CN112521497B

CN112521497B - Preparation and application of myoglobin monoclonal antibody

Info

Publication number: CN112521497B
Application number: CN202011500965.9A
Authority: CN
Inventors: 项美华; 李小平; 刘清泉; 余铭恩; 吴琼杉
Original assignee: HANGZHOU XIANZHI BIOTECHNOLOGY CO Ltd
Current assignee: HANGZHOU XIANZHI BIOTECHNOLOGY CO Ltd
Priority date: 2020-12-17
Filing date: 2020-12-17
Publication date: 2022-05-20
Anticipated expiration: 2040-12-17
Also published as: CN112521497A

Abstract

The invention belongs to the field of biotechnology. The invention provides a recombinant protein, wherein an amino acid sequence of the recombinant protein is formed by repeated tandem connection of two dominant epitopes of Myoglobin (Myoglobin, Myo), an escherichia coli preferred codon is adopted to convert the amino acid sequence of the recombinant protein into a corresponding nucleotide sequence, the nucleotide sequence is chemically synthesized, and a recombinant expression vector is constructed, so that the expression quantity of the recombinant protein in escherichia coli is improved. The invention also relates to a phage library established by using the recombinant protein immunized mouse, a corresponding myoglobin single-chain antibody scfv sequence is obtained by panning and screening, the obtained scfv sequence is constructed into a complete mouse IgG1 antibody sequence expression vector, a monoclonal antibody is expressed by transiently transferring HEK293F cells, the monoclonal antibody is purified, and the optimal monoclonal antibody pairing combination is determined by an immunofluorescence orthogonal experiment.

Description

Preparation and application of myoglobin monoclonal antibody

Technical Field

The invention belongs to the field of biotechnology. Specifically, the invention expresses a novel recombinant protein, relates to establishment of a phage library by using the recombinant protein to immunize a mouse, obtains a specific single-chain antibody scfv sequence by screening, and also relates to establishment of the obtained scfv sequence into a eukaryotic expression vector to express a myoglobin (Myo) monoclonal antibody, and application of the scfv sequence to early diagnosis of Acute Myocardial Infarction (AMI).

Background

Acute Myocardial Infarction (AMI) is one of common diseases seriously harming human health, and the incidence rate of the AMI is obviously increased in China in recent years. Myoglobin (Myo) is one of the earliest elevated markers of myocardial injury when AMI occurs, and the content of Myo in serum begins to increase within 2h of onset, reaching a peak within 6-9 h. In addition, Myo is one of the best noninvasive indexes for judging whether the acute myocardial infarction patient has reperfusion, and if myoglobin is increased by nearly 4 times 90min after the start of thrombolytic therapy, the infarcted coronary artery is completely patent can be accurately judged. Therefore, the detection of myoglobin has important significance for the diagnosis of acute myocardial infarction, prediction of myocardial infarction area, guidance of thrombolytic therapy and prognosis.

At present, the diagnosis of myocardial infarction mainly depends on electrocardiogram, magnetic resonance, coronary angiography and the like, special equipment and instruments are needed, professional operation is needed, and on-site detection cannot be carried out. Therefore, the preparation of Myo monoclonal antibody for myoglobin Myo specific recognition detection becomes a main mode for early diagnosis. The conventional Myo monoclonal antibody is prepared by preparing Balb/c mouse ascites from a Myo monoclonal cell strain and purifying the monoclonal antibody by using Protein A affinity chromatography. However, the yield of ascites of a single mouse is uncertain, and the individual difference is large, so that the obtained myoglobin Myo monoclonal antibody has large batch difference, and the detection accuracy is poor.

Disclosure of Invention

The design purpose is as follows: in order to overcome the defects of the traditional preparation of the monoclonal antibody, the monoclonal antibody is prepared by designing and synthesizing recombinant Myo protein and establishing a phage library and expressing eukaryotic cells, the time is greatly shortened compared with the traditional preparation of the monoclonal antibody, and the obtained monoclonal antibody has high stability and good uniformity and greatly reduces the batch difference.

The design scheme is as follows: in order to achieve the above design objectives. The application: (1) the myoglobin (Myo) is taken as a target antigen, two dominant epitopes of the antigen are analyzed and selected, and the sequence comparison result shows that the two selected epitopes are all myoglobin (Myo) common epitopes and have no obvious homology with other protein sequences. (2) In order to enhance the immune effect and shorten the preparation time of the monoclonal antibody, two selected dominant antigen epitopes are connected in series and used for immunizing a mouse. (3) In order to improve the expression quantity of the recombinant protein, the amino acid sequence of the recombinant protein is converted into a corresponding nucleotide sequence by adopting an escherichia coli preferred codon. (5) And chemically synthesizing the nucleotide sequence obtained in the last step, carrying out enzyme digestion connection, inserting the synthesized nucleotide fragment into an expression vector PET-32a (+), and constructing a recombinant myoglobin (Myo) expression vector. (6) And transforming the Escherichia coli ER2566 competent cells by the recombinant myoglobin (Myo) expression vector, and screening to obtain a recombinant protein expression strain. (7) After the recombinant protein expression strain is cultured in a large scale, the strain is broken by ultrasonic waves and centrifuged at low temperature, and the supernatant of the solution is taken to pass through a nickel agarose affinity chromatography column for affinity chromatography and eluted to obtain the purified recombinant myoglobin (Myo). (8) After the Balb/c mice are immunized by the recombinant Myo protein for multiple times, spleen separated lymphocytes are taken to establish a single-chain antibody scfv phage display library, and the recombinant Myo protein is used for multiple rounds of panning and screening to finally obtain a single-chain antibody scfv sequence capable of being combined with the recombinant Myo. (9) The scfv sequence was constructed into a complete murine IgG1 expression vector and HEK293 cells were used to express monoclonal antibodies, which were purified using Protein a affinity chromatography and labeled with fluorescent microspheres, respectively. (10) Orthogonal experimental screening showed that 5E6 mab coating paired with 1a5 mab labeling for optimal detection of myoglobin (Myo) combinations.

Detailed Description

Although the following embodiments describe the design concept of the present invention in more detail, these descriptions are only simple words for describing the design concept of the present invention, and are not intended to limit the design concept of the present invention, and any combination, addition or modification without departing from the scope of the design concept of the present invention will fall within the scope of the present invention.

Example 1: selection of dominant myoglobin (Myo) epitopes

Myoglobin (Myo) is used as a target antigen, the hydrophilicity and the antigenicity of an antigen epitope sequence of the myoglobin are analyzed by using biological software DNAssist2.0, and an A dominant antigen epitope and a B dominant antigen epitope are selected. Meanwhile, the sequence comparison result shows that the selected A, B dominant antigen epitope sequences have broad spectrum and are common epitopes of all myoglobin (Myo); and the A, B epitope has no obvious homology with other protein sequences and exists only in a myoglobin (Myo) sequence.

Example 2: tandem connection of myoglobin (Myo) dominant epitopes

In order to enhance the stimulation of the selected epitope to the mouse immune system to be beneficial to the subsequent experiment, A, B two dominant epitope sequences of myoglobin (Myo) are respectively repeated and then connected through flexible fragments (four continuous glycines) to obtain the amino acid sequence of the recombinant protein.

Example 3: optimized nucleotide sequence for encoding recombinant Myo protein

In order to improve the expression amount of the recombinant protein in the escherichia coli, on the premise that the amino acid sequence of the recombinant protein is not changed, the amino acid sequence of the encoded recombinant protein is converted into a corresponding nucleotide sequence according to the preferred codon of the escherichia coli, and the nucleotide sequences corresponding to enzyme cutting sites BamHI and EcoRI are respectively added at the upstream and the downstream of the nucleotide sequence, and then the nucleotide sequence is synthesized by Anhui limited of a general biological system. The synthesized target gene is cloned in pMD25-T vector (Takara Bio-engineering Co., Ltd.).

Example 4: construction of recombinant Myo protein expression vector

The pMD25-T vector containing the target gene and the PET-32a (+) vector (Novagen, Germany) are subjected to double enzyme digestion at 37 ℃ for 12 hours respectively by restriction enzymes BamHI and EcoRI (Bao bioengineering Dalian Co., Ltd.), the enzyme digestion products are subjected to 1% agarose gel electrophoresis respectively, and the target gene and the PET-32a (+) vector are recovered by gel cutting respectively (the gel recovery kit used by the invention is from Hangzhou, Inc. of Seika biotechnology). The recovered target gene and a PET-32a (+) vector are connected for 12 hours at 4 ℃ by using T4 ligase (Baozoigaojii Co., Ltd.), a connection product is transformed into DH5 alpha competent cells (Hangzhou Jixian to Biotechnology Co., Ltd.), the cells are coated on an LB plate containing ampicillin resistance (50 mu g/mL), after the cells are cultured for 12 hours at 37 ℃ at constant temperature, a single clone strain is picked on the plate to an LB liquid culture medium containing ampicillin resistance (50 mu g/mL), after the cells are cultured for 12 hours at 37 ℃ at constant temperature by a shaking table, a plasmid purification kit (plasmid extraction and collection kits used by the invention are all from Hangzhou Co., Ltd. of Seiki Biotechnology) is adopted to extract plasmids, and a correct recombinant expression vector is obtained after BamHI and EcoRI double enzyme digestion identification.

Example 5: construction of recombinant Myo antigen expression strains

E.coli ER2566 competent cells were transformed with the constructed recombinant expression vector, spread on LB plates containing ampicillin resistance (50. mu.g/mL), and cultured overnight at 37 ℃. The next day, the monoclonal strains on the plates were picked up to LB liquid medium containing ampicillin resistance (50. mu.g/mL), shake-cultured at 37 ℃ for 8 hours, and then added with an inducer isopropyl thio-beta-D-galactoside (final concentration of 1.0mmol/L) for induction expression for 4 hours to prepare protein electrophoresis samples. The result of 9% polyacrylamide gel electrophoresis shows that the recombinant protein is successfully expressed to obtain the recombinant Myo antigen expression strain.

Example 6: purification of recombinant Myo proteins

Inoculating the recombinant Myo protein expression strain to an LB liquid culture medium, adding ampicillin to a final concentration of 50 mu g/mL, carrying out shake culture at a constant temperature of 37 ℃ for 8 hours, and then carrying out shake culture on the strain by using an LB liquid culture medium containing 50 mu g/mL ampicillin according to a ratio of 1: diluting at a ratio of 100, subpackaging into bacteria culture bottles, shake culturing at 37 deg.C until OD600 is 0.8, adding inducer isopropylthio-beta-D-galactoside to final concentration of 1.0mmol/L, and further culturing and inducing for 4 hr. And (3) after the thalli are collected by centrifugation, carrying out ultrasonic bacteria breaking at a low temperature of 4 ℃, taking the supernatant after low-temperature centrifugation, passing the supernatant through a nickel-agarose affinity chromatography column, and washing and eluting to finally obtain the purified recombinant Myo protein.

Example 7: construction of Single chain antibody scfv phage library

4-6-week-old female Balb/c mice were taken, and basal immunization was performed on each mouse by subcutaneous multi-point injection of 100. mu.g of recombinant Myo protein emulsified in Freund's complete adjuvant, for a total of 400. mu.l/mouse. A second booster immunization was performed 20 days later by taking 80. mu.g of recombinant Myo protein and emulsifying with Freund's incomplete adjuvant, 400. mu.l/mouse, and injecting subcutaneously at multiple points. Third boost after 15 days, the procedure was the same as for the second boost. After 20 days, 120. mu.g of recombinant Myo antigen was intraperitoneally injected, and 72 hours later, blood was taken from the orbit, and the mice were sacrificed, and their spleens were removed to isolate mouse spleen lymphocytes using a mouse spleen lymphocyte isolation kit (Tianjin, a third-class biologicals science, Inc.). Extracting total RNA from separated lymphocytes by using an RNA extraction kit (Tiangen Biochemical technology Co., Ltd.), performing reverse transcription by using a reverse transcription kit (Takara) to synthesize cDNA, amplifying genes of a heavy chain variable region and a light chain variable region by using a mouse single-chain antibody scfv universal degenerate primer, performing 1% agarose gel electrophoresis on PCR products, respectively cutting gel to recover target genes, linking the recovered target genes into scfv by overlap PCR, performing 1% agarose gel electrophoresis on the PCR products, performing enzyme digestion on the gel-cutting recovered target genes by using NotI and SfiI, connecting the recovered target genes by using T4 ligase and pCANTAB5e (Beijing Baokou Weiyangshen Biotech Co., Ltd.) carriers according to a certain proportion at 4 ℃ for 12 hours, recovering the connecting products by using the gel recovery kit to remove enzymes and buffer substances in the connecting products, and transferring the recovered products into a transgenic state of Escherichia coli TG1 by using a bacterial electrotransformation instrument (biorad) for multiple times, and spread on a 2 XYT-AG plate containing ampicillin resistance (50. mu.g/mL) and 2% glucose, incubated at 30 ℃ for 12 hours, then an appropriate amount of 2 XYT medium was taken, colonies on the plate were all scraped off with a sterile glass rod, and a cell suspension was collected, which was a constructed phage antibody library.

Example 8: panning and screening of Single chain antibodies scfv

A predetermined amount of the bacterial suspension was removed from the phage antibody library and inoculated into 2 XYT-AG culture medium so that OD600 was 0.3. After OD600 reached 0.5, the helper phage M13K07 was added to superinfect at 37 ℃ and 250rpm for about 1h, with the infection ratio M13K07/TG1 being 20: 1. 3300g after shaking at 37 ℃ and 250rpm for 1h, the bacteria were pelleted by centrifugation at 4 ℃ for 10min, and the supernatant was carefully discarded. The bacteria were resuspended in ampicillin-resistant (50. mu.g/mL) and kanamycin-resistant (50. mu.g/mL) 2 XYT-AK medium and cultured overnight at 30 ℃ with shaking at 250 rpm. The next day, 10800g, centrifugation at 4 ℃ for 20min precipitated bacteria. The supernatant was transferred to a clean centrifuge tube and 1/5 volumes of PEG/NaCl were added and mixed for 2h in an ice bath. 10800g, centrifugation at 4 ℃ for 20min precipitated cells, carefully removed supernatant, dried, pellet resuspended in PBS, and filtered through 0.45 μm membrane to remove bacterial debris for the panning step. The purified recombinant Myo antigen was diluted with coating solution to 8. mu.g/ml coated immune tubes (Thermo), 4ml per immune tube, coated overnight at 4 ℃. The next day, the coating solution and unadsorbed antigen were discarded, washed 3 times with sterile PBST, 5ml of blocking solution was added to each immune tube, and incubated at 37 ℃ for 2 h. The blocking solution was discarded, and after 3 washes with sterile PBST, the phage obtained by PEG precipitation was added to immune tubes, 4ml of each immune tube was added, and incubated at 37 ℃ for 1 h. The liquid in the tube was discarded, washed 10 times with sterile PBST and 10 times with sterile PBS, and the bound phage were eluted by adding 1ml of 100mM triethylamine, and immediately neutralized by adding 500. mu.l of 1M Tris-HCl, pH 7.4. The neutralized phage was added to a certain amount of TG1 E.coli in the logarithmic growth phase for superinfection, which is the first panning enrichment process. After 3 rounds of panning, Myo-specific scfv were enriched. The phage after the last round of elution and neutralization is infected with TG1 colibacillus and then spread on a 2 XYT-AG plate, after 12 hours of constant temperature culture at 30 ℃, 400-once monoclonal colonies are randomly picked out to a 96-hole deep-hole plate, a certain amount of M13K07 helper phage is added for superinfection after 2 hours of oscillation at 250rpm and 37 ℃ in a 2 XYT-AG culture medium, after 1 hour of oscillation at 250rpm and 37 ℃, the supernatant is centrifugally removed, and 2 XYT-AK culture medium containing ampicillin resistance (50 mu g/mL) and kanamycin resistance (50 mu g/mL) is added for 30 ℃ and 250rpm overnight culture. The following day a monoclonal ELISA screening was performed, with the following screening steps:

coating: diluting Myo recombinant protein with coating solution to a final concentration of 1 μ g/mL, adding an enzyme label plate (Shenzhen Jinlau actual Co., Ltd.) into a 100 μ L/well, and washing with washing solution for 1 time by a DEM-3 type plate washing machine (Daan Gene of Zhongshan university Co., Ltd.) after overnight at 4 ℃;

and (3) sealing: adding sealing liquid into 200 μ L/hole, sealing at 37 deg.C for 2 hr, and washing with washing liquid for 1 time;

sample adding: adding overnight induction expressed bacterial culture supernatant and control serum, incubating at 100 μ L/well for 1h at 37 deg.C, washing with washing solution for 3 times by plate washing machine;

adding an enzyme-labeled antibody: adding a fresh diluted rabbit anti-M13 phage HRP enzyme-labeled secondary antibody (purchased from Beijing Yiqiao Shenzhou Biotechnology Co., Ltd.) into a 100 mu L/hole, incubating at 37 ℃ for 30 minutes, and washing with a washing solution for 4 times by a plate washing machine;

adding a color development liquid: adding 50 mu L of color development liquid A and 50 mu L of color development liquid B into each hole, and carrying out light-proof color development for 10 minutes at 37 ℃;

and (3) terminating the reaction: add 2M H at 50. mu.L/well₂SO₄；

And (4) judging a result: the OD was read after blank wells were zeroed at 450nm on a microplate reader. Sera from immunized mice were used as positive controls. The result shows that 14 positive clones have higher OD value, 5 strains of scfv sequences are obtained by sequencing, and the sequences are respectively 1A5, 3D3, 4G4, 5E6 and 5G 10. The relevant solution formulation is as follows:

coating liquid: na (Na)₂CO₃ 1.5g，NaHCO₃2.9g, plus ddH₂O was metered to 1000mL (pH 9.6).

Sealing liquid: na (Na)₂HPO₄.12H₂O 2.68g，NaH₂PO₄.2H₂O0.39 g, NaCl 8.5g, bovine serum albumin 20g, plus ddH₂O was metered to 1000mL (pH 7.4).

Washing liquid: na (Na)₂HPO₄.12H₂O 2.68g，NaH₂PO₄.2H₂O0.39 g, NaCl 8.5g, Tween-200.5 mL, ddH₂O was metered to 1000mL (pH 7.4).

Color developing solution A: 200mg TMB dissolved in 100mL absolute ethanol, ddH added₂And O is metered to 1000 mL.

Color developing solution B: citric acid 2.1g, Na₂HPO₄.12H₂O71 g, plus ddH₂And O is metered to 1000 mL.

When in use: 1mL of developing solution A +1mL of developing solution B + 0.4. mu.L of 30% H₂O₂

Stopping liquid: 2M H₂SO₄21.7mL of concentrated H₂SO₄Add ddH₂And O is metered to 1000 mL.

Example 8: construction of eukaryotic expression vector and transient expression and purification of HEK293F cell

The 5 Myo single-chain antibody scfv sequences were constructed into complete mouse IgG1 antibody sequences, i.e., the heavy chain variable region and the light chain variable region in scfv were bridged with the heavy chain constant region and the light chain constant region of mouse IgG1, respectively, by PCR, and then inserted into pcDNA3.1 (Novagen, Germany) plasmids, respectively. The constructed heavy chain plasmid and light chain plasmid were co-transfected into HEK293F cells by PEI, expressed for 7 days at 37 ℃, 5% carbon dioxide, in a cell shaker at 120rpm, and then centrifuged to precipitate, and the supernatant was collected and filtered through a 0.45 μm filter. The agarose affinity medium Protein A column (Nanjing King Shirui Biotech Co., Ltd.) was equilibrated with 50mL of PBS (pH7.4) to a computer nucleic acid Protein detector (Shanghai Huxi analytical Instrument Co., Ltd.) to show that the absorbance was 0. After the supernatant was loaded, PBS was added and washed until the absorbance became 0, followed by elution with 0.1M glycine (pH3.0), and the effluent was collected and neutralized to about pH 7.0 with 500mM Tris-HCl (pH8.5) buffer to obtain purified monoclonal antibodies 1A5, 3D3, 4G4, 5E6, 5G 10.

Example 9: preparation of myoglobin (Myo) -labeled monoclonal antibody fluorescent microsphere pad

Fluorescent microspheres (Bangslab, Dragongreen) with a diameter of 190nm were selected, the concentration of microspheres was adjusted to 1% with 0.05M MES buffer pH4.5, and then the myoglobin (Myo) monoclonal antibody was labeled on the fluorescent microspheres by covalent coupling using carbodiimide (EDC) and succinimide (NHS) at an antibody concentration of 0.2 mg/ml. And spraying the prepared fluorescent microspheres on a fluorescent microsphere pad by using a quantitative film spraying instrument in an amount of 4 mul/cm, carrying out vacuum drying at 25 ℃ for 1-2 h, and placing the fluorescent microspheres in a drying environment for later use.

Example 10: preparation of nitrocellulose Membrane (NC Membrane)

Separately adjusting myoglobin (Myo) monoclonal antibodies (1a5, 3D3, 4G4, 5E6, 5G10) to a concentration of 0.4mg/mL with 0.01M pH7.4 PBS (phosphate buffered saline containing 5% sucrose and 0.05% tween-20), and separately spraying the resulting solutions on NC membranes to form test lines (T-lines); goat anti-mouse was adjusted to a concentration of 0.5mg/mL with 0.01M pH7.4 PBS (phosphate buffered saline containing 5% sucrose and 0.05% tween-20), and the resulting solution was sprayed on NC membrane to form a quality control region (line C). The film spraying amount of the two areas is 1 mu L/cm, the two areas are separated by 5mm, the distance between the quality control area and one end of the NC film is 2mm, and the two areas are dried at 37 ℃ overnight and then stored in a room-temperature dry environment for later use.

Example 11: preparation of fluorescent microsphere immunoassay card

Assembling the test strip: sequentially overlapping and sticking on a PVC bottom plate: (1) NC membrane sprayed with myoglobin monoclonal antibody (1A5, 3D3, 4G4, 5E6, 5G10) as detection area and goat anti-mouse IgG as quality control area; (2) a fluorescent microsphere pad sprayed with a fluorescent microsphere labeled myoglobin monoclonal antibody (1A5, 3D3, 4G4, 5E6, 5G 10); (3) filter paper and a sample pad, wherein the sample pad is a glass fiber membrane treated by 2% Tween-20; (4) and (3) cutting the water absorption paper into 4mm wide after the assembly is finished, installing a reagent card strip shell and compacting to obtain the fluorescent microsphere immunochromatography detection card.

Example 12: paired monoclonal antibody screening

Myoglobin (Myo) clinical serum samples and normal serum samples were loaded at 100 μ L/well and allowed to stand at room temperature for 15min, and then T, C line signals on the NC membrane were read and measured values T/(T + C) were calculated by a fluorescence analyzer (basic egg biotechnology, ltd.) as detailed in table 1.

The above table shows that the pairing of the 5E6 monoclonal antibody coating and the 1A5 monoclonal antibody labeled fluorescent microspheres is the optimal antibody pairing for detecting Myo.

SEQ ID NO 1: the variable region amino acid sequence of the light chain of an anti-myoglobin Myo-specific single-chain antibody scfv-1A 5;

SEQ ID NO 2: the amino acid sequence of the heavy chain variable region of an anti-myoglobin Myo-specific single-chain antibody scfv-1A 5;

SEQ ID NO 3: the variable region amino acid sequence of the light chain of an anti-myoglobin Myo-specific single-chain antibody scfv-5E 6;

SEQ ID NO 4: anti-myoglobin Myo-specific single chain antibody scfv-5E6 heavy chain variable region amino acid sequence;

SEQ ID NO 5: the Myo-specific single-chain antibody scfv-1A5 light chain variable region nucleotide sequence of the anti-myoglobin;

SEQ ID NO 6: the Myo-specific single-chain antibody scfv-1A5 heavy chain variable region nucleotide sequence of the anti-myoglobin;

SEQ ID NO 7: a Myo-specific single-chain antibody scfv-5E6 light chain variable region nucleotide sequence of anti-myoglobin;

SEQ ID NO 8: the Myo-specific single-chain antibody scfv-5E6 heavy chain variable region nucleotide sequence of anti-myoglobin;

sequence listing

<110> Hangzhou xian Zhi Biotechnology Co., Ltd

Preparation and application of <120> myoglobin monoclonal antibody

<160> 8

<170> SIPOSequenceListing 1.0

<210> 1

<211> 111

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Asp Ile Val Ile Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp

20 25 30

Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Asn Leu Glu Ser Gly Ile Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His

65 70 75 80

Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Asn

85 90 95

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

100 105 110

<210> 2

<211> 121

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Val Met His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe

50 55 60

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

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Tyr Tyr Gly Ser Ser Tyr Trp Tyr Phe Asp Val Trp Gly

100 105 110

Ala Gly Thr Ser Val Thr Val Ser Ser

115 120

<210> 3

<211> 112

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Asp Ile Gln Met Thr Gln Thr Pro Ala Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp

20 25 30

Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Asn Leu Glu Ser Gly Ile Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His

65 70 75 80

Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Asn

85 90 95

Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110

<210> 4

<211> 120

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Met Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Thr Gly Tyr Thr Phe Ser Ser Tyr

20 25 30

Trp Ile Glu Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp Ile

35 40 45

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

50 55 60

Lys Gly Lys Ala Thr Phe Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Val Arg Arg Thr Thr Ala Thr Asp Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Ser Val Thr Val Ser Ser

115 120

<210> 5

<211> 333

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

gacattgtga tcactcagtc tccagcttct ttggctgtgt ctctagggca gagggccacc 60

atctcctgca aggccagcca aagtgttgat tatgatggtg atagttatat gaactggtac 120

caacagaaac caggacagcc acccaaactc ctcatctatg ctgcatccaa tctagaatct 180

gggatcccag ccaggtttag tggcagtggg tctgggacag acttcaccct caacatccat 240

cctgtggagg aggaggatgc tgcaacctat tactgtcagc aaagtaatga ggatcggacg 300

ttcggtggag gcaccaagct ggaaataaaa cgt 333

<210> 6

<211> 363

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

gaggtccaac tgcaacaatc tggacctgag ctggtaaagc ctggggcttc agtgaagatg 60

tcctgcaagg cttctggata cacattcact agctatgtta tgcactgggt gaagcagaag 120

cctgggcagg gccttgagtg gattggatat attaatcctt acaatgatgg tactaagtac 180

aatgagaagt tcaaaggcaa ggccacactg acttcagaca aatcctccag cacagcctac 240

atggagctca gcagcctgac ctctgaggac tctgcggtct attactgtgc aagatcttac 300

tacggtagta gctactggta cttcgatgtc tggggcgcag ggacctcagt caccgtctcc 360

tcg 363

<210> 7

<211> 336

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

gatattcaga tgacacagac tccagcttct ttggctgtgt ctctagggca gagggccacc 60

atctcctgca aggccagcca aagtgttgat tatgatggtg atagttatat gaattggtac 120

caacagaaac caggacagcc acccaaactc ctcatctatg ctgcatccaa tctagaatct 180

gggatcccag ccaggtttag tggcagtggg tctgggacag acttcaccct caacatccat 240

cctgtggagg aggaggatgc tgcaacctat tactgtcagc aaagtaatga ggatccgtgg 300

acgttcggtg gaggcacaaa gttggaaata aaacgt 336

<210> 8

<211> 360

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

caggtccagc tgcagcagtc tggagctgag ctgatgaagc ctggggcctc agtgaagata 60

tcctgcaagg ctactggcta cacattcagt agctactgga tagagtgggt aaagcagagg 120

cctggacatg gccttgagtg gattggagag attttacctg gaagtggtag tactaactac 180

aatgagaagt tcaagggcaa ggccacattc actgcagata catcctccaa cacagcctac 240

atgcaactca gcagcctgac atctgaggac tctgccgtct attactgtgt cagaaggact 300

acggctacgg actatgctat ggactactgg ggtcaaggaa cctcagtcac cgtctcctcg 360

Claims

1. A group of antibodies for a myoglobin detection kit is characterized in that: comprises a labeled antibody and a coated antibody, wherein the labeled antibody is 1A5, and the coated antibody is 5E 6;

the amino acid sequence of the variable region of the light chain of the labeled antibody 1A5 is shown in SEQ ID NO. 1;

the amino acid sequence of the heavy chain variable region of the labeled antibody 1A5 is shown in SEQ ID NO. 2;

the amino acid sequence of the variable region of the light chain of the coating antibody 5E6 is shown as SEQ ID NO. 3;

the amino acid sequence of the heavy chain variable region of the coating antibody 5E6 is shown as SEQ ID NO. 4.

2. The antibody for myoglobin detection kit of claim 1, wherein: the nucleotide sequence of the coded light chain variable region of the labeled antibody 1A5 is shown as SEQ ID NO.5, and the nucleotide sequence of the coded heavy chain variable region is shown as SEQ ID NO. 6.

3. The antibody for myoglobin detection kit of claim 1, wherein: the nucleotide sequence of the coated antibody 5E6 for encoding the light chain variable region is shown in SEQ ID NO.7, and the nucleotide sequence for encoding the heavy chain variable region is shown in SEQ ID NO. 8.

4. A set of plasmid vectors characterized by: comprises a plasmid vector containing a light chain variable region nucleotide sequence shown in SEQ ID NO.5, a plasmid vector containing a heavy chain variable region nucleotide sequence shown in SEQ ID NO.6, a plasmid vector containing a light chain variable region nucleotide sequence shown in SEQ ID NO.7 and a plasmid vector containing a heavy chain variable region nucleotide sequence shown in SEQ ID NO. 8.

5. A method for preparing an antibody for myoglobin assay kit according to claim 1, wherein:

co-transfecting a plasmid vector containing a light chain variable region nucleotide sequence shown in SEQ ID No.5 and a plasmid vector containing a heavy chain variable region nucleotide sequence shown in SEQ ID No.6 to HEK293F cells, and expressing to obtain a marker antibody 1A 5;

the plasmid vector containing the nucleotide sequence of the light chain variable region shown in SEQ ID NO.7 and the plasmid vector containing the nucleotide sequence of the heavy chain variable region shown in SEQ ID NO.8 were co-transfected into HEK293F cells and expressed to obtain the coating antibody 5E 6.