A human-mouse chimeric monoclonal antibody against NF155 protein, its product, method for detecting NF155 protein, and applications thereof

Abstract

The present invention discloses a human-mouse chimeric monoclonal antibody against NF155 protein, its product, a method for detecting NF155 protein and applications thereof, belonging to the technical field of monoclonal antibody. The human-mouse chimeric monoclonal antibody against NF155 protein disclosed by the present invention is an NF155 antibody with strong specificity and high sensitivity. On the one hand, the antibody disclosed by the present invention can be used as a quality control product / control product / calibration product in a diagnostic kit instead of a human serum antibody; on the other hand, the antibody disclosed by the present invention can visualize and localize the NF155 protein; in addition, the antibody disclosed by the present invention can also be used as a ligand in an affinity system to prepare a chromatography column to realize the separation and purification of NF155 protein, etc.

Description

Technical Field

[0001] The present invention belongs to the technical field of monoclonal antibody, and particularly relates to a human-mouse chimeric monoclonal antibody of NF155 protein, its product and a method for detecting NF155 protein. Background Art

[0002] Neurofascin (NF) is a cell surface protein involved in the formation of nerve fascicles discovered in chickens by Ranthjen et al. in 1987. It belongs to a cell adhesion molecule and plays a crucial role in the formation and maintenance of the stability of the Nodes of Ranvier (also known as Ranvier nodes or nerve nodes, which are special structures on nerve fibers). Currently, five different NF polypeptide types, namely NF140, NF155, NF166, NF180, and NF186, have been discovered. NF155 is mainly present in glial cells. Its expression begins with the formation of myelin sheaths and remains highly expressed during the myelin sheath formation period. As oligodendrocytes gradually develop and mature, NF155 migrates from the cell body to the ends of cell processes and finally distributes in the lipid rafts in the paranodal regions of the myelin sheaths in the mature central nervous system. NF155 plays an important role in the early stage of the formation of the axon initial segment. In addition, NF155 also plays an important role in the formation of the myelin sheath structure and the repair process after injury. It ensures the stability of the myelin sheath structure by interacting with the caspr / contactin complex expressed on the axonal membrane. The research by Mathey et al. also found that the repair function of NF155 after myelin sheath injury can be used as a new target for the treatment of autoantibody-mediated axonal injury (MATHEY E K, DERFUSS T, STORCH M K, et al. Neurofascin as a novel target for autoantibody-mediated axonal injury [J]. Journal of Experimental Medicine, 2007, 204(10): 2363-2372).

[0003] In 2012, NG et al. detected anti-NF155 antibodies mainly of the IgG4 subtype in patients with chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) (Ng J K M, Malotka J, Kawakami N, et al. Neurofascin as a target for autoantibodies in peripheral neuropathies[J]. Neurology, 2012, 79(23): 2241-2248.). At present, the diagnosis of CIDP related to NF155 antibodies in China depends on the detection of anti-NF155 antibodies.

[0004] The detection of autoantibodies has important clinical value for the diagnosis of autoimmune diseases, the judgment of disease progression, disease treatment and prognosis. In the detection system of autoantibodies, reference materials are an important part of the quality control of the detection system. Usually, the positive serum of patients can be used as a positive reference material. However, due to the small amount of positive serum of autoimmune diseases, high acquisition cost, difficulty in value determination and traceability, it is necessary to provide a positive reference material that can replace the positive serum of patients, and chimeric antibodies or humanized antibodies can meet such requirements. At present, some chimeric antibodies or humanized antibodies have been used as quality control products / control products / calibration products for in vitro diagnostic reagents internationally, such as the chimeric antibody against Toxoplasma gondii prepared by Abbott Diagnostic Laboratories.

[0005] However, the existing commercial NF155 antibodies are rabbit / mouse-derived mono / polyclonal antibodies, and such antibodies: (1) There are inevitably differences between different batches, and it is impossible to ensure the same sensitivity between each batch; (2) The sequences of the antibodies are not publicly available, and it is impossible to form low-cost and large-scale industrial batch production; (3) Their Fc regions are rabbit / mouse-derived sequences and cannot react with antibodies against human IgG. Summary of the Invention

[0006] In order to overcome the above-mentioned disadvantages of the prior art, the purpose of the present invention is to provide a human-mouse chimeric monoclonal antibody of NF155 protein, its product, a method for detecting NF155 protein and its application, so as to solve the technical problems that the existing mouse / rabbit-derived mono / polyclonal antibodies have batch-to-batch differences, cannot form low-cost and large-scale industrial production, and their Fc regions cannot react with antibodies against human IgG. At the same time, it can provide a new human-mouse chimeric monoclonal antibody of NF155 protein for identifying the expression and localization of NF155 protein and purifying NF155 protein.

[0007] To achieve the above object, the present invention is implemented by the following technical solutions:

[0008] The present invention discloses a human-mouse chimeric monoclonal antibody against NF155 protein, which comprises a murine light chain variable region VL, a murine heavy chain variable region VH, a human antibody light chain constant region, and a human antibody heavy chain constant region, wherein:

[0009] The variable region VL of the murine light chain comprises three light chain complementarity determining regions, and the amino acid sequences of the three light chain complementarity determining regions CDR1, CDR2, and CDR3 are respectively as shown in SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5;

[0010] The variable region VH of the murine heavy chain comprises three heavy chain complementarity determining regions, and the amino acid sequences of the three heavy chain complementarity determining regions CDR1, CDR2, and CDR3 are respectively as shown in SEQ ID NO.8, SEQ ID NO.9, and SEQ ID NO.10;

[0011] The amino acid sequence of the human antibody light chain constant region is as shown in SEQ ID NO.13;

[0012] The amino acid sequence of the human antibody heavy chain constant region is as shown in SEQ ID NO.14.

[0013] Furthermore, the amino acid sequence of the light chain variable region VL of the human-mouse chimeric monoclonal antibody is as shown in SEQ ID NO.1, and the amino acid sequence of the heavy chain variable region VH of the human-mouse chimeric monoclonal antibody is as shown in SEQ ID NO.6.

[0014] Still further, the nucleotide sequence encoding the light chain variable region VL of the human-mouse chimeric monoclonal antibody against NF155 protein is as shown in SEQ ID NO.2; the nucleotide sequence encoding the heavy chain variable region VH of the human-mouse chimeric monoclonal antibody against NF155 protein is as shown in SEQ ID NO.7.

[0015] Still further, the amino acid sequence of the light chain of the human-mouse chimeric monoclonal antibody against NF155 protein is as shown in SEQ ID NO.15; the amino acid sequence of the heavy chain of the human-mouse chimeric monoclonal antibody against NF155 protein is as shown in SEQ ID NO.16.

[0016] Furthermore, the human-mouse chimeric monoclonal antibody comprises a light chain of complementarity determining regions having an amino acid sequence with at least 80% identity to the sequence of the variable region VL of the light chain, and the human-mouse chimeric monoclonal antibody comprises a heavy chain of complementarity determining regions having an amino acid sequence with at least 80% identity to the sequence of the variable region VH of the heavy chain; and the complementary domain of the light chain and the complementary domain of the heavy chain retain the ability to bind to the NF155 protein.

[0017] Still further, the above antibody has 3 complementarity determining regions of the light chain comprising amino acid sequences having at least 90% sequence identity to the sequences comprising QATQDIVKTLN (SEQ ID NO.3), YATELAE (SEQ ID NO.4), and LQFYEFPFT (SEQ ID NO.5), and has 3 complementarity determining regions of the heavy chain comprising amino acid sequences having at least 90% sequence identity to the sequences comprising SSWIH (SEQ ID NO.8), YINPNSDYTKYNQKFKD (SEQ ID NO.9), and DGTYVFDY (SEQ ID NO.10); and wherein the light chain complementary domain and the heavy chain complementary domain retain the ability to bind to NF155.

[0018] Still further, the above antibody has 3 complementarity determining regions of the light chain comprising amino acid sequences having at least 95% sequence identity to the sequences comprising QATQDIVKTLN (SEQ ID NO.3), YATELAE (SEQ ID NO.4), and LQFYEFPFT (SEQ ID NO.5), and has 3 complementarity determining regions of the heavy chain comprising amino acid sequences having at least 95% sequence identity to the sequences comprising SSWIH (SEQ ID NO.8), YINPNSDYTKYNQKFKD (SEQ ID NO.9), and DGTYVFDY (SEQ ID NO.10); and wherein the light chain complementary domain and the heavy chain complementary domain retain the ability to bind to the NF155 protein.

[0019] Furthermore, the above antibodies have a light chain with 3 complementarity-determining regions comprising amino acid sequences having at least 99% sequence identity with the sequences including QATQDIVKTLN (SEQ ID NO.3), YATELAE (SEQ ID NO.4), and LQFYEFPFT (SEQ ID NO.5), and a heavy chain with 3 complementarity-determining regions comprising amino acid sequences having at least 99% sequence identity with the sequences including SSWIH (SEQ ID NO.8), YINPNSDYTKYNQKFKD (SEQ ID NO.9), and DGTYVFDY (SEQ ID NO.10); and wherein the light chain complementary domains and the heavy chain complementary domains retain the NF155 protein-binding ability.

[0020] The present invention also discloses a nucleic acid molecule encoding the human-mouse chimeric monoclonal antibody of the above-mentioned NF155 protein.

[0021] The present invention also discloses a vector comprising a nucleotide sequence encoding the human-mouse chimeric monoclonal antibody of the above-mentioned NF155 protein.

[0022] The present invention also discloses a host cell comprising the above-mentioned vector.

[0023] The present invention discloses the use of the above-mentioned human-mouse chimeric monoclonal antibody, vector or host cell of the NF155 protein in the preparation of a reagent / kit for detecting the NF155 protein.

[0024] The present invention discloses the use of the above-mentioned human-mouse chimeric monoclonal antibody, vector or host cell of the NF155 protein in the preparation of a reagent / kit for human chronic inflammatory demyelinating polyneuropathy and / or prognosis judgment.

[0025] The present invention also discloses a method for detecting the NF155 protein or NF155 polypeptide, which comprises contacting a sample to be tested with the above-mentioned human-mouse chimeric monoclonal antibody of the NF155 protein to detect the NF155 protein or NF155 polypeptide in the sample.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The present invention discloses a human - mouse chimeric monoclonal antibody against NF155 protein, which is an NF155 antibody with high specificity and sensitivity. In the prior art, since murine / rabbit polyclonal antibodies are directly purified from the sera of immunized mice / rabbits, there are certain differences among immunized mice / rabbits at different times, making it difficult to ensure that the antibody types are exactly the same among different batches. Moreover, the packaging specifications of commercial murine / rabbit monoclonal / polyclonal antibodies are small and the prices are expensive, resulting in too high long - term purchase costs. Therefore, the human - mouse chimeric monoclonal antibody against NF155 protein prepared by molecular biological means in the present invention can be expressed and purified on a large scale by using genetic engineering technology, and a large amount of antibodies can be obtained at one time to solve the technical problems of batch - to - batch differences, inability to form low - cost large - scale industrial production, and the Fc region of which cannot react with anti - human IgG antibodies existing in current murine / rabbit monoclonal / polyclonal antibodies. From an application perspective, on the one hand, the antibody disclosed in the present invention can be used as a quality control product / control product / calibration product in a diagnostic kit instead of human serum antibodies; on the other hand, the antibody disclosed in the present invention can visualize and localize the NF155 protein; in addition, the antibody disclosed in the present invention can also be used as a ligand in an affinity system to prepare a chromatography column to achieve the separation and purification of NF155 protein, etc. At the same time, in view of the fact that the signal pattern generated by the binding of the human - mouse chimeric antibody 4c7 against NF155 protein to the NF155 - overexpressing cell smear is similar to the signal pattern generated by positive blood, a large number of fluorescence pictures can be obtained through multiple experiments, enriching the picture library of anti - NF155 antibody - positive, providing a large number of different - style signals for the development of an automatic immunofluorescence image processing system, and improving the accuracy of automatic interpretation. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 is the recombinant NF155 protein purified by prokaryotic expression;

[0029] Figure 2 is the measurement result graph of the binding titer of the sera of 3 mice with NF155 protein;

[0030] Figure 3 is the measurement of the binding titer of the supernatant of hybridoma 4c7 with NF155 protein by ELISA;

[0031] Figure 4 is the detection of the binding of the supernatant of hybridoma 4c7 with NF155 protein by immunofluorescence; among them, A the primary antibody incubated is the serum of mouse No. 3 after immunization with NF155 protein; B the primary antibody incubated is the commercial antibody against NF155 protein; C the primary antibody incubated is the supernatant of hybridoma 4c7;

[0032] Figure 5 is the purification of the human - mouse chimeric antibody 4c7;

[0033] Figure 6Typing and identification of human-mouse chimeric antibody 4c7;

[0034] Figure 7 Verification of the specificity of human-mouse chimeric antibody 4c7;

[0035] Figure 8 Comparison of the signal patterns between human-mouse chimeric antibody 4c7 and serum. Detailed implementation manners

[0036] To enable those skilled in the art to better understand the solution of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts shall fall within the protection scope of the present invention.

[0037] It should be noted that the terms "first", "second", etc. in the description and claims of the present invention and the above-mentioned drawings are used to distinguish similar objects, and do not necessarily need to be used to describe a specific order or sequence. It should be understood that such data can be interchanged under appropriate circumstances so that the embodiments of the present invention described here can be implemented in an order other than those illustrated or described here. In addition, the terms "comprising" and "having" and any variations thereof are intended to cover non-exclusive inclusion. For example, a process, method, system, product or device comprising a series of steps or units does not necessarily have to be limited to those steps or units clearly listed, but may include other steps or units not clearly listed or inherent to these processes, methods, products or devices.

[0038] Unless otherwise defined, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by those of ordinary skill in the art.

[0039] The term "antibody" includes polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized monoclonal antibodies, fully humanized monoclonal antibodies, single-chain antibodies or portions thereof capable of binding to their antigen, such as Fab fragments or fragments generated from a Fab expression library.

[0040] The term "primary antibody" is an antibody that can specifically bind to a specific antigen (non-antibody antigen). It is usually produced by B cells under antigen stimulation, has high specificity, and can recognize and bind to a specific antigenic epitope on the target antigen. The primary antibody can be a monoclonal antibody (mAb) or a polyclonal antibody (pAb). The primary antibody has a wide range of applications in the fields of immunoassay, disease diagnosis, treatment, and scientific research. For example, in an ELISA experiment, the primary antibody can be used as a probe to specifically bind to the target protein, thereby achieving the detection of the target protein.

[0041] The term "secondary antibody" refers to an antibody that can bind to a primary antibody, i.e., an antibody against an antibody. It is usually produced by the immune system of a heterologous animal and is an immunoglobulin against the primary antibody. The main function of the secondary antibody is to detect the presence of the primary antibody and amplify the signal of the primary antibody. In practical applications, a secondary antibody that is species-specifically matched to the target primary antibody is usually selected, and secondary antibodies are widely used in immunoassay techniques such as immunohistochemistry, flow cytometry, and Western blot. For example, in an immunohistochemistry experiment, the secondary antibody can bind to the primary antibody and emit a signal through its conjugated labeling substance (such as a fluorescent dye), thereby achieving the localization and detection of the target antigen.

[0042] Among them, the term "monoclonal antibody (MAb)": refers to a population of antibodies containing only one species composed of the unique light chain gene product and the unique heavy chain gene product. In all molecules of the antibody population, the complementarity-determining regions (CDRs) of the monoclonal antibody are the same and can bind to the antigen-binding site of a specific epitope of the antigen.

[0043] Among them, the term "chimeric antibody" means an antibody in which the sequences of the heavy chain variable region and the light chain variable region (VH and VL) are derived from a non-human animal species and in which the constant region sequences (CH and CL) are derived from humans. The sequences of the variable regions are preferably derived from animal species such as mice, rats, rabbits, etc. that allow for the easy preparation of hybridomas.

[0044] Among them, the term "humanized monoclonal antibody" refers to replacing the CDR regions of a human antibody with the CDR regions from an antibody of a non-human animal species, also known as a CDR-grafted antibody. The CDR sequences of the antibody of the non-human animal species are preferably derived from animal species such as mice, rats, rabbits, etc. that allow for the easy preparation of hybridomas.

[0045] Among them, the term "polypeptide": refers to a protein, a protein fragment, and a fragment or analogue of a polypeptide sequence. Protein fragments and analogues are considered to be types belonging to polypeptides. Further, according to the present invention, examples of polypeptides include the light chain immunoglobulin molecule represented as SEQ ID NO: 1, the heavy chain immunoglobulin molecule represented as SEQ ID NO: 6, and the CDRs represented as SEQ ID NOs: 3, 4, 5, 8, 9, and 10, antibody molecules formed by a combination of a heavy chain immunoglobulin molecule and a light chain immunoglobulin molecule, such as a κ light chain immunoglobulin molecule (and vice versa), and their fragments and analogues.

[0046] Among them, the NF155 (Neuro-oncological ventral antigen 1, NF155) protein: is a protein that exists in glial cells and is mainly expressed in the central nervous system. The NF155 protein described in the present invention refers to a polypeptide, fragment, or analogue that retains the binding ability of the NF155 protein.

[0047] Herein, the term "amino acid": The 20 conventional amino acids and their abbreviations used in the present invention are used according to convention.

[0048] The monoclonal antibodies of the present invention also include antibodies having "equivalent binding properties" to the antibody, and the antibodies with "equivalent binding properties" can bind to the NF155 protein or polypeptide.

[0049] Specifically, it is considered that changes in the amino acid sequence of the antibody having "equivalent binding properties" are encompassed by the present invention, provided that the changes in the amino acid sequence of the antibody having "equivalent binding properties" remain at least 75%, more preferably at least 80%, 90%, 95% and most preferably 99%. This includes certain percentages therebetween, such as 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% sequence identity.

[0050] Herein, the term "gene" or "DNA" or "nucleic acid molecule": includes not only double-stranded DNA, but also the corresponding single-stranded DNA, the sense strand and the antisense strand constituting the double-stranded DNA. It is not particularly limited by its length. Unless otherwise specified, the genes (DNA) in this specification include double-stranded DNA (including human genomic DNA), single-stranded DNA (plus strand) (including cDNA), single-stranded DNA having a sequence complementary to the plus strand (complementary strand), and fragments thereof. According to the present invention, examples of nucleic acid molecules include the nucleotide sequence (SEQ ID NO.2) encoding the light chain variable region VL of the human-mouse chimeric monoclonal antibody against the above-mentioned NF155 protein; the nucleotide sequence (SEQ ID NO.7) encoding the heavy chain variable region VH of the above-mentioned NF155 monoclonal antibody; the nucleotide sequence encoding the human Ig Kappa light chain constant region as shown in SEQ ID NO.11, and its corresponding amino acid sequence as shown in SEQ ID NO.13; the nucleotide sequence encoding the human IgG4 heavy chain constant region as shown in SEQ ID NO.12, and its corresponding amino acid sequence as shown in SEQ ID NO.14. The nucleic acid molecules of the present invention can be synthesized, for example, by standard chemical synthesis methods and / or recombinant methods, or produced semi-synthetically, for example, by a combination of chemical synthesis and recombinant methods.

[0051] The present invention also relates to variants of the above nucleic acid molecules. The nucleic acid molecule variants can be naturally occurring variants, such as naturally occurring allelic variants, or they can be non-naturally occurring variants. These non-naturally occurring variants of nucleic acid molecules can be prepared by mutagenesis techniques, including those applied to nucleic acid molecules, cells or organisms.

[0052] The vector of the present invention includes the above nucleic acid molecule. Many suitable vectors are known to those skilled in the field of molecular biology, and the selection depends on the desired function. There is no particular limitation on the vector of the present invention, and it can be a vector capable of replicating and / or expressing polynucleotides in eukaryotic or prokaryotic cells including mammalian cells (such as human, monkey, rabbit, rat, hamster or mouse cells), plant cells, yeast cells, insect cells and bacterial cells (such as Escherichia coli (E. coli)). Non-limiting examples of vectors include pBAD, pQE-12, pGEX, pBluescript, pET-series expression vectors, pCAI-n, pPOW3.0, pJOE, pBBR1-MCS series, pJB861, pBSMuL, pBC2, pUCPKS, pTACT1, pTRE, pCAL-n-EK, pESP-1, pOP13CAT, pREP, pCEP4, pMC1neo, pXT1, pSG5, EBO-pSV2neo, pBPV-1, pFUSE, pRSVgpt, pRSVneo, pIZD35, pRc / CMV, pcDNA1, pcDNA3.1, pSPORT1, pGEMHE, pLXIN, pSIR, pIRES-EGFP, pEAK-10, pTriEx-Hygro, pCINeo, pAO815, pPIC9K and pPIC3.5K.

[0053] The host cell of the present invention includes the above nucleic acid molecule or vector.

[0054] In an embodiment of the present invention, the host cells for introducing the vector include prokaryotic cells and eukaryotic cells, and the above cells include but are not limited to bacterial cells such as Escherichia coli; yeast cells; fungal cells such as Pichia pastoris, Saccharomyces cerevisiae; insect cells such as Sf9 cells, Sf21 cells, Tn-368 cells, High Five cells; animal cells such as CHO cells, COS, NSO, 293T, HT-1080, BHK (baby hamster kidney cells), HEK (human embryonic kidney cells), Expi293F, PERC.6 (human retinal cells); plant cells. Any cell known to those skilled in the art that can be used as a mammalian host cell can be used in the present field.

[0055] The present invention provides a method for detecting NF155 protein or polypeptide, the method comprising contacting a sample with the above human-mouse chimeric monoclonal antibody to thereby detect the NF155 protein or polypeptide in the sample.

[0056] In the present invention, the method for detecting or determining the amount of a protein or polypeptide can be any known immunological detection technique, including immunagglutination assay, immunoprecipitation assay, enzyme-linked immunosorbent assay, fluorescence immunoassay, immunoelectron microscopy, immunoblotting assay, immunomagnetic bead assay. An immunological detection technique is a method for detecting or determining the amount of an antibody or antigen using a labeled antigen or antibody. Biological samples that can be tested by the above immunological detection or determination include, but are not limited to, plasma, whole blood, dried whole blood, serum, tissue, cells, or extracts of tissue and cells.

[0057] In an embodiment of the present invention, the enzyme immunoassay includes, but is not limited to, indirect immunofluorescence, direct ELISA, dot blot (linear blot). By way of example, a method includes adsorbing a human NF155 polypeptide (antigen) onto a solid-phase support NC membrane, blocking the solid phase with a blocking protein (e.g., skim milk powder, bovine serum albumin, etc.), contacting and incubating an anti-human NF155 human-mouse chimeric monoclonal antibody (test antibody) with the solid phase, removing the unreacted antibody, and adding a labeled second antibody that specifically reacts with the test antibody to the solid phase to determine the amount of the label on the solid phase.

[0058] The products of the present invention include the above-mentioned human-mouse chimeric monoclonal antibody, nucleic acid molecule, vector, or host cell.

[0059] As an alternative embodiment, the product is a kit; the kit includes the antibody prepared by the present invention. As another alternative embodiment, the kit of the present invention includes a diagnostic composition, and the diagnostic composition includes at least one detectable labeling substance. As the labeling substance, a radioactive isotope, an enzyme, a fluorescent substance, a luminescent substance, etc. can be used. The enzyme can be β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase, etc.; the fluorescent substance can be fluorescamine, fluorescein isothiocyanate, etc.; the luminescent substance can be luminol, luminol derivatives, luciferin, lucigenin, etc.

[0060] As an alternative embodiment, the kit includes a protein immunoassay kit; the protein immunoassay kit includes the above-mentioned monoclonal antibody or its fragment.

[0061] The present invention provides the application of the above-mentioned human-mouse chimeric monoclonal antibody, nucleic acid molecule, vector, host cell, or product in detecting NF155 protein or polypeptide products.

[0062] Wherein, the term "large-scale extraction of recombinant plasmid" refers to a method of inserting a target gene into the multiple cloning site of a plasmid to construct a new recombinant plasmid and then extracting these plasmids in large quantities through a series of steps.

[0063] The present invention will be further described in detail below with reference to the accompanying drawings:

[0064] Example 1 Preparation of Murine Monoclonal Antibody

[0065] 1. Preparation of NF155 Recombinant Protein

[0066] The human NF155 gene sequence with the GenBank accession number NM_001160331.2 was retrieved from the GenBank sequence database. This gene sequence was synthesized by GenScript Corporation onto the pET30a vector at the insertion sites of EcoR I and Xba I to obtain the NF155 recombinant vector. The constructed and correctly sequenced recombinant plasmid was transformed into Escherichia coli Arctic Express (Agilent) expression competent cells. A single colony was picked and inoculated into LB medium, and the bacteria were shaken at 37°C until the OD value reached 0.5 - 1. IPTG was added at a final concentration of 0.5 mM for induction, and the expression was carried out overnight at 16°C. The bacterial cells were collected, sonicated, and the supernatant was collected after centrifugation. The NF155 protein expressed by pET-30a was purified using Ni-NTA (QIAGEN), and the purified recombinant protein was designated as NF155. The purification results are as Figure 1 shown.

[0067] 2. Immunization of Mice with Antigen

[0068] Three Balb / c mice were immunized with the above-prepared NF155 recombinant protein as the antigen. The immunization was carried out three times, and the antigen dosage used for immunizing the mice was 40 μg / mouse. At the primary immunization, the antigen was mixed with Freund's adjuvant in equal volumes and thoroughly ground into a water-in-oil emulsion, and 100 μL (40 μg antigen) of the mixed antigen was injected intraperitoneally. The second immunization was carried out two weeks after the primary immunization. 40 μg of antigen was mixed with incomplete Freund's adjuvant at a ratio of 1:1, and 100 μL (40 μg antigen) of the mixed antigen was injected into the right posterior calf muscle. The third immunization was carried out two weeks after the second immunization, and the dosage, method, and route of antigen injection were the same as those of the second immunization. After a 2-week interval, the mice were boosted by intraperitoneal injection of antigen. Three days later, mouse tail blood was collected and its titer was detected using ELISA.

[0069] The specific detection process is as follows: Coat the purified recombinant NF155 protein overnight at 4°C with a carbonate buffer solution with a pH value of 9.6, 100 ng / well; the next day, wash 3 times with PBST, 3 minutes each time, and pat dry; seal the wells completely with 2% BSA and incubate at 37°C for 1 hour; wash 3 times with PBST, 3 minutes each time, and pat dry; Dilute the sera of 3 mice in a serial dilution with PBS at dilutions of 1:50, 1:150, 1:450, 1:1350, 1:4050, 1:12150, 1:36450, 1:109350. Add 100 μL to each well and incubate at 37°C for 1 hour. Wash 3 times with PBST, 3 minutes each time, and pat dry; Dilute goat anti-mouse IgG-HRP 1:5000 (Catalog No. 115-035-003, Jackson), incubate at 37°C for 30 minutes. Wash 3 times with PBST, after patting dry, develop color with TMB for 10 minutes, terminate with 2M H2SO4, and measure the absorbance at 450 nm. The results are as Figure 2 shown. According to the detection results, comprehensively considering the titer and specificity data of the serum antibodies, select mouse No. 3 for subsequent experiments.

[0070] 3. Cell fusion and hybridoma culture

[0071] After intraperitoneal injection of antigen for booster immunization, sacrifice the animal 3 days later and collect splenocytes for cell fusion. Prepare a 37°C water bath in the laminar flow hood. Add 5 - 6×10 7 mouse myeloma cells SP2 / 0 cells in the logarithmic growth phase with good growth status and splenocytes to a 50 mL centrifuge tube at a ratio of 1:10, and mix well. Centrifuge at 500 g for 10 minutes, aspirate the supernatant, gently flick the bottom of the centrifuge tube to slightly loosen the cell pellet; slowly drip 1 mL of pre-warmed 45% PEG1450 solution at 37°C within 90 s; and continuously shake the centrifuge tube gently; the whole process is carried out in a 37°C water bath. Then gradually add DMEM medium to the cell mixture, drop by drop 1 mL in the first minute, 2 mL in the second minute, 3 mL in the third minute, 4 mL in the fourth minute, 5 mL in the fifth minute, and shake while adding in a 37°C water bath. Then incubate at 37°C for 15 minutes, centrifuge at 500 g for 5 minutes, and discard the supernatant. Add 5 mL of DMEM medium containing HAT (Thermo, Catalog No.: 21060017), gently suspend the precipitated cells, and finally supplement the DMED medium containing HAT to about 100 mL. Aliquot into a 96-well cell culture plate pre-coated with macrophages, 100 μL / well, and then place the culture plate in a 37°C, 5% CO2 incubator for culture.

[0072] 4. Screening of positive hybridomas

[0073] Positive clones were selected by ELISA. Observe the growth of hybridoma cells. Seven days later, when the cell culture supernatant turns yellow, aspirate an appropriate amount of cell supernatant for ELISA antibody detection; according to the ELISA results, select the clones with high OD values, more than twice that of the negative control, seed them in 96-well plates for the first subcloning screening; after seven to ten days, perform ELISA antibody detection again, select the clones with high OD values, seed them in 96-well plates for the second subcloning screening to make about 1 cell per well; after culturing for seven to ten days, perform ELISA antibody detection again, select the clones with high OD values, seed them in 96-well plates for the third subcloning screening to make about 1 cell per well. After three screenings, one clone with good signal was initially selected and named hybridoma 4c7. The culture supernatant of hybridoma 4c7 and the original solution diluted at 1:3, 1:9, 1:27, 1:81, 1:243, 1:729, 1:2187 were used for ELISA titer detection. The results showed that the antibody in the supernatant of the selected hybridoma 4c7 could still be detected after being diluted 2187 times, and the antibody in the supernatant secreted by this hybridoma had good binding ability with NF155 protein( Figure 3 ). The antibody in the supernatant secreted by hybridoma 4c7 was named murine monoclonal antibody 4c7.

[0074] The positive clones screened by ELISA were verified by indirect immunofluorescence assay. Preparation of cell slides overexpressing NF155 protein: By molecular biology methods, the NF155 gene was ligated to pCDNA3.1 to obtain a recombinant plasmid. The recombinant plasmid was transfected into 293T cells with a cell density of 30% - 40% (a 6 cm * 6 cm cell slide had been placed in a 10 cm cell culture dish in advance) using PEI transfection reagent (the transfection reagent was purchased from thermo company and transfected according to the instruction manual). 48 h after transfection, add acetone and fix at 4°C for 5 min, wash twice with PBS, dry, and cut the 6 cm * 6 cm cell slide into pieces of 0.25 cm * 0.25 cm size for standby. Dilute the supernatant of hybridoma 4c7, the commercial antibody of NF155 (abcam), and the blood of mouse No. 3 after immunization 200 times as the primary antibody and incubate them on the cell slides overexpressing NF155 protein, incubate at room temperature for 1 h, wash 3 times with PBST, 5 min each time; incubate with Alexa Fluor 594-labeled goat anti-mouse secondary antibody (Jackson) for 40 min, wash 3 times with PBST, 5 min each time, and take pictures with a fluorescence microscope. The results are as Figure 4 shown. The supernatant of hybridoma 4c7 produced signals similar to those of the commercial antibody, further indicating that the murine monoclonal antibody in the supernatant of hybridoma 4c7 can not only recognize the prokaryotic NF155 protein coated on the ELISA plate, but also recognize the NF155 protein expressed in eukaryotic cells.

[0075] 5. Identification of the Subtype of Murine Monoclonal Antibody 4c7

[0076] The identification of the subtype of murine monoclonal antibody was performed by indirect ELISA. The specific procedure of indirect ELISA was as follows: NF155 antigen (100 ng / well) was coated, and after blocking with 2% BSA, it was incubated with the supernatant of hybridoma 4c7 obtained in Example 1 at 37 °C for 1 hour, washed 3 times with PBST, and then incubated with different commercially available HRP-labeled rabbit anti-mouse (total IgG, IgG1, IgG2a, IgG2b, IgG3, IgM, Ig kappa chain, Ig lambda chain) secondary antibodies diluted 1:5000 at 37 °C for 30 min. After washing 3 times with PBST, TMB was used for color development for 5 min. The results of indirect ELISA all showed that the heavy chain subtype of the murine monoclonal antibody in the supernatant of hybridoma 4c7 was IgG1, and the light chain subtype was Kappa (Table 1).

[0077] Table 1 Identification of the Subtype of Murine Monoclonal Antibody 4c7

[0078]

[0079] Example 2 Identification of the Sequence of Anti-NF155 Murine Monoclonal Antibody 4c7

[0080] After the hybridoma 4c7 for antibody production was cultured to a certain scale (cell number > 3×10 6 cells), the cells were lysed with Trizol, and total RNA of the hybridoma cell lysate was extracted using the Quick-RNA MicroPrep Kit. RNA was separated by agarose gel electrophoresis, and then 5' RACE was performed using the Clontech SMARTer RACE 5' / 3' kit to obtain cDNA. Using this as a template, PCR amplification was carried out with Max DNA Polymerase, the cDNA of the antibody V region was extracted, and submitted to a sequencing company for sequencing. At the same time, the CDR regions of the amino acid sequence of murine monoclonal antibody 4c7 were marked using the Kabat method.

[0081] Table 2 Sequence of Murine Monoclonal Antibody 4c7

[0082]

[0083]

[0084]

[0085]

[0086] Example 3 Preparation and Subtype Identification of Anti-NF155 Human-Mouse Chimeric Antibody 4c7

[0087] Since the anti-NF155 antibody present in the samples of CIDP patients mainly shows the IgG4 subtype, in order to prepare the quality control product / control product / calibration product in the kit for detecting anti-NF155 autoantibody, in this example, when constructing the recombinant vector, the variable heavy chain VH of the murine monoclonal antibody 4c7 was linked to the human IgG4 heavy chain constant region sequence for recombinant expression.

[0088] Referring to the sequences in the Kabat database, by molecular biology methods, using the Seamless Cloning and Assembly Kit of TransGen Biotech, the nucleotide sequence of the variable light chain VL of the murine monoclonal antibody 4c7 encoding the NF155 protein (shown as SEQ ID NO.2) was ligated to the human kappa light chain constant region sequence SEQ ID NO.11 into the vector pcDNA3.1, labeled as pcDNA3.1-NF155-VL. The nucleotide sequence of the variable heavy chain VH of the murine monoclonal antibody 4c7 encoding the NF155 protein SEQ ID NO.7 was ligated to the human IgG4 heavy chain constant region sequence SEQ ID NO.12 into the vector pcDNA3.1, labeled as pcDNA3.1-NF155-VH. The ligated recombinant plasmids were sent to Sangon Biotech for sequencing, and the sequenced recombinant plasmids were amplified in large scale for standby. Using PEI according to the Thermo instruction manual, the human-mouse chimeric antibody recombinant plasmids pcDNA3.1-NF155-VL and pcDNA3.1-NF155-VH were co-transfected into Expi293F cells (Thermo) for transient expression. After 3 days of transfection, the supernatant was collected and purified using protein A resin. The protein concentration was measured by BCA method to be 0.1 mg / mL (Solarbio). The obtained recombinant antibody was named NF155 human-mouse chimeric antibody 4c7 ( Figure 5 ). To further determine that the obtained recombinant human-mouse chimeric antibody is of IgG4 type, indirect immunofluorescence was used to identify the subtype of NF155 human-mouse chimeric antibody 4c7: The human-mouse chimeric antibody 4c7 was incubated on the cell smear overexpressing NF155 and incubated at room temperature for 1 hour, and then incubated with different kinds of FITC-labeled rabbit anti-human secondary antibodies (total IgG, IgG1, IgG2, IgG3, IgG4) diluted 1:200 at room temperature for 30 min. After washing 3 times with PBST, pictures were taken under a fluorescence microscope. The results of indirect immunofluorescence showed that the heavy chain subtype of the recombinant human-mouse chimeric antibody 4c7 was IgG4 ( Figure 6 ).

[0089] Example 4 Application of Anti-NF155 Human-Mouse Chimeric Antibody

[0090] 1. Verification of the specificity of anti-NF155 human-mouse chimeric antibody by indirect immunofluorescence

[0091] The NF186 protein and the NF155 protein are different types of neurofascin proteins, and the amino acid sequence similarity between the two proteins is 89.72%. Therefore, in this example, it was investigated whether the NF155 human-mouse chimeric antibody prepared by the present invention can be used to distinguish between the NF155 protein and the NF186 protein.

[0092] Refer to the process of preparing cell smears overexpressing NF155 in Reference Example 1 to prepare cell smears overexpressing NF186 (sequence number: NM_001005388.3). Take the purified human-mouse chimeric antibody 4c7 antibody of Example 3 and the mouse serum No. 3 of Example 1, and refer to the steps of the indirect immunofluorescence method in Example 1 to incubate the human-mouse chimeric antibody 4c7 antibody (incubate the FITC-labeled mouse anti-human IgG4 secondary antibody, sigma) and the mouse serum No. 3 (incubate the Alexa 594-labeled goat anti-mouse secondary antibody, Jackson) at a dilution ratio of 1:200 as the primary antibody and incubate them on cell smears overexpressing pCDNA3.1, overexpressing NF155, and overexpressing NF186 respectively to verify the specificity of the human-mouse chimeric antibody 4c7 antibody. As Figure 7 can be seen, although there is a signal when the mouse serum No. 3 binds to the cell smear overexpressing NF186, the human-mouse chimeric antibody 4c7 antibody does not react with the cell smears overexpressing pCDNA3.1 and overexpressing NF186. Thus, it can be known that the human-mouse chimeric antibody 4c7 antibody has good specificity.

[0093] 2. Compare the signals generated by the anti-NF155 human-mouse chimeric antibody and the positive serum by indirect immunofluorescence

[0094] Take the cell smears overexpressing NF155 prepared in Example 1, and incubate the sera of 3 patients positive for anti-NF155 antibody and the anti-NF155 human-mouse chimeric antibody 4c7 obtained in Example 3 of the present application respectively. The secondary antibody is incubated with the FITC-labeled mouse anti-human IgG4 secondary antibody of Sigma Corporation, and the staining results are as Figure 8 shown. As Figure 8It can be seen that the anti-NF155 human-mouse chimeric antibody 4c7 can not only recognize the overexpressed NF155 protein and bind to the anti-human IgG4 antibody, but also the signal pattern generated is similar to that of the anti-NF155 antibody positive serum, both being an irregular flaky pattern. Therefore, the modified human-mouse chimeric antibody, on the one hand, can replace the human serum antibody as a quality control product / control / calibrator in the diagnostic kit; on the other hand, the antibody disclosed in the present invention can visualize and localize the NF155 protein; in addition, the antibody disclosed in the present invention can also be used as a ligand in the affinity system to prepare a chromatography column to achieve the separation and purification of the NF155 protein, etc. At the same time, in view of the fact that the signal pattern generated by the binding of the anti-NF155 human-mouse chimeric antibody 4c7 to the overexpressed NF155 cell smear is similar to the signal pattern generated by the positive blood, a large number of fluorescence images can be obtained through multiple experiments to enrich the picture library of anti-NF155 antibody positivity, providing a large number of different styles of signals for the development of the immunofluorescence image automatic processing system and improving the accuracy of automatic interpretation.

[0095] Therefore, the present invention develops new human-mouse / human-rabbit chimeric antibodies against NF155 protein, which can, to a certain extent, replace the function of serum and contribute to the development of a neuroimmune autoantibody detection kit with controllable quality; at the same time, the mouse monoclonal antibody or human-mouse / human-rabbit chimeric antibody against NF155 protein can also be used to visualize and localize the NF155 protein; the mouse monoclonal antibody or human-mouse / human-rabbit chimeric antibody against NF155 protein can also be used as a ligand in the affinity system to prepare a chromatography column to achieve applications such as the separation and purification of the NF155 protein.

[0096] The above content is only to illustrate the technical idea of the present invention and cannot be used to limit the protection scope of the present invention. Any modification made on the basis of the technical solution according to the technical idea proposed by the present invention falls within the protection scope of the claims of the present invention.

Claims

1. A human-mouse chimeric monoclonal antibody against NF155 protein, characterized in that it comprises a murine light chain variable region VL, a murine heavy chain variable region VH, a human antibody light chain constant region, and a human antibody heavy chain constant region, wherein: The variable region VL of the murine light chain comprises three light chain complementarity-determining regions, and the amino acid sequences of the three light chain complementarity-determining regions CDR1, CDR2, and CDR3 are shown in SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5 respectively; The variable region VH of the murine heavy chain comprises three heavy chain complementarity-determining regions, and the amino acid sequences of the three heavy chain complementarity-determining regions CDR1, CDR2, and CDR3 are shown in SEQ ID NO.8, SEQ ID NO.9, and SEQ ID NO.10 respectively; The amino acid sequence of the human antibody light chain constant region is as described in SEQ ID NO.13; The amino acid sequence of the human antibody heavy chain constant region is as described in SEQ ID NO.

14.

2. The human-mouse chimeric monoclonal antibody of the NF155 protein according to claim 1, characterized in that, The amino acid sequence of the light chain variable region VL of the human-mouse chimeric monoclonal antibody against NF155 protein is shown in SEQ ID NO.1, and the nucleotide sequence encoding the light chain variable region VL of the human-mouse chimeric monoclonal antibody against NF155 protein is shown in SEQ ID NO.2; the amino acid sequence of the heavy chain variable region VH of the human-mouse chimeric monoclonal antibody against NF155 protein is shown in SEQ ID NO.6, and the nucleotide sequence encoding the heavy chain variable region VH of the human-mouse chimeric monoclonal antibody against NF155 protein is shown in SEQ ID NO.

7.

3. The human-mouse chimeric monoclonal antibody of the NF155 protein according to claim 1, characterized in that, The amino acid sequence of the light chain of the human-mouse chimeric monoclonal antibody against NF155 protein is shown in SEQ ID NO.15; the amino acid sequence of the heavy chain of the human-mouse chimeric monoclonal antibody against NF155 protein is shown in SEQ ID NO.

16.

4. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the human-mouse chimeric monoclonal antibody against NF155 protein according to any one of claims 1 to 3.

5. A carrier, characterized in that, The vector comprises a nucleotide sequence encoding the human-mouse chimeric monoclonal antibody against NF155 protein according to any one of claims 1 to 3.

6. A host cell that is not a plant cell, characterized in that, The host cell of non-plant cells comprises the vector according to claim 5.

7. Use of the human-mouse chimeric monoclonal antibody against NF155 protein according to any one of claims 1 to 3, the vector according to claim 5, or the host cell of non-plant cells according to claim 6 in the preparation of a reagent / kit for detecting NF155 protein.

8. Use of the human-mouse chimeric monoclonal antibody against NF155 protein according to any one of claims 1 to 3, the vector according to claim 5, or the host cell of non-plant cells according to claim 6 in the preparation of a reagent / kit for the diagnosis and / or prognosis judgment of human chronic inflammatory demyelinating polyneuropathy.

9. A method for detecting NF155 protein or NF155 polypeptide for non-disease diagnosis purposes, characterized in that, Contact the sample to be tested with the human-mouse chimeric monoclonal antibody according to any one of claims 1 to 3 to detect NF155 protein or NF155 polypeptide in the sample.

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