CN112608385B

CN112608385B - Preparation of canine Brain Natriuretic Peptide (BNP) monoclonal antibody

Info

Publication number: CN112608385B
Application number: CN202011507501.0A
Authority: CN
Inventors: 武戌青; 曹丹琴; 陈安琪; 余铭恩; 吴琼杉
Original assignee: HANGZHOU XIANZHI BIOTECHNOLOGY CO Ltd
Current assignee: HANGZHOU XIANZHI BIOTECHNOLOGY CO Ltd
Priority date: 2020-12-18
Filing date: 2020-12-18
Publication date: 2022-05-20
Anticipated expiration: 2040-12-18
Also published as: CN112608385A

Abstract

The invention belongs to the field of biotechnology. The invention relates to a polypeptide containing two dominant epitopes of canine Brain Natriuretic Peptide (BNP), which enhances the immune effect by immunizing a mouse by coupling the polypeptide with KLH protein. The invention also provides a recombinant protein which comprises two dominant epitopes of canine BNP, in order to improve the yield of the recombinant protein in a prokaryotic expression system, the amino acid sequence of the recombinant protein is converted into a corresponding nucleotide sequence by adopting preferred codons of escherichia coli, and the nucleotide sequence is chemically synthesized to construct a recombinant protein expression vector. The invention also relates to a method for establishing a phage library by using the polypeptide-coupled KLH protein immunized mouse, obtaining a corresponding canine BNP single-chain antibody scfv sequence through panning and screening, constructing the obtained scfv sequence into a complete mouse IgG1 antibody sequence expression vector, expressing a monoclonal antibody through transient HEK293F cells, purifying the monoclonal antibody, and determining the optimal monoclonal antibody pairing combination through an immunofluorescence orthogonal experiment.

Description

Preparation of canine Brain Natriuretic Peptide (BNP) monoclonal antibody

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a novel canine Brain Natriuretic Peptide (BNP) recombinant protein. It also relates to the establishment of phage library by polypeptide coupling KLH protein immune mice, screening to obtain specific single chain antibody scfv sequence, and also relates to the construction of obtained scfv sequence into eukaryotic expression vector to express monoclonal antibody of anti-canine Brain Natriuretic Peptide (BNP) recombinant protein, and the application in early diagnosis of canine heart failure.

Background

Heart failure (heart failure), also known as cardiac insufficiency, is not an independent disease, is often caused in the course of other diseases, and is a serious dysfunction syndrome occurring in the whole body due to weakened myocardial contractility and insufficient blood output from the heart. The disease rate is high, the harm is large, and the disease can be divided into acute disease and chronic disease. Acute heart failure is often caused by temporary overload of the heart. Chronic heart failure, the disease progresses slowly, and the course of disease can last for months or years. The sick dog has the advantages of mental depression, easy fatigue, dyspnea, cyanosis of mucous membrane, frequent symmetrical edema at the tail end of the limbs, and reduction or disappearance of edema after exercise; the pulse is rapid and thready, the heart sound is weakened, heart noise and arrhythmia can be heard, the treatment is not timely, and the death can be caused by anoxia and shock.

For a long time, in addition to animal characterization such as respiration and pulse, and images such as X-ray, electrocardiogram and echocardiogram to examine the morphological structure of heart cavity and blood vessel, the diagnosis of heart failure of pets requires examination of ions and enzymes related to heart function such as blood routine, blood biochemistry, ions, blood gas and the like, so as to make a comprehensive judgment on the disease. However, these inspections are not only relatively time consuming, but also expensive. In recent years, Brain Natriuretic Peptide (BNP) has come to be used for the diagnosis of heart diseases, and this new laboratory test indicator is attracting much attention for the clinical diagnosis of heart failure.

In the case of heart disease, the renin-angiotensin-aldosterone system is over-stimulated for long periods of time, resulting in cardiac overload, volume overload and congestive heart failure. Natriuretic peptides antagonize this effect by stimulating natriuresis, increasing renal blood flow, inducing diuresis, and promoting vasodilation and improving diastolic function. The levels of atrial natriuretic peptides (atrial natriuretic peptides, ANP) and B-type natriuretic peptides (brain natriuretic peptides, BNP) are increased in the circulatory system, both released in the form of pro-bodies and then enzymatically cleaved into an active C-terminal fragment (C-ANP and C-BNP) and an inactive N-terminal fragment (NT-proANP and NT-proBNP). The half-life of C-ANP and C-BNP is very difficult to measure the concentration transiently, and the half-life of NT-proANP and NT-proBNP is longer and more stable, thereby being beneficial to collection and sample processing and being more feasible in clinical determination. Therefore, BNP has wide application in the aspects of prevention screening, curative effect detection, prognosis evaluation and the like of heart failure.

At present, the immunological detection method is a mainstream direction in the market due to simple and convenient operation, time saving and rapidness and easy judgment of results. The method needs to prepare the canine brain natriuretic peptide monoclonal antibody, immunogen used for preparing the monoclonal antibody conventionally is complete protein expressed by genetic engineering technology, and due to base codons, the protein is difficult to express in escherichia coli and has extremely low expression quantity, so that subsequent purification work is difficult to carry out, and the preparation of the monoclonal antibody is hindered. And because of the homology of the amino acid sequence of the epitope, the prepared monoclonal antibody has poor specificity and high homology with other proteins, and can also cause the distortion of the detection result. In addition, the traditional preparation method of the monoclonal antibody by the mouse ascites has long preparation period and large difference among different batches, and various foreign proteins are often mixed in the ascites and can be used after purification. The processing mechanism of the eukaryotic expression product is closest to the natural form in vivo, the bioactivity is easy to retain, and the expression product can be properly modified and distributed regionally; the monoclonal antibody can be rapidly and flexibly prepared by a transient transfection mode, so that the preparation period is greatly shortened, the stability is strong, and the batch difference is small. Therefore, the monoclonal antibody required by the invention is expressed by a eukaryotic system.

Disclosure of Invention

The design purpose is as follows: in order to solve the defects of long period, low purity and large batch difference of the traditional monoclonal antibody prepared from mouse ascites, the monoclonal antibody is prepared by designing and synthesizing canine BNP recombinant protein and establishing a phage library and eukaryotic cell expression, so that the time is saved, and the specific recognition and detection on the canine BNP are improved.

The design scheme is as follows: in order to achieve the above design objectives. The application: (1) the canine Brain Natriuretic Peptide (BNP) is taken as a target antigen, two dominant antigen epitopes of the antigen are analyzed and selected, and the sequence comparison result shows that the two selected antigen epitopes are common epitopes of all the canine Brain Natriuretic Peptide (BNP) and have no obvious homology with other protein sequences. (2) Two selected dominant antigens are connected in series and coupled with KLH protein, and the two dominant antigens are used for immunizing mice, so that the immune effect can be effectively enhanced, and the preparation time of the monoclonal antibody can be shortened. (3) And connecting the two selected dominant antigen epitope sequences through flexible fragments (four continuous glycines) respectively and then repeating to form the recombinant canine brain natriuretic peptide protein amino acid sequence. (4) The recombinant protein amino acid sequence is converted into a corresponding nucleotide sequence by adopting an escherichia coli preferred codon, so that the expression quantity of the recombinant protein in escherichia coli can be improved. (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 canine Brain Natriuretic Peptide (BNP) recombinant protein expression vector. (6) And transforming the Escherichia coli ER2566 competent cells by the canine Brain Natriuretic Peptide (BNP) recombinant protein 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, the supernatant of the solution is taken to pass through a nickel agarose affinity chromatography column for affinity chromatography, and the purified canine Brain Natriuretic Peptide (BNP) recombinant protein is obtained by elution. (8) After a Balb/c mouse is immunized for multiple times by using polypeptide coupling KLH protein containing two dominant antigen epitopes of canine Brain Natriuretic Peptide (BNP), spleen of the Balb/c mouse is taken to separate lymphocytes for establishing a single-chain antibody scfv phage display library, and a single-chain antibody scfv sequence capable of being combined with the canine BNP recombinant protein is finally obtained by carrying out multiple rounds of panning and screening. (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 shows that the 4H5 monoclonal antibody coating and the 6D2 monoclonal antibody are labeled and matched to be the best to detect the combination of the canine Brain Natriuretic Peptide (BNP).

Compared with the background technology, the invention adopts the preferred codon of the escherichia coli to optimize the nucleotide sequence corresponding to the recombinant protein, thereby greatly improving the expression level of the recombinant protein in the escherichia coli; secondly, the KLH coupled protein serving as the immunogen only contains two specific dominant antigen epitopes of the canine Brain Natriuretic Peptide (BNP), so that the immune efficiency is improved, the finally obtained monoclonal antibody can be ensured to only specifically recognize the canine Brain Natriuretic Peptide (BNP), and the detection sensitivity is improved; thirdly, the eukaryotic expression canine Brain Natriuretic Peptide (BNP) monoclonal antibody is used for rapidly preparing the monoclonal antibody in a transient transfection mode, which not only can greatly shorten the preparation period, but also ensures the natural activity of the protein through surface modification.

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: dominant epitope selection of canine Brain Natriuretic Peptide (BNP)

Dog Brain Natriuretic Peptide (BNP) is taken as a target antigen, the hydrophilicity and the antigenicity of an antigen epitope sequence of the BNP 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 canine Brain Natriuretic Peptide (BNP); and the A, B epitope has no obvious homology with other protein sequences and only exists in a canine Brain Natriuretic Peptide (BNP) sequence.

Example 2: synthesis of polypeptide containing dominant epitope of canine Brain Natriuretic Peptide (BNP)

In order to enhance the activation effect of the selected epitope on the mouse immune system and shorten the preparation time of the monoclonal antibody, two dominant epitope sequences of kyunonatriuretic peptide A, B are chemically synthesized and connected in series, and cysteine (synthesized by Nanjing Kingsrei Biotech Co., Ltd.) is connected at the tail end to obtain the polypeptide.

Example 3: polypeptide-coupled KLH proteins

20mg of SMCC was dissolved in 2ml of DMF (dimethylformamide), 0.8ml of KLH was added to a 25ml round-bottomed flask, and 1 XPBS (pH7.2) was added thereto to give a final protein concentration of 15 mg/ml. The dissolved SMCC solution was slowly added dropwise to a 120mg KLH protein system and the reaction was stirred at room temperature for 1 hour. Free SMCC was removed by dialysis against 1L of 1 XPBS (pH7.4) solution at 4 ℃ for 6 hours. The KLH protein after dialysis was poured into a 50ml centrifuge tube to give a volume of 20 ml. 417ul of the KLH-SMCC solution was removed and transferred to a 5ml centrifuge tube. 3.0mg of canine BNP polypeptide was dissolved in 0.6ml of 1 XPBS (pH 7.2). And detecting sulfydryl in the polypeptide by using an Ellman reagent, wherein the OD value is 0.18, dripping the polypeptide liquid into a KLH-SMCC tube, and uniformly mixing the polypeptide liquid by using a vertical mixer at room temperature for reacting for 4 hours. The OD value detected by Ellman reagent is 0.02, and the reagent does not show yellow color, so that the canine BNP coupled KLH protein is obtained.

Example 4: tandem connection of dominant epitopes of canine Brain Natriuretic Peptide (BNP)

In order to enhance the stimulation of the selected antigen epitope to the mouse immune system to be beneficial to the subsequent experiment, A, B two dominant antigen epitope sequences of canine Brain Natriuretic Peptide (BNP) are respectively connected through flexible fragments (four continuous glycines) to obtain a recombinant protein amino acid sequence.

Example 5: optimizing nucleotide sequence for coding canine BNP recombinant 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 6: construction of expression vector of canine BNP recombinant protein

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 was ligated with PET-32a (+) vector at a certain ratio at 4 ℃ for 12 hours using T4 ligase (Bao bioengineering, Dalian Co., Ltd.). The ligation product was transformed into DH5 α competent cells (Hangzhou to Biotechnology, Inc.), spread on LB plates containing ampicillin resistance (50 μ g/mL), cultured at 37 ℃ for 12 hours, on which monoclonal strains were picked up to LB liquid medium containing ampicillin resistance (50 μ g/mL), cultured at 37 ℃ for 12 hours on a shaker, and after cultured at 37 ℃ for 12 hours, plasmids were extracted using plasmid purification kits (plasmid extraction and collection kits used in the present invention are all from Hangzhou, Inc., of Seiki Biotechnology), and the correct recombinant expression vector was obtained after BamHI and EcoRI double digestion.

Example 7: construction of canine BNP recombinant protein expression strain

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 ℃. On the next day, the monoclonal strains on the plate 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 a protein electrophoresis sample. And (3) the result of 9% polyacrylamide gel electrophoresis shows that the recombinant protein is successfully expressed to obtain the recombinant canine BNP expression strain.

Example 8: purification of canine BNP recombinant protein

Inoculating the recombinant canine BNP 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 using the LB liquid culture medium containing 50 mu g/mL ampicillin to mix the strain according to the 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 centrifugally collected, carrying out ultrasonic bacteria breaking at a low temperature of 4 ℃, carrying out low-temperature centrifugation, taking the supernatant, passing the supernatant through a nickel-agarose affinity chromatography column, washing and eluting to finally obtain the purified canine BNP recombinant protein.

Example 9: construction of Single chain antibody scfv phage library

Immune: 4-6 weeks old female Balb/c mice were taken, and basal immunization was performed on each mouse by subcutaneous multi-point injection of 100ug of canine BNP polypeptide coupled with KLH protein emulsified in Freund's complete adjuvant for a total of 400 ul/mouse. After 20 days, a second booster immunization was performed by emulsifying 80ug of canine BNP polypeptide coupled with KLH protein with Freund's incomplete adjuvant, and injecting 400 ul/mouse subcutaneously at multiple points. Third boost after 15 days, the procedure was the same as for the second boost.

Preparing scfv: after 20 days, 120ug of canine BNP polypeptide was administered by intraperitoneal injection coupled with KLH protein, after 72 hours, blood was taken from the orbit, and the mice were sacrificed, and their spleens were isolated using a mouse spleen lymphocyte isolation kit (Tianjin Co., Ltd.). Extracting total RNA from the separated lymphocytes by using an RNA extraction kit (Tiangen Biochemical technology Co., Ltd.), carrying out 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 universal degenerate primer of a murine single-chain antibody scfv, respectively carrying out 1% agarose gel electrophoresis on PCR products, respectively cutting gel to recover target genes, and linking the recovered target genes into scfv by overlap PCR.

Constructing a phage antibody library: the PCR product is subjected to 1% agarose gel electrophoresis, after the target gene is cut by NotI and SfiI, the PCR product is cut by gel and recovered, T4 ligase and a pCANTAB5e (Beijing Baokou Weishi Ansheng Biotech limited company) carrier are used for connecting for 12 hours at 4 ℃ according to a certain proportion, the connecting product is recovered by a gel recovery kit to remove the enzyme and buffer substances in the connecting product, the recovered product is electrically transferred into escherichia coli TG1 for multiple times by a bacterial electric converter (biorad) to be electrically transferred into competence, and is coated on a 2 XYT-AG plate containing ampicillin resistance (50 mu g/mL) and 2% glucose, after the product is cultured for 12 hours at the constant temperature of 30 ℃, a proper amount of 2 XYT culture medium is taken, the colony on the plate is completely scraped by a sterile glass rod, and thallus suspension is collected, so that the phage antibody library is constructed.

Example 10: 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 (50ug/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 method comprises the following specific steps:

the purified canine BNP recombinant protein is diluted by coating liquid to 8ug/ml coated immune tubes (Thermo), each immune tube has 4ml, and the immune tubes are coated overnight at 4 ℃.

The next day, discarding the coating solution and unadsorbed antigen, and washing with sterile PBST for 3 times; 5ml of blocking solution was added to each immunization tube and incubated at 37 ℃ for 2 h.

Thirdly, the sealing liquid is discarded, the bacteriophage obtained by PEG precipitation is added into an immune tube after 3 times of aseptic PBST washing, 4ml is added into each immune tube, and the immune tubes are incubated for 1h at 37 ℃.

Fourthly, liquid in the immune tube is discarded, the immune tube is washed for 10 times by sterile PBST, then the immune tube is washed for 10 times by sterile PBS, 1ml of 100mM triethylamine is added to elute the combined phage, and then 500ul of 1M Tris-HCl is added immediately for neutralization with 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.

Fifthly, BNP specificity scfv is enriched after 3 rounds of panning. The phage after the last round of elution and neutralization were infected with TG1 E.coli, spread on 2 XYT-AG plates, and cultured at 30 ℃ for 12 hours.

Sixthly, randomly picking up 600 monoclonal colonies of 400-.

Seventhly, centrifuging to remove supernatant, adding 2 XYT-AK culture medium containing ampicillin resistance (50. mu.g/mL) and kanamycin resistance (50. mu.g/mL) for overnight culture at 30 ℃ and 250 rpm.

Eighthly, carrying out monoclonal ELISA screening on the next day, wherein the screening steps are as follows:

coating: diluting the canine BNP recombinant protein with a coating solution to a final concentration of 1 mu g/mL, adding an enzyme label plate (Shenzhen Jinlau actual Co., Ltd.) into a 100 mu L/well, and washing the protein 1 time by using a washing solution through a DEM-3 type plate washing machine (Daan Gene of Zhongshan university Co., Ltd.) after the mixture is over night 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 the OD value of 10 positive clones is higher, and 5 strains of scfv sequences, 2A9, 4H5, 5C1, 5G3 and 6D2, are obtained by sequencing. The relevant solution formulation is as follows:

coating liquid: na (Na)₂CO₃ 1.5g，NaHCO₃2.9g, plus ddH₂O was added 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, add ddH₂O was metered to 1000mL (pH 7.4).

Color developing solution A: 200mg TMB in 100mL absolute ethanol, ddH₂And 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 ddH₂O is added to 1000 mL.

Example 11: construction of eukaryotic expression vector and transient expression and purification of HEK293F cell

The 5 canine BNP single-chain antibody scfv sequences were respectively constructed into complete murine IgG1 antibody sequences, i.e., the heavy chain variable region and the light chain variable region in scfv were respectively bridged with murine IgG1 heavy chain constant region and light chain constant region by PCR, and then were respectively inserted into pcDNA3.1 (Novagen, Germany) plasmids. 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 an equilibration buffer PBS (pH7.4) to a computer nucleic acid Protein detector (Shanghai Huxi analytical Instrument Co., Ltd.) to show an absorbance of 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 2A9, 4H5, 5C1, 5G3, and 6D 2.

Example 12: preparation of monoclonal antibody fluorescent microsphere pad marked with canine Brain Natriuretic Peptide (BNP)

The concentration of fluorescent microspheres (Bangslab Co.) having a diameter of 190nm was adjusted to 1% with 0.5M MES buffer (pH4.5), and then 5 monoclonal antibodies previously screened were labeled onto the fluorescent microspheres using covalent coupling of 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 13: preparation of nitrocellulose Membrane (NC Membrane)

5 monoclonal antibodies (2a9, 4H5, 5C1, 5G3, 6D2) were individually adjusted 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 the resulting solutions were individually sprayed on an NC membrane 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 14: preparation of fluorescent microsphere immunoassay card

Assembling the test strip: sequentially overlapping and sticking on a PVC bottom plate: (1) NC films which are respectively sprayed with canine Brain Natriuretic Peptide (BNP) recombinant protein monoclonal antibodies (2A9, 4H5, 5C1, 5G3 and 6D2) as detection areas and goat anti-mouse IgG as quality control areas; (2) fluorescent microsphere pads respectively sprayed with fluorescent microsphere labeled canine Brain Natriuretic Peptide (BNP) recombinant protein monoclonal antibodies (2A9, 4H5, 5C1, 5G3 and 6D 2); (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 15: paired monoclonal antibody screening

The canine heart failure clinical serum sample and the normal canine serum sample were loaded at 100. mu.L/well, left at room temperature for 15min, and then the T, C line signals on the NC membrane were read by a fluorescence analyzer (Geneva Biotech Co., Ltd.) and the measured values T/(T + C) were calculated, as detailed in Table 1.

The above table shows that the pairing of the 4H5 monoclonal antibody coating and the 6D2 monoclonal antibody labeled fluorescent microspheres is the optimal antibody pairing of canine BNP.

SEQ ID NO 1: the variable region amino acid sequence of the light chain of a single-chain antibody scfv-4H5 specific to the canine brain natriuretic peptide recombinant protein;

SEQ ID NO 2: the heavy chain variable region amino acid sequence of a single-chain antibody scfv-4H5 specific for the anti-canine natriuretic peptide recombinant protein;

SEQ ID NO 3: the variable region amino acid sequence of a light chain of a single-chain antibody scfv-6D2 specific to the canine natriuretic peptide recombinant protein;

SEQ ID NO 4: the heavy chain variable region amino acid sequence of a single-chain antibody scfv-6D2 specific for the anti-canine natriuretic peptide recombinant protein;

SEQ ID NO 5: the anti-canine brain natriuretic peptide recombinant protein specific single-chain antibody scfv-4H5 light chain variable region nucleotide sequence;

SEQ ID NO 6: a heavy chain variable region nucleotide sequence of a single-chain antibody scfv-4H5 specific for the anti-canine brain natriuretic peptide recombinant protein;

SEQ ID NO 7: the anti-canine brain natriuretic peptide recombinant protein specific single-chain antibody scfv-6D2 light chain variable region nucleotide sequence;

SEQ ID NO 8: the anti-canine brain natriuretic peptide recombinant protein specific single-chain antibody scfv-6D2 heavy chain variable region nucleotide sequence;

sequence listing

<110> Hangzhou xian Zhi Biotechnology Co., Ltd

<120> preparation of canine Brain Natriuretic Peptide (BNP) monoclonal antibody

<160> 8

<170> SIPOSequenceListing 1.0

<210> 1

<211> 108

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Asp Ile Leu Met Thr Gln Thr His Lys Phe Met Ser Thr Ser Val Gly

1 5 10 15

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

20 25 30

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

35 40 45

Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

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

65 70 75 80

Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys Arg

100 105

<210> 2

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

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

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

35 40 45

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

50 55 60

Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser 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

Ala Arg Ser Pro Gly Ser Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ala

115

<210> 3

<211> 112

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

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

1 5 10 15

Gln Ser Val Thr Ile Ser Cys Arg Thr Ser Glu Ser Val Glu Tyr Tyr

20 25 30

Gly Thr Ser Leu Met Gln Trp Tyr Gln Gln Lys Pro Arg Gln Pro Pro

35 40 45

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

50 55 60

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

65 70 75 80

Pro Val Glu Glu Asp Asp Ile Ala Val Tyr Phe Cys Gln Gln Ser Arg

85 90 95

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

100 105 110

<210> 4

<211> 126

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

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

1 5 10 15

Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Tyr

20 25 30

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

35 40 45

Gly Val Met Trp Ala Gly Gly Ile Thr Asn Tyr Asn Leu Ala Leu Met

50 55 60

Ser Arg Leu Thr Ile Thr Lys Asp Ser Ser Lys Thr Gln Val Phe Leu

65 70 75 80

Lys Ile Asn Ser Leu Gln Thr Asp Asp Thr Ala Met Tyr Tyr Cys Ala

85 90 95

Arg Gly Gly Thr Arg Tyr Phe Gly Glu Ser Tyr Tyr Leu Asp Tyr Trp

100 105 110

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

115 120 125

<210> 5

<211> 324

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

gacattttga tgacccaaac tcacaaattc atgtccacat cagtaggaga cagggtcagc 60

atcacctgca aggccagtca ggatgtgagt actgctgtag cctggtatca acagaaacca 120

ggacaatctc ctaaactact gatttactcg gcatcctacc ggtacactgg agtccctgat 180

cgcttcactg gcagtggatc tgggacggat ttcactttca ccatcagcag tgtgcaggct 240

gaagacctgg cagtttatta ctgtcagcaa cattatagta ctccgtacac gttcggaggg 300

gggaccaagc tggagctgaa acgt 324

<210> 6

<211> 348

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

caggtgcagc tgaagcagtc tggggctgaa ctggcaagac ctggggcctc agtgaagatg 60

tcctgcaagg cttctggcta cacctttact agctacacga tgcactgggt aaaacagagg 120

cctggacagg gtctggaatg gattggatac attaatccta gcagtggtta tactaattac 180

aatcagaagt tcaaggacaa ggccacattg actgcagaca aatcctccag cacagcctac 240

atgcaactga gcagcctgac atctgaggac tctgcagtct attactgtgc aagatccccc 300

gggtcgtttg cttactgggg ccaagggact ctggtcactg tctctgcg 348

<210> 7

<211> 336

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

gacatcgtcg tcacacagtc ccccgctagc ctggccgtgt ctctgggcca gtccgtgacc 60

atctcctgtc ggacctctga gtctgtggaa tactacggca cctccctgat gcagtggtac 120

cagcagaagc ctcggcagcc tccaaaactg ctgatcaatg gcgcctccaa cgtggaatcc 180

ggagttcccg cccgcttctc cggctccggc tctggcaccg agttctctct gaacatccat 240

cctgtggaag aggacgacat cgctgtgtac ttctgccagc aatctagaaa ggccccttgg 300

accttcggcg gcggaaccaa gctggatatc aagaga 336

<210> 8

<211> 378

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

caggtccagc tgaaagaatc cggacctggt ctgatggctc cttctcagtc tctgtccatc 60

acctgtaccg tctctggctt cagcctgagc tcctacggcg tgcattgggt gcgccaacct 120

ccaggccaag gactggagtg gctgggcgtg atgtgggccg gcggcatcac caattacaac 180

ctggccctga tgtcccggct gaccatcacc aaggactcct ccaagaccca agtgttcctg 240

aagatcaaca gcctgcagac cgacgacacc gccatgtact actgcgccag aggcggaacc 300

agatacttcg gcgagtctta ctacctggac tactggggcc agggcacccc tctgaccgtg 360

tccagtgcta agacaacg 378

Claims

1. A group of antibodies for a canine brain natriuretic peptide detection kit is characterized in that: comprises a coating antibody and a labeled antibody, wherein the coating antibody is 4H5, and the labeled antibody is 6D 2;

the amino acid sequence of the variable region of the light chain of the coating antibody 4H5 is shown in SEQ ID NO. 1;

the amino acid sequence of the heavy chain variable region of the coating antibody 4H5 is shown as SEQ ID NO. 2;

the amino acid sequence of the variable region of the light chain of the labeled antibody 6D2 is shown in SEQ ID NO. 3;

the heavy chain variable region amino acid sequence of the labeled antibody 6D2 is shown in SEQ ID NO. 4.

2. The antibody for a canine brain natriuretic peptide detection kit of claim 1, wherein: the nucleotide sequence of the coated antibody 4H5 for encoding the light chain variable region is shown in SEQ ID NO.5, and the nucleotide sequence for encoding the heavy chain variable region is shown in SEQ ID NO. 6.

3. The antibody for a canine brain natriuretic peptide detection kit of claim 1, wherein: the nucleotide sequence of the coded light chain variable region of the labeled antibody 6D2 is shown as SEQ ID NO.7, and the nucleotide sequence of the coded heavy chain variable region is shown as 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 a canine brain natriuretic peptide detection 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 an envelope antibody 4H 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 are co-transfected into HEK293F cells, and the marker antibody 6D2 is obtained by expression.