CN109678950B - spine 1 antigen, antibody capable of specifically binding to spine 1, functional fragment thereof, application and product thereof - Google Patents
spine 1 antigen, antibody capable of specifically binding to spine 1, functional fragment thereof, application and product thereof Download PDFInfo
- Publication number
- CN109678950B CN109678950B CN201910014180.1A CN201910014180A CN109678950B CN 109678950 B CN109678950 B CN 109678950B CN 201910014180 A CN201910014180 A CN 201910014180A CN 109678950 B CN109678950 B CN 109678950B
- Authority
- CN
- China
- Prior art keywords
- antibody
- 1cdr
- cdr
- spine
- seq
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/81—Protease inhibitors
- C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
- C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
- C07K14/8135—Kazal type inhibitors, e.g. pancreatic secretory inhibitor, ovomucoid
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/38—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against protease inhibitors of peptide structure
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57415—Specifically defined cancers of breast
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57419—Specifically defined cancers of colon
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57446—Specifically defined cancers of stomach or intestine
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57449—Specifically defined cancers of ovaries
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/54—F(ab')2
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/81—Protease inhibitors
- G01N2333/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
- G01N2333/811—Serine protease (E.C. 3.4.21) inhibitors
- G01N2333/8135—Kazal type inhibitors, e.g. pancreatic secretory inhibitor or ovomucoid
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2410/00—Assays, e.g. immunoassays or enzyme assays, involving peptides of less than 20 animo acids
Abstract
The invention relates to the technical field of biology, and particularly provides a spine 1 antigen, an antibody capable of specifically binding spine 1, a functional fragment thereof, application thereof and a product thereof. The spine 1 antigen provided by the invention comprises: the polypeptide derived from any one of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and the other one has good immunogenicity. The antigen respectively obtains a first antibody, a second antibody and a third antibody, and hybridoma cell strains capable of respectively generating the three antibodies are obtained, and the antibody has high titer, good sensitivity and stable structure; the hybridoma can stably and efficiently produce high-quality antibody and functional fragment of spine 1, and can be used as an engineering strain for industrial production. The invention also provides the application of the antibody and the functional fragment thereof and a product using the antibody and the functional fragment thereof.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a spine 1 antigen, an antibody capable of specifically binding spine 1, a functional fragment thereof, application thereof and a product thereof.
Background
Serine protease inhibitors (SERPINs) are the most widely distributed and largest-scale protease inhibitor superfamily, and the serine protease inhibitor Kazal1 type (serine protease inhibitor Kazal type 1, SPINK1) is taken as a member of the family, and has attracted extensive attention in recent years, and is a secreted polypeptide consisting of 56 amino acid residues, and the main function is to inhibit the activity of trypsin.
Studies have shown that SPINK1 is a potential tumor biomarker in vivo, is expressed in many types of tumors, and can be used to identify patients at risk and poor prognosis of tumors. SPINK1 is highly expressed in various tumor cells, including liver cancer, ovarian cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, bladder cancer, gastric cancer or renal cancer, etc. The detection and research of SPINK1 are of great significance to the regulation mechanism and early diagnosis of various tumors.
At present, the research on SPINK1 mainly focuses on the detection of related expression level, regulation mechanism and nucleic acid level, but most of the fields of SPINK1 protein detection are in the exploration stage, the effect is not ideal, effective antigens are still unclear, and meanwhile, few products of antibodies for detecting SPINK1 protein have the defects of high cost and low titer (the minimum action concentration is 0.8 ug/ml).
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The first purpose of the invention is to provide a spin 1 antigen, so as to relieve the technical problem of lacking effective antigen for the detection of spin 1 protein in the prior art.
The second purpose of the invention is to provide an antibody and a functional fragment thereof, which can specifically bind to spin 1, so as to solve the technical problems of high cost and low titer of the prior spin 1 antibody.
The third object of the present invention is to provide a hybridoma producing an antibody and a functional fragment thereof capable of specifically binding to spin 1, which can stably and efficiently produce a high-quality antibody and a functional fragment thereof of spin 1 and can be industrially produced as an engineered strain.
The fourth objective of the invention is to provide an isolated nucleic acid molecule and a vector, so as to obtain the antibody of spin 1 and functional fragments thereof through efficient and rapid expression.
The fifth object of the present invention is to provide the use of the protein 1 antigen or antibody and functional fragments thereof
It is a sixth object of the present invention to provide a composition and kit to alleviate the lack of diagnostic or therapeutic products targeting spike 1 in the prior art.
In order to achieve the above purpose of the present invention, the following technical solutions are adopted:
a spin 1 antigen which is a polypeptide according to any one of the following a) -f):
a)RQTSILIQKSGPC(SEQ ID NO.1)；
b) a polypeptide which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO.1, has the function of a spink1 epitope and is derived from a);
c)MKVTGIFLLSALALLSLS(SEQ ID NO.2)；
d) c) the polypeptide which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO.2 and has the function of the epitope of the spine 1 and is derived from the c);
e)GNTGADSLGREAKCYNE(SEQ ID NO.3)；
f) a polypeptide which is derived from the e) and has the function of the epitope of the spin 1 by substituting and/or deleting and/or adding one or more amino acid residues of the amino acid sequence shown in the SEQ ID NO. 3.
An antibody and a functional fragment thereof capable of specifically binding to spin 1, which is any one of the following a1) -c 1):
a1) a first antibody of SEQ ID No.1, said first antibody and functional fragments thereof comprising a light chain and a heavy chain;
the light chain has a light chain CDR consisting of 1CDR-L1, 1CDR-L2, 1 CDR-L3; the heavy chain has a heavy chain CDR consisting of 1CDR-H1, 1CDR-H2, 1 CDR-H3;
the amino acid sequences of the 1CDR-L1, the 1CDR-L2 and the 1CDR-L3 are respectively shown as SEQ ID No.4, 5 and 6, and the amino acid sequences of the 1CDR-H1, the 1CDR-H2 and the 1CDR-H3 are respectively shown as SEQ ID No.7, 8 and 9;
b1) a second antibody of SEQ ID No.2, said second antibody and functional fragments thereof comprising a light chain and a heavy chain;
the light chain has a light chain CDR consisting of 2 CDR-L1, 2 CDR-L2, 2 CDR-L3; the heavy chain has a heavy chain CDR consisting of 2 CDR-H1, 2 CDR-H2, 2 CDR-H3;
the amino acid sequences of the 2 CDR-L1, the 2 CDR-L2 and the 2 CDR-L3 are respectively shown as SEQ ID No.10, 11 and 12, and the amino acid sequences of the 2 CDR-H1, the 2 CDR-H2 and the 2 CDR-H3 are respectively shown as SEQ ID No.13, 14 and 15;
c1) a third antibody of SEQ ID No.2, said third antibody and functional fragments thereof comprising a light chain and a heavy chain;
the light chain has light chain CDRs consisting of 3 CDR-L1, 3 CDR-L2, 3 CDR-L3; the heavy chain has a heavy chain CDR consisting of 3 CDR-H1, 3 CDR-H2, 3 CDR-H3;
the amino acid sequences of the 3 CDR-L1, the 3 CDR-L2 and the 3 CDR-L3 are respectively shown as SEQ ID NO.16, 17 and 18, and the amino acid sequences of the 3 CDR-H1, the 3 CDR-H2 and the 3 CDR-H3 are respectively shown as SEQ ID NO.19, 20 and 21.
Further, the antibodies and functional fragments thereof include spine 1 chimeric antibodies and functional fragments thereof;
preferably, the antibody comprises the sequence of a constant region of any one of bovine, equine, dairy cow, porcine, ovine, caprine, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, goose, turkey, chicken, banister or human IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD;
preferably, said functional fragment comprises F (ab')2、Fab’、One or more of Fab, Fv, scFv, diabody, and antibody minimal recognition unit.
A hybridoma cell producing the antibody and functional fragments thereof capable of specifically binding to spin 1 is any one of the following a2) -c 2):
a2) the hybridoma cell producing the first antibody has the preservation number of CGMCC No. 15196;
b2) hybridoma cell producing the second antibody with the preservation number of CGMCC No. 15698;
c2) the hybridoma cell producing the third antibody has the preservation number of CGMCC No. 15699.
An isolated nucleic acid molecule selected from the group consisting of:
a3) DNA or RNA encoding the above antibody and functional fragments thereof;
b3) a nucleic acid complementary to the nucleic acid defined in a 3).
A vector comprising the nucleic acid molecule described above.
The spink1 antigen is applied to a4) or b4) as follows:
a4) preparing an antibody capable of specifically binding to spin 1;
b4) screening drugs taking spike 1 protein as a target.
The application of the antibody and the functional fragment thereof in a5) -d 5) as follows:
a5) detecting the expression level of spin 1 protein;
b5) the carrier is used as a drug taking the spine 1 protein as a target;
c5) for purifying or isolating spink1 protein;
d5) the method is used for preparing products for detecting spine 1 protein;
preferably, the spin 1 protein is derived from cattle, horses, dairy cows, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, camels, donkeys, deer, mink, chickens, ducks, geese, turkeys or humans;
preferably, the method for detecting in a5) comprises a western blot method, immunohistochemistry, immunostaining, or ELISA method;
preferably, the sample detected in a5) comprises whole blood, serum, plasma, urine or tumor vesicles;
preferably, the medicament in b5) is used for treating liver cancer, ovarian cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, bladder cancer, stomach cancer or kidney cancer;
preferably, the product in d5) comprises an ELISA kit, a colloidal gold kit or a magnetic bead method kit.
A composition comprising the above antibody and functional fragments thereof and at least one diagnostic and/or therapeutic agent;
the antibodies and functional fragments thereof are conjugated to diagnostic and/or therapeutic agents to form immunoconjugates.
A kit containing the antibody and functional fragments thereof is an ELISA kit, a colloidal gold kit or a magnetic bead method kit, and is used for detecting spin 1 protein.
Compared with the prior art, the invention has the beneficial effects that:
the spine 1 antigen provided by the invention has strong antigen activity, and the selection of peptide chains rather than recombinant proteins as antigens is more favorable for locking antigenic determinants, thereby providing more theoretical support for subsequent ELISA pairing and antibody drug research. SEQ ID NO.1 is aa 67-79 segment of spine 1 protein, and the polypeptide chain is a high hydrophilic region and can generate high titer antibody; SEQ ID NO.2 is aa 1-18 segment of spine 1 protein, and the polypeptide chain can increase specificity of recognizing hepatoma cells; SEQ ID NO.3 is aa 19-35 of the spine 1 protein, the polypeptide chain is more exposed to the outside of the spine 1 protein and more complementary to the C-terminal antibody in the ELAISA double antibody sandwich. The spine 1 antigen provided by the invention has good antigenic activity, is efficient and accurate when being used for preparing spine 1 protein antibodies or screening drugs taking spine 1 protein as a target spot, and has important industrial application value.
The antibody and the functional fragment thereof provided by the invention can be specifically combined with spin 1 protein, have high titer, good sensitivity and stable structure, can be applied to detection, identification, separation and purification of spin 1 protein, and can also be used as a carrier taking spin 1 protein as a target spot.
The hybridoma provided by the invention can generate the antibody with the CDR sequence and the functional fragment thereof, can stably and efficiently generate the high-quality antibody of spin 1 and the functional fragment thereof, and can be used as an engineering strain for industrial production.
The nucleic acid molecule provided by the invention can encode the antibody and the functional fragment thereof or is complementary with the encoded nucleic acid, and the nucleic acid molecule can be applied to genetic engineering to construct a vector or a host cell containing the nucleic acid molecule so as to efficiently and rapidly express the antibody and the functional fragment thereof of the spin 1.
The composition provided by the invention is an immunoconjugate formed by coupling the antibody and the functional fragment thereof with a diagnostic agent and/or a therapeutic agent. The method is used for identifying, purifying, detecting and separating the spin 1 protein, or researching and medicines taking the spin 1 as a target. Due to the high specificity and potency of the antibodies and functional fragments thereof, the compositions can be applied in various fields and scenarios.
The kit provided by the invention comprises but is not limited to an ELISA kit, a colloidal gold kit or a magnetic bead method kit, is used for detecting spin 1 protein, and has the advantages of high sensitivity, low cost and accurate result.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments or the prior art descriptions will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a SDS-PAGE result of a primary antibody in example 8 of the present invention, wherein M: marker, 1 and 2: a first antibody;
FIG. 2 is a SDS-PAGE detection result of a second antibody in example 9 of the present invention;
FIG. 3 is a SDS-PAGE result of a third antibody in example 10 of the present invention, wherein M: maker, 1: a third antibody;
FIG. 4 is a graph showing the results of western blot detection of the first antibody in example 11 of the present invention, wherein the ratios of 1-79: whole protein of spine 1 expressed by liver cancer cell lines (theoretical molecular weight 8.5KD), 24-79: the whole protein of spin 1 (with GST label and theoretical molecular weight of 33KD) expressed by plant cells;
fig. 5 is a graph showing the result of western blot detection of the third antibody in example 12 of the present invention, wherein 1: whole protein of spine 1 expressed by hepatoma cells; 2: blank control.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer.
A spin 1 antigen which is a polypeptide according to any one of the following a) -f):
a)RQTSILIQKSGPC(SEQ ID NO.1)；
b) a polypeptide which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO.1, has the function of a spink1 epitope and is derived from a);
c)MKVTGIFLLSALALLSLS(SEQ ID NO.2)；
d) c) the polypeptide which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO.2 and has the function of the epitope of the spine 1 and is derived from the c);
e)GNTGADSLGREAKCYNE(SEQ ID NO.3)；
f) a polypeptide which is derived from the e) and has the function of the epitope of the spin 1 by substituting and/or deleting and/or adding one or more amino acid residues of the amino acid sequence shown in the SEQ ID NO. 3.
The spine 1 antigen provided by the invention has good immunogenicity, and the selection of peptide chains rather than recombinant proteins as antigens is more favorable for locking antigenic determinants, thereby providing more theoretical support for subsequent ELISA pairing and antibody drug research. SEQ ID NO.1 is aa 67-79 segment of spine 1 protein, the polypeptide chain is a high hydrophilic region, and the antigen and the derived antigen thereof can generate high-titer antibodies; SEQ ID NO.2 is aa 1-18 segment of spine 1 protein, the expression molecular weight (about 10kd) of spine 1 in the liver cancer cell is greater than the expected molecular weight (about 6.3kd), and the result shows that the molecular weight change is probably caused by incomplete excision of an N-terminal signal peptide chain in the liver cancer cell, so that the polypeptide chain and the derived antigen thereof can increase the specificity of recognizing the liver cancer cell; SEQ ID NO.3 is aa 19-35 of the spine 1 protein, the polypeptide chain is more exposed to the outside of the spine 1 protein and more complementary to the C-terminal antibody in the ELAISA double antibody sandwich. The spine 1 antigen provided by the invention has good antigenic activity, is economic and effective, and provides a new idea for preparing spine 1 protein antibodies or screening drugs taking spine 1 protein as a target. Specific humoral immune response aiming at the spin 1 protein can be induced and generated in a mouse animal model, and the obtained antibody has high titer and good quality.
An antibody and a functional fragment thereof capable of specifically binding to spin 1, which is any one of the following a1) -c 1):
a1) a first antibody of SEQ ID No.1, the first antibody and functional fragments thereof comprising a light chain and a heavy chain;
the light chain has light chain CDRs consisting of 1CDR-L1, 1CDR-L2, 1 CDR-L3; the heavy chain has a heavy chain CDR consisting of 1CDR-H1, 1CDR-H2, 1 CDR-H3;
the amino acid sequences of 1CDR-L1, 1CDR-L2 and 1CDR-L3 are respectively shown as SEQ ID NO.4, 5 and 6, and the amino acid sequences of 1CDR-H1, 1CDR-H2 and 1CDR-H3 are respectively shown as SEQ ID NO.7, 8 and 9;
b1) a second antibody of SEQ ID No.2, the second antibody and functional fragments thereof including a light chain and a heavy chain;
the light chain has light chain CDRs consisting of 2 CDR-L1, 2 CDR-L2, 2 CDR-L3; the heavy chain has heavy chain CDRs consisting of 2 CDR-H1, 2 CDR-H2, 2 CDR-H3;
the amino acid sequences of 2 CDR-L1, 2 CDR-L2 and 2 CDR-L3 are respectively shown in SEQ ID No.10, 11 and 12, and the amino acid sequences of 2 CDR-H1, 2 CDR-H2 and 2 CDR-H3 are respectively shown in SEQ ID No.13, 14 and 15;
c1) a third antibody of SEQ ID No.2, the third antibody and functional fragments thereof comprising a light chain and a heavy chain;
the light chain has light chain CDRs consisting of 3 CDR-L1, 3 CDR-L2, 3 CDR-L3; the heavy chain has heavy chain CDRs consisting of 3 CDR-H1, 3 CDR-H2, 3 CDR-H3;
the amino acid sequences of 3 CDR-L1, 3 CDR-L2 and 3 CDR-L3 are respectively shown in SEQ ID NO.16, 17 and 18, and the amino acid sequences of 3 CDR-H1, 3 CDR-H2 and 3 CDR-H3 are respectively shown in SEQ ID NO.19, 20 and 21.
It is well known in the art that both the binding specificity and avidity of an antibody are determined primarily by the CDR sequences, and that variants with similar biological activity can be obtained by readily altering the amino acid sequence of the non-CDR regions according to well-established, well-known techniques of the art.
The antibody and the functional fragment thereof provided by the invention can be specifically combined with spin 1 protein, have high titer, good sensitivity and stable structure, can be applied to detection, identification, separation and purification of spin 1 protein, and can also be used as a carrier taking spin 1 protein as a target spot.
In some embodiments of the invention, antibodies and functional fragments thereof include spine 1 chimeric antibodies and functional fragments thereof.
In some embodiments of the invention, the antibody comprises a sequence of a constant region of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD of a cow, horse, dairy cow, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, goose, turkey, game hen or human.
In some embodiments of the invention, the functional fragment comprises F (ab')2One or more of, Fab', Fab, Fv, scFv, diabody, and antibody minimal recognition unit.
"functional fragment" as referred to herein particularly refers to an antibody fragment having the same specificity for spink1 as the parent antibody. In addition to the above functional fragments, any fragment having an increased half-life is also included.
scFv (sc ═ single chain), bispecific antibodies (diabodies).
These functional fragments typically have the same binding specificity as the antibody from which they are derived. As the person skilled in the art deduces from the description of the invention, the antibody fragment of the invention may be obtained by methods such as enzymatic digestion (including pepsin or papain) and/or by chemical reduction cleavage of disulfide bonds.
Antibody fragments can also be obtained by peptide synthesis by recombinant genetic techniques also known to those skilled in the art or by, for example, automated peptide synthesizers, such as those sold by Applied BioSystems and the like.
A hybridoma cell producing the antibody and functional fragments thereof capable of specifically binding to spin 1 is any one of the following a2) -c 2):
a2) the hybridoma cell producing the first antibody has the preservation number of CGMCC No. 15196;
b2) hybridoma cell producing the second antibody with the preservation number of CGMCC No. 15698;
c2) the hybridoma cell producing the third antibody has the preservation number of CGMCC No. 15699.
The hybridoma cell KZ-017-E6-2017-1226 is preserved in the ordinary microorganism center of China Committee for culture Collection of microorganisms 27.12.2017, the preservation unit address is No.3 of the No.1 Xilu Beijing Kogyo of the Beijing city, the preservation number is CGMCC No.15196, and the classification name of the strain is hybridoma cell strain.
The hybridoma cell KZ-017-E6-2017-1226 produces a first antibody capable of specifically binding to spine 1 and comprises a light chain and a heavy chain;
the light chain has light chain CDRs consisting of 1CDR-L1, 1CDR-L2, 1 CDR-L3; the heavy chain has a heavy chain CDR consisting of 1CDR-H1, 1CDR-H2, 1 CDR-H3;
the amino acid sequences of 1CDR-L1, 1CDR-L2 and 1CDR-L3 are respectively shown in SEQ ID NO.4, 5 and 6, and the amino acid sequences of 1CDR-H1, 1CDR-H2 and 1CDR-H3 are respectively shown in SEQ ID NO.7, 8 and 9. The specific sequence information is shown in table 1 below:
TABLE 1CDR information of the first antibody
Hybridoma cell KZN 1-3C 8-E8-2018-one 0507 is preserved in the common microorganism center of China Committee for culture Collection of microorganisms at 05.08.2018, the preservation unit address is No.3 of Xilu No.1 Bichen of the Chaoyang district in Beijing, the preservation number is CGMCC No.15698, and the classification name of the strain is hybridoma cell strain.
The hybridoma cell KZN 1-3C 8-E8-2018-0507 produced secondary antibody capable of specifically binding to spine 1 comprises a light chain and a heavy chain;
the light chain has light chain CDRs consisting of 2 CDR-L1, 2 CDR-L2, 2 CDR-L3; the heavy chain has heavy chain CDRs consisting of 2 CDR-H1, 2 CDR-H2, 2 CDR-H3;
the amino acid sequences of 2 CDR-L1, 2 CDR-L2 and 2 CDR-L3 are respectively shown in SEQ ID No.10, 11 and 12, and the amino acid sequences of 2 CDR-H1, 2 CDR-H2 and 2 CDR-H3 are respectively shown in SEQ ID No.13, 14 and 15. The specific sequence information is shown in table 2 below:
TABLE 2CDR information of the second antibody
| Sequence of | Serial number | |
| 2CDR－L1 | SSVSY | SEQ ID NO.10 |
| 2CDR－L2 | DTS | SEQ ID NO.11 |
| 2CDR－L3 | QQWRSNPPT | SEQ ID NO.12 |
| 2CDR－H1 | GFTFSSYG | SEQ ID NO.13 |
| 2CDR－H2 | INSNGGST | SEQ ID NO.14 |
| 2CDR－H3 | ARKITAWFAY | SEQ ID NO.15 |
Hybridoma cell KZN 3-5D 7-D6-2018-one 0508 is preserved in the common microorganism center of China Committee for culture Collection of microorganisms at 05.08.2018, the preservation unit address is No.3 of Xilu No.1 Bichen of the Shangyang district of Beijing city, the preservation number is CGMCC No.15699, and the classification name of the strain is hybridoma cell strain.
The hybridoma cell KZN 3-5D 7-D6-2018-0508 produces a third antibody capable of specifically binding to spin 1 comprising a light chain and a heavy chain;
the light chain has light chain CDRs consisting of 3 CDR-L1, 3 CDR-L2, 3 CDR-L3; the heavy chain has heavy chain CDRs consisting of 3 CDR-H1, 3 CDR-H2, 3 CDR-H3;
the amino acid sequences of 3 CDR-L1, 3 CDR-L2 and 3 CDR-L3 are respectively shown in SEQ ID NO.16, 17 and 18, and the amino acid sequences of 3 CDR-H1, 3 CDR-H2 and 3 CDR-H3 are respectively shown in SEQ ID NO.19, 20 and 21. The specific sequence information is shown in table 3 below:
TABLE 3CDR information for the third antibody
| Sequence of | Serial number | |
| 3CDR－L1 | QSVDYEGNSY | SEQ ID NO.16 |
| 3CDR－L2 | VAS | SEQ ID NO.17 |
| 3CDR－L3 | QQSNEDPWT | SEQ ID NO.18 |
| 3CDR－H1 | GYPFSRYW | SEQ ID NO.19 |
| 3CDR－H2 | IYPGDGDT | SEQ ID NO.20 |
| 3CDR－H3 | ARKGMLTPVDY | SEQ ID NO.21 |
The hybridoma provided by the invention can generate the antibody with the CDR sequence and the functional fragment thereof, can stably and efficiently generate the high-quality antibody of spin 1 and the functional fragment thereof, and can be used as an engineering strain for industrial production.
An isolated nucleic acid molecule selected from the group consisting of:
a3) DNA or RNA encoding the above antibody and functional fragments thereof;
b3) a nucleic acid complementary to the nucleic acid defined in a 3).
A vector comprising a nucleic acid as described above.
The invention further comprises at least one nuclear construct, preferably a vector, further preferably an expression vector, such as a plasmid, encoding a nucleic acid molecule as described above.
A host cell transformed with a vector as described above. In a preferred embodiment, the host cell is a eukaryotic cell, such as a mammalian cell.
The spink1 antigen is applied to a4) or b4) as follows:
a4) preparing an antibody capable of specifically binding to spin 1;
b4) screening drugs taking spike 1 protein as a target.
The application of the antibody and the functional fragment thereof in a5) -d 5) as follows:
a5) detecting the expression level of spin 1 protein;
b5) the carrier is used as a drug taking the spine 1 protein as a target;
c5) for purifying or isolating spink1 protein;
d5) the method is used for preparing products for detecting spine 1 protein.
In some embodiments of the invention, the source of spin 1 protein is a cow, horse, dairy cow, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, goose, turkey, chicken fighting, or human.
In some embodiments of the present invention, the method for detecting the expression level of spin 1 protein by using the above-mentioned antibody and functional fragment thereof comprises western blot method, immunohistochemistry, immunostaining, or ELISA method.
In some embodiments of the present invention, the sample for detecting the expression level of spin 1 protein using the above-described antibodies and functional fragments thereof includes whole blood, serum, plasma, urine, or tumor vesicles.
In some embodiments of the present invention, a drug targeting the spin 1 protein can be developed on the basis of the above-mentioned antibody and functional fragments thereof, and the drug is used for treating liver cancer, ovarian cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, bladder cancer, stomach cancer, kidney cancer, etc.
In some embodiments of the present invention, the product for identifying and detecting the spin 1 protein comprises an ELISA kit, a colloidal gold kit or a paramagnetic bead method kit, etc.
A composition comprising the above antibody and functional fragments thereof and at least one diagnostic and/or therapeutic agent, wherein the antibody and functional fragments thereof are conjugated to the diagnostic and/or therapeutic agent to form an immunoconjugate. The composition is used for identification, purification, detection and separation of the spine 1 protein, or research and medicines taking spine 1 as a target. Due to the high specificity and potency of the antibodies and functional fragments thereof, the compositions can be applied in various fields and scenarios.
In some preferred embodiments of the invention, the diagnostic agent is selected from: one or more of a radionuclide, a radiocontrast agent, a paramagnetic ion, a metal, a fluorescent label, a chemiluminescent label, an ultrasound contrast agent, or a photosensitizer.
In some embodiments of the invention, the radionuclide comprises110In、111In、177Lu、18F、52Fe、62Cu、64Cu、67Cu、67Ga、68Ga、86Y、90Y、89Zr、94mTc、94Tc、99mTc、120I、123I、124I、125I、131I、154－158Gd、32P、11C、13N、15O、186Re、188Re、51Mn、52mMn、55Co、72As、75Br、76Br、82mRb or83Sr.
In some embodiments of the invention, the paramagnetic ion comprises one or more of chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III), or erbium (III).
In some embodiments of the invention, the fluorescent label comprises Alexa 350, Alexa 405, Alexa 430, Alexa 488, Alexa 555, Alexa 647, AMCA, aminoacridine, BODIPY 630/650, BODIPY 650/665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, 5-carboxy-4 ', 5' -dichloro-2 ', 7' -dimethoxyfluorescein, 5-carboxy-2 ', 4', 5 ', 7' -tetrachlorofluorescein, 5-carboxyfluorescein, 5-carboxyrhodamine, 6-carboxytetramethylrhodamine, Cascade Blue, Cy2, Cy3, Cy5, Cy7, 6-FAM, dansyl chloride, fluorescein, HEX, 6-JOE, NBD (7-nitrobenz-2-oxa-1, 3-oxadiazole), Oregeon 488, Oregeon 500, Oregon Green, Oreba 500, Blue Green, terephthalic acid, isophthalic acid, or terephthalic acid or mixtures thereof, Cresol fast violet, cresol blue violet, brilliant cresol blue, p-aminobenzoic acid, erythrosine, phthalocyanine, azomethine, cyanine, xanthine, succinyl fluorescein, rare earth metal cryptate, tripyridyldiamine europium, europium cryptate or chelate, diamine, bispyanin, LaJolla blue dye, allophycocyanin, allocyanonin B, phycocyanin C, phycocyanin R, thiamine, phycoerythrin R, REG, rhodamine green, rhodamine isothiocyanate, rhodamine red, ROX, TAMRA, TET, TRIT (tetramethylrhodamine isothiol), tetramethylrhodamine, or texas red.
In some preferred embodiments of the invention, the therapeutic agent is selected from: one or more of a naked antibody, cytotoxic agent, drug, radionuclide, boron atom, immunomodulator, anti-apoptotic agent, photoactive therapeutic agent, immunoconjugate or oligonucleotide.
In some embodiments of the invention, the drug is selected from methotrexate, fluorouracil, mercaptopurine, hydroxyurea, cytarabine, mechlorethamine, cyclophosphamide, thiotepa, cisplatin, mitomycin, bleomycin, camptothecin, podophyllotoxin, actinomycin D, doxorubicin, daunorubicin, vinblastine, paclitaxel, cephalotaxus alkaloids, or L-asparaginase.
In some embodiments of the invention, the oligonucleotide is selected from one or more of shRNA, miRNA, or siRNA.
In some embodiments of the invention, the immunomodulator is selected from: one or more of a cytokine, chemokine, stem cell growth factor, lymphotoxin, hematopoietic factor, Colony Stimulating Factor (CSF), interferon, erythropoietin, thrombopoietin, Tumor Necrosis Factor (TNF), Interleukin (IL), granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage-colony stimulating factor (GM-CSF), or stem cell growth factor.
In some embodiments of the invention, the radionuclide is selected from111In、111At、177Lu、211Bi、212Bi、213Bi、211At、62Cu、67Cu、90Y、125I、131I、133I、32P、33P、47Sc、111Ag、67Ga、153Sm、161Tb、152Dy、166Dy、161Ho、166Ho、186Re、188Re、189Re、211Pb、212Pb、223Ra、225Ac、77As、89Sr、99Mo、105Rh、149Pm、169Er、194Ir、58Co、80mBr、99mTc、103mRh、109Pt、119Sb、189mOs、192Ir、219Rn、215Po、221Fr、255Fm、11C、13N、15O、75Br、198Au、199Au、224Ac、77Br、113mIn、95Ru、97Ru、103Ru、105Ru、107Hg、203Hg、121mTe、122mTe、125mTe、165Tm、167Tm、168Tm、197Pt、109Pd、142Pr、143Pr、161Tb、57Co、58Co、51Cr、59Fe、75Se、201Tl、76Br and169one or more of Yb.
The kit containing the antibody and the functional fragment thereof can be, but is not limited to, an ELISA kit, a colloidal gold kit or a magnetic bead method kit, and is used for detecting the spin 1 protein.
In order to facilitate a further understanding of the present invention, the technical solutions of the present invention will now be described in detail with reference to the preferred embodiments.
Unless otherwise specified, all instruments or reagents used in the examples of the present invention are commercially available. The test methods used in the examples of the present invention are all conventional methods unless otherwise specified.
The antigen screening process, the acquisition of the primary antibody and hybridoma cells expressing the same, and the measurement of the antibody titer will be described in detail below, taking the spin 1 antigen of SEQ ID NO.1 as an example.
EXAMPLE 1 polypeptide Synthesis
Protein structure prediction is carried out on spine 1 protein through I-TASSER protein structure prediction software, and as a result, the aa 67-79 region is a high hydrophilic region and is expected to have strong antigen activity. The polypeptide chain KZ-017 intercepted by the synthetic spink1 (spink 1aa 67-79, the specific sequence is: RQTSILIQKSGPC) has the following purity: greater than 90% with 5mg coupled KLH and the remaining about 5mg used in ELISA. The KLH-coupled polypeptide antigen and polypeptide were diluted to 1mg/ml with sterile PBS and stored at-20 ℃ for further use. The selection of the peptide chain rather than the recombinant protein as the antigen is more helpful for locking antigenic determinants, and provides more theoretical support for subsequent ELISA pairing and antibody drug research.
Example 2 animal immunization
Immunization of 5 Male BALB/c mice for 6-8 weeks
1. Primary immunization: KLH-conjugated polypeptide was mixed with equal volume of Freund's complete adjuvant, emulsified and then immunized subcutaneously, with 400ul of emulsion injected subcutaneously per mouse.
2. And (3) second immunization: 1mg/ml KZ-017-KLH antigen with equal volume of Freund's incomplete adjuvant 1: 1 mixing, emulsifying, and injecting 200ul of emulsion into the abdominal cavity/mouse.
3. And (3) third immunization: and (4) carrying out simultaneous immunization.
4. Blood is taken from the eye vein, placed at room temperature for 2h, centrifuged at 4000rpm for 5min, and serum is taken.
5. And (3) coating KZ-017 polypeptide on a 96-well plate, and detecting the serum titer of the immune mice by ELISA.
ELISA step:
1) the amount of polypeptide coated was 100 ng/well, 100 ul/well, diluted with PBS, and left overnight at 4 ℃.
2) Pouring out, adding sealing liquid (10% skimmed milk powder, dissolved in PBS) 300 ul/well, 37 deg.C, and 2 hr.
3) Pouring out, patting the liquid dry, and freezing and storing at-20 ℃ for later use.
4) And taking out the coated plate in a refrigerator, putting the plate to room temperature, adding 100 ul/hole of serum diluent, and keeping the temperature at 37 ℃ for 1 h.
5) PBST (0.1% Tween) wash plate 3 times.
6) Then, 1ul of secondary antibody (horseradish enzyme-labeled goat anti-mouse) was added, diluted with 10ml of PBS, 100 ul/well, 37 ℃ and 45 min.
7) PBST wash plate 3 times.
8) Adding 100ul of TMB per hole, and keeping the temperature at 37 ℃ for about 15 min.
9) And adding 50ul of 2M hydrochloric acid stop solution per hole to stop the reaction.
10) OD value was measured at a wavelength of 450 nm.
The ELISA results show that: the serum titer of the mice immunized by KZ-017-KLH can be finally immunized.
6. Final immunity: mice # 1 and # 2 were selected and injected intraperitoneally with 200ug KZ-017-KLH antigen per mouse, 200ul antigen mixed with 200ul PBS.
Example 3 cell fusion
1. Mouse myeloma cells were recovered and cultured (10% FBS + DMEM medium).
2. Taking spleen of a terminally immunized mouse, preparing a single cell suspension by using a culture medium, and counting.
3. According to the following steps: 5 mixing myeloma cells and mouse spleen cells, and adding PEG for cell fusion.
4. 100 ul/well 2 XHAT complete medium was plated into 96 well plates and 100ul fused cell liquid/well was added.
5. The fusion cell culture medium was replaced with 1 × HAT half.
Example 4 ELISA screening of cell well supernatants
1. The amount of polypeptide coated was 100 ng/well, 100 ul/well, diluted in PBS, and incubated overnight at 4 ℃. Pouring off, adding sealing solution (10% skimmed milk powder, dissolved in PBS) 300 ul/well, 37 deg.C, 2 h.
3. Pouring out, patting the liquid dry, and freezing and storing at-20 ℃ for later use.
4. The coated plate was taken out of the freezer and allowed to stand at room temperature, 100 ul/well of cell supernatant was added, 37 ℃ and 1 hour.
5. PBST (0.1% Tween) plates 3 times.
6. Add secondary antibody (horseradish enzyme labeled goat anti-mouse) 1ul, 10ml PBS dilution, 100 ul/hole. 37 ℃ for 45 min.
7. PBST wash plate 3 times.
8. Adding TMB 100 ul/hole, 37 deg.C, 15 min.
9. The reaction was stopped by adding 50ul of 2M HCl buffer solution per well.
10. OD was measured at a wavelength of 450 nm.
The ELISA results show that: there were multiple highly positive cloning wells. And (3) selecting 5 strains of the fused well cells with high OD value, few clones and good cell states, and carrying out the next step of limited dilution well screening of monoclonal hybridoma.
EXAMPLE 5 screening of monoclonal hybridoma cell lines and hybridoma cell line Collection
1. Feeder cells were prepared from Balb/c or Kunming healthy mice (both male and female).
2. Limiting dilution of selected fusion well cells: the cells were diluted in 1 XHT medium and the cell suspension per well was added to feeder cells in 96 wells at concentrations of 0.75, 1.5, and 3 cells per 100 ul/well, 5% CO2And cultured at 37 ℃.
3. After 10 days, the growth of the clones is observed under a microscope, ELISA is used for detecting the clone holes with cells, and the holes with high positive value and single clone are selected (limited dilution is carried out if necessary until all the clone holes are positive).
4. The selected monoclonal hybridoma cells were expanded in culture and frozen.
As a result: and (5) amplifying and freezing and storing KZ-017-E6-2017-1226.
5. The hybridoma is preserved in 2017 at 27.12.12 to China general microbiological culture Collection center, and the number of the preservation registration accession number is as follows: CGMCC No. 15196.
EXAMPLE 6 antibody production purification
1. Pretreatment of BALB/c mice: mice were treated with sterilized liquid paraffin, and injected intraperitoneally with 0.5 mL/mouse, and hybridoma cells were injected 7-10 days later.
2. Diluting hybridoma cells into 1-2 × 10 with DMEM basic culture solution6One mouse per mL, and 0.5mL per mouse per intraperitoneal injection.
3. Ascites collection: after 7 days, the ascites generation condition of the mice is observed every day, if the abdomen is obviously enlarged and the skin is tense when the mice are touched by hands, the ascites can be collected once every one to two days. This was repeated until the mice died naturally. Ascites was centrifuged (5 min at 2000 rpm), the uppermost layer of adipose tissue was aspirated, and cellular components and other precipitates were removed.
4. Ascites purification: the pretreated ascites fluid was incubated using Protein G column and eluted. Finally obtaining the purified monoclonal antibody.
Example 7 hybridoma CDR sequencing
Amplifying mouse VH & VL from KZ-017-E6-2017-1226 cells, and connecting a target gene into a T vector and sequencing to obtain a CDR sequence of a first antibody:
light chain antigen binding region:
1CDR－L1：ENIYSY(SEQ ID NO.4)
1CDR－L2：NAK(SEQ ID NO.5)
1CDR－L3：QHHYGIPWT(SEQ ID NO.6)
heavy chain antigen binding region:
1CDR－H1：GFTFSSFG(SEQ ID NO.7)
1CDR－H2：ISSGSSTL(SEQ ID NO.8)
1CDR－H3：DHGHRLL(SEQ ID NO.9)
example 8 antibody subtype and potency assay
The first antibody subtype is IgG, kappa.
The purified antibody is loaded on SDS-PAGE gel for two times to carry out purity determination, and the purity of the primary antibody is more than 95 percent through Image J analysis. The results are shown in FIG. 1.
The primary antibody concentration was 0.6mg/ml as determined by NanoDrop, the titer was 1: 80000, the minimal action concentration of the primary antibody is 0.0075ug/ml, which is superior to the commercially available antibody (the minimal action concentration is 0.8 ug/ml). The results of the antibody titer measurements are shown in the following table:
it should be noted that: positive controls were fusion sera, dilutions 1: 1000, negative control healthy mouse serum 1: 1000 dilution.
Example 9 spin 1 antigen of SEQ ID NO.2
Based on the same method as in example 1, KZN1 (spink 1aa 1-18, specific sequence: MKVTGIFLLSALALLSLS) which is a polypeptide chain intercepted by spink1 was synthesized, and the reason why the polypeptide chain was selected as an antigen to make an antibody was that: the research shows that the expression molecular weight (about 10kd) of the spine 1 in the hepatoma cells is larger than the expected molecular weight (about 6.3kd), and the sequencing result shows that the molecular weight change is probably caused by that the N-terminal signal peptide chain in the hepatoma cells is not completely cut off. Therefore, the sequence is selected to increase the specificity of recognizing hepatoma cells.
The experiment of KZN1 was conducted according to the experimental procedures of examples 2-5, and as a result, hybridoma cell KZN 1-3C 8-E8-2018-: CGMCC No. 15698.
The secondary antibody was purified and CDR sequenced with reference to the experimental procedures of examples 6-7:
light chain antigen binding region:
2CDR－L1：SSVSY(SEQ ID NO.10)
2CDR－L2：DTS(SEQ ID NO.11)
2CDR－L3：QQWRSNPPT(SEQ ID NO.12)
heavy chain antigen binding region:
2CDR－H1：GFTFSSYG(SEQ ID NO.13)
2CDR－H2：INSNGGST(SEQ ID NO.14)
2CDR－H3：ARKITAWFAY(SEQ ID NO.15)
the second antibody subtype was IgM, kappa.
The purified secondary antibody was loaded on SDS-PAGE gels and purity determination was repeated with greater than 90% by ImageJ analysis. The results are shown in FIG. 2.
The concentration of the second antibody was 0.18mg/ml as determined by NanoDrop, the titer was 1: 4000, i.e.the second antibody had a minimum action concentration of 0.045 ug/ml. The results of the antibody titer measurements are shown in the following table:
| KZN1－3C8－E8－2018－0507 | |
| 1：500 | 1.095 |
| 1：1000 | 0.878 |
| 1：2000 | 0.528 |
| 1：4000 | 0.267 |
| 1：8000 | 0.197 |
| 1：16000 | 0.178 |
| PBS | 0.183 |
example 10 the spin 1 antigen of SEQ ID NO.3
Based on the same method as in example 1, KZN3 (spink 1aa 19-35, specific sequence: GNTGADSLGREAKCYNE) which is a polypeptide chain intercepted by spink1 was synthesized, and the reason why the polypeptide chain was selected as an antigen to make an antibody was that: the peptide chain is more easily exposed to the outer side of spin 1 protein, has strong antigenicity, and the determined N-terminal peptide chain provides more information for antibody drug development and is more easily complementary with C-terminal antibody in an ELAISA double-antibody sandwich method.
The experiment of KZN1 was conducted according to the experimental procedures of examples 2-5, and as a result, hybridoma cell KZN 3-5D 7-D6-2018-: CGMCC No. 15699.
The secondary antibody was purified and CDR sequenced with reference to the experimental procedures of examples 6-7:
light chain antigen binding region:
3CDR－L1：QSVDYEGNSY(SEQ ID NO.16)
3CDR－L2：VAS(SEQ ID NO.17)
3CDR－L3：QQSNEDPWT(SEQ ID NO.18)
heavy chain antigen binding region:
3CDR－H1：GYPFSRYW(SEQ ID NO.19)
3CDR－H2：IYPGDGDT(SEQ ID NO.20)
3CDR－H3：ARKGMLTPVDY(SEQ ID NO.21)
the third antibody subtype is IgG1, kappa.
The purified third antibody is loaded on SDS-PAGE gel for two repeated purity determination, and the purity is more than 90% by Image J analysis. The results are shown in FIG. 3.
The third antibody concentration was 0.3mg/ml as determined by NanoDrop, the titer was 1: 640000, the minimal effect concentration of the third antibody was 0.00046875 ug/ml. The results of the antibody titer measurements are shown in the following table:
| KZN3－5D7－D6－2018－0508 | KZN3－5D7－D6－2018－0508 | |
| 1：10000 | 3.654 | 3.575 |
| 1：20000 | 3.637 | 3.546 |
| 1：40000 | 3.595 | 3.557 |
| 1：80000 | 3.559 | 3.516 |
| 1：160000 | 3.485 | 3.486 |
| 1：320000 | 3.168 | 2.982 |
| 1：640000 | 2.205 | 2.08 |
| PBS | 0.178 | 0.168 |
example 11 Western blot of primary antibodies in KZ-017-E6-2017-containing 1226 cells
1.SDS－PAGE
5% concentrated gel + 15% separation gel (containing 10% glycerol), 80V 100min, 110V 60 min. The loading volume was 10. mu.L.
Sample treatment: blood sample of liver cancer patient, normal blood sample, precipitating macromolecular protein with equal volume of acetonitrile, collecting supernatant, and boiling at 100 deg.C for 3-5 min.
The Spink1 protein is cooked for 3-5 min at 100 ℃. The loading amount was 4. mu.g.
2. Rotary film
And wet rotating at 100V for 40 min.
3. Sealing of
5% skim milk (PBST) was then shake-sealed for 2h at room temperature.
4. Primary antibody incubation
Primary antibody was diluted with 5% skim milk (PBST) at 1. mu.g/mL overnight at 4 ℃.
5. Washing three times, 5min each time.
6. Incubation with secondary antibody
Dilution of goat anti-mouse-HRP with PBST was 1: 5000, shaking at room temperature for 1 h.
7. Washing three times, 5min each time.
8. Exposure method
ECL exposure and observation of the bands, the results are shown in FIG. 4. The test result shows that the first antibody can simultaneously recognize the spine 1 protein expressed by the liver cancer cells and the plant cells.
Example 12 Western blot of the third antibody in KZN 3-5D 7-D6-2018-0508 cells
1.SDS－PAGE
5% concentrated gel + 15% separation gel (containing 10% glycerol), 80V 100min, 110V 60 min. The loading volume is 10. mu.L.
Sample treatment: blood samples of liver cancer patients and normal blood samples are obtained by precipitating macromolecular protein with acetonitrile of the same volume, taking supernatant, and opening a cover at 100 ℃ to boil the sample for 3-5 min.
The Spink1 protein is cooked for 3-5 min at 100 ℃. The loading amount was 4. mu.g.
2. Rotary film
Wet-rotating at 100V for 40 min.
3. Sealing of
5% skim milk (PBST) was then shake-sealed for 2h at room temperature.
4. Primary antibody incubation
Primary antibody was diluted with 5% skim milk (PBST) at 1. mu.g/mL overnight at 4 ℃.
5. Washing three times, 5min each time.
6. Incubation with secondary antibody
Dilution of goat anti-mouse-HRP with PBST was 1: 5000, shaking at room temperature for 1 h.
7. Washing three times, 5min each time.
8. Exposure to light
ECL exposure and strip observation are carried out, the result is shown in FIG. 5, and the test result shows that the kzn3 antibody can recognize spin 1 protein expressed by liver cancer cells.
While particular embodiments of the present invention have been illustrated and described, it would be obvious that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.
SEQUENCE LISTING
<110> tertiary amines Seao (Tianjin) Biotechnology Limited
TIANJIN REASONBIO Co.,Ltd.
<120> spin 1 antigen, antibody capable of specifically binding spin 1, functional fragment thereof, application thereof and product thereof
<160> 21
<170> PatentIn version 3.5
<210> 1
<211> 13
<212> PRT
<213> Artificial sequence
<400> 1
Arg Gln Thr Ser Ile Leu Ile Gln Lys Ser Gly Pro Cys
1 5 10
<210> 2
<211> 18
<212> PRT
<213> Artificial sequence
<400> 2
Met Lys Val Thr Gly Ile Phe Leu Leu Ser Ala Leu Ala Leu Leu Ser
1 5 10 15
Leu Ser
<210> 3
<211> 17
<212> PRT
<213> Artificial sequence
<400> 3
Gly Asn Thr Gly Ala Asp Ser Leu Gly Arg Glu Ala Lys Cys Tyr Asn
1 5 10 15
Glu
<210> 4
<211> 6
<212> PRT
<213> Artificial sequence
<400> 4
Glu Asn Ile Tyr Ser Tyr
1 5
<210> 5
<211> 3
<212> PRT
<213> Artificial sequence
<400> 5
Asn Ala Lys
1
<210> 6
<211> 9
<212> PRT
<213> Artificial sequence
<400> 6
Gln His His Tyr Gly Ile Pro Trp Thr
1 5
<210> 7
<211> 8
<212> PRT
<213> Artificial sequence
<400> 7
Gly Phe Thr Phe Ser Ser Phe Gly
1 5
<210> 8
<211> 8
<212> PRT
<213> Artificial sequence
<400> 8
Ile Ser Ser Gly Ser Ser Thr Leu
1 5
<210> 9
<211> 7
<212> PRT
<213> Artificial sequence
<400> 9
Asp His Gly His Arg Leu Leu
1 5
<210> 10
<211> 5
<212> PRT
<213> Artificial sequence
<400> 10
Ser Ser Val Ser Tyr
1 5
<210> 11
<211> 3
<212> PRT
<213> Artificial sequence
<400> 11
Asp Thr Ser
1
<210> 12
<211> 9
<212> PRT
<213> Artificial sequence
<400> 12
Gln Gln Trp Arg Ser Asn Pro Pro Thr
1 5
<210> 13
<211> 8
<212> PRT
<213> Artificial sequence
<400> 13
Gly Phe Thr Phe Ser Ser Tyr Gly
1 5
<210> 14
<211> 8
<212> PRT
<213> Artificial sequence
<400> 14
Ile Asn Ser Asn Gly Gly Ser Thr
1 5
<210> 15
<211> 10
<212> PRT
<213> Artificial sequence
<400> 15
Ala Arg Lys Ile Thr Ala Trp Phe Ala Tyr
1 5 10
<210> 16
<211> 10
<212> PRT
<213> Artificial sequence
<400> 16
Gln Ser Val Asp Tyr Glu Gly Asn Ser Tyr
1 5 10
<210> 17
<211> 3
<212> PRT
<213> Artificial sequence
<400> 17
Val Ala Ser
1
<210> 18
<211> 9
<212> PRT
<213> Artificial sequence
<400> 18
Gln Gln Ser Asn Glu Asp Pro Trp Thr
1 5
<210> 19
<211> 8
<212> PRT
<213> Artificial sequence
<400> 19
Gly Tyr Pro Phe Ser Arg Tyr Trp
1 5
<210> 20
<211> 8
<212> PRT
<213> Artificial sequence
<400> 20
Ile Tyr Pro Gly Asp Gly Asp Thr
1 5
<210> 21
<211> 11
<212> PRT
<213> Artificial sequence
<400> 21
Ala Arg Lys Gly Met Leu Thr Pro Val Asp Tyr
1 5 10
Claims (11)
1. A spin 1 antigen, which is a polypeptide comprising: RQTSILIQKSGPC is added.
2. An antibody and functional fragments thereof capable of specifically binding to spin 1, comprising a light chain and a heavy chain;
the light chain has a light chain CDR consisting of 1CDR-L1, 1CDR-L2, 1 CDR-L3; the heavy chain has a heavy chain CDR consisting of 1CDR-H1, 1CDR-H2, 1 CDR-H3;
the amino acid sequences of 1CDR-L1, 1CDR-L2 and 1CDR-L3 are respectively shown as SEQ ID NO.4, 5 and 6, and the amino acid sequences of 1CDR-H1, 1CDR-H2 and 1CDR-H3 are respectively shown as SEQ ID NO.7, 8 and 9.
3. The antibody and functional fragments thereof according to claim 2, wherein the antibody and functional fragments thereof comprise a spine 1 chimeric antibody and functional fragments thereof.
4. The antibody and functional fragments thereof of claim 2, wherein the antibody comprises the sequence of any one of the constant regions of bovine, equine, porcine, ovine, caprine, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, goose, or human IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD.
5. A hybridoma cell producing the antibody and functional fragments thereof capable of specifically binding to spine 1 according to any one of claims 2 to 4, wherein the hybridoma cell has a preservation number of CGMCC number 15196.
6. An isolated nucleic acid molecule selected from the group consisting of:
a3) DNA or RNA encoding the antibody of any one of claims 2 to 4 and functional fragments thereof;
b3) a nucleic acid complementary to the nucleic acid defined in a 3).
7. A vector comprising the nucleic acid molecule of claim 6.
8. Use of the antibody and functional fragments thereof according to any one of claims 2 to 4 in b5) -c 5) as follows:
b5) for purifying or isolating spink1 protein;
c5) the method is used for preparing products for detecting spine 1 protein.
9. The use of claim 8, wherein the spin 1 protein is derived from cattle, horses, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, camels, donkeys, deer, mink, chickens, ducks, geese or humans.
10. The use of claim 8, wherein the product of c5) comprises an ELISA kit, a colloidal gold kit or a magnetic bead kit.
11. A kit comprising the antibody and functional fragments thereof according to any one of claims 2 to 4, wherein the kit is an ELISA kit, a colloidal gold kit or a magnetic bead assay kit, and the kit is used for detecting the spin 1 protein.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910014180.1A CN109678950B (en) | 2019-01-08 | 2019-01-08 | spine 1 antigen, antibody capable of specifically binding to spine 1, functional fragment thereof, application and product thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910014180.1A CN109678950B (en) | 2019-01-08 | 2019-01-08 | spine 1 antigen, antibody capable of specifically binding to spine 1, functional fragment thereof, application and product thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109678950A CN109678950A (en) | 2019-04-26 |
| CN109678950B true CN109678950B (en) | 2022-05-10 |
Family
ID=66191928
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910014180.1A Active CN109678950B (en) | 2019-01-08 | 2019-01-08 | spine 1 antigen, antibody capable of specifically binding to spine 1, functional fragment thereof, application and product thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109678950B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP3996816A1 (en) * | 2019-07-08 | 2022-05-18 | Imcare Biotech, LLC | Anti-serine protease inhibitor kazal (spik) antibodies, immunoconjugates, and methods of use |
| JP2022547574A (en) * | 2019-09-11 | 2022-11-14 | インケア バイオテック， エルエルシー | Epitopes of Anti-Serine Protease Inhibitor KAZAL (SPIK) Antibodies |
| CN116063535B (en) * | 2022-09-14 | 2023-09-26 | 北京市疾病预防控制中心 | Antibody or antigen binding fragment thereof, and preparation method and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010099577A1 (en) * | 2009-03-04 | 2010-09-10 | The University Of Queensland | Cancer biomarkers and uses therefor |
| CN101988110A (en) * | 2009-08-07 | 2011-03-23 | 芮屈生物技术（上海）有限公司 | In-situ hybridization detection kit for Spink1 genes and detection method and application thereof |
| CN103233063A (en) * | 2006-11-08 | 2013-08-07 | 密歇根大学董事会 | SPINK1 as a prostate cancer marker and uses thereof |
-
2019
- 2019-01-08 CN CN201910014180.1A patent/CN109678950B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103233063A (en) * | 2006-11-08 | 2013-08-07 | 密歇根大学董事会 | SPINK1 as a prostate cancer marker and uses thereof |
| WO2010099577A1 (en) * | 2009-03-04 | 2010-09-10 | The University Of Queensland | Cancer biomarkers and uses therefor |
| CN101988110A (en) * | 2009-08-07 | 2011-03-23 | 芮屈生物技术（上海）有限公司 | In-situ hybridization detection kit for Spink1 genes and detection method and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| Abstract 712：Antitumor activity of SPINK1 monoclonal antibody in a subset of SPINK1 positive ETS-negative prostate cancer;Ateeq等;《Cancer Research》;20101231;摘要 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109678950A (en) | 2019-04-26 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP7317272B2 (en) | TIGIT Antibodies, Antigen-Binding Fragments Thereof, and Medical Uses Thereof This application is based on and claims priority from Application No. CN201710908565.3 filed on September 29, 2019. The disclosure of which is incorporated herein by reference in its entirety. | |
| CN109937212B (en) | B7-H3 antibody, antigen binding fragment thereof and medical application thereof | |
| US11760795B2 (en) | Monoclonal antibodies to growth and differentiation factor 15 (GDF-15), and uses thereof for treating cancer cachexia and cancer | |
| TWI708788B (en) | Bispecific antibody | |
| CN110914304B (en) | CD96 antibody, antigen binding fragment thereof and medical application | |
| CN109678950B (en) | spine 1 antigen, antibody capable of specifically binding to spine 1, functional fragment thereof, application and product thereof | |
| EP2558501A1 (en) | Monoclonal antibodies against her2 antigens, and uses therefor | |
| WO2018105560A1 (en) | Claudin 5 ANTIBODY, AND MEDICINE CONTAINING SAID ANTIBODY | |
| CN114591434B (en) | anti-Siglec 15 antibody and preparation method and application thereof | |
| JP6749901B2 (en) | Monoclonal anti-GPC-1 antibody and uses thereof | |
| CN113227148B (en) | anti-GPC 3 antibody, antigen-binding fragment thereof, and medical use thereof | |
| WO2019098133A1 (en) | Monoclonal antibody and use thereof | |
| JP4651495B2 (en) | Monoclonal antibody specifically reacting with ISG15 protein, hybridoma producing the same, and method for detecting cancer and virus infection | |
| CN113880948B (en) | anti-CA 724 antibody or antigen-binding fragment thereof, and preparation method and application thereof | |
| CN115925866A (en) | Monoclonal antibody for breast cancer detection and kit thereof | |
| US11634481B2 (en) | B-cell epitope of Trichinella spiralis cysteine protease inhibitor, hybridoma cell line, monoclonal antibody and uses thereof | |
| CN111434686B (en) | Anti-human PBX1 monoclonal antibody, preparation method thereof and application thereof in clinical diagnosis of recurrent abortion | |
| CN116262786A (en) | Single-domain antibody aiming at FOLR1, derivative protein and application thereof | |
| CN115505043A (en) | Antibodies specifically binding glycosylated CEACAM5 | |
| CN114761042A (en) | IL-38 specific antibodies | |
| CN110407942B (en) | Single domain antibodies against KN044 | |
| US20220411527A1 (en) | Compositions and methods for transferrin receptor 1 targeting |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |