CN111018977A

CN111018977A - Recombinant antibody of anti-human cardiac troponin I

Info

Publication number: CN111018977A
Application number: CN201811180367.0A
Authority: CN
Inventors: 崔鹏; 何志强; 孟媛; 钟冬梅; 韩日才
Original assignee: Dongguan Pengzhi Biotechnology Co Ltd
Current assignee: Dongguan Pengzhi Biotechnology Co Ltd
Priority date: 2018-10-10
Filing date: 2018-10-10
Publication date: 2020-04-17
Anticipated expiration: 2038-10-10
Also published as: CN111018977B

Abstract

The present invention relates to a novel isolated binding protein comprising a cTnI antigen binding domain and studies on the preparation, use, etc. of the binding protein. The binding protein has strong activity and high affinity with human cTnI protein, and can be widely applied to the field of detection of the cTnI protein.

Description

Recombinant antibody of anti-human cardiac troponin I

Technical Field

The invention relates to the technical field of immunity, in particular to a recombinant antibody of an anti-human cardiac troponin I.

Background

Before the 80's of the 20 th century, the World Health Organization (WHO) has used the activity of the myocardial zymogram as one of the diagnostic criteria for Acute Myocardial Infarction (AMI). At the end of the 80's 20 th century, researchers found that troponin (Tn) had higher sensitivity and specificity than biomarkers such as phosphocreatine kinase (CK), phosphocreatine kinase isoenzyme (CK-MB), lactate dehydrogenase, and aspartate aminotransferase. The cardiac troponin I (cTnI) is only present in cardiac muscle, is a marker of cardiac muscle cells, can affect the contraction and relaxation functions of the heart by abnormal change, can be used for diagnosing myocardial necrosis, judging cardiac muscle injury and the like, becomes one of the markers with the strongest damage sensitivity and specificity of the cardiac muscle cells, and is a main biochemical marker which is generally accepted to rapidly diagnose AMI and Acute Coronary Syndrome (ACS) and assist ACS risk stratification and reflect the prognosis of ACS.

The cTnI content in normal human blood is generally lower than 0.3 mu g/L. When the integrity of the cell membrane of the cardiomyocytes is damaged by ischemia or hypoxia, the free cTnI can rapidly penetrate the cell membrane and enter the blood stream. Therefore, the rapid, sensitive and accurate determination of cTnI and its change trend in human blood at the early stage of onset has important clinical significance for the diagnosis of acute myocardial infarction, risk stratification of acute coronary syndrome, monitoring of myocardial damage caused by various factors, and the like. The clinical methods for detecting the cTnI level include enzyme-linked immunosorbent assay (ELISA), chemiluminescence, colloidal gold and the like, and different methods have respective advantages and disadvantages, but all require specific monoclonal antibodies aiming at the cTnI.

The existing cTnI antibody has low activity and poor affinity, and cannot be well applied to the detection of cTnI protein, so that the field has strong demand for an antibody which can effectively and specifically bind and detect cTnI.

Disclosure of Invention

The present invention relates to a novel isolated binding protein comprising a cTnI antigen binding domain and studies on the preparation, use, etc. of the binding protein.

The antigen binding domain comprises at least one complementarity determining region selected from the group consisting of amino acid sequences set forth below; or; has at least 80% sequence identity with the complementarity determining regions of the amino acid sequence described below and has K with cardiac troponin I_D≤1.43×10^-9(ii) affinity of (a);

CDR-VH1 is A-S-G-F-X1-F-X2-T-F-A-M-S, wherein,

x1 is S or T, X2 is I, S or L;

CDR-VH2 is I-S-S-G-G-S-Y-T-X1-Y-P-D-X2-V-K-G, wherein,

x1 is Y, A or F, X2 is T or S;

CDR-VH3 is A-R-R-X1-Y-G-X2-S-W-X3-S-Y, wherein,

x1 is P or A, X2 is E or D, X3 is P or F;

the CDR-VL1 is K-S-S-Q-S-X1-L-N-S-N-N-Q-X2-N-Y-L-A, wherein,

x1 is I or L, X2 is R or K;

the complementarity determining region CDR-VL2 is Y-F-A-S-X1-R-X2-S-G-V-P-D, wherein,

x1 is S or T, X2 is D or E;

the CDR-VL3 is Q-X1-H-X2-S-X3-P-L-T-F-G, wherein,

x1 is N or Q, X2 is F, Y or V, and X3 is I or L.

An important advantage is that the binding protein is highly active and has a high affinity for the human cTnI protein.

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 can be further subdivided into adjacent regions separated by CDRs (FR1, FR2, FR3 and FR 4).

Typically, the variable domains VL/VH of the heavy and light chains are obtained by linking the CDRs and FRs numbered as follows in a combinatorial arrangement: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR 4.

As used herein, the term "purified" or "isolated" in relation to a polypeptide or nucleic acid means that the polypeptide or nucleic acid is not in its native medium or native 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 set forth in seq id nos; or; has at least 80% sequence identity with the complementarity determining regions of the amino acid sequence described below and has K with cardiac troponin I_D≤1.43×10^-9(ii) affinity of (a);

CDR-VH1 is A-S-G-F-X1-F-X2-T-F-A-M-S, wherein,

x1 is S or T, X2 is I, S or L;

CDR-VH2 is I-S-S-G-G-S-Y-T-X1-Y-P-D-X2-V-K-G, wherein,

x1 is Y, A or F, X2 is T or S;

CDR-VH3 is A-R-R-X1-Y-G-X2-S-W-X3-S-Y, wherein,

x1 is P or A, X2 is E or D, X3 is P or F;

the CDR-VL1 is K-S-S-Q-S-X1-L-N-S-N-N-Q-X2-N-Y-L-A, wherein,

x1 is I or L, X2 is R or K;

x1 is S or T, X2 is D or E;

the CDR-VL3 is Q-X1-H-X2-S-X3-P-L-T-F-G, wherein,

x1 is N or Q, X2 is F, Y or V, and X3 is I or L.

In some embodiments, the antigen binding domain is linked toThe complementarity determining region of the amino acid sequence 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 and has K with cardiac troponin I_D≤1.43×10^-9mol/L，K_DThe value can also be selected to be 9.0 × 10^- ¹⁰mol/L、8.0×10^-10mol/L、7.0×10^-10mol/L、6.0×10^-10mol/L、5.0×10^-10mol/L、4.0×10^- ¹⁰mol/L、3.0×10^-10mol/L、2.0×10^-10mol/L、1.0×10^-10mol/L or 8.60X 10^-11(ii) affinity of (a); or 8.60 × 10^-11≤K_D≤1.43×10^-9mol/L。

Wherein the affinity is determined according to the method of the present specification.

In some embodiments:

in the complementarity determining region CDR-VH1, X1 is T;

in the complementarity determining region CDR-VH2, X1 is F;

in the CDR-VH3, X1 is P;

in the complementarity determining region CDR-VL1, X2 is K;

in the complementarity determining region CDR-VL2, X1 is T;

in the complementarity determining region CDR-VL3, X2 is Y.

In some embodiments, in the complementarity determining region CDR-VH1, X2 is I.

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 L.

In some embodiments, in the complementarity determining region CDR-VH2, X2 is T.

In some embodiments, in the complementarity determining region CDR-VH2, X2 is S.

In some embodiments, in the complementarity determining region CDR-VH3, X2 is E and X3 is P.

In some embodiments, in the complementarity determining region CDR-VH3, X2 is E and X3 is F.

In some embodiments, in the complementarity determining region CDR-VH3, X2 is D and X3 is P.

In some embodiments, in the complementarity determining region CDR-VH3, X2 is D and X3 is F.

In some embodiments, in the complementarity determining region CDR-VL1, X1 is I.

In some embodiments, in the complementarity determining region CDR-VL1, X1 is L.

In some embodiments, in the complementarity determining region CDR-VL2, X2 is D.

In some embodiments, in the complementarity determining region CDR-VL2, X2 is E.

In some embodiments, in the complementarity determining region CDR-VL3, X1 is N and X3 is I.

In some embodiments, in the complementarity determining region CDR-VL3, X1 is N and X3 is L.

In some embodiments, in the complementarity determining region CDR-VL3, X1 is Q and X3 is I.

In some embodiments, in the complementarity determining region CDR-VL3, X1 is Q and X3 is L.

In some embodiments, the mutation site of each complementarity determining region is selected from any one of the following combinations of mutations:

in some embodiments, the binding protein includes at least 3 CDRs (e.g., 3 CDRs of a heavy chain or 3 CDRs of a light chain); 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 "functional fragment" as defined in the present invention particularly refers to an antibody fragment having the same specificity for cTnI 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 a binding protein as described above for the preparation of a diagnostic agent or kit for the diagnosis of acute myocardial infarction, acute coronary syndrome, pulmonary infarction, unstable angina pectoris, myocardial injury.

According to one aspect of the invention, the invention also relates to a method for detecting troponin I antigen in a test sample, comprising:

a) contacting a troponin I antigen in the test sample with a binding protein as defined above to form an immune complex under conditions sufficient for an antibody/antigen binding reaction to occur; and

b) detecting the presence of said immune complex, the presence of said complex being indicative of the presence of said troponin I antigen in said 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 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 cTnI;

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, which second antibody binds to the troponin I 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 shows 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, Alexa488, 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-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 trisbipyridinediamine, europium cryptate or chelate, diamine, bispyanine, La Jolla Blue dye, allophycocyanin, allocyanin 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 cardiac troponin I.

S10, constructing an expression plasmid:

wherein, the restriction enzyme, Prime Star DNA polymerase in this example was purchased from Takara;

the MagExtractor-RNA extraction kit was purchased from TOYOBO; BD SMART^TMThe RACE cDNAamplification 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;

s11, design and synthesis of primers:

5' RACE upstream primers for heavy and light chain amplification:

amplification of Heavy Chain and Light Chain 5' RACE primers:

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’

amplifying a downstream primer of a light chain gene:

mkR：

5’>TTTTCCTTTTGAATTCCTAACACTCATTCCTGTTGAAGC<3’。

downstream primers for amplification of heavy chain genes:

mHR：

5’>TTTTCCTTTTGAATTCTCATTTACCAGGAGAGTGGGAGA<3’。

s12, antibody variable region gene cloning and sequencing:

RNA is extracted from hybridoma cell strains secreting Anti-cTnI 11C4 monoclonal antibody, first strand cDNA synthesis is carried out by SMARTER II A Oligopuleotide and 5' -CDS primers in SMARTERTMRACE cDNA Amplification Kit kit and kit, obtained first strand cDNA products are used as PCR amplification templates, Light Chain gene is amplified by Universal Primer A Mix (UPM), Nested Universal Primer A (NUP) and mkR primers, heavyChain gene is amplified by Universal Primer A Mix (UPM), Nested Universal Primer A (NUP) and mHR primers, target band of about 0.8KB is amplified by the Primer pair of heavyChain, target band of about 1.4KB is amplified by the Primer pair of heavyChain, the products are purified and recovered by agarose gel electrophoresis, added into pMD-18 KB DNA polymerase after rTaq DNA polymerase reaction, transferred into pMD-18 KB, clone of Invitrogen vector is obtained, and clone of individual clone of the heavChain gene is detected after Invitrogen 5 α clone.

Sequence analysis of S13, CTNI 11C4 antibody variable region genes:

putting the gene sequence obtained by sequencing in 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 fragment amplified by the Light Chain, the VL gene sequence is 342bp, belongs to VkII gene family, and a leader peptide sequence of 57bp is arranged in front of the VL gene sequence; in the gene fragment amplified by the HeavyChain primer pair, the VH gene sequence is 357bp, belongs to a VH1 gene family, and has a leader peptide sequence of 57bp in front.

S14, construction of recombinant antibody expression plasmid:

pcDNA^TM3.4

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 CTNI 11C4 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:

C11C4-HF：

5’>CAGCAAGCTTGCCGCCACCATGGAATGCAGCTGTGTCATGCTCTTCTTC<3’；

C11C4-HR:5’>CATCGAATTCTTATCATTTACCAGGAGAGTGGGAGA<3’；

C11C4-LF:

5’>CATCAAGCTTGCCGCCACCATGAAGTTGCCTGTTAGGCTGTTGG<3’；

C11C4-LR:

5’>CAGCGAATTCTTACTAACACTCATTCCTGTTGAAGC<3’；

a0.75 KB Light Chain gene fragment and a 1.42KB 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.

S20, screening stable cell strains:

s21, diluting the plasmid to 400ng/ml with ultrapure water, adjusting the CHO cells to 1.43X 107cells/ml, mixing 100. mu.l of plasmid and 700. mu.l of cells in a centrifuge tube, transferring into an electric rotating cup, electrically rotating, sampling and counting on days 3, 5 and 7, and collecting and detecting on day 7.

The coating solution dilutes the corresponding antigen to the designated concentration, each well is 100 mu L, 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), and drying at 37 deg.C for 1 hr in each well; adding diluted cell supernatant at 100 μ L/well, 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 100 mu L per well at 37 ℃ for 30 min; washing with washing solution for 5 times, and drying; adding a developing solution A (50 muL/hole), adding a developing solution B (50 muL/hole), and keeping for 10 min; adding stop solution into the mixture, wherein the concentration of the stop solution is 50 mu L/hole; OD readings were taken at 450nm (reference 630nm) on the microplate reader. And (4) calculating the content of the antibody in the cell supernatant by taking the concentration of the standard substance and the OD value as a standard curve.

S22, recombinant antibody expression plasmid linearization:

the following reagents were prepared: 50 mul Buffer, 100 mul DNA/tube, 10 mul Puv I enzyme, and sterile water to 500 mul, water bath enzyme digestion overnight at 37 ℃; 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, stably transfecting the recombinant antibody expression plasmid, and performing pressurized screening on stable cell strains:

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, 100 mul of plasmid is mixed with 700 mul of cells, and the mixture is transferred into an electric rotating cup and is electrically rotated, and the next day is counted; 25 u mol/L MSX 96 hole pressure culture about 25 days.

Observing the marked clone holes with cells under a microscope, and recording the confluence degree; taking culture supernatant, and sending the culture supernatant to a sample for 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.

S30, production of recombinant antibody:

s31, cell expanding culture:

after the cells are recovered, 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 amount meets the production requirement, the production is carried out by strictly controlling the inoculation density to be about 50 ten thousand cells/ml.

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. After purification, 500mg of recombinant antibody was obtained, and 4. mu.g of the purified antibody was subjected to reducing SDS-PAGE, which revealed two bands, one of which was a 28KD light chain (SEQ ID NO: 11) and the other was a 50KD heavy chain (SEQ ID NO: 12).

Example 2

Although the antibody obtained in example 1 (having light and heavy chains having sequences shown in SEQ ID NOS: 11 and 12) had the ability to bind cTnI, the inventors performed the above-described mutation on the CDR sites in WT to obtain a more active antibody.

Upon analysis, the complementarity determining region (WT) of the heavy chain:

CDR-VH1 is A-S-G-F-S (X1) -F-I (X2) -T-F-A-M-S;

CDR-VH2 is I-S-S-G-G-S-Y-T-A (X1) -Y-P-D-T (X2) -V-K-G;

CDR-VH3 is A-R-R-A (X1) -Y-G-E (X2) -S-W-P (X3) -S-Y;

complementarity determining regions of the light chain:

CDR-VL1 is K-S-S-Q-S-I (X1) -L-N-S-N-N-Q-R (X2) -N-Y-L-A;

CDR-VL2 is Y-F-A-S-S (X1) -R-D (X2) -S-G-V-P-D;

CDR-VL3 is Q-N (X1) -H-F (X2) -S-I (X3) -P-L-T-F-G;

wherein, X1, X2 and X3 are all mutation sites.

TABLE 1 mutant sites associated with antibody Activity

Detecting the activity of the antibody after mutation, diluting CTNI quality control product recombinant antigen to 0.5ug/ml by using a coating solution, coating the antigen by using a microplate, 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 cTnI monoclonal antibody, 100 uL/hole, 37 ℃,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. Some of the results are as follows:

TABLE 2 antibody Activity assay data

	WT	Mutation 1	Mutation 2	Mutation 3	Mutation 4	Mutation 5
							Original multiple	1.564	2.449	2.098	0.878	0.421	-
Diluting by 5 times	1.959	2.365	2.167	0.542	0.028	-
							Diluting by 25 times	1.859	2.301	2.185	0.031	-	-
Diluting 125 times	1.577	2.279	1.803	-	-	-
							Diluting by 625 times	1.195	1.973	1.433	-	-	-
Diluted 3125 times	0.658	1.383	1.044	-	-	-
							Diluting 15625 times	0.359	0.902	0.679	-	-	-
Blank hole	0.016	0.056	0.032	-	-	-

"-" indicates no activity.

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

Affinity assay

Using AMC sensors, purified antibodies with PBST diluted to 10ug/ml, CTNI quality control recombinant protein PBST gradient dilution:

1000nmol、500nmol、250nmol、125nmol、62.5nmol、31.25nmol、15.625nmol、0nmol；

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 800s, regenerating the sensor with 10mM GLY solution pH 1.69 and buffer 3, and outputting the data.

Table 4 affinity assay data

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

Table 6 affinity assay data

	K_D(M)	K_on(1/Ms)	Koff(1/S)
				WT	3.45E-10	4.55E+05	1.57E-04
WT 1-1	1.43E-09	3.85E+04	5.51E-05
				WT 1-4	1.04E-09	3.75E+04	3.90E-05
WT 1-7	7.28E-10	1.82E+05	1.32E-04
				WT 1-20	9.98E-10	4.88E+05	4.87E-04

From the analyses in tables 5 and 6, the association between the mutation site and other sites was not significant on the premise that the antibody activity was ensured.

SEQUENCE LISTING

<110> Dongguan City of Pengzhi Biotech Co., Ltd

<120> recombinant antibody against human cardiac troponin I

<160>12

<170>PatentIn version 3.3

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<211>23

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

1 5 10 15

Gln Lys Val Thr Met Ser Cys

20

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

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Arg Phe Ile Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

1 5 10 15

Ser Val Gln Ala Glu Asp Leu Ala Asp Tyr Phe Cys

20 25

<210>4

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Ala Gly Thr Lys Leu Glu Leu Lys

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

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

20 25 30

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Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala

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35 40 45

Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu

65 70 75 80

Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser

85 90 95

Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys

100 105

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

1 5 10 15

Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu

50 55 60

Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Gln Thr Val

65 70 75 80

Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys

85 90 95

Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro

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Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu

115 120 125

Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser

130 135 140

Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu

145 150 155 160

Val His Thr Ala Gln Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr

165 170 175

Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn

180 185 190

Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro

195 200 205

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

210 215 220

Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val

225 230 235 240

Ser Leu Thr Cys Met Ile Thr Asn Phe Phe Pro Glu Asp Ile Thr Val

245 250 255

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

260 265 270

Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn

275 280 285

Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val

290 295 300

Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His

305 310 315 320

Ser Pro Gly

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

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

35 40 45

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

50 55 60

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

65 70 75 80

Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Asp Tyr Phe Cys Gln Asn

85 90 95

His Phe Ser Ile Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu

100 105 110

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

115 120 125

Glu Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn

130 135 140

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

145 150 155 160

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

165 170 175

Ser Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr

180 185 190

Glu Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr

195 200 205

Ser Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys

210 215 220

<210>12

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<213> Artificial sequence

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

1 5 10 15

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

20 25 30

Ala Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Ser Gly Gly Ser Tyr Thr Ala Tyr Pro Asp Thr Val

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Arg Ala Tyr Gly Glu Ser Trp Pro Ser Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ala Ala Lys Thr Thr Pro Pro Ser Val Tyr

115 120 125

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

130 135 140

Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp

145 150 155 160

Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser

180 185 190

Thr Trp Pro Ser Gln Thr Val Thr Cys Asn Val Ala His Pro Ala Ser

195 200 205

Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys

210 215 220

Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro

225 230 235 240

Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr

245 250 255

Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser

260 265 270

Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Lys Pro Arg

275 280 285

Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile

290 295 300

Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn

305 310 315 320

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

325 330 335

Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu

340 345 350

Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asn Phe

355 360 365

Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala

370 375 380

Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr

385 390 395 400

Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly

405 410 415

Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His

420 425 430

Thr Glu Lys Ser Leu Ser His Ser Pro Gly

435 440

Claims

1. 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 with the complementarity determining regions of the amino acid sequence described below and has K with cardiac troponin I_D≤1.43×10^-9(ii) affinity of (a);

CDR-VH1 is A-S-G-F-X1-F-X2-T-F-A-M-S, wherein,

x1 is S or T, X2 is I, S or L;

CDR-VH2 is I-S-S-G-G-S-Y-T-X1-Y-P-D-X2-V-K-G, wherein,

x1 is Y, A or F, X2 is T or S;

CDR-VH3 is A-R-R-X1-Y-G-X2-S-W-X3-S-Y, wherein,

x1 is P or A, X2 is E or D, X3 is P or F;

the CDR-VL1 is K-S-S-Q-S-X1-L-N-S-N-N-Q-X2-N-Y-L-A, wherein,

x1 is I or L, X2 is R or K;

x1 is S or T, X2 is D or E;

the CDR-VL3 is Q-X1-H-X2-S-X3-P-L-T-F-G, wherein,

x1 is N or Q, X2 is F, Y or V, X3 is I or L;

preferably:

in the complementarity determining region CDR-VH1, X1 is T;

in the complementarity determining region CDR-VH2, X1 is F;

in the CDR-VH3, X1 is P;

in the complementarity determining region CDR-VL1, X2 is K;

in the complementarity determining region CDR-VL2, X1 is T;

in the complementarity determining region CDR-VL3, X2 is Y;

preferably, in the complementarity determining region CDR-VH1, X2 is I;

preferably, in the complementarity determining region CDR-VH1, X2 is S;

preferably, in the complementarity determining region CDR-VH1, X2 is L;

preferably, in the complementarity determining region CDR-VH2, X2 is T;

preferably, in the complementarity determining region CDR-VH2, X2 is S;

preferably, in the complementarity determining region CDR-VH3, X2 is E, X3 is P;

preferably, in the complementarity determining region CDR-VH3, X2 is E, X3 is F;

preferably, in the complementarity determining region CDR-VH3, X2 is D, X3 is P;

preferably, in the complementarity determining region CDR-VH3, X2 is D, X3 is F;

preferably, in the complementarity determining region CDR-VL1, X1 is I;

preferably, in the complementarity determining region CDR-VL1, X1 is L;

preferably, in the complementarity determining region CDR-VL2, X2 is D;

preferably, in the complementarity determining region CDR-VL2, X2 is E;

preferably, in the complementarity determining region CDR-VL3, X1 is N, X3 is I;

preferably, in the complementarity determining region CDR-VL3, X1 is N, X3 is L;

preferably, in the complementarity determining region CDR-VL3, X1 is Q, X3 is I;

preferably, in the complementarity determining region CDR-VL3, X1 is Q, and X3 is L;

preferably, the mutation site of each complementarity determining region is selected from any one of the following combinations of mutations:

2. the isolated binding protein comprising an antigen binding domain according to claim 1, wherein at least 3 CDRs are included in the binding protein; alternatively, the binding protein comprises at least 6 CDRs;

preferably, the binding protein is a nanobody, F (ab')₂One of, Fab', Fab, Fv, scFv, diabody, and antibody minimal recognition unit;

preferably, the binding protein comprises light chain framework regions FR-L1, FR-L2, FR-L3 and FR-L4 which have the sequences shown in SEQ ID NO. 1-4 in sequence, and/or heavy chain framework regions FR-H1, FR-H2, FR-H3 and FR-H4 which have the sequences shown in SEQ ID NO. 5-8 in sequence.

3. The isolated binding protein comprising an antigen binding domain according to claim 1 or 2, wherein the binding protein further comprises an antibody constant region sequence;

preferably, 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;

preferably, the species of the constant region is from a cow, horse, cow, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, goose, turkey, chicken fight, or human;

preferably, 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.

4. An isolated nucleic acid molecule which is DNA or RNA encoding the binding protein of any one of claims 1 to 3.

5. A vector comprising the nucleic acid molecule of claim 4.

6. A host cell transformed with the vector of claim 5.

7. A method of producing the binding protein of any one of claims 1 to 3, comprising the steps of:

culturing the host cell of claim 6 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.

8. Use of the binding protein of any one of claims 1 to 3 for the preparation of a diagnostic agent or kit for the diagnosis of acute myocardial infarction, acute coronary syndrome, pulmonary infarction, unstable angina, myocardial injury.

9. A method of detecting troponin I antigen in a test sample comprising:

a) contacting a troponin I antigen in the test sample with the binding protein of claim 3 under conditions sufficient for an antibody/antigen binding reaction to occur to form an immune complex; and

preferably, in step a), a second antibody is further included in the immune complex, the second antibody binding to the binding protein;

preferably, in step a), a second antibody is further included in the immune complex, said second antibody binding to the troponin I antigen.

10. A kit comprising the binding protein of any one of claims 1 to 3.