIAGATLGYDQRYQALENLGRIYLKLGNIASAEKTFKQALLANRDSYISMLELAEIFYLQQ；
the complete sequence of the gene for the protein coding for the surface protein of Acinetobacter baumannii (Fhue and Pilf) is:
CATATGTTCGATGGCAACTACCTGGACCCGGTAGAAGGTAACTCTACTGAAGTTGGTCTCAAATCCGCTTGGTTTGACGGCCGTCTGAACGGTACCCTGGCTCTGTACCACATCAAACAGGACAACCTGGCACAGGAAGCGGGCCAAGTTACCCGTAACGGTGTTAAAGAGACTTACTACCGTGCAGCGAAAGGCGCTACATCCGAAGGTTTTGAAGTTGAAGTATCAGGTCAGATCACTCCAGATTGGAACATCACCGCAGGTTACTCTCAATTTTCTGCTAAGGACGCGAACGATGCGGACGTAAACACTCAGCTTCCGCGTAAAATGATCCAGAGCTTCACTACCTATAAACTGCCTGGTAAACTGGAAAACATCACAGTTGGCGGTGGCGTAAACTGGCAGTCTTCTACCTACGTGAACGCAAAAAACCCGAAAAAAGTAATCGAAAAAGTTGAGCAGGGTGACTACGCTCTGGTAAACCTGATGGCGCGTTACCAGATCACTAAGGACTTTTCTGCACAGCTTAACATTAACAACGTTTTCGGTGGTTCTGGTGGTTCTGGTGGTTCTGGTGGTTCTGCGGTTAAGGTACGCACTCAACTGGCGGCTGAATATATCCGTTCAGGTGATCTGGACTCCGCGAAACGCTCCCTGGACCAGGCCCTGAGCGTTGACTCTCGTGACGCGACAGCAAACATGATGATGGGCATCCTGCTGCAGCAGGAGGGCTCTAAATCTAACCTGGAGAAAGCGGAGCACTACTTCAAACGTGCTATCAGTTCTGAACCGGATAACGCTCAGGCGCGCAACAACTATGGTACCTATCTGTACCAGATGGAACGTTATAACGACGCGATTGAACAGTTTCGTATCGCAGGTGCGACCCTGGGTTATGATCAGCGTTATCAGGCGCTGGAAAACCTGGGCCGCATCTACCTGAAGCTGGGTAACATCGCCAGCGCTGAAAAAACTTTCAAGCAGGCACTGCTGGCGAACCGTGACTCCTACATCTCTATGCTGGAGCTGGCTGAAATCTTTTACCTGCAGCAGTAAGGATCC。
2. method for the preparation of acinetobacter baumannii surface proteins (Fhue and Pilf) according to claim 1, characterized in that: the method comprises the following steps:
respectively obtaining peptide segments with the most abundant antigenic epitopes in the extracellular domains of the Acinetobacter baumannii surface protein Fhue and the surface protein Pilf, finding out the gene coding sequence of the peptide segments, optimizing the gene coding sequence of the peptide segments, and connecting the optimized gene coding sequence of the peptide segments by using the coding sequence of flexible connecting peptide to form a fusion gene; the Acinetobacter baumannii surface protein Fhue and the surface protein Pilf have access numbers of KMV27515 and AJF80497 in an NCBI protein database respectively; the sequence of the flexible connecting peptide is ggsggsggsggs; simultaneously, enzyme cutting site NdeI is introduced into the 5 'end of the fusion gene, and termination signal TAA and enzyme cutting site BamHI are introduced into the 3' end of the fusion gene, and then a complete gene sequence is chemically synthesized and is marked as FhuPil;
the complete sequence of the gene of the FhuPil is as follows:
CATATGTTCGATGGCAACTACCTGGACCCGGTAGAAGGTAACTCTACTGAAGTTGGTCTCAAATCCGCTTGGTTTGACGGCCGTCTGAACGGTACCCTGGCTCTGTACCACATCAAACAGGACAACCTGGCACAGGAAGCGGGCCAAGTTACCCGTAACGGTGTTAAAGAGACTTACTACCGTGCAGCGAAAGGCGCTACATCCGAAGGTTTTGAAGTTGAAGTATCAGGTCAGATCACTCCAGATTGGAACATCACCGCAGGTTACTCTCAATTTTCTGCTAAGGACGCGAACGATGCGGACGTAAACACTCAGCTTCCGCGTAAAATGATCCAGAGCTTCACTACCTATAAACTGCCTGGTAAACTGGAAAACATCACAGTTGGCGGTGGCGTAAACTGGCAGTCTTCTACCTACGTGAACGCAAAAAACCCGAAAAAAGTAATCGAAAAAGTTGAGCAGGGTGACTACGCTCTGGTAAACCTGATGGCGCGTTACCAGATCACTAAGGACTTTTCTGCACAGCTTAACATTAACAACGTTTTCGGTGGTTCTGGTGGTTCTGGTGGTTCTGGTGGTTCTGCGGTTAAGGTACGCACTCAACTGGCGGCTGAATATATCCGTTCAGGTGATCTGGACTCCGCGAAACGCTCCCTGGACCAGGCCCTGAGCGTTGACTCTCGTGACGCGACAGCAAACATGATGATGGGCATCCTGCTGCAGCAGGAGGGCTCTAAATCTAACCTGGAGAAAGCGGAGCACTACTTCAAACGTGCTATCAGTTCTGAACCGGATAACGCTCAGGCGCGCAACAACTATGGTACCTATCTGTACCAGATGGAACGTTATAACGACGCGATTGAACAGTTTCGTATCGCAGGTGCGACCCTGGGTTATGATCAGCGTTATCAGGCGCTGGAAAACCTGGGCCGCATCTACCTGAAGCTGGGTAACATCGCCAGCGCTGAAAAAACTTTCAAGCAGGCACTGCTGGCGAACCGTGACTCCTACATCTCTATGCTGGAGCTGGCTGAAATCTTTTACCTGCAGCAGTAAGGATCC；
the protein sequence encoded by the FhuPil gene is as follows:
MFDGNYLDPVEGNSTEVGLKSAWFDGRLNGTLALYHIKQDNLAQEAGQVTRNGVKETYYRAAKGATSEGFEVEVSGQITPDWNITAGYSQFSAKDANDADVNTQLPRKMIQSFTTYKLPGKLENITVGGGVNWQSSTYVNAKNPKKVIEKVEQGDYALVNLMARYQITKDFSAQLNINNVFGGSGGSGGSGGSAVKVRTQLAAEYIRSGDLDSAKRSLDQALSVDSRDATANMMMGILLQQEGSKSNLEKAEHYFKRAISSEPDNAQARNNYGTYLYQMERYNDAIEQFRIAGATLGYDQRYQALENLGRIYLKLGNIASAEKTFKQALLANRDSYISMLELAEIFYLQQ；
the protein sequence coded by the FhuPil gene is 509-688aa of the surface protein Fhue of acinetobacter baumannii and 28-184aa of the surface protein Pilf; the two protein sequences are connected by flexible connecting peptide ggsggsggs;
cloning the FhuPil gene complete sequence into prokaryotic expression vector pET-28a (+) by a conventional method, transferring the gene complete sequence into E.coliBL21(DE3) bacteria, inducing the recombinant escherichia coli expression by IPTG, and using Ni to2+And (3) purifying the recombinant His-FhuPil protein by affinity chromatography.
3. A method for preparing polyclonal antibodies against acinetobacter baumannii surface proteins (Fhue and Pilf), characterized in that: the method comprises the steps of taking the recombinant His-FhuPil protein as an immune antigen as claimed in claim 2, mixing the recombinant His-FhuPil protein with Freund's adjuvant, repeatedly and artificially immunizing healthy New Zealand white rabbits, drawing blood for titer measurement, separating and purifying high-titer recombinant protein antibodies, and finally obtaining the polyclonal antibody against the surface proteins (Fhue and Pilf) of the acinetobacter baumannii.
4. An acinetobacter baumannii surface protein (OmpA and ponA), characterized in that: the protein sequences of the acinetobacter baumannii surface proteins (OmpA and ponA) are as follows:
MLGYTFQDTQHNNGGKDGELTNGPELQDDLFVGAALGIELTPWLGFEAEYNQVKGDVDGLAAGAEYKQKQINGNFYVTSDLITKNYDSKIKPYVLLGAGHYKYEIPDLSYHNDEEGTLGNAGVGAFWRLNDALSLRTEARGTYNEAAAAKEAAAAKGSIEAIVGGYNFYQSKFNRALQGWRQPGSTIKPFLYALALERGMTPYSMVNDSPITIGKWTPKNSDGRYLGMIPLRRALYLSRNTVSVRLLQTVGIERTRQLFMDFGLQEDQIPRNYTIALGTPQVLPIQMATGYATFANGGYRVQPHFIQRIEDAYGKVIYEAKPEY；
the complete sequence of the gene for the protein encoding the surface protein of acinetobacter baumannii (OmpA and ponA) is:
CATATGCTGGGTTACACCTTCCAGGATACTCAGCACAACAACGGCGGTAAAGACGGTGAACTGACCAACGGTCCGGAACTGCAGGACGACCTGTTCGTTGGTGCTGCGCTGGGTATCGAACTGACTCCGTGGCTGGGTTTCGAAGCCGAGTATAACCAGGTTAAGGGTGACGTGGATGGCCTGGCAGCGGGTGCTGAATACAAACAGAAACAGATCAACGGTAACTTCTACGTTACCAGCGACCTGATTACCAAGAACTATGACTCTAAAATCAAACCGTATGTTCTGCTGGGTGCGGGCCACTACAAATACGAAATCCCGGACCTTTCCTATCACAACGACGAGGAAGGCACTCTGGGTAACGCGGGTGTTGGTGCTTTCTGGCGTCTGAACGACGCTCTGTCTCTGCGTACCGAAGCTCGTGGTACCTATAACGAAGCTGCTGCTGCTAAAGAAGCTGCTGCTGCTAAAGGCTCTATCGAAGCTATCGTAGGTGGTTACAACTTCTACCAGTCCAAGTTTAACCGTGCGCTCCAGGGCTGGCGCCAGCCGGGCTCTACCATTAAACCTTTCCTGTACGCTCTGGCTCTGGAACGTGGCATGACCCCGTACAGCATGGTAAACGATTCTCCGATCACTATTGGTAAATGGACCCCAAAAAATTCTGACGGCCGTTACCTGGGTATGATCCCGCTGCGTCGCGCTCTGTACCTGTCCCGTAACACTGTATCCGTTCGTCTGCTGCAGACTGTTGGCATCGAACGTACCCGCCAACTGTTTATGGATTTCGGTCTGCAGGAAGACCAGATTCCACGTAACTACACTATCGCTCTGGGCACTCCGCAGGTACTGCCGATCCAGATGGCTACCGGCTACGCTACTTTCGCTAATGGCGGCTACCGTGTTCAGCCACATTTCATCCAGCGTATCGAAGACGCGTATGGTAAAGTAATTTACGAAGCTAAACCGGAATATAAGGATCC。
5. a method for preparing Acinetobacter baumannii surface proteins (OmpA and ponA) according to claim 4, characterized in that: the method comprises the following steps:
respectively obtaining peptide segments with the most abundant antigenic epitopes in the surface protein OmpA and the surface protein ponA extracellular domain of the acinetobacter baumannii, finding out the gene coding sequence of the peptide segment, optimizing the gene coding sequence of the peptide segment, and connecting the optimized gene coding sequence of the peptide segment by using the coding sequence of rigid connecting peptide to form a fusion gene; the Acinetobacter baumannii surface protein OmpA and the surface protein ponA have access numbers in an NCBI protein database of AJF83030 and ADX05080 respectively; the sequence of the rigid linker peptide is eaaakaaaak; simultaneously, enzyme cutting site NdeI is introduced into the 5 'end of the fusion gene, and termination signal TAA and enzyme cutting site BamHI are introduced into the 3' end of the fusion gene, and then a complete gene sequence is chemically synthesized and is marked as OmpPon;
the complete gene sequence of OmpPon is as follows:
CATATGCTGGGTTACACCTTCCAGGATACTCAGCACAACAACGGCGGTAAAGACGGTGAACTGACCAACGGTCCGGAACTGCAGGACGACCTGTTCGTTGGTGCTGCGCTGGGTATCGAACTGACTCCGTGGCTGGGTTTCGAAGCCGAGTATAACCAGGTTAAGGGTGACGTGGATGGCCTGGCAGCGGGTGCTGAATACAAACAGAAACAGATCAACGGTAACTTCTACGTTACCAGCGACCTGATTACCAAGAACTATGACTCTAAAATCAAACCGTATGTTCTGCTGGGTGCGGGCCACTACAAATACGAAATCCCGGACCTTTCCTATCACAACGACGAGGAAGGCACTCTGGGTAACGCGGGTGTTGGTGCTTTCTGGCGTCTGAACGACGCTCTGTCTCTGCGTACCGAAGCTCGTGGTACCTATAACGAAGCTGCTGCTGCTAAAGAAGCTGCTGCTGCTAAAGGCTCTATCGAAGCTATCGTAGGTGGTTACAACTTCTACCAGTCCAAGTTTAACCGTGCGCTCCAGGGCTGGCGCCAGCCGGGCTCTACCATTAAACCTTTCCTGTACGCTCTGGCTCTGGAACGTGGCATGACCCCGTACAGCATGGTAAACGATTCTCCGATCACTATTGGTAAATGGACCCCAAAAAATTCTGACGGCCGTTACCTGGGTATGATCCCGCTGCGTCGCGCTCTGTACCTGTCCCGTAACACTGTATCCGTTCGTCTGCTGCAGACTGTTGGCATCGAACGTACCCGCCAACTGTTTATGGATTTCGGTCTGCAGGAAGACCAGATTCCACGTAACTACACTATCGCTCTGGGCACTCCGCAGGTACTGCCGATCCAGATGGCTACCGGCTACGCTACTTTCGCTAATGGCGGCTACCGTGTTCAGCCACATTTCATCCAGCGTATCGAAGACGCGTATGGTAAAGTAATTTACGAAGCTAAACCGGAATATAAGGATCC；
the protein sequence encoded by the OmpPon gene is:
MLGYTFQDTQHNNGGKDGELTNGPELQDDLFVGAALGIELTPWLGFEAEYNQVKGDVDGLAAGAEYKQKQINGNFYVTSDLITKNYDSKIKPYVLLGAGHYKYEIPDLSYHNDEEGTLGNAGVGAFWRLNDALSLRTEARGTYNEAAAAKEAAAAKGSIEAIVGGYNFYQSKFNRALQGWRQPGSTIKPFLYALALERGMTPYSMVNDSPITIGKWTPKNSDGRYLGMIPLRRALYLSRNTVSVRLLQTVGIERTRQLFMDFGLQEDQIPRNYTIALGTPQVLPIQMATGYATFANGGYRVQPHFIQRIEDAYGKVIYEAKPEY；
the protein sequence coded by the OmpPon gene is 31-173aa of the Acinetobacter baumannii surface protein OmpA and 406-572aa of the surface protein ponA; the middle of the two protein sequences is connected by rigid connecting peptide eaaakaaaak;
cloning OmpPon gene complete sequence into prokaryotic expression vector pET-28a (+) by conventional method, transferring into E.coliBL21(DE3) bacteria, inducing recombinant Escherichia coli expression with IPTG, and using Ni2+And (3) purifying the recombinant His-OmpPon protein by an affinity chromatography method.
6. A method for preparing an anti-acinetobacter baumannii surface protein (OmpA and ponA) polyclonal antibody, characterized in that: the method comprises the following steps:
taking the recombinant His-OmpPon protein as an immune antigen according to claim 5, mixing the recombinant His-OmpPon protein with Freund's adjuvant, then repeatedly and artificially immunizing healthy New Zealand white rabbits, drawing blood for titer determination, separating and purifying high-titer recombinant protein antibodies, finally obtaining polyclonal antibodies against Acinetobacter baumannii OmpA and ponA proteins, and diluting the polyclonal antibodies with a confining liquid to a final concentration of 20 mu g/mL; the sealing liquid comprises the following components in percentage by weight: 1.4g/L of disodium hydrogen phosphate, 0.2g/L of sodium dihydrogen phosphate, 8.5g/L of sodium chloride and 10g/L of bovine serum albumin, wherein the pH value of the sealing liquid is 7.4.
7. A high-sensitivity acinetobacter baumannii antigen Elisa assay kit is characterized in that: the high-sensitivity acinetobacter baumannii antigen Elisa determination kit comprises an enzyme label plate coated with a polyclonal antibody of anti-acinetobacter baumannii surface protein (Fhue and Pilf), an anti-acinetobacter baumannii surface protein (OmpA and ponA) polyclonal antibody, acinetobacter baumannii positive control, negative control, washing liquid, enzyme-labeled secondary antibody, enzyme chromogenic substrate and termination liquid; the acinetobacter baumannii positive control is inactivated acinetobacter baumannii bacterial liquid; the negative control is inactivated escherichia coli liquid; the washing solution comprises the following components in percentage by weight: 1.4g/L of disodium hydrogen phosphate, 0.2g/L of sodium dihydrogen phosphate, 8.5g/L of sodium chloride and 200.5mL/L of Tween, wherein the pH value of the washing solution is 7.4; the enzyme-labeled secondary antibody is goat anti-rabbit IgG labeled by horseradish peroxidase; the enzyme chromogenic substrate is TMB chromogenic solution; the stop solution is a 1M HCL solution.
8. A preparation method of a high-sensitivity Acinetobacter baumannii antigen Elisa assay kit is characterized by comprising the following steps: the high-sensitivity acinetobacter baumannii antigen Elisa determination kit comprises an enzyme label plate coated with a polyclonal antibody of anti-acinetobacter baumannii surface protein (Fhue and Pilf), an anti-acinetobacter baumannii surface protein (OmpA and ponA) polyclonal antibody, acinetobacter baumannii positive control, negative control, washing liquid, enzyme-labeled secondary antibody, enzyme chromogenic substrate and termination liquid; the acinetobacter baumannii positive control is inactivated acinetobacter baumannii bacterial liquid; the negative control is inactivated escherichia coli liquid; the washing solution comprises the following components in percentage by weight: 1.4g/L of disodium hydrogen phosphate, 0.2g/L of sodium dihydrogen phosphate, 8.5g/L of sodium chloride and 200.5mL/L of Tween, wherein the pH value of the washing solution is 7.4; the enzyme-labeled secondary antibody is goat anti-rabbit IgG labeled by horseradish peroxidase; the enzyme chromogenic substrate is TMB chromogenic solution; the stop solution is 1M HCL solution;
the preparation method of the ELISA plate for coating the polyclonal antibody of the anti-Acinetobacter baumannii surface protein (Fhue and Pilf) comprises the following steps:
1) preparing polyclonal antibodies against acinetobacter baumannii surface proteins (Fhue and Pilf);
2) coating of anti-acinetobacter baumannii surface protein (Fhue and Pilf) polyclonal antibody:
diluting the polyclonal antibody against Acinetobacter baumannii surface protein (Fhue and Pilf) prepared in the step 1) to the concentration of 10 mu g/mL by using PBS buffer solution, coating a 96-hole EIA high-efficiency binding enzyme standard plate according to the amount of 100 mu L/hole, and carrying out 2 hours at 37 ℃; taking out, washing the plate for three times by using 250 mu L of washing liquid, and spin-drying; using a washing solution containing 1% BSA as a blocking solution, adding an enzyme label plate according to the amount of 250 mu L/hole, and blocking for 1 hour at 37 ℃; taking out, washing the plate with 250 μ L of washing solution for 3 times, each time for one minute, spin-drying, and storing in sealed condition;
wherein the PBS buffer solution comprises the following components in percentage by weight: 1.4g/L of disodium hydrogen phosphate, 0.2g/L of sodium dihydrogen phosphate and 8.5g/L of sodium chloride; the pH of the PBS buffer was 7.4;
the washing solution comprises the following components in percentage by weight: 1.4g/L of disodium hydrogen phosphate, 0.2g/L of sodium dihydrogen phosphate, 8.5g/L of sodium chloride and 200.5mL/L of Tween, wherein the pH value of the washing solution is 7.4;
the blocking solution was an aqueous solution of a washing solution containing 1% BSA, and the pH of the blocking solution was 7.4.
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