CN115433280A - Preparation and application of anti-CD 22 rabbit recombinant monoclonal antibody - Google Patents

Abstract

The invention relates to preparation and application of a CD22 rabbit recombinant monoclonal antibody, wherein the obtained antibody has good specificity, and most importantly, the antibody has high affinity, can identify CD22 protein on the surface of a natural cell, and can be used for flow-type and ELISA detection applications.

Description

Preparation and application of anti-CD 22 rabbit recombinant monoclonal antibody

Technical Field

The invention belongs to the field of antibody preparation in biotechnology, and particularly relates to a rabbit recombinant monoclonal antibody capable of specifically binding CD22 protein and application thereof.

Background

CD22 belongs to type I transmembrane glycoprotein, is a member of sialic acid binding immunoglobulin-like lectin family, is also an inhibitory co-receptor of B cell receptors, and has the function of negatively regulating B cell activation signals. CD22 is capable of binding specifically to glycoprotein ligands containing alpha-2,6 linked sialic acid, antigen-activating BCR, also rapidly phosphorylating tyrosine in the tyrosine inhibitory motif of the immunoreceptor in the CD22 cytoplasmic domain, and activating downstream signaling molecules to inhibit calcium influx and attenuate BCR signaling. CD22 is relatively specifically expressed on the surface of B cells and is now a good target for modulating B cell immunity and treating certain B cell tumors.

In the current state of the art, some types of anti-CD 22 antibodies have been developed. For example, chinese patent application CN2020100889800 developed a chimeric antigen receptor targeting humanized CD22 and its use. Although the antibody also has a certain application prospect, the requirement on the antibody in the flow-type and ELISA detection application is higher, and the antibody does not necessarily completely meet the requirement of the current detection, so that the development of monoclonal antibodies with stronger affinity and more epitopes is still needed.

Disclosure of Invention

The invention aims to provide an anti-CD 22 rabbit recombinant monoclonal antibody, which is selected from one or more of the following rabbit recombinant monoclonal antibodies:

rabbit recombinant monoclonal antibody designated 8B 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. 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 9 A3: 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 rabbit recombinant monoclonal antibody named 10F9 has the amino acid sequences shown in SEQ ID No. 19, SEQ ID No. 20 and SEQ ID No. 21 as the heavy chain complementarity 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 complementarity determining regions CDR1, CDR2 and CDR 3;

on the basis of the technical scheme, the rabbit recombinant monoclonal antibody named as 8B5 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 9A3 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 10F9 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;

on the basis of the technical scheme, the rabbit recombinant monoclonal antibody named as 8B5 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 9A3 is an amino acid sequence shown in SEQ ID NO. 18;

the SCFV sequence of the rabbit recombinant monoclonal antibody named 10F9 is an amino acid sequence shown in SEQ ID NO. 27;

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 rabbit recombinant monoclonal antibody that binds to CD22 protein.

The invention also provides a vector comprising the nucleic acid molecule.

The invention also provides a host cell which contains the rabbit recombinant monoclonal antibody combined with the CD22 protein, 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 rabbit recombinant monoclonal antibody which binds to the CD22 protein, the nucleic acid molecule, the carrier, the host cell and the conjugate, and the accessory component is selected from a pharmaceutically acceptable carrier or excipient and other optional bioactive substances.

The invention also provides the application of the rabbit recombinant monoclonal antibody combined with the CD22 protein, 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 rabbit recombinant monoclonal antibody combined with the CD22 protein.

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

at present, no rabbit-derived monoclonal antibody for CD22 exists, and because the affinity of the rabbit-derived antibody is higher than that of the rabbit-derived monoclonal antibody, and the immunization process of rabbits is more complicated compared with that of mice, antibodies with more epitopes can be obtained. After a rabbit is immunized by the CD22 extracellular region protein, the anti-CD 22 rabbit recombinant monoclonal antibody is prepared by a B cell cloning technology, and importantly, the anti-CD 22 rabbit recombinant monoclonal antibody has high affinity and good specificity, can identify the CD22 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 CD22 target.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the description below are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 shows the SDS electrophoretogram of CD22 protein.

FIG. 2 shows the result of PCR amplification of heavy and light chains of anti-CD 22 rabbit recombinant monoclonal antibody.

FIG. 3 shows SDS electrophorograms of expression purified rabbit recombinant monoclonal antibodies to CD22.

FIGS. 4-6 show the specificity of FACs to detect CD22 rabbit recombinant monoclonal antibodies.

FIG. 7 shows that FACs detect the binding capacity of the CD22 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 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, as used herein, are all terms and routine procedures used extensively in the relevant arts. 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.

Example (b):

(1) Preparation of CD22 protein: the invention constructs CD22 FC fusion tag protein eukaryotic expression plasmid, transfects 293F cell for 5 days, collects cell culture medium supernatant, purifies by using protein A resin, and identifies the concentration of protein, and the identification result of protein expression purification is shown in figure 1.

(2) Obtaining peripheral blood mononuclear cells (namely PBMC cells) of the immune animals: new Zealand white rabbits were selected as immunized animals. The first immunization is carried out by emulsifying 250 mu g of CD22 protein and an equal volume of complete Freund's adjuvant, injecting subcutaneous back of New Zealand white rabbit at multiple points, carrying out the second immunization 21 days later, emulsifying 120 mu g of protein and an equal volume of incomplete Freund's adjuvant, carrying out subcutaneous injection on the back of New Zealand white rabbit, carrying out the third immunization 21 days later, emulsifying 120 mu g of protein and an equal volume of incomplete Freund's adjuvant, and carrying out subcutaneous injection on the back of New Zealand white rabbit. After one week of interval after the third immunization, peripheral blood was collected aseptically.

(3) Acquisition of CD 22-specific B lymphocytes: separating PBMC cells from the collected peripheral blood by using lymphocyte separation liquid; coupling CD22 protein to magnetic beads according to the immune magnetic bead operation instruction; and (3) incubating the magnetic beads coupled with the CD22 protein and the separated PBMC cells for 50min at room temperature, putting the mixture into a magnetic frame, immersing all the magnetic beads into 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 CD22 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-well cell culture plate, adding 1640 culture medium containing 10% Fetal Bovine Serum (FBS) and 2 mu g/ml human IL2, and adding 5% CO at 37 DEG C ₂ Cultured under the conditions for 6 days. Harvesting machineAnd collecting the culture medium supernatant for antibody identification.

And (3) indirect ELISA identification:

coating CD22 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, B cell supernatant was added to 50. Mu.l/well, and incubated at 37 ℃ for 1h. 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 washed 5 times with phosphate Tween buffer (PBST), 2 min/time. The wash was discarded, patted dry, and substrate was added for color development. The B cell clones marked 8B5,9A3, 10F9 were identified as positive by ELISA and the results are shown in Table 1.

TABLE 1 ELISA test results

Clone number	OD450
		8B5	1.93
9A3	2.12
		10F9	1.86

(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 of 5-: an upstream primer 5-: denaturation was carried out at 94 ℃ for 3min, (1 min at 95 ℃, 30s at 56 ℃ and 1min at 72 ℃) for 30 cycles, and finally extension was carried out at 72 ℃ for 10min (the PCR amplification results are shown in FIG. 2). And (3) 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 labeled as 8B5,9A3 and 10F9 are shown in Table 2.

TABLE 2 amino acid sequence of specific antibodies

SEQ ID NO.	Name of	form/Source	Types of	Sequence of
					1	8B5	VH CDR1	aa	GIDLSSYA
2		VH CDR2	aa	VTYAGSA
					3		VH CDR3	aa	ARGVDNFYTLPDAFDP
4		VL CDR1	aa	QSISRF
					5		VL CDR2	aa	RAS
6		VL CDR3	aa	QQGDTYINVDNP
					7		VH	aa	QEQLKESGGGLVTPGGSLTLTCTVSGIDLSSYAMGWVRQAPGKGLEYIGIVTYAGSAYYASWARGRFTISKTSTTVDLKITSPTTEDTA TYFCARGVDNFYTLPDAFDPWGPGTLVTVS
8		VL	aa	AYDMTQTPASVSAAVGGTVTINCQASQSISRFLSWYQQKPGQRPKLLIYRASTLASGVSSRFEGSGSGTQFTLTISGVECADAATYYCQ QGDTYINVDNPFGGGTEVVVK
					9		scfv	aa	QEQLKESGGGLVTPGGSLTLTCTVSGIDLSSYAMGWVRQAPGKGLEYIGIVTYAGSAYYASWARGRFTISKTSTTVDLKITSPTTEDTA TYFCARGVDNFYTLPDAFDPWGPGTLVTVSAYDMTQTPASVSAAVGGTVTINCQASQSISRFLSWYQQKPGQRPKLLIYRASTLASGVS SRFEGSGSGTQFTLTISGVECADAATYYCQQGDTYINVDNPFGGGTEVVVK
10	9A3	VH CDR1	aa	GIDLSSYW
					11		VH CDR2	aa	IWSDDNT
12		VH CDR3	aa	ARGYVGDI
					13		VL CDR1	aa	QNIYSG
14		VL CDR2	aa	GAS
					15		VL CDR3	aa	QQTYTSDNVDNP
16		VH	aa	QSVEESGGRLVTPGTPLTLTCTVSGIDLSSYWMNWVRQAPGKGLEWIGIIWSDDNTYYANWAKGRFTISKTSSTTVDLTITSPTTEDTA TYFCARGYVGDIWGPGTLITVS
					17		VL	aa	ALVMTQTPSSVDVAVGGTVTIRCQASQNIYSGLAWYQQKPGQPPKLLIYGASNLASGVSSRFKGSGSGTQFTLTISGVECADAATYYCQ QTYTSDNVDNPFGGGTEVVVE
18		scfv	aa	QSVEESGGRLVTPGTPLTLTCTVSGIDLSSYWMNWVRQAPGKGLEWIGIIWSDDNTYYANWAKGRFTISKTSSTTVDLTITSPTTEDTA TYFCARGYVGDIWGPGTLITVSALVMTQTPSSVDVAVGGTVTIRCQASQNIYSGLAWYQQKPGQPPKLLIYGASNLASGVSSRFKGSGS GTQFTLTISGVECADAATYYCQQTYTSDNVDNPFGGGTEVVVE
					19	10F9	VH CDR1	aa	GFSLNTDA
20		VH CDR2	aa	IGSSGSA
					21		VH CDR3	aa	ARFTGYGGYGRGGMDP
22		VL CDR1	aa	ESVHNNNW
					23		VL CDR2	aa	KAS
24		VL CDR3	aa	QATYNSGGWYVA
					25		VH	aa	QSVEESGGRLVTPGTPLTLTCTVSGFSLNTDAMIWVRQAPGKGLEYIGIIGSSGSAFFASWAKGRFTISKTSTTVDLTIISPTTEDTAT YFCARFTGYGGYGRGGMDPWGPGTLVTVSS
26		VL	aa	AQVLTQTPSSVSAAVGGTVTINCQSSESVHNNNWLSWYQQKPGQPPKLLIYKASTLASGVPSRFSGSGSGTQFTLTISGVQCDDAATYY CQATYNSGGWYVAFGGGTEVVVK
					27		scfv	aa	QSVEESGGRLVTPGTPLTLTCTVSGFSLNTDAMIWVRQAPGKGLEYIGIIGSSGSAFFASWAKGRFTISKTSTTVDLTIISPTTEDTAT YFCARFTGYGGYGRGGMDPWGPGTLVTVSSAQVLTQTPSSVSAAVGGTVTINCQSSESVHNNNWLSWYQQKPGQPPKLLIYKASTLASG VPSRFSGSGSGTQFTLTISGVQCDDAATYYCQATYNSGGWYVAFGGGTEVVVK

(6) Production and identification of CD22 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 CD22 protein with the concentration of 1 mu g/ml, 100 mu l/hole, and incubating for 16h at 4 ℃; the next day, the coating solution was discarded, and the cells were blocked 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 with PBS containing 0.5% Tween-20 for 5 times and 2 min/time, adding a secondary goat anti-rabbit IgG-HRP antibody diluted by 5000 times, and incubating at 37 ℃ 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 Table 2.

TABLE 2 ELISA test results

Dilution factor	8B5	9A3	10F9
				1K	Overflow	3.9542	Overflow
5K	Overflow	Overflow	Overflow
				25K	3.5169	3.1039	3.3671
125K	1.9231	1.3259	1.3943
				625K	0.5284	0.3816	0.4051
3125k	0.2027	0.1796	0.1714
				15625k	0.1459	0.1204	0.1246
blank	0.1162	0.1326	0.1123

b) Flow-based specificity determination method

CD22 full-length protein plasmids were transfected with 293 cells for testing. Namely, the transfected 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 flow test specificity results are shown in FIGS. 4-6. The analysis results show that the screened antibody can specifically recognize CD22 on the cell membrane.

(c) Flow-type 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 flow machine. The binding results of the flow assay are shown in FIG. 7. Flow-type binding analysis showed that the screened antibodies 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.

Sequence listing

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<120> preparation and application of anti-CD 22 rabbit recombinant monoclonal antibody

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<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 24

Gln Ala Thr Tyr Asn Ser Gly Gly Trp Tyr Val Ala

1 5 10

<210> 25

<211> 119

<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 Thr Cys Thr Val Ser Gly Phe Ser Leu Asn Thr Asp Ala

20 25 30

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

35 40 45

Ile Ile Gly Ser Ser Gly Ser Ala Phe Phe Ala Ser Trp Ala Lys Gly

50 55 60

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

65 70 75 80

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

85 90 95

Gly Tyr Gly Gly Tyr Gly Arg Gly Gly Met Asp Pro Trp Gly Pro Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 26

<211> 112

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 26

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

1 5 10 15

Gly Thr Val Thr Ile Asn Cys Gln Ser Ser Glu Ser Val His Asn Asn

20 25 30

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

35 40 45

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

50 55 60

Ser Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Gly Val

65 70 75 80

Gln Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Gln Ala Thr Tyr Asn Ser

85 90 95

Gly Gly Trp Tyr Val Ala Phe Gly Gly Gly Thr Glu Val Val Val Lys

100 105 110

<210> 27

<211> 231

<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 Thr Cys Thr Val Ser Gly Phe Ser Leu Asn Thr Asp Ala

20 25 30

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

35 40 45

Ile Ile Gly Ser Ser Gly Ser Ala Phe Phe Ala Ser Trp Ala Lys Gly

50 55 60

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

65 70 75 80

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

85 90 95

Gly Tyr Gly Gly Tyr Gly Arg Gly Gly Met Asp Pro Trp Gly Pro Gly

100 105 110

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

115 120 125

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

130 135 140

Ser Glu Ser Val His Asn Asn Asn Trp Leu Ser Trp Tyr Gln Gln Lys

145 150 155 160

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

165 170 175

Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Gln Phe

180 185 190

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

195 200 205

Cys Gln Ala Thr Tyr Asn Ser Gly Gly Trp Tyr Val Ala Phe Gly Gly

210 215 220

Gly Thr Glu Val Val Val Lys

225 230

Claims (10)

1. A rabbit recombinant monoclonal antibody that binds to CD22 protein, characterized by: one or more rabbit recombinant monoclonal antibodies selected from the group consisting of:

rabbit recombinant monoclonal antibody designated 1D 12: 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;

rabbit recombinant monoclonal antibody designated 1H 2: 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 2B3 are respectively the amino acid sequences shown in 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 in SEQ ID NO. 22, SEQ ID NO. 23 and SEQ ID NO. 24.

2. The rabbit recombinant monoclonal antibody that binds to a CD22 protein of claim 1, wherein:

the heavy chain variable region sequence of the rabbit recombinant monoclonal antibody named as 1D12 is an amino acid sequence shown by SEQ ID NO. 7, and the light chain variable region sequence is an amino acid sequence shown by SEQ ID NO. 8;

the rabbit recombinant monoclonal antibody named as 1H2 has a heavy chain variable region sequence of an amino acid sequence shown as SEQ ID NO. 16 and a light chain variable region sequence of an amino acid sequence shown as SEQ ID NO. 17;

the rabbit recombinant monoclonal antibody named as 2B3 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.

3. The rabbit recombinant monoclonal antibody that binds to CD22 protein according to claim 1 or 2, characterized in that:

the SCFV sequence of the rabbit recombinant monoclonal antibody named as 1D12 is an amino acid sequence shown in SEQ ID NO. 9;

the SCFV sequence of the rabbit recombinant monoclonal antibody named as 1H2 is an amino acid sequence shown in SEQ ID NO. 18;

the SCFV sequence of the rabbit recombinant monoclonal antibody named as 2B3 is an amino acid sequence shown as SEQ ID NO. 27.

4. The rabbit recombinant monoclonal antibody that binds to a CD22 protein of 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 encoding a heavy chain complementarity determining region or a light chain complementarity determining region of the rabbit recombinant monoclonal antibody that binds to CD22 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 binding to CD22 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 CD22 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 a rabbit recombinant monoclonal antibody that binds to a CD22 protein according to any one of claims 1 to 4, a nucleic acid molecule according to claim 5, a vector according to claim 6, a host cell according to claim 7, or a conjugate according to claim 8, for the preparation of a medicament or a test agent for the treatment of a disease.

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