CN110540596A - Moraxella catarrhalis Elisa detection kit based on moraxella catarrhalis surface protein antibody and preparation method thereof - Google Patents

Abstract

The invention relates to an Elisa detection kit for rapidly detecting Moraxella catarrhalis and a preparation method thereof. The kit comprises: an enzyme label plate for coating anti-Moraxella catarrhalis M35 and Olpa protein polyclonal antibody, anti-Moraxella catarrhalis McaP and OmpCD protein polyclonal antibody, Moraxella catarrhalis positive control, negative control, washing liquid, enzyme-labeled secondary antibody, enzyme chromogenic substrate and stop solution. The kit provided by the invention is used for carrying out combined detection on four specific surface proteins of the Moraxella catarrhalis, so that the sensitivity, specificity and repeatability of the Moraxella catarrhalis detection are greatly improved, and the kit can be used for non-diagnostic detection and research work on the Moraxella catarrhalis.

Description

Moraxella catarrhalis Elisa detection kit based on moraxella catarrhalis surface protein antibody and preparation method thereof

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 Moraxella catarrhalis Elisa detection kit based on a Moraxella catarrhalis surface protein antibody and a preparation method thereof.

Background

Moraxella catarrhalis (MC for short) was first discovered in 1896, and was called Micrococcus catarrhalis (Micrococcus catarrhalis) later also called Neisseria catarrhalis (Neisseria catarrhalis) and Branhamella catarrhalis (Branhamella catarrhalis). Moraxella catarrhalis has been thought in the past to be a normal inhabitant of the upper respiratory tract that is not pathogenic to humans. However, in more than 20 years of research, the bacterium can cause upper respiratory tract infection of children and old people, and is also an important pathogenic bacterium causing lower respiratory tract infection of adults, the 3 rd most common pathogenic bacterium causing maxillary sinusitis, otitis media, pneumonia of children and chronic lower respiratory tract infection of adults is only second to haemophilus influenzae and streptococcus pneumoniae, and the incidence rate of the bacterium is increased year by year, particularly in patients with chronic obstructive pulmonary disease. With the wide application of antibiotics, hormones and immunosuppressants, the relationship between parasitic bacteria of human bodies and hosts is changed, and the nosocomial infection rate is obviously increased. Since the pathogenic bacteria of lung infection are closely related to the flora colonized by the oropharynx of the patient, Moraxella catarrhalis may be pathogenic under appropriate conditions. The chronic obstructive pulmonary disease patient is weak after long-term illness, and the long-term application of broad-spectrum antibiotics and hormones reduces the respiratory tract defense function and the immunity of the organism, so that bacteria enter the lower respiratory tract to cause 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. In addition, false positives are often caused by the sensitivity of this technique. 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, 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 Moraxella catarrhalis M35, Olpa, McaP and OmpCD proteins are important molecules on the cell surface, have high conservation, strong specificity, strong antigenicity and high surface exposure, and are ideal detection targets. In the research, surface proteins M35, Olpa, McaP, OmpCD and the like with interspecific specificity are selected as detection targets, recombinant antigens are prepared in a gene recombination mode, and then polyclonal antibodies with good specificity are prepared and are applied to the preparation of a Moraxella catarrhalis Elisa detection kit.

Disclosure of Invention

In order to solve the technical problems in the background art, the invention provides the moraxella catarrhalis Elisa detection kit based on the moraxella catarrhalis surface protein antibody, which can improve the sensitivity, specificity and repeatability of the moraxella catarrhalis detection, is simple to operate, is low in cost, and can quickly and quickly detect the moraxella catarrhalis, and the preparation method thereof.

In order to achieve the purpose, the invention adopts the following technical scheme:

A Moraxella catarrhalis Elisa detection kit based on Moraxella catarrhalis surface protein antibody is characterized in that: the Moraxella catarrhalis Elisa detection kit based on the Moraxella catarrhalis surface protein antibody comprises an enzyme label plate coated with a polyclonal antibody of anti-Moraxella catarrhalis surface proteins (M35 and Olpa), an anti-Moraxella catarrhalis surface protein (McaP and OmpCD) polyclonal antibody, a Moraxella catarrhalis positive control, a negative control, a washing solution, an enzyme-labeled secondary antibody, an enzyme chromogenic substrate and a termination solution; the Moraxella catarrhalis positive control is inactivated Moraxella catarrhalis 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 Moraxella catarrhalis Elisa detection kit based on a Moraxella catarrhalis surface protein antibody is characterized in that: the Moraxella catarrhalis Elisa detection kit based on the Moraxella catarrhalis surface protein antibody comprises an enzyme label plate coated with a polyclonal antibody of anti-Moraxella catarrhalis surface proteins (M35 and Olpa), an anti-Moraxella catarrhalis surface protein (McaP and OmpCD) polyclonal antibody, a Moraxella catarrhalis positive control, a negative control, a washing solution, an enzyme-labeled secondary antibody, an enzyme chromogenic substrate and a termination solution; the Moraxella catarrhalis positive control is inactivated Moraxella catarrhalis 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-Moraxella catarrhalis surface protein (M35 and Olpa) comprises the following steps:

1) Preparation of polyclonal antibodies against Moraxella catarrhalis surface proteins (M35 and Olpa):

1.1) respectively obtaining peptide fragments with most abundant antigenic epitopes in the extracellular domains of Moraxella catarrhalis surface protein M35 and surface protein Olpa, finding out the gene coding sequence of the peptide fragments, optimizing the gene coding sequence of the peptide fragments, and connecting the optimized gene coding sequence of the peptide fragments with the coding sequence of flexible connecting peptide to form a fusion gene; the accession numbers of the Moraxella catarrhalis surface protein M35 and the surface protein Olpa in the NCBI protein database are AY905613 and DQ996463 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 M35 olp;

The complete sequence of the M35olp gene is:

CATATGGAGGATACTAACGAGTCTGGTCTGAACAGCAACACCTCTCGTATCGGCTTTAAGGGTAGCGA AGCTCTGAACGCAAACACCGACGTAGTTTACCAGCTGGAGTACAAAATCGACATCGATGCTGACCGTGGTGACAAC TTCAAATCTCGTGATACTTACCTGGGCCTGGCACATAAACAGTACGGTACTCTGCTGGCTGGCCGCCTGACCACTA TCGATGATTCTGTGGATTTCGCATCTATGCTGGAAGACAACAACGTTGCAGACATCGGCCCGACCTTCAACGCTCC GCGTGCAAACAACGCATTTGCTTACGTGTCTCCGGAATACAACGGTACCCAGTTTCTGGCCATGTATGCTTTCGAT TCCGATACCGACAAAGGTGGCCTGGCGAAAGATGACCAGTTCGGTGTTGGCGCTACTTACTCTACCGGTCCAATCA ACGCTGGCGCAACTTACATCCACTACGGCGATGACAGCCACATTCGCCTGGGTGGTTCTGGTGGTTCTGGTGGTTC TGGTGGTTCTGGTTACTACGTACAGGCGGACCTGGGCTACTCCAACCTGGAAGCCAAATACAACGACAACCGCCCG AACGACAACAAGCTCGAAGACAAAGGTCTGGGCTACGGTATCTCTTTCGGCAAAGATACTGACCTGGCTCGCTACG CGCTGGACTACAAAAACTACGGTAAGATCAACTCCTCTTACACCTACCAGCCGCAGACTAACACCTCTATCACCTC TTCTGGCGAACTGAAAGCACAGTCCCTGGGTCTGAGCGCGATCTACGATTTCGACACCGTATCCGGTTTCACCCCG TACGCAGGCGCTCGTGTTGACATCAACCAGATCAAATCTACCGTTTCCACCGTAACTACCACCGCAGCTGCACAGT AAGGATCC；

the protein sequence encoded by the M35olp gene is:

MEDTNESGLNSNTSRIGFKGSEALNANTDVVYQLEYKIDIDADRGDNFKSRDTYLGLAHKQYGTLLAG RLTTIDDSVDFASMLEDNNVADIGPTFNAPRANNAFAYVSPEYNGTQFLAMYAFDSDTDKGGLAKDDQFGVGATYS TGPINAGATYIHYGDDSHIRLGGSGGSGGSGGSGYYVQADLGYSNLEAKYNDNRPNDNKLEDKGLGYGISFGKDTD LARYALDYKNYGKINSSYTYQPQTNTSITSSGELKAQSLGLSAIYDFDTVSGFTPYAGARVDINQIKSTVSTVTTT AAAQ；

the protein sequence coded by the M35olp gene is 50-213aa of the Moraxella catarrhalis surface protein M35 and 26-148aa of the surface protein Olpa; the two protein sequences are connected by flexible connecting peptide ggsggsggs;

1.2) cloning the complete sequence of M35olp gene into a prokaryotic expression vector pET-28a (+) by a conventional method, transferring the complete sequence into E.coli BL21(DE3) bacteria, inducing recombinant escherichia coli expression by IPTG, and purifying recombinant His-M35olp 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 high-titer recombinant protein antibodies and purifying to finally obtain polyclonal antibodies against Moraxella catarrhalis surface proteins (M35 and Olpa);

2) Coating of polyclonal antibodies against Moraxella catarrhalis surface proteins (M35 and Olpa):

Diluting the polyclonal antibody against Moraxella catarrhalis surface proteins (M35 and Olpa) prepared in the step 1) to the concentration of 10 mu g/mL by using PBS buffer, coating a 96-well EIA high-efficiency binding enzyme standard plate according to the amount of 100 mu L/well, 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 polyclonal antibody against the surface protein (McaP and OmpCD) of the catarrhal bacteria comprises the following steps:

1) Obtaining a peptide segment with the most abundant antigenic epitopes in the extracellular domains of the Moraxella catarrhalis surface protein McaP and the surface protein OmpCD, finding out a gene coding sequence of the peptide segment, optimizing the gene coding sequence of the peptide segment, and connecting the optimized gene coding sequence of the peptide segment by using a coding sequence of rigid connecting peptide to form a fusion gene; the access numbers of the Moraxella catarrhalis surface protein McaP and the surface protein OmpCD in the NCBI protein database are EF075940 and AAA66180 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 Mcaomp;

the complete gene sequence of Mcap is as follows:

CATATGCAGGACTATCACCTCAGCGACTCCATTACCGCTGACGTTAAGCAGACTGGCATCGGTCTTTA TCATCGCCACGACTTCGACCACCTGCGTATTTCTGCAAACCTGGGCGTAGATCACCTGTCTACTCAGAGTCTGCGT CAAATCATGTGGGACGGTGAAAACCGCAACCACCAGGCACATGCTACCGGCCAGCGTCGTTATGCATCCGTACAGG GTTCCTACGGTTTCACTCAGAACAACGTTACCTACCGCCCATACGTAGGCATCCACGCACAGGACATCAAACAGAA CCGCTTCTTCGAAAACCAGCCGAACCTCAGCACTGCTCTGTCCTTCAAGCTGCCGGACCACCAGAGCCTGCAGGCT GATATCGGCGTTAACATCGATTACGCAATGAACGACAAACTGAACCTGCTGGCTGGTCTGGGCTATCAGCATGAAT TCAAAGACAACAACAAAACTGTGGAAACCGCGGTTCTGTCTAACCGTGATTACCATCGCTCCTTCGTTACTACCGT ACCGTTCGACAAAAAACACACCACCCACGCTCACCTCGGTGCTACTTTAGCGCTGGGCAACAACACCCACCTGGAA GCTGCTGCTGCTAAAGAAGCTGCTGCTGCTAAAAAAATCCTGCGTACTGGTAAGAAACTGGAACTGGACGCTACCA ACGCGCCGGCTCCTGCAAACGGTGGCGTTGCGCTCGACTCCGAACTGTGGACCGGCGCTGCCATCGGTATCGAACT GACTCCGTCTACTCAGTTCCAGGTTGAATACGGTATCTCCAACCGCGACGCTAAATCTTCCGACAAATCTGCCCAC CGCTTCGATGCAGAACAGGAAACCATCTCTGGCAACTTCCTGATCGGTACTGAACAGTTCTCCGGCTACAACCCAA CCAACAAATTCAAACCTTACGTACTGGTTGGCGCTGGTCAGTCTAAAATTAAGGTGAACGCAATCGACGGCTACAC TGCAGAAGTTGCGAACGGTCAGAACATTGCGAAAGACCAGGCAGTGAAAGCAGGCCAGGAAGTTGCGGAATCCAAA GACACCATCGGTAACTAAGGATCC；

The protein sequence coded by the Mcpacomp gene is as follows:

MQDYHLSDSITADVKQTGIGLYHRHDFDHLRISANLGVDHLSTQSLRQIMWDGENRNHQAHATGQRRY ASVQGSYGFTQNNVTYRPYVGIHAQDIKQNRFFENQPNLSTALSFKLPDHQSLQADIGVNIDYAMNDKLNLLAGLG YQHEFKDNNKTVETAVLSNRDYHRSFVTTVPFDKKHTTHAHLGATLALGNNTHLEAAAAKEAAAAKKILRTGKKLE LDATNAPAPANGGVALDSELWTGAAIGIELTPSTQFQVEYGISNRDAKSSDKSAHRFDAEQETISGNFLIGTEQFS GYNPTNKFKPYVLVGAGQSKIKVNAIDGYTAEVANGQNIAKDQAVKAGQEVAESKDTIGN；

The protein sequence coded by the Mcpacomp gene is 429-625aa of the Moraxella catarrhalis surface protein McaP and 49-194aa of the surface protein OmpCD; the middle of the two protein sequences is connected by rigid connecting peptide eaaakaaaak;

2) cloning the complete gene sequence of Mcap into prokaryotic expression vector pET-28a (+) by a conventional method, transferring the complete gene sequence into E.coliBL21(DE3) bacteria, inducing recombinant escherichia coli expression by IPTG, and purifying recombinant His-Mcap 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-Moraxella catarrhalis McaP and OmpCD protein polyclonal antibodies, and diluting the polyclonal antibodies with confining liquid to the final concentration of 20 mug/mL;

the sealing liquid comprises the following components in percentage by weight: 1.4g/L of disodium hydrogen phosphate, 0.2g/L of sodium dihydrogen phosphate, 8.5g/L of sodium chloride and 10g/L of bovine serum albumin, wherein the pH value of the sealing liquid is 7.4.

Compared with the prior art, the invention has the following advantages:

(1) the invention adopts structural analysis, gene optimization and other modes to construct two brand new fusion genes, and successfully obtains soluble recombinant M35/Olpa fusion protein and McaP/OmpCD 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 invention firstly utilizes the four protein exposed regions on the surface of the Moraxella catarrhalis to prepare the antibody with high titer, more antigen binding sites, strong capture force, no site competition problem and high detection sensitivity of the kit. The detection sensitivity of the kit to the Moraxella catarrhalis standard strain ATCC46327 reaches 1 × 103CFU/mL, is obviously higher than that of the traditional microbial detection method, and has the advantages of rapidness, high efficiency and the like.

(3) The Elisa kit has good specificity, and a result of a specificity experiment carried out by 6 Moraxella catarrhalis strains and 17 Moraxella non-catarrhalis standard strains (containing most common respiratory pathogens) shows that the test strip has good specificity and stability, can detect all the tested Moraxella catarrhalis, has no cross reaction with all the Moraxella non-catarrhalis 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 moraxella catarrhalis M35 and OlpA protein:

1.1) cloning of M35olp fusion Gene of Moraxella catarrhalis

obtaining peptide segments with most abundant antigen epitopes in the extracellular domains of Moraxella catarrhalis surface proteins M35 and Olpa (the access numbers in the NCBI protein database are AY905613 and DQ996463 respectively), finding out gene coding sequences thereof, optimizing the gene coding sequences thereof, and connecting the two sequences by using the coding sequences of flexible connecting peptides (ggsgggsggsggs) 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 M35 olp. The complete gene sequence and the coded amino acid sequence are shown in a sequence table. Specifically, the protein sequences coded by the M35olp gene are 50-213aa of the Moraxella catarrhalis surface protein M35 and 26-148aa of the surface protein Olpa, 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 M35olp 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-M35olp expression vector. The construction of the expression vector is verified to be correct by enzyme digestion and sequence determination. The vector expresses recombinant M35olp fusion protein.

1.2) expression and purification of Moraxella catarrhalis M35olp 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. Single colonies transformed with pET-M35olp and 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 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 cells were washed 3 times with 50mL Buffer A (50mM Na3PO4, 0.5M NaCl; pH7.4) and resuspended in 50mL loading Buffer (50mM Na3PO4, 0.5M NaCl; 5mM imidazole, pH7.4) and sonicated 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 M35olp 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 30% of the total bacterial protein. The wild type M35olp which is not subjected to gene optimization is expressed in the same manner, and the result shows that the expression product only accounts for 5 percent of the total protein, which indicates that the gene optimization is successful and the effect is outstanding. The sonicated supernatant obtained above was filtered through a 0.45 μ M 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. Thus obtaining the Moraxella catarrhalis M35olp fusion protein.

1.3) preparation of polyclonal antibodies against Moraxella catarrhalis M35 and Olpa protein

1.3.1) mixing the Moraxella catarrhalis M35olp 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 for each rabbit is 2ml, and the total amount of antigen is 2 mg/rabbit. Then, the emulsion formed by the M35olp fusion protein and Freund's incomplete adjuvant is used for immunization once every two weeks for 5 times, and the dosage of the antigen is the same as that of the primary immunization. Large amount of blood is taken 3-5 days after five-immunization, placed at 37 ℃ for 1 hour, then placed in a refrigerator at 4 ℃ overnight, and serum is taken every other day.

1.3.2) determination of the potency of the polyclonal antibody

the M35olp 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 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, polyclonal antibodies against Moraxella catarrhalis M35 and Olpa protein were prepared.

Example 2

preparation of polyclonal antibodies against Moraxella catarrhalis McaP and OmpCD proteins:

1.1) cloning of Moraxella catarrhalis Mcpacomp fusion Gene

Obtaining peptide segments with the most abundant antigenic epitopes in the extracellular domains of Moraxella catarrhalis surface proteins McaP and OmpCD (the access numbers in the NCBI protein database are EF075940 and AAA66180 respectively), finding out the gene coding sequences thereof, optimizing the gene coding sequences thereof, and connecting the two sequences by using the coding sequences of rigid connecting peptide (eaaakaeaak) to form a fusion gene. 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 Mcaomp. The complete gene sequence and the coded amino acid sequence are shown in a sequence table. Specifically, the protein sequences encoded by the Mcaapomp gene are 429-625aa of the Moraxella catarrhalis surface protein McaP and 49-194aa of the surface protein OmpCD, and a rigid connecting peptide (eaaakaaaak) is connected between the two protein sequences. 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 Mcacomp 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-Mcacomp expression vector. The construction of the expression vector is verified to be correct by enzyme digestion and sequence determination. The vector expresses recombinant Mcapomp fusion protein.

1.2) expression and purification of Moraxella catarrhalis Mcpacomp 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. Individual colonies transformed with pET-Mcaomp 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 500mL 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 cells were washed 3 times with 50mL Buffer A (50mM Na3PO4, 0.5M NaCl; pH7.4) and resuspended in 50mL loading Buffer (50mM Na3PO4, 0.5M NaCl; 5mM imidazole, pH7.4) and sonicated 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 Mcapomp fusion protein was found to be present in the bacterial cells in a partially solubilized form (the other portion was present in the form of inclusion bodies). Thin-layer scanning showed that the recombinant protein accounted for more than 30% of the total bacterial protein. The wild Mcpacomp which is not subjected to gene optimization is expressed in the same manner, and as a result, the expression fails, no product appears, and the gene optimization is successful and the effect is remarkable. 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 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 40mM imidazole, and the target protein was found to have a purity of 90% or more, and was adjusted to 0.2mg/mL for use after measuring the protein concentration with the bradford kit. Thus obtaining the Moraxella catarrhalis Mcpacomp fusion protein.

1.3) preparation of polyclonal antibodies against Moraxella catarrhalis McaP and OmpCD proteins

1.3.1) mixing the Moraxella catarrhalis Mcaapomp 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 for each rabbit is 2ml, and the total amount of antigen is 2 mg/rabbit. And then immunizing once every two weeks by using emulsion formed by Mcap fusion protein and Freund's incomplete adjuvant, wherein the immunization is carried out for 5 times totally, and the dosage of the antigen is the same as that of the primary immunization. Large amount of blood is taken 3-5 days after five-immunization, placed at 37 ℃ for 1 hour, then placed in a refrigerator at 4 ℃ overnight, and serum is taken every other day.

1.3.2) determination of the potency of the polyclonal antibody

the Mcap 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 serum antibody level 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, polyclonal antibodies against Moraxella catarrhalis McaP and OmpCD proteins are prepared.

Example 3

moraxella catarrhalis Elisa detection kit

1. Composition of Moraxella catarrhalis Elisa detection kit

An ELISA plate coated with anti-Moraxella catarrhalis M35 and Olpa protein polyclonal antibody, anti-Moraxella catarrhalis McaP and OmpCD protein polyclonal antibody, Moraxella catarrhalis positive control, negative control, washing liquid, enzyme-labeled secondary antibody, enzyme chromogenic substrate and stop buffer form the Moraxella catarrhalis Elisa detection kit.

(1) ELISA plate coated with anti-Moraxella catarrhalis M35 and Olpa protein polyclonal antibody

anti-Moraxella catarrhalis M35 and the Olpa protein polyclonal antibody (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 for 2 hours at 37 ℃. 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 Moraxella catarrhalis McaP and OmpCD proteins

The anti-Moraxella catarrhalis McaP and OmpCD protein polyclonal antibodies described in example 2 were prepared as a 20. mu.g/mL solution using the above blocking solution, and packaged in an amount of 5mL per kit.

(3) enzyme-labeled secondary antibody: the enzyme-labeled secondary antibody is horseradish peroxidase-labeled goat anti-rabbit IgG which is purchased from Beijing Ceh Biotechnology Co., Ltd in the example, is product number 030005-G, has a concentration of 1mg/mL, and is diluted 3000 times by PBS buffer solution when in use. The mixture was packaged in an amount of 0.1mL per kit.

(4) enzyme chromogenic substrate: the enzyme chromogenic substrate preparation process is as follows:

Solution A: (configuration amount 1L)

1. 3.14g of citric acid (containing 1 molecule of crystal water and having a molecular weight of 210.14g) and 11.56g of sodium acetate (containing 3 molecules of crystal water and having a molecular weight of 136.0) were weighed and dissolved in 970mL of double distilled water to prepare an aqueous solution of sodium acetate having a pH of 5.0.

2. weighing 0.08g of phenacetin, adding 30mL of double distilled water, heating to 100 ℃, and adding the mixture into the solution in the first step after completely dissolving.

3. Then 0.5g of carbamide peroxide is added and mixed evenly.

and B, liquid B: (configuration amount 1L)

Adding 500mL of methanol into a 2L beaker, adding 1.27g of 3,3,5, 5-tetramethylbenzidine TMB (SIGMA), heating at 60 ℃ to dissolve, and adding 500mL of glycerol.

A, B liquid 1: 1, mixing to prepare an enzyme chromogenic substrate. The solution A and the solution B were packaged in an amount of 5mL each per kit.

(5) Positive and negative controls

The positive control is formaldehyde-inactivated moraxella catarrhalis (ATCC46327), which was prepared as follows: taking Moraxella catarrhalis liquid cultured by a 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 × 109 CFU/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 medium, counting by plate, centrifuging, collecting thallus, resuspending thallus with physiological saline, and adjusting thallus concentration to 1 × 109 CFU/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) Washing liquid: the specific preparation method of the PBST solution is as described in (1). Packaging the obtained product in an amount of 200mL per kit.

(7) Stopping liquid: 1M HCl solution prepared with double distilled water. Packaging the obtained product in an amount of 10mL per kit.

example 4

use method of Moraxella catarrhalis 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 of anti-Moraxella catarrhalis McaP and OmpCD proteins: adding anti-Moraxella catarrhalis McaP and OmpCD protein polyclonal antibody working solution in the kit, incubating for 1 hour at 37 ℃, washing the plate for 3 times by using 250 mu L of PBST washing solution, and drying by spinning.

4) Adding enzyme-labeled secondary antibody: the enzyme-labeled secondary antibody in the kit described in example 3 was diluted 1:3000 with PBS buffer to prepare a working solution, and corresponding enzyme-labeled wells were added at 50. mu.L/well, incubated at 37 ℃ for 1 hour, and then washed 3 times with 250. mu.L of PBST washing solution, and spun-dried.

5) adding 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 Moraxella catarrhalis antigen in the clinical pharynx swab is judged to be positive, otherwise, the Moraxella catarrhalis 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 Moraxella catarrhalis Elisa detection kit

1) specific assay

in order to verify the specificity of the Moraxella catarrhalis Elisa test kit of the present invention, 6 Moraxella catarrhalis standard strains and 17 Moraxella non-catarrhalis standard strains, both at a concentration of 2X 105CFU/mL, were tested according to the kit compositions and methods described in examples 3 and 4, and are shown in Table 1. The result shows that the detection results of the kit are positive for all 6 catamaran standard strains, and the detection results of the kit are negative for other 17 respiratory common pathogenic microorganisms. The kit showed good specificity.

TABLE 1

strain name	Results of the detection
		moraxella catarrhalis ATCC46327	Positive for
Moraxella catarrhalis ATCC25240	Positive for
		Moraxella catarrhalis ATCC25238	Positive for
Moraxella catarrhalis ATCC49143	Positive for
		Moraxella catarrhalis ATCC8176	Positive for
Moraxella catarrhalis ATCC49264	Positive for
		mycoplasma pneumoniae ATCC15531	Negative of
Pseudomonas aeruginosa ATCC27853	Negative of
		Human Haemophilus influenzae ATCC49247	Negative of
Acinetobacter baumannii ATCC19606	negative of
		Haemophilus parainfluenza ATCC7901	Negative of
Legionella pneumophila ATCC33152	Negative of
		Streptococcus pyogenes ATCC19615	Negative of
Staphylococcus aureus ATCC25923	Negative of
		streptococcus pneumoniae ATCC49619	Negative of
Klebsiella pneumoniae ATCC700603	negative of
		Enterobacter cloacae ATCC13047	negative of
escherichia coli ATCC25922	Negative of
		Candida ATCC10231	Negative of
influenza A virus ATCCVR-1743	negative of
		Influenza B virus ATCCVR-790	Negative of
Respiratory syncytial virus ATCCVR26	Negative of
		Adenovirus ATCCVR-3	Negative of

Meanwhile, the test of 118 clinical isolates of Moraxella catarrhalis with the concentration of 2X 105CFU/mL by using the kit shows positive results, and shows that the kit has high detection coverage on the clinically isolated Moraxella catarrhalis.

2) Sensitivity assay

The Moraxella catarrhalis ATCC46327 strain is inoculated in a sheep blood chocolate culture medium, after the culture is carried out for 24 hours at 35 ℃, the gradient dilution is carried out by 10 times by normal saline, meanwhile, the plate counting is carried out, the thallus solution with the thallus concentration of 108-102CFU/mL is obtained, then 100 mu L of the bacteria solution is dripped on an enzyme label plate, and the detection is carried out according to the kit composition and the method described in the embodiment 3 and the embodiment 4. The result shows that the detection sensitivity of the kit is 103 CFU/mL.

Sequence listing

<110> Hubei university of industry

<120> Moraxella catarrhalis Elisa detection kit based on Moraxella catarrhalis surface protein antibody and preparation method thereof

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 912

<212> DNA

<213> complete Gene sequence of M35olp (M35olp)

<400> 1

catatggagg atactaacga gtctggtctg aacagcaaca cctctcgtat cggctttaag 60

ggtagcgaag ctctgaacgc aaacaccgac gtagtttacc agctggagta caaaatcgac 120

atcgatgctg accgtggtga caacttcaaa tctcgtgata cttacctggg cctggcacat 180

aaacagtacg gtactctgct ggctggccgc ctgaccacta tcgatgattc tgtggatttc 240

gcatctatgc tggaagacaa caacgttgca gacatcggcc cgaccttcaa cgctccgcgt 300

gcaaacaacg catttgctta cgtgtctccg gaatacaacg gtacccagtt tctggccatg 360

tatgctttcg attccgatac cgacaaaggt ggcctggcga aagatgacca gttcggtgtt 420

ggcgctactt actctaccgg tccaatcaac gctggcgcaa cttacatcca ctacggcgat 480

gacagccaca ttcgcctggg tggttctggt ggttctggtg gttctggtgg ttctggttac 540

tacgtacagg cggacctggg ctactccaac ctggaagcca aatacaacga caaccgcccg 600

aacgacaaca agctcgaaga caaaggtctg ggctacggta tctctttcgg caaagatact 660

gacctggctc gctacgcgct ggactacaaa aactacggta agatcaactc ctcttacacc 720

taccagccgc agactaacac ctctatcacc tcttctggcg aactgaaagc acagtccctg 780

ggtctgagcg cgatctacga tttcgacacc gtatccggtt tcaccccgta cgcaggcgct 840

cgtgttgaca tcaaccagat caaatctacc gtttccaccg taactaccac cgcagctgca 900

cagtaaggat cc 912

<210> 2

<211> 300

<212> PRT

<213> protein sequence of M35olp (M35olp)

<400> 2

Met Glu Asp Thr Asn Glu Ser Gly Leu Asn Ser Asn Thr Ser Arg Ile

1 5 10 15

Gly Phe Lys Gly Ser Glu Ala Leu Asn Ala Asn Thr Asp Val Val Tyr

20 25 30

Gln Leu Glu Tyr Lys Ile Asp Ile Asp Ala Asp Arg Gly Asp Asn Phe

35 40 45

Lys Ser Arg Asp Thr Tyr Leu Gly Leu Ala His Lys Gln Tyr Gly Thr

50 55 60

Leu Leu Ala Gly Arg Leu Thr Thr Ile Asp Asp Ser Val Asp Phe Ala

65 70 75 80

Ser Met Leu Glu Asp Asn Asn Val Ala Asp Ile Gly Pro Thr Phe Asn

85 90 95

Ala Pro Arg Ala Asn Asn Ala Phe Ala Tyr Val Ser Pro Glu Tyr Asn

100 105 110

Gly Thr Gln Phe Leu Ala Met Tyr Ala Phe Asp Ser Asp Thr Asp Lys

115 120 125

Gly Gly Leu Ala Lys Asp Asp Gln Phe Gly Val Gly Ala Thr Tyr Ser

130 135 140

Thr Gly Pro Ile Asn Ala Gly Ala Thr Tyr Ile His Tyr Gly Asp Asp

145 150 155 160

Ser His Ile Arg Leu Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly

165 170 175

Ser Gly Tyr Tyr Val Gln Ala Asp Leu Gly Tyr Ser Asn Leu Glu Ala

180 185 190

Lys Tyr Asn Asp Asn Arg Pro Asn Asp Asn Lys Leu Glu Asp Lys Gly

195 200 205

Leu Gly Tyr Gly Ile Ser Phe Gly Lys Asp Thr Asp Leu Ala Arg Tyr

210 215 220

Ala Leu Asp Tyr Lys Asn Tyr Gly Lys Ile Asn Ser Ser Tyr Thr Tyr

225 230 235 240

Gln Pro Gln Thr Asn Thr Ser Ile Thr Ser Ser Gly Glu Leu Lys Ala

245 250 255

Gln Ser Leu Gly Leu Ser Ala Ile Tyr Asp Phe Asp Thr Val Ser Gly

260 265 270

Phe Thr Pro Tyr Ala Gly Ala Arg Val Asp Ile Asn Gln Ile Lys Ser

275 280 285

Thr Val Ser Thr Val Thr Thr Thr Ala Ala Ala Gln

290 295 300

<210> 3

<211> 1080

<212> DNA

<213> Gene sequence of Mcpacomp (Mcpacomp)

<400> 3

catatgcagg actatcacct cagcgactcc attaccgctg acgttaagca gactggcatc 60

ggtctttatc atcgccacga cttcgaccac ctgcgtattt ctgcaaacct gggcgtagat 120

cacctgtcta ctcagagtct gcgtcaaatc atgtgggacg gtgaaaaccg caaccaccag 180

gcacatgcta ccggccagcg tcgttatgca tccgtacagg gttcctacgg tttcactcag 240

aacaacgtta cctaccgccc atacgtaggc atccacgcac aggacatcaa acagaaccgc 300

ttcttcgaaa accagccgaa cctcagcact gctctgtcct tcaagctgcc ggaccaccag 360

agcctgcagg ctgatatcgg cgttaacatc gattacgcaa tgaacgacaa actgaacctg 420

ctggctggtc tgggctatca gcatgaattc aaagacaaca acaaaactgt ggaaaccgcg 480

gttctgtcta accgtgatta ccatcgctcc ttcgttacta ccgtaccgtt cgacaaaaaa 540

cacaccaccc acgctcacct cggtgctact ttagcgctgg gcaacaacac ccacctggaa 600

gctgctgctg ctaaagaagc tgctgctgct aaaaaaatcc tgcgtactgg taagaaactg 660

gaactggacg ctaccaacgc gccggctcct gcaaacggtg gcgttgcgct cgactccgaa 720

ctgtggaccg gcgctgccat cggtatcgaa ctgactccgt ctactcagtt ccaggttgaa 780

tacggtatct ccaaccgcga cgctaaatct tccgacaaat ctgcccaccg cttcgatgca 840

gaacaggaaa ccatctctgg caacttcctg atcggtactg aacagttctc cggctacaac 900

ccaaccaaca aattcaaacc ttacgtactg gttggcgctg gtcagtctaa aattaaggtg 960

aacgcaatcg acggctacac tgcagaagtt gcgaacggtc agaacattgc gaaagaccag 1020

gcagtgaaag caggccagga agttgcggaa tccaaagaca ccatcggtaa ctaaggatcc 1080

<210> 4

<211> 356

<212> PRT

<213> protein sequence of Mcpacomp (Mcpacomp)

<400> 4

Met Gln Asp Tyr His Leu Ser Asp Ser Ile Thr Ala Asp Val Lys Gln

1 5 10 15

Thr Gly Ile Gly Leu Tyr His Arg His Asp Phe Asp His Leu Arg Ile

20 25 30

Ser Ala Asn Leu Gly Val Asp His Leu Ser Thr Gln Ser Leu Arg Gln

35 40 45

Ile Met Trp Asp Gly Glu Asn Arg Asn His Gln Ala His Ala Thr Gly

50 55 60

Gln Arg Arg Tyr Ala Ser Val Gln Gly Ser Tyr Gly Phe Thr Gln Asn

65 70 75 80

Asn Val Thr Tyr Arg Pro Tyr Val Gly Ile His Ala Gln Asp Ile Lys

85 90 95

Gln Asn Arg Phe Phe Glu Asn Gln Pro Asn Leu Ser Thr Ala Leu Ser

100 105 110

Phe Lys Leu Pro Asp His Gln Ser Leu Gln Ala Asp Ile Gly Val Asn

115 120 125

Ile Asp Tyr Ala Met Asn Asp Lys Leu Asn Leu Leu Ala Gly Leu Gly

130 135 140

Tyr Gln His Glu Phe Lys Asp Asn Asn Lys Thr Val Glu Thr Ala Val

145 150 155 160

Leu Ser Asn Arg Asp Tyr His Arg Ser Phe Val Thr Thr Val Pro Phe

165 170 175

Asp Lys Lys His Thr Thr His Ala His Leu Gly Ala Thr Leu Ala Leu

180 185 190

Gly Asn Asn Thr His Leu Glu Ala Ala Ala Ala Lys Glu Ala Ala Ala

195 200 205

Ala Lys Lys Ile Leu Arg Thr Gly Lys Lys Leu Glu Leu Asp Ala Thr

210 215 220

Asn Ala Pro Ala Pro Ala Asn Gly Gly Val Ala Leu Asp Ser Glu Leu

225 230 235 240

Trp Thr Gly Ala Ala Ile Gly Ile Glu Leu Thr Pro Ser Thr Gln Phe

245 250 255

Gln Val Glu Tyr Gly Ile Ser Asn Arg Asp Ala Lys Ser Ser Asp Lys

260 265 270

Ser Ala His Arg Phe Asp Ala Glu Gln Glu Thr Ile Ser Gly Asn Phe

275 280 285

Leu Ile Gly Thr Glu Gln Phe Ser Gly Tyr Asn Pro Thr Asn Lys Phe

290 295 300

Lys Pro Tyr Val Leu Val Gly Ala Gly Gln Ser Lys Ile Lys Val Asn

305 310 315 320

Ala Ile Asp Gly Tyr Thr Ala Glu Val Ala Asn Gly Gln Asn Ile Ala

325 330 335

Lys Asp Gln Ala Val Lys Ala Gly Gln Glu Val Ala Glu Ser Lys Asp

340 345 350

Thr Ile Gly Asn

355

Claims (8)

1. A Moraxella catarrhalis surface protein (M35 and Olpa), comprising: the protein sequences of the Moraxella catarrhalis surface proteins (M35 and Olpa) are:

MEDTNESGLNSNTSRIGFKGSEALNANTDVVYQLEYKIDIDADRGDNFKSRDTYLGLAHKQYGTLLAGRLTT IDDSVDFASMLEDNNVADIGPTFNAPRANNAFAYVSPEYNGTQFLAMYAFDSDTDKGGLAKDDQFGVGATYSTGPI NAGATYIHYGDDSHIRLGGSGGSGGSGGSGYYVQADLGYSNLEAKYNDNRPNDNKLEDKGLGYGISFGKDTDLARY ALDYKNYGKINSSYTYQPQTNTSITSSGELKAQSLGLSAIYDFDTVSGFTPYAGARVDINQIKSTVSTVTTTAAAQ；

The complete sequence of the gene for the protein encoding the Moraxella catarrhalis surface proteins (M35 and Olpa) is:

CATATGGAGGATACTAACGAGTCTGGTCTGAACAGCAACACCTCTCGTATCGGCTTTAAGGGTAGCGAAGCT CTGAACGCAAACACCGACGTAGTTTACCAGCTGGAGTACAAAATCGACATCGATGCTGACCGTGGTGACAACTTCA AATCTCGTGATACTTACCTGGGCCTGGCACATAAACAGTACGGTACTCTGCTGGCTGGCCGCCTGACCACTATCGA TGATTCTGTGGATTTCGCATCTATGCTGGAAGACAACAACGTTGCAGACATCGGCCCGACCTTCAACGCTCCGCGT GCAAACAACGCATTTGCTTACGTGTCTCCGGAATACAACGGTACCCAGTTTCTGGCCATGTATGCTTTCGATTCCG ATACCGACAAAGGTGGCCTGGCGAAAGATGACCAGTTCGGTGTTGGCGCTACTTACTCTACCGGTCCAATCAACGC TGGCGCAACTTACATCCACTACGGCGATGACAGCCACATTCGCCTGGGTGGTTCTGGTGGTTCTGGTGGTTCTGGT GGTTCTGGTTACTACGTACAGGCGGACCTGGGCTACTCCAACCTGGAAGCCAAATACAACGACAACCGCCCGAACG ACAACAAGCTCGAAGACAAAGGTCTGGGCTACGGTATCTCTTTCGGCAAAGATACTGACCTGGCTCGCTACGCGCT GGACTACAAAAACTACGGTAAGATCAACTCCTCTTACACCTACCAGCCGCAGACTAACACCTCTATCACCTCTTCT GGCGAACTGAAAGCACAGTCCCTGGGTCTGAGCGCGATCTACGATTTCGACACCGTATCCGGTTTCACCCCGTACG CAGGCGCTCGTGTTGACATCAACCAGATCAAATCTACCGTTTCCACCGTAACTACCACCGCAGCTGCACAGTAAGG ATCC。

2. Process for the preparation of Moraxella catarrhalis surface proteins (M35 and Olpa) according to claim 1, characterized in that: the method comprises the following steps:

Respectively obtaining peptide segments with most abundant antigenic epitopes in the Moraxella catarrhalis surface protein M35 and the surface protein Olpa extracellular domain, 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 Moraxella catarrhalis surface protein M35 and the surface protein Olpa in the NCBI protein database are AY905613 and DQ996463 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 M35 olp;

The complete sequence of the M35olp gene is:

CATATGGAGGATACTAACGAGTCTGGTCTGAACAGCAACACCTCTCGTATCGGCTTTAAGGGTAGCGAAGCT CTGAACGCAAACACCGACGTAGTTTACCAGCTGGAGTACAAAATCGACATCGATGCTGACCGTGGTGACAACTTCA AATCTCGTGATACTTACCTGGGCCTGGCACATAAACAGTACGGTACTCTGCTGGCTGGCCGCCTGACCACTATCGA TGATTCTGTGGATTTCGCATCTATGCTGGAAGACAACAACGTTGCAGACATCGGCCCGACCTTCAACGCTCCGCGT GCAAACAACGCATTTGCTTACGTGTCTCCGGAATACAACGGTACCCAGTTTCTGGCCATGTATGCTTTCGATTCCG ATACCGACAAAGGTGGCCTGGCGAAAGATGACCAGTTCGGTGTTGGCGCTACTTACTCTACCGGTCCAATCAACGC TGGCGCAACTTACATCCACTACGGCGATGACAGCCACATTCGCCTGGGTGGTTCTGGTGGTTCTGGTGGTTCTGGT GGTTCTGGTTACTACGTACAGGCGGACCTGGGCTACTCCAACCTGGAAGCCAAATACAACGACAACCGCCCGAACG ACAACAAGCTCGAAGACAAAGGTCTGGGCTACGGTATCTCTTTCGGCAAAGATACTGACCTGGCTCGCTACGCGCT GGACTACAAAAACTACGGTAAGATCAACTCCTCTTACACCTACCAGCCGCAGACTAACACCTCTATCACCTCTTCT GGCGAACTGAAAGCACAGTCCCTGGGTCTGAGCGCGATCTACGATTTCGACACCGTATCCGGTTTCACCCCGTACG CAGGCGCTCGTGTTGACATCAACCAGATCAAATCTACCGTTTCCACCGTAACTACCACCGCAGCTGCACAGTAAGG ATCC；

the protein sequence encoded by the M35olp gene is:

MEDTNESGLNSNTSRIGFKGSEALNANTDVVYQLEYKIDIDADRGDNFKSRDTYLGLAHKQYGTLLAGRLTT IDDSVDFASMLEDNNVADIGPTFNAPRANNAFAYVSPEYNGTQFLAMYAFDSDTDKGGLAKDDQFGVGATYSTGPI NAGATYIHYGDDSHIRLGGSGGSGGSGGSGYYVQADLGYSNLEAKYNDNRPNDNKLEDKGLGYGISFGKDTDLARY ALDYKNYGKINSSYTYQPQTNTSITSSGELKAQSLGLSAIYDFDTVSGFTPYAGARVDINQIKSTVSTVTTTAAAQ；

the protein sequence coded by the M35olp gene is 50-213aa of the Moraxella catarrhalis surface protein M35 and 26-148aa of the surface protein Olpa; the two protein sequences are connected by flexible connecting peptide ggsggsggs;

Cloning the complete sequence of M35olp gene into prokaryotic expression vector pET-28a (+) by a conventional method, transferring the complete sequence into E.coli BL21(DE3) bacteria, inducing recombinant escherichia coli expression by IPTG, and purifying the recombinant His-M35olp protein by Ni2+ affinity chromatography.

3. A method for the preparation of polyclonal antibodies against moraxella catarrhalis surface proteins (M35 and OlpA), characterized in that: the method comprises the steps of taking the recombinant His-M35olp protein as an immune antigen as claimed in claim 2, mixing with Freund's adjuvant, repeatedly and artificially immunizing healthy New Zealand white rabbits, performing titer measurement by blood drawing, separating and purifying high-titer recombinant protein antibodies, and finally obtaining the polyclonal antibody against Moraxella catarrhalis surface proteins (M35 and Olpa).

4. A Moraxella catarrhalis surface protein (McaP and OmpCD), comprising: the protein sequences of the Moraxella catarrhalis surface proteins (McaP and OmpCD) are:

MQDYHLSDSITADVKQTGIGLYHRHDFDHLRISANLGVDHLSTQSLRQIMWDGENRNHQAHATGQRRYASVQ GSYGFTQNNVTYRPYVGIHAQDIKQNRFFENQPNLSTALSFKLPDHQSLQADIGVNIDYAMNDKLNLLAGLGYQHE FKDNNKTVETAVLSNRDYHRSFVTTVPFDKKHTTHAHLGATLALGNNTHLEAAAAKEAAAAKKILRTGKKLELDAT NAPAPANGGVALDSELWTGAAIGIELTPSTQFQVEYGISNRDAKSSDKSAHRFDAEQETISGNFLIGTEQFSGYNP TNKFKPYVLVGAGQSKIKVNAIDGYTAEVANGQNIAKDQAVKAGQEVAESKDTIGN；

the complete sequence of the gene for the protein encoding the Moraxella catarrhalis surface proteins (McaP and OmpCD) is:

CATATGCAGGACTATCACCTCAGCGACTCCATTACCGCTGACGTTAAGCAGACTGGCATCGGTCTTTATCAT CGCCACGACTTCGACCACCTGCGTATTTCTGCAAACCTGGGCGTAGATCACCTGTCTACTCAGAGTCTGCGTCAAA TCATGTGGGACGGTGAAAACCGCAACCACCAGGCACATGCTACCGGCCAGCGTCGTTATGCATCCGTACAGGGTTC CTACGGTTTCACTCAGAACAACGTTACCTACCGCCCATACGTAGGCATCCACGCACAGGACATCAAACAGAACCGC TTCTTCGAAAACCAGCCGAACCTCAGCACTGCTCTGTCCTTCAAGCTGCCGGACCACCAGAGCCTGCAGGCTGATA TCGGCGTTAACATCGATTACGCAATGAACGACAAACTGAACCTGCTGGCTGGTCTGGGCTATCAGCATGAATTCAA AGACAACAACAAAACTGTGGAAACCGCGGTTCTGTCTAACCGTGATTACCATCGCTCCTTCGTTACTACCGTACCG TTCGACAAAAAACACACCACCCACGCTCACCTCGGTGCTACTTTAGCGCTGGGCAACAACACCCACCTGGAAGCTG CTGCTGCTAAAGAAGCTGCTGCTGCTAAAAAAATCCTGCGTACTGGTAAGAAACTGGAACTGGACGCTACCAACGC GCCGGCTCCTGCAAACGGTGGCGTTGCGCTCGACTCCGAACTGTGGACCGGCGCTGCCATCGGTATCGAACTGACT CCGTCTACTCAGTTCCAGGTTGAATACGGTATCTCCAACCGCGACGCTAAATCTTCCGACAAATCTGCCCACCGCT TCGATGCAGAACAGGAAACCATCTCTGGCAACTTCCTGATCGGTACTGAACAGTTCTCCGGCTACAACCCAACCAA CAAATTCAAACCTTACGTACTGGTTGGCGCTGGTCAGTCTAAAATTAAGGTGAACGCAATCGACGGCTACACTGCA GAAGTTGCGAACGGTCAGAACATTGCGAAAGACCAGGCAGTGAAAGCAGGCCAGGAAGTTGCGGAATCCAAAGACA CCATCGGTAACTAAGGATCC。

5. Process for the preparation of Moraxella catarrhalis surface proteins (McaP and OmpCD) 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 extracellular domains of the Moraxella catarrhalis surface protein McaP and the surface protein OmpCD, 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 Moraxella catarrhalis surface protein McaP and the surface protein OmpCD in the NCBI protein database are EF075940 and AAA66180 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 Mcaomp;

the complete gene sequence of Mcap is as follows:

CATATGCAGGACTATCACCTCAGCGACTCCATTACCGCTGACGTTAAGCAGACTGGCATCGGTCTTTATCAT CGCCACGACTTCGACCACCTGCGTATTTCTGCAAACCTGGGCGTAGATCACCTGTCTACTCAGAGTCTGCGTCAAA TCATGTGGGACGGTGAAAACCGCAACCACCAGGCACATGCTACCGGCCAGCGTCGTTATGCATCCGTACAGGGTTC CTACGGTTTCACTCAGAACAACGTTACCTACCGCCCATACGTAGGCATCCACGCACAGGACATCAAACAGAACCGC TTCTTCGAAAACCAGCCGAACCTCAGCACTGCTCTGTCCTTCAAGCTGCCGGACCACCAGAGCCTGCAGGCTGATA TCGGCGTTAACATCGATTACGCAATGAACGACAAACTGAACCTGCTGGCTGGTCTGGGCTATCAGCATGAATTCAA AGACAACAACAAAACTGTGGAAACCGCGGTTCTGTCTAACCGTGATTACCATCGCTCCTTCGTTACTACCGTACCG TTCGACAAAAAACACACCACCCACGCTCACCTCGGTGCTACTTTAGCGCTGGGCAACAACACCCACCTGGAAGCTG CTGCTGCTAAAGAAGCTGCTGCTGCTAAAAAAATCCTGCGTACTGGTAAGAAACTGGAACTGGACGCTACCAACGC GCCGGCTCCTGCAAACGGTGGCGTTGCGCTCGACTCCGAACTGTGGACCGGCGCTGCCATCGGTATCGAACTGACT CCGTCTACTCAGTTCCAGGTTGAATACGGTATCTCCAACCGCGACGCTAAATCTTCCGACAAATCTGCCCACCGCT TCGATGCAGAACAGGAAACCATCTCTGGCAACTTCCTGATCGGTACTGAACAGTTCTCCGGCTACAACCCAACCAA CAAATTCAAACCTTACGTACTGGTTGGCGCTGGTCAGTCTAAAATTAAGGTGAACGCAATCGACGGCTACACTGCA GAAGTTGCGAACGGTCAGAACATTGCGAAAGACCAGGCAGTGAAAGCAGGCCAGGAAGTTGCGGAATCCAAAGACA CCATCGGTAACTAAGGATCC；

The protein sequence coded by the Mcpacomp gene is as follows:

MQDYHLSDSITADVKQTGIGLYHRHDFDHLRISANLGVDHLSTQSLRQIMWDGENRNHQAHATGQRRYASVQ GSYGFTQNNVTYRPYVGIHAQDIKQNRFFENQPNLSTALSFKLPDHQSLQADIGVNIDYAMNDKLNLLAGLGYQHE FKDNNKTVETAVLSNRDYHRSFVTTVPFDKKHTTHAHLGATLALGNNTHLEAAAAKEAAAAKKILRTGKKLELDAT NAPAPANGGVALDSELWTGAAIGIELTPSTQFQVEYGISNRDAKSSDKSAHRFDAEQETISGNFLIGTEQFSGYNP TNKFKPYVLVGAGQSKIKVNAIDGYTAEVANGQNIAKDQAVKAGQEVAESKDTIGN；

the protein sequence coded by the Mcpacomp gene is 429-625aa of the Moraxella catarrhalis surface protein McaP and 49-194aa of the surface protein OmpCD; the middle of the two protein sequences is connected by rigid connecting peptide eaaakaaaak;

Cloning the complete gene sequence of Mcap into prokaryotic expression vector pET-28a (+) by a conventional method, transferring the complete gene sequence into E.coli BL21(DE3) bacteria, inducing recombinant Escherichia coli expression by IPTG, and purifying the recombinant His-Mcap protein by Ni2+ affinity chromatography.

6. a method for preparing polyclonal antibodies against moraxella catarrhalis surface proteins (McaP and OmpCD), characterized in that: the method comprises the steps of taking the recombinant His-Mcaapomp 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 measurement, separating and purifying high-titer recombinant protein antibodies, finally obtaining polyclonal antibodies against Moraxella catarrhalis McaP and OmpCD proteins, and diluting with a confining liquid to a final concentration of 20 mug/mL; the sealing liquid comprises the following components in percentage by weight: 1.4g/L of disodium hydrogen phosphate, 0.2g/L of sodium dihydrogen phosphate, 8.5g/L of sodium chloride and 10g/L of bovine serum albumin, wherein the pH value of the sealing liquid is 7.4.

7. a Moraxella catarrhalis Elisa detection kit based on Moraxella catarrhalis surface protein antibody is characterized in that: the Moraxella catarrhalis Elisa detection kit based on the Moraxella catarrhalis surface protein antibody comprises an enzyme label plate coated with a polyclonal antibody of anti-Moraxella catarrhalis surface proteins (M35 and Olpa), an anti-Moraxella catarrhalis surface protein (McaP and OmpCD) polyclonal antibody, a Moraxella catarrhalis positive control, a negative control, a washing solution, an enzyme-labeled secondary antibody, an enzyme chromogenic substrate and a termination solution; the Moraxella catarrhalis positive control is inactivated Moraxella catarrhalis 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 a Moraxella catarrhalis Elisa detection kit based on the Moraxella catarrhalis surface protein antibody of claim 7, characterized in that: the Moraxella catarrhalis Elisa detection kit based on the Moraxella catarrhalis surface protein antibody comprises an enzyme label plate coated with a polyclonal antibody of anti-Moraxella catarrhalis surface proteins (M35 and Olpa), an anti-Moraxella catarrhalis surface protein (McaP and OmpCD) polyclonal antibody, a Moraxella catarrhalis positive control, a negative control, a washing solution, an enzyme-labeled secondary antibody, an enzyme chromogenic substrate and a termination solution; the Moraxella catarrhalis positive control is inactivated Moraxella catarrhalis 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-Moraxella catarrhalis surface protein (M35 and Olpa) comprises the following steps:

1) Preparing polyclonal antibodies against Moraxella catarrhalis surface proteins (M35 and Olpa);

2) Coating of polyclonal antibodies against Moraxella catarrhalis surface proteins (M35 and Olpa):

Diluting the polyclonal antibody against Moraxella catarrhalis surface proteins (M35 and Olpa) prepared in the step 1) to the concentration of 10 mu g/mL by using PBS buffer, coating a 96-well EIA high-efficiency binding enzyme standard plate according to the amount of 100 mu L/well, 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.