Mouse anti-human elli2 monoclonal antibody, its preparation method and application

Abstract

This invention belongs to the field of biotechnology, providing a mouse anti-human ELL2 monoclonal antibody, its preparation method, and its applications. This invention successfully obtained a 2E1 anti-human ELL2 monoclonal antibody and established a hybridoma cell line capable of stably producing this antibody. The binding affinity (KD) of the obtained 2E1 monoclonal antibody to human ELL2 protein reached the picomolar level (approximately 10^-3 kDa). ‑12 The antibody (M) exhibits extremely strong binding ability. Cross-reactivity tests confirmed that it binds only to human ELL2, and shows no binding signal with highly homologous human ELL1, ELL3, and mouse ELL2, thus completely solving the specificity problem of existing technologies.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for preparing a highly specific and high-affinity mouse monoclonal antibody against human transcription elongation factor ELL2 (Eleven-Nineteen Lysine-Rich Leukemia 2), as well as the resulting hybridoma cell line and the application of the antibody. Background Technology

[0002] ELL2, a transcription elongation factor, is a core regulatory protein in the RNA polymerase II-mediated gene transcription elongation process. Its key function is to bind to RNA polymerase II, significantly enhancing the rate and duration of transcription elongation, effectively preventing premature transcription termination, and thus ensuring efficient and complete messenger RNA (mRNA) synthesis. This function is crucial in physiological processes requiring rapid and large-scale synthesis of specific proteins, especially in the terminal differentiation stage of adaptive immune responses, where ELL2 is a master regulator of plasma cell differentiation and function. Plasma cells, as the body's "antibody factories," have the ability to efficiently synthesize and secrete immunoglobulins (Ig), which is fundamentally regulated by ELL2. The absence of ELL2 directly leads to plasma cell developmental defects and severely insufficient antibody production, thereby impairing the body's immune defense function.

[0003] In recent years, with the application of high-throughput omics technologies such as single-cell RNA sequencing, the clinicopathological significance of ELL2 has been further revealed. In sepsis, a fatal systemic inflammatory response syndrome triggered by infection, the patient's immune system undergoes a dynamic transition from excessive inflammation to immune paralysis, the latter being a major cause of later death and secondary infections. Numerous cutting-edge studies have indicated that ELL2 mRNA expression levels are significantly and persistently downregulated in peripheral blood mononuclear cells (PBMCs) and B lymphocyte populations of sepsis patients, and this downregulation shows a strong negative correlation with disease severity, the formation of immunosuppression, and poor patient prognosis (such as high mortality). Therefore, ELL2 has transformed from a basic transcription factor into a highly promising novel prognostic biomarker and potential immunotherapeutic target, providing a new perspective for precise sepsis subtyping, risk assessment, and therapeutic intervention.

[0004] Despite the growing importance of ELL2, a significant technological gap exists in the supporting research tools, particularly high-quality specific antibodies, greatly hindering the translation from basic research to clinical application. The shortcomings of existing technologies are mainly reflected in the following aspects:

[0005] (1) Severe lack of specificity: The ELL protein family includes multiple members such as ELL1, ELL2, and ELL3, which share high homology in amino acid sequences with each other and with homologous proteins from different species (such as human and mouse ELL2). Currently, most commercially available antibodies are rabbit polyclonal antibodies or monoclonal antibodies that have not been rigorously validated. These antibodies are often prepared using recombinant proteins expressed in prokaryotic systems as immunogens. Antibodies produced by this method often cannot accurately distinguish ELL2 from other family members, leading to severe cross-reactions in applications such as Western blotting and immunohistochemistry, resulting in high background noise and significantly reducing the reliability of experimental results, failing to truly reflect the expression level and localization of ELL2 protein.

[0006] (2) Limited application scenarios and inability to identify native conformation proteins: As a nuclear protein, ELL2's function depends on its precise localization within the cell nucleus. Therefore, experimental techniques capable of detecting ELL2 protein in situ, such as immunohistochemistry (IHC) and immunofluorescence (IF), are crucial for understanding its pathophysiological functions at the tissue and cellular levels. However, existing antibodies perform poorly, or even fail to function at all, in these applications requiring the identification of native conformation proteins. This limitation means that although we know that ELL2 mRNA levels are decreased in the immune cells of sepsis patients, we cannot directly observe the corresponding changes in protein levels within the cell nucleus, greatly restricting a deeper understanding of its mechanism of action.

[0007] (3) Translational Medicine Bottleneck: The cornerstone of transforming ELL2 from an investigational biomarker into a clinical diagnostic tool is the ability to perform accurate and reliable protein-level quantification and localization analysis. For example, the development of immunohistochemistry-based companion diagnostic reagents to screen patient populations that may benefit from ELL2-targeted therapy relies entirely on a clinically validated, high-performance monoclonal antibody. The lack of existing tools constitutes a significant obstacle to the clinical translation of ELL2.

[0008] In summary, there is an urgent need in the field for a monoclonal antibody that can specifically recognize human ELL2, effectively distinguish its family homologs from mouse orthologs, and perform exceptionally well in key applications requiring recognition of native protein conformations (especially IHC / IF). Summary of the Invention

[0009] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a hybridoma cell line capable of stably secreting high-affinity, high-specificity anti-human ELL2 monoclonal antibodies, along with its preparation method and applications. This method aims to generate a novel monoclonal antibody that specifically recognizes the native conformation of the human ELL2 protein, effectively avoiding cross-reactivity with other members of the ELL family and murine homologous proteins. It is applicable to various immunological detection platforms, including Western blotting, flow cytometry, immunocytochemistry, and immunohistochemistry, providing a key tool for basic research and clinical translational applications related to ELL2.

[0010] The technical solution provided by this invention is as follows:

[0011] A mouse anti-human ELL2 monoclonal antibody, the antibody comprising a heavy chain variable region and a light chain variable region;

[0012] The heavy chain variable region includes CDR-H1, CDR-H2 and CDR-H3, and the light chain variable region includes CDR-L1, CDR-L2 and CDR-L3;

[0013] The amino acid sequence of the CDR-H1 is shown in SEQ ID NO.5;

[0014] The amino acid sequence of the CDR-H2 is shown in SEQ ID NO.6;

[0015] The amino acid sequence of the CDR-H3 is shown in SEQ ID NO.7;

[0016] The amino acid sequence of CDR-L1 is shown in SEQ ID NO.8;

[0017] The amino acid sequence of the CDR-L2 is shown in SEQ ID NO.9;

[0018] The amino acid sequence of the CDR-L3 is shown in SEQ ID NO.10.

[0019] Furthermore, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.2; the amino acid sequence of the light chain variable region is shown in SEQ ID NO.4.

[0020] Furthermore, the heavy chain amino acid sequence is shown in SEQ ID NO.11; the light chain amino acid sequence is shown in SEQ ID NO.12.

[0021] The present invention also provides a nucleic acid molecule that encodes the mouse anti-human ELL2 monoclonal antibody as described above.

[0022] Furthermore, the sequences of the nucleic acid molecule include SEQ ID NO.1 and SEQ ID NO.3;

[0023] The sequence SEQ ID NO.1 encodes the heavy chain variable region of the antibody described;

[0024] The sequence SEQ ID NO.3 encodes the light chain variable region of the antibody described.

[0025] The present invention also provides an expression vector containing the above-described nucleic acid molecules.

[0026] The present invention also provides a host cell containing the above-described expression vector.

[0027] The present invention also provides a method for preparing the above-mentioned mouse anti-human ELL2 monoclonal antibody, characterized by comprising the following steps:

[0028] An expression vector containing a nucleic acid molecule expressing the mouse anti-human ELL2 monoclonal antibody was prepared.

[0029] The obtained expression vector was transfected into eukaryotic host cells and cultured.

[0030] The mouse anti-human ELL2 monoclonal antibody was isolated and purified.

[0031] The present invention also provides the application of the above-mentioned mouse anti-human ELL2 monoclonal antibody in the preparation of sepsis drugs.

[0032] The present invention also provides the application of the above-mentioned mouse anti-human ELL2 monoclonal antibody in the preparation of ELL2 detection reagents.

[0033] The core of the technical solution adopted in this invention lies in an innovative, multi-stage immunization strategy and a rigorous screening process.

[0034] (1) Rational antigen design and preparation: Through bioinformatics analysis, the amino acid sequences of human ELL1, ELL2, ELL3 and mouse ELL2 were compared to screen for short peptide sequences that are highly specific to human ELL2 protein and have good immunogenicity. The synthesized specific peptides were conjugated with the carrier protein keyhole hemocyanin (KLH) as the initial immunogen.

[0035] (2) Multimodal immunization strategy: A multi-site repeated immunization (RIMMS) protocol was adopted. First, multiple immunizations were performed using the aforementioned peptide-KLH conjugate to initiate and expand B cells targeting the human ELL2-specific epitope. Subsequently, booster immunizations were performed using full-length recombinant human ELL2 protein expressed in eukaryotic cells to screen B cell clones capable of recognizing the conformational epitope. Finally, before cell fusion, a shock immunization was performed using live cells stably overexpressing human ELL2 to enrich B cells capable of recognizing ELL2 protein in its native state on the cell surface and intracellularly.

[0036] (3) Hybridoma preparation and screening: Spleen cells from immunized mice were electrofused with SP2 / 0 myeloma cells, and hybridoma cells were screened in HAT selective medium. Through multiple rounds of functional tests such as ELISA, flow cytometry and immunocytochemistry, the secreted antibodies were strictly screened and subcloned, and finally the hybridoma cell line 2E1, which can stably secrete the target monoclonal antibody, was obtained.

[0037] Beneficial effects

[0038] (1) Extremely high affinity and specificity: This invention successfully obtained the 2E1 anti-human ELL2 monoclonal antibody and established a hybridoma cell line capable of stably producing the antibody. The binding affinity constant (KD) of the obtained 2E1 monoclonal antibody to human ELL2 protein reached the picomolar level (approximately 10). -12 The antibody (M) exhibits extremely strong binding ability. Cross-reactivity tests confirmed that it binds only to human ELL2, and shows no binding signal with highly homologous human ELL1, ELL3, and mouse ELL2, thus completely solving the specificity problem of existing technologies.

[0039] (2) Wide platform applicability: The 2E1 antibody has been fully validated and has shown excellent performance in a variety of mainstream immunoassay platforms, including enzyme-linked immunosorbent assay (ELISA), Western blotting, flow cytometry (FCM), immunocytochemistry (ICC), and immunohistochemistry (IHC).

[0040] (3) Breakthrough native conformation recognition capability: Especially in immunocytochemistry and immunohistochemistry experiments, the 2E1 antibody can clearly and specifically recognize endogenous ELL2 in paraffin sections of human tonsil tissue and cultured cells, and accurately locate it in the cell nucleus with a clean background. This performance is generally unattainable by existing commercial antibodies, solving a key technical bottleneck in the field.

[0041] (4) Great scientific research and clinical application value: The 2E1 antibody provided by this invention is a powerful tool for studying the function and regulatory mechanism of ELL2 protein and its role in diseases such as sepsis, and lays a solid foundation for developing ELL2-based clinical diagnostic kits (such as IHC companion diagnostics) and targeted therapy strategies. Attached Figure Description

[0042] Figure 1 This is a flowchart illustrating the technical route for preparing anti-human ELL2 monoclonal antibodies according to the present invention.

[0043] Figure 2 The image shows the results of the successful construction of the 293T-hELL2 cell line stably overexpressing human ELL2, as verified by flow cytometry.

[0044] Figure 3 The figure shows the results of detecting the titer of anti-human ELL2 antibody in the serum of immunized mice using the indirect ELISA method.

[0045] Figure 4 The graph shows the affinity assay for the 2E1 monoclonal antibody.

[0046] Figure 5 The binding assay results of the 2E1 monoclonal antibody were used to verify its specificity, showing that it does not cross-react with ELL1, ELL3 and mouse ELL2.

[0047] Figure 6 The dose-response curve of 2E1 monoclonal antibody in enzyme-linked immunosorbent assay (ELISA);

[0048] Figure 7 The results of detecting endogenous ELL2 protein in cell lysates using the 2E1 monoclonal antibody in Western blotting.

[0049] Figure 8 The results of intracellular ELL2 expression were detected by flow cytometry (FCM) using the 2E1 monoclonal antibody.

[0050] Figure 9 The results of staining 293T-hELL2 cells with the 2E1 monoclonal antibody in immunocytochemistry (ICC) show clear nuclear localization;

[0051] Figure 10 This is the result of staining paraffin sections of human tonsil tissue with the 2E1 monoclonal antibody in immunohistochemistry (IHC). Detailed Implementation

[0052] Example 1

[0053] Design and synthesis of human ELL2-specific antigen peptides

[0054] To obtain a specific immune response against human ELL2 (hELL2) and avoid cross-reactivity with other homologous proteins, this embodiment employed rational antigen design. Bioinformatics software such as DNASTAR was used to perform multiple sequence alignment analysis on the full-length amino acid sequences of human ELL (UniProt accession number: P55199), human ELL2 (UniProt accession number: O00472), human ELL3 (UniProt accession number: Q9HB65), and mouse ELL2 (UniProt accession number: Q3UKU1) (sequence alignment details are provided below). Based on immunogenicity prediction, three unique sequences significantly different from other homologous proteins were screened within the 400-550 amino acid region of the human ELL2 protein. These three sequences were selected as immunogenic peptides and synthesized by Nanjing GenScript Biotech Co., Ltd. Furthermore, their immunogenicity was enhanced by conjugating them to the carrier protein keyhole lipoteihemocyanin (KLH) via the addition of a cysteine ​​(C) residue at their terminal ends. The specific sequence is as follows:

[0055] SEQ ID NO. 19 (amino acid positions 420-437): SIYEDQQDKYTSRTSLET;

[0056] SEQ ID NO. 20 (amino acid positions 459-467): HKKSKKKSK;

[0057] SEQ ID NO. 21 (amino acid positions 499-510): LNNSSPNSSGGV.

[0058] Example 2

[0059] Construction of a stable 293T-hELL2 cell line overexpressing human ELL2

[0060] To obtain the ELL2 protein in its native conformation as an immunogen and screening tool, this embodiment constructed a cell line stably expressing human ELL2. Lentiviral expression vectors containing the human ELL2 gene were packaged into lentiviral particles using lentiviral transfection technology. Viral supernatant was collected to infect 293T cells, and single-clone selection was performed using limiting dilution to ultimately establish a stable 293T-hELL2 cell line overexpressing human ELL2. Flow cytometry analysis confirmed that this cell line successfully and efficiently expressed the human ELL2 protein (…). Figure 2 (Table 1) can be used for subsequent shock immunization and functional screening.

[0061] Table 1. Flow cytometry results of the 293T-hELL2 cell line

[0062]

[0063] Example 3

[0064] Mouse immunization and serum titer detection

[0065] This embodiment employs a highly efficient multimodal immunization strategy.

[0066] Animals and Immunization Protocol: Six-week-old female Balb / c mice were selected and a rapid immunization protocol with repeated multisite immunization (RIMMS) was adopted.

[0067] Peptide Immunization Phase: Equal amounts of the three KLH-conjugated peptides synthesized in Example 1 were mixed and emulsified with Freund's complete adjuvant, followed by initial subcutaneous multi-site immunization of mice. Subsequently, booster immunizations were administered every 2-3 days using the mixed peptides and Freund's incomplete adjuvant, for a total of 5 times.

[0068] Recombinant protein booster immunization: During the 6th booster immunization, the immunogen was replaced with 50 μg of human ELL2-his recombinant protein expressed in the eukaryotic system (Huamei Biotechnology, CSB-YP007609HU), emulsified with Freund's incomplete adjuvant and then injected.

[0069] Serum titer detection: 3-4 days after the last immunization, blood was collected via the submandibular vein, and serum antibody titers were detected using an indirect ELISA method. Results ( Figure 3 The results showed that mice 2 and 3 produced a strong antibody response against human ELL2, with serum dilution titers exceeding 1:27,000, meeting the requirements for subsequent cell fusion.

[0070] Cellular shock immunization: Mice with the highest titers, number 2 and 3, were selected and injected intraperitoneally with 1×10⁻⁶ cells three days before cell fusion. 7 In Example 2, the 293T-hELL2 live cells constructed were subjected to a final shock immunization to maximize the enrichment of B cells capable of recognizing native conformation proteins.

[0071] Example 4

[0072] Preparation, fusion and screening of hybridoma cells

[0073] Cell fusion: Spleens were aseptically harvested from mice after shock immunization, and spleen cell suspensions were prepared. Spleen cells were mixed with logarithmically growing SP2 / 0 myeloma cells at a ratio of 2:1, and cell fusion was performed using a BTX ECM2001 electrofusion instrument.

[0074] Screening and culture: The fused cells were resuspended in selective medium containing HAT, seeded into 96-well plates, and cultured in a 37°C, 5% CO2 incubator. After 3-5 days, the medium was replaced with HT medium for continued culture.

[0075] Primary screening and subcloning: After the hybridoma clones have grown to a certain density, the culture supernatant is collected and primary screening is performed using an indirect ELISA method to identify positive clones that can secrete anti-human ELL2 antibodies. All positive clones are subjected to at least two rounds of subcloning using a limiting dilution method to ensure their monoclonal nature.

[0076] Secondary screening and cell line selection: After screening, a total of 76 hybridoma cell lines capable of stably secreting antibodies were obtained. Six candidate clones with optimal growth status and antibody secretion levels were selected for performance comparison across multiple platforms, including ELISA, flow cytometry (FACS), and immunocytochemistry (ICC). Ultimately, the clone named "2E1" was selected due to its superior overall performance across all assays. Biophysical analysis showed that the 2E1 antibody had an affinity as high as the picomolar level (KD≈10). -12 M, Figure 4 It has excellent specificity, recognizing only human ELL2 and not binding to human ELL1, ELL3, or mouse ELL2. Figure 5 ).

[0077] Example 5

[0078] Application detection of 2E1 antibody - enzyme-linked immunosorbent assay (ELISA)

[0079] To verify the performance of the 2E1 antibody on the ELISA platform, 1 µg / mL of recombinant human ELL2-his protein was coated onto 96-well microplates. After blocking, serially diluted purified 2E1 antibody was added for incubation. HRP-labeled goat anti-mouse IgG secondary antibody was then added, and the plates were developed using TMB substrate. Results ( Figure 6 The results showed that the 2E1 antibody exhibited a typical dose-dependent binding curve, with a calculated half-maximal effective concentration (EC50) of 0.025 µg / mL. It also showed a high plateau in the high concentration range, indicating that it has high affinity and high titer, making it suitable for establishing a highly sensitive quantitative detection method.

[0080] Example 6

[0081] Application of 2E1 antibody in detection - Western blotting

[0082] To verify the ability of the 2E1 antibody to recognize denatured proteins, whole-cell lysates of human hepatocellular carcinoma line HepG2 and human cervical carcinoma line HeLa were extracted. After SDS-PAGE electrophoresis and PVDF membrane transfer, the cells were incubated with 5 µg / mL 2E1 antibody as the primary antibody. Results ( Figure 7The results showed that in the lysates of both cell lines, the 2E1 antibody successfully detected a clear, single protein band with a molecular weight of approximately 80 kDa, which is completely consistent with the theoretical molecular weight of human ELL2 protein. This result demonstrates that the 2E1 antibody has good specificity and can accurately detect endogenous ELL2 protein in cell samples.

[0083] Example 7

[0084] Application of 2E1 antibody detection - flow cytometry (FACS)

[0085] To evaluate the antibody's ability to detect intracellular proteins, intracellular staining was performed on 293T-hELL2 cells stably overexpressing human ELL2. After fixation and permeabilization, cells were incubated with either 2E1 antibody or an isotype control antibody, and then stained with fluorescently labeled secondary antibodies. Flow cytometry analysis results ( Figure 8 The results showed that, compared with the isotype control group, cells stained with the 2E1 antibody exhibited a very strong fluorescence signal peak shift, indicating that the antibody can be effectively used in flow cytometry to specifically detect intracellular ELL2.

[0086] Example 8

[0087] Applications of 2E1 antibody detection - immunocytochemistry (ICC) and immunohistochemistry (IHC)

[0088] 1) Immunocytochemistry (ICC) detection:

[0089] 293T-hELL2 cells were cultured on coverslips, fixed, and permeabilized, then incubated with 10 µg / mL 2E1 antibody. Results were detected using fluorescent secondary antibody (immunofluorescence assay) and HRP-conjugated secondary antibody and DAB substrate (DAB colorimetric assay). Figure 9 The results showed that both the green fluorescent signal and the brownish-yellow DAB precipitate were clearly and specifically localized within the cell nucleus, while no obvious signal was observed in the cytoplasm. This result is completely consistent with the biological function of ELL2 as a nuclear transcription factor, demonstrating the antibody's high specificity and ability to precisely localize at the cellular level.

[0090] 2) Immunohistochemical (IHC) detection:

[0091] To verify the antibody's performance in clinically relevant samples, paraffin-embedded sections of human tonsil tissue were selected for IHC staining. Tonsils are rich in B lymphocytes and plasma cells, making them an ideal model for studying ELL2 function. After dewaxing, hydration, and antigen retrieval, the sections were incubated with a 1:500 dilution of 2E1 antibody and developed using the DAB method. Results ( Figure 10The results showed that the 2E1 antibody produced a clear and strong positive staining signal in the tonsil tissue. The signal appeared as brownish granular precipitates, clearly localized within the cell nucleus, against a clean background. This result represents a significant breakthrough in this invention, demonstrating the superior performance of the 2E1 antibody and its complete suitability for IHC analysis of paraffin-embedded tissues, overcoming the major shortcomings of existing technologies.

[0092] Example 9

[0093] Gene sequence determination of antibody from hybridoma cell line 2E1

[0094] Mouse monoclonal antibody 2E1 sequencing workflow:

[0095] Hybridoma cell line 2E1 was cultured and total RNA was extracted. After reverse transcription to synthesize cDNA, the heavy chain (VH) and light chain (VL) variable region genes were amplified by PCR using degenerate primers. The purified PCR products were then cloned into the pTZ57R / T vector. Positive clones were verified by blue-white screening and colony PCR. Finally, the positive clones were sequenced, and the complete VL and VH coding sequences were obtained through sequence assembly and analysis. The sequencing results are as follows:

[0096] Antibody heavy chain variable region nucleic acid sequence (SEQ ID NO.1)

[0097] CAGGAGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAACTATGCGATGTCTTGGGTGCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCGCGATTAGTGGTAGTGGTGGTAGTACCTACTACGCC GACAGCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACACTCTACCTGCAAATGAACAGCCTGAGAGCCGAGGACACAGCCGTGTATTACTGTGCGAGAGGTAACTACGGTTACTATTACTGGTACTTCGACGTCTGGGGCCAGGGTACCCTGGTCACCGTCTCCTCA.

[0098] The amino acid sequence of the variable region of the antibody heavy chain (SEQ ID NO.2) is: QEQLVESGGGLVQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKGLEWVAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGNYGYYDYWYFDVWGQGTLVTVSS.

[0099] Antibody light chain variable region nucleic acid sequence (SEQ ID NO.3) GACATCCAGCTGACCCAGTCTCCAGCCATCATGTCTGCATCTCCAGGAGAAAAGGTGACCATGACCTGCAGTTCTAGTTCTAGCGTGTCTTACTATGGTTACTGGCAGCAGAAACCTGGGACTTCTCCAAAATGGGATATTCTACCAAAACTG GCATCTGGACCTGTGCCAGCATTTTCTGGCTCAGGCTCAGGCACCTCCATCTCTCACCATCAGCAGCCTGAAGGCTGAGGACGCAGCCACCTACTACTGCCAGCAGTGGTCTAGTAACCCCTATACCTTCGGCCAAGGGACCAAGCTGGAAATCAAA.

[0100] The amino acid sequence of the variable region of the antibody light chain (SEQ ID NO.4) is: DIQLTQSPAIMSASPGEKVTMTCSSSSSVSYMYWYQQKPGTSPKWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPYTFGQGTKLEIK.

[0101] Antibody variable region sequence:

[0102] CDR-H1 (SEQ ID NO.5)

[0103] GFTFSNYAM.

[0104] CDR-H2 (SEQ ID NO.6)

[0105] AISGSGGSTYYADSVKG.

[0106] CDR-H3 (SEQ ID NO.7)

[0107] GNYGYYDYWYFDV.

[0108] CDR-L1 (SEQ ID NO.8)

[0109] SSSSVSYMY。

[0110] CDR-L2 (SEQ ID NO.9)

[0111] DTSKLAS。

[0112] CDR-L3 (SEQ ID NO.10)

[0113] QQWSSNPYT。

[0114] The complete heavy chain amino acid sequence of antibody 2E1 (SEQ ID NO.11)

[0115] QEQLVESGGGLVQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKGLEWVAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGNYGYYDYWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。

[0116] The complete light chain amino acid sequence of antibody 2E1 (SEQ ID NO.12)

[0117] DIQLTQSPAIMSASPGEKVTTMTCSSSSSVSYMYWYQQKPGTSPKWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPYTFGQGTKLEIKR TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0118] The complete nucleic acid sequence of the antibody 2E1 heavy chain (SEQ ID NO.13)

[0119]

[0120] The complete nucleic acid sequence of the light chain of antibody 2E1 (SEQ ID NO.14)

[0121] GACATCCAGCTGACCCAGTCTCCAGCCATCATGTCTGCATCTCCAGGAGAAAAGGTGACCATGACCTGCAGTTCTAGTTCTAGCGTGTCTTACTATGGTTACTGGCAGCAGAAACCTGGACCTTCTCCAAAATGGGATATTCTACCAAACTGGCATCTGGACCTGTGCCAGCATTTTTCTGGCTCAGGCTCAGGCACCTCCATCTCTCACCATCAGCAGCCTGAAGGCTGAGGACGCAGCCACCTACTACTGCCAGCAGTGGTCTAGTAACCCCTATACCTTCGGCCAAGGGACCAGCTGGAATTCAAACGGACCGTCGCCGCCCCCTCCGTGTTTATCTTCCCCCCCTCCGACGAGCAGCTGAAGTCCGGCACCGCCTCCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGTCCGGCAACTCCCAGGAGAGCGTGACCGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAGGTGACCCACCAGGGCCTGAGCTCCCCCGTGACCAAGAGCTTCAACAGGGGCGAGTGC。

[0122] The amino acid sequence of human ELL1 (SEQ ID NO. 15)

[0123] MAALKEDRSYGLSCGRVSDGSKVSVFHVKLTDSALRAFESYRARQDSVSLRPSIRFQGSQGHISIPQPDCPAEARTFSFYLSNIGRDNPQGSFDCIQQYVSSHGEVHLDCLGSIQDKITVCATDDSYQKARQSMAQAEEETRSRSAIVIKAGGRYLGKKVQFRKPAPGATDAVPSRKRATPINLASAIRKSGASAVSGGSGVSQRPFRDRVLHLLALRPYRKAELLLRLQKDGLTQADKDALDGLLQQVANMSAKDGTCTLQDCMYKDVQKDWPGYSEGDQQLLKRVLVRKLCQPQSTGSLLGDPAASSPPGERGRSASPPQKRLQPPDFIDPLANKKPRISHFTQRAQPAVNGKLGVPNGREALLPTPGPPASTDTLSSSTHLPPRLEPPRAHDPLADVSNDLGHSGRDCEHGEAAAPAPTVRLGLPLLTDCAQPSRPHGSPSRSKPKKKSKKHKDKERAAEDKPRAQLPDCAPATHATPGAPADTPGLNGTCSVSSVPTSTSETPDYLLKYAAISSSEQRQSYKNDFNAEYSEYRDLHARIERITRRFTQLDAQLRQLSQGSEEYETTRGQILQEYRKIKKTNTNYSQEKHRCEYLHSKLAHIKRLIAEYDQRQLQAWP。

[0124] Amino acid sequence of human ELL2 (SEQ ID NO. 16)

[0125] MAAGGTGGLREEQRYGLSCGRLGQDNITVLHVKLTETAIRALETYQSHKNLIPFRPSIQFQGLHGLVKIPKNDPLNEVHNFNFYLSNVGKDNPQGSFDCIQQTFSSSGASQLNCLGFIQDKITVCATNDSYQMTRERMTQAEEESRNRSTKVIKPGGPYVGKRVQIRKAPQAVSDTVPERKRSTPMNPANTIRKTHSSSTISQRPYRDRVIHLLALKAYKKPELLARLQKDGVNQKDKNSLGAILQQVANLNSKDLSYTLKDYVFKELQRDWPGYSEIDRRSLESVLSRKLNPSQNAAGTSRSESPVCSSRDAVSSPQKRLLDSEFIDPLMNKKARISHLTNRVPPTLNGHLNPTSEKSAAGLPLPPAAAAIPTPPPLPSTYLPISHPPQIVNSNSNSPSTPEGRGTQDLPVDSFSQNDSIYEDQQDKYTSRTSLETLPPGSVLLKCPKPMEENHSMSHKKSKKKSKKHKEKDQIKKHDIETIEEKEEDLKREEEIAKLNNSSPNSSGGVKEDCTASMEPSAIELPDYLIKYIAIVSYEQRQNYKDDFNAEYDEYRALHARMETVARRFIKLDAQRKRLSPGSKEYQNVHEEVLQEYQKIKQSSPNYHEEKYRCEYLHNKLAHIKRLIGEFDQQQAESWS。

[0126] Amino acid sequence of human ELL3 (SEQ ID NO. 17)

[0127] MEELQEPLRGQLRLCFTQAARTSLLLLRLNDAALRALQECQRQQVRPVIAFQGHRGYLRLPGPGWSCLFSFIVSQCCQEGAGGSLDLVCQRFLRSGPNSLHCLGSLRERLIIWAAMDSIPAPSSVQGHNLTEDARHPESWQNTGGYSEGDAVSQPQMALEEVSVSDPLASNQGQSLPGSSREHMAQWEVRSQTHVPNREPVQALPSSASRKRLDKKRSVPVATVELEEKRFRTLPLVPSPLQGLTNQDLQEGEDWEQEDEDMDPRLEHSSSVQEDSESPSPEDIPDYLLQYRAIHSAEQQHAYEQDFETDYAEYRILHARVGTASQRFIELGAEIKRVRRGTPEYKVLEDKIIQEYKKFRKQYPSYREEKRRCEYLHQKLSHIKGLILEFEEKNRGS。

[0128] Amino acid sequence of murine ELL2 (SEQ ID NO. 18)

[0129] 。

Claims

1. A mouse anti-human ELL2 monoclonal antibody, characterized in that, The antibody comprises a heavy chain variable region and a light chain variable region; The heavy chain variable region includes complementarity-determining regions CDR-H1, CDR-H2 and CDR-H3, and the light chain variable region includes complementarity-determining regions CDR-L1, CDR-L2 and CDR-L3. The amino acid sequence of the CDR-H1 is shown in SEQ ID NO.5; The amino acid sequence of the CDR-H2 is shown in SEQ ID NO.6; The amino acid sequence of the CDR-H3 is shown in SEQ ID NO.7; The amino acid sequence of CDR-L1 is shown in SEQ ID NO.8; The amino acid sequence of the CDR-L2 is shown in SEQ ID NO.9; The amino acid sequence of the CDR-L3 is shown in SEQ ID NO.

10.

2. The mouse anti-human ELL2 monoclonal antibody according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.2; the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

4.

3. The mouse anti-human ELL2 monoclonal antibody according to claim 1, characterized in that, The heavy chain amino acid sequence is shown in SEQ ID NO.11; the light chain amino acid sequence is shown in SEQ ID NO.

12.

4. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes a mouse anti-human ELL2 monoclonal antibody as described in any one of claims 1 to 3.

5. The nucleic acid molecule according to claim 4, characterized in that, The sequences of the nucleic acid molecule include SEQ ID NO.1 and SEQ ID NO.3; The sequence SEQ ID NO.1 encodes the heavy chain variable region of the antibody described; The sequence SEQ ID NO.3 encodes the light chain variable region of the antibody described.

6. An expression carrier, characterized in that, The expression vector contains the nucleic acid molecule as described in claim 4 or 5.

7. A host cell, characterized in that, The host cell contains the expression vector as described in claim 6.

8. The method for preparing the mouse anti-human ELL2 monoclonal antibody according to any one of claims 1 to 3, characterized in that, It includes the following steps: An expression vector containing a nucleic acid molecule expressing the mouse anti-human ELL2 monoclonal antibody was prepared. The obtained expression vector was transfected into eukaryotic host cells and cultured. The mouse anti-human ELL2 monoclonal antibody was isolated and purified.

9. The use of the mouse anti-human ELL2 monoclonal antibody as described in any one of claims 1 to 3 in the preparation of ELL2 detection reagents.

Images