CN111378043A

CN111378043A - Human-mouse chimeric anti-Siglec-15 whole-molecule IgG with neutralization function and preparation method and application thereof

Info

Publication number: CN111378043A
Application number: CN202010103042.3A
Authority: CN
Inventors: 唐奇; 毛圆; 唐小军; 冯振卿; 朱进; 肖雪筠
Original assignee: Nanjing Medical University
Current assignee: Shenzhen small molecule New Drug Innovation Center Co.,Ltd.
Priority date: 2020-02-19
Filing date: 2020-02-19
Publication date: 2020-07-07
Anticipated expiration: 2040-02-19
Also published as: CN111378043B

Abstract

The invention discloses a human-mouse chimeric anti-Siglec-15 full-molecular IgG with a neutralization function, a preparation method and application thereof, belonging to the field of biological pharmacy; the invention uses Siglec-15 specific polypeptide to immunize a mouse, adopts a hybridoma technology to prepare a mouse anti-Siglec-15 monoclonal antibody, and adopts a genetic engineering technology and an antibody engineering technology to prepare recombinant human-mouse chimeric anti-Siglec-15 full-molecular IgG; the chimeric antibody can effectively recognize the specific amino acid fragment of the extracellular region of Siglec-15 and inhibit the combination of the Siglec-15 protein and the Sialyl-Tn protein; the invention discloses a human-mouse chimeric anti-Siglec-15 whole-molecule IgG, which comprises a light chain variable region and a heavy chain variable region, wherein the amino acid sequence of the light chain variable region is shown as SEQ ID NO.5, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO.6, or a conservative variant obtained by conservative mutation of the sequence through one or more amino acid additions, deletions, substitutions and modifications; the invention also discloses a DNA molecule, an expression vector, a host cell and application of the whole IgG antibody.

Description

Human-mouse chimeric anti-Siglec-15 whole-molecule IgG with neutralization function and preparation method and application thereof

Technical Field

The invention belongs to the field of biological pharmacy, and relates to a human-mouse chimeric anti-Siglec-15 whole-molecule IgG antibody, a DNA molecule, an expression vector, a host cell and application of the whole-molecule IgG antibody.

Background

Sialic acid binds to the Ig-like lectin family, the Siglecs family, and is a classical immunoglobulin-like lectin protein family. Under physiological conditions, only 15 Siglec molecules are expressed on the surface of myeloid cells and immune cells that mediate immunosuppression, and have immunosuppressive properties. Siglecs, when recognizing sialic acid containing glycans expressed by all mammalian cells, can help immune cells to distinguish between self and non-self. Sialylated pathogens can also disrupt and promote immune responses through Siglec-dependent interactions, or down-regulate immune cell responses and escape immune surveillance. Siglecs play an important role in the regulation of immune cell activation and inhibitory receptors, which can influence host-pathogen interactions, neurodegeneration, autoimmune diseases regulating osteoclast differentiation and cancer. Due to its limited expression on immune cells, endocytic properties and the ability to modulate receptor signaling, it has become an important target for cell-directed therapies.

Siglec-15, among others, acts as a cell surface transmembrane receptor and consists of an immunoglobulin (Ig) -like domain, a transmembrane domain, and a short cytoplasmic tail. The immunoglobulin-like domain consists of 2 extracellular Ig domains, including an N-terminal V-set domain containing a sialic acid binding site and a type 2 constant region (IgC2) region. It associates with the tyrosine activation motif (ITAM) adaptor molecule DAP12 through a positively charged amino acid in its transmembrane domain and activates the receptor by recruiting SYK kinase.

Siglec-15 differs from other family members according to phylogenetic analysis. It shows a similar domain composition and high homology to B7-H1 as well as many other B7 family members. More importantly, the expression of Siglec-15 and B7-H1 in human lung cancer tissues is mutually exclusive, so Siglec-15 may have a unique regulatory mechanism in addition to the immunomodulatory function as may be the B7 family members: siglec-15 was found to bind to no other receptors other than TCR and CD44, and did not interact with B7-H1, PD-1, B7-1, or any other known B7 family ligand or receptor. The characteristics of high expression of abnormal polysaccharide components on the surface of cancer cells and secretion of polysaccharide polymers also enable the tumor polysaccharide map to be used as a clinical cancer biomarker and widely used for cancer diagnosis. The property of Siglecs to recognize tumor-associated sTn antigens has led to an increasing number of therapeutic drugs targeting Siglecs or glycosylated ligands being developed in succession and used for the treatment of various siglec-associated diseases, such as lymphoma, leukemia and autoimmune diseases. The micro-domains of the tumor cell surface sialoglycan lead to immune escape of the cancer, resulting in enhanced tumor growth, invasion and metastatic capacity.

Siglec-15 is expressed on Tumor Associated Macrophages (TAMs) in various human tumor tissues in vitro experiments further demonstrated its expression in macrophage colony stimulating factor (M-CSF) -induced M2-like macrophages significantly increased Siglec-15 binds to the 12kDa linker protein DNAX activator protein (DAP12) on the binding determinant Lys (274) of the transmembrane region and transduces signals to spleen tyrosine kinase (Syk). Syk inhibitors treat THP-1 cells or replace Siglec-15Lys (274Lys) with ALA, thereby disrupting the molecular interaction between Siglec-15 and DAP12 and significantly inhibiting the secretion of TGF- β by THp-1 cells.

Most of the antibodies using Siglec-15 as a target point, which are published at present, are mainly used for diagnosis and scientific research, and there is no antibody which can be clearly combined with a specific amino acid region of the extracellular region of Siglec-15 and has the function of inhibiting or blocking the combination of Siglec-15. The antibody prepared by the invention can be effectively combined with the specific fragment of the extracellular region of the Siglec-15 protein, and can block the combination of the antibody and the sTn protein, thereby showing good neutralization property, and no anti-Siglec-15 antibody patent report with related functions exists.

Disclosure of Invention

The invention aims to provide a human-mouse chimeric anti-Siglec-15 antibody IgG with a neutralization function, a nucleic acid and amino acid sequence of a CDR region of the antibody, a nucleic acid and amino acid sequence of a variable region of the antibody, a whole-molecule amino acid sequence of the antibody, an antigen-binding epitope amino acid sequence, a method for preparing a mouse anti-Siglec-15 antibody, a method for preparing the human-mouse chimeric anti-Siglec-15 whole-molecule IgG and application of the human-mouse chimeric anti-Siglec-15 antibody IgG in inhibiting a binding reaction of Siglec-15 and sTn protein.

The invention provides a human-mouse chimeric anti-Siglec-15 full-molecular IgG, wherein the nucleic acid sequence of an antibody light chain variable region is shown in SEQ ID No.1, and the nucleic acid sequence of an antibody heavy chain variable region is shown in SEQ ID No. 2. The amino acid sequence of the light chain variable region of the anti-Siglec-15 antibody is shown as SEQ ID NO.3, and the amino acid sequence of the heavy chain variable region of the anti-Siglec-15 antibody is shown as SEQ ID NO. 4. The full-length nucleic acid sequence (containing a constant region) of the anti-Siglec-15 antibody light chain is SEQ ID NO.5, and the amino acid sequence is SEQ ID NO. 7; the heavy chain full-length nucleic acid sequence (containing a constant region) is SEQ ID NO.6, and the amino acid sequence is SEQ ID NO. 8. The nucleic acid sequence of the anti-Siglec-15 antibody light chain antigen complementary region CDR is SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO. 11; the amino acid sequence is shown in SEQ ID NO.15, SEQ ID NO.16 and SEQ ID NO. 17. The nucleic acid sequence of the anti-Siglec-15 antibody heavy chain antigen complementary region CDR is SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO. 14; the amino acid sequence is shown in SEQ ID NO.18, SEQ ID NO.19 and SEQ ID NO. 20. The antigen binding epitope amino acid sequence is at least one of the following amino acid sequences: SEQ ID NO.21, SEQ ID NO.22 and SEQ ID NO. 23.

The invention provides a human-mouse chimeric anti-Siglec-15 whole-molecule IgG antibody, which comprises a light chain variable region and a heavy chain variable region, wherein the nucleic acid sequence of the light chain variable region is shown in SEQ ID NO.1, and the nucleic acid sequence of the heavy chain variable region is shown in SEQ ID NO. 2.

Further, the amino acid sequence of the light chain variable region is shown in SEQ ID NO.3, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 4.

Further, the nucleic acid sequences of the antigen complementary region CDR of the light chain variable region are respectively shown as SEQ ID NO.9, SEQ ID NO.10 and SEQ ID NO. 11; the nucleic acid sequences of the antigen complementary region CDR of the heavy chain variable region are respectively shown in SEQ ID NO.12, SEQ ID NO.13 and SEQ ID NO. 14.

Further, the amino acid sequences of the antigen complementary region CDR of the light chain variable region are respectively shown as SEQ ID NO.15, SEQ ID NO.16 and SEQ ID NO. 17; the amino acid sequences of the antigen complementary region CDR of the heavy chain variable region are respectively shown as SEQ ID NO.18, SEQ ID NO.19 and SEQ ID NO. 20.

The invention provides a DNA molecule which encodes the heavy chain or/and the light chain of the whole IgG antibody.

Further, the light chain nucleic acid sequence is shown as SEQ ID NO.5, and the heavy chain nucleic acid sequence is shown as SEQ ID NO. 6.

Further, the light chain amino acid sequence is shown as SEQ ID NO.7, and the heavy chain amino acid sequence is shown as SEQ ID NO. 8.

The invention provides an expression vector, which comprises the DNA molecule and an expression regulation and control sequence operatively connected with the DNA molecule.

The invention provides a method for expressing a protein in a eukaryotic cell, wherein the method is used for expressing a nucleic acid and an amino acid sequence containing the antibody.

The invention provides a human-mouse chimeric anti-Siglec-15 whole-molecule IgG antibody, wherein a characteristic epitope in the whole-molecule IgG antibody is combined with A37-A180 in a Siglec-15 protein molecule, and the combination of the Siglec-15 protein and Sialyl-Tn protein is blocked; the characteristic epitope is at least one of the following amino acid sequences: SEQ ID NO.21, SEQ ID NO.22, SEQ ID NO. 23.

The invention provides application of a full-molecular IgG antibody in preparing a tumor diagnostic reagent or a therapeutic drug for inhibiting Siglec-15 functions, wherein the full-molecular IgG antibody is the human-mouse chimeric anti-Siglec-15 full-molecular IgG.

The invention provides a medicament for inhibiting biological functions of Siglec-15, which contains the human-mouse chimeric anti-Siglec-15 whole-molecule IgG.

Has the advantages that:

currently, humanized antibody technology and genetic engineering technology have matured and become important methods for altering the immunogenicity and functional characteristics of antibody molecules themselves, while maintaining the specific recognition specificity of the antibody. The currently published antibodies related to Siglec-15 are mainly mice, and specific epitopes bound to Siglec-15 are not clear, so that the antibodies are mainly used for clinical detection and development. The anti-Siglec-15 antibody prepared by the invention has higher binding activity and affinity, can be specifically combined with the Siglec-15 (A37-A180) amino acid segment, blocks the combination of the Siglec-15 protein and the Sialyl-Tn protein, and shows good neutralization property. At present, no patent report about anti-Siglec-15 humanized antibody with blocking function and definite epitope exists.

Drawings

FIG. 1 is a graph showing the results of the ELISA experiment in example 5;

FIG. 2 is a graph showing the results of SDS-PAGE in example 5;

FIG. 3 is a graph showing the result of Western-blot detection in example 5;

FIG. 4 is a graph showing the results of the affinity assay for Biacore T100 antibody in example 5; wherein KD is 8.532 e-8;

FIG. 5 is a graph showing the results of detection of epipe Mapping in example 5;

FIG. 6 is a graph showing the results of competitive inhibition of rsTn protein by the S154EA6 antibody in example 6;

FIG. 7 is a graph showing the results of competitive ELISA of the S154EA6 antibody with an epitope in example 6;

FIG. 8 is the effect of the S154EA6 antibody on the proliferation of different tumor cells in example 6;

Detailed Description

Siglec-15 extracellular region peptide fragment design.

2. Preparation of murine anti-Siglec-15 hybridoma cells.

3. Screening, strain determination and identification of murine anti-Siglec-15 antibodies.

4. Preparing, expressing and affinity purifying human and mouse chimeric anti-Siglec-15 whole IgG.

5. Characterization of human murine chimeric anti-Siglec-15 Whole molecule antibodies.

6. And (3) detecting the competitive inhibition effect of the anti-Siglec-15 whole-molecule IgG on the Sialyl-Tn protein.

Example 1: siglec-15 extracellular region peptide fragment design

Siglec-15 has a total of 328 amino acids, and the extracellular region consists of an N-terminal V-set domain containing a sialic acid binding site (A49-A165) and a type 2 constant region (IgC2) region (A168-A251), wherein the IgV region is a key site for the binding of Siglec-15 to Sialyl-Tn. Siglec-15A37-A180 amino acid is used as a template to design the polypeptide of the Overlap, which is respectively named A1-A12 and B1-B11. After polypeptide synthesis, parts were mixed and coupled to OVA and KLH, respectively.

A1	HSSPAQRWSMQV	B1	RWSMQVPPEVSA
				A2	PPEVSAEAGDAA	B2	EAGDAAVLPCTF
A3	VLPCTFTHPHRH	B3	THPHRHYDGPLT
				A4	YDGPLTAIWRAG	B4	AIWRAGEPYAGP
A5	EPYAGPQVFRCA	B5	QVFRCAAARGSE
				A6	AARGSELCQTAL	B6	LCQTALSLHGRF
A7	SLHGRFRLLGNP	B7	RLLGNPRRNDLS
				A8	RRNDLSLRVERL	B8	LRVERLALADDR
A9	ALADDRRYFCRV	B9	RYFCRVEFAGDV
				A10	EFAGDVHDRYES	B10	HDRYESRHGVRL
A11	RHGVRLHVTAAP	B11	HVTAAPRIVNIS
				A12	RIVNISVLPSPA

Example 2: preparation of murine anti-Siglec-15 hybridoma cells

The recombinant Siglec-15 whole gene protein is customized according to the human Siglec-15 gene. Immunization is carried out on the belly of a pure line BALB/c mouse by subcutaneous injection by using the protein and mixed polypeptide coupled with OVA as immunogen, each time the immunization is carried out for 100 mu g/ml, and the immunization is carried out for five times, wherein the recombinant Siglec-15 whole gene protein is used for 1, 3 and 5 times, and the mixed polypeptide coupled with OVA is used for 2 and 4 times. When positive serum with OD larger than 2 can be detected by peripheral serum, cell fusion is carried out, and mixed polypeptide coupled with OVA is used for enhancing immunity 3 days before fusion. On the day of fusion, mouse spleen was taken, and prepared into a single cell suspension using DMEM medium (GIBCO, USA), spleen cells and SP2/0 mouse myeloma cells were fused in the presence of 50% PEG (pH 8.0), cultured for 7 days using HAT selective medium (98 ml of DMEM medium, 1ml of HT stock solution, 1ml of A stock solution), and cultured using HT medium (99 ml of DMEM medium, HT1ml) instead.

Example 3: screening, strain determination and identification of murine anti-Siglec-15 antibodies

Enzyme-linked immunosorbent assay (ELISA) detection and screening are carried out according to the growth condition of the hybridoma cells, and the specific method is shown in the following. And (4) coating the plate with the recombinant Siglec-15 protein, detecting cells of the positive hole, performing cloning culture again, and determining a positive clone strain after 3 times of subcloning. And then, detecting the positive clone strain by using a Siglec-15 mixed polypeptide plate coupled with KLH. And after the monoclonal cells in all the wells are detected to be positive by Siglec-15-KLH and negative by KLH, taking the wells, performing amplification culture, and partially freezing and storing.

The ELISA method is used for screening monoclonal antibodies positive to Siglec-15 and negative to normal mouse serum, and the specific method is as follows:

(1) chemically synthesized Siglec-15-KLH polypeptide was coated on a 96-well plate of ELISA, diluted to 20. mu.g/ml with coating solution (0.1M carbonate buffer, pH9.6), added in 100. mu.l per well, overnight at 4 ℃;

(2) washing with PBST washing solution (PBS containing 0.05% Tween) for 5 times, adding 10% BSA (200 μ l/well) for blocking, incubating at 37 deg.C for 2h, and washing for 5 times for later use;

(3) adding 50 μ l hybridoma cell culture supernatant into each well, incubating at 37 deg.C for 1h with serum of splenectomized mice as positive control (1: 1000 dilution), and serum of normal mice as negative control (1: 1000 dilution);

(4) will be measured at a rate of 1: mu.l/well of a 5000-diluted goat anti-mouse Ig-HRP secondary antibody (Thermo Co.) was added to the wells, incubated at 37 ℃ for 1h, and washed 5 times with PBST;

(5) adding 100 mul/hole of peroxidase substrate color development solution, stopping reaction with 2M sulfuric acid at room temperature for 15 minutes; detecting with a full-wavelength microplate reader (Thermo labsystems, USA), comparing with color with dual wavelength of 450nm/630nm, obtaining Siglec-15 positive clone with detection result 2.5 times larger than OD value of normal mouse serum, and selecting 1 hybridoma cell stably secreting anti-Siglec-15 antibody, named 4E6A, and named S154E 6A.

The subtype of the antibody was determined using the monoclonal antibody subtype determination kit (ISO-2KT) from Sigma, USA, and the result showed that the subtype of the anti-Siglec-15 antibody E4A6 was IgG 1.

Example 4: preparation, expression and purification of human-mouse chimeric anti-Siglec-15 whole-molecule IgG

Recovering Siglec-15 hybridoma cells 4E6A, extracting total RNA of the cells according to Trizol Reagent Kit instructions, and obtaining cDNA by an RT-PCR method; 19 VHforward and 17 V.kappa.forward primers, 4 VH reverse and 3 V.kappa.reverse primers were designed with reference to the statistics of the Phage Display and BLAST databases, and the primer sequences were as follows:

Vκ5’forward primers

Vκ-1：5’-GGGCCCAGGCGGCCGAGCTCGAYATCCAGCTGACTCAGCC-3’

Vκ-2：5’-GGGCCCAGGCGGCCGAGCTCGAYATTGTTCTCWCCCAGTC-3’

Vκ-3：5’-GGGCCCAGGCGGCCGAGCTCGAYATTGTGMTMACTCAGTC-3’

Vκ-4：5’-GGGCCCAGGCGGCCGAGCTCGAYATTGTGYTRACACAGTC-3’

Vκ-5：5’-GGGCCCAGGCGGCCGAGCTCGAYATTGTRATGACMCAGTC-3’

Vκ-6：5’-GGGCCCAGGCGGCCGAGCTCGAYATTMAGATRAMCCAGTC-3’

Vκ-7：5’-GGGCCCAGGCGGCCGAGCTCGAYATTCAGATGAYDCAGTC-3’

Vκ-8：5’-GGGCCCAGGCGGCCGAGCTCGAYATYCAGATGACACAGAC-3’

Vκ-9：5’-GGGCCCAGGCGGCCGAGCTCGAYATTGTTCTCAWCCAGTC-3’

Vκ-10：5’-GGGCCCAGGCGGCCGAGCTCGAYATTGWGCTSACCCAATC-3’

Vκ-11：5’-GGGCCCAGGCGGCCGAGCTCGAYATTSTRATGACCCARTC-3’

Vκ-12：5’-GGGCCCAGGCGGCCGAGCTCGAYATTKTGATGACCCARAC-3’

Vκ-13：5’-GGGCCCAGGCGGCCGAGCTCGAYATTGTGATGACBCAGKC-3’

Vκ-14：5’-GGGCCCAGGCGGCCGAGCTCGAYATTGTGATAACYCAGGA-3’

Vκ-15：5’-GGGCCCAGGCGGCCGAGCTCGAYATTGTGATGACCCAGWT-3’

Vκ-16：5’-GGGCCCAGGCGGCCGAGCTCGAYATTGTGATGACACAACC-3’

Vκ-17：5’-GGGCCCAGGCGGCCGAGCTCGAYATTTTGCTGACTCAGTC-3’

Vκ3’reverse primers

VκR1：5’-AGATGGTGCAGCCACAGTTCGTTTKATTTCCAGYTTGGTCCC-3’

VκR2：5’-AGATGGTGCAGCCACAGTTCGTTTTATTTCCAACTTTGTCCC-3’

VκR3：5’-AGATGGTGCAGCCACAGTTCGTTTCAGCTCCAGCTTGGTCCC-3’

VH 5’forward primers

VH1：5’-GCTGCCCAACCAGCCATGGCCCTCGAGGTRMAGCTTCAGGAGTC-3’

VH 2：5’-GCTGCCCAACCAGCCATGGCCCTCGAGGTBCAGCTCAGCAGTC-3’

VH 3：5’-GCTGCCCAACCAGCCATGGCCCTCGAGGTGCAGCTGAAGSASTC-3’

VH 4：5’-GCTGCCCAACCAGCCATGGCCCTCGAGGTCCARCTGCAACARTC-3’

VH 5：5’-GCTGCCCAACCAGCCATGGCCCTCGAGGTYCAGCTBCAGCARTC-3’

VH 6：5’-GCTGCCCAACCAGCCATGGCCCTCGAGGTYCARCTGCAGCAGTC-3’

VH 7：5’-GCTGCCCAACCAGCCATGGCCCTCGAGGTCCACGTGAAGCAGTC-3’

VH 8：5’-GCTGCCCAACCAGCCATGGCCCTCGAGGTGAASSTGGTGGAATC-3’

VH 9：5’-GCTGCCCAACCAGCCATGGCCCTCGAGGTGAWGYTGGTGGAGTC-3’

VH 10：5’-GCTGCCCAACCAGCCATGGCCCTCGAGGTGCAGSKGGTGGAGTC-3’

VH 11：5’-GCTGCCCAACCAGCCATGGCCCTCGAGGTGCAMCTGGTGGAGTC-3’

VH 12：5’-GCTGCCCAACCAGCCATGGCCCTCGAGGTGAAGCTGATGGARTC-3’

VH 13：5’-GCTGCCCAACCAGCCATGGCCCTCGAGGTGCARCTTGTTGAGTC-3’

VH 14：5’-GCTGCCCAACCAGCCATGGCCCTCGAGGTRAAGCTTCTCGAGTC-3’

VH 15：5’-GCTGCCCAACCAGCCATGGCCCTCGAGGTGAARSTTGAGGAGTC-3’

VH 16：5’-GCTGCCCAACCAGCCATGGCCCTCGAGGTTACTCTRAAAGWGTSTG-3’

VH 17：5’-GCTGCCCAACCAGCCATGGCCCTCGAGGTCCAACTVCAGCARCC-3’

VH 18：5’-GCTGCCCAACCAGCCATGGCCCTCGAGGTGAACTTGGAAGTGTC-3’

VH 19：5’-GCTGCCCAACCAGCCATGGCCCTCGAGGTGAAGGTCATCGAGTC-3’

VH 3’reverse primers

VH R1：5’-CGATGGGCCCTTGGTGGAGGCTGAGGAGACGGTGACCGTGGT-3’

VH R2：5’-CGATGGGCCCTTGGTGGAGGCTGAGGAGACTGTGAGAGTGGT-3’

VH R3：5’-CGATGGGCCCTTGGTGGAGGCTGCAGAGACAGTGACCAGAGT-3’

VH R4：5’-CGATGGGCCCTTGGTGGAGGCTGAGGAGACGGTGACTGAGGT-3’

after the primers are mixed evenly, the cDNA of the prepared clone 4E6A is taken as a template, and the variable region sequence of the antibody is amplified; the amplification condition is Step one at 95 ℃ for 4 min; step two, 30s at 95 ℃, 30s at 58 ℃, 45s at 72 ℃ and 35 cycles; extending for 10min at the temperature of Step three72 ℃; step four at 4 ℃ for 20 min; the PCR product is subjected to nucleic acid electrophoresis recovery, purified and amplified gene fragment, recombined and connected to pMD-18T, transformed into Escherichia coli BL21(DE3), and sequenced to obtain the sequences of the light chain and heavy chain variable regions.

(1)PCR

The reaction system is as follows:

the reaction conditions were as follows:

(2) performing 2% agarose gel electrophoresis, observing a target band under ultraviolet, cutting and recovering gel;

(3) purifying the target DNA fragment by using the gel recovery kit, and eluting by using deionized water;

(4) double restriction enzyme IgG expression plasmid

The IgG expression plasmids pFUSE-CHIg-hG1, pFUSE-CLIg-hk (from Invivogen) contained base coding sequences for the heavy and light chain (Kappa) constant regions of human origin of IgG 1.

The double enzyme digestion reaction system of pFUSE-CHIg-hG1 and pFUSE-CLIg-hk template vectors is as follows:

the reaction conditions are as follows: the cleavage was carried out overnight at 37 ℃.

b, carrying out 1% agarose gel electrophoresis, and recovering the cutting gel under ultraviolet;

c. purifying the target DNA fragment by using the gel recovery kit, and eluting by using deionized water;

(5) infusion PCR recombinant expression plasmid

Heavy chain IF-PCR amplification primers:

F：5’-TACAGGTGTCCACTCGCTAgatCAGGTGCAGCTGCAGCAG3’

R：5’-GGAGGACACGGTCACCAGGCCCTTGGTGGATGC-3’

light chain IF-PCR amplification primers:

F：5’-CTTACAGACGCTCGCTGCGACATCGTGATGACCCAG-3’

R：5’-TGCAGCCACCGTACGCTTGATCTCCAGCTTGGT-3’

the reaction system is as follows:

the reaction conditions are as follows: incubate at 50 ℃ for 15 min.

5 mul of reaction solution was taken to transform competent bacteria, spread on the corresponding resistant plates, and the next day clones were picked for sequencing. And cloning and preserving strains with correct sequencing results, carrying out amplification culture, and extracting plasmids.

(6) Eukaryotic expression of full-molecular anti-Siglec-15 IgG

a. 50. mu.g of the recombinant heavy chain plasmid was placed in 1mL of Opti-MEM medium, 50. mu.g of the light chain plasmid was placed in 1mL of Opti-MEM medium, 200. mu.L of 293Fectin was placed in 2.8mL of Opti-MEM medium, and the three mixtures were allowed to stand at room temperature for 5 min.

b. After the two plasmid mixed solutions are uniformly mixed, 500 mu L of Opti-MEM culture medium is added and uniformly mixed, the mixed solution of the transfection reagent 293Fectin is directly added, and the mixture is uniformly mixed and then stands for 20 min. During the treatment, 293F cells were centrifugedResuspend with 293F Expression Medium, count and calculate cell viability ratio with Trypan blue, aspirate 1.00 × 10⁸The cells were placed in a flask and made up to 94mL with 293F Expression Medium.

At the end of c.20min, 6mL of the DNA, 293Fectin complex was added to the prepared 293F cells.

d. Culturing the cells in a shaking incubator under 8% CO₂Cell supernatants were collected after 6 days at 120rmp, 37 ℃.

(7) Purification of anti-Siglec-15 Whole-molecule IgG

The collected cell culture supernatant was filtered through 0.45 μm and 0.22 μm filters while the balance and the eluate were filtered. The Protein was purified using the AKATApurifier100 Protein purification system according to the standard procedure for Protein A purification, loading at a flow rate of 1ml/min, and eluting at a flow rate of 1.5 ml/min.

Example 5: characterization of human murine chimeric anti-Siglec-15 Whole molecule antibodies

1) Enzyme-linked immunosorbent assay

a) Diluting the Siglec-15 recombinant protein with coating solution (0.1M carbonate buffer, pH9.6) to 2. mu.g/mL coated ELISA 96-well plates, adding 100. mu.L per well, and standing overnight at 4 ℃;

b) blocking with PBST (PBS containing 0.5% Tween20) 5% skim milk-wash buffer, incubating at 37 ℃ for 2 h;

c) PBST wash 5 times;

d) add 100. mu.l of anti-Siglec-154 EA6 whole IgG diluted in two fold per well (2. mu.g/mL starting concentration, 14 concentration gradient dilutions) and incubate for 2h at 37 ℃;

e) mu.l of goat anti-human secondary antibody (1: 4000 dilution), and incubating for 1h at 37 ℃;

f) PBST wash 5 times;

g) adding peroxidase substrate color developing solution, stopping reaction with 2M sulfuric acid at room temperature for 15min, and detecting protein absorbance value by using a computer.

The results are shown in FIG. 1, and the Siglec-154EA6 antibody can obviously react with the recombinant Siglec-15 protein.

2) SDS-PAGE detection of purified Siglec-15 antibodies

a) Preparing 10% polyacrylamide gel;

b) adding 5 × sample buffer solution into the untransfected 293F cell supernatant, the cell supernatant before purification, the whole-molecule Siglec-15 antibody after ultrafiltration, the flow-through liquid collected after purification and the control human IgG respectively, mixing uniformly, and carrying out boiling water bath for 10 min;

c) loading sample, performing electrophoresis, concentrating gel at 80V, separating gel, performing electrophoresis at 110V until bromophenol blue comes out, and stopping electrophoresis;

d) coomassie blue stained gel for 30min, PBST was destained to a clear background and the bands were clearly visible (FIG. 2).

3)Western-blot

a) Lysis of cells HFL-1 that are positive for Siglec-15, A375, U87MG, Raji, THP-1, HCT-8 and negative for Siglec-15;

b) performing 10% SDS-PAGE electrophoresis;

c) transferring the protein to a nitrocellulose membrane by adopting an electric transfer mode;

d) the membrane was incubated with 2. mu.g/mL Siglec-15 antibody for 1h at room temperature;

e) 1: HRP-goat anti-human IgG (Beijing Cedar) and ECL luminescence kit (Pierce, USA) were exposed to a gel imaging system (Bio-Rad) at 4000 dilutions, and the results are shown in FIG. 3.

4) Affinity assays

The coupling conditions were optimized according to isoelectric point and protocol of BiacoreX100 control soft, and sodium acetate was selected as the coupling dilution buffer for slope optimization.

a) The Siglec-154EA6 antibody sample was diluted to 25ug/ml with this buffer and coupled to a CM5 chip (GE # BR 100012);

b) presetting a coupling level of 1500 RU;

c) dilution of mAb series samples with Running buffer pH7.4 to a range of concentrations of 0uM, 5nM, 10nM, 20nM, 40nM, 80 nM;

d) setting the sample injection time to be 180s, the dissociation time to be 10min, and using 50mM pH2.2Gly-HCl for the regeneration buffer solution;

e) the on-board test was performed according to the protocol of BiacoreX100 control soft. The affinity KD of the detected antibody was 5.719e (-8) (see FIG. 4).

5)Epitope mapping

a) Diluting Siglec-15 specific polypeptides (A1-A12 and B1-B11) to 2 mu g/mL by using a coating solution (0.1M carbonate buffer solution, pH9.6), sequentially coating the Siglec-15 specific polypeptides to an ELISA 96 well plate according to the group A and the group B, adding 100 mu L of the Siglec-15 specific polypeptides to each well, setting 3 multiple wells, and standing overnight at 4 ℃;

c) PBST wash 5 times;

d) add 100. mu.l of anti-Siglec-154 EA6 whole IgG diluted in two fold per well (2. mu.g/mL starting concentration, 4 concentration gradient dilutions) and incubate for 2h at 37 ℃;

f) PBST wash 5 times;

g) adding peroxidase substrate color development solution, stopping reaction with 2M sulfuric acid at room temperature for 15 min;

h) the absorbance of the protein was measured by a full-wavelength microplate reader, and the results are shown in FIG. 5.

Example 6: competitive inhibition of anti-Siglec-15 whole IgG on sTn protein 1) competitive detection of anti-Siglec-15 and sTn protein

a) Coating a 96-well plate (2 mu g/ml) with recombinant GST-Siglec-15 protein, diluting to 20 mu g/ml, 100 mu l/well with the coating solution, and standing overnight at 4 ℃;

b) washing with PBST for 5 times (5 min/time);

c) adding 5% skimmed milk (200 μ l/well), sealing, incubating at 37 deg.C for 2h, washing for 5 times for later use;

d) recombinant sTn protein was added at concentrations of 0, 0.02, 0.04, 0.08, 0.16, 0.32. mu.g/ml, while an equal amount of anti-Siglec-15 antibody E4A6 (0.5. mu.g/ml) was added per well. Finally adding 100 mul of sample into each hole, and setting 3 multiple holes for reaction;

e) PBS instead of anti-Siglec-15 antibody E4A6 was used as a negative control; serum from immunized mice was used as a positive control (1:200 dilution); incubating at 37 ℃ for 1.5 h;

f) washing with PBST for 5-6 times (5 min/time);

g)1, adding 100 mu l/hole of HRP-labeled goat anti-human IgG diluted at 2000, and incubating for 1.5h at 37 ℃;

h) PBST wash 5 times;

i) using Pierce TMB color kit, 50. mu.l of TMB, H was added to each well₂O₂50 μ l, developing at room temperature for 15min, stopping the reaction with 2M sulfuric acid;

j) the detection is carried out by a microplate reader, and the color comparison is carried out by using dual wavelength 450nm/630nm, and the result is shown in figure 6.

2) Competitive binding of anti-Siglec-15 to a polypeptide

a) Culturing human lymphoma cells Raji, selecting cells in logarithmic growth phase, digesting with 0.25% trypsin to prepare single cell suspension, and adding 1 × 10 per well³Inoculating to a 96-well plate, 100 μ l/well; placing at 37 ℃ with 5% CO₂Culturing for 24h in a constant-temperature moisture-preserving incubator;

b) washing with PBS for 2 times;

c) adding 100 μ l of fixing solution (50% acetone, 50% isopropanol) into each well, standing at room temperature for 15 min;

d) washing with PBS for 5 times;

e) adding 5% skimmed milk (200 μ l/well), sealing, incubating at 37 deg.C for 2h, washing for 5 times for later use;

f) the synthesized Siglec-15 specific polypeptides A6, A9, B8 and B9 were added at concentrations of 0, 0.1, 0.2, 0.4, 0.8, 1.6 and 3.2. mu.g/ml, respectively, and an equal amount of anti-Siglec-15 antibody E4A6 (5. mu.g/ml) was added to each well, and a sample volume of 100. mu.l was finally added to each well, and 3 wells were set for reaction;

g) serum of an immunized mouse is taken as a positive control (diluted 1: 200) and incubated for 1.5h at 37 ℃;

h) washing with PBST for 5-6 times (5 min/time);

i)1, 100 mu l/hole of HRP-labeled goat anti-human IgG diluted by 4000, and incubating for 1.5h at 37 ℃;

j) PBST wash 5 times; using Pierce TMB color kit, 50. mu.l of TMB, H was added to each well₂O₂50 μ l, developing at room temperature for 15min, stopping the reaction with 2M sulfuric acid;

k) and (3) detecting by using a microplate reader, carrying out color comparison by using a dual wavelength of 450nm/630nm, and obtaining a statistical result as shown in figure 7.

3) Effect of anti-Siglec-15 on tumor cell proliferation

a) The experimental group is prepared by cultured human malignant melanoma cells A375, malignant glioblastoma cells U87MG, monocyte leukemia cells THP-1, lymphoma cells Raji and colon cancer cells HCT-8; human lung cell HFL-1 as negative control group;

b) cells from logarithmic growth phase were selected, digested with 0.25% trypsin and prepared as single cell suspensions, 1 × 10 per well³Inoculating to a 96-well plate, 100 μ l/well;

c) placing at 37 ℃ with 5% CO₂Culturing for 24h in a constant-temperature moisture-preserving incubator;

d) the anti-Siglec-15 antibody E4A6 was added at a concentration of 0, 1.25, 2.5, 5, 7.5, 10. mu.g/ml, while the same amount of recombinant sTn protein (1. mu.g/ml) was added per well; finally adding 100 mul of sample into each hole, and setting 3 multiple holes for reaction;

e)37℃5％CO₂culturing overnight;

f) collecting cell culture supernatants at 24, 48, 72 and 96 hours respectively;

g) the cell proliferation inhibition rate was measured using CCK8 cell proliferation assay kit, and the results are shown in fig. 8.

The above-mentioned embodiments are merely preferred embodiments of the present invention, and should not be construed as limiting the present invention, and the scope of the present invention should be defined by the claims, and equivalents including technical features of the claims, i.e., equivalent modifications within the scope of the present invention.

Sequence listing

<110> Nanjing university of medical science

<120> human-mouse chimeric anti-Siglec-15 whole-molecule IgG with neutralization function, and preparation method and application thereof

<160>23

<170>SIPOSequenceListing 1.0

<210>1

<211>345

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>1

gacatcgtga tgacccaggc cgccttctcc aaccccgtga ccctgggcac ctccgcctcc 60

atctcctgcc gctcctccaa gtcctcccag tccctgctgg actcctccgg caacacctac 120

ctgtactggt tcctgcagaa gcccggccag tccccccagc tgctgatcta caacgccaag 180

aacctggcct ccggcgtgcc cgaccgcttc tccggctccg gctccggcac cgacttcacc 240

ctgcgcatct cccgcgtgga ggccgaggac gtgggcgtgt actactgctg gcagggcacc 300

cactacccct tcaccttcgg ctccggcacc aagctggaga tcaag 345

<210>2

<211>369

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>2

caggtgcagc tgcagcagtc cggcgccgag ctggtgcgcc ccggcgcctc cgtgaccctg 60

tcctgcaagg cctccggcta caccttcacc cgctacgaca tgcactgggt gaagcagacc 120

ccccgccagg gcctggagtg gatcggcggc atcgaccccg agaccggcgg caccaagtac 180

aaccagaagt tcaagggcaa ggccatcctg accgccgaca agtcctcctc caccgcctac 240

atggagctgc gctccctgac ctccgaggac tccgccgtgt actactgcgc ccgcttctac 300

tactcccact ccaactacga cgtgggcttc gcctactggg gccagggcac cctggtgacc 360

gtgtcctcc 369

<210>3

<211>115

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>3

Asp Ile Val Met Thr Gln Ala Ala Phe Ser Asn Pro Val Thr Leu Gly

1 5 10 15

Thr Ser Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Ser Gln Ser Leu

20 25 30

Leu Asp Ser Ser Gly Asn Thr Tyr Leu Tyr Trp Phe Leu Gln Lys Pro

35 40 45

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

50 55 60

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

65 70 75 80

Leu Arg Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys

85 90 95

Trp Gln Gly Thr His Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu

100 105 110

Glu Ile Lys

115

<210>4

<211>123

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>4

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 9095

Ala Arg Phe Tyr Tyr Ser His Ser Asn Tyr Asp Val Gly Phe Ala Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210>5

<211>669

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>5

gacatcgtga tgacccaggc cgccttctcc aaccccgtga ccctgggcac ctccgcctcc 60

atctcctgcc gctcctccaa gtcctcccag tccctgctgg actcctccgg caacacctac 120

ctgtactggt tcctgcagaa gcccggccag tccccccagc tgctgatcta caacgccaag 180

aacctggcct ccggcgtgcc cgaccgcttc tccggctccg gctccggcac cgacttcacc 240

ctgcgcatct cccgcgtgga ggccgaggac gtgggcgtgt actactgctg gcagggcacc 300

cactacccct tcaccttcgg ctccggcacc aagctggaga tcaagcgtac ggtggctgca 360

ccatctgtct tcatcttccc gccatctgat gagcagttga aatctggaac tgcctctgtt 420

gtgtgcctgc tgaataactt ctatcccaga gaggccaaag tacagtggaa ggtggataac 480

gccctccaat cgggtaactc ccaggagagt gtcacagagc aggacagcaa ggacagcacc 540

tacagcctca gcagcaccct gacgctgagc aaagcagact acgagaaaca caaagtctac 600

gcctgcgaag tcacccatca gggcctgagc tcgcccgtca caaagagctt caacagggga 660

gagtgttag 669

<210>6

<211>1362

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>6

caggtgcagc tgcagcagtc cggcgccgag ctggtgcgcc ccggcgcctc cgtgaccctg 60

tcctgcaagg cctccggcta caccttcacc cgctacgaca tgcactgggt gaagcagacc 120

ccccgccagg gcctggagtg gatcggcggc atcgaccccg agaccggcgg caccaagtac 180

aaccagaagt tcaagggcaa ggccatcctg accgccgaca agtcctcctc caccgcctac 240

atggagctgc gctccctgac ctccgaggac tccgccgtgt actactgcgc ccgcttctac 300

tactcccact ccaactacga cgtgggcttc gcctactggg gccagggcac cctggtgacc 360

gtgtcctccg catccaccaa gggcccatct gtcttccccc tggccccatc ctccaagagc 420

acctctggcg gcacagctgc cctgggctgc ctggtgaagg actacttccc tgagcctgtg 480

acagtgtcct ggaactctgg cgccctgacc agcggcgtgc acaccttccc tgctgtgctc 540

cagtcctctg gcctgtactc cctgagcagc gtggtgacag tgccatccag cagcctgggc 600

acccagacct acatctgcaa tgtgaaccac aagcccagca acaccaaggt ggacaagcgg 660

gtggagccca agtcctgtga caagacccac acctgccccc catgccccgc ccctgagctg 720

ctgggcggcc catctgtctt cctgttcccc cccaagccca aggacaccct gatgatctcc 780

cggacccccg aggtgacctg tgtggtggtg gatgtgagcc atgaggaccc cgaggtgaag 840

ttcaactggt atgtggatgg cgtggaggtg cacaacgcca agaccaagcc ccgggaggag 900

cagtacaaca gcacctaccg ggtggtgagc gtgctgacag tgctgcatca ggactggctg 960

aatggcaagg agtacaagtg caaggtgtcc aacaaggccc tgcctgcccc cattgagaag 1020

accatctcca aggccaaggg ccagccccgg gagccccagg tctacaccct gcccccctcc 1080

cgggaggaga tgaccaagaa ccaggtgagc ctgacctgcc tggtgaaggg cttctacccc 1140

agcgacattg ctgtggagtg ggagagcaac ggccagcctg agaacaacta caagaccacc 1200

ccccctgtgc tggactctga tggctccttc ttcctgtaca gcaagctgac agtggacaag 1260

agccggtggc agcagggcaa tgtcttctcc tgctctgtga tgcatgaggc cctgcacaac 1320

cactacaccc agaagagcct gtccctgtcc cccggcaagt ga 1362

<210>7

<211>223

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>7

Asp Ile Val Met Thr Gln Ala Ala Phe Ser Asn Pro Val Thr Leu Gly

1 5 10 15

Thr Ser Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Ser Gln Ser Leu

20 25 30

Leu Asp Ser Ser Gly Asn Thr Tyr Leu Tyr Trp Phe Leu Gln Lys Pro

35 40 45

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

50 55 60

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

6570 75 80

Leu Arg Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys

85 90 95

Trp Gln Gly Thr His Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu

100 105 110

Glu Ile Lys Ala Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro

115 120 125

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

130 135 140

Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp

145 150 155 160

Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp

165 170 175

Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys

180 185 190

Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln

195 200 205

Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<210>8

<211>454

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>8

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Phe Tyr Tyr Ser His Ser Asn Tyr Asp Val Gly Phe Ala Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Cys Ala Ser Thr Lys

115 120 125

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

130 135 140

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

145 150 155 160

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

165 170 175

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

180 185 190

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn

195 200 205

Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro

210 215 220

Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

225 230 235 240

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

245 250 255

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

260 265 270

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

275 280 285

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn

290 295 300

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

305 310 315 320

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro

325 330 335

Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

340 345 350

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn

355 360 365

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

370 375 380

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

385 390 395 400

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

405 410 415

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

420 425 430

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

435 440 445

Ser Leu Ser Pro Gly Lys

450

<210>9

<211>42

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>9

aagtcctccc agtccctgct ggactcctcc ggcaacacct ac 42

<210>10

<211>9

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>10

aacgccaag 9

<210>11

<211>27

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>11

tggcagggca cccactaccc cttcacc 27

<210>12

<211>24

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>12

ggctacacct tcacccgcta cgac 24

<210>13

<211>24

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>13

atcgaccccg agaccggcgg cacc 24

<210>14

<211>48

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>14

gcccgcttct actactccca ctccaactac gacgtgggct tcgcctac 48

<210>15

<211>14

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>15

Lys Ser Ser Gln Ser Leu Leu Asp Ser Ser Gly Asn Thr Tyr

1 5 10

<210>16

<211>3

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>16

Asn Ala Lys

1

<210>17

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>17

Trp Gln Gly Thr His Tyr Pro Phe Thr

1 5

<210>18

<211>8

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>18

Gly Tyr Thr Phe Thr Arg Tyr Asp

1 5

<210>19

<211>8

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>19

Ile Asp Pro Glu Thr Gly Gly Thr

1 5

<210>20

<211>16

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>20

Ala Arg Phe Tyr Tyr Ser His Ser Asn Tyr Asp Val Gly Phe Ala Tyr

1 5 10 15

<210>21

<211>12

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>21

Ala Leu Ala Asp Asp Arg Arg Tyr Phe Cys Arg Val

1 5 10

<210>22

<211>12

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>22

Leu Arg Val Glu Arg Leu Ala Leu Ala Asp Asp Arg

1 5 10

<210>23

<211>12

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>23

Arg Tyr Phe Cys Arg Val Glu Phe Ala Gly Asp Val

1 5 10

Claims

1. A human-mouse chimeric anti-Siglec-15 whole-molecule IgG antibody, which comprises a light chain variable region and a heavy chain variable region, and is characterized in that: the nucleic acid sequence of the light chain variable region is shown in SEQ ID NO.1, and the nucleic acid sequence of the heavy chain variable region is shown in SEQ ID NO. 2.

2. The human murine chimeric anti-Siglec-15 whole IgG antibody of claim 1, wherein: the amino acid sequence of the light chain variable region is shown in SEQ ID NO.3, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 4.

3. A human murine chimeric anti-Siglec-15 whole IgG antibody according to claim 1 or 2, characterized in that: the nucleic acid sequences of the antigen complementary region CDR of the light chain variable region are respectively shown in SEQ ID NO.9, SEQ ID NO.10 and SEQ ID NO. 11; the nucleic acid sequences of the antigen complementary region CDR of the heavy chain variable region are respectively shown in SEQ ID NO.12, SEQ ID NO.13 and SEQ ID NO. 14.

4. A human murine chimeric anti-Siglec-15 whole IgG antibody according to claim 1 or 2, characterized in that: the amino acid sequences of the antigen complementary region CDR of the light chain variable region are respectively shown as SEQ ID NO.15, SEQ ID NO.16 and SEQ ID NO. 17; the amino acid sequences of the antigen complementary region CDR of the heavy chain variable region are respectively shown as SEQ ID NO.18, SEQ ID NO.19 and SEQ ID NO. 20.

5. A DNA molecule encoding the heavy chain or/and the light chain of a whole molecule IgG antibody according to any one of claims 1-4.

6. The DNA molecule of claim 5, wherein: the light chain nucleic acid sequence is shown as SEQ ID NO.5, and the heavy chain nucleic acid sequence is shown as SEQ ID NO. 6.

7. The DNA molecule of claim 5, wherein: the light chain amino acid sequence is shown as SEQ ID NO.7, and the heavy chain amino acid sequence is shown as SEQ ID NO. 8.

8. An expression vector, characterized in that: comprising the DNA molecule of claim 5 or 6 or 7 and an expression control sequence operably linked to the DNA molecule.

9. A method of expressing a protein in a eukaryotic cell, comprising: the method is used for expressing nucleic acid and amino acid sequences containing the antibody of claims 1-4.

10. The human murine chimeric anti-Siglec-15 whole IgG antibody of any of claims 1-2, characterized by: the characteristic epitope in the whole molecular IgG antibody is combined with A37-A180 in the Siglec-15 protein molecule, and the combination of the Siglec-15 protein and the Sialyl-Tn protein is blocked; the characteristic epitope is at least one of the following amino acid sequences: SEQ ID NO.21, SEQ ID NO.22, SEQ ID NO. 23.

11. The application of the whole-molecule IgG antibody in preparing tumor diagnostic reagents or therapeutic drugs for inhibiting Siglec-15 functions is characterized in that: the whole IgG antibody is the human murine chimeric anti-Siglec-15 whole IgG of any of claims 1-4.

12. A medicament for inhibiting Siglec-15 biological function, comprising: the medicament contains the human murine chimeric anti-Siglec-15 whole IgG of any one of claims 1 to 4.