Monoclonal antibody for identifying proinsulin and application thereof

Abstract

The invention provides a monoclonal antibody for identifying proinsulin. The amino acid sequence of a light chain CDR3 of the monoclonal antibody is shown as SEQ ID NO: 7, SEQ ID NO: 13 or SEQ ID NO: 19; the amino acid sequence of the heavy chain CDR3 of the monoclonal antibody is as shown in SEQ ID NO: 10, SEQ ID NO: 16 or SEQ ID NO: 22. The monoclonal antibody can specifically recognize the specific epitope of the proinsulin, has the advantages of high recognition specificity, good affinity, high titer and the like, and has wide application value.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a monoclonal antibody for recognizing proinsulin and application thereof. Background Art

[0002] Proinsulin is an important precursor in the biosynthesis of insulin, with unique biological characteristics and clinical significance. The biosynthesis of insulin starts from the insulin precursor in the cytoplasm - preproinsulin. The newly synthesized preproinsulin contains 110 amino acids. Under the guidance of its amino-terminal signal peptide, it enters the endoplasmic reticulum through transmembrane translocation and is subsequently cleaved by signal peptidase to form another insulin precursor - proinsulin consisting of 86 amino acids. In the endoplasmic reticulum, proinsulin rapidly undergoes oxidative folding to form three pairs of disulfide bonds that are highly conserved in evolution and its normal spatial conformation. Normally folded proinsulin is further transported from the endoplasmic reticulum to the Golgi apparatus for further processing and cleavage to form insulin and C-peptide stored in secretory granules.

[0003] Proinsulin is composed of two parts, insulin and C-peptide, and has dual immune activity. This means that proinsulin can bind to both insulin antibodies and C-peptide antibodies. Although proinsulin is similar to insulin in molecular structure, its glucose-lowering activity is only 5%-10% of that of insulin. Under physiological conditions, only a very small amount of proinsulin will be released into the blood. In pathological conditions, such as diabetes, pancreatic tumors, polycystic ovary syndrome, or chronic renal insufficiency, the body produces insulin resistance or synthesizes a large amount of insulin, and the level of proinsulin in the blood will increase. Preliminary studies by the research and development team of the present invention also found that delayed proinsulin secretion can be used as an earlier and more sensitive clinical indicator for evaluating pancreatic islet dysfunction.

[0004] At present, proinsulin detection reagents can be used to diagnose diseases such as diabetes, insulinoma and insulin resistance syndrome, as well as accurately determine the physiological function of pancreatic β cells. However, the means of diagnosing abnormal proinsulin expression are not perfect enough, and it is very necessary to develop a new monoclonal antibody and detection reagent that can recognize specific epitopes of proinsulin. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present invention discloses a monoclonal antibody for identifying proinsulin and its application. The monoclonal antibody has the advantages of good affinity and high titer, and has strong specificity for identifying proinsulin in biological samples.

[0006] In order to achieve the above technical objectives, in the first aspect, the present invention proposes a monoclonal antibody that recognizes proinsulin, the amino acid sequence of the light chain CDR3 of the monoclonal antibody is shown in SEQ ID NO:7, SEQ ID NO:13 or SEQ ID NO:19; the amino acid sequence of the heavy chain CDR3 of the monoclonal antibody is shown in SEQ ID NO:10, SEQ ID NO:16 or SEQ ID NO:22.

[0007] In a further example of the present invention, the amino acid sequence of the light chain CDR1 of the monoclonal antibody is shown as SEQ ID NO:8, SEQ ID NO:14 or SEQ ID NO:20; and / or, the amino acid sequence of the light chain CDR2 of the monoclonal antibody is shown as SEQ ID NO:9, SEQ ID NO:15 or SEQ ID NO:21.

[0008] In a further example of the present invention, the amino acid sequence of the heavy chain CDR1 of the monoclonal antibody is shown as SEQ ID NO:11, SEQ ID NO:17 or SEQ ID NO:23; and / or, the amino acid sequence of the heavy chain CDR2 of the monoclonal antibody is shown as SEQ ID NO:12, SEQ ID NO:18 or SEQ ID NO:24.

[0009] It should be noted that the partial mutations introduced into the hypervariable region of the monoclonal antibody of the present invention do not affect the binding of the antibody molecule to the antigen. In a further example of the present invention, the amino acid sequence of the light chain variable region of the monoclonal antibody is as shown in SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5; or SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5 is substituted and / or deleted and / or added with one or more amino acids and has the same function as the protein shown in SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5; or SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5 has at least 90% homology (such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, etc. but not limited to the exemplified values) and has the same function as the protein shown in SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5.

[0010] Furthermore, the light chain amino acid sequence of the monoclonal antibody includes the light chain variable region and constant region SEQ ID NO: 35.

[0011] In a further example of the present invention, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is as shown in SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6; or an amino acid sequence in which one or more amino acids are substituted and / or deleted and / or added to SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6 and has the same function as the protein shown in SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6; or an amino acid sequence that has at least 90% homology (such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, etc. but not limited to the exemplified values) with SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6 and has the same function as the protein shown in SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6.

[0012] Furthermore, the heavy chain amino acid sequence of the monoclonal antibody includes a heavy chain variable region and a constant region SEQ ID NO: 36.

[0013] In a further example of the present invention, the monoclonal antibody is a whole antibody or an antigen-binding portion of a whole antibody; further, the whole antibody is of IgG1 type; further, the antigen-binding portion is a Fab fragment, a F(ab')2 fragment, a single-chain antibody or a full-length antibody. In a further preferred example of the present invention, the monoclonal antibody is a full-length antibody.

[0014] In a further example of the present invention, the amino acid sequence of proinsulin recognized by the monoclonal antibody is shown as SEQ ID NO:25, SEQ ID NO:26 or SEQ ID NO:27.

[0015] In a further example of the present invention, three monoclonal antibodies are provided, namely monoclonal antibody 1, monoclonal antibody 2 and monoclonal antibody 3, wherein the amino acid sequences of the light chain variable region and the heavy chain variable region of monoclonal antibody 1 are SEQ ID NO: 1 and SEQ ID NO: 2, respectively, the amino acid sequences of the light chain variable region and the heavy chain variable region of monoclonal antibody 2 are SEQ ID NO: 3 and SEQ ID NO: 4, respectively, and the amino acid sequences of the light chain variable region and the heavy chain variable region of monoclonal antibody 3 are SEQ ID NO: 5 and SEQ ID NO: 6, respectively.

[0016] In a second aspect, the present invention provides a nucleic acid molecule encoding the above-mentioned monoclonal antibody recognizing proinsulin.

[0017] Furthermore, the nucleotide sequence encoding the light chain variable region of monoclonal antibody 1 is shown in SEQ ID NO:28, and the nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO:29; the nucleotide sequence encoding the light chain variable region of monoclonal antibody 2 is shown in SEQ ID NO:30, and the nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO:31; the nucleotide sequence encoding the light chain variable region of monoclonal antibody 3 is shown in SEQ ID NO:32, and the nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO:33.

[0018] In a third aspect, the present invention proposes the use of the above-mentioned monoclonal antibody recognizing proinsulin in detecting proinsulin in a biological sample.

[0019] The present invention is not limited to the specific types of the biological samples, such as human tissues, cells, supernatants, serum, plasma, etc. Those skilled in the art can use the monoclonal antibodies for recognizing proinsulin of the present invention to detect biological samples containing antigens as needed, and the protection scope of the present invention is not limited thereby.

[0020] In the fourth aspect, the present invention proposes the use of the above-mentioned monoclonal antibody that recognizes proinsulin in the preparation of an in vitro insulin immunoassay reagent, a kit or a pharmaceutical composition; by specifically applying the above-mentioned monoclonal antibody that recognizes proinsulin to a specific kit, the proinsulin content in a biological sample can be determined.

[0021] Furthermore, the kit also includes any one or a combination of at least two of an ELISA plate, a coating buffer, a substrate, a substrate color developing solution or a reaction termination solution.

[0022] Furthermore, the pharmaceutical composition also includes a pharmaceutically acceptable carrier and / or diluent.

[0023] In addition, the present invention is not limited to the method for preparing the hybridoma mother cell for producing the above monoclonal antibody, and those skilled in the art can choose accordingly. Further, the method for preparing the hybridoma mother cell for producing the above monoclonal antibody can optionally include the following steps:

[0024] (1) Polypeptide synthesis: A polypeptide with an amino acid sequence as shown in SEQ ID NO: 25, SEQ ID NO: 26 or SEQ ID NO: 27 was synthesized by chemical synthesis and purified by HPLC.

[0025] (2) Antigen preparation: Using patented IEF (Immunogenicity Enhancement Factors), VLP (Virus-Like Particles) is combined with traditional KLH carrier protein.

[0026] (3) Pre-immunization test: Five female BALB / c mice aged 8-12 weeks were selected, blood was collected from the eye sockets and serum was separated. The serum titer was detected by ELISA, and the coating antigen was a conjugate of peptide and BSA.

[0027] (4) Mouse immunization: Use peptide and KLH conjugate as immunogen for immunization, and after the last injection of immunization, take blood from the mice to start serum titer detection, the detection method is the same as the pre-immunization detection. Only mice with antiserum titer greater than 10K can enter the sorting stage, otherwise they will be immunized once more.

[0028] (5) Antibody discovery: Use conventional hybridoma preparation procedures to obtain hybridoma cell lines, use pre-immune detection methods to detect the supernatant of hybridoma cell lines, and use supernatant purified antibodies for retesting. Select a cell line with the highest ELISA titer to obtain the hybridoma mother cell.

[0029] In addition, the present invention is not limited to the method for producing the above-mentioned monoclonal antibodies, and those skilled in the art can choose accordingly. Further, the method for preparing the monoclonal antibody can be selected as follows: (1) hybridoma production: cell production is carried out using the standard production process of serum-free medium, and the antibody is separated and purified using the Protein A / G affinity column chromatography method; (2) ascites production: antibodies are produced using the standard production process of ascites, and antibodies are separated and purified using the Protein A / G affinity column chromatography method; (3) recombinant production: light and heavy chain nucleic acids of hybridoma cell lines are obtained through molecular biological operations, agarose gel verification, and constructed into expression vectors using restriction endonucleases and T4 ligases, and plasmids are amplified using a prokaryotic expression system. Cell production is carried out using transient transfection and the method of constructing a stable cell line, and the antibody is separated and purified using the Protein A / G affinity column chromatography method.

[0030] The amino acid sequences involved in the present invention include:

[0031] (1) Monoclonal antibody 1

[0032] SEQ ID NO: 1 (light chain variable region):

[0033] DIVLTQSPLSLPVSLGDQASISCRSSQNIVHSNGKTYLEWYLQKPGQSPKLLIYNIYNRF SGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPYTFGGGTKLEIK

[0034] SEQ ID NO:7 (light chain CDR3):

[0035] FQSHPY

[0036] SEQ ID NO:8 (light chain CDR1):

[0037] RSSQNIVHSNGKTYLE

[0038] SEQ ID NO:9 (light chain CDR2):

[0039] NIYNRFS

[0040] SEQ ID NO:2 (heavy chain variable region):

[0041] QVQLQQSGAEVVKPGASVKMSCKASGYSFTSYNMHWIKQTPGQGLEWIGGIYPGNG ESSYNQKFKGKATMTADKSSNTAYMQFSSLTSEDSAVYYCARGDGFDYWGQGTTLTVSS

[0042] SEQ ID NO: 10 (heavy chain CDR3):

[0043] GDJ

[0044] SEQ ID NO:11 (heavy chain CDR1):

[0045] SYNMH

[0046] SEQ ID NO: 12 (heavy chain CDR2):

[0047] GIYPGNGESSYNQKFKG

[0048] (2) Monoclonal antibody 2

[0049] SEQ ID NO:3 (light chain variable region):

[0050] DIVLTQSPASLAVSLGQRATISCKASQSVDYNGDSYMNWYQQKPGQPPTLLIYVASKLE SGIPARFSGSGSGTDFTLNIHPVEEEDVATYYCQQTNEEPLTFGAGTKLELK

[0051] SEQ ID NO: 13 (light chain CDR3):

[0052] QQTNEEPLT

[0053] SEQ ID NO: 14 (light chain CDR1):

[0054] KASQSVDYNGDSYMN

[0055] SEQ ID NO: 15 (light chain CDR2):

[0056] VASKLES

[0057] SEQ ID NO:4 (heavy chain variable region):

[0058] QVQLEQSGPELVKPGASVRISCKASKASGYTFTSYYVHWVRQRPGQGLEWIGWIHPGNINS KYNEKFKDKATLTADKSSTTAYMQLSSLTSEDSAVYFCSRSVDYWGQGTTLTVSS

[0059] SEQ ID NO: 16 (heavy chain CDR3):

[0060] SVDY

[0061] SEQ ID NO: 17 (heavy chain CDR1):

[0062] YV

[0063] SEQ ID NO: 18 (heavy chain CDR2):

[0064] WIHPGNINSKYNEKFKD

[0065] (3) Monoclonal antibody 3

[0066] SEQ ID NO:5 (light chain variable region):

[0067] DIVLTQSTALMAASPGEKVTITCSVSSSISSSYLHWYQQKSETSPKPWIYGTSNLASGVP VRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSNYPLTFGAGTKLELK

[0068] SEQ ID NO: 19 (light chain CDR3):

[0069] QQWSNYPLT

[0070] SEQ ID NO:20 (light chain CDR1):

[0071] SVSSSISSSYLH

[0072] SEQ ID NO:21 (light chain CDR2):

[0073] GTSNLAS

[0074] SEQ ID NO:6 (heavy chain variable region):

[0075] QVQLEQSGGGLVQPKGSLKLSCAASGFNFNTNGMNWVRQAPGKGLEWVARIRSKSNNYATYYADSVKDRFTISRDDSQSMLYLQMNNLKTEDTAMYYCVRDGGYGAWFAYWGQGSLVTVSA

[0076] SEQ ID NO:22 (heavy chain CDR3):

[0077] DGGYGAWFAY

[0078] SEQ ID NO:23 (heavy chain CDR1):

[0079] TNGMN

[0080] SEQ ID NO:24 (heavy chain CDR2):

[0081] RIRSKSNNYATYYADSVKD

[0082] In addition, the amino acid sequence of the light chain constant region is SEQ ID NO: 34 (light chain constant region):

[0083] RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTD QDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0084] The amino acid sequence of the heavy chain constant region is SEQ ID NO: 35 (heavy chain constant region):

[0085] ASTTPPSVYPLAPGSANSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHT AQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMNTNGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

[0086] In addition, the amino acid sequences of SEQ ID NO:25, SEQ ID NO:26 or SEQ ID NO:27 in the present invention are respectively:

[0087] SEQ ID NO:25:

[0088] PLALEGSLQKRGIV

[0089] SEQ ID NO:26:

[0090] PKTRREAEDLQVGQ

[0091] SEQ ID NO:27:

[0092] KSRREVEDPQVEQLEC

[0093] The nucleic acid molecule sequences involved in the present invention include:

[0094] The nucleotide sequence encoding the light chain variable region of monoclonal antibody 1 is shown in SEQ ID NO: 28, and the nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID NO: 29;

[0095] SEQ ID NO:28:

[0096] GACATTGTGCTCACCCAATCTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAACATTGTACATAGTAATGGAAAGACCTATTTAGAGTGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTACAATATTTACAACCGGTTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTACTGTTTTCAAGGTTCACATGTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA

[0097] SEQ ID NO:29:

[0098] CAGGTCCAGCTGCAGCAGTCAGGGGCTGAGGTGGTGAAGCCTGGGGCCTCAGTGAAGATGTCCTGCAAGGCTTCTGGCTACTCATTTACCAGTTACAATATGCACTGGATAAAGCAGACACCTGGACAGGGCCTGGAATGGATTGGAGGTATTTATCCAGGAAATGGTGAAAGTTCCTACAATCAGAAGTTCAAAGGCAAGGCCACAATGACTGCAGACAAATCCTCCAACACAGCCTACATGCAGTTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTATTGTGCAAGAGGGGACGGTTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA

[0099] The nucleotide sequence encoding the light chain variable region of monoclonal antibody 2 is shown in SEQ ID NO:30, and the nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID NO:31:

[0100] SEQ ID NO:30:

[0101] GACATTTGTGCTCACCCAGTCTCCAGCCTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCACCATCTCCTGCAAGGCCAGCCAAAGTGTTGATTATAATGGTGATAGTTATATGAACTGGTACCAACAGAAACCGGGACAGCCACCCACACTCCTCATCTATGTTGCAT CCAAGCTAGAATCTGGGATCCCAGCCAGGTTTAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGTTGCAACCTATTACTGTCAGCAAACTAATGAGGAACCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA

[0102] SEQ ID NO:31:

[0103] CAAGTTCAGCTGGAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAGGATATCCTGCAAGGCTTCTGGCTACACCTTCACAAGCTACTATGTTCACTGGGTGAGGCAGAGGCCTGGACAGGGACTTGAGTGGATTGGATGGATTCATCCTGGAAATATTA ATTCTAAATACAATGAGAAATTCAAGGACAAGGCCACACTGACTGCAGACAAATCCTCCACCACAGCCTACATGCAGCTCAGCAGCCTGACCTCTGAGGACTCTGCGGTCTATTTCTGTTCAAGATCGGTTGACTACTGGGGCCAAGGGACCACTCTCACAGTCTCCTCA

[0104] The nucleotide sequence encoding the light chain variable region of monoclonal antibody 3 is shown in SEQ ID NO:32, and the nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID NO:33:

[0105] SEQ ID NO:32:

[0106] GATATTGTGCTGACCCAGTCTACAGCACTCATGGCTGCATCTCCAGGGGAAGGTCACCATCACCTGCAGTGTCAGCTCAAGTATAAGTTCCAGCTACTTGCACTGGTACCAGCAGAAGTCAGAAACCTCCCCCAAACCCTGGATTTATGGCACATCCAAC CTGGCTTCTGGAGTCCCTGTTCGCTTCAGTGGCAGTGGATCTGGGACCTCTTATTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGTCAACAGTGGAGTAATTACCCACTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA

[0107] SEQ ID NO:33:

[0108] CAAGTTCAGCTGGAGCAGTCTGGTGGAGGATTGGTGCAGCCTAAAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTTAACTTCAATACCAATGGCATGAACTGGGTCCGCCAGGCCCCAGGAAAGGGTTTGGAGTGGGTTGCTCGCATAAGAAGTAAAAGTAATAATTATGCAACAT ATTATGCCGATTCAGTGAAAGACAGGTTCACCATCTCCAGAGATGATTCACAAAGCATGCTCTATCTGCAAATGAACAACTTGAAAACTGAGGACACAGCCATGTATTACTGTGTGAGAGACGGGGGTTACGGGGCCTGGTTTGCTTACTGGGGCCAAGGGTCTCTGGTCACTGTCTCTGCA

[0109] Compared with the prior art, the monoclonal antibody for recognizing proinsulin of the present invention can specifically recognize specific antigen epitopes in human / mouse proinsulin, has strong recognition specificity, good affinity, high titer, and has wide application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0110] The drawings in the specification, which constitute a part of this application, are used to provide a further understanding of the present invention. The illustrative embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0111] Figure 1The results of protein immunoblotting experiments of monoclonal antibody 1 of the present invention in Example 6 are shown; wherein, the left figure is the result of using monoclonal antibody 1 of the present invention as a primary antibody to recognize protein, and the right figure is the result of using human insulin antibody as a primary antibody to recognize protein; lane 1 represents supernatant protein secreted by human insulinoma cells under a non-reduced state, lane 2 represents supernatant protein secreted by human insulinoma cells under a reduced state, and lane M represents marker (Thermo Fisher #26616);

[0112] Figure 2 The results of protein immunoblotting experiments of monoclonal antibody 2 of the present invention in Example 7 are shown; wherein, the left figure shows the results of using monoclonal antibody 2 of the present invention as a primary antibody to recognize a protein, and the right figure shows the results of using human insulin antibody as a primary antibody to recognize a protein; lane 1 represents the supernatant protein secreted by human insulinoma cells under a non-reduced state, lane 2 represents the supernatant protein secreted by human insulinoma cells under a reduced state, and lane M represents a marker (Thermo Fisher #26616);

[0113] Figure 3 It shows that the monoclonal antibody 3 of the present invention can specifically recognize mouse proinsulin protein, but not insulin, in Example 8. The left figure shows the result of using the monoclonal antibody 3 of the present invention as the primary antibody to recognize the protein, and the right figure shows the result of using the mouse insulin antibody as the primary antibody to recognize the protein; lane 1 represents mouse islet cell protein; lane M represents marker (ThermoFisher #26616).

[0114] Note: The red arrow in the figure represents the location where proinsulin protein is detected, and the blue arrow represents the location where insulin protein is detected. DETAILED DESCRIPTION

[0115] In order to facilitate the understanding of the present invention, the present invention will be described more comprehensively below, and preferred embodiments of the present invention are given. However, it should be understood that these embodiments are only used for more detailed description and should not be understood as limiting the present invention in any form, i.e., not intended to limit the scope of protection of the present invention.

[0116] Unless otherwise defined, the technical terms used in the following examples have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. The test reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the experimental methods, unless otherwise specified, are all conventional methods.

[0117] Example 1 Preparation of hybridoma cell lines of anti-proinsulin monoclonal antibodies

[0118] (1) Preparation of immunogens: A polypeptide (amino acid sequence such as SEQ ID NO: 25) is coupled to a KLH (keyhole limpet hemocyanin) carrier protein using a conventional coupling method to prepare an immune antigen, and the polypeptide concentration is controlled to be ≥ 0.25 mg / mL (thiol value ≥ 1.0);

[0119] Similarly, another polypeptide (amino acid sequence such as SEQ ID NO: 25) was coupled to the BSA carrier protein using a conventional coupling method to prepare a screening antigen, and the polypeptide concentration was controlled to be ≥ 0.25 mg / mL (thiol value ≥ 1.0). Before immunization, the immunization antigen was mixed with Freund's complete adjuvant in a ratio of 1:1 and fully emulsified.

[0120] (2) Immunization: Five healthy 8-week-old BALB / c mice were selected as immunization subjects (the pre-immunization serum ELISA titer was tested before immunization. It was best if the pre-immunization serum did not have a color reaction with the screening antigen, or the OD value of the undiluted serum ELISA test was less than 2.1 times that of the PBS group).

[0121] The immunization scheme is to perform a second immunization in the third week after the first immunization, and a third immunization in the second week after the second immunization. The total immunization cycle is 7 weeks. The immunization dose for each mouse is 100 μL each time. The serum ELISA titer is tested one week after the third immunization (using the screening antigen coated plate). When the titer is greater than 256k, the immune antigen is used for a shock. Cell fusion is completed 2-3 days after the shock. If the titer is not reached, an additional immunization is performed, and the shock is performed again after the titer is reached.

[0122] (3) Fusion screening: The mice to be fused are killed using the carbon dioxide method, the spleen is collected aseptically, and the spleen cell suspension is prepared and counted after the red blood cells are lysed. The thymus of 3-4 week old mice is collected aseptically, and the thymic cells are plated in 20-40 96-well plates using HAT medium, with 100 μL per well. Myeloma cells in the logarithmic growth phase are selected, collected and counted aseptically to ensure that the cell viability is greater than 90%. Splenocytes and myeloma cells are mixed in a certain ratio (such as 3:1-5:1), and cell fusion is performed using the electrofusion method.

[0123] After fusion, place the cell suspension in HAT selective medium and mix evenly to make the cell density less than 15 cells / mL, and divide it into 20-40 prepared 90-well plates, 100 μL per well (200 μL in total). After the cells grow for 1-2 weeks, take 100 μL of supernatant from the wells where the cells have successfully fused by microscopy for ELISA detection (using screening antigen coating), and select the wells with the highest ELISA value for subcloning. Try to make the subcloned cells as single cells in 96 wells. Subclone screening also uses the ELISA method to detect the supernatant until the whole plate is tested and the ELISA is positive.

[0124] (4) Identification: From the subclones that are positive on the whole plate, select 3-5 cell lines with the best growth status by microscopic examination, and use 50mL culture system to culture for 1-3 weeks. When the cell viability is less than 40%, collect the supernatant, purify the supernatant using the Protein A / G affinity purification method, and use the BCA kit to calibrate the antibody concentration. Detect the EC50 values ​​of these 3-5 antibody strains in the ELISA test. The best cell line is the final hybridoma cell line of anti-proinsulin monoclonal antibody.

[0125] Example 2 Production of anti-proinsulin monoclonal antibody hybridoma cells

[0126] The cell lines were acclimated to serum-free suspension culture using the culture component replacement method and cultured for 10 5 The inoculation density was gradually inoculated into a 1L culture bottle (330mL culture medium) for shake flask culture. When the sugar content of the culture medium was lower than 10mmol / mL, glucose solution was added to control the sugar content of the culture medium at 10-25mmol / mL. In addition, when the density was greater than 2×10 6 When the cell viability was less than 40%, the supernatant was collected.

[0127] Place the prepared Protein A column on the AKTA device, use 10mM PBS (pH=7.4±0.05) to balance the column, hang the column at 10mL / min, and collect the flow-through liquid for secondary column hanging. Use pH=5.5 glycine-HCl buffer to clean impurities at a flow rate of 5mL / min, and the cleaning volume is 2-5 times the column volume. After cleaning, use the balancing buffer (10mM PBS at pH=7.4) to balance the column. Use pH=2.5 glycine-HCl buffer to elute the antibody at a rate of 5mL / min. Refer to the supernatant antibody content obtained by ELISA method and control the antibody concentration at 3-5mg / mL. Immediately add an equal volume of glycine-NaHCO after elution. 3 Buffer solution (pH = 9.5 ± 0.1) to neutralize the acidic environment.

[0128] The antibody buffer system was replaced with 10 mM PBS (pH=7.4±0.05) using an ultrafiltration centrifuge tube, and the concentration was calibrated using a BCA kit and controlled at 2.0 mg / mL.

[0129] Please note that if the antibody is not used immediately, it is necessary to add an appropriate amount of preservative (such as sodium azide, etc.) and store it in a -80℃ refrigerator.

[0130] Example 3 Recombinant production of anti-proinsulin monoclonal antibodies

[0131] The monoclonal cell line obtained in Example 1 was cultured to 75 cm 2 The bottom of the carbon dioxide cell culture flask was filled to 80%, and the cells were collected and resuspended in nuclease-free water containing RNase inhibitors, and centrifuged again to collect the cells. A portion of them was subjected to single-cell sequencing to obtain antibody sequence information.

[0132] The other part uses a 5'RACE kit to extract total mRNA, reverse transcribe it into cDNA, and amplify the antibody light and heavy chain variable region deoxyribonucleotide sequences.

[0133] According to the kit instructions, the pcDNA5.0 plasmid and the antibody light and heavy chains were digested with restriction endonucleases. The digestion products were recovered and purified using a nucleic acid recovery kit. The purified products were verified by dextran gel electrophoresis and connected using a T4 ligase kit to construct the antibody light and heavy chains into the plasmids respectively, so that the variable and constant regions of the light and heavy chains were intact on the plasmids.

[0134] The plasmid was transferred into E. coli (DH5α) competent cells using competent cells and heat shock method, the plasmid was amplified, and the plasmid was extracted using an endotoxin-free plasmid extraction kit.

[0135] The linearized light and heavy chain plasmids were simultaneously transformed into CHO cells using PEI transformation or electroporation.

[0136] Stable transfected cells were screened by standard stable transfection cell line screening methods, or cell supernatants were produced by transient transfection methods.

[0137] The antibody production of the stable transfectant was achieved according to the method in Example 2.

[0138] The antibody purification method in Example 2 was used to purify the supernatant of cells expressing transient transfection.

[0139] In this example, a specific monoclonal antibody 1 against the partial sequence of SEQ ID NO: 25 (PLALEGSLQKRGIV) of human insulin C-peptide-A chain connection was prepared. The monoclonal antibody was of IgG1 type and its light chain was of kappa type.

[0140] The sequence of its light chain variable region is SEQ ID NO:1, and the amino acid sequences of the three hypervariable regions CDR1, CDR2, and CDR3 of the light chain variable region are shown in SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:7, respectively, wherein CDR1 is located at positions 24-39 of the light chain variable region, CDR2 is located at positions 55-61 of the light chain variable region, and CDR3 is located at positions 94-102 of the light chain variable region; the heavy chain variable region is SEQ ID NO:2, and the amino acid sequences of the three hypervariable regions CDR1, CDR2, and CDR3 of the heavy chain variable region are shown in SEQ ID NO:11, SEQ ID NO:112, and SEQ ID NO:10, respectively, wherein CDR1 is located at positions 31-35 of the heavy chain variable region, CDR2 is located at positions 50-66 of the heavy chain variable region, and CDR3 is located at positions 99-104 of the heavy chain variable region.

[0141] Its light chain variable region can be combined with SEQ ID NO: 34 to form a complete light chain; its heavy chain variable region can be combined with SEQ ID NO: 35 to form a complete heavy chain.

[0142] Example 4 Preparation of proinsulin monoclonal antibody 2

[0143] Using the experimental methods of Examples 1-3 above, a specific monoclonal antibody 2 against the partial sequence SEQID NO: 26 (PKTRREAEDLQVGQ) of the human proinsulin B chain-C peptide connection can be obtained. The monoclonal antibody is of IgG1 type, and the light chain is of kappa type.

[0144] The sequence of its light chain variable region is SEQ ID NO:3, and the amino acid sequences of the three hypervariable regions CDR1, CDR2, and CDR3 of the light chain variable region are shown in SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:13, respectively, wherein CDR1 is located at positions 24-38 of the light chain variable region, CDR2 is located at positions 54-60 of the light chain variable region, and CDR3 is located at positions 93-101 of the light chain variable region; the heavy chain variable region is SEQ ID NO:4, and the amino acid sequences of the three hypervariable regions CDR1, CDR2, and CDR3 of the heavy chain variable region are shown in SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:16, respectively, wherein CDR1 is located at positions 31-35 of the heavy chain variable region, CDR2 is located at positions 50-66 of the heavy chain variable region, and CDR3 is located at positions 99-102 of the heavy chain variable region.

[0145] Its light chain variable region can be combined with SEQ ID NO: 34 to form a complete light chain; its heavy chain variable region can be combined with SEQ ID NO: 35 to form a complete heavy chain.

[0146] The antibody production of the stable transfectant was achieved according to the method in Example 2.

[0147] The antibody purification method in Example 2 was used to purify the supernatant of cells expressing transient transfection.

[0148] Example 5 Preparation of proinsulin monoclonal antibody 3

[0149] Using the experimental methods of Examples 1-3 above, a specific monoclonal antibody 3 against the partial sequence SEQ ID NO: 27 (KSRREVEDPQVEQLEC) of mouse proinsulin B chain-C peptide connection can be obtained. The monoclonal antibody is of IgG1 type, and the light chain is of kappa type.

[0150] The sequence of its light chain variable region is SEQ ID NO:5, and the amino acid sequences of the three hypervariable regions CDR1, CDR2, and CDR3 of the light chain variable region are shown in SEQ ID NO:20, SEQ ID NO:21, and SEQ ID NO:19, respectively, wherein CDR1 is located at positions 24-35 of the light chain variable region, CDR2 is located at positions 51-57 of the light chain variable region, and CDR3 is located at positions 90-98 of the light chain variable region; the heavy chain variable region is SEQ ID NO:6, and the amino acid sequences of the three hypervariable regions CDR1, CDR2, and CDR3 of the heavy chain variable region are shown in SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:22, respectively, wherein CDR1 is located at positions 31-35 of the heavy chain variable region, CDR2 is located at positions 50-68 of the heavy chain variable region, and CDR3 is located at positions 101-110 of the heavy chain variable region.

[0151] Its light chain variable region can be combined with SEQ ID NO: 34 to form a complete light chain; its heavy chain variable region can be combined with SEQ ID NO: 35 to form a complete heavy chain.

[0152] The antibody production of the stable transfectant was achieved according to the method in Example 2.

[0153] The antibody purification method in Example 2 was used to purify the supernatant of cells expressing transient transfection.

[0154] Example 6

[0155] The supernatant of the immortalized human insulinoma cells was collected, 60 μL of the supernatant was added to 20 μL of 4×NPLDS SAMPLE BUF (Thermo Fisher, NP0007), mixed, and boiled at 95°C for 5 min to prepare a protein sample under a non-reduced state; 36 μL of the above sample was added to 4 μL of 1 M reducing agent DTT (Sigma, D9163), mixed, and boiled at 95°C for 5 min to prepare a protein sample under a reduced state.

[0156] The specific protein immunoblotting experiment operation is as described in the previous article published by the present invention team (e.g. Yang J, et al. PNAS; 2022; 119 (45): e2204443119, etc.), and 30 μL of the above non-reduced and reduced protein samples are taken for protein immunoblotting experiments, and the primary antibodies used are monoclonal antibody 1 of the present invention and human insulin antibody. The results are shown in Figure 1 shown.

[0157] Figure 1 The left figure in the figure shows the result of using the monoclonal antibody 1 prepared in Example 1-3 of the present invention as the primary antibody recognition protein, and the right figure shows the result of using the human insulin antibody as the primary antibody recognition protein. Figure 1 Comparative analysis of the middle left figure and the right figure shows that the monoclonal antibody 1 of the present invention can specifically recognize proinsulin protein and only recognize proinsulin protein, especially proinsulin protein in a reduced state, while the human insulin antibody as a primary antibody recognition protein can simultaneously recognize proinsulin and insulin protein; this result verifies that the monoclonal antibody 1 of the present invention has no cross-reaction with insulin protein during the process of recognizing proinsulin protein, and has excellent specificity for recognizing proinsulin.

[0158] Example 7

[0159] The supernatant of the immortalized human insulinoma cells was collected, 60 μL of the supernatant was added to 20 μL of 4×NPLDS SAMPLE BUF (Thermo Fisher, NP0007), mixed, and boiled at 95°C for 5 min to prepare a protein sample under a non-reduced state; 36 μL of the above sample was added to 4 μL of 1 M reducing agent DTT (Sigma, D9163), mixed, and boiled at 95°C for 5 min to prepare a protein sample under a reduced state.

[0160] The specific protein immunoblotting experiment operation is as described in the previous article published by the present invention team (e.g. Yang J, et al. PNAS; 2022; 119 (45): e2204443119, etc.), and 30 μL of the above non-reduced and reduced protein samples are taken for protein immunoblotting experiments, and the primary antibodies used are monoclonal antibody 2 of the present invention and human insulin antibody. The results are shown in Figure 2 shown.

[0161] Figure 2 The left figure in the figure shows the result of using the monoclonal antibody 2 of the present invention as the primary antibody recognition protein, and the right figure shows the result of using the human insulin antibody as the primary antibody recognition protein. Figure 2As can be seen from the left and right figures in the figure, the monoclonal antibody 2 of the present invention can specifically recognize proinsulin protein but not insulin protein, especially recognize proinsulin protein in a reduced state, while the human insulin antibody as a primary antibody recognition protein can simultaneously recognize proinsulin and insulin protein; this result confirms that the monoclonal antibody 2 of the present invention has no cross-reaction with insulin protein in the process of recognizing proinsulin protein and has excellent specificity for recognizing proinsulin.

[0162] Example 8

[0163] 60 pancreatic islets from wild-type mice were extracted, 30 μL of protein lysis buffer was added and lysed on ice for 20 min. After centrifugation at 13500 rpm and 4°C for 15 min, 27 μL of supernatant was taken, 9 μL of 4×NP LDS SAMPLE BUF (Thermo Fisher, NP0007) and 4 μL of 1M reducing agent DTT (Sigma, D9163) were added, mixed, and boiled at 95°C for 5 min.

[0164] The specific islet extraction and protein immunoblotting experiments were performed as described in the previous articles published by the present invention team (e.g. Yang J, et al. PNAS; 2022; 119(45): e2204443119, etc.). 30 μL of the above protein sample was taken for protein immunoblotting experiments, and the primary antibodies used were monoclonal antibody 3 of the present invention and mouse insulin antibody. The results are shown in Figure 3 shown.

[0165] Figure 3 The left figure shows the result of using the monoclonal antibody 3 of the present invention as the primary antibody to recognize the protein, and the right figure shows the result of using the mouse-derived insulin antibody as the primary antibody to recognize the protein; the results show that the monoclonal antibody 3 of the present invention can specifically recognize the proinsulin protein and does not recognize the insulin protein, while the mouse-derived insulin antibody can recognize both the proinsulin and the insulin protein; this result verifies that the monoclonal antibody 3 of the present invention has no cross-reaction with the insulin protein and can specifically recognize the proinsulin protein.

[0166] It should be noted that the above contents are further detailed descriptions of the present invention in combination with specific implementation methods, and it cannot be determined that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, several simple improvements can be made without departing from the concept of the present invention, which should be regarded as falling within the scope of protection of the present invention.

Claims

1. A monoclonal antibody that recognizes proinsulin, characterized in that: The amino acid sequence of the light chain CDR3 of the monoclonal antibody is shown in SEQ ID NO:7, SEQ ID NO:13 or SEQ ID NO:19; The amino acid sequence of the heavy chain CDR3 of the monoclonal antibody is shown in SEQ ID NO:10, SEQ ID NO:16 or SEQ ID NO:

22.

2. The monoclonal antibody for recognizing proinsulin according to claim 1, characterized in that: The amino acid sequence of the light chain CDR1 of the monoclonal antibody is shown in SEQ ID NO:8, SEQ ID NO:14 or SEQ ID NO:20; And / or, the amino acid sequence of the light chain CDR2 of the monoclonal antibody is shown as SEQ ID NO:9, SEQ ID NO:15 or SEQ ID NO:

21.

3. The monoclonal antibody for recognizing proinsulin according to claim 1, characterized in that: The amino acid sequence of the heavy chain CDR1 of the monoclonal antibody is shown in SEQ ID NO:11, SEQ ID NO:17 or SEQ ID NO:23; and / or the amino acid sequence of the heavy chain CDR2 of the monoclonal antibody is shown in SEQ ID NO:12, SEQ ID NO:18 or SEQ ID NO:

24.

4. The monoclonal antibody for recognizing proinsulin according to claim 1, characterized in that: The amino acid sequence of the light chain variable region of the monoclonal antibody is as shown in SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5; or an amino acid sequence in which one or more amino acids are substituted and / or deleted and / or added to SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5 and has the same function as the protein shown in SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5; or an amino acid sequence that has at least 90% homology with SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5 and has the same function as the protein shown in SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:

5.

5. The monoclonal antibody for recognizing proinsulin according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region of the monoclonal antibody is as shown in SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6; or an amino acid sequence in which one or more amino acids are substituted and / or deleted and / or added to SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6 and has the same function as the protein shown in SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6; or an amino acid sequence that has at least 90% homology with SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6 and has the same function as the protein shown in SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:

6.

6. The monoclonal antibody for recognizing proinsulin according to claim 1, characterized in that: The monoclonal antibody is a whole antibody or an antigen-binding portion of a whole antibody; Preferably, the whole antibody is of IgG1 type; Preferably, the antigen binding portion is a Fab fragment, a F(ab')2 fragment, a single chain antibody or a full-length antibody.

7. The monoclonal antibody recognizing proinsulin according to claim 1, characterized in that: The amino acid sequence of proinsulin recognized by the monoclonal antibody is shown in SEQ ID NO:25, SEQ ID NO:26 or SEQ ID NO:

27.

8. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the monoclonal antibody recognizing proinsulin according to any one of claims 1 to 7.

9. Use of the monoclonal antibody recognizing proinsulin according to any one of claims 1 to 7 in detecting proinsulin in a biological sample.

10. Use of the monoclonal antibody recognizing proinsulin according to any one of claims 1 to 7 in the preparation of an in vitro insulin immunoassay reagent, kit or pharmaceutical composition.

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