CN115433279A

CN115433279A - Preparation and application of targeting CD123 rabbit recombinant monoclonal antibody

Info

Publication number: CN115433279A
Application number: CN202110628001.0A
Authority: CN
Inventors: 马东晖; 洪淑娟; 梅芬; 蔡萌
Original assignee: Suzhou Dima Biotechnology Co ltd
Current assignee: Suzhou Dima Biotechnology Co ltd
Priority date: 2021-06-05
Filing date: 2021-06-05
Publication date: 2022-12-06

Abstract

The invention relates to preparation and application of a targeted CD123 rabbit recombinant monoclonal antibody, wherein the obtained antibody has good specificity, and most importantly, the antibody has high affinity, can identify CD123 protein on the surface of a natural cell, and can be used for flow-type and ELISA detection applications.

Description

Preparation and application of targeting CD123 rabbit recombinant monoclonal antibody

Technical Field

The invention belongs to the field of antibody preparation in biotechnology, and particularly relates to preparation and application of a targeted CD123 rabbit recombinant monoclonal antibody.

Background

CD123 is an alpha subunit of IL-3R, the ligand of the alpha subunit is IL-3, and belongs to type I single transmembrane protein, the total length of amino acids is 378 amino acids, and the extracellular region has 287 amino acids, namely 19-305 amino acids. Studies have shown that many hematological tumors express excessive amounts of CD123, particularly Acute Myeloid Leukemia (AML), acute B-Cell lymphoblastic Leukemia (B-ALLs), hairy Cell Leukemia (hair Cell Leukemia), blastic plasmacytoid dendritic Cell tumor (BPDCN), and the like. Flow cytometry is widely applied clinically at present due to its advantages of sensitivity, rapidness, quantifiability and the like. The detection of CD123 by flow cytometry requires the presence of a well-specific antibody targeting CD123. In addition, the expression level of CD123 in the initial diagnosis of acute myelogenous leukemia patients can be used as a reference for clinically evaluating the treatment response of patients and judging the clinical prognosis. Therefore, the development of antibodies targeting CD123 has important value for diagnostic and therapeutic applications.

Antibodies targeting CD123 are all murine in the current art. For example, chinese patent application CN 2020102295811 discloses that the monoclonal antibody against CD123 is murine CD123 monoclonal antibody produced by means of phage library displaying mouse single chain antibody. Although the antibodies also have a certain application prospect, monoclonal antibodies with stronger affinity and more epitopes still need to be developed.

Disclosure of Invention

The invention aims to provide a recombinant monoclonal antibody capable of targeting CD123 rabbit, which is selected from one or more of the following recombinant monoclonal antibodies of rabbit:

rabbit recombinant monoclonal antibody designated 1E 4: the amino acid sequences of the heavy chain complementarity determining regions CDR1, CDR2 and CDR3 are respectively the amino acid sequences shown in SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3; and the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain complementary determining region are respectively the amino acid sequences shown in SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6;

rabbit recombinant monoclonal antibody designated 1E 5: the amino acid sequences of the heavy chain complementary determining regions CDR1, CDR2 and CDR3 are respectively the amino acid sequences shown in SEQ ID NO 10, SEQ ID NO 11 and SEQ ID NO 12, and the amino acid sequences of the light chain complementary determining regions CDR1, CDR2 and CDR3 are respectively the amino acid sequences shown in SEQ ID NO 13, SEQ ID NO 14 and SEQ ID NO 15;

the amino acid sequences of the heavy chain complementary determining regions CDR1, CDR2 and CDR3 of the rabbit recombinant monoclonal antibody named as 1E6 are respectively the amino acid sequences shown as SEQ ID NO. 19, SEQ ID NO. 20 and SEQ ID NO. 21, and the amino acid sequences of the light chain complementary determining regions CDR1, CDR2 and CDR3 are respectively the amino acid sequences shown as SEQ ID NO. 22, SEQ ID NO. 23 and SEQ ID NO. 24;

the rabbit recombinant monoclonal antibody named as 5D3 has the amino acid sequences shown in SEQ ID No. 28, SEQ ID No. 29 and SEQ ID No. 30 as the heavy chain complementary determining regions CDR1, CDR2 and CDR3, and the amino acid sequences shown in SEQ ID No. 22, SEQ ID No. 23 and SEQ ID No. 24 as the light chain complementary determining regions CDR1, CDR2 and CDR 3;

on the basis of the technical scheme, the rabbit recombinant monoclonal antibody named as 1E4 has a heavy chain variable region sequence of an amino acid sequence shown as SEQ ID NO. 7 and a light chain variable region sequence of an amino acid sequence shown as SEQ ID NO. 8;

the rabbit recombinant monoclonal antibody named as 1E5 has the heavy chain variable region sequence as shown in SEQ ID No. 16 and the light chain variable region sequence as shown in SEQ ID No. 17;

the rabbit recombinant monoclonal antibody named as 1E6 has the heavy chain variable region sequence as shown in SEQ ID No. 25 and the light chain variable region sequence as shown in SEQ ID No. 26;

the rabbit recombinant monoclonal antibody named 5D3 has the heavy chain variable region sequence of the amino acid sequence shown in SEQ ID NO. 34 and the light chain variable region sequence of the amino acid sequence shown in SEQ ID NO. 35;

on the basis of the technical scheme, the rabbit recombinant monoclonal antibody named as 1E4 has an SCFV sequence as an amino acid sequence shown in SEQ ID NO. 9;

the SCFV sequence of the rabbit recombinant monoclonal antibody named as 1E5 is an amino acid sequence shown as SEQ ID NO. 18;

the SCFV sequence of the rabbit recombinant monoclonal antibody named 1E6 is an amino acid sequence shown in SEQ ID NO. 27;

the SCFV sequence of the rabbit recombinant monoclonal antibody named 5D3 is an amino acid sequence shown in SEQ ID NO. 36;

on the basis of the technical scheme, the light chain constant region of the rabbit recombinant monoclonal antibody is a kappa chain, and the heavy chain constant region is an IgG type.

The invention also provides a nucleic acid molecule comprising a nucleic acid sequence capable of encoding a heavy chain complementarity determining region or a light chain complementarity determining region of a recombinant monoclonal antibody targeted to a CD123 rabbit.

The present invention also provides a vector containing the above-described nucleic acid molecule.

The invention also provides a host cell which contains the knot-targeted CD123 rabbit recombinant monoclonal antibody, the nucleic acid molecule or the vector.

The invention also provides a conjugate containing the antibody.

The invention also provides a pharmaceutical composition, which comprises a main component and an auxiliary component, wherein: the main component is one or more of the CD123 rabbit-targeted recombinant monoclonal antibody, the nucleic acid molecule, the vector, the host cell and the conjugate, and the accessory component is selected from pharmaceutically acceptable carriers or excipients and other optional bioactive substances.

The invention also provides the application of the targeted CD123 rabbit recombinant monoclonal antibody, the nucleic acid molecule, the vector, the host cell and the conjugate in preparing a medicament or a detection reagent for treating diseases.

The invention also provides a kit, which comprises the targeted CD123 rabbit recombinant monoclonal antibody.

Compared with the prior art, the invention has the following advantages and beneficial effects:

the CD123 targeting monoclonal antibody on the market is mainly of a murine source, and because the affinity of the rabbit source antibody is higher than that of the murine source, and the immunization process of rabbits is more complicated than that of mice, antibodies with more epitopes can be obtained. After the rabbit is immunized by the CD123 extracellular region protein, the target CD123 rabbit recombinant monoclonal antibody is prepared by a B cell cloning technology, and importantly, the target CD123 rabbit recombinant monoclonal antibody has high affinity and good specificity, can identify the CD123 protein on the surface of a natural cell, and can be used for flow-type and ELISA detection applications. In addition, other diagnostically and therapeutically relevant agents may be developed in the future for the CD123 target.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.

FIG. 1 shows the SDS electrophoretogram of CD123 protein.

FIG. 2 shows the result of PCR amplification of heavy and light chains of anti-CD 123 rabbit recombinant monoclonal antibody.

FIG. 3 shows SDS electrophorograms of expression purified CD123 rabbit recombinant monoclonal antibodies.

FIG. 4 shows the binding capacity of the recombinant monoclonal antibody of CD123 rabbit detected by ELISA.

FIGS. 5-8 show the specificity of FACs to detect CD123 rabbit recombinant monoclonal antibody.

FIG. 9 shows that FACs detect the binding capacity of CD123 rabbit recombinant monoclonal antibody.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.

The invention is further described below: in the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art. Also, protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology related terms, and laboratory procedures used herein are all terms and conventional procedures used extensively in the relevant art. Meanwhile, in order to better understand the present invention, the definitions and explanations of related terms are provided below.

The term "antibody" as referred to herein includes whole antibodies and any antigen-binding fragment (i.e., "antigen-binding portion") or single chain thereof. An "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains that are linked to each other by disulfide bonds, or an antigen-binding portion thereof. Each heavy chain is composed of a heavy chain variable region and a heavy chain constant region. The proteins or fragments thereof involved in the present invention may be naturally purified products, or chemically synthesized products, or produced from eukaryotic hosts (e.g., mammalian cells) using recombinant techniques. The raw materials and reagents used in the invention can be purchased from the market.

The technical scheme of the invention is further illustrated by the following embodiments.

The embodiment is as follows:

(1) Preparation of CD123 protein: the invention constructs eukaryotic expression plasmid of CD123 FC fusion tag protein, after transfecting 293F cells for 5 days, collects cell culture medium supernatant, purifies by using protein A resin, identifies the concentration of the protein, analyzes the obtained CD123 protein by SDS-PAGE mode, and the result is shown in figure 1, and the obtained protein about 80-90kda is the CD123 protein.

(2) Obtaining peripheral blood mononuclear cells (namely PBMC cells) of the immune animals: new Zealand white rabbits were selected as the immunized animal. The first immunization is carried out by emulsifying 250 mu g of CD123 protein and isovolumic complete Freund's adjuvant, injecting multipoint on the subcutaneous back of New Zealand white rabbit, carrying out the second immunization 21 days later, emulsifying 120 mu g of protein and isovolumic incomplete Freund's adjuvant, injecting the dorsal part of New Zealand white rabbit subcutaneously, carrying out the third immunization 21 days later, emulsifying 120 mu g of protein and isovolumic incomplete Freund's adjuvant, and injecting the dorsal part of New Zealand white rabbit subcutaneously. After one week of interval after the third immunization, peripheral blood was collected aseptically.

(3) Acquisition of CD 123-specific B lymphocytes: separating PBMC cells from the collected peripheral blood by using lymphocyte separating liquid; coupling the CD123 protein to magnetic beads according to the operation instructions of the immunomagnetic beads; and (3) incubating the CD123 protein coupled magnetic beads and the separated PBMC cells for 50min at room temperature, putting the mixture into a magnetic frame, immersing the magnetic beads at the bottom after 5min, discarding the supernatant, adding sterile PBS to wash the cells, repeatedly washing the cells for 3 times, and finally separating the cells to obtain the CD123 specific B lymphocytes.

(4) Identification of B lymphocytes: diluting the separated B lymphocyte by a certain multiple, placing the diluted B lymphocyte into a 96-hole cell culture plate, and adding a solution containing 10% of fetus1640 medium of bovine serum (FBS) and 2. Mu.g/ml human IL2, 5% CO at 37 ℃ ₂ Cultured under the conditions for 6 days. And collecting the culture medium supernatant to identify the antibody.

And (3) indirect ELISA identification:

coating CD123 protein with the concentration of 1 mu g/ml, incubating for 16h at 4 ℃ in 100 mu l/hole; the next day, the coating solution was discarded and blocked with 1% Bovine Serum Albumin (BSA) in PBS, 150. Mu.l/well, and incubated at 37 ℃ for 1h. The blocking solution was discarded, and B cell supernatant was added thereto at 50. Mu.l/well and incubated at 37 ℃ for 1 hour. The B cell supernatant was discarded, the plate was washed 5 times with PBS containing 0.5wt.% Tween-20 for 2 min/time, and a 5000-fold diluted secondary goat anti-rabbit IgG-HRP antibody was added and incubated at 37 ℃ for 1h. The secondary antibody was discarded and the plate was washed 5 times 2 min/time with phosphate Tween buffer (PBST). The wash was discarded, patted dry, and substrate was added for color development. The B cell clones marked as 1E4,1E5,1E6 were identified as positive by ELISA and the results are shown in Table 1.

TABLE 1 ELISA test results

Clone number	OD450
		1E4	2.10
1E5	1.87
		1E6	1.53
5D3	1.77

(5) Cloning of antibody genes

Collecting B cells which are identified as positive, extracting RNA by using a conventional RNA extraction method, performing reverse transcription to obtain cDNA, and performing reverse transcription by using gene primers of antibody heavy chains: an upstream primer 5 'CAGTCGCTGGAGGAGTCCGG-3' and a downstream primer 5 'CCATTGGTGAGGGGTGCCCGAG-3', and the primers of the antibody light chain gene are as follows: an upstream primer 5-: denaturation was carried out at 94 ℃ for 3min, (95 ℃ for 1min,56 ℃ for 30s,72 ℃ for 1 min) for 30 cycles, and final extension at 72 ℃ for 10min (PCR amplification results are shown in FIG. 2). And recovering the PCR product by using a DNA gel purification and recovery kit. Cloning heavy and light chain genes of the rabbit recombinant monoclonal antibody to an expression vector, then transforming, verifying a single colony by colony PCR, and carrying out gene sequencing on a positive colony to obtain a gene sequence of the specific antibody. The amino acid sequence of each region of the specific antibody can be obtained by translating the gene sequence of the specific antibody according to the codon. The amino acid sequences of the specific antibodies finally obtained by B lymphocytes marked 1E4,1E5,1E6,5D3 are shown in Table 2.

TABLE 2 amino acid sequence of specific antibodies

SEQ ID NO.	Naming of	form/Source	Types of	Sequence of
					1	1E4	VH CDR1	aa	GFSLSSYY
2		VH CDR2	aa	IYDSGST
					3		VH CDR3	aa	ARTGSIYYIWLDL
4		VL CDR1	aa	QTIYKNND
					5		VL CDR2	aa	EAS
6		VL CDR3	aa	LGGYDDDDDNT
					7		VH	aa	QSLEESGGRLVTPGTPLTLTCTASGFSLSSYYMSWVRQAPGKGLEYIGIIYDSGSTWYANWAKGRFTISKTSTAVYLEITSPTTEDTATYFCARTGSIYYIWLD LWGQGTLVTVSS
8		VL	aa	AAVLTQTPSPVSAAVGGSVTISCQSSQTIYKNNDLAWYQQKPGQPPKLLIYEASKLASGVPSRFSGSGSGTQFTLTISGVQCDDAATYYCLGGYDDDDDNTFGG GTEVVVK
					9		scfv	aa	QSLEESGGRLVTPGTPLTLTCTASGFSLSSYYMSWVRQAPGKGLEYIGIIYDSGSTWYANWAKGRFTISKTSTAVYLEITSPTTEDTATYFCARTGSIYYIWL DLWGQGTLVTVSS AAVLTQTPSPVSAAVGGSVTISCQSSQTIYKNNDLAWYQQKPGQPPKLLIYEASKLASGVPSRFSGSGSGTQFTLTISGVQCDDAAT YYCLGGYDDDDDNTFGGGTEVVVK
10	1E5	VH CDR1	aa	GFTISSNA
					11		VH CDR2	aa	IDSSGSA
12		VH CDR3	aa	AGGSNI
					13		VL CDR1	aa	ESIYSY
14		VL CDR2	aa	LAS
					15		VL CDR3	aa	QSSYFSSGSSYGNT
16		VH	aa	QSVKESAGGLFKPTDTLTLTCTASGFTISSNAITWVRQAPGNGLEWIGGIDSSGSAYYASWAKSRSTITRNTNLNTVTLKMTSLTAADTATYFCAGGSNIWGPG TLVTVSS
					17		VL	aa	DIVMTQTPSSVSAAVGGTVTIKCQASESIYSYLSWYQQKPGQPPKLLIYLASTLDSGVPSRFKGSGSGTQFTLTISDLECADAATYYCQSSYFSSGSSYGNTFG GGTEVVVK
18		scfv	aa	QSVKESAGGLFKPTDTLTLTCTASGFTISSNAITWVRQAPGNGLEWIGGIDSSGSAYYASWAKSRSTITRNTNLNTVTLKMTSLTAADTATYFCAGGSNIWGP GTLVTVSS DIVMTQTPSSVSAAVGGTVTIKCQASESIYSYLSWYQQKPGQPPKLLIYLASTLDSGVPSRFKGSGSGTQFTLTISDLECADAATYYCQSSYFS SGSSYGNTFGGGTEVVVK
					19	1E6	VH CDR1	aa	GFDISSYS
20		VH CDR2	aa	GIIGGST
					21		VH CDR3	aa	ARFAGTSSTDYYDL
22		VL CDR1	aa	QSIDSY
					23		VL CDR2	aa	DTS
24		VL CDR3	aa	QNYWIITTFG
					25		VH	aa	QSVEESGGRLVTPGTPLTLACTVSGFDISSYSMSWVRQAPGKGLEWIGSGIIGGSTYYASWAKGRFTISKTSTTVDLKITSPTTEDTATYFCARFAGTSSTDYY DLWGQGTLVTVSS
26		VL	aa	TQTPSSVEAAVGGTVTIKCQASQSIDSYLAWYQQKPGQPPKLLIYDTSTLASGVSSRFKGSGSGTQFTLTISDLECADAATYYCQNYWIITTFGFGGGTEVVVK
					27		scfv	aa	QSVEESGGRLVTPGTPLTLACTVSGFDISSYSMSWVRQAPGKGLEWIGSGIIGGSTYYASWAKGRFTISKTSTTVDLKITSPTTEDTATYFCARFAGTSSTDY YDLWGQGTLVTVSS TQTPSSVEAAVGGTVTIKCQASQSIDSYLAWYQQKPGQPPKLLIYDTSTLASGVSSRFKGSGSGTQFTLTISDLECADAATYYCQNYW IITTFGFGGGTEVVVK
28	5D3	VH CDR1	aa	GFSLSNLR
					29		VH CDR2	aa	IISDSSGTT
30		VH CDR3	aa	ASRVNITSYDLSL
					31		VL CDR1	aa	KSVYNNKW
32		VL CDR2	aa	SAS
					33		VL CDR3	aa	AGGYSGSSDTYS
34		VH	aa	GGAEGGLVKPGGSLELCCKASGFSLSNLRVNWVRQAPGKGLEWIGCIISDSSGTTSYASWVNGRFTLSRDIDQNTGCLQLNSLTVADTAMYYCASRVNITSYDL SLWGQGTLVTVSS
					35		VL	aa	AAVLTQTPSPVSAAVGGTVSISCQSSKSVYNNKWLSWFQQKPGQPPKLLIYSASTLASGVPSRFSGSGSGTDFTLTISDVQCDDAATYYCAGGYSGSSDTYSFG GGTEVVVK
36		scfv	aa	GGAEGGLVKPGGSLELCCKASGFSLSNLRVNWVRQAPGKGLEWIGCIISDSSGTTSYASWVNGRFTLSRDIDQNTGCLQLNSLTVADTAMYYCASRVNITSYDL SLWGQGTLVTVSSAAVLTQTPSPVSAAVGGTVSISCQSSKSVYNNKWLSWFQQKPGQPPKLLIYSASTLASGVPSRFSGSGSGTDFTLTISDVQCDDAATYYCA GGYSGSSDTYSFGGGTEVVVK

(6) Production and characterization of CD123 Rabbit recombinant monoclonal antibody

The expression plasmid of heavy-light chain gene of antibody is CO-transfected into 293 cells at 37 ℃ and 5% CO ₂ Culturing for 72h under the culture condition, collecting cell supernatant, purifying the antibody by using proteinA resin, and carrying out SDS electrophoresis identification on the purified antibody, wherein the result is shown in figure 3.

The purified antibody is subjected to functional identification, and the functional identification is as follows:

a) ELISA reaction

Coating CD123 protein with the concentration of 1 mu g/ml, incubating for 16h at 4 ℃ in 100 mu l/hole; the next day, the coating solution was discarded, and blocking was performed with PBS containing 1% BSA, 150. Mu.l/well, and incubated at 37 ℃ for 1h. The blocking solution was discarded, and the diluted antibodies at different fold ratios were added, 50. Mu.l/well, and incubated at 37 ℃ for 1h. Discarding the antibody, washing the plate 5 times and 2 min/time with PBS containing 0.5% Tween-20, adding secondary goat anti-rabbit IgG-HRP antibody diluted 5000 times, and incubating at 37 deg.C for 1h. The secondary antibody was discarded and the plate was washed 5 times with PBST, 2 min/time. The wash was discarded, patted dry, and substrate was added for color development. The ELISA assay results are shown in FIG. 4.

b) Flow-based specificity determination method

CD123 full-length protein plasmids were transfected with 293 cells for testing. Namely, transfected cells are collected in a centrifuge tube, the cells are washed twice by sterile PBS, the Fc receptor confining liquid is adopted to confine Fc receptors on the surfaces of the cells, and the cells are incubated for 30min at 4 ℃. Cells were harvested by centrifugation, washed twice with PBS containing 0.5wt.% BSA, added with different concentrations of antibody and incubated at 4 ℃ for 30min. The cells were washed twice with PBS containing 0.5wt.% BSA and finally incubated with goat anti-rabbit IgG-488 fluorescent secondary antibody at 4 ℃ for 30min. After washing the cells twice with PBS containing 0.5wt.% BSA, 200 μ l PBS containing 0.5wt.% BSA was added to resuspend the cells and tested on the flow machine. The flow test specificity results are shown in FIGS. 5-8. The analysis result shows that the screened antibody can specifically recognize CD123 on the cell membrane.

c) Flow-based binding force determination method

The assay was performed with Raji cells. Namely, the cells are collected in a centrifuge tube, the cells are washed twice by sterile PBS, the Fc receptor blocking solution is adopted to block the Fc receptor on the cell surface, and the cells are incubated for 30min at 4 ℃. Cells were harvested by centrifugation, washed twice with PBS containing 0.5wt.% BSA, added with different concentrations of antibody and incubated at 4 ℃ for 30min. The cells were washed twice with PBS containing 0.5wt.% BSA and finally incubated with goat anti-rabbit IgG-488 fluorescent secondary antibody at 4 ℃ for 30min. After washing the cells twice with PBS containing 0.5wt.% BSA, 200 μ l PBS containing 0.5wt.% BSA was added to resuspend the cells and tested on the machine with a flow machine. The binding results of the flow assay are shown in FIG. 9. Flow-type binding assays showed that the antibodies screened had different binding capacity.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are also included in the scope of the present invention.

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<213> Artificial Sequence (Artificial Sequence)

<400> 18

Gln Ser Val Lys Glu Ser Ala Gly Gly Leu Phe Lys Pro Thr Asp Thr

1 5 10 15

Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Thr Ile Ser Ser Asn Ala

20 25 30

Ile Thr Trp Val Arg Gln Ala Pro Gly Asn Gly Leu Glu Trp Ile Gly

35 40 45

Gly Ile Asp Ser Ser Gly Ser Ala Tyr Tyr Ala Ser Trp Ala Lys Ser

50 55 60

Arg Ser Thr Ile Thr Arg Asn Thr Asn Leu Asn Thr Val Thr Leu Lys

65 70 75 80

Met Thr Ser Leu Thr Ala Ala Asp Thr Ala Thr Tyr Phe Cys Ala Gly

85 90 95

Gly Ser Asn Ile Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser Asp

100 105 110

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

115 120 125

Thr Val Thr Ile Lys Cys Gln Ala Ser Glu Ser Ile Tyr Ser Tyr Leu

130 135 140

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

145 150 155 160

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

165 170 175

Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Asp Leu Glu Cys Ala

180 185 190

Asp Ala Ala Thr Tyr Tyr Cys Gln Ser Ser Tyr Phe Ser Ser Gly Ser

195 200 205

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

210 215 220

<210> 19

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 19

Gly Phe Asp Ile Ser Ser Tyr Ser

1 5

<210> 20

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 20

Gly Ile Ile Gly Gly Ser Thr

1 5

<210> 21

<211> 14

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 21

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

1 5 10

<210> 22

<211> 6

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 22

Gln Ser Ile Asp Ser Tyr

1 5

<210> 23

<211> 3

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 23

Asp Thr Ser

1

<210> 24

<211> 10

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 24

Gln Asn Tyr Trp Ile Ile Thr Thr Phe Gly

1 5 10

<210> 25

<211> 117

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 25

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

1 5 10 15

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

20 25 30

Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly

35 40 45

Ser Gly Ile Ile Gly Gly Ser Thr Tyr Tyr Ala Ser Trp Ala Lys Gly

50 55 60

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

65 70 75 80

Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Phe Ala

85 90 95

Gly Thr Ser Ser Thr Asp Tyr Tyr Asp Leu Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 26

<211> 104

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 26

Thr Gln Thr Pro Ser Ser Val Glu Ala Ala Val Gly Gly Thr Val Thr

1 5 10 15

Ile Lys Cys Gln Ala Ser Gln Ser Ile Asp Ser Tyr Leu Ala Trp Tyr

20 25 30

Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Asp Thr Ser

35 40 45

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

50 55 60

Thr Gln Phe Thr Leu Thr Ile Ser Asp Leu Glu Cys Ala Asp Ala Ala

65 70 75 80

Thr Tyr Tyr Cys Gln Asn Tyr Trp Ile Ile Thr Thr Phe Gly Phe Gly

85 90 95

Gly Gly Thr Glu Val Val Val Lys

100

<210> 27

<211> 221

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 27

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

1 5 10 15

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

20 25 30

Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly

35 40 45

Ser Gly Ile Ile Gly Gly Ser Thr Tyr Tyr Ala Ser Trp Ala Lys Gly

50 55 60

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

65 70 75 80

Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Phe Ala

85 90 95

Gly Thr Ser Ser Thr Asp Tyr Tyr Asp Leu Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Thr Gln Thr Pro Ser Ser Val Glu Ala Ala Val

115 120 125

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

130 135 140

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu

145 150 155 160

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

165 170 175

Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Asp Leu Glu

180 185 190

Cys Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Asn Tyr Trp Ile Ile Thr

195 200 205

Thr Phe Gly Phe Gly Gly Gly Thr Glu Val Val Val Lys

210 215 220

<210> 28

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 28

Gly Phe Ser Leu Ser Asn Leu Arg

1 5

<210> 29

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 29

Ile Ile Ser Asp Ser Ser Gly Thr Thr

1 5

<210> 30

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 30

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

1 5 10

<210> 31

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 31

Lys Ser Val Tyr Asn Asn Lys Trp

1 5

<210> 32

<211> 3

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 32

Ser Ala Ser

1

<210> 33

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 33

Ala Gly Gly Tyr Ser Gly Ser Ser Asp Thr Tyr Ser

1 5 10

<210> 34

<211> 117

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 34

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

1 5 10 15

Cys Cys Lys Ala Ser Gly Phe Ser Leu Ser Asn Leu Arg Val Asn Trp

20 25 30

Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly Cys Ile Ile

35 40 45

Ser Asp Ser Ser Gly Thr Thr Ser Tyr Ala Ser Trp Val Asn Gly Arg

50 55 60

Phe Thr Leu Ser Arg Asp Ile Asp Gln Asn Thr Gly Cys Leu Gln Leu

65 70 75 80

Asn Ser Leu Thr Val Ala Asp Thr Ala Met Tyr Tyr Cys Ala Ser Arg

85 90 95

Val Asn Ile Thr Ser Tyr Asp Leu Ser Leu Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 35

<211> 112

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 35

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

1 5 10 15

Gly Thr Val Ser Ile Ser Cys Gln Ser Ser Lys Ser Val Tyr Asn Asn

20 25 30

Lys Trp Leu Ser Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu

35 40 45

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

50 55 60

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

65 70 75 80

Gln Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Ala Gly Gly Tyr Ser Gly

85 90 95

Ser Ser Asp Thr Tyr Ser Phe Gly Gly Gly Thr Glu Val Val Val Lys

100 105 110

<210> 36

<211> 229

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 36

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

1 5 10 15

Cys Cys Lys Ala Ser Gly Phe Ser Leu Ser Asn Leu Arg Val Asn Trp

20 25 30

Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly Cys Ile Ile

35 40 45

Ser Asp Ser Ser Gly Thr Thr Ser Tyr Ala Ser Trp Val Asn Gly Arg

50 55 60

Phe Thr Leu Ser Arg Asp Ile Asp Gln Asn Thr Gly Cys Leu Gln Leu

65 70 75 80

Asn Ser Leu Thr Val Ala Asp Thr Ala Met Tyr Tyr Cys Ala Ser Arg

85 90 95

Val Asn Ile Thr Ser Tyr Asp Leu Ser Leu Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ala Val Leu Thr Gln Thr Pro Ser Pro Val

115 120 125

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

130 135 140

Ser Val Tyr Asn Asn Lys Trp Leu Ser Trp Phe Gln Gln Lys Pro Gly

145 150 155 160

Gln Pro Pro Lys Leu Leu Ile Tyr Ser Ala Ser Thr Leu Ala Ser Gly

165 170 175

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

180 185 190

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

195 200 205

Gly Gly Tyr Ser Gly Ser Ser Asp Thr Tyr Ser Phe Gly Gly Gly Thr

210 215 220

Glu Val Val Val Lys

225

Claims

1. A rabbit recombinant monoclonal antibody that binds to CD123 protein, characterized by: one or more of the following rabbit recombinant monoclonal antibodies:

rabbit recombinant monoclonal antibody designated 1E 4: the amino acid sequences of the heavy chain complementary determining regions CDR1, CDR2 and CDR3 are respectively the amino acid sequences shown in SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3; and the amino acid sequences of the light chain complementarity determining regions CDR1, CDR2 and CDR3 are respectively the amino acid sequences shown in SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6;

the rabbit recombinant monoclonal antibody named as 1E6 has the amino acid sequences shown in SEQ ID No. 19, SEQ ID No. 20 and SEQ ID No. 21 as the heavy chain complementary determining region CDR1, CDR2 and CDR3, and the amino acid sequences shown in SEQ ID No. 22, SEQ ID No. 23 and SEQ ID No. 24 as the light chain complementary determining region CDR1, CDR2 and CDR 3;

the amino acid sequences of the heavy chain complementarity determining regions CDR1, CDR2 and CDR3 of the rabbit recombinant monoclonal antibody named 5D3 are respectively the amino acid sequences shown in SEQ ID NO. 28, SEQ ID NO. 29 and SEQ ID NO. 30, and the amino acid sequences of the light chain complementarity determining regions CDR1, CDR2 and CDR3 are respectively the amino acid sequences shown in SEQ ID NO. 22, SEQ ID NO. 23 and SEQ ID NO. 24.

2. The rabbit recombinant monoclonal antibody that binds to CD123 protein according to claim 1, wherein:

the rabbit recombinant monoclonal antibody named as 1E4 has the amino acid sequence shown in SEQ ID NO. 7 as the heavy chain variable region sequence and the amino acid sequence shown in SEQ ID NO. 8 as the light chain variable region sequence;

the rabbit recombinant monoclonal antibody named as 1E5 has the heavy chain variable region sequence of the amino acid sequence shown in SEQ ID NO. 16 and the light chain variable region sequence of the amino acid sequence shown in SEQ ID NO. 17;

the rabbit recombinant monoclonal antibody named as 1E6 has a heavy chain variable region sequence of an amino acid sequence shown as SEQ ID NO. 25 and a light chain variable region sequence of an amino acid sequence shown as SEQ ID NO. 26;

the rabbit recombinant monoclonal antibody named as 5D3 has the heavy chain variable region sequence as shown in SEQ ID No. 34 and the light chain variable region sequence as shown in SEQ ID No. 35.

3. The rabbit recombinant monoclonal antibody that binds to CD123 protein according to claim 1 or 2, characterized in that:

on the basis of the technical scheme, the SCFV sequence of the rabbit recombinant monoclonal antibody named as 1E4 is an amino acid sequence shown as SEQ ID NO. 9;

the SCFV sequence of the rabbit recombinant monoclonal antibody named as 1E5 is an amino acid sequence shown in SEQ ID NO. 18;

the SCFV sequence of the rabbit recombinant monoclonal antibody named as 1E6 is an amino acid sequence shown in SEQ ID NO. 27;

the SCFV sequence of the rabbit recombinant monoclonal antibody named 5D3 is the amino acid sequence shown in SEQ ID NO. 36.

4. The rabbit recombinant monoclonal antibody that binds to CD123 protein according to claim 1, wherein: the light chain constant region of the rabbit recombinant monoclonal antibody is a kappa chain, and the heavy chain constant region is an IgG type.

5. A nucleic acid molecule, characterized in that: comprising a nucleic acid sequence capable of encoding a heavy chain complementarity determining region or a light chain complementarity determining region of a rabbit recombinant monoclonal antibody that binds CD123 protein according to any one of claims 1-4.

6. A carrier, characterized by: comprising the nucleic acid molecule of claim 5.

7. A host cell, characterized in that: the host cell comprising the rabbit recombinant monoclonal antibody that binds to a CD123 protein according to any one of claims 1 to 4, the nucleic acid molecule of claim 5, or the vector of claim 6.

8. A conjugate, characterized by: comprising the antibody of any one of claims 1 to 4.

9. A pharmaceutical composition characterized by: contains a main component and an auxiliary component, wherein the main component contains one or more of the rabbit recombinant monoclonal antibody which binds to the CD123 protein and is described in any one of claims 1 to 4, the nucleic acid molecule described in claim 5, the vector described in claim 6, the host cell described in claim 7, and the conjugate described in claim 8, and the auxiliary component is selected from pharmaceutically acceptable carriers or excipients, and other optional bioactive substances.

10. Use of the rabbit recombinant monoclonal antibody that binds to CD123 protein according to any one of claims 1 to 4, the nucleic acid molecule of claim 5, the vector of claim 6, the host cell of claim 7, or the conjugate of claim 8 for the preparation of a medicament or a detection reagent for the treatment of a disease.