CN110540598B - Haemophilus influenzae Elisa detection kit based on surface protein antibody of haemophilus influenzae and preparation method - Google Patents

Haemophilus influenzae Elisa detection kit based on surface protein antibody of haemophilus influenzae and preparation method Download PDF

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CN110540598B
CN110540598B CN201811566010.6A CN201811566010A CN110540598B CN 110540598 B CN110540598 B CN 110540598B CN 201811566010 A CN201811566010 A CN 201811566010A CN 110540598 B CN110540598 B CN 110540598B
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haemophilus influenzae
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CN110540598A (en
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杨波
李�杰
胡征
王毅
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Hubei University of Technology
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/285—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Pasteurellaceae (F), e.g. Haemophilus influenza
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
    • C07K16/1242—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Pasteurellaceae (F), e.g. Haemophilus influenza
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Abstract

The invention relates to an Elisa detection kit for rapidly detecting haemophilus influenzae and a preparation method thereof. The kit comprises: the kit comprises an enzyme label plate coated with anti-Haemophilus influenzae D15 and Pe protein polyclonal antibody, anti-Haemophilus influenzae Pcp and PilA protein polyclonal antibody, a positive control and a negative control of Haemophilus influenzae, 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 joint detection on four specific surface proteins of Haemophilus influenzae, so that the sensitivity, specificity and repeatability of Haemophilus influenzae detection are greatly improved, and the kit can be used for non-diagnostic detection and research work on Haemophilus influenzae.

Description

Haemophilus influenzae Elisa detection kit based on surface protein antibody of haemophilus influenzae and preparation method
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 Haemophilus influenzae Elisa detection kit based on a Haemophilus influenzae surface protein antibody and a preparation method thereof.
Background
Haemophilus influenzae (Hi) is a gram-negative bacterium without motility. It was discovered in 1892 by the fuverbo in epidemic cold. It is generally an aerobic organism, but can grow into a facultative anaerobic organism. Haemophilus influenzae was originally mistaken for the cause of influenza, but until 1933 this misunderstanding was not eliminated when the viral etiology of influenza was discovered. Haemophilus influenzae has generally six strains, which are called type a, type b (also called type B), type c, type d, type e and type f. Naturally occurring diseases caused by haemophilus influenzae occur only in humans. In infants and children, haemophilus influenzae b causes bacteremia and acute bacterial meningitis. Occasionally it causes cellulitis, osteomyelitis and joint infections. Since 1990, the prevalence of HiB disorders has decreased to 1.3 childhood infections per hundred thousand children in the United states after the use of HiB-conjugated vaccines. However, HiB remains the leading cause of lower respiratory tract disease in infants and children in developing countries. Haemophilus influenzae without a capsule can cause ear infections (e.g. otitis media), eye infections (conjunctivitis) and sinusitis in children, and associated pneumonia. Such bacteria have strict nutrient requirements for culture and tend to parasitize in the upper airway, so that false negatives and false positives often occur in culture.
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 components is critical to the production of highly specific antibodies. The Haemophilus influenzae Pe, D15, PCP and PilA proteins are important molecules located on the cell surface, and the protein is high in conservation, specificity, antigenicity and surface exposure, and is an ideal detection target. In the research, surface proteins Pe, D15, PCP, PilA and the like with interspecies specificity are selected as detection targets, recombinant antigens are prepared in a gene recombination mode, and then a polyclonal antibody with good specificity is prepared and is applied to preparation of an Elisa detection kit for haemophilus influenzae.
Disclosure of Invention
In order to solve the technical problems in the background art, the invention provides the haemophilus influenzae Elisa detection kit based on the haemophilus influenzae surface protein antibody, and the preparation method thereof, wherein the haemophilus influenzae detection kit can improve the sensitivity, specificity and repeatability of haemophilus influenzae detection, is simple to operate, is low in cost, and can quickly and rapidly detect haemophilus influenzae.
In order to achieve the purpose, the invention adopts the following technical scheme:
a Haemophilus influenzae Elisa detection kit based on a Haemophilus influenzae surface protein antibody is characterized in that: the haemophilus influenzae Elisa detection kit based on the haemophilus influenzae surface protein antibody comprises an enzyme label plate coated with a polyclonal antibody of anti-haemophilus influenzae surface protein (D15 and Pe), an anti-haemophilus influenzae surface protein (Pcp and PilA) polyclonal antibody, a haemophilus influenzae positive control, a negative control, a washing solution, an enzyme-labeled secondary antibody, an enzyme chromogenic substrate and a stop solution; the positive control of the haemophilus influenzae is inactivated haemophilus influenzae 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 Haemophilus influenzae Elisa detection kit based on a Haemophilus influenzae surface protein antibody is characterized in that: the haemophilus influenzae Elisa detection kit based on the haemophilus influenzae surface protein antibody comprises an enzyme label plate coated with a polyclonal antibody of anti-haemophilus influenzae surface protein (D15 and Pe), an anti-haemophilus influenzae surface protein (Pcp and PilA) polyclonal antibody, a haemophilus influenzae positive control, a negative control, a washing solution, an enzyme-labeled secondary antibody, an enzyme chromogenic substrate and a stop solution; the positive control of the haemophilus influenzae is inactivated haemophilus influenzae 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 coated with the polyclonal antibody of the anti-Haemophilus influenzae surface protein (D15 and Pe) comprises the following steps:
1) preparation of polyclonal antibodies against haemophilus influenzae surface proteins (D15 and Pe):
1.1) respectively obtaining peptide segments with most abundant antigenic epitopes in the surface protein D15 and the extracellular domain of the surface protein Pe of the haemophilus influenzae, 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 accession numbers of the haemophilus influenzae surface protein D15 and the surface protein Pe in the NCBI protein database are AAX87955 and AGT37361 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 a 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 D15 pe;
the complete sequence of the D15pe gene is as follows:
CATATGCAGCGCGTAACCGACAACGACGTTGCAAACATCGTACGTTCTCTGTTTGTGTCCGGCCGTTTCGATGACGTTAAAGCTCACCAGGAAGGTGATGTTCTGGTTGTTTCTGTTGTTGCAAAATCCATCATCAGTGACGTTAAAATCAAGGGCAACTCTATCATCCCAACTGAAGCTCTGAAGCAGAACCTGGACGCTAACGGTTTTAAAGTAGGTGATGTTCTGATCCGTGAAAAGCTGAACGAATTCGCTAAATCCGTTAAGGAGCATTATGCGAGTGTTGGTCGTTACAACGCTACCGTAGAACCGATTGTTAACACTCTGCCGAACAACCGCGCGGAAATCCTGATCCAGATCAACGAAGATGATAAAGCAAAACTGGCTTCTCTGACCTTCAAAGGTAACGAATCTGTATCCTCATCCACTCTTCAGGAACAGATGGAACTGCAGCCGGACTCTTGGTGGAAGCTGTGGGGCAACAAATTCGAAGGCGCGCAGTTCGAAAAAGACCTGCAATCTATTCGTGATTATTACCTGAACAACGGTTATGCAAAAGGTGGTTCTGGTGGTTCTGGTGGTTCTGGTGGTTCTCTGGTAAAAAACGTTAACTACTACATCGACTCTGAATCCATCTGGGTTGATAACCAGGAACCACAGATCGTACACTTCGACGCTGTTGTTAACCTCGATAAAGGCCTGTACGTTTACCCGGAACCAAAGCGTTACGCTCGTTCTGTTCGTCAGTACAAAATCCTTAACTGTGCGAACTACCATCTGACCCAGGTACGTACCGACTTTTACGACGAATTCTGGGGTCAGGGCCTGCGCGCAGCTCCGAAAAAACAGAAAAAACACTAAGGATCC；
the protein sequence coded by the D15pe gene is:
MQRVTDNDVANIVRSLFVSGRFDDVKAHQEGDVLVVSVVAKSIISDVKIKGNSIIPTEALKQNLDANGFKVGDVLIREKLNEFAKSVKEHYASVGRYNATVEPIVNTLPNNRAEILIQINEDDKAKLASLTFKGNESVSSSTLQEQMELQPDSWWKLWGNKFEGAQFEKDLQSIRDYYLNNGYAKGGSGGSGGSGGSLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKH；
the protein sequence coded by the D15Pe gene is 50-233aa of the Haemophilus influenzae surface protein D15 and 43-130aa of the surface protein Pe; the two protein sequences are connected by flexible connecting peptide ggsggsggs;
1.2) cloning the complete gene sequence of D15pe into prokaryotic expression vector pET-28a (+) by a conventional method, transferring into E.coli BL21(DE3) bacteria, inducing recombinant escherichia coli expression by IPTG, and using Ni2+Purifying the recombinant His-D15pe protein by affinity chromatography; the recombinant protein is used as an immune antigen, mixed with Freund's adjuvant, repeatedly and artificially immunized to obtain new Zealand white rabbit, the blood is drawn for titer determination, high-titer recombinant protein antibody is separated andpurifying to obtain polyclonal antibody against Haemophilus influenzae surface protein (D15 and Pe);
2) coating of polyclonal antibodies against haemophilus influenzae surface proteins (D15 and Pe):
diluting the anti-Haemophilus influenzae surface protein (D15 and Pe) polyclonal antibody 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-Haemophilus influenzae surface protein (Pcp and PilA) polyclonal antibody comprises the following steps:
1) respectively obtaining peptide segments with the most abundant antigenic epitopes in the extracellular domains of the haemophilus influenzae surface protein Pcp and the surface protein PilA, finding out the gene coding sequences of the peptide segments, optimizing the gene coding sequences of the peptide segments, and connecting the optimized gene coding sequences of the peptide segments by using the coding sequences of rigid connecting peptides to form fusion genes; the access numbers of the haemophilus influenzae surface protein Pcp and the surface protein PilA in the NCBI protein database are AAX88288 and AAX12377 respectively; the sequence of the rigid linker peptide is eaaakaaaak; simultaneously, introducing a restriction enzyme site NdeI at the 5 'end of the fusion gene, introducing a termination signal TAA and a restriction enzyme site BamHI at the 3' end of the fusion gene, and then chemically synthesizing a whole gene sequence which is marked as pcppil;
the complete sequence of the gene of pcppil is:
CATATGCGCCCGGTTAAAATCCAGGCTGATAACCAGGGTGTAATCGGCACCCTGGGCGGCGGCGCTCTGGGCGGTATCGCAGGTTCTGCTATCGGCGGCGGTCGCGGCCAGGTTATTGCCGCTGTAGTAGGCGCTATCGGTGGTGCGGTGGCCGGTTCTAAAATCGAAGAAAAGGTTTCCCAGGTAAACGGTGCTGAGCTGGTAATCAAAAAAGACGATGGCCAGGAAATAGTTGTTGTACAGAAAGCCGACTCTTCCTTTGTAGCAGGTCGTCGCGTGCGCATCGTTGGTGGTGGGTCTAACCTGAACGTCTCTGTTCTGGAAGCTGCTGCTGCTAAAGAAGCTGCTGCTGCTAAACTGATTGAACTGATGATCGTGATCGCGATCATCGCAATTCTGGCGACCATCGCAATCCCGTCTTACCAGAACTACACTAAAAAAGCTGCTGTCTCCGAACTGCTGCAAGCATCTGCTCCGTACAAAGCTGACGTTGAACTCTGCGTTTACTCTACGAACGAGACCACCAACTGCACTGGTGGCAAAAACGGTATTGCGGCTGACATCACCACTGCTAAGGGTTACGTAAAATCTGTTACCACCTCTAACGGCGCAATCACTGTGAAAGGGGACGGCACCCTGGCTAACATGGAATACATTCTGCAGGCTACCGGTAACGCAGCGACCGGCGTGACCTGGACTACCACCTGCAAAGGCTAAGGATCC；
the protein sequence encoded by the pcppil gene is:
MRPVKIQADNQGVIGTLGGGALGGIAGSAIGGGRGQVIAAVVGAIGGAVAGSKIEEKVSQVNGAELVIKKDDGQEIVVVQKADSSFVAGRRVRIVGGGSNLNVSVLEAAAAKEAAAAKLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNETTNCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQATGNAATGVTWTTTCKG；
the protein sequence coded by the pcppil gene is 50-154aa of a surface protein Pcp of the haemophilus influenzae and 17-133aa of a surface protein PilA; the middle of the two protein sequences is connected by rigid connecting peptide eaaakaaaak;
2) cloning the complete gene sequence of Pcppil into prokaryotic expression vector pET-28a (+) by a conventional method, transferring the cloned complete gene sequence into E.coli BL21(DE3) bacteria, inducing recombinant escherichia coli expression by IPTG, and purifying the recombinant His-Pcpppil protein by Ni2+ 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, finally obtaining anti-Haemophilus influenzae Pcp and PilA protein polyclonal antibodies, and diluting the antibodies 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 the soluble recombinant D15/Pe fusion protein and the Pcp/PilA fusion protein are successfully obtained 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 haemophilus influenzae 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 a haemophilus influenzae standard strain ATCC49247 reaches 1 x 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 specific experiments carried out by using 6 strains of Haemophilus influenzae strains and 17 strains of non-Haemophilus influenzae standard strains (containing most common respiratory pathogens) show that the test strip has good specificity and stability, can detect all tested Haemophilus influenzae strains, has no cross reaction with all non-Haemophilus influenzae 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 haemophilus influenzae D15 and Pe protein:
1.1) cloning of the fusion Gene of Haemophilus influenzae D15pe
The peptide segment with the most abundant antigenic epitopes in the extracellular domains of the haemophilus influenzae surface proteins D15 and Pe (the accession numbers in the NCBI protein database are AAX87955 and AGT37361 respectively) is obtained, the gene coding sequence is found, the gene coding sequence is optimized, and the two sequences are connected by the coding sequence of flexible connecting peptide (ggsgggsgggs) to form the 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 D15 pe. The complete gene sequence and the coded amino acid sequence are shown in a sequence table. Specifically, the protein sequence coded by the D15Pe gene is 50-233aa of the Haemophilus influenzae surface protein D15 and 43-130aa of the surface protein Pe, 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 carrying out double enzyme digestion on the vector pET-28a (+) by NdeI and BamHI, connecting the D15pe gene obtained after double enzyme digestion into the pET-28a (+) vector according to a conventional molecular biological method, and transforming Escherichia coli TOP10 to construct a pET-D15pe expression vector. The construction of the expression vector is verified to be correct by enzyme digestion and sequence determination. The vector expresses a recombinant D15pe fusion protein.
1.2) expression and purification of Haemophilus influenzae D15pe fusion protein
Culturing the correctly identified positive clone bacteria, extracting plasmids, transferring into competent E.coli BL21(DE3) according to a conventional technology, coating the bacterial liquid on an LB flat plate containing 50 mu g/mL kanamycin after the conversion is finished, and screening expression strains according to a conventional method. A single colony transformed with pET-D15pe and having the ability to express a foreign protein was 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 37 ℃ until OD600 became 0.6, added with 1mol/L of 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 mixture was treated with 50mL of 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 D15pe fusion protein was found to be present in the bacterial cells in partially solubilized form (the other portion was present as inclusion bodies). Thin-layer scanning showed that the recombinant protein accounted for more than 20% of the total bacterial protein. When wild type D15pe which was not gene-optimized was expressed in the same manner as described above, no expression product was detected, indicating that the gene optimization effect was prominent. 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 for recombinant D15pe protein. 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 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. Thus, a Haemophilus influenzae D15pe fusion protein was prepared.
1.3) preparation of polyclonal antibody against Haemophilus influenzae D15 and Pe protein
1.3.1) mixing the Haemophilus influenzae D15pe fusion protein prepared in the step (1.2) with Freund's complete adjuvant, emulsifying to obtain immunogen for immunizing 2 male New Zealand rabbits, wherein the total amount of subcutaneous injection per 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 D15pe fusion protein and Freund incomplete adjuvant, wherein the immunization is performed for 5 times in total, 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 D15pe fusion protein was used as a coating antigen, the coating concentration was 5. mu.g/ml, each well was coated with 100. mu.l, and the serum antibody level was measured by indirect ELISA. 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 above, it shows 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 polyclonal antibody against the haemophilus influenzae D15 and the Pe protein is prepared.
Example 2
Preparation of polyclonal antibodies against Haemophilus influenzae Pcp and PilA proteins:
1.1) cloning of the fusion Gene of Haemophilus influenzae pcppil
The method comprises the steps of obtaining peptide segments with most abundant antigen epitopes in extracellular domains of haemophilus influenzae surface proteins Pcp and PilA (the access numbers in an NCBI protein database are AAX88288 and AAX12377 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, 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 pcppil. The complete gene sequence and the coded amino acid sequence are shown in a sequence table. Specifically, the protein sequence coded by the pcppil gene is 50-154aa of the Haemophilus influenzae surface protein Pcp and 17-133aa of the surface protein PilA, 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. Meanwhile, NdeI and BamHI are adopted to carry out double enzyme digestion on a vector pET-28a (+), and pcppil gene obtained after double enzyme digestion is connected into the pET-28a (+) vector according to a conventional molecular biology method, and escherichia coli TOP10 is transformed to construct a pET-pcppil expression vector. The construction of the expression vector is verified to be correct by enzyme digestion and sequence determination. The vector expresses a recombinant Pcppil fusion protein.
1.2) expression and purification of Haemophilus influenzae Pcppel fusion proteins
Culturing the correctly identified positive clone bacteria, extracting plasmids, transferring into competent E.coli BL21(DE3) according to a conventional technology, coating the bacterial liquid on an LB flat plate containing 50 mu g/mL kanamycin after the conversion is finished, and screening expression strains according to a conventional method. Individual colonies transformed with pET-pcppil 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, 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 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 mixture was treated with 50mL of 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 Pcppil fusion protein was found to be present in the thallus in partially solubilized form (another portion is present in inclusion bodies). Thin-layer scanning showed that the recombinant protein accounted for more than 30% of the total bacterial protein. The wild-type pcppil gene which was not gene-optimized was expressed in the same manner as described above, and as a result, it was found that the expression product accounted for only 5% of the total protein, indicating that the gene optimization effect was outstanding. 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 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, a Haemophilus influenzae Pcppel fusion protein is prepared.
1.3) preparation of polyclonal antibody against Haemophilus influenzae Pcp and PilA proteins
1.3.1) mixing the Haemophilus influenzae Pcppel 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 per 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 Pcppel fusion protein and Freund's incomplete adjuvant, wherein the immunization is performed 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 Pcppel 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 above, it shows 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 polyclonal antibody against the haemophilus influenzae Pcp and the PilA protein is prepared.
Example 3
Haemophilus influenzae Elisa detection kit
1. Haemophilus influenzae Elisa detection kit composition
The haemophilus influenzae Elisa detection kit comprises an enzyme label plate coated with anti-haemophilus influenzae D15 and a Pe protein polyclonal antibody, an anti-haemophilus influenzae Pcp and PilA protein polyclonal antibody, a haemophilus influenzae positive control, a negative control, a washing solution, an enzyme-labeled secondary antibody, an enzyme chromogenic substrate and a stop solution.
(1) ELISA plate coated with anti-influenza haemophilus D15 and Pe protein polyclonal antibody
The anti-H.influenzae D15 and the polyclonal antibody against the Pe protein (prepared in example 1) were diluted to a concentration of 10. mu.g/mL with PBS buffer, and a 96-well EIA high-efficiency binding ELISA plate (model: Corning costar 2592) was coated at 100. mu.L/well and then 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 Haemophilus influenzae Pcp and PilA protein
The anti-Haemophilus influenzae Pcp and PilA protein polyclonal antibody as described in example 2 was formulated with the above blocking solution into a 20. mu.g/mL 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 was formaldehyde inactivated haemophilus influenzae (ATCC49247) prepared as follows: taking Haemophilus influenzae liquid cultured in chocolate liquid culture medium, counting by a flat plate, centrifugally collecting thalli, re-suspending the thalli by physiological saline and adjusting the concentration of the thalli to 1 × 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. 5mL of the bacterial solution was added with analytically pure formaldehyde to a final concentration of 1% and inactivated overnight at 4 ℃. 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
Application method of Haemophilus influenzae Elisa detection kit
1) Treating a sample to be detected: 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 a control sample and a sample to be detected: 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 Haemophilus influenzae Pcp and PilA protein: adding working solution of polyclonal antibody against Haemophilus influenzae Pcp and PilA protein in the kit, incubating at 37 deg.C for 1 hr at 50 μ L/well, washing the plate with 250 μ L PBST washing solution for 3 times, and spin-drying.
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 an enzyme chromogenic substrate: add freshly prepared enzyme chromogenic substrate in the kit, 50. mu.L/well, develop for 15 minutes.
6) Adding a stop solution: the reaction was stopped by adding 50. mu.L of stop solution to each well.
7) Measurement of OD450nm value: the microplate was placed in a microplate reader to determine the OD450nm value.
8) And (4) judging a result: 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 Haemophilus influenzae antigen in the clinical pharynx swab is judged to be positive, otherwise, the Haemophilus influenzae 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 Haemophilus influenzae Elisa detection kit
1) Specific assay
To verify the specificity of the Haemophilus influenzae Elisa test kit of the present invention, the kit composition and method described in examples 3 and 4 were performed at a concentration of 2X 1056 strains of Haemophilus influenzae and 17 non-Haemophilus influenzae of CFU/mL were tested and shown in Table 1. The result shows that the detection results of the kit are positive for all 6 strains of haemophilus influenzae strains, and the detection results of the kit are negative for other 17 strains of common respiratory pathogenic microorganisms. The kit showed good specificity.
TABLE 1
Figure GDA0002973268660000121
Figure GDA0002973268660000131
At the same time, the concentration is 2 multiplied by 105The test of 121 human haemophilus influenzae clinical isolates of CFU/mL by using the kit shows positive results, and shows that the kit has high detection coverage on the human haemophilus influenzae clinically isolated.
2) Sensitivity assay
Inoculating Haemophilus influenzae ATCC49247 strain to sheep blood chocolate culture medium, culturing at 35 deg.C for 24 hr, diluting with normal saline 10 times in 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> Haemophilus influenzae Elisa detection kit based on Haemophilus influenzae surface protein antibody and preparation method thereof
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 867
<212> DNA
<213> complete Gene sequence of D15pe (D15pe)
<400> 1
catatgcagc gcgtaaccga caacgacgtt gcaaacatcg tacgttctct gtttgtgtcc 60
ggccgtttcg atgacgttaa agctcaccag gaaggtgatg ttctggttgt ttctgttgtt 120
gcaaaatcca tcatcagtga cgttaaaatc aagggcaact ctatcatccc aactgaagct 180
ctgaagcaga acctggacgc taacggtttt aaagtaggtg atgttctgat ccgtgaaaag 240
ctgaacgaat tcgctaaatc cgttaaggag cattatgcga gtgttggtcg ttacaacgct 300
accgtagaac cgattgttaa cactctgccg aacaaccgcg cggaaatcct gatccagatc 360
aacgaagatg ataaagcaaa actggcttct ctgaccttca aaggtaacga atctgtatcc 420
tcatccactc ttcaggaaca gatggaactg cagccggact cttggtggaa gctgtggggc 480
aacaaattcg aaggcgcgca gttcgaaaaa gacctgcaat ctattcgtga ttattacctg 540
aacaacggtt atgcaaaagg tggttctggt ggttctggtg gttctggtgg ttctctggta 600
aaaaacgtta actactacat cgactctgaa tccatctggg ttgataacca ggaaccacag 660
atcgtacact tcgacgctgt tgttaacctc gataaaggcc tgtacgttta cccggaacca 720
aagcgttacg ctcgttctgt tcgtcagtac aaaatcctta actgtgcgaa ctaccatctg 780
acccaggtac gtaccgactt ttacgacgaa ttctggggtc agggcctgcg cgcagctccg 840
aaaaaacaga aaaaacacta aggatcc 867
<210> 2
<211> 285
<212> PRT
<213> protein sequence of D15pe (D15pe)
<400> 2
Met Gln Arg Val Thr Asp Asn Asp Val Ala Asn Ile Val Arg Ser Leu
1 5 10 15
Phe Val Ser Gly Arg Phe Asp Asp Val Lys Ala His Gln Glu Gly Asp
20 25 30
Val Leu Val Val Ser Val Val Ala Lys Ser Ile Ile Ser Asp Val Lys
35 40 45
Ile Lys Gly Asn Ser Ile Ile Pro Thr Glu Ala Leu Lys Gln Asn Leu
50 55 60
Asp Ala Asn Gly Phe Lys Val Gly Asp Val Leu Ile Arg Glu Lys Leu
65 70 75 80
Asn Glu Phe Ala Lys Ser Val Lys Glu His Tyr Ala Ser Val Gly Arg
85 90 95
Tyr Asn Ala Thr Val Glu Pro Ile Val Asn Thr Leu Pro Asn Asn Arg
100 105 110
Ala Glu Ile Leu Ile Gln Ile Asn Glu Asp Asp Lys Ala Lys Leu Ala
115 120 125
Ser Leu Thr Phe Lys Gly Asn Glu Ser Val Ser Ser Ser Thr Leu Gln
130 135 140
Glu Gln Met Glu Leu Gln Pro Asp Ser Trp Trp Lys Leu Trp Gly Asn
145 150 155 160
Lys Phe Glu Gly Ala Gln Phe Glu Lys Asp Leu Gln Ser Ile Arg Asp
165 170 175
Tyr Tyr Leu Asn Asn Gly Tyr Ala Lys Gly Gly Ser Gly Gly Ser Gly
180 185 190
Gly Ser Gly Gly Ser Leu Val Lys Asn Val Asn Tyr Tyr Ile Asp Ser
195 200 205
Glu Ser Ile Trp Val Asp Asn Gln Glu Pro Gln Ile Val His Phe Asp
210 215 220
Ala Val Val Asn Leu Asp Lys Gly Leu Tyr Val Tyr Pro Glu Pro Lys
225 230 235 240
Arg Tyr Ala Arg Ser Val Arg Gln Tyr Lys Ile Leu Asn Cys Ala Asn
245 250 255
Tyr His Leu Thr Gln Val Arg Thr Asp Phe Tyr Asp Glu Phe Trp Gly
260 265 270
Gln Gly Leu Arg Ala Ala Pro Lys Lys Gln Lys Lys His
275 280 285
<210> 3
<211> 723
<212> DNA
<213> complete Gene sequence of pcppil (pcppil)
<400> 3
catatgcgcc cggttaaaat ccaggctgat aaccagggtg taatcggcac cctgggcggc 60
ggcgctctgg gcggtatcgc aggttctgct atcggcggcg gtcgcggcca ggttattgcc 120
gctgtagtag gcgctatcgg tggtgcggtg gccggttcta aaatcgaaga aaaggtttcc 180
caggtaaacg gtgctgagct ggtaatcaaa aaagacgatg gccaggaaat agttgttgta 240
cagaaagccg actcttcctt tgtagcaggt cgtcgcgtgc gcatcgttgg tggtgggtct 300
aacctgaacg tctctgttct ggaagctgct gctgctaaag aagctgctgc tgctaaactg 360
attgaactga tgatcgtgat cgcgatcatc gcaattctgg cgaccatcgc aatcccgtct 420
taccagaact acactaaaaa agctgctgtc tccgaactgc tgcaagcatc tgctccgtac 480
aaagctgacg ttgaactctg cgtttactct acgaacgaga ccaccaactg cactggtggc 540
aaaaacggta ttgcggctga catcaccact gctaagggtt acgtaaaatc tgttaccacc 600
tctaacggcg caatcactgt gaaaggggac ggcaccctgg ctaacatgga atacattctg 660
caggctaccg gtaacgcagc gaccggcgtg acctggacta ccacctgcaa aggctaagga 720
tcc 723
<210> 4
<211> 237
<212> PRT
<213> protein sequence of pcppil (pcppil)
<400> 4
Met Arg Pro Val Lys Ile Gln Ala Asp Asn Gln Gly Val Ile Gly Thr
1 5 10 15
Leu Gly Gly Gly Ala Leu Gly Gly Ile Ala Gly Ser Ala Ile Gly Gly
20 25 30
Gly Arg Gly Gln Val Ile Ala Ala Val Val Gly Ala Ile Gly Gly Ala
35 40 45
Val Ala Gly Ser Lys Ile Glu Glu Lys Val Ser Gln Val Asn Gly Ala
50 55 60
Glu Leu Val Ile Lys Lys Asp Asp Gly Gln Glu Ile Val Val Val Gln
65 70 75 80
Lys Ala Asp Ser Ser Phe Val Ala Gly Arg Arg Val Arg Ile Val Gly
85 90 95
Gly Gly Ser Asn Leu Asn Val Ser Val Leu Glu Ala Ala Ala Ala Lys
100 105 110
Glu Ala Ala Ala Ala Lys Leu Ile Glu Leu Met Ile Val Ile Ala Ile
115 120 125
Ile Ala Ile Leu Ala Thr Ile Ala Ile Pro Ser Tyr Gln Asn Tyr Thr
130 135 140
Lys Lys Ala Ala Val Ser Glu Leu Leu Gln Ala Ser Ala Pro Tyr Lys
145 150 155 160
Ala Asp Val Glu Leu Cys Val Tyr Ser Thr Asn Glu Thr Thr Asn Cys
165 170 175
Thr Gly Gly Lys Asn Gly Ile Ala Ala Asp Ile Thr Thr Ala Lys Gly
180 185 190
Tyr Val Lys Ser Val Thr Thr Ser Asn Gly Ala Ile Thr Val Lys Gly
195 200 205
Asp Gly Thr Leu Ala Asn Met Glu Tyr Ile Leu Gln Ala Thr Gly Asn
210 215 220
Ala Ala Thr Gly Val Thr Trp Thr Thr Thr Cys Lys Gly
225 230 235

Claims (8)

1. Haemophilus influenzae surface proteins D15 and Pe, comprising: the protein sequences of the Haemophilus influenzae surface proteins D15 and Pe are as follows:
MQRVTDNDVANIVRSLFVSGRFDDVKAHQEGDVLVVSVVAKSIISDVKIKGNSIIPTEALKQNLDANGFKVGDVLIREKLNEFAKSVKEHYASVGRYNATVEPIVNTLPNNRAEILIQINEDDKAKLASLTFKGNESVSSSTLQEQMELQPDSWWKLWGNKFEGAQFEKDLQSIRDYYLNNGYAKGGSGGSGGSGGSLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKH；
the complete sequence of the gene for encoding the proteins of the Haemophilus influenzae surface proteins D15 and Pe is:
CATATGCAGCGCGTAACCGACAACGACGTTGCAAACATCGTACGTTCTCTGTTTGTGTCCGGCCGTTTCGATGACGTTAAAGCTCACCAGGAAGGTGATGTTCTGGTTGTTTCTGTTGTTGCAAAATCCATCATCAGTGACGTTAAAATCAAGGGCAACTCTATCATCCCAACTGAAGCTCTGAAGCAGAACCTGGACGCTAACGGTTTTAAAGTAGGTGATGTTCTGATCCGTGAAAAGCTGAACGAATTCGCTAAATCCGTTAAGGAGCATTATGCGAGTGTTGGTCGTTACAACGCTACCGTAGAACCGATTGTTAACACTCTGCCGAACAACCGCGCGGAAATCCTGATCCAGATCAACGAAGATGATAAAGCAAAACTGGCTTCTCTGACCTTCAAAGGTAACGAATCTGTATCCTCATCCACTCTTCAGGAACAGATGGAACTGCAGCCGGACTCTTGGTGGAAGCTGTGGGGCAACAAATTCGAAGGCGCGCAGTTCGAAAAAGACCTGCAATCTATTCGTGATTATTACCTGAACAACGGTTATGCAAAAGGTGGTTCTGGTGGTTCTGGTGGTTCTGGTGGTTCTCTGGTAAAAAACGTTAACTACTACATCGACTCTGAATCCATCTGGGTTGATAACCAGGAACCACAGATCGTACACTTCGACGCTGTTGTTAACCTCGATAAAGGCCTGTACGTTTACCCGGAACCAAAGCGTTACGCTCGTTCTGTTCGTCAGTACAAAATCCTTAACTGTGCGAACTACCATCTGACCCAGGTACGTACCGACTTTTACGACGAATTCTGGGGTCAGGGCCTGCGCGCAGCTCCGAAAAAACAGAAAAAACACTAAGGATCC。
2. a process for preparing haemophilus influenzae surface proteins D15 and Pe 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 Haemophilus influenzae surface protein D15 and the surface protein Pe extracellular domain, finding out peptide segment gene coding sequences with the most abundant antigenic epitopes in the Haemophilus influenzae surface protein D15 and the surface protein Pe extracellular domain, optimizing the peptide segment gene coding sequences, and connecting the optimized peptide segment gene coding sequences by using the coding sequences of flexible connecting peptides to form a fusion gene; the accession numbers of the haemophilus influenzae surface protein D15 and the surface protein Pe in the NCBI protein database are AAX87955 and AGT37361 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 a 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 D15 pe;
the complete sequence of the D15pe gene is as follows: CATATGCAGCGCGTAACCGACAACGACGTTGCAAACATCGTACGTTCTCTGTTTGTGTCCGGCCGTTTCGATGACGTTAAAGCTCACCAGGAAGGTGATGTTCTGGTTGTTTCTGTTGTTGCAAAATCCATCATCAGTGACGTTAAAATCAAGGGCAACTCTATCATCCCAACTGAAGCTCTGAAGCAGAACCTGGACGCTAACGGTTTTAAAGTAGGTGATGTTCTGATCCGTGAAAAGCTGAACGAATTCGCTAAATCCGTTAAGGAGCATTATGCGAGTGTTGGTCGTTACAACGCTACCGTAGAACCGATTGTTAACACTCTGCCGAACAACCGCGCGGAAATCCTGATCCAGATCAACGAAGATGATAAAGCAAAACTGGCTTCTCTGACCTTCAAAGGTAACGAATCTGTATCCTCATCCACTCTTCAGGAACAGATGGAACTGCAGCCGGACTCTTGGTGGAAGCTGTGGGGCAACAAATTCGAAGGCGCGCAGTTCGAAAAAGACCTGCAATCTATTCGTGATTATTACCTGAACAACGGTTATGCAAAAGGTGGTTCTGGTGGTTCTGGTGGTTCTGGTGGTTCTCTGGTAAAAAACGTTAACTACTACATCGACTCTGAATCCATCTGGGTTGATAACCAGGAACCACAGATCGTACACTTCGACGCTGTTGTTAACCTCGATAAAGGCCTGTACGTTTACCCGGAACCAAAGCGTTACGCTCGTTCTGTTCGTCAGTACAAAATCCTTAACTGTGCGAACTACCATCTGACCCAGGTACGTACCGACTTTTACGACGAATTCTGGGGTCAGGGCCTGCGCGCAGCTCCGAAAAAACAGAAAAAACACTAAGGATCC, respectively;
the protein sequence coded by the D15pe gene is: MQRVTDNDVANIVRSLFVSGRFDDVKAHQEGDVLVVSVVAKSIISDVKIKGNSIIPTEALKQNLDANGFKVGDVLIREKLNEFAKSVKEHYASVGRYNATVEPIVNTLPNNRAEILIQINEDDKAKLASLTFKGNESVSSSTLQEQMELQPDSWWKLWGNKFEGAQFEKDLQSIRDYYLNNGYAKGGSGGSGGSGGSLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKH, respectively;
the protein sequence coded by the D15Pe gene is 50-233aa of the Haemophilus influenzae surface protein D15 and 43-130aa of the surface protein Pe; the two protein sequences are connected by flexible connecting peptide ggsggsggs;
cloning the complete gene sequence of D15pe into prokaryotic expression vector pET-28a (+) by conventional method, transferring into E.coli BL21DE3 strain, inducing recombinant Escherichia coli expression with IPTG, and using Ni2+Affinity chromatography purified recombinant D15pe protein.
3. A method for preparing anti-Haemophilus influenzae surface protein D15 and Pe polyclonal antibody, which is characterized by comprising the following steps: the method comprises the steps of taking the recombinant D15Pe protein as an immune antigen as claimed in claim 2, mixing the immune antigen with Freund's adjuvant, repeatedly and artificially immunizing healthy New Zealand white rabbits, drawing blood to perform titer measurement, separating high-titer recombinant protein antibodies and purifying to finally obtain the anti-Haemophilus influenzae surface protein D15 and Pe polyclonal antibodies.
4. A Haemophilus influenzae surface protein Pcp and PilA, characterized in that: the protein sequences of the Haemophilus influenzae surface proteins Pcp and PilA are as follows:
MRPVKIQADNQGVIGTLGGGALGGIAGSAIGGGRGQVIAAVVGAIGGAVAGSKIEEKVSQVNGAELVIKKDDGQEIVVVQKADSSFVAGRRVRIVGGGSNLNVSVLEAAAAKEAAAAKLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNETTNCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQATGNAATGVTWTTTCKG；
the complete sequence of the gene for encoding the proteins of the Haemophilus influenzae surface proteins Pcp and PilA is:
CATATGCGCCCGGTTAAAATCCAGGCTGATAACCAGGGTGTAATCGGCACCCTGGGCGGCGGCGCTCTGGGCGGTATCGCAGGTTCTGCTATCGGCGGCGGTCGCGGCCAGGTTATTGCCGCTGTAGTAGGCGCTATCGGTGGTGCGGTGGCCGGTTCTAAAATCGAAGAAAAGGTTTCCCAGGTAAACGGTGCTGAGCTGGTAATCAAAAAAGACGATGGCCAGGAAATAGTTGTTGTACAGAAAGCCGACTCTTCCTTTGTAGCAGGTCGTCGCGTGCGCATCGTTGGTGGTGGGTCTAACCTGAACGTCTCTGTTCTGGAAGCTGCTGCTGCTAAAGAAGCTGCTGCTGCTAAACTGATTGAACTGATGATCGTGATCGCGATCATCGCAATTCTGGCGACCATCGCAATCCCGTCTTACCAGAACTACACTAAAAAAGCTGCTGTCTCCGAACTGCTGCAAGCATCTGCTCCGTACAAAGCTGACGTTGAACTCTGCGTTTACTCTACGAACGAGACCACCAACTGCACTGGTGGCAAAAACGGTATTGCGGCTGACATCACCACTGCTAAGGGTTACGTAAAATCTGTTACCACCTCTAACGGCGCAATCACTGTGAAAGGGGACGGCACCCTGGCTAACATGGAATACATTCTGCAGGCTACCGGTAACGCAGCGACCGGCGTGACCTGGACTACCACCTGCAAAGGCTAAGGATCC。
5. method for the preparation of the haemophilus influenzae surface proteins Pcp and PilA 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 Pcp of haemophilus influenzae and the extracellular domain of the surface protein PilA, finding out the gene coding sequences of the peptide segments with the most abundant antigenic epitopes in the surface protein Pcp of haemophilus influenzae and the extracellular domain of the surface protein PilA, optimizing the gene coding sequences of the peptide segments, and connecting the optimized gene coding sequences of the peptide segments by using the coding sequences of rigid connecting peptides to form fusion genes; the access numbers of the haemophilus influenzae surface protein Pcp and the surface protein PilA in the NCBI protein database are AAX88288 and AAX12377 respectively; the sequence of the rigid linker peptide is eaaakaaaak; simultaneously, introducing a restriction enzyme site NdeI at the 5 'end of the fusion gene, introducing a termination signal TAA and a restriction enzyme site BamHI at the 3' end of the fusion gene, and then chemically synthesizing a whole gene sequence which is marked as pcppil;
the complete sequence of the gene of pcppil is: CATATGCGCCCGGTTAAAATCCAGGCTGATAACCAGGGTGTAATCGGCACCCTGGGCGGCGGCGCTCTGGGCGGTATCGCAGGTTCTGCTATCGGCGGCGGTCGCGGCCAGGTTATTGCCGCTGTAGTAGGCGCTATCGGTGGTGCGGTGGCCGGTTCTAAAATCGAAGAAAAGGTTTCCCAGGTAAACGGTGCTGAGCTGGTAATCAAAAAAGACGATGGCCAGGAAATAGTTGTTGTACAGAAAGCCGACTCTTCCTTTGTAGCAGGTCGTCGCGTGCGCATCGTTGGTGGTGGGTCTAACCTGAACGTCTCTGTTCTGGAAGCTGCTGCTGCTAAAGAAGCTGCTGCTGCTAAACTGATTGAACTGATGATCGTGATCGCGATCATCGCAATTCTGGCGACCATCGCAATCCCGTCTTACCAGAACTACACTAAAAAAGCTGCTGTCTCCGAACTGCTGCAAGCATCTGCTCCGTACAAAGCTGACGTTGAACTCTGCGTTTACTCTACGAACGAGACCACCAACTGCACTGGTGGCAAAAACGGTATTGCGGCTGACATCACCACTGCTAAGGGTTACGTAAAATCTGTTACCACCTCTAACGGCGCAATCACTGTGAAAGGGGACGGCACCCTGGCTAACATGGAATACATTCTGCAGGCTACCGGTAACGCAGCGACCGGCGTGACCTGGACTACCACCTGCAAAGGCTAAGGATCC, respectively;
the protein sequence encoded by the pcppil gene is: MRPVKIQADNQGVIGTLGGGALGGIAGSAIGGGRGQVIAAVVGAIGGAVAGSKIEEKVSQVNGAELVIKKDDGQEIVVVQKADSSFVAGRRVRIVGGGSNLNVSVLEAAAAKEAAAAKLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNETTNCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQATGNAATGVTWTTTCKG, respectively;
the protein sequence coded by the pcppil gene is 50-154aa of a surface protein Pcp of the haemophilus influenzae and 17-133aa of a surface protein PilA; the middle of the two protein sequences is connected by rigid connecting peptide eaaakaaaak;
cloning pcppil gene complete sequence into prokaryotic expression vector pET-28a (+) according to conventional method, transferring into E.coli BL21DE3 strain, inducing recombinant Escherichia coli expression with IPTG, and using Ni2+The recombinant Pcpsil protein was purified by affinity chromatography.
6. A method for preparing polyclonal antibodies against Haemophilus influenzae surface protein Pcp and PilA, which is characterized by comprising the following steps: the method comprises the following steps: taking the recombinant Pcppel protein as an immune antigen as claimed in claim 5, mixing with Freund's adjuvant, repeatedly and artificially immunizing healthy New Zealand white rabbits, drawing blood for titer determination, separating high-titer recombinant protein antibodies and purifying to finally obtain polyclonal antibodies against Haemophilus influenzae Pcp and PilA protein, and diluting with a sealing solution to a final concentration of 20 μ 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 Haemophilus influenzae Elisa detection kit based on a Haemophilus influenzae surface protein antibody is characterized in that: the Haemophilus influenzae surface protein antibody-based Elisa detection kit comprises an enzyme label plate coated with polyclonal antibodies against Haemophilus influenzae surface protein D15 and Pe prepared according to claim 3, polyclonal antibodies against Haemophilus influenzae surface protein Pcp and PilA prepared according to claim 5, a positive control, a negative control, a washing solution, an enzyme-labeled secondary antibody, an enzyme chromogenic substrate and a stop solution; the positive control of the haemophilus influenzae is inactivated haemophilus influenzae 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 method for preparing the haemophilus influenzae Elisa detection kit based on the haemophilus influenzae surface protein antibody according to claim 7, characterized in that: the Haemophilus influenzae surface protein antibody-based Elisa detection kit comprises an enzyme label plate coated with polyclonal antibodies against Haemophilus influenzae surface protein D15 and Pe prepared according to claim 3, polyclonal antibodies against Haemophilus influenzae surface protein Pcp and PilA prepared according to claim 5, a positive control, a negative control, a washing solution, an enzyme-labeled secondary antibody, an enzyme chromogenic substrate and a stop solution; the positive control of the haemophilus influenzae is inactivated haemophilus influenzae 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-Haemophilus influenzae surface protein D15 and Pe comprises the following steps:
1) preparing anti-haemophilus influenzae surface protein D15 and Pe polyclonal antibody;
2) coating of anti-haemophilus influenzae surface protein D15 and Pe polyclonal antibody:
diluting the anti-Haemophilus influenzae surface protein D15 and the Pe polyclonal antibody 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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