CN111349172B - Recombinant antibody of anti-human creatine kinase isoenzyme CK-MB - Google Patents

Recombinant antibody of anti-human creatine kinase isoenzyme CK-MB Download PDF

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CN111349172B
CN111349172B CN201811577526.0A CN201811577526A CN111349172B CN 111349172 B CN111349172 B CN 111349172B CN 201811577526 A CN201811577526 A CN 201811577526A CN 111349172 B CN111349172 B CN 111349172B
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cdr
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崔鹏
何志强
孟媛
钟冬梅
周全兴
梁碧
游辉
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Dongguan Pengzhi Biotechnology Co Ltd
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Abstract

The invention relates to a novel separated binding protein containing creatine kinase isoenzyme CK-MB antigen binding structural domain, and researches on the aspects of preparation, application and the like of the binding protein. The binding protein has strong activity and high affinity with human creatine kinase isoenzyme CK-MB protein, and can be widely applied to the field of detection of the creatine kinase isoenzyme CK-MB protein.

Description

Recombinant antibody of anti-human creatine kinase isoenzyme CK-MB
Technical Field
The invention relates to the technical field of immunity, in particular to a recombinant antibody for resisting human creatine kinase isoenzyme CK-MB.
Background
There are four isozyme forms of creatine kinase isozymes (CK): muscle type (MM), brain type (BB), hybrid type (MB), and mitochondrial type (MiMi). MM type is mainly present in various muscle cells, and is a dimer composed of two identical subunits, BB type is mainly present in brain cells, MB type is mainly present in cardiac muscle cells, and MiMi type is mainly present in cardiac muscle and skeletal muscle mitochondria. CKMB is generally divided into two allotypes of MB 1 and MB 2, wherein the CKMB mainly exists in the form of MB 2 in myocardial cells, and the MB 2 is released once the myocardial cells are damaged, so that the CKMB level in serum is rapidly increased within a short time, the increase time is usually within 6h of the onset of the disease and reaches a peak value within about 24h, and gradually decreases after 72h until the normal level is recovered, which indicates that the CK-MB can reflect the myocardial damage condition at an early stage. Because the sensitivity and the specificity of CKMB in the diagnosis of acute myocardial infarction are higher, after years of deep research and clinical analysis, the increase of CKMB in serum becomes a well-known important index for diagnosing acute myocardial infarction and confirming the existence of myocardial necrosis, has higher diagnosis accuracy and can be popularized and used in clinic. Especially, the use of thrombolytic therapy and emergency percutaneous coronary intervention for patients with acute myocardial infarction in hospitals of all levels is becoming widespread, which requires the necessity of early diagnosis of acute myocardial infarction.
At present, an immunosuppression method is mostly adopted in a clinical laboratory for measuring CKMB, and the method is simple and rapid, but has poor specificity. The detection principle is that the activity of the M subunit is inhibited through an anti-CK-M antibody, the activity of the B subunit is detected, because the content of CKBB in a healthy human body is very small and can be directly ignored, and the activity of CKMB in blood can be obtained by multiplying the detected result by 2. The method has the advantages of simple operation, high detection speed, low cost and the like, and is widely applied to clinical laboratory inspection. However, it was later found that muscle breakdown and some non-cardiac diseases can lead to an increase in the B subunit content, leading to a false increase in CKMB, and therefore, the enzyme quality method has a more significant advantage in terms of accuracy.
The enzyme mass method adopts a chemical immune luminescence technology for quantitative detection, mainly adopts a double-antibody sandwich method, so that an anti-human CKMB antibody is coated on a solid phase carrier such as magnetic particles, and the like, and a marker of the anti-human CKMB antibody is an enzyme-labeled anti-human CKMB antibody for quantitative analysis and detection of CKMB. The detection method has higher specificity to antigen-antibody reaction, has no cross reaction with CKBB and CKMM, has higher sensitivity, and is not influenced by factors such as giant CK and other enzymes in serum.
Monoclonal antibodies with high sensitivity and specificity to CKMB are required to achieve rapid and accurate detection of CKMB. At present, the domestic monoclonal antibody for detecting CKMB is mostly from mouse ascites or purchased from foreign countries, and the sensitivity, specificity and batch stability of the monoclonal antibody have defects.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The invention relates to a novel separated binding protein containing creatine kinase isoenzyme CK-MB antigen binding structural domain, and researches on the aspects of preparation, application and the like of the binding protein.
Wherein the antigen binding domain comprises at least one complementarity determining region selected from the group consisting of amino acid sequences set forth in SEQ ID NO: or; has at least 80% sequence identity to a complementarity determining region of the amino acid sequence shown below and has K with creatine kinase isoenzyme CK-MBD≤3.66×10-8Affinity of mol/L;
CDR-VH1 is G-Y-X1-F-X2-D-Y-W, wherein,
x1 is A or G, X2 is S, Y or T;
CDR-VH2 is Q-X1-Y-P-G-D-X2-D-T-X3-Y-N, wherein,
x1 is L or I, X2 is P, A or G, X3 is N or Q;
CDR-VH3 is A-X1-V-Y-P-X2-F-T-Y-X3, wherein,
x1 is K or R, X2 is S, Y or T, X3 is F or W;
the complementarity determining region CDR-VL1 is K-X1-S-Q-X2-V-S-T-A-X3-A, wherein,
x1 is A or P, X2 is E or D, X3 is I, V or L;
the complementarity determining region CDR-VL2 is S-X1-S-X2-R-Y-T, wherein,
x1 is A or P, X2 is T, Y or S;
the CDR-VL3 is Q-X1-H-Y-S-S-P-X2-T-X3-G, wherein,
x1 is N or Q, X2 is L, V or I, and X3 is W or F.
An important advantage is that the binding protein is highly active and has a high affinity for the human creatine kinase isoenzyme CK-MB.
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 description of the embodiments or the prior art 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 an electrophoretogram of a monoclonal antibody against human creatine kinase isoenzyme CK-MB according to an embodiment of the present invention.
Detailed Description
The present invention may be understood more readily by reference to the following description of certain embodiments of the invention and the detailed description of the examples included therein.
Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such embodiments are necessarily varied. It is also to be understood that the terminology used in the description is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.
Noun definitions
"isolated binding protein comprising an antigen binding domain" broadly refers to all proteins/protein fragments that comprise a CDR region. The term "antibody" includes polyclonal and monoclonal antibodies and antigenic compound-binding fragments of these antibodies, including Fab, F (ab') 2, Fd, Fv, scFv, diabodies and minimal recognition units of antibodies, as well as single chain derivatives of these antibodies and fragments. The type of antibody can be selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, IgD. Furthermore, the term "antibody" includes naturally occurring antibodies as well as non-naturally occurring antibodies, including, for example, chimeric (chimeric), bifunctional (bifunctional) and humanized (humanized) antibodies, as well as related synthetic isomeric forms (isoforms). The term "antibody" is used interchangeably with "immunoglobulin".
The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of the antibody. The variable domain of the heavy chain may be referred to as "VH". The variable domain of the light chain may be referred to as "VL". These domains are usually the most variable parts of an antibody and contain an antigen binding site. The light or heavy chain variable region (VL or VH) is composed of framework regions interrupted by three hypervariable regions, termed "complementarity determining regions" or "CDRs". The extent of the framework regions and CDRs has been precisely defined, for example, in Kabat (see Sequences of Proteins of Immunological Interest), E.Kabat et al, U.S. department of Health and Human Services (U.S.. department of Health and Human Services), (1983), and Chothia. The framework regions of the antibody, which constitute the combination of the essential light and heavy chains, serve to locate and align the CDRs, which are primarily responsible for binding to the antigen.
As used herein, the "framework" or "FR" regions mean the regions of the antibody variable domain excluding those defined as CDRs. Each antibody variable domain framework may be further subdivided into adjacent regions separated by CDRs (FR1, FR2, FR3 and FR 4).
In general, the variable domains VL/VH of the heavy and light chains can be obtained by linking the CDRs and FRs, numbered as follows, in a combined arrangement: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR 4.
The term "purified" or "isolated" in relation to a polypeptide or nucleic acid, as used herein, means that the polypeptide or nucleic acid is not in its natural medium or in its natural form. Thus, the term "isolated" includes a polypeptide or nucleic acid that is removed from its original environment, e.g., from its natural environment if it is naturally occurring. For example, an isolated polypeptide is generally free of at least some proteins or other cellular components that are normally bound to or normally mixed with it or in solution. Isolated polypeptides include the naturally-produced polypeptide contained in a cell lysate, the polypeptide in purified or partially purified form, recombinant polypeptides, the polypeptide expressed or secreted by a cell, and the polypeptide in a heterologous host cell or culture. In connection with a nucleic acid, the term isolated or purified indicates, for example, that the nucleic acid is not in its natural genomic context (e.g., in a vector, as an expression cassette, linked to a promoter, or artificially introduced into a heterologous host cell).
Exemplary embodiments of the invention
The present invention relates to an isolated binding protein comprising an antigen binding domain, wherein the antigen binding domain comprises at least one complementarity determining region selected from the group consisting of amino acid sequences recited in seq id nos: or; has at least 80% sequence identity to a complementarity determining region of the amino acid sequence shown below and has K with creatine kinase isoenzyme CK-MBD≤3.66×10-8Affinity of mol/L;
CDR-VH1 is G-Y-X1-F-X2-D-Y-W, wherein,
x1 is A or G, X2 is S, Y or T;
CDR-VH2 is Q-X1-Y-P-G-D-X2-D-T-X3-Y-N, wherein,
x1 is L or I, X2 is P, A or G, X3 is N or Q;
CDR-VH3 is A-X1-V-Y-P-X2-F-T-Y-X3, wherein,
x1 is K or R, X2 is S, Y or T, X3 is F or W;
the complementarity determining region CDR-VL1 is K-X1-S-Q-X2-V-S-T-A-X3-A, wherein,
x1 is A or P, X2 is E or D, X3 is I, V or L;
the CDR-VL2 is S-X1-S-X2-R-Y-T, wherein,
x1 is A or P, X2 is T, Y or S;
the CDR-VL3 is Q-X1-H-Y-S-S-P-X2-T-X3-G, wherein,
x1 is N or Q, X2 is L, V or I, and X3 is W or F.
In some embodiments, the antigen binding domain has at least 85%, or 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99% sequence identity to a complementarity determining region of an amino acid sequence having K with creatine kinase isoenzyme CK-MBD≤3.66×10-8mol/L,KDThe value can also be selected to be 1 × 10-9mol/L、2×10-9mol/L、3×10-9mol/L、4×10-9mol/L、4.5×10-9mol/L、5×10-9mol/L、6×10- 9mol/L、7×10-9mol/L、8×10-9mol/L、9×10-9mol/L、1×10-10mol/L、3×10-10mol/L、5×10- 10mol/L、7×10-10mol/L、9×10-10mol/L or 1X 10-8mol/L;
Or 8.99X 10-10mol/L≤KD≤3.66×10-8mol/L;
Wherein the affinity is determined according to the method of the present specification.
In some embodiments:
in the CDR-VH1, X1 is A;
in the complementarity determining region CDR-VH2, X3 is N;
in the complementarity determining region CDR-VH3, X3 is W;
in the complementarity determining region CDR-VL1, X1 is A;
in the complementarity determining region CDR-VL2, X1 is P;
in the complementarity determining region CDR-VL3, X3 is F.
In some embodiments, in the complementarity determining region CDR-VH1, X2 is S.
In some embodiments, in the complementarity determining region CDR-VH1, X2 is Y.
In some embodiments, in the complementarity determining region CDR-VH1, X2 is T.
In some embodiments, in the complementarity determining region CDR-VH2, X1 is L.
In some embodiments, in the complementarity determining region CDR-VH2, X1 is I.
In some embodiments, in the complementarity determining region CDR-VH2, X2 is P.
In some embodiments, in the complementarity determining region CDR-VH2, X2 is a.
In some embodiments, in the complementarity determining region CDR-VH2, X2 is G.
In some embodiments, in the complementarity determining region CDR-VH3, X1 is K.
In some embodiments, in the complementarity determining region CDR-VH3, X1 is R.
In some embodiments, in the complementarity determining region CDR-VH3, X2 is S.
In some embodiments, in the complementarity determining region CDR-VH3, X2 is Y.
In some embodiments, in the complementarity determining region CDR-VH3, X2 is T.
In some embodiments, in the complementarity determining region CDR-VL1, X2 is E.
In some embodiments, in the complementarity determining region CDR-V L1, X2 is D.
In some embodiments, in the complementarity determining region CDR-VL1, X3 is I.
In some embodiments, in the complementarity determining region CDR-VL1, X3 is V.
In some embodiments, in the complementarity determining region CDR-VL1, X3 is L.
In some embodiments, in the complementarity determining region CDR-VL2, X2 is T.
In some embodiments, in the complementarity determining region CDR-VL2, X2 is Y.
In some embodiments, in the complementarity determining region CDR-VL2, X2 is S.
In some embodiments, in the complementarity determining region CDR-VL3, X1 is N.
In some embodiments, in the complementarity determining region CDR-VL3, X1 is Q.
In some embodiments, in the complementarity determining region CDR-VL3, X2 is L.
In some embodiments, in the complementarity determining region CDR-VL3, X2 is V.
In some embodiments, in the complementarity determining region CDR-VL3, X2 is I.
In some embodiments, the mutation site of each complementarity determining region is selected from any one of the following combinations of mutations:
Figure GDA0003511344150000061
Figure GDA0003511344150000071
in some embodiments, the binding protein includes at least 3 CDRs (e.g., 3 light chain CDRs or 3 heavy chain CDRs); alternatively, the binding protein comprises at least 6 CDRs.
In some embodiments, the binding protein is a whole antibody comprising a variable region and a constant region.
In some embodiments, the binding protein is a "functional fragment" of an antibody, e.g., a nanobody, F (ab')2Fab', Fab, Fv, scFv, diabody and antibody minimal recognition unit.
scFv (sc ═ single chain), bispecific antibodies (diabodies).
The term "functional fragment" as used herein refers in particular to an antibody fragment having the same specificity for creatine kinase isoenzyme CK-MB as the parent antibody. In addition to the above functional fragments, any fragment having an increased half-life is also included.
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.
In some embodiments, the binding protein comprises light chain framework regions FR-L1, FR-L2, FR-L3 and FR-L4 in the sequence shown in SEQ ID NOS: 1-4, and/or heavy chain framework regions FR-H1, FR-H2, FR-H3 and FR-H4 in the sequence shown in SEQ ID NOS: 5-8.
In addition to the amino acid sequences disclosed herein above, the framework regions may be derived from human species to constitute humanized antibodies.
In some embodiments, the binding protein further comprises an antibody constant region sequence.
In some embodiments, the constant region sequence is selected from the group consisting of sequences of any one of the constant regions of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, IgD.
In some embodiments, the species of the constant region is derived from 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, the constant region is derived from a mouse;
the light chain constant region sequence is shown as SEQ ID NO. 9;
the heavy chain constant region sequence is shown in SEQ ID NO 10.
According to one aspect of the invention, the invention also relates to an isolated nucleic acid molecule, which is DNA or RNA, encoding a binding protein as described above.
According to one aspect of the invention, the invention also relates to a vector comprising a nucleic acid molecule as described above.
The invention further comprises at least one nuclear construct, e.g. a plasmid, further an expression plasmid, encoding a nucleic acid molecule as described above, the construction of which vector will be described in one embodiment of the present application.
According to one aspect of the invention, the invention also relates to a host cell transformed with a vector as described above.
The host cell may be a eukaryotic cell, such as a mammalian cell.
In some embodiments, the host cell is a CHO cell.
According to one aspect of the invention, the invention also relates to a method for producing a binding protein as described above, said method comprising the steps of:
the host cells as described above are cultured in a medium and under suitable culture conditions, and the binding protein so produced is recovered from the medium or from the cultured host cells.
According to one aspect of the invention, the invention also relates to the use of the binding protein as described above for the preparation of a diagnostic agent for the diagnosis of myocardial infarction, viral myocarditis.
According to one aspect of the invention, the invention also relates to a method for detecting creatine kinase isoenzyme CK-MB in a test sample, comprising:
a) contacting creatine kinase isoenzyme, CK-MB, antigen in the test sample with a binding protein as described above under conditions sufficient for an antibody/antigen binding reaction to occur to form an immune complex; and
b) detecting the presence of the immune complex, the presence of the complex indicating the presence of the creatine kinase isoenzyme CK-MB in the test sample.
In this embodiment, the binding protein may be labeled with an indicator that indicates the strength of the signal, so that the complex is readily detected.
In some embodiments, in step a), a second antibody is further included in the immune complex, the second antibody binding to the binding protein.
In some embodiments, in step a), a second antibody is further included in the immune complex, the second antibody binding to the creatine kinase isoenzyme CK-MB;
in this embodiment, the binding protein forms a partner antibody with the second antibody in the form of a first antibody for binding to a different epitope of creatine kinase isoenzyme CK-MB;
the second antibody may be labeled with an indicator showing the intensity of the signal so that the complex is easily detected.
In some embodiments, in step a), a second antibody is further included in the immune complex, the second antibody binding to the creatine kinase isoenzyme CK-MB antigen;
in this embodiment, the binding protein serves as an antigen for the second antibody, which may be labeled with an indicator of signal intensity to allow the complex to be readily detected.
In some embodiments, the indicator that indicates signal intensity comprises any one of a fluorescent substance, a quantum dot, a digoxigenin-labeled probe, biotin, a radioisotope, a radiocontrast agent, a paramagnetic ion fluorescent microsphere, an electron dense substance, a chemiluminescent label, an ultrasound contrast agent, a photosensitizer, colloidal gold, or an enzyme.
In some embodiments, the fluorescent species include Alexa 350, Alexa 405, Alexa 430, Alexa 488, Alexa 555, Alexa 647, AMCA, aminoacridine, BODIPY 630/650, BODIPY650/665, 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-diazole), Any one of Oregon Green 488, Oregon Green 500, Oregon Green514, Pacific Blue, phthalic acid, terephthalic acid, isophthalic acid, cresol fast violet, cresol Blue violet, brilliant cresol Blue, p-aminobenzoic acid, erythrosine, phthalocyanine, azomethine, cyanine, xanthine, succinyl fluorescein, rare earth metal cryptate, europium tripyridyldiamine, europium cryptate or chelate, diamine, bispyanine, La Jolla 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, and Texas red.
In some embodiments, the radioisotope 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 and83sr.
In some embodiments, the enzyme comprises any one of horseradish peroxidase, alkaline phosphatase, and glucose oxidase.
In some embodiments, the fluorescent microspheres are: the polystyrene fluorescent microsphere is internally wrapped with rare earth fluorescent ion europium.
According to one aspect of the invention, the invention also relates to a kit comprising a binding protein as described above.
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. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
Example 1
This example provides an exemplary method for the preparation of recombinant antibodies against human creatine kinase isoenzyme CK-MB.
S1, constructing an expression plasmid:
restriction enzyme, Prime Star DNA polymerase in this example was purchased from Takara;
the MagExtractor-RNA extraction kit was purchased from TOYOBO;
BD SMARTTMRACE cDNA Amplification Kit was purchased from Takara;
pMD-18T vector was purchased from Takara;
the plasmid extraction kit is purchased from Tiangen corporation;
primer synthesis and gene sequencing were done by Invitrogen;
the Anti-CKMB3F10 monoclonal antibody is secreted as an existing hybridoma cell strain, and is recovered for later use.
S11, designing and synthesizing primers:
5' RACE upstream primers for heavy and light chain amplification:
SMARTER II A Oligonucleotide:
5’>AAGCAGTGGTATCAACGCAGAGTACXXXXX<3’;
5'-RACE CDS Primer(5'-CDS):5’>(T)25VN<3’(N=A,C,G,orT;V=A,G,orC);
Universal Primer A Mix(UPM):
5’>CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT<3’;
Nested Universal Primer A(NUP):
5’>AAGCAGTGGTATCAACGCAGAGT<3’;
mIgG CKR:5’>CTAACACTCATTCCTGTTGAAGCTCTTGACAAT<3’;
mIgG CHR:5’>TCATTTACCAGGAGAGTGGGAGAGGC<3’。
s12, antibody variable region gene cloning and sequencing:
RNA extracted from hybridoma cell strains secreting Anti-CKMB3F10 monoclonal antibodies is synthesized into first strand cDNA by using a SMARTERTM RACE cDNA Amplification Kit and SMARTER II AOligonucleotide and a 5' -CDS primer in the Kit, and an obtained first strand cDNA product is used as a PCR Amplification template. The Light Chain gene was amplified with Universal Primer A Mix (UPM), Nested Universal Primer A (NUP) and mkR primers, and the Heavy Chain gene was amplified with Universal Primer A Mix (UPM), Nested Universal Primer A (NUP) and mHR primers. The primer pair of Light Chain can amplify a target band about 0.7KB, and the primer pair of Heavy Chain can amplify a target band about 1.4 KB. The product was purified and recovered by agarose gel electrophoresis, and the product was subjected to A addition reaction with rTaq DNA polymerase, inserted into pMD-18T vector, transformed into DH 5. alpha. competent cells, and after colonies were grown, 4 clones of the Heavy Chain and Light Chain gene clones were each cloned and sent to Invitrogen corporation for sequencing.
Sequence analysis of S13, Anti-CKMB3F10 antibody variable region genes:
putting the gene sequence obtained by sequencing into an IMGT antibody database for analysis, and analyzing by using VNTI11.5 software to determine that the genes amplified by the heavy Chain primer pair and the Light Chain primer pair are correct, wherein in the gene segment amplified by the Light Chain, the VL gene sequence is 381bp, belongs to VkII gene family, and a leader peptide sequence of 60bp is arranged in front of the VL gene sequence; in the gene fragment amplified by the Heavy Chain primer pair, the VH gene sequence is 405bp, belongs to a VH1 gene family, and has a leader peptide sequence of 57bp in front.
S14, construction of recombinant antibody expression plasmid:
pcDNATM 3.4
Figure GDA0003511344150000111
vector is a constructed recombinant antibody eukaryotic expression vector, and multiple cloning enzyme cutting sites such as HindIII, BamHI, EcoRI and the like are introduced into the expression vector and named as pcDNA3.4A expression vector, and the vector is called as 3.4A expression vector for short in the following; according to the sequencing result of the antibody variable region gene in the pMD-18T, VL and VH gene specific primers of the Anti-CKMB3F10 antibody are designed, wherein two ends of the primers are respectively provided with HindIII and EcoRI enzyme cutting sites and protective bases, and the primers are as follows:
CKMB 3F10-HF:
5’>CCCAAGCTTGCCACCATGGAATGGCCTTGTATCTTTCTCTTCCTC<3’;
CKMB 3F10-HR:
5’>CCCGAATTCTCATTATTTACCAGGAGAGTGGGAGAGGCTCTTCTC<3’;
CKMB 3F10-LF:
5’>CCCAAGCTTGCCACCATGGAGTCACAGATTCAGGTCTTTGTATTC<3’;
CKMB 3F10-LR:
5’>CCCGAATTCTCATTAACACTCATTCCTGTTGAAGCTCTTGACAA<3’;
a0.7 KB Light Chain gene fragment and a 1.4KB Heavy Chain gene fragment were amplified by PCR amplification. The gene fragments of the Heavy Chain and the Light Chain are subjected to double enzyme digestion by HindIII/EcoRI respectively, the 3.4A vector is subjected to double enzyme digestion by HindIII/EcoRI, the Heavy Chain gene and the Light Chain gene are respectively connected into the 3.4A expression vector after the fragments and the vector are purified and recovered, and recombinant expression plasmids of the Heavy Chain and the Light Chain are respectively obtained.
S2. Stable cell strain screening
Transient transfection of recombinant antibody expression plasmid S21 in CHO cells, determination of expression plasmid activity
Plasmid was diluted to 400ng/ml with ultrapure water and CHO cells were conditioned at 1.43X 107cells/ml are put into a centrifuge tube, 100ul of plasmid is mixed with 700ul of cells, transferred into an electric rotating cup, electrically rotated, sampled and counted on days 3, 5 and 7, and sampled and detected on day 7.
Coating solution diluted CKMB (PAJ18) to the indicated concentration, 100uL per well, overnight at 4 ℃; the next day, washing with the washing solution for 2 times, and patting dry; adding blocking solution (20% BSA + 80% PBS), beating to dry at 37 deg.C for 1h and 120uL per well; adding diluted cell supernatant at 100 uL/well at 37 deg.C for 30min (partial supernatant for 1 h); washing with washing solution for 5 times, and drying; adding goat anti-mouse IgG-HRP (goat anti-mouse IgG-HRP) with the concentration of 100uL per well at 37 ℃ for 30 min; washing with washing solution for 5 times, and drying; adding a developing solution A (50 uL/hole), adding a developing solution B (50 uL/hole), and carrying out 10 min; adding stop solution into the mixture, wherein the concentration of the stop solution is 50 uL/hole; OD readings were taken at 450nm (reference 630nm) on the microplate reader. The results show that the reaction OD after the cell supernatant is diluted 1000 times is still larger than 1.0, and the reaction OD of the wells without the cell supernatant is smaller than 0.1, which indicates that the antibody generated after the plasmid is transiently transformed has activity on CKMB protein.
Linearization of S22 recombinant antibody expression plasmid
The following reagents were prepared: 50ul Buffer, 100 ug/tube DNA, 10ul Puv I enzyme and sterile water to 500ul, and performing enzyme digestion in water bath at 37 ℃ overnight; sequentially extracting with equal volume of phenol/chloroform/isoamyl alcohol (lower layer) 25:24:1 and then chloroform (water phase); precipitating with 0.1 volume (water phase) of 3M sodium acetate and 2 volumes of ethanol on ice, rinsing with 70% ethanol, removing organic solvent, re-melting with appropriate amount of sterilized water after ethanol is completely volatilized, and finally measuring concentration.
S23 recombinant antibody expression plasmid stable transfection, pressurized screening of stable cell strain
Plasmid was diluted to 400ng/ml with ultrapure water and CHO cells were conditioned at 1.43X 107cells/ml is put into a centrifuge tube, 100ul of plasmid is mixed with 700ul of cells, and the mixture is transferred into an electric rotating cup and is electrically rotated, and the next day is counted; 25umol/L MSX 96-well pressure culture for about 25 days.
Observing the marked clone holes with the cells under a microscope, and recording the confluence degree; taking culture supernatant, and carrying out sample detection; selecting cell strains with high antibody concentration and relative concentration, transferring the cell strains into 24 holes, and transferring the cell strains into 6 holes after 3 days; after 3 days, the seeds were kept and cultured in batches, and the cell density was adjusted to 0.5X 106cells/ml, 2.2ml, cell density 0.3X 106cell/ml, 2ml for seed preservation; and (4) 7 days, carrying out batch culture supernatant sample sending detection in 6 holes, and selecting cell strains with small antibody concentration and cell diameter to transfer TPP for seed preservation and passage.
S3. recombinant antibody production
S31 cell expanding culture
After cell recovery, the cells are cultured in a shaking flask with the specification of 125ml, the inoculation volume is 30ml, the culture medium is 100% Dynamis culture medium, and the cells are placed in a shaking table with the rotation speed of 120r/min, the temperature of 37 ℃ and the carbon dioxide of 8%. Culturing for 72h, inoculating and expanding culture at an inoculation density of 50 ten thousand cells/ml, wherein the expanding culture volume is calculated according to production requirements, and the culture medium is 100% Dynamis culture medium. Then the culture is expanded every 72 h. When the cell quantity meets the production requirement, the seeding density is strictly controlled to be about 50 ten thousand cells/ml for production.
S32 shake flask production and purification
Shake flask parameters: the rotating speed is 120r/min, the temperature is 37 ℃, and the carbon dioxide is 8 percent. Feeding in a flowing mode: daily feeding was started when the culture was carried out for 72h in a shake flask, 3% of the initial culture volume was fed daily to HyCloneTM Cell BoostTM Feed 7a, and one thousandth of the initial culture volume was fed daily to Feed 7b, up to day 12 (day 12 feeding). Glucose was supplemented with 3g/L on the sixth day. Samples were collected on day 13. Affinity purification was performed using a proteinA affinity column. Mu.g of the purified antibody was subjected to reducing SDS-PAGE, and 4. mu.g of an external control antibody was used as a control, and the electrophoretogram was shown in FIG. 1. Two bands were shown after reducing SDS-PAGE, 1 with 50kD Mr and 28kD Mr (light chain).
Example 2
Antibody affinity analysis and activity identification
The antibody obtained in example 1 was analyzed to have a light chain having a sequence shown in SEQ ID NO. 11 and a heavy chain having a sequence shown in 12.
Upon analysis, the complementarity determining region (WT) of the heavy chain:
CDR-VH1 is G-Y-G (X1) -F-S (X2) -D-Y-W;
CDR-VH2 is Q-L (X1) -Y-P-G-D-P (X2) -D-T-Q (X3) -Y-N;
CDR-VH3 is A-K (X1) -V-Y-P-S (X2) -F-T-Y-F (X3);
complementarity determining regions of the light chain:
CDR-VL1 is K-P (X1) -S-Q-E (X2) -V-S-T-A-I (X3) -A;
CDR-VL2 is S-A (X1) -S-T (X2) -R-Y-T;
CDR-VL3 is Q-N (X1) -H-Y-S-S-P-L (X2) -T-W (X3) -G;
wherein, X1, X2 and X3 are all the sites to be mutated.
TABLE 1 mutant sites associated with antibody Activity
Figure GDA0003511344150000131
Figure GDA0003511344150000141
The inventors performed the above-described mutation of the CDR sites in WT to obtain a more active antibody.
Diluting CKMB (PAJ18) to 1ug/ml by the coating solution to coat the micro-well plate, wherein each well is 100uL, and the temperature is kept overnight at 4 ℃; the next day, washing with the washing solution for 2 times, and patting dry; adding blocking solution (20% BSA + 80% PBS), beating to dry at 37 deg.C for 1h and 120uL per well; adding diluted CKMB monoclonal antibody at a concentration of 100 uL/well, and heating at 37 deg.C for 30min (partial supernatant for 1 h); washing with washing solution for 5 times, and drying; adding goat anti-mouse IgG-HRP (goat anti-mouse IgG-HRP) with the concentration of 100uL per well at 37 ℃ for 30 min; washing with washing solution for 5 times, and drying; adding a developing solution A (50 uL/hole), adding a developing solution B (50 uL/hole), and carrying out 10 min; adding stop solution into the mixture, wherein the concentration of the stop solution is 50 uL/hole; OD readings were taken at 450nm (reference 630nm) on the microplate reader.
TABLE 2 antibody Activity assay data
Sample concentration ng/ml WT Mutation 1 Mutation 2 Mutation 3 Mutation 4
12.35 1.753 2.189 2.037 2.089 1.964
4.12 0.937 1.331 1.210 1.249 1.187
1.37 0.511 0.652 0.602 0.615 0.572
0.46 0.260 0.346 0.337 0.337 0.324
0.15 0.138 0.172 0.183 0.192 0.162
0 0.087 0.105 0.097 0.086 0.100
As can be seen from the above table, the activity effect of mutation 1 is the best, so that mutation sites with better potency are screened by using mutation 1 as a framework sequence (ensuring that the activity of the antibody obtained by screening is similar to that of mutation 1, and the antibody activity is +/-10%), and partial results are as follows.
TABLE 3 mutation sites related to antibody affinity
Figure GDA0003511344150000142
Figure GDA0003511344150000151
Figure GDA0003511344150000161
Figure GDA0003511344150000171
Affinity assay
Using AMC sensors, purified antibodies were diluted to 10ug/ml with PBST and gradient diluted with PBST for CKMB (PAJ18) (200U/ml): 200U/ml, 100U/ml, 50U/ml, 25U/ml, 12.5U/ml, 6.25U/ml, 3.13U/ml, 0U/ml;
the operation flow is as follows: equilibrating in buffer 1(PBST) for 60s, immobilizing antibody in antibody solution for 300s, incubating in buffer 2(PBST) for 180s, binding in antigen solution for 420s, dissociating in buffer 2 for 1200s, regenerating the sensor with 10mM GLY solution pH 1.69 and buffer 3, and outputting the data. (KD represents the equilibrium dissociation constant, i.e.affinity; kon represents the association rate; kdis represents the dissociation rate. calculation assumes that CKMB (PAJ18) specimen 1U/ML corresponds to 2.5 nmol/ML.)
Table 4 affinity assay data
Figure GDA0003511344150000172
Figure GDA0003511344150000181
Figure GDA0003511344150000191
As can be seen from table 4, the mutation sites listed in table 3 have little effect on the affinity of the antibody.
To verify the above results, the above experiment was repeated using WT as a backbone sequence, and affinity verification of the mutation site was performed, and some results are as follows.
TABLE 5 mutations with WT as backbone
Figure GDA0003511344150000192
Table 6 affinity assay data
Figure GDA0003511344150000193
Figure GDA0003511344150000201
From the analyses in tables 5 and 6, the mutation sites were not much related to other sites while ensuring the antibody activity.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
SEQUENCE LISTING
<110> Dongguan City of Pengzhi Biotech Co., Ltd
<120> recombinant antibody against human creatine kinase isoenzyme CK-MB
<130> 66
<160> 12
<170> PatentIn version 3.3
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Claims (17)

1. An isolated binding protein comprising a CK-MB antigen binding domain, wherein the antigen binding domain comprises the amino acid sequence CDR-VH1, CDR-VH2, CDR-VH3, CDR-VL1, CDR-VL2, and CDR-VL 3;
CDR-VH1 is G-Y-X1-F-X2-D-Y-W, wherein X1 is A;
CDR-VH2 is Q-X1-Y-P-G-D-X2-D-T-X3-Y-N, wherein X3 is N;
CDR-VH3 is A-X1-V-Y-P-X2-F-T-Y-X3, wherein X3 is W;
CDR-VL1 is K-X1-S-Q-X2-V-S-T-A-X3-A, wherein X1 is A;
CDR-VL2 is S-X1-S-X2-R-Y-T, wherein X1 is P;
CDR-VL3 is Q-X1-H-Y-S-S-P-X2-T-X3-G, wherein X3 is F;
the mutation site of each complementarity determining region is selected from any one of the following combinations of mutations:
site of the body CDR-VH1X2 CDR-VH2X1/X2 CDR-VH3X1/X2 CDR-VL1X2/X3 CDR-VL2X2 CDR-VL3X1/X2 Mutant combination 1 S L/P K/S E/I T N/L Combination of mutations 2 Y L/G K/Y E/V Y N/V Combination of mutations 3 T L/A K/T E/L S N/I Combination of mutations 4 T I/P R/S D/I S Q/L Combination of mutations 5 S I/G R/Y D/V Y Q/V Combination of mutations 6 Y I/A R/T D/L T Q/I Mutant combination 7 Y L/P R/T E/V T N/I Combination of mutations 8 T L/G R/Y E/L Y Q/L Combination of mutations 9 S L/A R/S D/I S N/I Combination of mutations 10 S I/P K/T D/V S Q/L Combination of mutations 11 Y I/G K/Y E/I Y N/L Mutant combination 12 T I/A K/S D/L T N/V Mutant combinations 13 T L/P R/T D/V T N/V Combination of mutations 14 S I/P R/S D/L Y N/I Combination of mutations 15 Y L/A K/Y E/L S Q/L Mutant combinations 16 Y I/A R/Y D/I S Q/I Mutant combinations 17 T L/G K/T E/I Y Q/V Combination of mutations 18 S I/G K/S E/V T N/L Combination of mutations 19 T L/P K/T E/V T N/L Combination of mutations 20 S I/P R/S D/I Y N/V Mutant combination 21 Y L/A R/Y E/L S N/I Mutant combination 22 S I/A K/Y D/L S Q/L Mutant combination 23 Y L/G R/T D/V Y Q/V Mutant combinations 24 T I/G K/S E/I T Q/I Mutant combinations 25 S L/P K/S E/V T N/I Mutant combinations 26 Y I/P K/Y E/L Y Q/L Mutant combinations 27 T L/A K/T D/I S N/I Mutant combinations 28 T I/A R/S D/V S Q/L Mutant combinations 29 S L/G R/Y D/L Y N/L Combination of mutations 30 Y I/G R/T E/V T N/V Combination of mutations 31 S L/P R/T E/L T N/V Mutant combinations 32 Y I/P R/Y D/I Y N/I Mutant combinations 33 T L/A R/S D/V S Q/L Mutant combinations 34 T I/A K/T E/I S Q/I Combination of mutations 35 S L/P K/Y D/L Y Q/V Combination of mutations 36 Y L/G K/S D/V T N/L Mutant combinations 37 S L/A R/T D/L T N/L Combination of mutations 38 Y I/P R/S E/L Y N/V Mutant combinations 39 T I/G K/Y D/I S N/I Combination of mutations 40 T I/A R/Y E/I S Q/L Mutant combination 41 S L/P K/T E/V Y Q/V Combination of mutations 42 Y L/P K/S E/V T Q/I Mutant combinations 43 S L/G K/T D/I T N/I Mutant combinations 44 Y L/A R/S E/L Y Q/L Combination of mutations 45 T I/P R/Y D/L S N/I Mutant combinations 46 T I/G K/Y D/V S Q/L Mutant combinations 47 S I/A R/T E/V Y N/L
2. An isolated binding protein comprising a CK-MB antigen binding domain, wherein the antigen binding domain comprises the amino acid sequence CDR-VH1, CDR-VH2, CDR-VH3, CDR-VL1, CDR-VL2, and CDR-VL 3;
CDR-VH1 is G-Y-X1-F-X2-D-Y-W, wherein X1 is G;
CDR-VH2 is Q-X1-Y-P-G-D-X2-D-T-X3-Y-N, wherein X3 is Q;
CDR-VH3 is A-X1-V-Y-P-X2-F-T-Y-X3, wherein X3 is F;
CDR-VL1 is K-X1-S-Q-X2-V-S-T-A-X3-A, wherein X1 is P;
CDR-VL2 is S-X1-S-X2-R-Y-T, wherein X1 is A;
CDR-VL3 is Q-X1-H-Y-S-S-P-X2-T-X3-G, wherein X3 is W;
the mutation site of each complementarity determining region is selected from any one of the following combinations of mutations:
site of the body CDR-VH1X2 CDR-VH2X1/X2 CDR-VH3X1/X2 CDR-VL1X2/X3 CDR-VL2X2 CDR-VL3X1/X2 Combination 1 S L/P K/S E/I T N/L Combination 2 T L/G K/T E/L S N/I Combination 3 Y I/A R/Y D/V S Q/V Combination 4 S I/P R/Y E/L T Q/L Combination 5 T I/G R/S D/I S N/I Combination 6 Y L/P K/T D/V S Q/L Combination 7 S I/A K/S D/L T N/V Combination 8 Y L/G R/S D/L Y N/I Combination 9 T I/A K/Y E/L S Q/L Assembly 10 S L/G R/S D/I T N/V
3. The isolated binding protein comprising an antigen binding domain according to any one of claims 1 to 2, wherein said binding protein is F (ab')2Fab', Fab, Fv, scFv and diabody.
4. The isolated binding protein comprising an antigen binding domain according to any of claims 1 to 2, wherein the binding protein comprises the light chain framework regions FR-L1, FR-L2, FR-L3 and FR-L4 in sequence as shown in SEQ ID NO. 1-4, and/or the heavy chain framework regions FR-H1, FR-H2, FR-H3 and FR-H4 in sequence as shown in SEQ ID NO. 5-8.
5. The isolated binding protein comprising an antigen binding domain according to any one of claims 1 to 2, wherein the binding protein further comprises an antibody constant region sequence.
6. The binding protein according to claim 5, wherein said constant region sequence is selected from the group consisting of sequences of any one of the constant regions of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD.
7. The binding protein of claim 5, wherein the species of said constant region is derived from a cow, horse, pig, sheep, goat, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose, or human.
8. The binding protein of claim 7, wherein the species source of said constant region is a bovine.
9. The binding protein of claim 7, wherein said species source of the constant region is turkey or turkey.
10. The binding protein according to claim 7, wherein said constant region is derived from a mouse.
11. The binding protein according to claim 10, wherein the light chain constant region sequence is set forth in SEQ ID No. 9;
the heavy chain constant region sequence is shown in SEQ ID NO 10.
12. An isolated nucleic acid molecule which is DNA or RNA encoding the binding protein of any one of claims 1 to 11.
13. A vector comprising the nucleic acid molecule of claim 12.
14. A host cell transformed with the vector of claim 13.
15. A method of producing a binding protein according to any one of claims 1 to 11, comprising the steps of:
culturing the host cell of claim 14 in a culture medium and under suitable culture conditions, and recovering the binding protein so produced from the culture medium or from the cultured host cell.
16. Use of the binding protein of any one of claims 1 to 11 for the preparation of a diagnostic agent for the diagnosis of myocardial infarction, viral myocarditis.
17. A kit comprising the binding protein of any one of claims 1 to 11.
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