Monoclonal antibody against tmed10 protein and use thereof
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- JIANGSU LAISEN BIOTECHNOLOGY RES INST CO LTD
- Filing Date
- 2026-06-24
- Publication Date
- 2026-08-04
AI Technical Summary
前列腺特异性抗原（PSA）是当前最常用的筛查指标，但其特异性有限，存在过度诊断和假阳性等问题
[0019]The anti-TMED10 monoclonal antibody of the present invention has the characteristics of high specificity and strong affinity, and the resulting antibody titer reaches 1×10⁻⁶. 5 The anti-TMED10 monoclonal antibody obtained in this invention can specifically bind to the TMED10 protein and can be used to detect the expression level of TMED10 in the exosomes of prostate cancer patients' tissues or serum, providing a new tool for the diagnosis and treatment monitoring of prostate cancer and having broad market prospects.
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Figure CN122502484A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the fields of biotechnology and oncology medicine, specifically relating to monoclonal antibodies against TMED10 protein and their applications. Background Technology
[0002] Prostate cancer (PCa) is one of the most common malignant tumors of the male genitourinary system. Prostate-specific antigen (PSA) is currently the most commonly used screening indicator, but its specificity is limited, leading to problems such as overdiagnosis and false positives. Therefore, finding more specific and stable biomarkers is crucial for accurate diagnosis, risk stratification, and treatment decisions in prostate cancer.
[0003] TMED10 (also known as TMP21), a member of the p24 family, is a type I transmembrane protein located in the endoplasmic reticulum-Golgi apparatus intermediate compartment (ERGIC). It not only participates in protein transport and sorting but has also been found in recent years to be closely related to neurodegenerative diseases and tumorigenesis. Studies have shown that TMED10 exists in specific extracellular vesicle subtypes and has been identified in exosomes derived from prostate cancer cells, and it plays a potential role in regulating the tumor microenvironment and influencing chemotherapy resistance. Therefore, developing a high-affinity monoclonal antibody against TMED10 is of great significance for exploring the mechanisms of prostate cancer development and establishing new diagnostic methods. Summary of the Invention
[0004] To address some shortcomings in existing technologies, this invention provides a monoclonal antibody against TMED10 protein and its applications. This invention uses recombinant TMED10 protein as an immunogen to develop a monoclonal antibody against TMED10 protein. The amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID No:3, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID No:4. The monoclonal antibody achieves a titer of 1×10⁻⁶. 5 The monoclonal antibody possesses high specificity and high affinity; it can specifically bind to the TMED10 protein and can be used for cellular immunological detection, showing broad market prospects; the monoclonal antibody can be used to prepare immunological detection tools, which is of great significance for further research and development of biological diagnostic and therapeutic drugs targeting TMED10.
[0005] To achieve the above-mentioned technical objectives, the present invention employs the following technical means:
[0006] The present invention first provides a monoclonal antibody against TMED10 protein, wherein the light chain variable region of the monoclonal antibody includes the amino acid sequence shown in SEQ ID No:3, and the heavy chain variable region includes the amino acid sequence shown in SEQ ID No:4.
[0007] Preferably, the amino acid sequence of the TMED10 protein is shown in SEQ ID No:1.
[0008] Preferably, the gene encoding the TMED10 protein is shown in SEQ ID No:2.
[0009] The present invention also provides a polynucleotide encoding a monoclonal antibody against the above-mentioned anti-TMED10 protein.
[0010] This invention also provides a method for preparing the above-mentioned monoclonal antibody against TMED10 protein, the method comprising:
[0011] BALB / c mice were immunized with TMED10 recombinant protein. Then, splenic lymphocytes from the successfully immunized mice were fused with myeloma cells. Positive clones were screened to obtain hybridoma cell lines that specifically secrete anti-TMED10 monoclonal antibodies. Antibodies were then produced by in vivo ascites induction method to obtain the monoclonal antibody against TMED10 protein.
[0012] The present invention also provides a hybridoma cell line capable of specifically secreting anti-TMED10 monoclonal antibodies.
[0013] The present invention also provides the use of the above-mentioned monoclonal antibody against TMED10 protein and the above-mentioned polynucleotide-encoded monoclonal antibody in the detection of TMED10 protein for non-therapeutic and diagnostic purposes.
[0014] The present invention also provides the use of the above-mentioned monoclonal antibody against TMED10 protein and the above-mentioned polynucleotide-encoded monoclonal antibody in the preparation of immunological detection tools.
[0015] Preferably, the immunological detection tool includes a biological detection reagent or kit for detecting TMED10 protein.
[0016] The present invention also provides a reagent or kit comprising the above-described monoclonal antibody against TMED10 protein.
[0017] The present invention also provides the use of the above-described reagents or kits in the detection of TMED10 protein for non-therapeutic and diagnostic purposes.
[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0019] The anti-TMED10 monoclonal antibody of the present invention has the characteristics of high specificity and strong affinity, and the resulting antibody titer reaches 1×10⁻⁶. 5 The anti-TMED10 monoclonal antibody obtained in this invention can specifically bind to the TMED10 protein and can be used to detect the expression level of TMED10 in the exosomes of prostate cancer patients' tissues or serum, providing a new tool for the diagnosis and treatment monitoring of prostate cancer and having broad market prospects. Attached Figure Description
[0020] Figure 1 This is an SDS-PAGE electrophoresis image of the TMED10 recombinant protein.
[0021] Figure 2 This is an SDS-PAGE electrophoresis image of the purified anti-TMED10 protein monoclonal antibody.
[0022] Figure 3 This is a graph showing the immunotiter test results of monoclonal antibodies against TMED10 protein at different dilutions.
[0023] Figure 4 This is the specificity identification result of the monoclonal antibody against TMED10 protein (verified by Western blot).
[0024] Figure 5 This is a Western blot result of detecting TMED10 expression in different prostate cancer cell lines using a monoclonal antibody against TMED10 protein. Detailed Implementation
[0025] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the scope of protection of the present invention is not limited thereto. Unless otherwise specified, the methods, equipment, and materials used in the following embodiments are conventional methods, equipment, and materials in the art, and are all commercially available.
[0026] Example 1: Preparation of monoclonal antibody against TMED10 protein
[0027] (1) Expression and purification of TMED10 recombinant protein:
[0028] The nucleotide sequence of the gene encoding the TMED10 protein was obtained from a public database (Accession: NM_006827.6). The coding region (CDS) of the TMED10 protein is located at nucleotides 34 to 693. The extracellular segment corresponds to the N-terminal portion of the mature protein (amino acids 32 to 185), located before the transmembrane region. The nucleotide sequence encoding the extracellular segment corresponds to the interval in the CDS from codon 32 to codon 185, i.e., positions 127 to 588 of the mRNA sequence, with a sequence length of 462 bp, encoding 154 amino acids, and is designated as the TMED10 recombinant protein. The amino acid sequence of the TMED10 recombinant protein is shown in SEQ ID No:1, and the nucleotide sequence of the gene encoding the extracellular segment of this TMED10 recombinant protein is shown in SEQ ID No:2.
[0029] SEQ ID No:1
[0030] ISFHLPINSRKCLREEIHKDLLVTGAYEISDQSGGAGGLRSHLKITDSAGHILYSKEDATKGKFAFTTEDYDMFEVCFESKGTGRIPDQLVILDMKHGVEAKNYEEIAKVEKLKPLEVELRRLEDLSESIVNDFAYMKKREEEMRDTNESTNTRV
[0031] SEQ ID No:2
[0032] atctccttccatctgcccattaactctcgcaagtgcctccgtgaggagattcacaaggacctgctagtgactggcgcgtacgagatctccgaccagtctgggggcgctggcggcct gcgcagccacctcaagatcacagattctgctggccatattctctactccaaagaggatgcaaccaaggggaaatttgcctttaccactgaagattatgacatgtttgaagtgtgtt ttgagagcaagggaacagggcggatacctgaccaactcgtgatcctagacatgaagcatggagtggaggcgaaaaattacgaagagattgcaaaagttgagaagctcaaaccatta gaggtagagctgcgacgcctagaagacctttcagaatctattgttaatgattttgcctacatgaagaagagagaagaggagatgcgtgataccaacgagtcaacaaacactcgggt.
[0033] The nucleotide sequence of the gene encoding the extracellular segment of the TMED10 protein was codon-optimized, and the optimized codons were then transferred into the prokaryotic expression vector pET-30a to construct the recombinant plasmid pET-30a-TMED10. The recombinant plasmid pET-30a-TMED10 was transformed into *E. coli* BL21(DE3) using a 42℃ heat shock for 90 s method. The transformed bacterial culture was plated on LB agar containing kanamycin (final concentration 50 μg / mL) and incubated overnight at 37℃. Kanamycin-resistant plaques were picked and cultured in 500 mL LB agar until the OD of the bacterial culture reached 0.6–0.8. 0.5 mM IPTG was added to induce the expression of the target protein. After incubation at 25℃ for 12 h, the bacterial cells were collected, sonicated to lyse the bacteria, and the supernatant was collected by centrifugation. The collected supernatant was added to a Ni-NTA affinity chromatography column (purchased from Qiagen) and incubated overnight at 4°C. The supernatant was discarded, and unbound protein was washed away with Wash Buffer, followed by elution with Elution Buffer. The elution peak was collected and analyzed by SDS-PAGE electrophoresis. The results showed that high-purity TMED10 recombinant protein was obtained. Figure 1 ).
[0034] (2) Immunization and titer testing of BALB / c mice:
[0035] BALB / c mice were immunized with the TMED10 recombinant protein obtained in step (1) (purchased from Dongshan Kehui Trading Company, Wuzhong District). The first immunization used complete Freund's adjuvant, and the booster immunization used incomplete Freund's adjuvant. Blood was collected one week after each immunization, and serum titer was detected by indirect ELISA. Mice with the highest serum titer were selected for subsequent fusion experiments.
[0036] The method for detecting the serum titer is as follows:
[0037] (a) Dilute the obtained TMED10 recombinant protein to 1 μg / mL with carbonate buffer (pH 9.6, 0.05 mol / L) and add 100 μL to each well of a 96-well microplate. Coat overnight at 4°C. Remove the coated microplate, wash three times with TBS-T buffer, pat dry the microplate, and store at 4°C for later use.
[0038] (b) One week after the second immunization of mice, blood was collected from the tail vein of the mice. The serum was separated by centrifugation at 5000 g for 15 min. The serum was diluted with sample diluent (phosphate buffer containing 0.5% bovine serum albumin) at a gradient of 1:100, 1:1000, 1:10000, 1:100000, and 1:1000000. 100 μL of the diluent was added to each well of the ELISA plate to be tested. After incubation at 37°C for 1 h, the plate was washed 3 times with TBS-T buffer, dried, and then 100 μL of HRP-labeled goat anti-mouse secondary antibody (purchased from Jackson Immuno Research) diluted 1:5000 was added to each well. The plate was incubated at 37°C for 30 min.
[0039] (c) After incubation, remove the microplate, wash it 5 times with TBS-T buffer, add 100 μL of TMB substrate display solution to each well, and develop the color at 37°C in the dark for 10-15 min. Then add 50 μL of stop solution and read the absorbance at 450 nm wavelength using a microplate reader.
[0040] Select serum titers reaching 1:10 5 The mice were then immunized a third time, referring to "Modern Antibody Technology and Its Application" published by Peking University Press and "Experimental Guide to Antibody Preparation and Use" published by Science Press.
[0041] (3) Fusion and screening of hybridoma cells:
[0042] Feeder cells were prepared, and SP2 / 0 myeloma cells were prepared. Three to four days after the immunization, mouse spleen cells from the three immunizations in step (2) were fused with SP2 / 0 myeloma cells. The fused cells were plated (4 96-well plates), and hybridoma cells that produce monoclonal antibodies were screened using indirect ELISA. After two rounds of subclonal screening, hybridoma cell lines with the best antibody secretion were obtained and numbered and named: TMED10-mAb.
[0043] (4) Production and purification of monoclonal antibodies against TMED10 protein:
[0044] Mouse monoclonal antibody ascites was prepared using standard methods. The ascites antibody was purified using the octanoic acid-protein G method to obtain a monoclonal antibody against TMED10 protein. The monoclonal antibody against TMED10 protein was verified by SDS-PAGE electrophoresis. Figure 2 The figure shows clear antibody bands, indicating that the purity meets the requirements. The molecular weight of the prepared antibody IgG (H+L) is approximately 160 KD, of which the IgG heavy chain is approximately 55 KD and the IgG light chain is approximately 25 KD.
[0045] Long-term preservation of monoclonal cells may lead to the loss of positive clones due to instability and contamination after multiple passages. In this embodiment, molecular biology techniques were used to amplify the heavy chain variable region (mVH) and light chain variable region (mVL) genes of positive monoclonal cell lines using the Mouse Ig-Primer Set kit (Merck Millipore). Sequence identification was performed, and the amino acid sequence of the light chain variable region of hybridoma cells was obtained as shown in SEQ ID No:3, and the amino acid sequence of the heavy chain variable region was shown in SEQ ID No:4.
[0046] SEQ ID No:3
[0047] DIVGTQTPLSLPVSLGEQASISCTVSNQLNYMHWYLNKPGQSPRLLIYLSTNVGCGVPERFSGSGSGTDFTLKITRVEAEDLGVYFCNSFTCNAPTFAGGTKLDIKR
[0048] SEQ ID No:4
[0049] QVQIKQSGPDLVKPGASVKMSCKTSGYTFASNFLKFVKQRTGQGIEWIGDIQPASGKTNTNEKWKQKATLTAEKTSNTAYMHLSSLTSEDSAVFFCVIYNTFKLEYWAQGTTITVSS.
[0050] Example 2: Determination of anti-TMED10 monoclonal antibody titer and specificity
[0051] (1) Determination of the titer of monoclonal antibodies:
[0052] The TMED10 protein was diluted to 1 μg / mL with 0.05 mol / L carbonate buffer at pH 9.6. The diluted protein was then added to 100 μL of each well in a 96-well microplate and incubated overnight at 4°C for later use.
[0053] The monoclonal antibody against TMED10 protein obtained in Example 1 was diluted 1:10 with sample dilution buffer (phosphate buffer containing 0.5% bovine serum albumin). 3 1:10 4 1:10 5 1:10 6 Dilute 100 μL / well and incubate at 37℃ for 1 h. Remove the ELISA plate, wash 3 times with TBS-T, blot dry, and add 100 μL of 1:5000 diluted HRP-labeled goat anti-mouse secondary antibody to each well. Incubate at 37℃ for 30 min. After washing 5 times with TBS-T, add 100 μL of TMB substrate display solution to each well and incubate at 37℃ in the dark for 10-15 min. Then add 50 μL of stop solution to terminate the reaction. Read the absorbance at 450 nm using an ELISA reader. The results are as follows: Figure 3 As shown in the figure, the titer of the monoclonal antibody against TMED10 protein is 1×10⁻⁶. 5 .
[0054] (2) Monoclonal antibody specific recognition and identification:
[0055] The recombinant TMED10 protein, vascular endothelial growth factor A (VEGFA), T-cell immune receptor with Ig and ITIM domains (TIGIT), heparin-binding epidermal growth factor (HB-EGF), and heat shock protein 90 (HSP90) were diluted to 1 μg / mL using carbonate buffer, coated onto an ELISA plate, and a blank control was set up. The blank control group consisted of 1 μg / mL BSA protein. The immunotiter of the monoclonal antibody against TMED10 protein was detected by indirect ELISA using the same method as step (2) in Example 1, in order to identify the specificity of the monoclonal antibody. The detection results are as follows: Figure 4 As shown.
[0056] As can be seen from the figure, the monoclonal antibody against TMED10 protein showed no cross-reactivity with VEGFA, TIGIT, HB-EGF, and HSP90 proteins, indicating that the monoclonal antibody has good specificity.
[0057] Example 3: Western blot experiment of monoclonal antibody against TMED10 protein
[0058] Human prostate cancer cell line DU145 (Chinese Academy of Sciences Cell Bank: Catalog No.: THu222) was collected, and cells were lysed with RIPA lysis buffer. The cells were centrifuged at 5000 g at 4℃ for 20 min, and the supernatant was collected. Protein concentration was detected using a BCA kit (purchased from Beyotime Biotechnology Co., Ltd.).
[0059] The collected supernatant was separated by 12% SDS-PAGE electrophoresis. The PAGE gel (purchased from Beyotime Biotechnology Co., Ltd.) was then placed on a PVDF membrane and transferred at 120 mA for 240 min. The PVDF membrane was then blocked in 5 wt% skim milk at room temperature for 1 h, and incubated overnight at 4°C with a 1:5000 dilution of the anti-TMED10 protein monoclonal antibody obtained in Example 1 as the primary antibody. The PVDF membrane was removed, washed three times with PBS-T for 5 min each time, and incubated for 1 h at room temperature with a 1:5000 dilution of HRP-labeled goat anti-mouse secondary antibody. The secondary antibody was discarded, and the membrane was washed five times with PBS-T for 5 min each time. The membrane was then added to a chromogenic solution for light-protected development, and the experimental results were photographed and recorded. The results are as follows: Figure 5 As shown.
[0060] from Figure 5 As can be seen from the above, the monoclonal antibody against TMED10 protein prepared in Example 1 can specifically recognize TMED10 protein and can be used to detect TMED10 protein in prostate cancer cells, indicating that the monoclonal antibody against TMED10 produced in this invention has high specificity.
[0061] In summary, the monoclonal antibody against TMED10 protein described in this invention achieves a titer of 1×10⁻⁶. 5 The monoclonal antibody possesses high specificity and high affinity; it can specifically bind to the TMED10 protein and can be used for cellular immunological detection, showing broad market prospects; the monoclonal antibody can be used to prepare immunological detection tools, which is of great significance for further research and development of biological diagnostic and therapeutic drugs targeting TMED10.
[0062] The embodiments described above are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments. Any obvious improvements, substitutions or modifications that can be made by those skilled in the art without departing from the essence of the present invention shall fall within the protection scope of the present invention.
Claims
1. A monoclonal antibody against TMED10 protein, characterized in that, The amino acid sequence of the light chain variable region of the monoclonal antibody against TMED10 protein is shown in SEQ ID No:3, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID No:
4.
2. The monoclonal antibody against TMED10 protein according to claim 1, characterized in that, The amino acid sequence of the TMED10 recombinant protein is shown in SEQ ID No:
1.
3. The monoclonal antibody against TMED10 protein according to claim 1, characterized in that, The nucleotide sequence encoding the TMED10 recombinant protein is shown in SEQ ID No:
2.
4. A polynucleotide encoding a monoclonal antibody against the TMED10 protein as described in any one of claims 1-3.
5. A hybridoma cell line capable of specifically secreting a monoclonal antibody against the anti-TMED10 protein as described in any one of claims 1-3.
6. Use of the monoclonal antibody against TMED10 protein according to any one of claims 1-3, and the polynucleotide-encoded monoclonal antibody according to claim 4, in the detection of TMED10 protein for non-therapeutic and diagnostic purposes.
7. Use of the monoclonal antibody against TMED10 protein as described in any one of claims 1-3, and the polynucleotide-encoded monoclonal antibody as described in claim 4, in the preparation of immunological detection tools.
8. The use according to claim 7, characterized in that, The immunological detection tools include biological detection reagents or kits for detecting TMED10 protein.
9. A reagent or kit, characterized in that, The reagent or kit contains a monoclonal antibody against the TMED10 protein as described in any one of claims 1-3.
10. Use of the reagent or kit of claim 9 in the detection of TMED10 protein for non-therapeutic and non-diagnostic purposes.