CN114773462B

CN114773462B - Recombinant single-chain antibody for detecting bovine CRP protein and application thereof

Info

Publication number: CN114773462B
Application number: CN202210378339.XA
Authority: CN
Inventors: 包福祥; 吴玘瑶; 王志; 蒋强; 付山; 刘艳龙; 程艺; 张慧敏
Original assignee: Inner Mongolia Agricultural University
Current assignee: Inner Mongolia Agricultural University
Priority date: 2022-04-12
Filing date: 2022-04-12
Publication date: 2023-07-07
Anticipated expiration: 2042-04-12
Also published as: CN114773462A

Abstract

The invention relates to a recombinant single-chain antibody for detecting bovine CRP protein and application thereof. The recombinant single chain antibody for detecting bovine CRP protein comprises C2 antibody and/or C5 antibody, wherein the C2 antibody and the C5 antibody respectively comprise heavy chain variable region (VH) and light chain variable region (VL), and the VH and the VL are connected with peptide S (G) ₄ S) ₃ The VH and VL of the C2 and C5 antibodies, respectively, include framework region FR and complementarity determining region CDR. The antibody of the invention can be used for detecting bovine C-reactive protein and has the advantages of good stability, good specificity, high expression quantity and the like.

Description

Recombinant single-chain antibody for detecting bovine CRP protein and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a recombinant single-chain antibody for detecting bovine CRP protein and application thereof.

Background

One of the C-reactive proteins (CRP), the acute phase protein, is widely distributed in vertebrates, and two different pentapeptide proteins are isolated from bovine serum by calcium-dependent affinity chromatography on high-pyruvate agarose gel. One of these proteins cross-reacts with some rabbit anti-human CRP antisera and is therefore designated bovine CRP. Its biological functions include: the two activities of anti-inflammatory and pro-inflammatory have multiple effects. CRP levels increase dramatically with injury, infection and inflammation and decrease rapidly with remission. CRP is therefore considered as a sensitive indicator for detecting bovine inflammation.

At present, the research on the utilization value of bovine CRP is less at home and abroad, and particularly, no report on recombinant single-chain antibody for detecting bovine CRP protein exists.

Disclosure of Invention

In view of the problems of the prior art, the invention provides a recombinant single-chain antibody for detecting bovine CRP, a preparation method and application thereof, and develops a stable and well-specific antibody for detecting bovine CRP so as to diagnose bovine inflammation. The recombinant single-chain antibody provided by the invention also provides a wide application prospect and has important significance for detection and diagnosis of bovine inflammation and research of bovine CRP.

The technical scheme for solving the technical problems is as follows:

recombinant single chain antibodies for detecting bovine CRP protein, including C2 antibodies and/or C5 antibodies;

the C2 antibody comprises a heavy chain variable region VH and a light chain variable region VL; the C2 antibodies VH and VL include framework region FR and complementarity determining region CDR, respectively;

the FR of the C2 antibody VH comprises an amino acid sequence shown as SEQ ID NO.1, an amino acid sequence shown as SEQ ID NO.3, an amino acid sequence shown as SEQ ID NO.5 and an amino acid sequence shown as SEQ ID NO. 7; the CDR of the C2 antibody VH comprises an amino acid sequence shown in SEQ ID NO.2, an amino acid sequence shown in SEQ ID NO.4 and an amino acid sequence shown in SEQ ID NO. 6;

the FR of the C2 antibody VL comprises an amino acid sequence shown as SEQ ID NO.8, an amino acid sequence shown as SEQ ID NO.10, an amino acid sequence shown as SEQ ID NO.12 and an amino acid sequence shown as SEQ ID NO. 14; the CDR of the VL of the C2 antibody comprises an amino acid sequence shown in SEQ ID NO.9, an amino acid sequence shown in SEQ ID NO.11 and an amino acid sequence shown in SEQ ID NO. 13;

the C5 antibody comprises a heavy chain variable region VH and a light chain variable region VL; c5 antibodies VH and VL each comprise framework regions FR and complementarity determining regions CDR;

the FR of the C5 antibody VH comprises an amino acid sequence shown as SEQ ID NO.17, an amino acid sequence shown as SEQ ID NO.19, an amino acid sequence shown as SEQ ID NO.21 and an amino acid sequence shown as SEQ ID NO. 23; the CDRs of the C5 antibody VH comprise an amino acid sequence shown as SEQ ID NO.18, an amino acid sequence shown as SEQ ID NO.20 and an amino acid sequence shown as SEQ ID NO. 22;

the FR of the C5 antibody VL comprises an amino acid sequence shown as SEQ ID NO.24, an amino acid sequence shown as SEQ ID NO.26, an amino acid sequence shown as SEQ ID NO.28 and an amino acid sequence shown as SEQ ID NO. 30; the CDRs of the C5 antibody VL comprise the amino acid sequence shown in SEQ ID NO.25, the amino acid sequence shown in SEQ ID NO.27 and the amino acid sequence shown in SEQ ID NO. 29.

Further, recombinant single chain antibodies for detecting bovine CRP protein, including C2 antibodies and/or C5 antibodies;

the FR of the C2 antibody VH comprises FR1, FR2, FR3 and FR4, the amino acid sequence of FR1 of the C2 antibody VH comprises the amino acid sequence shown in SEQ ID NO.1, the amino acid sequence of FR2 of the C2 antibody VH comprises the amino acid sequence shown in SEQ ID NO.3, the amino acid sequence of FR3 of the C2 antibody VH comprises the amino acid sequence shown in SEQ ID NO.5, and the amino acid sequence of FR4 of the C2 antibody VH comprises the amino acid sequence shown in SEQ ID NO. 7; the CDR of the C2 antibody VH comprises CDR1, CDR2 and CDR3, the amino acid sequence of the CDR1 of the C2 antibody VH comprises the amino acid sequence shown in SEQ ID NO.2, the amino acid sequence of the CDR2 of the C2 antibody VH comprises the amino acid sequence shown in SEQ ID NO.4, and the amino acid sequence of the CDR3 of the C2 antibody VH comprises the amino acid sequence shown in SEQ ID NO. 6;

the FR of the C2 antibody VL comprises FR1, FR2, FR3 and FR4, the amino acid sequence of FR1 of the C2 antibody VL comprises the amino acid sequence shown in SEQ ID NO.8, the amino acid sequence of FR2 of the C2 antibody VL comprises the amino acid sequence shown in SEQ ID NO.10, the amino acid sequence of FR3 of the C2 antibody VL comprises the amino acid sequence shown in SEQ ID NO.12, and the amino acid sequence of FR4 of the C2 antibody VL comprises the amino acid sequence shown in SEQ ID NO. 14; the CDR of the C2 antibody VL comprises CDR1, CDR2 and CDR3, the amino acid sequence of the CDR1 of the C2 antibody VL comprises the amino acid sequence shown in SEQ ID NO.9, the amino acid sequence of the CDR2 of the C2 antibody VL comprises the amino acid sequence shown in SEQ ID NO.11, and the amino acid sequence of the CDR3 of the C2 antibody VL comprises the amino acid sequence shown in SEQ ID NO. 13;

the FR of the C5 antibody VH comprises FR1, FR2, FR3 and FR4, the amino acid sequence of FR1 of the C5 antibody VH comprises the amino acid sequence shown in SEQ ID NO.17, the amino acid sequence of FR2 of the C5 antibody VH comprises the amino acid sequence shown in SEQ ID NO.19, the amino acid sequence of FR3 of the C5 antibody VH comprises the amino acid sequence shown in SEQ ID NO.21, and the amino acid sequence of FR4 of the C5 antibody VH comprises the amino acid sequence shown in SEQ ID NO. 23; the CDR of the C5 antibody VH comprises CDR1, CDR2 and CDR3, the amino acid sequence of the CDR1 of the C5 antibody VH comprises the amino acid sequence shown in SEQ ID NO.18, the amino acid sequence of the CDR2 of the C5 antibody VH comprises the amino acid sequence shown in SEQ ID NO.20, and the amino acid sequence of the CDR3 of the C5 antibody VH comprises the amino acid sequence shown in SEQ ID NO. 22;

the FR of the C5 antibody VL comprises FR1, FR2, FR3 and FR4, the amino acid sequence of FR1 of the C5 antibody VL comprises the amino acid sequence shown in SEQ ID NO.24, the amino acid sequence of FR2 of the C5 antibody VL comprises the amino acid sequence shown in SEQ ID NO.26, the amino acid sequence of FR3 of the C5 antibody VL comprises the amino acid sequence shown in SEQ ID NO.28, and the amino acid sequence of FR4 of the C5 antibody VL comprises the amino acid sequence shown in SEQ ID NO. 30; the CDR of the C5 antibody VL comprises CDR1, CDR2 and CDR3, the amino acid sequence of CDR1 of the C5 antibody VL comprises the amino acid sequence shown in SEQ ID No.25, the amino acid sequence of CDR2 of the C5 antibody VL comprises the amino acid sequence shown in SEQ ID No.27, and the amino acid sequence of CDR3 of the C5 antibody VL comprises the amino acid sequence shown in SEQ ID No. 29.

For example: the portions of the VH chain of the C2 antibody may be arranged in the following order: FR1 (SEQ ID NO. 1) of C2 antibody VH to CDR1 (SEQ ID NO. 2) of C2 antibody VH to FR2 (SEQ ID NO. 3) of C2 antibody VH to CDR2 (SEQ ID NO. 4) of C2 antibody VH to FR3 (SEQ ID NO. 5) of C2 antibody VH to CDR3 (SEQ ID NO. 6) of C2 antibody VH to FR4 (SEQ ID NO. 7) of C2 antibody VH; the portions of the VL chain of the C2 antibody may be arranged in the following order: FR1 (SEQ ID NO. 8) of C2 antibody VL to CDR1 (SEQ ID NO. 9) of C2 antibody VL to FR2 (SEQ ID NO. 10) of C2 antibody VL to CDR2 (SEQ ID NO. 11) of C2 antibody VL to FR3 (SEQ ID NO. 12) of C2 antibody VL to CDR3 (SEQ ID NO. 13) of C2 antibody VL to FR4 (SEQ ID NO. 14) of C2 antibody VL.

The portions of the C5 antibody VH chain may be arranged in the following order: FR1 (SEQ ID NO. 17) of C5 antibody VH to CDR1 (SEQ ID NO. 18) of C5 antibody VH to FR2 (SEQ ID NO. 19) of C5 antibody VH to CDR2 (SEQ ID NO. 20) of C5 antibody VH to FR3 (SEQ ID NO. 21) of C5 antibody VH to CDR3 (SEQ ID NO. 22) of C5 antibody VH to FR4 (SEQ ID NO. 23) of C5 antibody VH; the portions of the VL chain of the C5 antibody may be arranged in the following order: FR1 of C5 antibody VL (SEQ ID NO. 24) -CDR 1 of C5 antibody VL (SEQ ID NO. 25) -FR 2 of C5 antibody VL (SEQ ID NO. 26) -CDR 2 of C5 antibody VL (SEQ ID NO. 27) -FR 3 of C5 antibody VL (SEQ ID NO. 28) -CDR 3 of C5 antibody VL (SEQ ID NO. 29) -FR 4 of C5 antibody VL (SEQ ID NO. 30).

The beneficial effects of adopting the technical scheme include:

the antibody provided by the invention is obtained by immunizing a Balb/c mouse with prokaryotic expression bovine CRP protein, extracting cDNA (complementary deoxyribonucleic acid) obtained by total RNA reverse transcription from spleen cells of the mouse, performing PCR (polymerase chain reaction) amplification, establishing a recombinant single-chain antibody library, and specifically screening the antibody library by a phage display technology. The C2 antibody and the C5 antibody have high binding force with antigen, have the advantages of good stability, good specificity, high expression quantity and the like, and can be used for detecting bovine CRP so as to develop detection, diagnosis and research reagents and products for bovine inflammation.

In addition to the sequences described above, the C2 antibodies and C5 antibodies may also include other sequences.

Further, VH and VL are linked by a flexible linker peptide.

For example, the flexible connecting peptide may be flexible connecting peptide S (G ₄ S) _n Wherein n is an integer, and the flexible linking peptide may be a flexible linkingPeptide S (G) ₄ S) ₃ Other flexible connecting peptides are also possible.

Further, the nucleotide sequence of the C2 antibody comprises the nucleotide sequence shown as SEQ ID NO. 15;

and/or the amino acid sequence of the C2 antibody comprises the amino acid sequence shown in SEQ ID No. 16;

and/or the nucleotide sequence of the C5 antibody comprises the nucleotide sequence shown as SEQ ID NO. 31;

and/or the amino acid sequence shown in SEQ ID NO.32.

The beneficial effects of adopting the technical scheme include:

the recombinant single-chain antibody gene is transferred into escherichia coli, a recombinant single-chain antibody strain which is efficiently expressed in the escherichia coli is established, the gene sequence of each recombinant single-chain antibody strain is analyzed by utilizing sequence comparison software, and finally, the amino acid sequences of two recombinant single-chain antibodies aiming at bovine CRP proteins are obtained. The antibody of the sequence has the advantages of good stability, good specificity, high expression quantity and the like.

The present invention provides a nucleic acid encoding the above antibody.

Further, the nucleotide sequence of the nucleic acid includes the nucleotide sequence shown as SEQ ID NO. 15;

and/or a sequence in which one or several nucleotides are added, substituted, deleted or inserted into the nucleotide sequence shown in SEQ ID NO. 15;

and/or comprises the nucleotide sequence shown as SEQ ID NO. 31;

and/or a sequence in which one or several nucleotides are added, substituted, deleted or inserted in the nucleotide sequence shown in SEQ ID NO. 31.

The invention provides a vector comprising the nucleic acid. The vector can be a cloning vector or an expression vector. The cloning vector carries nucleic acid encoding the antibody, so that the purpose of replication and amplification can be achieved. Expression elements on the expression vectors may express the antibodies described above.

The invention provides a host cell comprising the vector. The purpose of nucleic acid replication, amplification, expression and the like of the antibody carried on the vector can be realized through propagation of host cells.

The invention provides a reagent for detecting bovine CRP protein, which comprises the antibody.

The invention provides a kit comprising the reagent.

The kit may further comprise: ELISA coating liquid, BSA, HRP labeled Anti-HA-tag antibody, TMB chromogenic liquid and stop reaction liquid.

The invention provides application of one or more of the antibodies, nucleic acids, vectors, host cells, reagents and kits in detecting bovine CRP protein.

The invention provides application of one or more of the antibodies, nucleic acids, vectors, host cells, reagents and kits in preparing a reagent or a kit for diagnosing inflammation of cattle.

For example: the application of one or more of the above antibodies, nucleic acids, vectors, host cells, reagents and kits in preparing reagents or kits for diagnosing inflammation related to bovine CRP protein.

For example, one or a combination of several of the above antibodies, nucleic acids, vectors, host cells, reagents, kits can be used in the preparation of reagents or kits for detecting bovine CRP proteins, and can also be used in the preparation of reagents for detecting, diagnosing, studying bovine inflammation and products thereof. Has the advantages of good stability, good specificity and the like.

The combination of one or more of the antibodies, nucleic acids, vectors, host cells, reagents and kits can be applied to the preparation of a reagent or a kit for detecting bovine CRP protein and the preparation of a reagent or a kit for diagnosing bovine inflammation based on the principle that the C2 antibody and the C5 antibody can be specifically combined with bovine CRP protein respectively.

In application, ELISA and other methods can be used for detecting the combination of the single-chain antibody provided by the invention and the bovine CRP protein, thereby detecting the bovine CRP protein and inflammation related to the bovine CRP protein.

Drawings

FIG. 1 is an SDS-PAGE electrophoresis chart of a prokaryotic expression system for expression of bovine CRP protein gene and purification by nickel column affinity chromatography in example 1, M is a protein molecular weight marker, lane 1: prokaryotic expression of bovine CRP protein;

FIG. 2 is an electrophoresis chart of a recombinant single-chain antibody gene fragment added with HA-tag by a PCR method in example 4, M is a DNA molecular weight marker, lane 1: c2 single chain antibody, lane2: c5 single chain antibodies;

FIG. 3 is an SDS-PAGE electrophoresis of a bovine CRP specific recombinant single-chain antibody purified by nickel column affinity chromatography in example 4, M is a protein molecular weight marker, lane 1: c2 single chain antibody, lane2: c5 single chain antibodies;

FIG. 4 is a schematic illustration of the ELISA method for identifying specific binding of recombinant single chain antibodies to bovine CRP protein in example 5.

Detailed Description

The principles and features of the present invention are described below with reference to the drawings, the examples are illustrated for the purpose of illustrating the invention and are not to be construed as limiting the scope of the invention.

The pMECS plasmid was given a benefit from the university of brucella free, belgium Serge Muyldermans. pET-22b vector and pET-25b vector plasmids were maintained by the laboratory where the inventors were located.

The above materials are available only to the public for repeating the experiments described in the examples of the present invention for non-commercial purposes only to demonstrate the effects described in the examples.

Coli Transetta (DE 3) competence was purchased from Beijing full gold Biotechnology Co. Ni-NTA filler was purchased from Sangon Biotech. TMB color development was purchased from Promega Biotechnology. HRP-labeled Anti HA-tag antibodies were purchased from south kyo gold strey biotechnology limited. NotI and NcoI restriction endonucleases were purchased from New England Biolabs. E.coli TG1 competent cells, TG1 bacteria, helper phage M13K07 were purchased from GE company. ELISA coating solution, ELISA stop solution, ampicillin, kanamycin, BSA, PEG-8000, PBS buffer solution and triethylamine were purchased from Soy Corp.

According to the gene sequence of the bovine CRP, the gene encoding the mature peptide protein of the bovine CRP is synthesized by a chemical synthesis method, and restriction endonucleases NcoI and NotI sites are designed and added at the upstream and downstream of the gene fragment, so that the gene is synthesized by Sangon Biotech company.

The instruments, reagent materials, etc. according to the examples are conventional instruments, reagents, materials, etc. existing in the prior art, and are commercially available in a normal manner unless otherwise specified. The experimental methods, detection methods, and the like in the examples described below are conventional experimental methods, detection methods, and the like that are known in the prior art unless otherwise specified.

The invention discloses two recombinant single-chain antibodies for detecting bovine CRP protein and a preparation method thereof. According to the invention, prokaryotic expression bovine CRP protein is adopted to immunize a Balb/c mouse, spleen cells of the Balb/c mouse are used as antibody gene sources, cDNA obtained by total RNA reverse transcription is extracted from the spleen cells of the mouse, and a recombinant single-chain antibody library is established after PCR amplification. And (3) establishing a single-chain antibody library aiming at CRP, coating the expressed and purified bovine CRP protein on an ELISA plate to serve as an antigen, and specifically screening the antibody library by a phage display technology. The exogenous DNA sequence is integrated into phage genes in phage display technology, so that the infection characteristic of phage is maintained, and target protein is expressed on the surface of phage. The desired DNA fragment is integrated into the gene of M13 phage coat protein iii or coat protein viii, providing multivalent display by processing and packaging of the phage itself, using the method of major coat protein iii. The recombinant single-chain antibody with high binding force between the gene sequences of the C2 and C5 single-chain antibodies and the antigen is obtained, the gene sequences of the single-chain antibody strains are analyzed, and the amino acid sequences of the recombinant scFv chains aiming at the bovine CRP are respectively shown as SEQ ID NO.16 and SEQ ID NO.32. The antibody disclosed by the invention has good stability and specificity, and can be used for detecting bovine CRP.

EXAMPLE 1 construction of a specific Single-Domain heavy chain phage antibody library

(1) Bovine CRP gene (GeneBank Sequence ID: AB 255049.1) was ligated with prokaryotic expression vector (pET-25 b plasmid) and transformed into E.coli (Transetta (DE 3) competent cells) for induction expression under conditions including: according to the inoculation amount of 1 percent, the bacterial liquid is inoculated into an induction culture medium,culturing at 37deg.C and 300rpm to obtain bacterial liquid OD ₆₀₀ When the temperature reaches between 0.6 and 0.8, the temperature is reduced to 30 ℃,220rpm, and the low-temperature induction expression is carried out for 10 hours; and then the Ni-NTA filler is used for purifying the expressed recombinant protein, and the figure 1 is the identification result of the prokaryotic expression purified bovine CRP protein SDS-PAGE.

(2) For primary immunization, 50 mug of bovine CRP protein expressed in the prophase and the equivalent volume of Freund's complete adjuvant (namely, the volume ratio is 1:1) are placed in a vortex oscillator to be fully oscillated and emulsified, and mice (Balb/c mice, purchased from Si Bei Fu (Beijing) biotechnology Co., ltd.) are injected into the abdominal cavity.

Subsequent immunization was performed using 50 μg of bovine CRP protein expressed from the prophase prokaryotes in combination with freund's incomplete adjuvant at 14 days intervals for a total of 5 immunizations. The method of immunization is that mice are used for intraperitoneal injection, and the immunization method is the same.

(3) After euthanasia of mice, spleen of the mice is taken for grinding, the mice are dissolved in PBS buffer solution, total RNA of the mice is extracted by a Trizol method, the total RNA is reversely transcribed into cDNA, the cDNA is taken as a template, VH and VL genes of the mice antibodies are amplified by PCR, and single-chain antibody gene scFv fragments are obtained by SOE-PCR amplification.

(4) After double restriction of NotI and NcoI restriction enzymes, a single-chain antibody gene fragment is connected with a pMECS plasmid vector which is subjected to the same restriction enzyme, a connection product (the fragment size is about 750 bp) is transformed into E.coli TG1 competent cells to construct a CRP specific phage antibody library, and the library capacity of the antibody library is determined to be 1.3x10 ⁸ 。

EXAMPLE 2 enrichment and screening of CRP Single chain antibodies

1) Inoculating the constructed CRP specific phage antibody library bacterial liquid into 100mL 2 XYT liquid culture medium according to the inoculation amount of 1%, shaking and culturing for 2h at 37 ℃ at 250rpm, adding helper phage M13K07 (the phage infection amount of Escherichia coli is 20 times of that of Escherichia coli), and shaking and culturing for 1h at 37 ℃ at 250 rpm.

2 XYT liquid medium: weighing 7.5g of yeast extract, 3.75g of NaCl and 12g of tryptone, adding 600ml of distilled water, fully stirring and dissolving, fixing the volume to 750ml, dripping NaOH, adjusting the pH value to 7.0, sealing, sterilizing at high pressure for 25min, and preserving at 4 ℃.

(2) Placing the cultured bacterial liquid at 4 ℃ for 2000rpm for 10min; the supernatant was discarded, and the cells were resuspended and allowed to settle into 40mL of 2 XYT liquid medium (ampicillin-containing 100. Mu.g/mL and kanamycin-containing 100. Mu.g/mL), and cultured overnight at 37℃and 220 rpm.

(3) Subpackaging the bacterial liquid into a centrifuge tube, centrifuging for 25min at 4 ℃ and 7197g, adding 20% PEG-8000/NaCl into the supernatant, precipitating recombinant phage, and standing for 30min.

(4) Phage 4 ℃,7197g, centrifugal 25min, removing supernatant, drying, using 1mL PBS buffer solution (PBS for short) to re-suspend sediment, namely phage antibody library. Phage antibody library was added to an ELISA plate pre-coated with CRP protein at 15. Mu.g/mL, sealed with sealing film, and bound for 1h at 37 ℃.

(5) After 5 times of PBST washing, 100. Mu.L of 100mM triethylamine elution buffer was added to each well and incubated at room temperature for 10min. Reinfecting the eluted recombinant phage with TG1 bacteria, adding auxiliary phage for saving (the number ratio of phage to bacteria is 20:1), and repeating the same enrichment and screening steps for five times to obtain recombinant phage infection E.coli TG1.

EXAMPLE 3PHAGE-ELISA screening of bovine CRP protein-specific phages

(1) The recombinant phage infects E.coli TG1 and is spread on 2 XYT-AG solid medium (Amp and glucose are added in the 2 XYT solid medium formula, amp 100. Mu.g/mL, glucose 2%), inverted in a 37 ℃ incubator, and cultured overnight.

(2) Randomly picking 92 monoclonals, inoculating 400 mul of 2 XYT/AG liquid culture medium, adding diluted 20 times auxiliary phage M13K07, shaking and culturing at 37 ℃ and 250rpm for 2 hours, centrifuging, discarding supernatant, drying, resuspending the precipitate with 400 mul of 2 XYT/AG liquid culture medium, shaking and culturing for 16 hours, centrifuging, taking 300 mul of supernatant, adding 120 mul of 20% PEG/NaCl, fully mixing, and standing at 4 ℃ for 30 minutes;

(3) After 30min, centrifuge at 4deg.C and 10000rpm for 20min, discard supernatant, drain, re-suspend precipitate with 100 μLPBS for Phage ELISA identification;

(4) Adding 50 mu L of PBS heavy suspension into an ELISA plate pre-coated with 5 mu g/mL CRP protein, and combining for 2 hours at room temperature, wherein a negative control hole is helper phage M13K07, and a blank control is PBS;

(5) Adding 100 mu L of TMB color development liquid into each hole after PBST washing, developing color for 10min in dark, adding 50 mu L of ELISA stop solution, measuring OD405 nm value, and judging positive when the OD405 nm value of the test group exceeds the negative control value by more than 2.1 times;

(6) Positive clones were inoculated into 5mL LB liquid containing 100. Mu.g/mL ampicillin to extract plasmids and sequenced;

analyzing the gene sequences of all clone strains according to the sequence comparison software DNAStar, regarding the strains with the same gene sequence as the same clone strain, regarding the strains with different sequences as different clone strains, and finally screening to obtain 2 single-chain antibodies which are respectively named as C2 antibody and C5 antibody;

in the C2 antibody, the heavy chain (VH) and the light chain (VL) of the antibody are connected with the peptide S (G) through flexibility ₄ S) ₃ And (5) connection. The gene sequence of the C2 antibody VH chain includes: FR1 (SEQ ID NO. 1) of C2 antibody VH to CDR1 (SEQ ID NO. 2) of C2 antibody VH to FR2 (SEQ ID NO. 3) of C2 antibody VH to CDR2 (SEQ ID NO. 4) of C2 antibody VH to FR3 (SEQ ID NO. 5) of C2 antibody VH to CDR3 (SEQ ID NO. 6) of C2 antibody VH to FR4 (SEQ ID NO. 7) of C2 antibody VH; the gene sequence of the VL chain of the C2 antibody includes: FR1 (SEQ ID NO. 8) of C2 antibody VL to CDR1 (SEQ ID NO. 9) of C2 antibody VL to FR2 (SEQ ID NO. 10) of C2 antibody VL to CDR2 (SEQ ID NO. 11) of C2 antibody VL to FR3 (SEQ ID NO. 12) of C2 antibody VL to CDR3 (SEQ ID NO. 13) of C2 antibody VL to FR4 (SEQ ID NO. 14) of C2 antibody VL; the gene sequence of the scFv of the whole C2 antibody is shown as SEQ ID NO.15, and the amino acid sequence is shown as SEQ ID NO. 16.

In the C5 antibody, the heavy chain (VH) and the light chain (VL) of the antibody are connected with the peptide S (G) through flexibility ₄ S) ₃ And (5) connection. The gene sequence of the C5 antibody VH chain includes: FR1 (SEQ ID NO. 17) of C5 antibody VH to CDR1 (SEQ ID NO. 18) of C5 antibody VH to FR2 (SEQ ID NO. 19) of C5 antibody VH to CDR2 (SEQ ID NO. 20) of C5 antibody VH to FR3 (SEQ ID NO. 21) of C5 antibody VH to CDR3 (SEQ ID NO. 22) of C5 antibody VH to FR4 (SEQ ID NO. 23) of C5 antibody VH; the gene sequence of the VL chain of the C5 antibody includes: FR1 (SEQ ID NO. 24) of C5 antibody VL to CDR1 (SEQ ID NO. 25) of C5 antibody VL to FR2 (SEQ ID NO. 26) of C5 antibody VL to C5 antibodyCDR2 of the bulk VL (SEQ ID NO. 27) -FR 3 of the C5 antibody VL (SEQ ID NO. 28) -CDR 3 of the C5 antibody VL (SEQ ID NO. 29) -FR 4 of the C5 antibody VL (SEQ ID NO. 30); the gene sequence of the scFv of the whole C5 antibody is SEQ ID NO.31, and the amino acid sequence thereof is SEQ ID NO.32.

EXAMPLE 4 expression and purification of bovine CRP protein recombinant Single chain antibody

(1) Positive clones from the results of the Phage ELISA of example 3 were inoculated in 5mL of 2 XYT-A broth (100. Mu.g/mL Amp added to 2 XYT broth) at an inoculum size of 1%, respectively, and cultured overnight.

(2) The next day, plasmid extraction and gene sequencing are carried out on the cultured bacterial liquid, after sequence analysis, single-chain antibody gene fragments are cut off from plasmids by utilizing restriction endonucleases NotI and NcoI, a single-chain antibody gene is taken as a template, HA-tag is added on the fragments by a PCR method, and FIG. 2 is a result diagram of the addition of the HA-tag to the single-chain antibody gene fragments, and then pET-22b vectors are connected to obtain connection products;

(3) Converting the connection product obtained in the step (2) into E.coli Transetta (DE 3) competent, performing low-temperature induced expression at 30 ℃ for 12 hours, then performing ultrasonic disruption on bacterial liquid, collecting supernatant and precipitate, performing SDS-PAGE analysis on the expression condition, purifying the disrupted supernatant by using Ni-NTA nickel column packing, collecting purified single-chain antibodies, wherein FIG. 3 is the purified single-chain antibodies, namely a C2 single-chain antibody and a C5 single-chain antibody;

EXAMPLE 5ELISA identification of recombinant Single-chain antibodies

(1) Coating antigen: prokaryotic expression of CRP protein was diluted to 20. Mu.g/ml using ELISA coating solution, 100. Mu.L per well, overnight at 4 ℃. Standing at room temperature for 30min; removing liquid in the holes, beating the holes on paper, adding 300 mu L of PBST into each hole, vibrating and washing at 37 ℃ and 220rpm for 3 minutes, and repeating washing for 5 times;

(2) 300 mu L of 3% BSA is added to each well, the mixture is placed at 37 ℃ for 1.5 hours and washed;

(3) Diluting the single-chain antibody protein to 20 mug/ml, and incubating at 37 ℃ for 1.5h with 100 mu L of each well; e.coli disruption solution transformed with pET-22b empty vector was used as negative control, and PBS was used as blank control.

(4) 100 μl 1:500 dilution of HRP-labeled Anti-HA-tag antibody was added per well. Incubating for 1h at 37 ℃;

(5) Adding 100 mu L of TMB color development liquid into each hole, and developing color in dark for 10min;

(6) 50. Mu.L of ELISA stop solution was added to each well, and the OD450nm value was measured. And judging whether the test sample is combined with CRP protein according to the measured OD450nm value, judging positive (namely, can be combined with CRP protein) if the OD450nm value of the test group exceeds the OD450nm value of the negative control by more than 2.1 times, and judging negative (namely, can not be combined with CRP protein) if the OD450nm value of the test group is less than the OD450nm value of the negative control by 2.1 times.

Fig. 4 is a result of the ELISA to identify the binding activity of C2 and C5 antibodies to bovine CRP protein, and it can be seen from fig. 4 that C2 antibodies and C5 antibodies can bind to bovine CRP protein, and that the specific binding activity of C5 antibodies to bovine CRP protein is higher than that of C2 antibodies.

According to the invention, prokaryotic expression bovine CRP protein is adopted to immunize a Balb/c mouse, spleen cells of the Balb/c mouse are used as antibody gene sources, and a recombinant single-chain antibody library aiming at CRP is established. The antibody obtained by the general hybridoma method has complex and time-consuming operation process, and the invention directly obtains the single-chain antibody of the mouse from the lymphocyte of the mouse by a three-cycle PCR method, so that the operation is simple and the time consumption is less. Coating the expressed and purified bovine CRP protein on an ELISA plate to be used as a screening antigen, performing immune screening on the recombinant single-chain antibody library by utilizing a phage display technology to obtain recombinant single-chain antibody genes aiming at the bovine CRP protein, and obtaining nucleotide and amino acid sequences of the recombinant single-chain antibody genes by sequencing. Transferring the recombinant single-chain antibody genes into escherichia coli, establishing recombinant single-chain antibody strains which are efficiently expressed in the escherichia coli, and analyzing the gene sequence of each recombinant single-chain antibody strain by utilizing sequence comparison software. Finally, two amino acid sequences of the recombinant single-chain antibody aiming at the bovine CRP protein are obtained. The method can prepare a large amount of single-chain antibodies with higher purity in an escherichia coli expression system. After the single-chain antibody is identified by ELISA method, the single-chain antibody has better binding activity to bovine CRP protein. Experiments prove that the single-chain antibody prepared by the invention has good stability and specificity; and also proves that the recombinant single-chain antibody can be specifically combined with bovine CRP protein, and can be applied to detection of bovine CRP protein and diagnosis of bovine inflammation.

Although not exhaustive, those skilled in the art can choose from the prior art.

The foregoing disclosure is merely illustrative of specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art will readily appreciate variations or substitutions within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

Sequence listing

<110> university of inner Mongolia agriculture

<120> recombinant single-chain antibody for detecting bovine CRP protein and use thereof

<160> 32

<170> SIPOSequenceListing 1.0

<210> 1

<211> 25

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<230> FR1 of C2 antibody VH

<400> 1

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

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser

20 25

<210> 2

<211> 8

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<230> CDR1 of C2 antibody VH

<400> 2

Gly Tyr Thr Phe Pro Ser Tyr Trp

1 5

<210> 3

<211> 17

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<230> FR2 of C2 antibody VH

<400> 3

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

1 5 10 15

Glu

<210> 4

<211> 8

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<230> CDR2 of C2 antibody VH

<400> 4

Ile Asp Pro Ser Asp Asn Tyr Thr

1 5

<210> 5

<211> 38

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<230> FR3 of C2 antibody VH

<400> 5

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

1 5 10 15

Ser Ser Ser Thr Ala Tyr Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp

20 25 30

Thr Ala Val Tyr Tyr Cys

35

<210> 6

<211> 12

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<230> CDR3 of C2 antibody VH

<400> 6

Ala Arg Ser Ile Leu Arg Pro Tyr Tyr Phe Asp Tyr

1 5 10

<210> 7

<211> 11

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<230> FR4 of C2 antibody VH

<400> 7

Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

1 5 10

<210> 8

<211> 26

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<230> FR1 of C2 antibody VL

<400> 8

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

1 5 10 15

Glu Lys Val Thr Met Thr Cys Ser Ala Ser

20 25

<210> 9

<211> 5

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<230> CDR1 of C2 antibody VL

<400> 9

Ser Ser Val Ser Tyr

1 5

<210> 10

<211> 17

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<230> FR2 of C2 antibody VL

<400> 10

Met His Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile

1 5 10 15

Tyr

<210> 11

<211> 3

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<230> CDR2 of C2 antibody VL

<400> 11

Asp Thr Ser

1

<210> 12

<211> 36

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<230> FR3 of C2 antibody VL

<400> 12

Asn Leu Ala Pro Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly

1 5 10 15

Asn Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Gly Glu Asp Ala Ala

20 25 30

Thr Tyr Tyr Cys

35

<210> 13

<211> 9

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<230> CDR3 of C2 antibody VL

<400> 13

Gln Gln Trp Asn Ser Tyr Pro Phe Thr

1 5

<210> 14

<211> 11

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<230> FR4 of C2 antibody VL

<400> 14

Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg

1 5 10

<210> 15

<211> 726

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<230> C2 antibody scFv

<400> 15

caggtcaaac tgcaggagtc tggggctgcg cttgtgaagc ctggggcttc agtgaagttg 60

tcctgcaagg cttctggcta caccttcccc agctactgga tgcagtgggt gaggcagagg 120

cctggacaag gccttgagtg ggtcggagag attgatcctt ctgataatta tactaactac 180

aatcaaaagt tcaagggcaa ggccacactg actgtagaca catcctccag cacagcctac 240

ctgcagctca gcagcctgac atctgaggac actgccgtct attactgtgc aagatctata 300

ctacggccgt actactttga ctactggggc caagggacca cggtcaccgt ctcctcatca 360

ggtggaggcg gttctggcgg aggtggctca ggcggtggag gctcggacat tgagctcacc 420

cagtctccag caatcatgtc tgcatctcca ggggagaagg tcaccatgac ctgcagtgcc 480

agctcaagtg taagttacat gcactggtac cagcagaagt caggcacctc ccccaaaaga 540

tggatttatg acacatccaa cctggctcct ggagtcccag ctcgcttcag tggcagtggg 600

tctgggaact cttattctct cacaatcagc agcatggagg gtgaagatgc tgccacttat 660

tactgccagc agtggaatag ttacccattc acgttcggtg ctggcaccaa gttggaactc 720

aaacgg 726

<210> 16

<211> 242

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<230> C2 antibody scFv

<400> 16

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

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Pro Ser Tyr

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Ser Ile Leu Arg Pro Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Val Thr Val Ser Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly

115 120 125

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

130 135 140

Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Ser Ala

145 150 155 160

Ser Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Ser Gly Thr

165 170 175

Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Asn Leu Ala Pro Gly Val

180 185 190

Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Asn Ser Tyr Ser Leu Thr

195 200 205

Ile Ser Ser Met Glu Gly Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln

210 215 220

Trp Asn Ser Tyr Pro Phe Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu

225 230 235 240

Lys Arg

<210> 17

<211> 25

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<230> FR1 of C5 antibody VH

<400> 17

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

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser

20 25

<210> 18

<211> 8

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<230> CDR1 of C5 antibody VH

<400> 18

Gly Tyr Thr Phe Thr Ser Tyr Trp

1 5

<210> 19

<211> 17

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<230> FR2 of C5 antibody VH

<400> 19

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

1 5 10 15

Tyr

<210> 20

<211> 8

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<230> CDR2 of C5 antibody VH

<400> 20

Ile Asn Pro Ser Thr Gly Tyr Thr

1 5

<210> 21

<211> 38

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<230> FR3 of C5 antibody VH

<400> 21

Glu Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Val Asp Arg

1 5 10 15

Ser Ser Asn Thr Ala Tyr Leu His Leu Ser Ser Leu Thr Ser Glu Asp

20 25 30

Thr Ala Val Tyr Phe Cys

35

<210> 22

<211> 12

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<230> CDR3 of C5 antibody VH

<400> 22

Ala Arg Arg Gly Tyr Tyr Ser Pro Trp Phe Ala Tyr

1 5 10

<210> 23

<211> 11

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<230> FR4 of C5 antibody VH

<400> 23

Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

1 5 10

<210> 24

<211> 26

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<230> FR1 of C5 antibody VL

<400> 24

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

1 5 10 15

Glu Lys Val Thr Ile Thr Cys Ser Ala Ser

20 25

<210> 25

<211> 5

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<230> CDR1 of C5 antibody VL

<400> 25

Ser Ser Val Ser Tyr

1 5

<210> 26

<211> 17

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<230> FR2 of C5 antibody VL

<400> 26

Met His Trp Phe Gln Gln Lys Pro Gly Thr Ser Pro Lys Leu Trp Ile

1 5 10 15

Tyr

<210> 27

<211> 3

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<230> CDR2 of C5 antibody VL

<400> 27

Ser Thr Ser

1

<210> 28

<211> 36

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<230> FR3 of C5 antibody VL

<400> 28

Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly

1 5 10 15

Asn Ser Tyr Ser Leu Thr Ile Ser Arg Met Glu Ala Glu Asp Ala Ala

20 25 30

Thr Tyr Tyr Cys

35

<210> 29

<211> 9

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<230> CDR3 of C5 antibody VL

<400> 29

Gln Gln Arg Ser Ser Tyr Pro Phe Thr

1 5

<210> 30

<211> 11

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<230> FR4 of C5 antibody VL

<400> 30

Phe Gly Ser Gly Thr Lys Leu Glu Met Lys Arg

1 5 10

<210> 31

<211> 726

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<230> C5 antibody scFv

<400> 31

caggtccagc tgcaggagtc aggggctgag ctggtgaggc ctgggtcctc agtgaagatg 60

tcctgcaagg cttctggcta cacctttact agctactgga tgcactgggt aaaacagagg 120

cctggacagg gtctggaatg gattggatac attaatccta gcactggtta tactgagtac 180

aatcagaagt tcaaggacaa ggccacattg actgttgaca gatcttccaa cacagcctac 240

ctgcacctca gcagcctgac atctgaggac actgccgtct atttctgtgc aaggaggggg 300

tactatagtc cctggtttgc ttactggggc caagggacca cggtcaccgt ctcctcatca 360

ggtggaggcg gttctggcgg aggtggctca ggcggtggag gctcggacat tgagctcacc 420

cagtctccag caatcatgtc tgcatctcca ggggagaagg tcaccataac ctgcagtgcc 480

agctcaagtg taagttacat gcactggttc cagcagaagc caggcacttc tcccaaactc 540

tggatttata gcacatccaa cctggcctct ggagtccctg ctcgcttcag tggcagtggg 600

tctgggaact cttattctct cacaatcagc cgaatggagg ctgaagatgc tgccacttat 660

tactgccagc aaaggagtag ttacccattc acgttcggct cggggaccaa gctggagatg 720

aaacgg 726

<210> 32

<211> 242

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<230> C5 antibody scFv

<400> 32

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Lys Asp Lys Ala Thr Leu Thr Val Asp Arg Ser Ser Asn Thr Ala Tyr

65 70 75 80

Leu His Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Arg Gly Tyr Tyr Ser Pro Trp Phe Ala Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Val Thr Val Ser Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly

115 120 125

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

130 135 140

Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Ile Thr Cys Ser Ala

145 150 155 160

Ser Ser Ser Val Ser Tyr Met His Trp Phe Gln Gln Lys Pro Gly Thr

165 170 175

Ser Pro Lys Leu Trp Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val

180 185 190

Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Asn Ser Tyr Ser Leu Thr

195 200 205

Ile Ser Arg Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln

210 215 220

Arg Ser Ser Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Met

225 230 235 240

Lys Arg

Claims

1. Recombinant single chain antibody for detecting bovine CRP protein, characterized in that it comprises a C2 antibody and/or a C5 antibody;

the FR of the C2 antibody VH comprises FR1, FR2, FR3 and FR4, the amino acid sequence of FR1 of the C2 antibody VH is shown as SEQ ID NO.1, the amino acid sequence of FR2 of the C2 antibody VH is shown as SEQ ID NO.3, the amino acid sequence of FR3 of the C2 antibody VH is shown as SEQ ID NO.5, and the amino acid sequence of FR4 of the C2 antibody VH is shown as SEQ ID NO. 7; the CDR of the C2 antibody VH comprises CDR1, CDR2 and CDR3, the amino acid sequence of the CDR1 of the C2 antibody VH is shown as SEQ ID NO.2, the amino acid sequence of the CDR2 of the C2 antibody VH is shown as SEQ ID NO.4, and the amino acid sequence of the CDR3 of the C2 antibody VH is shown as SEQ ID NO. 6;

the FR of the C2 antibody VL comprises FR1, FR2, FR3 and FR4, the amino acid sequence of FR1 of the C2 antibody VL is shown as SEQ ID NO.8, the amino acid sequence of FR2 of the C2 antibody VL is shown as SEQ ID NO.10, the amino acid sequence of FR3 of the C2 antibody VL is shown as SEQ ID NO.12, and the amino acid sequence of FR4 of the C2 antibody VL is shown as SEQ ID NO. 14; the CDR of the C2 antibody VL comprises a CDR1, a CDR2 and a CDR3, the amino acid sequence of the CDR1 of the C2 antibody VL is shown as SEQ ID NO.9, the amino acid sequence of the CDR2 of the C2 antibody VL is shown as SEQ ID NO.11, and the amino acid sequence of the CDR3 of the C2 antibody VL is shown as SEQ ID NO. 13;

the FR of the C5 antibody VH comprises FR1, FR2, FR3 and FR4, the amino acid sequence of FR1 of the C5 antibody VH is shown as SEQ ID NO.17, the amino acid sequence of FR2 of the C5 antibody VH is shown as SEQ ID NO.19, the amino acid sequence of FR3 of the C5 antibody VH is shown as SEQ ID NO.21, and the amino acid sequence of FR4 of the C5 antibody VH is shown as SEQ ID NO. 23; the CDR of the C5 antibody VH comprises CDR1, CDR2 and CDR3, the amino acid sequence of the CDR1 of the C5 antibody VH is shown as SEQ ID NO.18, the amino acid sequence of the CDR2 of the C5 antibody VH is shown as SEQ ID NO.20, and the amino acid sequence of the CDR3 of the C5 antibody VH is shown as SEQ ID NO. 22;

the FR of the C5 antibody VL comprises FR1, FR2, FR3 and FR4, the amino acid sequence of FR1 of the C5 antibody VL is shown as SEQ ID NO.24, the amino acid sequence of FR2 of the C5 antibody VL is shown as SEQ ID NO.26, the amino acid sequence of FR3 of the C5 antibody VL is shown as SEQ ID NO.28, and the amino acid sequence of FR4 of the C5 antibody VL is shown as SEQ ID NO. 30; the CDR of the C5 antibody VL comprises CDR1, CDR2 and CDR3, the amino acid sequence of the CDR1 of the C5 antibody VL is shown as SEQ ID NO.25, the amino acid sequence of the CDR2 of the C5 antibody VL is shown as SEQ ID NO.27, and the amino acid sequence of the CDR3 of the C5 antibody VL is shown as SEQ ID NO. 29.

2. The antibody of claim 1, wherein VH and VL are linked by a flexible linker peptide.

3. The antibody of claim 1 or 2, wherein the nucleotide sequence of the C2 antibody comprises the nucleotide sequence shown in SEQ ID No. 15;

and/or the amino acid sequence of the C5 antibody comprises the amino acid sequence shown in SEQ ID NO.32.

4. A nucleic acid encoding the antibody of any one of claims 1-3.

5. The nucleic acid of claim 4, wherein the nucleotide sequence of the nucleic acid comprises the nucleotide sequence set forth in SEQ ID NO. 15;

and/or comprises the nucleotide sequence shown as SEQ ID NO. 31.

6. A vector comprising the nucleic acid of claim 4 or 5.

7. A host cell comprising the vector of claim 6.

8. A reagent for detecting bovine CRP protein comprising the antibody of any one of claims 1-3.

9. A kit comprising the reagent of claim 8.

10. Use of one or a combination of several of the antibodies of any one of claims 1-3, the nucleic acid of claim 4 or 5, the vector of claim 6, the host cell of claim 7, the reagent of claim 8, the kit of claim 9 in (1) or (2);

(1) Preparing a reagent or a kit for detecting bovine CRP protein;

(2) Preparing a reagent or a kit for diagnosing inflammation of cattle.