CN112794900B

CN112794900B - cBIN1 antibodies and uses thereof

Info

Publication number: CN112794900B
Application number: CN202011626540.2A
Authority: CN
Inventors: 周杨钊; 周康
Original assignee: Second Xiangya Hospital of Central South University
Current assignee: Second Xiangya Hospital of Central South University
Priority date: 2020-12-31
Filing date: 2020-12-31
Publication date: 2022-10-25
Anticipated expiration: 2040-12-31
Also published as: CN112794900A

Abstract

The invention relates to the technical field of immunization, in particular to a cBIN1 antibody and application thereof. The three CDR regions of the heavy chain of the cBIN1 antibody provided by the invention have amino acid sequences shown as SEQ ID NO 1, 2 and 3 respectively; the three CDR regions of the light chain have amino acid sequences shown in SEQ ID NO 4, 5 and 6, respectively. The antibody has good binding capacity with antigen, and the reagent prepared by the antibody can realize accurate detection of target antigen.

Description

cBIN1 antibodies and uses thereof

Technical Field

The invention relates to the technical field of immunization, in particular to a cBIN1 antibody and application thereof.

Background

Heart failure is a complex group of clinical syndromes caused by abnormal changes in the structure and/or function of the heart due to a variety of causes that cause dysfunction in the systolic and/or diastolic function of the ventricles. Heart failure is a severe manifestation or late stage of various heart diseases, with high mortality and readmission rates. The data of all places in recent years show that the prevalence rate of heart failure in China has increased remarkably to 2% -3%, and about 1000 ten thousand of patients with current symptoms.

Heart failure the heart muscle has its core pathological changes that the calcium ion transport capacity is weakened, causing the contraction capacity of the heart muscle to be limited. The cause of calcium ion transport impairment characteristic of heart failure is excitation-contraction (E-C) decoupling and asynchronous calcium release. Under physiological conditions, the depolarization of the membrane potential induces a synchronous calcium release, which allows calcium ions to pass through the cell membrane and trigger more calcium release, i.e., a calcium-induced-calcium-release (CICR) process, in the sarcoplasmic reticulum. The CICR process, both healthy and ideal, relies on close cooperation between ryanodine receptors (RyRs) located on the sarcoplasmic omentum at the junctions between cardiomyocytes and L-type calcium channels (LTCCs) on the transverse-tube (T-tube) membrane. LTCCs in the transverse tubule form dimeric complexes with RyRs on the sarcoplasmic omentum of the junction, which affect the transport of extra-cytosolic calcium ions. In the pathological hypertrophy process caused by the increase of pressure load of cardiac muscle due to various reasons, disorder and reconstruction of a large number of transverse tubular structures are generated, and the destruction of the dimeric structures is accompanied, so that the asynchronous CICR process is finally caused, and the hypertrophy compensation is gradually developed into heart failure. In addition to a series of reconstructive changes in the morphology of the cross tubes during heart failure, the process of the damaged LTCC migrating to the cross tubes and forming binary complexes with RyRs is also disturbed, ga ²⁺ The metastatic capacity decreases, which in turn leads to E-C decoupling and decreased cardiac function.

BIN protein (Bar protein coding gene) can be divided into Amphihysini I, ampII (also called Bin 1), bin2 and Bin 3. As a protein superfamily containing BAR domains, bin1 contains an N-terminal BAR domain (N-BAR) characterized by being encoded by exons 1 to 9. Interestingly, the gene of Bin1 with 20 exons has different cellular functions due to the different splicing protocols for the production of different Bin1 proteins. The myocardial transverse-tube endocardial heart bridging integrator 1 (cBIN1) is a BIN1 protein subtype with 13, 17 exons.

Researches show that the transverse tube micro-domain formed by the cBIN1 has the function of promoting and maintaining the formation of the LTCC-RyR binary complex. The existing research results show that the micro-domain with the cBin1 can promote the formation of an effective LTCC-RyR binary complex through the following 4 mechanisms: 1) Promoting forward transport of LTCCs through the microdomains; 2) Promoting aggregation of LTCCs that have migrated to the cross tube into clusters; 3) Forming a protective slow dispersion area of extracellular calcium ions in the transverse tubular cavity to control the transfer power of the LTCCs; 4) And the RyRs in the cytoplasm are recalled to meet the sarcoplasmic reticulum of the junction area, so that a new binary complex can be formed with the LTCCs. Meanwhile, the cBin1 micro-domain plays a role in maintaining the steady state of the transverse cardiac tube through the capability of continuous membrane turnover and rapid recombination under acute stress stimulation. At the same time, studies have found that the cBin1 microdomains are lost due to reduced transcription of cBin1 under stimulation associated with heart failure, with a concomitant reduction in binary complex formation and impairment of myocardial contractility. That is, as the expression of cBin1 decreases, the microdomain structure of the transverse tubes becomes disordered, thereby reducing the contractility of the heart and the response to the beta receptor.

Therefore, cBIN1 is expected to be a novel marker of heart failure. If the expression level of cBIN1 in cardiac muscle or blood can be effectively detected, the structural integrity and the functional state of the heart can be objectively evaluated, an objective and reliable basis is provided for the evaluation and treatment of heart failure, and the treatment of the heart failure is guided. The development of sensitive antibodies for detecting cBIN1 can effectively facilitate the detection and evaluation of the marker, and provide a new reliable reference for clinical diagnosis and treatment of heart failure.

Disclosure of Invention

In view of the above, the technical problem to be solved by the present invention is to provide cBIN1 antibodies and uses thereof.

The invention provides a cBIN1 antibody, wherein a heavy chain of the antibody comprises three CDR regions, wherein the amino acid sequence of at least one CDR region has an amino acid sequence shown as SEQ ID NO. 1, 2 or 3, or a sequence with at least 80% sequence homology with the amino acid sequence; the light chain comprises three CDR regions, wherein the amino acid sequence of at least one CDR region has the amino acid sequence shown as SEQ ID NO. 4, 5 or 6, or a sequence with at least 80% of sequence homology with the CDR regions.

In the present invention, the three CDR regions of the heavy chain of the cBIN1 antibody have the amino acid sequences shown in SEQ ID NO 1, 2 and 3, respectively; the three CDR regions of the light chain have amino acid sequences shown in SEQ ID NO 4, 5 and 6, respectively.

In some embodiments of the invention, the amino acid sequences of the three CDR regions of the heavy chain of the cBIN1 antibody are set forth in SEQ ID NOs 1, 2 and 3, respectively; the amino acid sequences of the three CDR regions of the light chain are shown in SEQ ID NO 4, 5 and 6 in sequence.

Wherein, the sequence shown in SEQ ID NO. 1 is GFNIKDS;

the sequence shown in SEQ ID NO. 2 is DPEDDD;

the sequence shown in SEQ ID NO. 3 is STLVATPWFFDV;

the sequence shown in SEQ ID NO. 4 is SASQDINNYLN;

the sequence shown in SEQ ID NO. 5 is YTSTLHS;

the sequence shown in SEQ ID NO 6 is QQYTHNLPWT.

The cBIN1 antibody provided by the invention has a heavy chain comprising 4 FR regions, wherein the amino acid sequence of at least one FR region has the amino acid sequence shown as SEQ ID NO. 9, 10, 11 or 12, or a sequence with at least 80% sequence homology with the FR region; the light chain comprises 4 FR regions, wherein the amino acid sequence of at least one FR region has the amino acid sequence shown as SEQ ID NO 13, 14, 15 or 16, or a sequence with at least 80% of sequence homology with the FR region.

In the present invention, 4 FR regions of the heavy chain of the cBIN1 antibody have amino acid sequences shown as SEQ ID NO 9, 10, 11 or 12, respectively; the 4 FR regions of the light chain have amino acid sequences shown as SEQ ID NO 13, 14, 15 and 16 respectively.

In some embodiments of the invention, the amino acid sequences of the 4 FR regions of the heavy chain of the cBIN1 antibody are sequentially as set forth in SEQ ID NOs 9, 10, 11 or 12; the amino acid sequences of the 4 FR regions of the light chain are shown in SEQ ID NO 13, 14, 15 and 16.

Wherein the sequence shown in SEQ ID NO. 9 is EVQLQQSGAELVRPGASVKLSCTTS;

the sequence shown in SEQ ID NO. 10 is YMHWVKQRPEQGLEWIGRI;

the sequence shown in SEQ ID NO. 11 is AVYAPKFQDRATMTADTSSNTAYLHLS SLTSDDTAVYYCTT;

the sequence shown in SEQ ID NO. 12 is WGTGTTVTVSS;

the sequence shown in SEQ ID NO. 13 is DIQMTQTTSSLSASLGDRVTISC;

the sequence shown in SEQ ID NO. 14 is WYQQKPDGTVKLLIY.

The sequence shown in SEQ ID NO. 15 is VPSRFSGSGSGTDYSLTITNLE PEDFATYYC;

the sequence shown in SEQ ID NO 16 is FGGGTKLEIK.

In the present invention, the sequence having at least 80% sequence homology is an amino acid sequence obtained by substituting, deleting or adding one or more amino acids from the original sequence, wherein the plurality is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or 41.

In some embodiments, the heavy chain variable region of the cBIN1 antibody of the present invention has the amino acid sequence shown in SEQ ID No. 7; the light chain variable region has an amino acid sequence shown in SEQ ID NO. 8.

The invention also provides nucleotides encoding the antibodies.

The invention provides nucleotide sequences encoding the heavy chains of the antibodies.

The invention provides nucleotide sequences encoding the light chain of the antibody.

In the invention, the nucleotide for coding the heavy chain variable region of the antibody has a nucleotide sequence shown in SEQ ID NO. 17, or a nucleotide sequence which is obtained by substituting, deleting or adding one or more nucleotides and has the same or similar functions with the nucleotide sequence shown in SEQ ID NO. 17.

In some embodiments of the invention, the nucleotide sequence encoding the variable region of the antibody heavy chain is shown in SEQ ID NO. 17 or is the reverse complement of SEQ ID NO. 17.

In the invention, the nucleotide for coding the variable region of the antibody light chain has a nucleotide sequence shown in SEQ ID NO. 18, or a nucleotide sequence which is obtained by substituting, deleting or adding one or more nucleotides and has the same or similar functions with the nucleotide sequence shown in SEQ ID NO. 18.

In some embodiments of the invention, the nucleotide sequence encoding the variable region of the antibody light chain is as shown in SEQ ID NO. 18 or the reverse complement of SEQ ID NO. 18.

The present invention also provides an expression vector comprising a nucleotide encoding the cBIN1 antibody of the present invention.

The invention also provides a host cell transformed or transfected with the expression vector.

The preparation method of the cBIN1 antibody comprises the following steps: culturing the host cell of the invention and inducing the expression of the cBIN1 antibody.

The invention also provides said cBIN1 antibodies chemically or biologically labeled.

In the present invention, the chemical label is an isotope, an immunotoxin and/or a chemical drug; the biomarker is a biotin, avidin, or enzyme label. The enzyme label is preferably horseradish peroxidase or alkaline phosphatase. The immunotoxin is preferably aflatoxin, diphtheria toxin, pseudomonas aeruginosa exotoxin, ricin, abrin, mistletoe agglutinin, modeccin, PAP, nystatin, gelonin or luffa toxin.

The invention also provides a conjugate prepared by coupling the cBIN1 antibody with a solid medium or a semisolid medium. The solid medium or non-solid medium in the invention is selected from colloidal gold, polystyrene flat plate or bead.

The cBIN1 antibody or the conjugate disclosed by the invention is applied to preparation of a product for detecting cBIN1 expression.

The invention also provides a kit comprising the cBIN1 antibody or the conjugate.

The kit also comprises an ELISA detection reagent or a Western Blot detection reagent.

The invention also provides a method for detecting cBIN1, which adopts the kit provided by the invention to detect the cBIN1 in a sample by an ELISA method or a Western Blot method. The sample is a cardiac muscle sample or a blood sample.

The invention also provides a diagnostic method of heart failure, namely, the cBIN1 in the cardiac muscle or blood sample is detected by adopting the kit provided by the invention through an ELISA method or a WesternBlot method. The sample is a cardiac muscle sample or a blood sample.

The three CDR regions of the heavy chain of the cBIN1 antibody provided by the invention have amino acid sequences shown as SEQ ID NO 1, 2 and 3 respectively; the three CDR regions of the light chain have amino acid sequences shown in SEQ ID NO 4, 5 and 6, respectively. The antibody has good binding capacity with antigen, and the reagent prepared by the antibody can realize accurate detection of target antigen.

Drawings

FIG. 1 shows ELISA binding curves of antibodies to cBIN1 Isoform II2 and cBIN1 Isoform II 3;

FIG. 2 shows the effect of antibody binding to protein in WB assay.

Detailed Description

The invention provides cBIN1 antibodies and applications thereof, and can be realized by appropriately modifying process parameters by one skilled in the art by taking the contents in the text as reference. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

Unless otherwise indicated, the molecular biological experimental methods and immunoassay methods used in the present invention are essentially described by reference to j.sambrook et al, molecular cloning: a laboratory manual, 2 nd edition, cold spring harbor laboratory Press, 1989, and F.M. Ausubel et al, eds. Molecular biology laboratory Manual, 3 rd edition, john Wiley & Sons, inc., 1995; the use of restriction enzymes is in accordance with the conditions recommended by the product manufacturer. It will be appreciated by those skilled in the art that the examples describe the invention by way of example and are not intended to limit the scope of the invention as claimed.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. With regard to the definitions and terminology in this field, the expert can refer in particular to Current Protocols in Molecular Biology (Ausubel). Abbreviations for amino acid residues are standard 3-letter and/or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.

The term "antibody" refers to a protein composed of one or more polypeptides capable of specifically binding to an antigen. One form of antibody constitutes the basic building block of an antibody. This form is a tetramer, which is composed of two identical pairs of antibody chains, each pair having a light chain and a heavy chain. In each pair of antibody chains, the variable regions of the light and heavy chains, taken together, are responsible for binding to antigen, while the constant regions are responsible for the effector functions of the antibody.

The "variable region" of the heavy or light chain of the antibody is the N-terminal mature region of the chain. The types of antibodies currently known include kappa and lambda light chains, as well as alpha, gamma (IgG 1, igG2, igG3, igG 4), delta, epsilon and mu heavy chains or other class equivalents thereof. Full-length immunoglobulin "light chains" (about 25kDa or about 214 amino acids) comprise a single NH domain ₂ A variable region of approximately 110 amino acids at the end, and a kappa or lambda constant region at the COOH-terminus. Full length exempt fromThe immunoglobulin "heavy chain" (about 50kDa or about 446 amino acids) likewise comprises a variable region (about 116 amino acids), and one of the heavy chain constant regions, e.g., gamma (about 330 amino acids).

"antibody" includes any isotype of antibody or immunoglobulin, or antibody fragments that retain specific binding to an antigen, including but not limited to Fab, fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single chain antibodies, and fusion proteins comprising an antigen-binding portion of an antibody and a non-antibody protein. The antibody may be labeled and detected, for example, by a radioisotope, an enzyme capable of producing a detectable substance, a fluorescent protein, biotin, or the like. The antibodies can also be bound to a solid support, including but not limited to polystyrene plates or beads, and the like.

As used herein, "CDR region" or "CDR" refers to the hypervariable regions of the heavy and light chains of an immunoglobulin, as defined by Kabat et al (Kabat et al, sequences of proteins of immunological interest,5th Ed., U.S. department of Health and Human Services, NIH,1991, and later). There are three heavy chain CDRs and three light chain CDRs. As used herein, the term CDR or CDRs is intended to indicate one of these regions, or several or even all of these regions, which comprise the majority of the amino acid residues responsible for binding by the affinity of the antibody for the antigen or its recognition epitope, as the case may be.

The test materials adopted by the invention are all common commercial products and can be purchased in the market. The invention is further illustrated by the following examples:

example 1: synthesis of antigens for immunization

The antigen is a protein sequence LRKGPPVPPPPKHTPSKEVKQEQILSLFEDTFVPEISVTTPSQ corresponding to exon13 (cBIN 1 exon 13) of a human Cardiac bridging integrator 1, a polypeptide Gill biochemical company for immunization synthesizes the antigen by a Guo chemical method, and in order to improve the immunogenicity of the antigen, the C end of the polypeptide is coupled with Keyhole Limpet Hemocyanin (KLH) by distearate (PEG 4).

Example 2: production of anti-human cBIN1 exon13 antibody

To obtain murine anti-human cBIN1 exon13 antibodies, different strains of mice (Balb/c, shanghai chang organism; SJL, beijing vindoli, warrior) were immunized using the immunization strategy in table 1.1 (table 1). The antigen used was as described in example 1; adjuvants include: complete Freund's adjuvant CFA (InvivoGen, cat # vac-CFA-60), IFA (InvivoGen, cat # vac-IFA-60). Splenocytes from immunized mice were fused with mouse myeloma cells SP2/0 using polyethylene glycol 3 days after the final immunization to obtain B-cell fusions that both express antibody and proliferate indefinitely in vitro, and cultured in HAT selective medium. The fused hybridoma cells were plated in 96-well cell culture plates and positive clones were selected by primary screening for 2 rounds of subcloning.

TABLE 1 immunization strategy

Example 3: determination of variable region sequence of anti-human cBIN1 exon13 antibody

Hybridoma cells were collected by centrifugation at 5X 10 ⁶ ～10×10 ⁶ The cells were added with 1ml of TRIzol and 0.2ml of chloroform, vigorously shaken for 15 seconds, left at room temperature for 3 minutes, centrifuged to take the aqueous phase and 0.5ml of isopropanol, left at room temperature for 10 minutes, and the precipitate was collected, washed with ethanol and dried to obtain RNA. Adding template RNA and primers into an ice bath centrifuge tube, correctly matching the primers and the template, performing reverse transcription, and performing PCR amplification. dNTP/ddNTP mixtures 2.5. Mu.l are added into 4 microcentrifuge tubes respectively, and the mixtures are subjected to warm bath at 37 ℃ for 5min for standby. 1pmol of PCR amplified double-stranded DNA,10pmol of sequencing primer, 2. Mu.l of 5 Xsequencing buffer were added to an empty microcentrifuge tube, double distilled water was added to a total volume of 10. Mu.l, heating was carried out at 96 ℃ for 8min, cooling in ice bath for 1min, and centrifugation was carried out at 10000g for 10s. Mu.l of a pre-cooled labeling mix (dCTP, dGTP, dTTP 0.75. Mu. Mol/L each), alpha-32P-dATP 5. Mu. Ci, 1. Mu.l of 0.1mol/L DDT,2U of sequencing enzyme is added with water to 15 mu l, mixed evenly and placed on ice for 2min, and a newly synthesized DNA chain is marked. Mu.l of the labeled reaction mixture was added to 4 prepared microcentrifuge tubes and incubated at 37 ℃ for 5min, and 4. Mu.l of stop buffer was added to each tube. Samples were heat denatured in a water bath at 80 ℃ for 5min, 2. Mu.l of each lane was applied to the sequencing gel, the fragments were electrophoretically separated, and sequence information was collected.

The VH and VL sequences of the murine antibodies (designated 8G 1-D7-F5) were obtained as shown in the table below. Further, the CDR Sequences of murine mAb were determined using the method described by Kabat et al (Kabat et al, sequences of Proteins of Immunological Interest, fifth edition, public Health Service, national institutes of Health, bessedla, md. (1991), pp 647-669).

TABLE 2 sequence information of murine antibodies

TABLE 3 antibody sequences

4: evaluation of binding Activity of anti-human cBIN1 exon13 antibody

4.1 binding Activity of antibody (8G 1-D7-F5) in enzyme-linked immunosorbent assay (ELISA)

The antibody was diluted at 11 spots in a three-fold dilution at the starting concentration of 10. Mu.g/ml. The proteins cBIN1 Isoform II2 (cBIN 1 exon1-13 and 18-20) and cBIN1 Isoform II3 (cBIN 1 exon 1-12 and 18-20) recombinantly expressed by Ji Kai GeneChem, inc., respectively, were used as coating antigens, and the concentration of the coating antigen was 0.1. Mu.g/well coated plate. Diluted antibodies were added to an Elisa plate at 100ul per well. Incubate for 1h at room temperature and wash the plate. The secondary antibody was diluted to 1. TMBA solution and B solution were mixed at 1:1, and incubated at room temperature for 5min for color development at 100ul per well. After the color development was completed, 50ul 2% was added per well to terminate the reaction by H2SO4, and OD450 was read. GraphPadPrism 6 analyzes the data and calculates EC50 values for antibody binding to cBIN1 isofomm II2 and cBIN1 isofomm II 3. The antibody binding curves are shown in figure 1, and the binding EC50 for the antibody and both proteins are shown in table 3.

TABLE 3 ESLIA binding of antibodies to antigen EC50 (μ g/ml)

Antibodies	cBIN1IsoformII2	cBIN1IsoformII3
			8G1-D7-F5	0.058	Is not combined with
Positive control antibody	0.614

Antibody 8G1-D7-F5 binds with high affinity to cBIN1 isofomm II2 protein containing exon13 and does not bind to cBIN1 isofomm II3 protein lacking exon13, indicating that antibody 8G1-D7-F5 binds to cBIN in the region corresponding to its exon 13. The binding activity of antibody 8G1-D7-F5 in ELISA was 11-12 times higher than that of the positive control antibody (Novus, NBP 2-21689).

4.2 antibody binding Activity in immunoblotting experiments (WB)

The WB was run on a 12-percent SDS-PAGE gel at 120mA current using the protein cBIN1 Isoform II2 (cBIN 1 exon1-13 &18-20) recombinantly expressed by Ji Kai GeneChem, inc. After the electrophoresis is finished, the protein is transferred to the PVDF membrane by using a transfer electrophoresis device and electrotransfer for 150min under the constant current condition of 300mA at 4 ℃. Primary antibody incubation was performed at a concentration of 10. Mu.g/ml using antibody 1F3-D11-B and a positive control antibody (Novus, NBP 2-21689), respectively, with the blocked PVDF membrane at room temperature for 2 hours, with secondary antibody after washing, with the PVDF membrane at room temperature for 1.5 hours, and development after thorough washing. The results are shown in FIG. 2. As shown in fig. 2, the antibody 8G1-D7-F5 binds to the mbin 1 isofomm II2 protein containing exon13 with high affinity in WB, and the binding amount of the antibody 8G1-D7-F5 was significantly higher than that of the positive control antibody (Novus, NBP 2-21689).

The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.

Sequence listing

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Claims

An antibody cBIN1, characterized in that,

the amino acid sequences of three CDR regions of the heavy chain are respectively shown in SEQ ID NO. 1, 2 and 3;

the amino acid sequences of the three CDR regions of the light chain are respectively shown in SEQ ID NO. 4, 5 and 6.
2. The cBIN1 antibody according to claim 1,

the heavy chain variable region has an amino acid sequence shown as SEQ ID NO. 7;

the light chain variable region has an amino acid sequence shown in SEQ ID NO. 8.
3. A nucleic acid encoding the antibody of claim 1 or 2.
4. An expression vector comprising the nucleic acid of claim 3.
5. A host cell transformed or transfected with the expression vector of claim 4.
6. The method of preparing the cBIN1 antibody of claim 1 or 2, comprising: culturing the host cell of claim 5 to induce expression of the cBIN1 antibody.
7. The cBIN1 antibody of claim 1 or 2, chemically or biologically labeled.
8. A conjugate prepared by conjugating the cBIN1 antibody of claim 1, 2 or 7 to a solid or semi-solid medium.
9. Use of a cBIN1 antibody according to claim 1, 2 or 7 or a conjugate according to claim 8 in the preparation of a product for detecting cBIN1 expression.
10. A kit comprising the cBIN1 antibody of claim 1, 2 or 7 or the conjugate of claim 8.