CN110724201A - Rapid detection method for acinetobacter baumannii infection based on multiple epitope fusion antigen - Google Patents

Rapid detection method for acinetobacter baumannii infection based on multiple epitope fusion antigen Download PDF

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CN110724201A
CN110724201A CN201911053066.6A CN201911053066A CN110724201A CN 110724201 A CN110724201 A CN 110724201A CN 201911053066 A CN201911053066 A CN 201911053066A CN 110724201 A CN110724201 A CN 110724201A
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acinetobacter baumannii
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杨波
胡征
王毅
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Hubei University of Technology
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Abstract

The invention relates to a preparation method of an acinetobacter baumannii infection rapid detection card based on multiple epitope fusion antigens, which comprises a sample pad, a combination pad, a nitrocellulose membrane, a water absorption pad and a PVC (polyvinyl chloride) plate, wherein a colorful latex microsphere coupled multiple epitope fusion antigen polyclonal antibody of acinetobacter baumannii surface protein is sprayed on the combination pad, and the nitrocellulose membrane is coated with a detection line of the multiple epitope fusion antigen polyclonal antibody of the acinetobacter baumannii surface protein and a quality control line of a goat anti-rabbit IgG antibody. When the added sample contains acinetobacter baumannii, the acinetobacter baumannii firstly forms a compound with a latex-rabbit anti-acinetobacter baumannii surface protein polyclonal antibody, the compound is captured when the compound migrates to a detection line coated with the acinetobacter baumannii surface protein polyclonal antibody under the capillary action, and the detection line is in corresponding color, so that whether the sample contains the acinetobacter baumannii or not can be detected. The detection card has the advantages of rapidness, simplicity, high sensitivity and good specificity.

Description

Rapid detection method for acinetobacter baumannii infection based on multiple epitope fusion antigen
Technical Field
The invention belongs to the field of biological detection, and particularly relates to a rapid detection method for acinetobacter baumannii infection based on multiple epitope fusion antigens.
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. 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, immune latex test paper and other immunological detection techniques based on antibody have become an indispensable important means for detecting pathogenic microorganisms due to their characteristics of simplicity, rapidness, sensitivity, accuracy and practicality. Therefore, research and development of antibodies against pathogenic microorganisms having proprietary intellectual property rights are the basis for development of ELISA detection methods, latex microsphere labeling detection methods, and the like having proprietary intellectual property rights.
The choice of antigenic component is critical to the specificity of the assay. The Acinetobacter baumannii fhuE receptor, OmpA, PilF and PonA proteins are important molecules located on the surface of cells, and the surface proteins fhuecteptor, OmpA, PilF and PonA with interspecies specificity are selected as antigens in the research to prepare a polyclonal antibody with good specificity, and the polyclonal antibody is applied to the preparation of the Acinetobacter baumannii latex microsphere immunochromatography detection card.
Disclosure of Invention
The invention aims to develop a latex microsphere immunochromatography detection card for quickly and quickly detecting acinetobacter baumannii, which is simple to operate and low in cost, by using an immune latex labeling technology on the basis of a polyclonal antibody.
The purpose of the invention is realized by the following technical scheme:
a preparation method of an acinetobacter baumannii infection rapid detection card based on a multiple epitope fusion antigen is characterized by comprising the following steps: the method comprises the following steps:
1) preparation of acinetobacter baumannii multiple epitope fusion antigen (Fhue + OmpA) antibody:
respectively obtaining peptide segments with the most abundant antigenic epitopes in the surface protein Fhue of acinetobacter baumannii and the extracellular domain of the surface protein OmpA, 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 OmpA have access numbers of KMV27515 and AJF83030 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 FhuOmp;
the complete sequence of the FhuOmp gene is as follows:
CATATGTTCGATGGCAACTACCTGGACCCGGTAGAAGGTAACTCTACTGAAGTTGGTCTCAAATCCGCT
NdeITGGTTTGACGGCCGTCTGAACGGTACCCTGGCTCTGTACCACATCAAACAGGACAACCTGGCACAGGAAGCGGGCCAAGTTACCCGTAACGGTGTTAAAGAGACTTACTACCGTGCAGCGAAAGGCGCTACATCCGAAGGTTTTGAAGTTGAAGTATCAGGTCAGATCACTCCAGATTGGAACATCACCGCAGGTTACTCTCAATTTTCTGCTAAGGACGCGAACGATGCGGACGTAAACACTCAGCTTCCGCGTAAAATGATCCAGAGCTTCACTACCTATAAACTGCCTGGTAAACTGGAAAACATCACAGTTGGCGGTGGCGTAAACTGGCAGTCTTCTACCTACGTGAACGCAAAAAACCCGAAAAAAGTAATCGAAAAAGTTGAGCAGGGTGACTACGCTCTGGTAAACCTGATGGCGCGTTACCAGATCACTAAGGACTTTTCTGCACAGCTTAACATTAACAACGTTTTCGGTGGTTCTGGTGGTTCTGGTGGTTCTGGTGGTTCTCTGGGTTACACCTTCCAGGATACTCAGCACAACAACGGCGGTAAAGACGGTGAACTGACCAACGGTCCGGAACTGCAGGACGACCTGTTCGTTGGTGCTGCGCTGGGTATCGAACTGACTCCGTGGCTGGGTTTCGAAGCCGAGTATAACCAGGTTAAGGGTGACGTGGATGGCCTGGCAGCGGGTGCTGAATACAAACAGAAACAGATCAACGGTAACTTCTACGTTACCAGCGACCTGATTACCAAGAACTATGACTCTAAAATCAAACCGTATGTTCTGCTGGGTGCGGGCCACTACAAATACGAAATCCCGGACCTTTCCTATCACAACGACGAGGAAGGCACTCTGGGTAACGCGGGTGTTGGTGCTTTCTGGCGTCTGAACGACGCTCTGTCTCTGCGTACCGAAGCTCGTGGTACCTATAACTAAGGATCC；
BamHI
the protein sequence coded by the FhuOmp gene is as follows:
MFDGNYLDPVEGNSTEVGLKSAWFDGRLNGTLALYHIKQDNLAQEAGQVTRNGVKETYYRAAKG ATSEGFEVEVSGQITPDWNITAGYSQFSAKDANDADVNTQLPRKMIQSFTTYKLPGKLENITVGGGVNWQSSTYVNAKNPKKVIEKVEQGDYALVNLMARYQITKDFSAQLNINNVFGGSGGSGGSGGSLGYTFQDTQHNNGGKDGELTNGPELQDDLFVGAALGIELTPWLGFEAEYNQVKGDVDGLAAGAEYKQKQINGNFYVTSDLITKNYDSKIKPYVLLGAGHYKYEIPDLSYHNDEEGTLGNAGVGAFWRLNDALSLRTEARGTYN；
the protein sequence coded by the FhuOmp gene is 509-688aa of the surface protein Fhue of acinetobacter baumannii and 31-173aa of the surface protein OmpA; the two protein sequences are connected by flexible connecting peptide ggsggsggs; cloning the gene fragment into prokaryotic expression vector pET-28a (+) according to conventional method, inducing recombinant Escherichia coli expression by IPTG, and using Ni2+Purifying the recombinant His-FhuOmp protein by affinity chromatography; taking the recombinant protein as an immune antigen, mixing the immune antigen 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, and finally obtaining the Acinetobacter baumannii multiple epitope fusion antigen (Fhue + OmpA) antibody;
2) preparation of a multiple epitope fusion antigen (Pilf + ponA) antibody of Acinetobacter baumannii:
respectively obtaining peptide segments with the most abundant antigenic epitopes in the surface protein Pilf of the acinetobacter baumannii and the extracellular domain of the surface protein ponA, finding out the gene coding sequence of the peptide segment, optimizing the gene coding sequence of the peptide segment, and connecting the two segments of sequences by using the coding sequence of rigid connecting peptide to form a fusion gene; the Acinetobacter baumannii surface protein Pilf and the surface protein ponA have access numbers in an NCBI protein database of AJF80497 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 PilPon;
the complete sequence of the PilPon gene is as follows:
CATATGGCGGTTAAGGTACGCACTCAACTGGCGGCTGAATATATCCGTTCAGGTGATCTGGACTCCGCG
NdeIAAACGCTCCCTGGACCAGGCCCTGAGCGTTGACTCTCGTGACGCGACAGCAAACATGATGATGGGCATCCTGCTGCAGCAGGAGGGCTCTAAATCTAACCTGGAGAAAGCGGAGCACTACTTCAAACGTGCTATCAGTTCTGAACCGGATAACGCTCAGGCGCGCAACAACTATGGTACCTATCTGTACCAGATGGAACGTTATAACGACGCGATTGAACAGTTTCGTATCGCAGGTGCGACCCTGGGTTATGATCAGCGTTATCAGGCGCTGGAAAACCTGGGCCGCATCTACCTGAAGCTGGGTAACATCGCCAGCGCTGAAAAAACTTTCAAGCAGGCACTGCTGGCGAACCGTGACTCCTACATCTCTATGCTGGAGCTGGCTGAAATCTTTTACCTGCAGCAGGAAGCTGCTGCTGCTAAAGAAGCTGCTGCTGCTAAAGGCTCTATCGAAGCTATCGTAGGTGGTTACAACTTCTACCAGTCCAAGTTTAACCGTGCGCTCCAGGGCTGGCGCCAGCCGGGCTCTACCATTAAACCTTTCCTGTACGCTCTGGCTCTGGAACGTGGCATGACCCCGTACAGCATGGTAAACGATTCTCCGATCACTATTGGTAAATGGACCCCAAAAAATTCTGACGGCCGTTACCTGGGTATGATCCCGCTGCGTCGCGCTCTGTACCTGTCCCGTAACACTGTATCCGTTCGTCTGCTGCAGACTGTTGGCATCGAACGTACCCGCCAACTGTTTATGGATTTCGGTCTGCAGGAAGACCAGATTCCACGTAACTACACTATCGCTCTGGGCACTCCGCAGGTACTGCCGATCCAGATGGCTACCGGCTACGCTACTTTCGCTAATGGCGGCTACCGTGTTCAGCCACATTTCATCCAGCGTATCGAAGACGCGTATGGTAAAGTAATTTACGAAGCTAAACCGGAATATAAGGATCC；
BamHI
the PilPon-encoded protein sequence is:
MAVKVRTQLAAEYIRSGDLDSAKRSLDQALSVDSRDATANMMMGILLQQEGSKSNLEKAEHYFKRAISSEPDNAQARNNYGTYLYQMERYNDAIEQFRIAGATLGYDQRYQALENLGRIYLKLGNIASAEKTFKQALLANRDSYISMLELAEIFYLQQEAAAAKEAAAAKGSIEAIVGGYNFYQSKFNRALQGWRQPGSTIKPFLYALALERGMTPYSMVNDSPITIGKWTPKNSDGRYLGMIPLRRALYLSRNTVSVRLLQTVGIERTRQLFMDFGLQEDQIPRNYTIALGTPQVLPIQMATGYATFANGGYRVQPHFIQRIEDAYGKVIYEAKPEY；
the protein sequences coded by the PilPon gene are 28-184aa of surface protein Pilf of acinetobacter baumannii and 406-572aa of surface protein ponA, and the two protein sequences are connected by rigid connecting peptide eaakeaaak; cloning the gene fragment into prokaryotic expression vector pET-28a (+) according to conventional method, inducing recombinant Escherichia coli expression by IPTG, and using Ni2+Purifying the recombinant His-PilPon protein by affinity chromatography; taking the recombinant protein as an immune antigen, mixing the immune antigen with Freund's adjuvant, then repeatedly and artificially immunizing healthy New Zealand white rabbits, drawing blood for titer determination, separating and purifying a high-titer recombinant protein antibody, and finally obtaining an acinetobacter baumannii multiple epitope fusion antigen (Pilf + ponA) antibody;
3) preparing latex microsphere markers of the acinetobacter baumannii multiple epitope fusion antigen (Fhue + OmpA) antibody:
3.1) activation of the latex microspheres
Adding 9 mM MES (2- (N-morpholinyl) ethanesulfonic acid) buffer solution into 1mL of 10% colored carboxylated polystyrene latex microsphere solutionMixing, adding NHS (N-hydroxysuccinimide) and EDC (1- (3-dimethylaminopropyl) -3-ethyl 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) to a final concentration of 1mg/mL, slowly mixing at room temperature for 30min, centrifuging 19000g for 20min after incubation, removing supernatant, precipitating with 10mL borax buffer (0.1mol/L Na)2B4O7pH8.5), carrying out heavy suspension, oscillation and ultrasonic treatment to obtain activated latex microspheres; the MES buffer solution comprises the following components in percentage by weight: 0.1mol/LMES, pH of MES buffer is 8.5; the content of the components in the borax buffer solution is 0.1mol/L Na2B4O7The pH value of the borax buffer solution is 8.5; the grain size of the colored carboxylated polystyrene latex microspheres is 100 nm;
3.2) preparation of latex microsphere markers
Diluting the acinetobacter baumannii multiple epitope fusion antigen (Fhue + OmpA) antibody obtained in the step 1) into 1mg/mL by using borax buffer solution; adding 10mL of Acinetobacter baumannii multi-epitope fusion antigen (Fhue + OmpA) antibody into 10mL of activated latex microspheres, slowly mixing uniformly for 30 minutes, centrifuging for 10 minutes at 19000g, and removing supernatant; resuspending the precipitate with 10mL borax buffer solution containing 1% casein, repeating centrifugation for 1 time after ultrasonic pulverization, and removing supernatant; resuspending the precipitate by the same method, repeatedly centrifuging for 1 time after ultrasonic crushing, and removing supernatant; resuspending the precipitate with 10mL borax buffer solution containing 1% casein, namely the latex microsphere marker of the acinetobacter baumannii multiple epitope fusion antigen (Fhue + OmpA) antibody; the content of the components in the borax buffer solution is 0.1mol/LNa2B4O7The pH value of the borax buffer solution is 8.5;
4) preparation of the bonding pad:
spraying the latex microsphere marker of the acinetobacter baumannii multiple epitope fusion antigen (Fhue + OmpA) antibody obtained in the step 3) on a bonding pad made of a polyester fiber material, wherein the spraying amount of a polyester fiber film per square centimeter is 10 mu L of the latex microsphere marker; drying at 37 deg.C in environment with relative humidity not more than 30%, sealing at 25 deg.C, drying and storing;
5) preparation of antibody solid-phase nitrocellulose membrane:
diluting the Acinetobacter baumannii multiple epitope fusion antigen (Pilf + ponA) antibody obtained in the step 2) into 1.5mg/mL by using a borax buffer solution, and then coating the antibody on a detection line position on a nitrocellulose membrane by using a membrane spraying instrument as a detection line to capture the antibody, wherein the coating parameter is 1 mu L/cm; spraying goat anti-rabbit IgG on a quality control line position on a nitrocellulose membrane as a control line to capture an antibody, wherein the concentration is 1mg/mL, and the coating parameter is 1 mu L/cm; after coating, putting the nitrocellulose membrane in an environment with the relative humidity not more than 30%, drying at 37 ℃, sealing at 25 ℃, drying and storing; the content of the components in the borax buffer solution is 0.1mol/LNa2B4O7The pH value of the borax buffer solution is 8.5;
6) preparation of sample pad
Taking a glass cellulose membrane, soaking the glass cellulose membrane in a sample pad treatment solution for at least 3h, placing the sample pad treatment solution in a biological safety cabinet for ventilation drying at 37 ℃, cutting the sample pad treatment solution into required specifications, and sealing, drying and storing the sample pad treatment solution at 25 ℃; thus, a sample pad was prepared;
the sample pad treatment solution comprises the following components in percentage by weight: 0.01mol/L Na2B4O72g/L sodium chloride, 20g/L casein, 10ml/L Tween-20 and 10ml/L antifoaming agent S-17; the pH of the sample pad treatment solution was 8.5;
7) assembly of test card
Respectively sticking a water absorption pad, an antibody solid-phase nitrocellulose membrane, a combination pad and a sample pad which are made of water absorption filter paper materials on a PVC (polyvinyl chloride) base plate in sequence, wherein a quality control line on the nitrocellulose membrane is close to the end of the water absorption pad, a detection line is close to the end of the sample pad, cutting the nitrocellulose membrane into detection cards with certain widths, sealing and packaging the detection cards, and drying the detection cards for low-temperature storage; thus, the multiple epitope fusion antigen-based acinetobacter baumannii infection rapid detection card is prepared.
The invention has the advantages that:
(1) the invention adopts structural analysis, gene optimization and other modes to construct two brand new fusion genes, and successfully obtains soluble recombinant Fhue/OmpA fusion protein and Pilf/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, many targeted antigen sites, strong capture capacity, no site competition problem and high sensitivity of the detection card. The detection sensitivity of the kit to the Acinetobacter baumannii standard strain ATCC19606 reaches 2 multiplied by 104CFU/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 detection card has good specificity, and the specific experiment is carried out by using 6 Acinetobacter baumannii strains and 18 non-Acinetobacter baumannii standard strains (containing most common pathogens of respiratory tract), and the result shows that the detection card 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.
(4) The detection card can be preserved for two years at normal temperature, effectively prolongs the shelf life and reduces the storage condition; non-professional persons can finish the whole-process detection by using the detection test paper, the operation is simple, and the popularization of the method is facilitated; the whole detection process can be finished within 10min at the fastest speed, and is more suitable for bedside detection.
Drawings
FIG. 1 is a schematic diagram of an explosive structure of an Acinetobacter baumannii infection rapid detection card based on a multiple epitope fusion antigen provided by the invention;
FIG. 2 is a schematic structural diagram of an Acinetobacter baumannii infection rapid detection card based on a multiple epitope fusion antigen provided by the invention;
wherein:
1-sample pad; 2-a conjugate pad; 3-NC film; 4-absorbent pad; 5-PVC sheet.
Detailed Description
The present invention is further specifically described by the following examples.
Sources of the various materials used or employed in the present invention
1. Latex microspheres: the latex microspheres used in the invention are carboxylated modified polystyrene latex microspheres, are products of Shanghai Yan Biotech Co., Ltd, have the size of 100nm and the color of red, have the tolerance of the average diameter of the products within 10 percent, are in the form of 10 percent solid aqueous suspension, and have the product code of MSI-CAR100 NM.
2. Glass cellulose membrane: the thickness is 0.45-0.55mm, the water absorption capacity is 800mg/m2The glass fiber has a diameter of 0.6-3 μm and good hydrophilicity, and is available from Shanghai gold-labeled Biotech Co., Ltd (model number BT 50).
3. Polyester fiber film: has a thickness of 0.25-0.35mm, a climbing speed of 15-40mm/60s, excellent hydrophilicity, and is used for preparing a bonding pad, and is available from Shanghai gold-labeled Biotech Co., Ltd (model number VL 98).
4. Cellulose nitrate membrane: model number Millipore Corp SHF135, with liner plates, was purchased from Millipore corporation.
5. Water-absorbing filter paper: the thickness is 0.95mm, the water absorption speed is 60s/4cm, and the water absorption capacity is 700mg/cm2Has good water absorption and is used as a material for manufacturing the water absorption pad. Purchased from Shanghai gold Biotech, Inc. (model CH 37K).
6. A bottom plate: is made of high-whiteness PVC material, and is coated with a single-layer high-polymer pressure-sensitive adhesive SM31 purchased from Shanghai gold-labeled Biotech Co.
7. The microorganism samples used in the present invention were purchased from the American Type Culture Collection (ATCC).
8. pET28a (+): e.coli expression vectors, introduced from Novagen, USA.
9. Escherichia coli (e. coli) BL21(DE 3): purchased from northern Biotechnology, Inc., Shanghai.
10. Goat anti-rabbit IgG: is product of bioscience, Dr. Germany, product No. BA1039, and has a concentration of 1 mg/ml.
The methods used in the following examples are conventional methods unless otherwise specified.
Example 1
Preparation of acinetobacter baumannii multiple epitope fusion antigen (Fhue + OmpA) antibody:
1.1) cloning of Acinetobacter baumannii FhuOmp fusion Gene
The method comprises the steps of obtaining peptide segments with most abundant antigen epitopes in extracellular domains of acinetobacter baumannii surface proteins Fhue and OmpA (wherein accession numbers in an NCBI protein database are KMV27515 and AJF83030 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 flexible connecting peptides (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 FhuOmp. The complete gene sequence and the coded amino acid sequence are shown in a sequence table. Specifically, the protein sequences encoded by the FhuOmp gene are 509-688aa of the surface protein Fhue of acinetobacter baumannii and 31-173aa of the surface protein OmpA, 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 FhuOmp 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-FhuOmp expression vector. The construction of the expression vector is verified to be correct by enzyme digestion and sequence determination. The vector expresses the recombinant FhuOmp fusion protein.
1.2) expression and purification of Acinetobacter baumannii FhuOmp 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-FhuOmp having the ability to express foreign proteins 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 were inoculated into 100mL of LB medium containing 50. mu.g/mL of kanamycin, and cultured at 30 ℃ toWhen OD600 is 0.6, 1mol/L IPTG was added to a final concentration of 0.5mmol/L, and the mixture was 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 buffer A (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 FhuOmp fusion protein was found to be present in partially solubilized form in the bacterial cells (the other portion was present as inclusion bodies). Thin-layer scanning showed that the recombinant protein accounted for more than 30% of the total bacterial protein. The wild type FhuOmp 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 (GE healthcare 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 Buffer A +10mM imidazole, and collecting the elution peak;
1.2.8) eluting with Buffer A +20mM imidazole, and collecting the elution peak;
1.2.9) eluting with Buffer A +40mM imidazole, and collecting the elution peak;
1.2.10) eluting with Buffer A +100mM imidazole, and collecting the elution peak;
1.2.11) eluting with Buffer A +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 100mM 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 FhuOmp fusion protein.
1.3) preparation of Acinetobacter baumannii multiple epitope fusion antigen (Fhue + OmpA) antibody
1.3.1) mixing the acinetobacter baumannii FhuOmp 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, the emulsion formed by the FhuOmp fusion protein and Freund's incomplete adjuvant is used for immunization once every two weeks, 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 FhuOmp 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. Thus, the Acinetobacter baumannii multi-epitope fusion antigen (Fhue + OmpA) antibody is prepared.
Example 2
Preparation of a multiple epitope fusion antigen (Pilf + ponA) antibody of Acinetobacter baumannii:
2.1) cloning of the Acinetobacter baumannii PilPon fusion Gene
The method comprises the steps of obtaining peptide segments with most abundant antigenic epitopes in extracellular domains of acinetobacter baumannii surface proteins Pilf and ponA (the accession numbers in an NCBI protein database are AJF80497 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 peptides (eaaakaaaak) 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 PilPon. The complete gene sequence and the coded amino acid sequence are shown in a sequence table. Specifically, the protein sequence encoded by the PilPon gene is 28-184aa of surface protein Pilf of Acinetobacter baumannii and 406-572aa of surface protein ponA, and the two protein sequences are connected by 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 PilPon 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-PilPon expression vector. The construction of the expression vector is verified to be correct by enzyme digestion and sequence determination. The vector expresses recombinant PilPon fusion protein.
2.2) expression and purification of Acinetobacter baumannii PilPon fusion proteins
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-PilPon having the ability to express foreign proteins 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, cultured at 30 ℃ until OD600 became 0.6, added with 1mol/L of IPTG to a final concentration of 0.5mmol/L, and cultured with shaking at 37 ℃ to induce expression of the fusion protein. After induction for 4h, the thalli are collected by centrifugation for 10min at 8000 r/min. The resulting mycelia were inoculated with 50mM buffer A (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 PilPon 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 20% of the total bacterial protein. While the wild-type PilPon gene which is not sequence-optimized is expressed in the same manner as described above, not shown in the tableThe product is obtained, which shows that the gene optimization is successful and the effect is good. The sonicated supernatant obtained above was filtered through a 0.45 μm filter and purified by His Trap affinity columns (GE healthcare Co.) according to the method described in the specification. The specific method comprises the following steps:
(1) connecting a chromatography system, wherein the system comprises a sample inlet pipe, a peristaltic pump (Shanghai analytical instrument factory, model DHL-A), a chromatography column (product of GE healthcare company, trade name His Trap affinity column) and an ultraviolet detector (Shanghai analytical instrument factory, model HD1), the column volume is 2ml, and the ultraviolet detector is preheated for about 30min until the reading is stable;
(2) and (5) correcting T%: adjusting a brightness knob to display 100%;
(3) rotate the sensitivity to the appropriate position, typically 0.2A;
(4) equilibrating the chromatography system with the loading buffer until the reading stabilizes and then rotating "zero" to show "000";
(5) applying protein sample, controlling the flow rate within 5ml/min, and collecting penetration liquid;
(6) washing away unbound protein with a loading buffer, recording the reading during the process until the reading does not change any more, and collecting the eluate;
(7) eluting with Buffer A +10mM imidazole, and collecting an elution peak;
(8) eluting with Buffer A +20mM imidazole, and collecting an elution peak;
(9) eluting with Buffer A +40mM imidazole, and collecting an elution peak;
(10) eluting with Buffer A +60mM imidazole, and collecting an elution peak;
(11) eluting with Buffer A +100mM imidazole, and collecting an elution peak;
(12) eluting with Buffer A +150mM imidazole, and collecting an elution peak;
(13) taking 100ul of each elution peak sample to carry out SDS-PAGE electrophoresis;
(14) as a result, it was found that the target protein was eluted at 60mM imidazole and had a purity of 90% or more, and the concentration was adjusted to 0.2mg/mL for use after the protein concentration was measured with a bradford kit. Thus obtaining the Acinetobacter baumannii PilPon fusion protein.
2.3) preparation of Acinetobacter baumannii multiple epitope fusion antigen (Pilf + ponA) antibody
2.3.1) mixing the acinetobacter baumannii PilPon fusion protein prepared in the step 2.2) 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, the emulsion formed by the PilPon fusion protein and Freund's incomplete adjuvant is used for immunization once every two weeks, 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.
2.3.2) determination of the potency of the polyclonal antibody
The PilPon 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 the 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.
2.3.3) extraction of polyclonal antibodies
The antibodies were purified using a GE-HiTrap Protein A HP pre-packed column as described, in the following manner:
2.3.3.1) 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.
2.3.3.2) 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).
2.3.3.3) eluting 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)
2.3.3.4) the eluate was dialyzed against 50 volumes of Tris (10mM Tris-Cl, pH 8.0).
2.3.3.5) concentrating by ultrafiltration, adjusting the concentration to 5mg/ml, and storing at-70 deg.C for use. Thus, an acinetobacter baumannii multiple epitope fusion antigen (Pilf + ponA) antibody is prepared.
Example 3
Preparing latex microsphere markers of the acinetobacter baumannii multiple epitope fusion antigen (Fhue + OmpA) antibody:
3.1) activation of the latex microspheres
Taking 1mL of a 10% red carboxylated polystyrene latex microsphere (100nm) solution, adding 9mL of 2- (N-morpholinyl) ethanesulfonic acid (MES) buffer solution (0.1mol/LMES, pH8.5), and uniformly mixing; preparing 10mg/mL of N-hydroxysuccinimide (NHS) and 10mg/mL of 1- (3-dimethylaminopropyl) -3-ethyl-1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) solution by using MES buffer solution;
adding 1mL NHS solution and 1mL EDC solution into polystyrene latex microsphere (100nm) solution in sequence, slowly mixing at room temperature for 30min, centrifuging 19000g after incubation for 20min, removing supernatant, precipitating with 10mL borax buffer (0.1mol/L Na)2B4O7Ph8.5), resuspension, shaking, sonication (sonicator model: YJ92-IIDN, the power is 50W, the working time is 2s, the interval time is 3s, the alarm temperature is 60 ℃, and the total time is 30min) to obtain the activated latex microspheres.
3.2) preparation of latex microsphere markers
Using borax buffer (0.1mol/L Na)2B4O7pH8.5) the antibody against the Acinetobacter baumannii multiple epitope fusion antigen (Fhue + OmpA) obtained in step 1 was diluted to 1 mg/mL. 10mL of Acinetobacter baumannii multi-epitope fusion antigen (Fhue + OmpA) antibody is added into 10mL of activated latex microspheres, slowly and uniformly mixed for 30 minutes, then 19000g of the mixture is centrifuged for 10 minutes, and the supernatant is removed. The precipitate was resuspended in 10mL of borax buffer containing 1% casein, and after ultrasonication (model: YJ92-IIDN, power 50W, working time 2s, interval time 3s, alarm temperature 60 ℃ C., total time 30min), 19000g was centrifuged repeatedly 1 time (10 min) to remove the supernatant. The same method of precipitationResuspend, sonicate, repeat centrifugation at 19000g for 1 time (10 min), remove supernatant. And (3) resuspending the precipitate by using 10mL of borax buffer solution containing 1% casein, thus obtaining the latex microsphere marker of the acinetobacter baumannii multiple epitope fusion antigen (Fhue + OmpA) antibody.
Example 4
Preparation of the bonding pad:
the polyester fiber film was cut into pieces of 4cm × 0.8 cm/piece, and 32 μ L of the latex microsphere marker prepared in example 3 was dropped onto the cut pieces of the film. After spraying, drying at 37 ℃ for 12h in an environment with the relative humidity of 20%. Sealing, drying and storing at 25 ℃.
Example 5
Preparation of antibody solid-phase nitrocellulose membrane:
the nitrocellulose membrane was cut to a size of 4cm by 2.3 cm. Using borax buffer solution (0.1mol/L Na)2B4O7pH8.5) the Acinetobacter baumannii multiple epitope fusion antigen (Pilf + ponA) antibody obtained in example 2 and the goat anti-rabbit IgG were diluted to 1.5mg/mL and 1mg/mL, respectively; filling the diluted acinetobacter baumannii multiple epitope fusion antigen (Pilf + ponA) antibody into a BIODOT membrane scribing instrument spray head 1, setting the amount of 1.0 mu l/cm, and spraying the diluted antibody on a nitrocellulose membrane to form a detection line, wherein the edge distance between the detection line and the nitrocellulose membrane is 0.8 cm; and (3) filling the diluted goat anti-rabbit IgG into a nozzle 2 of a BIODOT membrane scribing instrument, setting the volume of 1.0 mu l/cm, spraying the diluted goat anti-rabbit IgG on a nitrocellulose membrane to be used as a quality control line, and setting the distance between the diluted goat anti-rabbit IgG and the detection line to be 0.7 cm. After coating, the nitrocellulose membrane is put in an environment with the relative humidity of 20 percent, dried for 12 hours at 37 ℃, sealed, dried and stored at 25 ℃.
Example 6
Preparation of sample pad:
preparing sample pad treating fluid with different formulas, observing the release effect of the latex microsphere labeled antibody, and optimizing through multiple orthogonal tests to obtain the optimal formula (0.01mol/L Na) of the sample pad treating fluid2B4O72g/L sodium chloride, 20g/L casein, 10ml/L Tween-20, 10ml/L antifoam S-17, pH 8.5). Taking a glass cellulose membrane, soaking the glass cellulose membrane in the sample pad treatment solution for 3h, and then placing the sample pad treatment solution in a biosafety placeAnd (3) after the air drying is carried out for 12h at the temperature of 37 ℃ in the cabinet, cutting the dried product into strips with the specification of 4cm multiplied by 3cm, sealing and drying at the temperature of 25 ℃ for storage, thus obtaining the sample pad. Thus, a sample pad was prepared. Tests prove that the use of the sample pad greatly improves the release rate of the latex microsphere labeled antibody on the bonding pad, and achieves better application effect.
EXAMPLE 7 absorbent pad cutting
The water-absorbing filter paper purchased from Shanghai gold-labeled Biotechnology Co., Ltd, model number CH37K, was cut into pieces of 4 cm. times.3 cm/strip for use.
EXAMPLE 8 tailoring of PVC sheets
A high-whiteness PVC sheet purchased from Shanghai gold-labeled Biotechnology Co., Ltd, model number SM31, was cut into 4cm by 8.5cm strips for use.
EXAMPLE 9 Assembly of test card
Referring to fig. 1 and 2, an NC film 3, a bonding pad 2, a water absorbent pad 4 and a sample pad 1 are sequentially adhered to a single-sided PVC plate 5, wherein the bonding pad 2 and the water absorbent pad 4 are laminated on the NC film 3, and are respectively overlapped with the NC film 3 by about 2mm, and the sample pad 1 is laminated on the bonding pad 2, and are overlapped with the bonding pad 2 by about 2 mm. The NC membrane 3 is marked with a detection line T and a quality control line C. Cutting the adhered detection board into detection cards with the width of 4mm by a cutting machine, and putting the prepared detection cards and a drying agent into an aluminum foil bag for sealing and storing.
Example 10 method of use of test cards:
10.1) treatment of the sample to be examined
The throat swab of the subject was obtained by a conventional method, and inserted into a sample processing solution (0.01 mol/LNa) containing 500. mu.L of the swab2B4O72g/L sodium chloride, 20ml/L Tween-20), the wall of the plastic tube is pressed to fully dissolve the sample on the swab.
10.2) adding the sample to be detected, and judging the result
Adding 100 mu L of sample into a randomly extracted and assembled detection card, combining Acinetobacter baumannii in sample liquid with an antibody marked by latex microspheres on a binding pad, combining the antibody with a detection line (T line) under the action of chromatography, and acting for 10min at room temperature, wherein two red lines, namely a detection line T line and a quality control line C line, appear in a positive result; if the sample does not contain the acinetobacter baumannii, a negative result only shows that a red line appears on the C line of the quality control line, and the sample does not contain the acinetobacter baumannii.
Example 11 measurement of the Performance of test cards:
11.1) specific analysis
In order to verify the specificity of the multiple epitope fusion antigen-based acinetobacter baumannii infection rapid detection card of the present invention, the composition and the using method of the detection card according to the embodiment 9 and the embodiment 10 are 2 × 10 for the concentration5CFU/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 card of the invention has positive detection results on all 6 Acinetobacter baumannii strains, and has negative detection results on other 17 common respiratory pathogenic microorganisms. The test card showed good specificity.
TABLE 1
Figure BDA0002255817590000151
At the same time, the concentration is 2 multiplied by 105The results of 120 CFU/mL clinical isolates of Acinetobacter baumannii detected by the detection card are positive, and the detection card shows high detection coverage on the clinically isolated Acinetobacter baumannii.
11.2) sensitivity determination
Inoculating Acinetobacter baumannii ATCC19606 strain in sheep blood chocolate culture medium, culturing at 35 deg.C for 36 hr, diluting with normal saline 10 times gradient, and counting to obtain 10-fold thallus concentration8-103CFU/mL of the cell solution, 100. mu.L of the cell solution was dropped on the sample pad, and the assay was performed according to the assay card composition and the method of use described in examples 9 and 10. The results show that the detection sensitivity of the detection card of the invention is 2 multiplied by 104CFU/mL。
11.3) stability test
The test card was dried and sealed, and then placed at 4 ℃, 25 ℃, 37 ℃ and used to test the physiological saline dilution of acinetobacter baumannii ATCC19606 after 6 months, 12 months, 18 months, 21 months and 24 months, respectively, and the results were observed.
After the detection card is dried and sealed, the detection card can still detect a strong positive result after being respectively placed at 4 ℃ and 25 ℃ for 6-24 months; positive results were also detected after 6-18 months at 37 ℃, but were reduced after 21-24 months of storage. Indicating that the test card can be stored at 4 ℃ or 25 ℃ for at least 2 years.
Finally, it is noted that the above-mentioned embodiments illustrate rather than limit the invention, and that, while the invention has been described with reference to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.
Sequence listing
<110> Hubei university of industry
<120> multiple epitope fusion antigen-based rapid detection method for acinetobacter baumannii infection
<141>2019-10-31
<160>4
<170>SIPOSequenceListing 1.0
<210>1
<211>1020
<212>DNA
<213> FhuOmp Gene sequence (FhuOmp)
<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 ctctgggtta caccttccag 600
gatactcagc acaacaacgg cggtaaagac ggtgaactga ccaacggtcc ggaactgcag 660
gacgacctgt tcgttggtgc tgcgctgggt atcgaactga ctccgtggct gggtttcgaa 720
gccgagtata accaggttaa gggtgacgtg gatggcctgg cagcgggtgc tgaatacaaa 780
cagaaacaga tcaacggtaa cttctacgtt accagcgacc tgattaccaa gaactatgac 840
tctaaaatca aaccgtatgt tctgctgggt gcgggccact acaaatacga aatcccggac 900
ctttcctatc acaacgacga ggaaggcact ctgggtaacg cgggtgttgg tgctttctgg 960
cgtctgaacg acgctctgtc tctgcgtacc gaagctcgtg gtacctataa ctaaggatcc 1020
<210>2
<211>336
<212>PRT
<213> FhuOmp protein sequence (FhuOmp)
<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 Leu Gly Tyr Thr Phe Gln Asp Thr Gln His Asn Asn Gly Gly Lys
195 200 205
Asp Gly Glu Leu Thr Asn Gly Pro Glu Leu Gln Asp Asp Leu Phe Val
210 215 220
Gly Ala Ala Leu Gly Ile Glu Leu Thr Pro Trp Leu Gly Phe Glu Ala
225 230 235 240
Glu Tyr Asn Gln Val Lys Gly Asp Val Asp Gly Leu Ala Ala Gly Ala
245 250 255
Glu Tyr Lys Gln Lys Gln Ile Asn Gly Asn Phe Tyr Val Thr Ser Asp
260 265 270
Leu Ile Thr Lys Asn Tyr Asp Ser Lys Ile Lys Pro Tyr Val Leu Leu
275 280 285
Gly Ala Gly His Tyr Lys Tyr Glu Ile Pro Asp Leu Ser Tyr His Asn
290 295 300
Asp Glu Glu Gly Thr Leu Gly Asn Ala Gly Val Gly Ala Phe Trp Arg
305 310 315 320
Leu Asn Asp Ala Leu Ser Leu Arg Thr Glu Ala Arg Gly Thr Tyr Asn
325 330 335
<210>4
<211>1025
<212>DNA
<213> PilPon Gene sequence (PilPon)
<400>4
catatggcgg ttaaggtacg cactcaactg gcggctgaat atatccgttc aggtgatctg 60
gactccgcga aacgctccct ggaccaggcc ctgagcgttg actctcgtga cgcgacagca 120
aacatgatga tgggcatcct gctgcagcag gagggctcta aatctaacct ggagaaagcg 180
gagcactact tcaaacgtgc tatcagttct gaaccggata acgctcaggc gcgcaacaac 240
tatggtacct atctgtacca gatggaacgt tataacgacg cgattgaaca gtttcgtatc 300
gcaggtgcga ccctgggtta tgatcagcgt tatcaggcgc tggaaaacct gggccgcatc 360
tacctgaagc tgggtaacat cgccagcgct gaaaaaactt tcaagcaggc actgctggcg 420
aaccgtgact cctacatctc tatgctggag ctggctgaaa tcttttacct gcagcaggaa 480
gctgctgctg ctaaagaagc tgctgctgct aaaggctcta tcgaagctat cgtaggtggt 540
tacaacttct accagtccaa gtttaaccgt gcgctccagg gctggcgcca gccgggctct 600
accattaaac ctttcctgta cgctctggct ctggaacgtg gcatgacccc gtacagcatg 660
gtaaacgatt ctccgatcac tattggtaaa tggaccccaa aaaattctga cggccgttac 720
ctgggtatga tcccgctgcg tcgcgctctg tacctgtccc gtaacactgt atccgttcgt 780
ctgctgcaga ctgttggcat cgaacgtacc cgccaactgt ttatggattt cggtctgcag 840
gaagaccaga ttccacgtaa ctacactatc gctctgggca ctccgcaggt actgccgatc 900
cagatggcta ccggctacgc tactttcgct aatggcggct accgtgttca gccacatttc 960
atccagcgta tcgaagacgc gtatggtaaa gtaatttacg aagctaaacc ggaatataag 1020
gatcc 1025
<210>4
<211>338
<212>PRT
<213> PilPon protein sequence (PilPon)
<400>4
Met Ala Val Lys Val Arg Thr Gln Leu Ala Ala Glu Tyr Ile Arg Ser
1 5 10 15
Gly Asp Leu Asp Ser Ala Lys Arg Ser Leu Asp Gln Ala Leu Ser Val
20 25 30
Asp Ser Arg Asp Ala Thr Ala Asn Met Met Met Gly Ile Leu Leu Gln
35 40 45
Gln Glu Gly Ser Lys Ser Asn Leu Glu Lys Ala Glu His Tyr Phe Lys
50 55 60
Arg Ala Ile Ser Ser Glu Pro Asp Asn Ala Gln Ala Arg Asn Asn Tyr
65 70 75 80
Gly Thr Tyr Leu Tyr Gln Met Glu Arg Tyr Asn Asp Ala Ile Glu Gln
85 90 95
Phe Arg Ile Ala Gly Ala Thr Leu Gly Tyr Asp Gln Arg Tyr Gln Ala
100 105 110
Leu Glu Asn Leu Gly Arg Ile Tyr Leu Lys Leu Gly Asn Ile Ala Ser
115 120 125
Ala Glu Lys Thr Phe Lys Gln Ala Leu Leu Ala Asn Arg Asp Ser Tyr
130 135 140
Ile Ser Met Leu Glu Leu Ala Glu Ile Phe Tyr Leu Gln Gln Glu Ala
145 150 155 160
Ala Ala Ala Lys Glu Ala Ala Ala Ala Lys Gly Ser Ile Glu Ala Ile
165 170 175
Val Gly Gly Tyr Asn Phe Tyr Gln Ser Lys Phe Asn Arg Ala Leu Gln
180 185 190
Gly Trp Arg Gln Pro Gly Ser Thr Ile Lys Pro Phe Leu Tyr Ala Leu
195 200 205
Ala Leu Glu Arg Gly Met Thr Pro Tyr Ser Met Val Asn Asp Ser Pro
210 215 220
Ile Thr Ile Gly Lys Trp Thr Pro Lys Asn Ser Asp Gly Arg Tyr Leu
225 230 235 240
Gly Met Ile Pro Leu Arg Arg Ala Leu Tyr Leu Ser Arg Asn Thr Val
245 250 255
Ser Val Arg Leu Leu Gln Thr Val Gly Ile Glu Arg Thr Arg Gln Leu
260 265 270
Phe Met Asp Phe Gly Leu Gln Glu Asp Gln Ile Pro Arg Asn Tyr Thr
275 280 285
Ile Ala Leu Gly Thr Pro Gln Val Leu Pro Ile Gln Met Ala Thr Gly
290 295 300
Tyr Ala Thr Phe Ala Asn Gly Gly Tyr Arg Val Gln Pro His Phe Ile
305 310 315 320
Gln Arg Ile Glu Asp Ala Tyr Gly Lys Val Ile Tyr Glu Ala Lys Pro
325 330 335
Glu Tyr

Claims (8)

1. An acinetobacter baumannii multiple epitope fusion antigen (Fhue + OmpA), characterized in that: the protein sequence of the acinetobacter baumannii multiple epitope fusion antigen (Fhue + OmpA) is as follows:
MFDGNYLDPVEGNSTEVGLKSAWFDGRLNGTLALYHIKQDNLAQEAGQVTRNGVKETYYRAAKGATSEGFEVEVSGQITPDWNITAGYSQFSAKDANDADVNTQLPRKMIQSFTTYKLPGKLENITVGGGVNWQSSTYVNAKNPKKVIEKVEQGDYALVNLMARYQITKDFSAQLNINNVFGGSGGSGGSGGSLGYTFQDTQHNNGGKDGELTNGPELQDDLFVGAALGIELTPWLGFEAEYNQVKGDVDGLAAGAEYKQKQINGNFYVTSDLITKNYDSKIKPYVLLGAGHYKYEIPDLSYHNDEEGTLGNAGVGAFWRLNDALSLRTEARGTYN；
the complete sequence of the nucleic acid gene for coding the acinetobacter baumannii multiple epitope fusion antigen (Fhue + OmpA) is as follows:
Figure FDA0002255817580000011
2. a preparation method of an acinetobacter baumannii multiple epitope fusion antigen (Fhue + OmpA) is characterized in that: the preparation method comprises the following steps: respectively obtaining peptide segments with the most abundant antigenic epitopes in the surface protein Fhue of acinetobacter baumannii and the extracellular domain of the surface protein OmpA, 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 OmpA have access numbers of KMV27515 and AJF83030 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 FhuOmp;
the complete sequence of the FhuOmp gene is as follows:
Figure FDA0002255817580000021
the protein sequence coded by the FhuOmp gene is 509-688aa of the surface protein Fhue of acinetobacter baumannii and 31-173aa of the surface protein OmpA; the middle of the two protein sequences is connected by flexible connecting peptide; cloning the gene fragment into prokaryotic expression vector pET-28a (+) according to conventional method, inducing recombinant Escherichia coli expression by IPTG, and using Ni2+Purifying the recombinant His-FhuOmp protein by affinity chromatography;
the protein sequence coded by the FhuOmp gene is as follows:
MFDGNYLDPVEGNSTEVGLKSAWFDGRLNGTLALYHIKQDNLAQEAGQVTRNGVKETYYRAAKGATSEGFEVEVSGQITPDWNITAGYSQFSAKDANDADVNTQLPRKMIQSFTTYKLPGKLENITVGGGVNWQSSTYVNAKNPKKVIEKVEQGDYALVNLMARYQITKDFSAQLNINNVFGGSGGSGGSGGSLGYTFQDTQHNNGGKDGELTNGPELQDDLFVGAALGIELTPWLGFEAEYNQVKGDVDGLAAGAEYKQKQINGNFYVTSDLITKNYDSKIKPYVLLGAGHYKYEIPDLSYHNDEEGTLGNAGVGAFWRLNDALSLRTEARGTYN。
3. a method for producing an Acinetobacter baumannii multiple epitope fusion antigen (Fhue + OmpA) antibody, characterized in that: the method comprises the following steps: the recombinant His-FhuOmp protein as claimed in claim 2 is used as an immune antigen, and is mixed with Freund's adjuvant, and then the healthy New Zealand white rabbits are repeatedly and artificially immunized, and then the blood is drawn for titer determination, and high titer recombinant protein antibodies are separated and purified, and finally the Acinetobacter baumannii multiple epitope fusion antigen (Fhue + OmpA) antibody is obtained.
4. An acinetobacter baumannii multiple epitope fusion antigen (Pilf + ponA), characterized in that: the protein sequence of the acinetobacter baumannii multiple epitope fusion antigen (Pilf + ponA) is as follows:
MAVKVRTQLAAEYIRSGDLDSAKRSLDQALSVDSRDATANMMMGILLQQEGSKSNLEKAEHYFKRAISSEPDNAQARNNYGTYLYQMERYNDAIEQFRIAGATLGYDQRYQALENLGRIYLKLGNIASAEKTFKQALLANRDSYISMLELAEIFYLQQEAAAAKEAAAAKGSIEAIVGGYNFYQSKFNRALQGWRQPGSTIKPFLYALALERGMTPYSMVNDSPITIGKWTPKNSDGRYLGMIPLRRALYLSRNTVSVRLLQTVGIERTRQLFMDFGLQEDQIPRNYTIALGTPQVLPIQMATGYATFANGGYRVQPHFIQRIEDAYGKVIYEAKPEY；
the complete sequence of the nucleic acid gene for encoding the acinetobacter baumannii multiple epitope fusion antigen (Pilf + ponA) is as follows:
Figure FDA0002255817580000031
Figure FDA0002255817580000041
5. a preparation method of an acinetobacter baumannii multiple epitope fusion antigen (Pilf + ponA) is characterized in that: the preparation method comprises the following steps: respectively obtaining peptide segments with the most abundant antigenic epitopes in the surface protein Pilf of the acinetobacter baumannii and the extracellular domain of the surface protein ponA, finding out the gene coding sequence of the peptide segment, optimizing the gene coding sequence of the peptide segment, and connecting the two segments of sequences by using the coding sequence of rigid connecting peptide to form a fusion gene; the Acinetobacter baumannii surface protein Pilf and the surface protein ponA have access numbers in an NCBI protein database of AJF80497 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 PilPon;
the complete sequence of the PilPon gene is as follows:
Figure FDA0002255817580000042
the PilPon-encoded protein sequence is:
MAVKVRTQLAAEYIRSGDLDSAKRSLDQALSVDSRDATANMMMGILLQQEGSKSNLEKAEHYFKRAISSEPDNAQARNNYGTYLYQMERYNDAIEQFRIAGATLGYDQRYQALENLGRIYLKLGNIASAEKTFKQALLANRDSYISMLELAEIFYLQQEAAAAKEAAAAKGSIEAIVGGYNFYQSKFNRALQGWRQPGSTIKPFLYALALERGMTPYSMVNDSPITIGKWTPKNSDGRYLGMIPLRRALYLSRNTVSVRLLQTVGIERTRQLFMDFGLQEDQIPRNYTIALGTPQVLPIQMATGYATFANGGYRVQPHFIQRIEDAYGKVIYEAKPEY；
the protein sequences coded by the PilPon gene are 28-184aa of surface protein Pilf of acinetobacter baumannii and 406-572aa of surface protein ponA, and the two protein sequences are connected by rigid connecting peptide; cloning the gene fragment into a prokaryotic expression vector pET-28a (+) according to a conventional method, inducing the recombinant Escherichia coli to express by IPTG, and purifying the recombinant His-PilPon protein by using Ni2+ affinity chromatography.
6. A method for producing an acinetobacter baumannii multiple epitope fusion antigen (Pilf + ponA) antibody, characterized in that: the method comprises the steps of taking the recombinant His-PilPon protein as an immune antigen as claimed in claim 5, mixing the immune antigen with Freund's adjuvant, then repeatedly and artificially immunizing healthy New Zealand white rabbits, drawing blood to perform titer measurement, separating and purifying high-titer recombinant protein antibodies, and finally obtaining the Acinetobacter baumannii multiple epitope fusion antigen (Pilf + ponA) antibody.
7. A rapid detection card for acinetobacter baumannii infection based on multiple epitope fusion antigens is characterized in that: the acinetobacter baumannii infection rapid detection card based on the multiple epitope fusion antigen comprises a latex microsphere marker coated with an acinetobacter baumannii multiple epitope fusion antigen (Fhue + OmpA) antibody and a nitrocellulose membrane coated with an acinetobacter baumannii multiple epitope fusion antigen (Pilf + ponA) antibody.
8. Method for the preparation of the multiple epitope fusion antigen based rapid detection card for acinetobacter baumannii infection according to claim 7, characterized in that: the method comprises the following steps:
1) preparing an acinetobacter baumannii multiple epitope fusion antigen (Fhue + OmpA) antibody and an acinetobacter baumannii multiple epitope fusion antigen (Pilf + ponA) antibody;
2) preparing latex microsphere markers of the acinetobacter baumannii multiple epitope fusion antigen (Fhue + OmpA) antibody:
2.1) activation of the latex microspheres
Taking 1mL of colored carboxylated polystyrene latex microsphere solution with the concentration of 10%, adding 9mLMES buffer solution, uniformly mixing, adding NHS and EDC until the final concentration of the two is 1mg/mL, slowly and uniformly mixing for 30 minutes at room temperature, centrifuging 19000g for 20 minutes after incubation is finished, removing supernatant, re-suspending the precipitate with 10mL of borax buffer solution, oscillating, and performing ultrasonic treatment to obtain activated latex microspheres; the MES buffer solution comprises the following components in percentage by weight: 0.1mol/LMES, pH of MES buffer is 8.5; the content of the components in the borax buffer solution is 0.1mol/LNa2B4O7The pH value of the borax buffer solution is 8.5; the grain size of the colored carboxylated polystyrene latex microspheres is 100 nm;
2.2) preparation of latex microsphere markers:
diluting the acinetobacter baumannii multiple epitope fusion antigen (Fhue + OmpA) antibody obtained in the step 1) into 1mg/mL by using borax buffer solution; adding 10mL of Acinetobacter baumannii multi-epitope fusion antigen (Fhue + OmpA) antibody into 10mL of activated latex microspheres, slowly mixing uniformly for 30 minutes, centrifuging for 10 minutes at 19000g, and removing supernatant; resuspending the precipitate with 10mL borax buffer solution containing 1% casein, repeating centrifugation for 1 time after ultrasonic pulverization, and removing supernatant; resuspending the precipitate by the same method, repeatedly centrifuging for 1 time after ultrasonic crushing, and removing supernatant; resuspending the precipitate with 10mL borax buffer solution containing 1% casein, namely the latex microsphere marker of the acinetobacter baumannii multiple epitope fusion antigen (Fhue + OmpA) antibody; the content of the components in the borax buffer solution is 0.1mol/L Na2B4O7The pH value of the borax buffer solution is 8.5;
3) preparation of the bonding pad:
spraying the latex microsphere marker of the acinetobacter baumannii multiple epitope fusion antigen (Fhue + OmpA) antibody obtained in the step 2) on a bonding pad made of a polyester fiber material, wherein the spraying amount of a polyester fiber film per square centimeter is 10 mu L of the latex microsphere marker; drying at 37 deg.C in environment with relative humidity not more than 30%, sealing at 25 deg.C, drying and storing;
4) preparation of antibody solid-phase nitrocellulose membrane:
diluting the Acinetobacter baumannii multiple epitope fusion antigen (Pilf + ponA) antibody obtained in the step 1) into 1.5mg/mL by using a borax buffer solution, and then coating the antibody on a detection line position on a nitrocellulose membrane by using a membrane spraying instrument as a detection line to capture the antibody, wherein the coating parameter is 1 mu L/cm; spraying goat anti-rabbit IgG on a quality control line position on a nitrocellulose membrane as a control line to capture an antibody, wherein the concentration is 1mg/mL, and the coating parameter is 1 mu L/cm; after coating, putting the nitrocellulose membrane in an environment with the relative humidity not more than 30%, drying at 37 ℃, sealing at 25 ℃, drying and storing; the content of the components in the borax buffer solution is 0.1mol/LNa2B4O7The pH value of the borax buffer solution is 8.5;
5) preparation of sample pad:
taking a glass cellulose membrane, soaking the glass cellulose membrane in a sample pad treatment solution for at least 3h, placing the sample pad treatment solution in a biological safety cabinet for ventilation drying at 37 ℃, cutting the sample pad treatment solution into required specifications, and sealing, drying and storing the sample pad treatment solution at 25 ℃; thus, a sample pad was prepared;
the sample pad treatment solution comprises the following components in percentage by weight: 0.01mol/LNa2B4O72g/L sodium chloride, 20g/L casein, 10ml/L Tween-20 and 10ml/L antifoaming agent S-17; the pH of the sample pad treatment solution was 8.5;
6) assembling the detection card:
respectively sticking a water absorption pad, an antibody solid-phase nitrocellulose membrane, a combination pad and a sample pad which are made of water absorption filter paper materials on a PVC (polyvinyl chloride) base plate in sequence, wherein a quality control line on the nitrocellulose membrane is close to the end of the water absorption pad, a detection line is close to the end of the sample pad, cutting the nitrocellulose membrane into detection cards with certain widths, sealing and packaging the detection cards, and drying the detection cards for low-temperature storage; thus, the multiple epitope fusion antigen-based acinetobacter baumannii infection rapid detection card is prepared.
CN201911053066.6A 2019-10-31 2019-10-31 Rapid detection method for acinetobacter baumannii infection based on multiple epitope fusion antigen Expired - Fee Related CN110724201B (en)

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