CN114163521A

CN114163521A - Monoclonal antibody for identifying hog cholera virus 2.1 subtype virulent strain and antibody thereof

Info

Publication number: CN114163521A
Application number: CN202111215602.5A
Authority: CN
Inventors: 潘春根; 黄攀; 张俊; 赵玉林; 王宁
Original assignee: Guangdong Haid Animal Husbandry And Veterinary Research Institute Co ltd; GUANGDONG HAID GROUP CO Ltd
Current assignee: Guangdong Haid Animal Husbandry And Veterinary Research Institute Co ltd; GUANGDONG HAID GROUP CO Ltd
Priority date: 2021-10-19
Filing date: 2021-10-19
Publication date: 2022-03-11
Anticipated expiration: 2041-10-19
Also published as: CN114163521B

Abstract

The invention belongs to the technical field of biology, and discloses a monoclonal antibody for identifying a hog cholera virus subtype 2.1 virulent strain and an antibody thereof. The monoclonal antibody can be specifically combined with a classical swine fever virus subtype 2.1 virulent strain, but is not combined with a classical swine fever virus vaccine strain, can be used for detecting classical swine fever virus subtype 2.1 virulent strain E2 protein, detecting classical swine fever virus subtype 2.1 virulent strain and identifying classical swine fever virus subtype 2.1 virulent strain and a classical swine fever virus vaccine strain C, and can also be used for identifying antibodies respectively aiming at the classical swine fever virus subtype 2.1 virulent strain and the classical swine fever virus vaccine strain C.

Description

Monoclonal antibody for identifying hog cholera virus 2.1 subtype virulent strain and antibody thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a monoclonal antibody for identifying a hog cholera virus subtype 2.1 virulent strain and an antibody thereof.

Background

Classical Swine Fever (CSF) is an acute, thermal and highly contagious infectious disease caused by Classical Swine Fever Virus (CSFV), which is classified as a type of infectious disease in China and specified by the world animal health Organization (OIE) as one of the animal infectious diseases that must be declared, in many Swine-breeding countries of the world, with different degrees of prevalence. Classical swine fever viruses have only one serotype, which can be divided into subtypes based on the gene encoding the virus. The currently popular subtypes in China are mostly 2.1 subtypes and 2.2 subtypes, wherein the 2.1 subtypes can be subdivided into four types of 2.1a, 2.1b, 2.1c, 2.1d and the like. The virulent strain of classical swine fever virus isolated in the 20 th century in China is named Shimen strain and belongs to the 1.1 subtype. The Shimen strain is continuously passaged on rabbit bodies to weaken the virulence thereof, and the vaccine strain widely used at present, also called hog cholera virus lapinized attenuated vaccine strain (or C strain vaccine strain) is obtained. The C strain vaccine strain has high cross reaction with Shimen strain and other subtype virulent strains in serology, and the positive classical swine fever virus antibodies which can not be clinically distinguished from swine herds in the prior art, such as an ELISA method for detecting the antibody level of the swine herds, are from virulent strain infection (the virulent strain in China is usually the Shimen strain of classical swine fever virus) or from immune vaccine C strain. In addition, the C-strain vaccine is widely used by swine herds in China, so that the swine fever antibody is always in a higher level, thereby causing great trouble for purification of swine fever virulent viruses. However, primers are designed according to the difference of conserved sequences of viruses, and the genotype of the classical swine fever virus is directly identified by using fluorescent quantitative PCR (polymerase chain reaction), so that the method is only suitable for detecting that a swinery is in a virus active stage or cannot identify the swinery with viruses at the late infection stage or recessive infection when the swinery is expelling the toxins. Therefore, the development of a detection method capable of distinguishing the hog cholera virus subtype 2.1 virulent strain, the C strain vaccine strain and the antibody thereof has important significance for the pig industry.

Disclosure of Invention

The first aspect of the present invention aims at providing a monoclonal antibody for identifying a virulent strain of classical swine fever virus subtype 2.1 and a vaccine strain C and/or an antibody of the virulent strain of classical swine fever virus subtype 2.1 and a vaccine strain C.

It is an object of the second aspect of the present invention to provide a nucleic acid molecule encoding the monoclonal antibody of the first aspect of the present invention.

It is an object of the third aspect of the invention to provide an expression cassette, a recombinant vector, a recombinant bacterium or a transgenic cell comprising the nucleic acid molecule of the second aspect of the invention.

The fourth aspect of the present invention is directed to the use of the monoclonal antibody of the first aspect of the present invention, the nucleic acid molecule of the second aspect of the present invention, or the expression cassette, recombinant vector, recombinant bacterium or transgenic cell of the third aspect of the present invention.

The fifth aspect of the present invention is to provide a hybridoma cell line.

In a sixth aspect, the present invention provides a kit comprising the monoclonal antibody of the first aspect of the present invention and/or the hybridoma cell line of the fifth aspect of the present invention.

It is an object of a seventh aspect of the invention to provide an in vitro method of non-diagnostic purpose.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

the first aspect of the invention provides a monoclonal antibody for identifying a virulent strain of classical swine fever virus subtype 2.1 and a vaccine strain C and/or an antibody of the virulent strain of classical swine fever virus subtype 2.1 and a vaccine strain C, wherein the monoclonal antibody is MM1 or MM 5;

the MM1 is specifically combined with an epitope formed by amino acid sequences at the 30 th to 39 th positions of the hog cholera virus subtype 2.1 virulent strain E2 protein;

the MM5 is specifically combined with an epitope formed by 19-23 amino acid sequences of hog cholera virus subtype 2.1 virulent strain E2 protein;

the amino acid sequence of the classical swine fever virus subtype 2.1 virulent strain E2 protein is shown in SEQ ID NO. 1.

Preferably, the MM1 specifically binds to an epitope consisting of amino acids 30, 35 and 39 of a virulent strain E2 protein of swine fever virus subtype 2.1;

the MM5 is specifically combined with an epitope consisting of 19 th and 23 th amino acids of the hog cholera virus subtype 2.1 virulent strain E2 protein.

Preferably, the MM1 comprises a heavy chain variable region and a light chain variable region;

the heavy chain variable region comprises CDR1, CDR2, CDR 3;

the amino acid sequence of the heavy chain variable region CDR1 is:

a) GYTFSRYW (SEQ ID NO. 47); or

b) An amino acid sequence which is shown in SEQ ID NO.47 and has the same or similar functions after being modified by substitution, deletion or addition of one or more amino acids;

the amino acid sequence of the heavy chain variable region CDR2 is:

a) ILPGSGSTN (SEQ ID NO. 48); or

b) The amino acid sequence shown in SEQ ID NO.48 is the amino acid sequence which is modified by substitution, deletion or addition of one or more amino acids and has the same or similar functions;

the amino acid sequence of the heavy chain variable region CDR3 is:

a) ASITTKAWFAY (SEQ ID NO. 49); or

b) An amino acid sequence which is shown in SEQ ID NO.49 and has the same or similar functions after being modified by substitution, deletion or addition of one or more amino acids;

the light chain variable region comprises CDR1, CDR2, CDR 3;

the amino acid sequence of the light chain variable region CDR1 is:

a) KSVSTS (SEQ ID NO. 50); or

b) The amino acid sequence shown in SEQ ID NO.50 is the amino acid sequence which is modified by substitution, deletion or addition of one or more amino acids and has the same or similar functions;

the amino acid sequence of the light chain variable region CDR2 is:

a) LVS (SEQ ID NO. 51); or

b) The amino acid sequence shown in SEQ ID NO.51 is the amino acid sequence which is modified by substitution, deletion or addition of one or more amino acids and has the same or similar functions;

the amino acid sequence of the light chain variable region CDR3 is:

a) QHIRELTRS (SEQ ID NO. 52); or

b) And the amino acid sequence shown in SEQ ID NO.52 is the amino acid sequence which is modified by substitution, deletion or addition of one or more amino acids and has the same or similar functions.

Preferably, the MM5 comprises a heavy chain variable region and a light chain variable region;

the heavy chain variable region comprises CDR1, CDR2, CDR 3;

the amino acid sequence of the heavy chain variable region CDR1 is:

a) GYTFTSYW (SEQ ID NO. 53); or

b) The amino acid sequence shown in SEQ ID NO.53 is the amino acid sequence which is modified by substitution, deletion or addition of one or more amino acids and has the same or similar functions;

the amino acid sequence of the heavy chain variable region CDR2 is:

a) IDPSDSYTR (SEQ ID NO. 54); or

b) The amino acid sequence shown in SEQ ID NO.54 is the amino acid sequence which is modified by substitution, deletion or addition of one or more amino acids and has the same or similar functions;

the amino acid sequence of the heavy chain variable region CDR3 is:

a) TREVVTPFDY (SEQ ID NO. 55); or

b) An amino acid sequence which is shown in SEQ ID NO.55 and has the same or similar functions after being modified by substitution, deletion or addition of one or more amino acids;

the light chain variable region comprises CDR1, CDR2, CDR 3;

the amino acid sequence of the light chain variable region CDR1 is:

a) KSVSTS (SEQ ID NO. 56); or

b) The amino acid sequence shown in SEQ ID NO.56 is the amino acid sequence which is modified by substitution, deletion or addition of one or more amino acids and has the same or similar functions;

the amino acid sequence of the light chain variable region CDR2 is:

a) LVS (SEQ ID NO. 57); or

b) An amino acid sequence which is shown in SEQ ID NO.57, is modified by substitution, deletion or addition of one or more amino acids and has the same or similar functions;

the amino acid sequence of the light chain variable region CDR3 is:

a) QHIRELTRS (SEQ ID NO. 58); or

b) And the amino acid sequence shown in SEQ ID NO.58 is the amino acid sequence which is modified by substitution, deletion or addition of one or more amino acids and has the same or similar functions.

the amino acid sequence of the heavy chain variable region is as follows:

a) an amino acid sequence shown as SEQ ID NO. 43; or

b) An amino acid sequence which is shown in SEQ ID NO.43 and has the same or similar functions after being modified by substitution, deletion or addition of one or more amino acids;

the amino acid sequence of the light chain variable region is as follows:

a) an amino acid sequence shown as SEQ ID NO. 44; or

b) And the amino acid sequence shown in SEQ ID NO.44 is the amino acid sequence which is modified by substitution, deletion or addition of one or more amino acids and has the same or similar functions.

the amino acid sequence of the heavy chain variable region is as follows:

a) an amino acid sequence shown as SEQ ID NO. 45; or

b) An amino acid sequence which is shown in SEQ ID NO.45 and has the same or similar functions after being modified by substitution, deletion or addition of one or more amino acids;

the amino acid sequence of the light chain variable region is as follows:

a) an amino acid sequence shown as SEQ ID NO. 46; or

b) And the amino acid sequence shown in SEQ ID NO.46 is the amino acid sequence which is modified by substitution, deletion or addition of one or more amino acids and has the same or similar functions.

Preferably, the MM1 is deposited at the Guangdong provincial microorganism culture collection center of No. 59 building 5 building of No. 100 college of Mieli Zhou, Guangzhou city on 24/9/2021, with the collection number being GDMCC NO: 61945, and the hybridoma cell strain BALB/c mouse hybridoma MM 1.

Preferably, the MM5 is deposited at the Guangdong provincial microorganism culture collection center of No. 59 building 5 building of No. 100 college of Mieli Zhou, Guangzhou city on 24/9/2021, with the collection number being GDMCC NO: 61946, and the hybridoma cell strain BALB/c mouse hybridoma MM 5.

Preferably, the MM1 belongs to the IgG2a subtype and the light chain is the kappa subtype.

Preferably, the MM5 belongs to the IgG1 subtype and the light chain is the kappa subtype.

In a second aspect of the invention, there is provided a nucleic acid molecule encoding a monoclonal antibody of the first aspect of the invention.

Preferably, when the monoclonal antibody is MM1, the nucleic acid molecule comprises: nucleic acid molecule a encoding the heavy chain variable region of MM1 and nucleic acid molecule B encoding the light chain variable region of MM 1;

the nucleotide sequence of the nucleic acid molecule A is shown as SEQ ID NO. 39; the nucleotide sequence of the nucleic acid molecule B is shown as SEQ ID NO. 40.

Preferably, when the monoclonal antibody is MM5, the nucleic acid molecule comprises: nucleic acid molecule C encoding the heavy chain variable region of MM5 and nucleic acid molecule D encoding the light chain variable region of MM 5;

the nucleotide sequence of the nucleic acid molecule C is shown as SEQ ID NO. 41; the nucleotide sequence of the nucleic acid molecule D is shown as SEQ ID NO. 42.

In a third aspect of the invention, there is provided an expression cassette, recombinant vector, recombinant bacterium or transgenic cell comprising the nucleic acid molecule of the second aspect of the invention.

Preferably, the transgenic cell does not comprise propagation material.

In a fourth aspect of the invention, there is provided use of a monoclonal antibody of the first aspect of the invention, a nucleic acid molecule of the second aspect of the invention, or an expression cassette, recombinant vector, recombinant bacterium or transgenic cell of the third aspect of the invention.

(1) Use of any one of (a) to (3) in any one of (c) to (p):

(1) a monoclonal antibody of the first aspect of the invention;

(2) a nucleic acid molecule of the second aspect of the invention;

(3) an expression cassette, recombinant vector, recombinant bacterium or transgenic cell of the third aspect of the invention;

(c) detecting the hog cholera virus subtype 2.1 virulent strain E2 protein in a non-diagnostic destination;

(d) preparing a product for detecting the hog cholera virus subtype 2.1 virulent strain E2 protein;

(e) detecting a virulent strain of classical swine fever virus subtype 2.1 in a non-diagnostic destination;

(f) preparing a product for detecting the virulent strain of the hog cholera virus subtype 2.1;

(g) identifying a hog cholera virus 2.1 subtype virulent strain and a C strain vaccine strain in a non-diagnosis destination;

(h) preparing a product for identifying the hog cholera virus 2.1 subtype virulent strain and the C strain vaccine strain;

(i) detecting a virulent strain E2 protein antibody of the subtype 2.1 of the classical swine fever virus in a non-diagnostic destination;

(j) preparing a product for detecting the hog cholera virus subtype 2.1 virulent strain E2 protein antibody;

(k) antibodies for detecting virulent strains of subtype 2.1 of hog cholera virus in a non-diagnostic destination;

(l) Preparing a product for detecting an antibody of a hog cholera virus subtype 2.1 virulent strain;

(m) identifying the classical swine fever virus subtype 2.1 virulent strain E2 protein antibody and the C strain vaccine strain E2 protein antibody in a non-diagnostic destination;

(n) preparing a product for identifying the hog cholera virus subtype 2.1 virulent strain E2 protein antibody and the C strain vaccine strain E2 protein antibody;

(o) identifying the hog cholera virus subtype 2.1 virulent strain antibody and the C strain vaccine strain antibody in a non-diagnostic destination;

(p) preparing a product for identifying the hog cholera virus subtype 2.1 virulent strain antibody and the C strain vaccine strain antibody.

Preferably, the amino acid sequence of the hog cholera virus subtype 2.1 virulent strain E2 protein is shown in SEQ ID NO. 1.

Preferably, the amino acid sequence of the C strain vaccine strain E2 protein is shown in SEQ ID NO. 3.

In a fifth aspect of the present invention, there is provided a hybridoma cell line, wherein any one of (q) to (r):

(q) the name "BALB/c mouse hybridoma MM 1", deposited at 24.9.2021 in Guangdong province collection of microorganisms in Lou 5 of Middledo 100, Middledo, Guangzhou city, with the deposit number GDMCC NO: 61945, respectively;

(r) named as "BALB/c mouse hybridoma MM 5", deposited at 24.9.2021 in Guangdong province microbial culture collection center of No. 59 building 5 of Mirabilitum 100, Mirabilitum, Guangzhou city, with the collection number GDMCC NO: 61946.

in a sixth aspect of the present invention, there is provided a kit comprising the monoclonal antibody of the first aspect and/or the hybridoma cell line of the fifth aspect of the present invention.

In a seventh aspect of the invention, there is provided an in vitro method of non-diagnostic interest, comprising the step of using a monoclonal antibody of the first aspect of the invention or a hybridoma cell line of the fifth aspect of the invention or a kit of the sixth aspect of the invention;

the in vitro method is any one of(s) to (y):

(s) detecting the hog cholera virus subtype 2.1 virulent strain E2 protein;

(t) detecting a virulent strain of the subtype 2.1 of the hog cholera virus;

(u) identifying the hog cholera virus 2.1 subtype virulent strain and the C strain vaccine strain;

(v) detecting hog cholera virus subtype 2.1 virulent strain E2 protein antibody;

(w) detecting an antibody of a virulent strain of classical swine fever virus subtype 2.1;

(x) Identifying a hog cholera virus subtype 2.1 virulent strain E2 protein antibody and a C strain vaccine strain E2 protein antibody;

(y) identifying the antibody of the hog cholera virus subtype 2.1 virulent strain and the antibody of the vaccine strain C.

Preferably, the amino acid sequence of the hog cholera virus virulent strain E2 protein is shown in SEQ ID NO. 1.

The invention has the beneficial effects that:

the invention provides a monoclonal antibody for identifying a classical swine fever virus subtype 2.1 virulent strain and a vaccine strain C and/or a classical swine fever virus subtype 2.1 virulent strain antibody and a vaccine strain C, wherein the monoclonal antibody can be specifically combined with a classical swine fever virus subtype 2.1 virulent strain but not combined with the vaccine strain C, and can be used for detecting a classical swine fever virus subtype 2.1 virulent strain E2 protein, detecting a classical swine fever virus subtype 2.1 virulent strain, a classical swine fever virus subtype 2.1 virulent strain and a vaccine strain C, detecting a classical swine fever virus subtype 2.1 virulent strain E2 protein antibody, detecting a classical swine fever virus subtype 2.1 virulent strain E2 protein antibody, identifying a classical swine fever virus subtype 2.1 virulent strain E2 protein antibody, and identifying a classical swine fever virus subtype virulent strain 2.1 virulent strain antibody and a vaccine strain C antibody.

Drawings

FIG. 1 is a schematic diagram of the design of the classical swine fever virus E2 recombinant protein and a graph showing the results of the recombinant protein expressed by insect cells in example 1: wherein, A is the complete genome of the classical swine fever virus and the structural schematic diagram of the E2 protein coded by the complete genome: the hog cholera virus subtype 2.1 virulent strain E2 protein (E2ab-C/S-2.1) and the C strain vaccine strain E2 protein (E2ab-C strain) comprise ab fragment of E2, and then His labels are respectively added for purifying the protein by a nickel column affinity chromatography, wherein a cysteine residue is mutated from E2ab-C/S-2.1 to be serine; b is the amino acid sequence comparison chart of E2ab fragments of hog cholera virus subtype 2.1 virulent strain, C strain vaccine strain and Shimen strain: the numbers in the figure represent the number of amino acids; c is the SDS electrophoresis and immunoblotting result of the recombinant protein.

FIG. 2 is a graph showing the results of the indirect ELISA assay for identifying the specificity of monoclonal antibodies (MM1, MM2, MM3, MM4, MM5, MM6, MM8, MM9, MM10, MM11, MM12, MM13, MM14, MM15, MM17, MM18, MM19, MM20, MM21, MM22) in example 1.

FIG. 3 is a graph showing the results of subtype identification of monoclonal antibodies (MM1, MM5, MM18, MM20) in example 1: wherein A is a result graph of the subtype of the monoclonal antibody in example 1; b is a graph of the subtype results of the monoclonal antibody light chain in example 1; c is the electrophoresis result of the monoclonal antibody in the non-denatured electrophoresis gel and the denatured electrophoresis gel in example 1: the monoclonal antibody appears as one IgG macromolecule in the non-denaturing electrophoresis gel, and the IgG molecule is broken down into two protein bands, heavy and light, in the denaturing electrophoresis, indicating that the monoclonal antibody has been successfully purified and has the correct molecular weight.

FIG. 4 is a graph showing the results of the specific recognition sites of the monoclonal antibodies (MM1, MM5) in classical swine fever virus E2 in example 2: wherein, A is a polypeptide fragment designed on the basis of the amino acid sequence of E2ab-C/S-2.1, E2ab-C strain and the E2ab fragment of Shimen strain and a position diagram on E2 ab; b is an amino acid sequence diagram of a polypeptide fragment designed on the basis of the amino acid sequence of the E2ab-C/S-2.1, E2ab-C strain and the amino acid sequence of the E2ab fragment of the Shimen strain; c is a graph of recognition results of different polypeptide fragments detected by MM1 and MM5 in an indirect ELISA experiment.

FIG. 5 is a graph of the results of the antigenic determinants recognized by the monoclonal antibodies (MM1, MM5) in classical swine fever virus E2 in example 2: wherein A is the amino acid of the polypeptide 2.1-1 of the virulent strain of the subtype 2.1 of the classical swine fever virus, and the result chart of the comparison of the sequences of 3 mutants 2.1-1-A, 2.1-1-B and 2.1-1-C of the polypeptide 2.1-1 and the amino acid sequence of the polypeptide C-1 from the vaccine strain of the strain C; b is a graph of the recognition result of indirect ELISA test detection MM5 on 2.1-1-A, 2.1-1-B and 2.1-1-C; c is an amino acid of the polypeptide 2.1-2 of the hog cholera virus subtype 2.1 virulent strain, and a result chart of the comparison of the 2 mutant 2.1-2-A and 2.1-2-B sequences of the polypeptide 2.1-2 and the amino acid sequence of the polypeptide C-2 derived from the vaccine strain of the strain C; d is a graph of the recognition result of the indirect ELISA test detection MM1 on 2.1-2-A and 2.1-2-B; e is a comparison result chart of the amino acid sequence of the polypeptide 2.1-2 of the hog cholera virus subtype 2.1 virulent strain and the amino acid sequences of 2.1-2-C, 2.1-2-D and 2.1-2-E; f is a graph of the recognition result of MM1 on 2.1-2-C, 2.1-2-D and 2.1-2-E detected by indirect ELISA experiment.

FIG. 6 is a graph showing the results of the ability of MM1 and MM5 to identify currently domestic circulating strains.

FIG. 7 is a graph showing the results of a competition ELISA to verify the specific recognition ability of MM1 and MM5 for classical swine fever virus subtype 2.1 virulent strain E2: wherein, A is a result graph of verifying the specificity recognition capability of MM1 and MM5 on rabbit serum of classical swine fever virus subtype 2.1 virulent strain E2 by competitive ELISA; b is a result graph of the competitive ELISA for verifying the specific recognition capability of the MM1 and MM5 on the standard swine fever positive serum of the vaccine strain C; p < 0.001.

FIG. 8 is a graph showing the results of gene amplification of the variable regions of monoclonal antibodies MM1 and MM 5: wherein A is a position diagram of variable region structures of mouse monoclonal antibody heavy chain and light chain genes and a primer group for PCR amplification on the genes; b is the gel electrophoresis image of the gene fragments of the heavy and light chains of monoclonal antibodies MM1 and MM 5.

Detailed Description

The present invention will be described in further detail with reference to specific examples.

It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. The materials, reagents and the like used in the present examples are commercially available reagents and materials unless otherwise specified.

EXAMPLE 1 preparation of monoclonal antibody for identifying virulent strains of subtype 2.1 of hog cholera virus

1. Expression of recombinant proteins

According to the design scheme A in FIG. 1, the genes encoding classical swine fever virus E2 (classical swine fever virus subtype 2.1 virulent strain E2 protein, strain C vaccine strain E2 protein) were inserted into the plasmid pFastBac in Bac-to-Bac insect baculovirus system (from Invitrogen) via the restriction enzyme sites BamHI and XhoI, respectively^TM1 inThen according to Bac-to-

Baculovir Expression System the recombinant viruses were packaged step by step using the instruction manual, and 2 recombinant proteins were expressed and purified after infecting insect cells H5. The specific method for protein purification is as follows:

(1) well-grown H5 cells were diluted to 40mL at a concentration of 3X 10⁶Per mL;

(2) inoculating the recombinant baculovirus, inoculating the recombinant baculovirus with an MOI value of 1, 27-28 ℃, and performing shake culture at 180 revolutions per minute;

(3) after culturing for 48 hours, observing the cell state every day, and when the cells become round and large and die, centrifugally collecting the cells;

(4) the cell pellet was added to 20mL PBS buffer (0.01M, Cat. No. P1010, available from Soley) and mixed by vortexing with 200. mu.L PMSF (100 ×) (10 mg/mL);

(5) transferring the mixed cell suspension into a 25mL beaker, and carrying out cell ultrasonication (amplitude 60A for 10min) under the ice bath condition;

(6) after the cell disruption is finished, transferring the cell disruption solution to a 50mL centrifuge tube, and centrifuging for 15min at 10000 rpm;

(7) taking 15mg of the inclusion body protein, adding 10mL of inclusion body denaturation liquid (Tris, 20 mM; NaCl, 500 mM; DTT, 1 mM; guanidine hydrochloride, 6M, pH 8.0) to ensure that the protein concentration is changed to 1.5mg/mL, and fully dissolving;

(8) the inclusion body was diluted with a renaturation solution (Tris, 20 mM; NaCl, 500 mM; 6M, pH 8.0) until the guanidine hydrochloride concentration became 1.5M, and the mixture was stirred while diluting, and then filtered through a 0.22 μ M filter.

(9) Using a 10KD 15mL ultrafiltration tube (

Ultra-15) concentration was carried out by centrifugation in a horizontal rotor at 4000g, and dilution was continued until the concentration of guanidine hydrochloride became 0.075M and the final volume after concentration was 500. mu.L.

(10) Transferring to a 1.5mL centrifuge tube, centrifuging at 12000rpm for 5min, collecting supernatant, measuring the concentration and storing.

The amino acid sequence of the classical swine fever virus subtype 2.1 virulent strain E2 protein (E2ab-C/S-2.1) is as follows: MSCKEDYRYAISSTNEIGPLGAEGLTTTWREYSHGLQLDDGTVRAICTAGSFKVIALTVVSRRYLASLHKRALPTSVTFELLFDGTSPAIEEMGDDFGFGLSPFDTTPVVKGKYNTTLLNGSAFYLVCPIGWTGVIECTAVSPTTLRTEVVKTFKREKPFPHHHHHHHG (SEQ ID NO.1), wherein the black underlined amino acid residue "S" is mutated by "C", and HHHHHHHG (SEQ ID NO.2) is a His tag; the amino acid sequence of the C strain vaccine strain E2 protein (E2ab-C strain) is as follows: MACKEDYRYAISSTDEIGLLGAGGLTTTWKEYNHDLQLNDGTVKASCVAGSFKVTALNVVSRRYLASLHKKALPTSVTFELLFDGTNPSTEEMGDDFRSGLCPFDTSPVVKGKYNTTLLNGSAFYLVCPIGWTGVIECTAVSPTTLRTEVVKTFRRDKPFPHHHHHHHG (SEQ ID NO.3), wherein HHHHHHHG (SEQ ID NO.2) is a His tag; SDS electrophoresis and immunoblotting were performed (primary antibody was a mouse anti-His monoclonal antibody purchased from Biotechnology engineering (Shanghai) Co., Ltd., cat # D191001; secondary antibody was HRP-labeled goat anti-mouse IgG purchased from Invitrogen, cat # 62-6520), and the results are shown in C in FIG. 1. In fig. 1, B aligns the a and B segments of E2 from different virus strains, compared with C strain vaccine strain and phylum strain virulent strain, the 2.1 subtype virulent strain has a plurality of different amino acids, and is mainly distributed in the first half: 2.1 subtype virulent strain sequence is derived from a strain with the number of KU375260.1 in GenBank, a vaccine strain sequence of a C strain is derived from a strain with the number of AF091507 in GenBank, and a virulent strain sequence of a Shimen strain is derived from a strain with the number of AF092448.2 in GenBank.

The gene sequence of the protein for coding the hog cholera virus subtype 2.1 virulent strain E2 is as follows: ATGTCCTGCAAGGAGGACTACCGCTACGCTATCTCCTCCACCAACGAGATCGGTCCTCTGGGTGCTGAAGGTCTGACTACCACCTGGCGCGAGTATTCCCATGGTCTGCAGCTGGACGATGGTACCGTGCGCGCTATTTGTACTGCTGGTTCCTTCAAGGTGATCGCTCTGAACGTGGTGTCCCGTCGTTATCTGGCTTCCCTGCATAAACGCGCTCTGCCTACTTCTGTGACCTTCGAGCTGCTGTTCGACGGTACCTCCCCTGCTATCGAGGAGATGGGTGACGACTTCGGTTTCGGTCTGTCCCCTTTCGACACTACCCCTGTGGTGAAGGGTAAGTACAACACCACCCTGCTGAACGGTTCCGCTTTCTACCTGGTGTGCCCTATCGGTTGGACCGGTGTGATCGAATGCACCGCTGTGTCCCCTACTACTCTGCGTACCGAAGTGGTGAAGACCTTCAAGCGCGAGAAGCCTTTCCCTCACCACCACCATCATCACCATGGTTAA (SEQ ID NO.4), the gene coding the C strain vaccine strain E2 protein is: ATGGCCTGCAAGGAAGATTACAGGTACGCAATATCGTCAACCGATGAGATAGGGCTACTTGGGGCCGGAGGTCTCACCACCACCTGGAAGGAATACAACCACGATTTGCAACTGAATGACGGGACCGTCAAGGCCAGTTGCGTGGCAGGTTCCTTTAAAGTCACAGCACTTAATGTGGTCAGTAGGAGGTATTTGGCGTCATTGCATAAGAAGGCTTTACCCACTTCCGTGACATTCGAGCTCCTGTTCGACGGGACCAACCCATCAACTGAGGAAATGGGAGATGACTTCAGGTCCGGGCTGTGCCCGTTTGATACGAGTCCTGTTGTTAAGGGAAAGTACAATACGACCTTGTTGAACGGTAGTGCTTTCTATCTTGTCTGCCCAATAGGGTGGACGGGTGTCATAGAGTGCACAGCAGTGAGCCCAACAACTCTGAGGACAGAAGTGGTAAAGACCTTCAGGAGAGACAAGCCCTTTCCGCACCACCACCATCATCACCATGGATAA (SEQ ID NO. 5).

2. Preparation of monoclonal antibodies

Emulsifying the recombinant protein E2ab-C/S-2.1 obtained in the step 1 with Freund' S adjuvant, injecting female Balb/C mice with 3-week interval, injecting once with 50 micrograms of recombinant protein each time, and immunizing 3 times in total. Blood is collected at 3 weeks after the last immunization to measure the serum titer, and the titer is selected to reach 1: 6400 or more, spleen cells of mice were taken and fused with mouse myeloma cells sp20, and hybridoma cell lines were prepared according to a conventional method. The indirect ELISA method is used for selecting cell strains with higher antibody titer aiming at E2ab-C/S-2.1 for further subcloning, and 20 monoclonal antibodies screened in total have better capability of recognizing E2ab-C/S-2.1 protein and are respectively named as: MM1, MM2, MM3, MM4, MM5, MM6, MM8, MM9, MM10, MM11, MM12, MM13, MM14, MM15, MM17, MM18, MM19, MM20, MM21, and MM 22. Then, the monoclonal antibody in the culture supernatant of the monoclonal cell strain is purified by using protein A, and the monoclonal cell strain is frozen.

3. Screening monoclonal antibody in indirect ELISA experiment

Monoclonal antibodies were screened by indirect ELISA assay: coating a 96-well plate (polystyrene plate (NUNC) with 5 micrograms per milliliter of recombinant proteins E2ab-C/S-2.1 and E2ab-C strain and 10 micrograms per milliliter of synthetic polypeptide (polypeptide sequences are shown as SEQ ID NO. 6-20 and SEQ ID NO. 22-26) prepared by Gill Biochemical (Shanghai) Co., Ltd.) respectively, wherein the product number is 468667 and MaxiSorp; purchased from Thermo corporation), 20 monoclonal antibodies, each at a concentration of 1. mu.g/ml (PBS as a control, pH 7.4, 1 liter containing each monoclonal antibody)Na₂HPO₄·12H₂O 3.63g，KH₂PO₄0.24g of NaCl 8g and KCl 0.2g), a secondary antibody of goat anti-mouse IgG (product number 62-6520, manufactured by Thermo company) marked with HRP and diluted by 3000 times, 1 liter of coating buffer solution containing 12.1g of Tris, and pH 8.8; the confining liquid is 5% skimmed milk, and is prepared on site; the washing solution was 1 liter of PBS plus Tween-201 mL. The stop solution is 2mol/L H₂SO₄The agent is prepared by dropwise adding 21.74mL of concentrated sulfuric acid (98%) into 178.26mL of double distilled water; TMB developing solution (TMB Substrate Kit) is purchased from Thermo company, and the product number is 34021; the results are shown in FIG. 2: compared with the control group, 16 antibodies in the 20 monoclonal antibodies can simultaneously recognize E2ab-C/S-2.1 and E2ab-C strain proteins, and the rest 4 antibodies: MM1, MM5, MM18 and MM20 can only recognize E2ab-C/S-2.1 but not E2ab-C strain, which indicates that the 4 monoclonal antibodies can specifically recognize E2 protein of the hog cholera virus subtype 2.1 virulent strain but cannot recognize E2 protein of the C vaccine strain, and MM1 recognizes the hog cholera virus subtype 2.1 virulent strain E2ab fragment with better specificity, and MM20 recognizes the hog cholera virus subtype 2.1 virulent strain E2ab fragment with the worst specificity.

4. Identification of subtypes of monoclonal antibodies

Subtype identification of the 4 monoclonal antibodies obtained in step 3 was performed by using an ELISA kit (purchased from Wuhan purity Biotechnology Co., Ltd., product No. C060101-L) (for details, see the description) for mouse monoclonal antibody Ig class/subclass/subtype identification, and the results are shown in FIG. 3A: MM1, MM18, and MM20 belong to the IgG2a subtype, MM5 belongs to the IgG1 subtype; as shown in fig. 3B: the light chains of the 4 monoclonal antibodies were all kappa subtypes. In addition, as shown in fig. 3C, native electrophoresis and denatured electrophoresis revealed that the protein molecular composition and molecular weight of the 4 monoclonal antibodies were both attached to mouse IgG.

As MM1, MM18 and MM20 belong to the same subtype, wherein the binding force of MM1 and E2ab-C strain is the lowest, MM1 and MM5 are selected as monoclonal antibodies for identifying virulent strains of subtype 2.1 of classical swine fever virus and vaccine strains of strain C, and hybridoma cell strains secreting MM1 and MM5 are preserved. Wherein, the hybridoma cell strain secreting MM1 is named as 'BALB/c mouse hybridoma cell MM 1', is preserved in Guangdong provincial microorganism strain preservation center of No. 59 floor 5 floor of Miao 100 college of Miehuo, Guangzhou city in 2021, 9 and 24 days, and the preservation number is GDMCC NO: 61945, respectively; the hybridoma cell strain secreting MM5 is named as 'BALB/c mouse hybridoma cell MM 5', is preserved in the Guangdong provincial microorganism strain preservation center of No. 59, No.5, of the Michelia furiosa No. 100 college of the prefecture, Guangzhou city at 9, 24 days in 2021, and the preservation number is GDMCC NO: 61946.

example 2 identification of the recognition site of a monoclonal antibody on E2

Based on the amino acid sequences of E2ab-C/S-2.1, E2ab-C strain and the E2ab fragment of Shimen strain respectively, a series of polypeptides are designed, the corresponding positions and sequences of the polypeptides on the E2 protein are shown as A in figure 4 and B in figure 4, and the specific amino acid sequences are as follows: the sequence of 2.1-1 is shown as SEQ ID NO.6, the sequence of 2.1-2 is shown as SEQ ID NO.7, the sequence of 2.1-3 is shown as SEQ ID NO.8, the sequence of 2.1-4 is shown as SEQ ID NO.9, the sequence of 2.1-5 is shown as SEQ ID NO.10, the sequence of 2.1-6 is shown as SEQ ID NO.11, the sequence of 2.1-7 is shown as SEQ ID NO.12, the sequence of C-1 is shown as SEQ ID NO.13, the sequence of C-2 is shown as SEQ ID NO.14, the sequence of C-3 is shown as SEQ ID NO.15, the sequence of C-4 is shown as SEQ ID NO.16, the sequence of C-5 is shown as SEQ ID NO.17, the sequence of SM-1 is shown as SEQ ID NO.18, the sequence of SM-2 is shown as SEQ ID NO.19, and the sequence of SM-3 is shown as SEQ ID NO. 20.

The indirect ELISA test is adopted to detect the recognition of the MM1 and MM5 on the polypeptides, and the result is shown as C in figure 4: MM1 specifically recognizes 2.1-2 polypeptides of a hog cholera virus subtype 2.1 virulent strain, MM5 specifically recognizes 2.1-1 polypeptides of a hog cholera virus subtype 2.1 virulent strain, and MM1 and MM5 do not recognize C-strain vaccine strains and Shimen strain-derived polypeptides.

Example 3 accurate identification of the antigenic determinant of a monoclonal antibody on E2

To analyze the antigenic determinants recognized by MM5, the amino acid sequences of polypeptides 2.1-1, 2.1-2 derived from a virulent strain of subtype 2.1 of hog cholera virus were aligned with the amino acid sequence of polypeptide C-1 derived from a vaccine strain of strain C, as shown in a in fig. 5: the black underlined part is non-conservative amino acid, and the swine fever virus 2.1 subtype virulent strain-derived polypeptide2.1-1 and polypeptide C-1 from C strain vaccine strain source have 2 amino acid difference numbers, span the length of 5 amino acids; since MM5 recognizes only the polypeptide 2.1-1, therefore "PLGAE(SEQ ID NO.21) "is the specific recognition site for MM 5. To further confirm the role of 2 amino acid residues P and E in antigenic determinants, 3 polypeptides 2.1-1-A, 2.1-1-B, and 2.1-1-C were synthesized, the amino acid sequences of which are respectively "ISSTNEIGPLGAGGLTT(SEQ ID NO.22)”，“ISSTNEIGLLGAEGLTT (SEQ ID NO.23) ", and" ISSTDEIGPLGAEGLTT (SEQ ID NO.24) ", wherein 2.1-1-A and 2.1-1-B respectively mutate the 'P' or 'E' in the specific recognition site to be consistent with the polypeptide C-1 from the vaccine strain of the C strain, and 2.1-1-C mutate the 'N' amino acid residue outside the specific recognition site to be 'D'. Indirect ELISA results (B in fig. 5) showed that amino acid "P", or "E", within the mutation specific recognition site significantly reduced the recognition ability of MM5, compared to the effect of amino acid "N" outside the mutation specific recognition site on MM5, which was smaller. This result shows "PLGAE"P" and "E" in "are both key amino acids in the antigenic determinant recognized by MM 5.

In order to analyze the antigenic determinants recognized by MM1, the amino acid sequence of the polypeptide 2.1-2 of the virulent strain of subtype 2.1 of classical swine fever virus is respectively aligned with the amino acid sequence of the polypeptide C-2 derived from the vaccine strain of strain C, as shown in C in FIG. 5, the black underlined part is a non-conservative amino acid, and the amino acid difference between the polypeptide 2.1-2 derived from the virulent strain of subtype 2.1 of classical swine fever virus and the polypeptide C-2 derived from the vaccine strain C is 5, and spans the length of 19 amino acids. In order to further refine the antibody recognition site, 2 polypeptides 2.1-2-A and 2.1-2-B are synthesized, and the amino acid sequences are respectively' TTTWREYSHGLQLDDGTVKAICVAGSFK (SEQ ID NO.25) "and" TTTWKEYSHDLQLNDGTVRAICTAGSFK (SEQ ID NO.26) ", wherein the polypeptide 2.1-2-A mutates the 2 last amino acids (with black and underlined) which are different from the polypeptide C-2 from the C strain vaccine strain into the amino acids which are consistent with the polypeptide C-2 from the C strain vaccine strain, and the polypeptide 2.1-2-B mutates the 3 first amino acids which are different from the polypeptide C-2 from the C strain vaccine strain into the amino acids which are consistent with the polypeptide C-2 from the C strain vaccine strainThe derived polypeptide C-2 is consistent, the recognition of 2.1-2-A and 2.1-2-B by MM1 is detected by using an indirect ELISA experiment, and the result is shown as D in FIG. 5: mutating the last 3 amino acids (polypeptide 2.1-2-A) does not affect the ability of MM1 to recognize E2ab-C/S-2.1, but mutating the first 3 amino acids (polypeptide 2.1-2-B) completely disrupts the recognition of E2ab-C/S-2.1 by MM 1; thus, the specific recognition site for MM1 is "REYSHGLQLD"(SEQ ID NO.27), wherein 3 amino acid residues" R, "" G, "and" D "are key recognition sites.

To further confirm the role of the 3 amino acid residues "R", "G", and "D" in the epitopes recognized by MM1 monoclonal antibody, 3 additional polypeptides 2.1-2-C, 2.1-2-D, and 2.1-2-E were synthesized, as shown in E in FIG. 5, the amino acid sequences were "TTTWREYSHGLQL-C," TTTWREYSHGLQL-D, "and" D "respectivelyNDGTVRAI(SEQ ID NO.28)”，“TTTWKEYSHGLQLDDGTVRAI (SEQ ID NO.29) ", and" TTTWREYSHDLQLDDGTVRAI (SEQ ID NO.30) ", and" R "," G ", and" D "were mutated to conform to the polypeptide C-2 derived from the vaccine strain of the C strain, respectively. Indirect ELISA results (F in fig. 5) showed that the 3 amino acid residues "R", "G", and "D" were all key sites recognized by MM1, in contrast to the greater interaction of "G" and "D" with MM 1.

Example 4 analysis of the ability of monoclonal antibodies MM1 and MM5 to identify currently endemic strains in China

According to the 2018 published paper of the Chinay embodied and described in the institute of veterinary and veterinary medicine, Shanghai of China academy of agricultural sciences, "Zhang H, Long C, Tian Z, Liu C, Chen J, Bai Y, Li Z, Xiang L, ZHai H, Wang Q, Peng J, An T, Kan Y, Yao L, Yang X, Cai X, Tong G.Complex characteristics and clinical analysis of the wlan embodied and described switch virus in China. Vet Res.2018Jun 25; 14(1) 204.doi:10.1186/s 12917-018. PMID: 29940930; PMCID 6019732. ", the sequences of E2 of multiple subtypes of CSFV were extracted from GenBank, including 9 subtypes of 1.1, 2.1a, 2.1b, 2.1c, 2.1d, 2.2, 2.3, 3.2, and 3.4. The results of the sequence alignment of the MM1 and MM5 recognition sites show that MM5 can specifically recognize all 4 types within subtype 2.1, namely: 2.1a, 2.1b, 2.1c, and 2.1 d. MM1 recognized both subtypes 2.1b and 2.1d (corresponding virus strain subtypes marked by grey boxes in fig. 6).

Example 5 Competition ELISA to verify the specificity of recognition of E2ab-C/S-2.1 by the monoclonal antibodies MM1 and MM5

1. Rabbit sera were prepared against virulent strain E2 of subtype 2.1: emulsifying the recombinant protein E2ab-C/S-2.1 with Freund' S adjuvant, immunizing male rabbit of about 1.5 kg, and boosting the immunization once every 3 weeks for 3 times; after 3 weeks, blood is collected from the heart, serum is separated, split charged and frozen at minus 40 ℃ after the recombinant protein E2ab-C/S-2.1 without adjuvant is immunized in the last time.

2. A competition ELISA experiment is used for detecting whether rabbit serum blocks the monoclonal antibody to recognize E2 ab-C/S-2.1: coating a 96-well plate with 5 micrograms of recombinant protein E2ab-C/S-2.1 per milliliter, wherein the blocking antibody is rabbit serum which is diluted by 5 times and aims at the virulent strain E2 of the subtype 2.1, and the nonimmune rabbit serum is used as a negative control; the primary antibody is monoclonal antibody MM1 or MM5, the concentration of each monoclonal antibody is 1 microgram per milliliter, and the secondary antibody is HRP-labeled goat anti-mouse IgG (manufactured by Thermo company, product number is 62-6520) diluted by 3000 times; the preparation methods of the sample diluent, the blocking solution, the washing solution, and the stop solution in the experiment, and the case of the display kit were the same as in example 1, and the results are shown in FIG. 7: rabbit serum against virulent strain E2 subtype 2.1 can block the binding of MM1 or MM5 to the recombinant protein E2 ab-C/S-2.1.

3. Competition ELISA experiments tested whether porcine sera against the C strain vaccine strain blocked MM1 or MM5 from recognizing E2 protein of the 2.1 subtype virulent strain: the experimental method is the same as above, namely 5 micrograms per milliliter of recombinant protein E2ab-C/S-2.1 is used for coating a 96-well plate, and standard swine fever positive serum and standard swine fever negative serum (purchased from Chinese veterinary drug inspection institute, with the cargo numbers of Z220 and Z221 respectively) for ELISA aiming at the strain vaccine strain are diluted by 2 times of blocking antibody; the primary antibody is monoclonal antibody MM1 or MM5, the concentration of each monoclonal antibody is 1 microgram per milliliter, and the secondary antibody is HRP-labeled goat anti-mouse IgG (manufactured by Thermo company, product number is 62-6520) diluted by 3000 times; the results are shown in FIG. 7: standard swine fever positive sera against the C strain vaccine strain failed to block the binding of MM1 or MM5 to the recombinant protein E2 ab-C/S-2.1.

The result shows that the ability of the monoclonal antibody MM1 or MM5 to recognize the virulent strain of swine fever virus subtype 2.1 can be blocked by corresponding serum, so that MM1 and MM5 can be used for identifying and distinguishing antibodies produced by the body stimulated by the virulent strain of swine fever virus subtype 2.1 or the vaccine strain of the strain C in a competitive ELISA experiment.

Example 6 amplification and sequencing of monoclonal antibody expressing genes in hybridoma cell lines

The hybridoma cell culture medium employed in this example was RPMI1640, purchased from Thermo Fisher, cat #: 11875093, containing 15% fetal bovine serum (manufactured by Gibco, cat # 10099-141C). Reverse transcription kit SuperScript^TMIV CellsDirect^TMcDNA Synthesis Kit ", purchased from Thermo Fisher, Cat #: 11750150. the PCR amplification reagent is "2 × High Fidelity PCR Master Mix", purchased from Biotechnology engineering (Shanghai) GmbH, Cat #: BN 39292-0001.

1. Amplification of cDNA of a Gene of interest

Culturing hybridoma cell lines secreting MM1 and MM5 to logarithmic growth phase, centrifuging to collect hybridoma cells, washing with precooled 4-degree PBS once, then resuspending with precooled PBS, and adjusting cell concentration to 2 × 10⁶one/mL, 5. mu.L of the cells were placed on ice, and lysate was added, the lysate composition being shown in Table 1.

TABLE 1 lysate Components

The lysis reaction was then incubated at room temperature for at least 15 minutes. Add 3. mu.L SuperScript^TMIV CellsDirect^TMStop Solution, incubation at room temperature for 10 min. Add 8. mu.L SuperScript to ice^TMIV RT Master Mix, make the final reaction volume of 40. mu.L (adjusted volume using nuclease free water). Then, reverse transcription was immediately performed. The reverse transcription conditions are shown in Table 2.

TABLE 2 reverse transcription conditions

Annealing	25℃	10min
			Reverse transcription	50℃	10min
Inactivating the enzyme	85℃	5min
			Final step	4℃	∞

The resulting cDNA can be stored at-20 ℃ for one week or immediately subjected to PCR amplification.

PCR reaction amplification of fragments of interest for sequencing

The primer design of PCR is shown as A in FIG. 8, and the specific sequence is as follows:

(1) mouse IgG heavy chain PCR upstream primer combination:

122Fh:5`-ATGGGATGGAGCTGTATCATCC-3`(1-22)(SEQ ID NO.31)；

3549Fh:5`-AGGAACTGCAGGTGTCC-3`(35-49)(SEQ ID NO.32)；

3756Fh:5`-CAGCTACAGGTGTCCACTCC-3`(37-56)(SEQ ID NO.33)；

mouse IgG heavy chain PCR downstream primer:

55375Rh:5`-AGAAGGTGTGCACACCGCTGGAC-3`(553-575)(SEQ ID NO.34)；

the numbering of the reference sequences at NCBI for heavy chain primer design is: x70423.1, BC092295.1, BC092271.1, BC057672.1, BC018280.1, and BC 003878.1. The position of the heavy chain primer in the mouse immunoglobulin heavy chain gene is shown in parentheses after the primer, and the reference sequence for the gene code is AF 466769.1.

(2) Mouse IgG light chain PCR upstream primer combination:

0124Fκ：5`-TGGACATGAGGGCYCCTGCTCAGT-3`(1-24)(SEQ ID NO.35)；

1541Fκ：5`-CAGTTCCTGTTTCTGTTARTGCTCTGG-3`(15-41)(SEQ ID NO.36)；

2443Fκ：5`-TGGGTGCTGCTGCTCTGGGT-3`(24-43)(SEQ ID NO.37)；

mouse IgG light chain PCR downstream primer:

46181Rκ：5`-ACTGAGGCACCTCCAGATGTT-3`(461-481)(SEQ ID NO.38)；

the numbering of the reference sequence at NCBI for the light chain primer design is: DQ078272.1, LC522515.1, MH208237.1, LC199874.1, LC026057.1, BC080787.1, BC094013.1, BC091750.1, BC028540.1, bc019474, and BC 002112.1. The position of the light chain primer in the mouse immunoglobulin light chain gene is shown in parentheses after the primer, and the reference sequence for the gene code is M35669.1.

The PCR reaction system is shown in Table 3.

TABLE 3 PCR reaction System

Reagent	Volume of
		2×High Fidelity PCR Master Mix	25μL
Primer pool	4μL
		cDNA	2μL
ddH₂O	Up to 50μL

Note: the primer pool comprises the following primers in the following dosage: heavy chain variable region amplification primer pool: 122Fh, 3549Fh, 3756Fh, 55375Rh, each 10 pmol; light chain variable region amplification primer pool: 0124Fκ, 1541Fκ, 2443Fκ, 46181Rκ each 10 pmol.

Antibody variable region amplification procedure: pre-denaturation at 95 ℃ for 30 seconds; denaturation at 95 ℃ for 15 seconds, annealing at 58 ℃ for 15 seconds, extension at 72 ℃ for 25 seconds, 35 cycles; extension at 72 ℃ for 5 min; infinity at 16 ℃.

The PCR product obtained by amplification using the above cDNA as a template was electrophoresed, and the result is shown in FIG. 8, B: DNA electrophoresis results show that the genes of the MM1 and MM5 antibodies have been successfully cloned; and the genes of MM1 and MM5 antibodies were sequenced, wherein the gene sequence of the heavy chain variable region of MM1 is AAGCCTGGGGCCTCAGTGAAGATATCCTGC AAGTCTACTGGCTACACATTCAGTAGGTACTGGATAGAGTGGGTAAAGCAGAGGCCTGGACATGGCCTTGAGTGGATTGGAGAGATTTTACCTGGAAGTGGAAGTACTAACTACAATGAGAAGTTCAAGGGAAAGGCCACATTCACTGCAGTTACATCCTCCAACACAGCCTACATGCAACTCAGCAGTCTGACATCTGAGGACTCTGCCGTCTATTACTGTGCAAGTATTACGACGAAGGCCTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCCAAAACAACAGCCCCATCGGTCTATCCACTGGCCCCTGTGTGTGGAGATACAACTGGCTCCTCGGTGACTCTAGGATGCCTGGTCAAGGGTTATTTCCCTGAGCCAGTGACC (SEQ ID NO. 39); the variable region gene sequence of the light chain of MM1 is ACACAGTCTCCTGCTTCCTTAGCTGTATCT CTGGGGCAGAGGGCCACCATCTCATACAGGGCCAGCAAAAGTGTCAGTACATCTGGCTATAGTTATATGCACTGGAACCAACAGAAACCAGGACAGCCACCCAGACTCCTCATCTATCTTGTATCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCACATTAGGGAGCTTACACGTTCGGAGGGGGGACCAAGCTGGAAA (SEQ ID NO. 40);the gene sequence of the heavy chain variable region of MM5 is AAGCCTGGGGCTTCAGTGAAGATGTCCTGC AAGGCTTCTGGCTACACCTTCACCAGCTACTGGATTCACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATCGGAGTGATTGATCCTTCAGATAGTTATACTAGGTACAATCAAAAGTTCAAGGGCAAGGCCACATTGACTGTAGACACATCGTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTACAAGAGAGGTAGTCACTCCCTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAGCCAAAACAACAGCCCCATCGGTCTATCCACTGGCCCCTGTGTGTGGAGATACAACTGGCTCCTCGGTGACTCTAGGATGCCTGGTCAAGGGTTATTTCCCTGAGCCAGTGACC (SEQ ID NO. 41); the variable region gene sequence of the light chain of MM5 is ACACAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATACAGGGCCAGCAAAAGTGTCAGTACATCTGGCTATAGTTATATGCACTGGAACCAACAGAAACCAGGACAGCCACCCAGACTCCTCATCTATCTTGTATCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCACATTAGGGAGCTTACACGTTCGGAGGGGGGACCAAGCTGGAAA (SEQ ID NO. 42). Meanwhile, the amino acid sequence of the heavy chain variable region of MM1 was determined to be KPGASVKISCKSTGYTFSRYWIEWVKQRPGHGLEWIGEILPGSGSTNYNEKFKGKATFTAVTSSNTAYMQLSSLTSEDSAVYYCASITTKAWFAYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVT (SEQ ID NO.43, the underlined parts are CDRs); MM1 light chain variable region amino acid sequence is TQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELTRSEGGPSWK (SEQ ID NO.44, CDR underlined); the heavy chain variable region amino acid sequence of MM5 is KPGASVKMSCKASGYTFTSYWIHWVKQRPGQGLEWIGVIDPSDSYTRYNQKFKGKATLTVDTSSSTAYMQLSSLTSEDSAVYYCTREVVTPFDYWGQGTTLTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPE PVT (SEQ ID NO.45, the underlined parts are CDRs); MM5 light chain variable region amino acid sequence is TQSPASLAVSLGQRATISYRASKSVS TSGYSYMHWNQQKPGQPPRLLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELTRSEGGPSWK (SEQ ID NO.46, CDR underlined).

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

SEQUENCE LISTING

<110> Guangdong sea group Ltd

GUANGDONG HAID ANIMAL HUSBANDRY AND VETERINARY RESEARCH INSTITUTE Co.,Ltd.

<120> monoclonal antibody for identifying classical swine fever virus subtype 2.1 virulent strain and antibody thereof

<130>

<160> 58

<170> PatentIn version 3.5

<210> 1

<211> 169

<212> PRT

<213> Artificial sequence

<400> 1

Met Ser Cys Lys Glu Asp Tyr Arg Tyr Ala Ile Ser Ser Thr Asn Glu

1 5 10 15

Ile Gly Pro Leu Gly Ala Glu Gly Leu Thr Thr Thr Trp Arg Glu Tyr

20 25 30

Ser His Gly Leu Gln Leu Asp Asp Gly Thr Val Arg Ala Ile Cys Thr

35 40 45

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

50 55 60

Leu Ala Ser Leu His Lys Arg Ala Leu Pro Thr Ser Val Thr Phe Glu

65 70 75 80

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

85 90 95

Phe Gly Phe Gly Leu Ser Pro Phe Asp Thr Thr Pro Val Val Lys Gly

100 105 110

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

115 120 125

Pro Ile Gly Trp Thr Gly Val Ile Glu Cys Thr Ala Val Ser Pro Thr

130 135 140

Thr Leu Arg Thr Glu Val Val Lys Thr Phe Lys Arg Glu Lys Pro Phe

145 150 155 160

Pro His His His His His His His Gly

165

<210> 2

<211> 8

<212> PRT

<213> Artificial sequence

<400> 2

His His His His His His His Gly

1 5

<210> 3

<211> 169

<212> PRT

<213> Artificial sequence

<400> 3

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

1 5 10 15

Ile Gly Leu Leu Gly Ala Gly Gly Leu Thr Thr Thr Trp Lys Glu Tyr

20 25 30

Asn His Asp Leu Gln Leu Asn Asp Gly Thr Val Lys Ala Ser Cys Val

35 40 45

Ala Gly Ser Phe Lys Val Thr Ala Leu Asn Val Val Ser Arg Arg Tyr

50 55 60

Leu Ala Ser Leu His Lys Lys Ala Leu Pro Thr Ser Val Thr Phe Glu

65 70 75 80

Leu Leu Phe Asp Gly Thr Asn Pro Ser Thr Glu Glu Met Gly Asp Asp

85 90 95

Phe Arg Ser Gly Leu Cys Pro Phe Asp Thr Ser Pro Val Val Lys Gly

100 105 110

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

115 120 125

Pro Ile Gly Trp Thr Gly Val Ile Glu Cys Thr Ala Val Ser Pro Thr

130 135 140

Thr Leu Arg Thr Glu Val Val Lys Thr Phe Arg Arg Asp Lys Pro Phe

145 150 155 160

Pro His His His His His His His Gly

165

<210> 4

<211> 510

<212> DNA

<213> Artificial sequence

<400> 4

atgtcctgca aggaggacta ccgctacgct atctcctcca ccaacgagat cggtcctctg 60

ggtgctgaag gtctgactac cacctggcgc gagtattccc atggtctgca gctggacgat 120

ggtaccgtgc gcgctatttg tactgctggt tccttcaagg tgatcgctct gaacgtggtg 180

tcccgtcgtt atctggcttc cctgcataaa cgcgctctgc ctacttctgt gaccttcgag 240

ctgctgttcg acggtacctc ccctgctatc gaggagatgg gtgacgactt cggtttcggt 300

ctgtcccctt tcgacactac ccctgtggtg aagggtaagt acaacaccac cctgctgaac 360

ggttccgctt tctacctggt gtgccctatc ggttggaccg gtgtgatcga atgcaccgct 420

gtgtccccta ctactctgcg taccgaagtg gtgaagacct tcaagcgcga gaagcctttc 480

cctcaccacc accatcatca ccatggttaa 510

<210> 5

<211> 510

<212> DNA

<213> Artificial sequence

<400> 5

atggcctgca aggaagatta caggtacgca atatcgtcaa ccgatgagat agggctactt 60

ggggccggag gtctcaccac cacctggaag gaatacaacc acgatttgca actgaatgac 120

gggaccgtca aggccagttg cgtggcaggt tcctttaaag tcacagcact taatgtggtc 180

agtaggaggt atttggcgtc attgcataag aaggctttac ccacttccgt gacattcgag 240

ctcctgttcg acgggaccaa cccatcaact gaggaaatgg gagatgactt caggtccggg 300

ctgtgcccgt ttgatacgag tcctgttgtt aagggaaagt acaatacgac cttgttgaac 360

ggtagtgctt tctatcttgt ctgcccaata gggtggacgg gtgtcataga gtgcacagca 420

gtgagcccaa caactctgag gacagaagtg gtaaagacct tcaggagaga caagcccttt 480

ccgcaccacc accatcatca ccatggataa 510

<210> 6

<211> 22

<212> PRT

<213> Artificial sequence

<400> 6

Arg Tyr Ala Ile Ser Ser Thr Asn Glu Ile Gly Pro Leu Gly Ala Glu

1 5 10 15

Gly Leu Thr Thr Thr Trp

20

<210> 7

<211> 28

<212> PRT

<213> Artificial sequence

<400> 7

Thr Thr Thr Trp Arg Glu Tyr Ser His Gly Leu Gln Leu Asp Asp Gly

1 5 10 15

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

20 25

<210> 8

<211> 32

<212> PRT

<213> Artificial sequence

<400> 8

Gly Ser Phe Lys Val Ile Ala Leu Asn Val Val Ser Arg Arg Tyr Leu

1 5 10 15

Ala Ser Leu His Lys Arg Ala Leu Pro Thr Ser Val Thr Phe Glu Leu

20 25 30

<210> 9

<211> 29

<212> PRT

<213> Artificial sequence

<400> 9

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

1 5 10 15

Gly Asp Asp Phe Gly Phe Gly Leu Ser Pro Phe Asp Thr

20 25

<210> 10

<211> 23

<212> PRT

<213> Artificial sequence

<400> 10

Ala Ile Glu Glu Met Gly Asp Asp Phe Gly Phe Gly Leu Ser Pro Phe

1 5 10 15

Asp Thr Thr Pro Val Val Lys

20

<210> 11

<211> 25

<212> PRT

<213> Artificial sequence

<400> 11

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

1 5 10 15

Tyr Leu Val Cys Pro Ile Gly Trp Thr

20 25

<210> 12

<211> 32

<212> PRT

<213> Artificial sequence

<400> 12

Gly Trp Thr Gly Val Ile Glu Cys Thr Ala Val Ser Pro Thr Thr Leu

1 5 10 15

Arg Thr Glu Val Val Lys Thr Phe Lys Arg Glu Lys Pro Phe Pro His

20 25 30

<210> 13

<211> 20

<212> PRT

<213> Artificial sequence

<400> 13

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

1 5 10 15

Thr Thr Thr Trp

20

<210> 14

<211> 28

<212> PRT

<213> Artificial sequence

<400> 14

Thr Thr Thr Trp Lys Glu Tyr Asn His Asp Leu Gln Leu Asn Asp Gly

1 5 10 15

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

20 25

<210> 15

<211> 32

<212> PRT

<213> Artificial sequence

<400> 15

Gly Ser Phe Lys Val Thr Ala Leu Asn Val Val Ser Arg Arg Tyr Leu

1 5 10 15

Ala Ser Leu His Lys Lys Ala Leu Pro Thr Ser Val Thr Phe Glu Leu

20 25 30

<210> 16

<211> 27

<212> PRT

<213> Artificial sequence

<400> 16

Thr Phe Glu Leu Leu Phe Asp Gly Thr Asn Pro Ser Thr Glu Glu Met

1 5 10 15

Gly Asp Asp Phe Arg Ser Gly Leu Cys Pro Phe

20 25

<210> 17

<211> 27

<212> PRT

<213> Artificial sequence

<400> 17

Ile Glu Cys Thr Ala Val Ser Pro Thr Thr Leu Arg Thr Glu Val Val

1 5 10 15

Lys Thr Phe Arg Arg Asp Lys Pro Phe Pro His

20 25

<210> 18

<211> 22

<212> PRT

<213> Artificial sequence

<400> 18

Arg Tyr Ala Leu Ser Ser Thr Asn Glu Ile Gly Leu Leu Gly Ala Gly

1 5 10 15

Gly Leu Thr Thr Thr Trp

20

<210> 19

<211> 28

<212> PRT

<213> Artificial sequence

<400> 19

Thr Thr Thr Trp Glu Glu Tyr Ser His Asp Leu Gln Leu Asn Asp Gly

1 5 10 15

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

20 25

<210> 20

<211> 32

<212> PRT

<213> Artificial sequence

<400> 20

Gly Ser Phe Lys Val Thr Ala Leu Asn Val Val Ser Arg Arg Tyr Leu

1 5 10 15

Ala Ser Leu His Lys Gly Ala Leu Leu Thr Ser Val Thr Phe Glu Leu

20 25 30

<210> 21

<211> 5

<212> PRT

<213> Artificial sequence

<400> 21

Pro Leu Gly Ala Glu

1 5

<210> 22

<211> 17

<212> PRT

<213> Artificial sequence

<400> 22

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

1 5 10 15

Thr

<210> 23

<211> 17

<212> PRT

<213> Artificial sequence

<400> 23

Ile Ser Ser Thr Asn Glu Ile Gly Leu Leu Gly Ala Glu Gly Leu Thr

1 5 10 15

Thr

<210> 24

<211> 17

<212> PRT

<213> Artificial sequence

<400> 24

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

1 5 10 15

Thr

<210> 25

<211> 28

<212> PRT

<213> Artificial sequence

<400> 25

Thr Thr Thr Trp Arg Glu Tyr Ser His Gly Leu Gln Leu Asp Asp Gly

1 5 10 15

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

20 25

<210> 26

<211> 28

<212> PRT

<213> Artificial sequence

<400> 26

Thr Thr Thr Trp Lys Glu Tyr Ser His Asp Leu Gln Leu Asn Asp Gly

1 5 10 15

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

20 25

<210> 27

<211> 10

<212> PRT

<213> Artificial sequence

<400> 27

Arg Glu Tyr Ser His Gly Leu Gln Leu Asp

1 5 10

<210> 28

<211> 21

<212> PRT

<213> Artificial sequence

<400> 28

Thr Thr Thr Trp Arg Glu Tyr Ser His Gly Leu Gln Leu Asn Asp Gly

1 5 10 15

Thr Val Arg Ala Ile

20

<210> 29

<211> 21

<212> PRT

<213> Artificial sequence

<400> 29

Thr Thr Thr Trp Lys Glu Tyr Ser His Gly Leu Gln Leu Asp Asp Gly

1 5 10 15

Thr Val Arg Ala Ile

20

<210> 30

<211> 21

<212> PRT

<213> Artificial sequence

<400> 30

Thr Thr Thr Trp Arg Glu Tyr Ser His Asp Leu Gln Leu Asp Asp Gly

1 5 10 15

Thr Val Arg Ala Ile

20

<210> 31

<211> 22

<212> DNA

<213> Artificial sequence

<400> 31

atgggatgga gctgtatcat cc 22

<210> 32

<211> 17

<212> DNA

<213> Artificial sequence

<400> 32

aggaactgca ggtgtcc 17

<210> 33

<211> 20

<212> DNA

<213> Artificial sequence

<400> 33

cagctacagg tgtccactcc 20

<210> 34

<211> 23

<212> DNA

<213> Artificial sequence

<400> 34

agaaggtgtg cacaccgctg gac 23

<210> 35

<211> 24

<212> DNA

<213> Artificial sequence

<400> 35

tggacatgag ggcycctgct cagt 24

<210> 36

<211> 27

<212> DNA

<213> Artificial sequence

<400> 36

cagttcctgt ttctgttart gctctgg 27

<210> 37

<211> 20

<212> DNA

<213> Artificial sequence

<400> 37

tgggtgctgc tgctctgggt 20

<210> 38

<211> 21

<212> DNA

<213> Artificial sequence

<400> 38

actgaggcac ctccagatgt t 21

<210> 39

<211> 432

<212> DNA

<213> Artificial sequence

<400> 39

aagcctgggg cctcagtgaa gatatcctgc aagtctactg gctacacatt cagtaggtac 60

tggatagagt gggtaaagca gaggcctgga catggccttg agtggattgg agagatttta 120

cctggaagtg gaagtactaa ctacaatgag aagttcaagg gaaaggccac attcactgca 180

gttacatcct ccaacacagc ctacatgcaa ctcagcagtc tgacatctga ggactctgcc 240

gtctattact gtgcaagtat tacgacgaag gcctggtttg cttactgggg ccaagggact 300

ctggtcactg tctctgcagc caaaacaaca gccccatcgg tctatccact ggcccctgtg 360

tgtggagata caactggctc ctcggtgact ctaggatgcc tggtcaaggg ttatttccct 420

gagccagtga cc 432

<210> 40

<211> 312

<212> DNA

<213> Artificial sequence

<400> 40

acacagtctc ctgcttcctt agctgtatct ctggggcaga gggccaccat ctcatacagg 60

gccagcaaaa gtgtcagtac atctggctat agttatatgc actggaacca acagaaacca 120

ggacagccac ccagactcct catctatctt gtatccaacc tagaatctgg ggtccctgcc 180

aggttcagtg gcagtgggtc tgggacagac ttcaccctca acatccatcc tgtggaggag 240

gaggatgctg caacctatta ctgtcagcac attagggagc ttacacgttc ggagggggga 300

ccaagctgga aa 312

<210> 41

<211> 429

<212> DNA

<213> Artificial sequence

<400> 41

aagcctgggg cttcagtgaa gatgtcctgc aaggcttctg gctacacctt caccagctac 60

tggattcact gggtgaagca gaggcctgga caaggccttg agtggatcgg agtgattgat 120

ccttcagata gttatactag gtacaatcaa aagttcaagg gcaaggccac attgactgta 180

gacacatcgt ccagcacagc ctacatgcag ctcagcagcc tgacatctga ggactctgcg 240

gtctattact gtacaagaga ggtagtcact ccctttgact actggggcca aggcaccact 300

ctcacagtct cctcagccaa aacaacagcc ccatcggtct atccactggc ccctgtgtgt 360

ggagatacaa ctggctcctc ggtgactcta ggatgcctgg tcaagggtta tttccctgag 420

ccagtgacc 429

<210> 42

<211> 312

<212> DNA

<213> Artificial sequence

<400> 42

acacagtctc ctgcttcctt agctgtatct ctggggcaga gggccaccat ctcatacagg 60

gccagcaaaa gtgtcagtac atctggctat agttatatgc actggaacca acagaaacca 120

ggacagccac ccagactcct catctatctt gtatccaacc tagaatctgg ggtccctgcc 180

aggttcagtg gcagtgggtc tgggacagac ttcaccctca acatccatcc tgtggaggag 240

gaggatgctg caacctatta ctgtcagcac attagggagc ttacacgttc ggagggggga 300

ccaagctgga aa 312

<210> 43

<211> 144

<212> PRT

<213> Artificial sequence

<400> 43

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

1 5 10 15

Phe Ser Arg Tyr Trp Ile Glu Trp Val Lys Gln Arg Pro Gly His Gly

20 25 30

Leu Glu Trp Ile Gly Glu Ile Leu Pro Gly Ser Gly Ser Thr Asn Tyr

35 40 45

Asn Glu Lys Phe Lys Gly Lys Ala Thr Phe Thr Ala Val Thr Ser Ser

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Ser Val Tyr Pro Leu Ala Pro Val Cys Gly Asp Thr Thr Gly Ser Ser

115 120 125

Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr

130 135 140

<210> 44

<211> 104

<212> PRT

<213> Artificial sequence

<400> 44

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

1 5 10 15

Ile Ser Tyr Arg Ala Ser Lys Ser Val Ser Thr Ser Gly Tyr Ser Tyr

20 25 30

Met His Trp Asn Gln Gln Lys Pro Gly Gln Pro Pro Arg Leu Leu Ile

35 40 45

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

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His Pro Val Glu Glu

65 70 75 80

Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ile Arg Glu Leu Thr Arg

85 90 95

Ser Glu Gly Gly Pro Ser Trp Lys

100

<210> 45

<211> 143

<212> PRT

<213> Artificial sequence

<400> 45

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

1 5 10 15

Phe Thr Ser Tyr Trp Ile His Trp Val Lys Gln Arg Pro Gly Gln Gly

20 25 30

Leu Glu Trp Ile Gly Val Ile Asp Pro Ser Asp Ser Tyr Thr Arg Tyr

35 40 45

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

50 55 60

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

65 70 75 80

Val Tyr Tyr Cys Thr Arg Glu Val Val Thr Pro Phe Asp Tyr Trp Gly

85 90 95

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

100 105 110

Val Tyr Pro Leu Ala Pro Val Cys Gly Asp Thr Thr Gly Ser Ser Val

115 120 125

Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr

130 135 140

<210> 46

<211> 104

<212> PRT

<213> Artificial sequence

<400> 46

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

1 5 10 15

Ile Ser Tyr Arg Ala Ser Lys Ser Val Ser Thr Ser Gly Tyr Ser Tyr

20 25 30

Met His Trp Asn Gln Gln Lys Pro Gly Gln Pro Pro Arg Leu Leu Ile

35 40 45

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

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His Pro Val Glu Glu

65 70 75 80

Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ile Arg Glu Leu Thr Arg

85 90 95

Ser Glu Gly Gly Pro Ser Trp Lys

100

<210> 47

<211> 8

<212> PRT

<213> Artificial sequence

<400> 47

Gly Tyr Thr Phe Ser Arg Tyr Trp

1 5

<210> 48

<211> 9

<212> PRT

<213> Artificial sequence

<400> 48

Ile Leu Pro Gly Ser Gly Ser Thr Asn

1 5

<210> 49

<211> 11

<212> PRT

<213> Artificial sequence

<400> 49

Ala Ser Ile Thr Thr Lys Ala Trp Phe Ala Tyr

1 5 10

<210> 50

<211> 6

<212> PRT

<213> Artificial sequence

<400> 50

Lys Ser Val Ser Thr Ser

1 5

<210> 51

<211> 3

<212> PRT

<213> Artificial sequence

<400> 51

Leu Val Ser

1

<210> 52

<211> 9

<212> PRT

<213> Artificial sequence

<400> 52

Gln His Ile Arg Glu Leu Thr Arg Ser

1 5

<210> 53

<211> 8

<212> PRT

<213> Artificial sequence

<400> 53

Gly Tyr Thr Phe Thr Ser Tyr Trp

1 5

<210> 54

<211> 9

<212> PRT

<213> Artificial sequence

<400> 54

Ile Asp Pro Ser Asp Ser Tyr Thr Arg

1 5

<210> 55

<211> 10

<212> PRT

<213> Artificial sequence

<400> 55

Thr Arg Glu Val Val Thr Pro Phe Asp Tyr

1 5 10

<210> 56

<211> 6

<212> PRT

<213> Artificial sequence

<400> 56

Lys Ser Val Ser Thr Ser

1 5

<210> 57

<211> 3

<212> PRT

<213> Artificial sequence

<400> 57

Leu Val Ser

1

<210> 58

<211> 9

<212> PRT

<213> Artificial sequence

<400> 58

Gln His Ile Arg Glu Leu Thr Arg Ser

1 5

Claims

1. A monoclonal antibody characterized by: the monoclonal antibody is MM1 or MM 5;

the MM1 comprises a MM1 heavy chain variable region and a MM1 light chain variable region;

the MM1 heavy chain variable region comprises CDR1, CDR2, CDR 3;

the amino acid sequence of the MM1 heavy chain variable region CDR1 is as follows:

a) GYTFSRYW (SEQ ID NO. 47); or

the amino acid sequence of the MM1 heavy chain variable region CDR2 is as follows:

a) ILPGSGSTN (SEQ ID NO. 48); or

the amino acid sequence of the MM1 heavy chain variable region CDR3 is as follows:

a) ASITTKAWFAY (SEQ ID NO. 49); or

the MM1 light chain variable region comprises CDR1, CDR2, CDR 3;

the amino acid sequence of the MM1 light chain variable region CDR1 is as follows:

a) KSVSTS (SEQ ID NO. 50); or

the amino acid sequence of the MM1 light chain variable region CDR2 is as follows:

a) LVS (SEQ ID NO. 51); or

the amino acid sequence of the MM1 light chain variable region CDR3 is as follows:

a) QHIRELTRS (SEQ ID NO. 52); or

b) The amino acid sequence shown in SEQ ID NO.52 is the amino acid sequence which is modified by substitution, deletion or addition of one or more amino acids and has the same or similar functions;

the MM5 comprises a MM5 heavy chain variable region and a MM5 light chain variable region;

the MM5 heavy chain variable region comprises CDR1, CDR2, CDR 3;

the amino acid sequence of the MM5 heavy chain variable region CDR1 is as follows:

a) GYTFTSYW (SEQ ID NO. 53); or

the amino acid sequence of the MM5 heavy chain variable region CDR2 is as follows:

a) IDPSDSYTR (SEQ ID NO. 54); or

the amino acid sequence of the MM5 heavy chain variable region CDR3 is as follows:

a) TREVVTPFDY (SEQ ID NO. 55); or

the MM5 light chain variable region comprises CDR1, CDR2, CDR 3;

the amino acid sequence of the MM5 light chain variable region CDR1 is as follows:

a) KSVSTS (SEQ ID NO. 56); or

the amino acid sequence of the MM5 light chain variable region CDR2 is as follows:

a) LVS (SEQ ID NO. 57); or

the amino acid sequence of the MM5 light chain variable region CDR3 is as follows:

a) QHIRELTRS (SEQ ID NO. 58); or

2. The monoclonal antibody according to claim 1,

the MM1 comprises a heavy chain variable region and a light chain variable region;

the amino acid sequence of the MM1 heavy chain variable region is as follows:

a) an amino acid sequence shown as SEQ ID NO. 43; or

the amino acid sequence of the MM1 light chain variable region is as follows:

a) an amino acid sequence shown as SEQ ID NO. 44; or

b) An amino acid sequence which is shown in SEQ ID NO.44 and has the same or similar functions after being modified by substitution, deletion or addition of one or more amino acids;

the amino acid sequence of the MM5 heavy chain variable region is as follows:

a) an amino acid sequence shown as SEQ ID NO. 45; or

the amino acid sequence of the MM5 light chain variable region is as follows:

a) an amino acid sequence shown as SEQ ID NO. 46; or

3. The monoclonal antibody according to claim 2,

the MM1 is preserved in Guangdong province microorganism strain preservation center of No. 59 building 5 building of No. 100 college of Mieli Zhonglu, Guangzhou city in 2021, 9 and 24 days, and the preservation number is GDMCC NO: 61945, and the hybridoma cell strain BALB/c mouse hybridoma MM1 is obtained by secretion;

the MM5 is preserved in Guangdong province microorganism strain preservation center of No. 59 building 5 building of No. 100 college of Mieli Zhonglu, Guangzhou city in 2021, 9 and 24 days, and the preservation number is GDMCC NO: 61946, and the hybridoma cell strain BALB/c mouse hybridoma MM 5.

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

5. An expression cassette, recombinant vector, recombinant bacterium or transgenic cell comprising the nucleic acid molecule of claim 4.

Use of any one of (1) to (3) in any one of (c) to (p):

(1) the monoclonal antibody of any one of claims 1-3;

(2) the nucleic acid molecule of claim 4;

(3) the expression cassette, recombinant vector, recombinant bacterium, or transgenic cell of claim 5;

7. Use according to claim 6, characterized in that:

the amino acid sequence of the classical swine fever virus subtype 2.1 virulent strain E2 protein is shown in SEQ ID NO. 1;

the amino acid sequence of the C strain vaccine strain E2 protein is shown in SEQ ID NO. 3.

8. A hybridoma cell line selected from any one of (q) to (r):

9. a kit comprising the monoclonal antibody of any one of claims 1 to 3 and/or the hybridoma cell line of claim 8.

10. An in vitro method for non-diagnostic purposes comprising the step of using the monoclonal antibody of any one of claims 1 to 3 or the hybridoma cell line of claim 8 or the kit of claim 9;

the in vitro method is any one of(s) to (y):

(s) detecting the hog cholera virus subtype 2.1 virulent strain E2 protein;

(t) detecting a virulent strain of the subtype 2.1 of the hog cholera virus;