A high-affinity rabbit monoclonal antibody against zebrafish Slc7a10a and its application
By designing zebrafish Slc7a10a peptides to immunize rabbits, screening and expressing high-affinity rabbit monoclonal antibodies, the problem of lacking high sensitivity and specificity in recognizing zebrafish Slc7a10a protein in existing technologies has been solved, achieving efficient detection and treatment effects.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- AFFILIATED HOSPITAL OF GUANGDONG MEDICAL UNIV
- Filing Date
- 2026-05-21
- Publication Date
- 2026-07-31
AI Technical Summary
Existing technologies lack antibodies with high sensitivity and specificity to recognize zebrafish Slc7a10a protein, making it difficult to effectively detect and target the inhibition of this protein's function, thus affecting the efficacy of amino acid metabolism-related diseases and tumor treatment.
Using zebrafish Slc7a10a peptide as an immunogen, New Zealand white rabbits were immunized. High-affinity rabbit monoclonal B cells were isolated and screened. High-affinity rabbit monoclonal antibodies were obtained by flow cytometry sorting and ELISA screening. Gene sequencing and recombinant expression were performed to verify their specificity and affinity.
An antibody with high sensitivity and specificity for recognizing zebrafish Slc7a10a protein was obtained, which can be used for detection and potential disease treatment, providing an efficient detection method and treatment strategy, especially for amino acid metabolism diseases, nervous system diseases and tumors.
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Figure CN122234211B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of monoclonal antibody development technology, and in particular to a high-affinity rabbit monoclonal antibody against zebrafish slc7a10a and its applications. Background Technology
[0002] Slc7a10a belongs to solute carrier family 7 (SLC7), whose members are mostly amino acid transporters involved in cellular amino acid uptake and metabolism. It mediates the transmembrane transport of neutral amino acids such as glycine and alanine, affecting intracellular amino acid homeostasis. Zebrafish Slc7a10a provides an animal model and therapeutic strategy for human amino acid metabolism-related diseases. It affects neural development by regulating glycine transport; its deficiency leads to motor coordination disorders and may be related to the pathogenesis of human epilepsy.
[0003] Tumor cells meet their rapid proliferation needs by upregulating amino acid transporters (such as Slc7a11a). Slc7a10a, as a neutral amino acid transporter, may participate in the amino acid supply of tumor cells, affecting tumor growth and drug resistance. Targeted inhibition of Slc7a10a function can cut off the "nutrient source" of tumor cells, enhancing the efficacy of chemotherapy or immunotherapy. Therefore, developing highly sensitive and specific antibodies that recognize zebrafish Slc7a10a is of great significance for the treatment of amino acid metabolism-related diseases, neurological disorders, and tumors. Summary of the Invention
[0004] This invention provides a high-affinity rabbit monoclonal antibody against zebrafish slc7a10a and its application, specifically achieved through the following techniques.
[0005] In a first aspect, this application provides a high-affinity rabbit monoclonal antibody against zebrafish slc7a10a, wherein the amino acid sequences of the heavy chain complementarity-determining regions CDR1, CDR2, and CDR3 of the high-affinity rabbit monoclonal antibody are shown in SEQ ID NO. 1-3, respectively, the amino acid sequences of the light chain complementarity-determining regions CDR1 and CDR3 are shown in SEQ ID NO. 4 and 5, respectively, and the amino acid sequence of the light chain complementarity-determining region CDR2 is RAS.
[0006] Furthermore, the amino acid sequence of the heavy chain variable region of the high-affinity rabbit monoclonal antibody is shown in SEQ ID NO.4, and the amino acid sequence of the heavy chain variable region of the high-affinity rabbit monoclonal antibody is shown in SEQ ID NO.9.
[0007] Furthermore, the amino acid sequence of the heavy chain of the high-affinity rabbit monoclonal antibody is shown in SEQ ID NO.5, and the amino acid sequence of the heavy chain of the high-affinity rabbit monoclonal antibody is shown in SEQ ID NO.10.
[0008] Furthermore, the amino acid sequence of the heavy chain encoding the high-affinity rabbit monoclonal antibody is shown in SEQ ID NO.6, and the amino acid sequence of the heavy chain of the high-affinity rabbit monoclonal antibody is shown in SEQ ID NO.11.
[0009] A second aspect of this application provides a nucleic acid molecule encoding the above-mentioned high-affinity rabbit monoclonal antibody against zebrafish slc7a10a.
[0010] A third aspect of this application provides a recombinant engineered cell or a recombinant engineered strain for expressing the above-mentioned high-affinity rabbit monoclonal antibody against zebrafish slc7a10a.
[0011] A fourth aspect of this application provides the use of the above-described high-affinity rabbit monoclonal antibody in the preparation of a product for detecting the amino acid transporter Slc7a10a not for the purpose of disease diagnosis and treatment.
[0012] A fifth aspect of this application provides a product for detecting the amino acid transporter Slc7a10a not for the purpose of disease diagnosis and treatment, the product comprising the aforementioned high-affinity rabbit monoclonal antibody against zebrafish slc7a10a.
[0013] It should be noted that the antibody specifically recognizing zebrafish Slc7a10a protein prepared in this application can be used to detect zebrafish Slc7a10a protein in the laboratory. For example, it can be detected using immunofluorescence and Western blotting.
[0014] Optionally, when the antibody is used to prepare a product for detecting the amino acid transporter Slc7a10a, the product can be in the form of common forms such as detection reagents, detection kits, test strips, detection probes, or detection chips.
[0015] A sixth aspect of this application provides the use of the above-mentioned high-affinity rabbit monoclonal antibody in the preparation of products for treating diseases, wherein the diseases are amino acid metabolic diseases, nervous system diseases, and tumors affected by the functional activity of the amino acid transporter Slc7a10a.
[0016] A seventh aspect of this application provides a product for treating diseases, the product comprising the above-mentioned high-affinity rabbit monoclonal antibody against zebrafish slc7a10a; the diseases being amino acid metabolic diseases, nervous system diseases, and tumors affected by the functional activity of the amino acid transporter Slc7a10a.
[0017] It should be noted that the antibody specifically recognizing the zebrafish Slc7a10a protein prepared in this application, when used to prepare products for treating diseases, primarily targets diseases affected by the physiological activity of the amino acid transporter Slc7a10a. Specifically, amino acid metabolic diseases include, but are not limited to, non-ketotic hyperglycinemia, hyperprolineemia, and urea cycle disorders; neurological diseases include, but are not limited to, epilepsy, schizophrenia, and neuropathic pain; and tumors include, but are not limited to, glioma, liver cancer, and colorectal cancer.
[0018] Compared with the prior art, the advantages of the present invention are as follows: The present invention utilizes rabbit single B cell antibody development technology to isolate zebrafish Slc7a10a-specific single B cells from rabbit peripheral blood immunized with zebrafish Slc7a10a polypeptide (extracellular region 210-223aa: Cys-KGHYDALEPQAAFE; extracellular region 288-299aa: Cys-SSMSPQELLESN). Through PCR amplification and sequencing analysis, an antibody that specifically recognizes zebrafish Slc7a10a protein is obtained.
[0019] Because the Slc7a10a protein has 12 transmembrane transport regions, antigen preparation is difficult, and there were previously no commercially available antibodies against this protein. Our company has developed the first antibody against the zebrafish Slc7a10a protein using peptide design and rabbit single B cell antibody development technology. Immunofluorescence detection was performed on zebrafish endogenous samples to verify the antibody's specificity, laying a solid foundation for the development of a highly sensitive immunofluorescence detection method for Slc7a10a. Attached Figure Description
[0020] Figure 1 This is the result of the ELISA titer assay for the monoclonal antibody 13D9.
[0021] Figure 2 This is the result of immunofluorescence detection of monoclonal antibody 13D9. Scale bar: 30 μm.
[0022] Figure 3 QC chart (Coomassie Brilliant Blue staining) for the final sample's non-reduced (reduced) SDS-PAGE analysis. MW. indicates protein marker. Detailed Implementation
[0023] The technical solution of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0024] I. Animal Immunization
[0025] To obtain a rabbit monoclonal antibody recognizing zebrafish Slc7a10a protein, this invention uses a self-designed zebrafish Slc7a10a polypeptide (extracellular region 210-223aa: Cys-KGHYDALEPQAAFE; extracellular region 288-299aa: Cys-SSMSPQELLESN) as an immunogen to immunize New Zealand white rabbits. Each rabbit was immunized with 250 μg. For the first immunization, the immunogen was mixed with an equal volume of complete Freund's adjuvant to form an emulsion, which was injected subcutaneously at multiple sites on the abdomen and back. Two weeks later, 250 μg of the immunogen was mixed with an equal volume of incomplete Freund's adjuvant to form an emulsion, which was also injected subcutaneously at multiple sites on the abdomen and back. Three booster immunizations were performed. After the three immunizations, serum titers were measured using ELISA. Rabbits with high serum titers were given a booster immunization with 250 μg of the immunogen injected subcutaneously at multiple sites. The spleen was harvested three days later.
[0026] II. Peripheral Blood PBMC
[0027] Peripheral anticoagulated blood was collected from rabbits aseptically, and PBMCs were separated using Ficoll density gradient centrifugation. The cells were washed twice with sterile PBS, and the suspended cells were counted. PBMCs can be stored in liquid nitrogen for long-term preservation.
[0028] III. Flow Cytometry Sorting and Culture of Single B Cells
[0029] (1) PBMCs were resuspended in PBS and the cell concentration was adjusted to 1×10⁻⁶. 7 B cells were stained with flow cytometry antibodies according to the resuspended cell volume. Goat Anti-rabbit IgM FITC and Slc7a10a peptide-BSA-APC were added at a ratio of 1:100. Note that blank control and single staining control should be set up. Incubate at room temperature in the dark for 30 min. Wash twice with PBS, centrifuge at 300 g for 5 min at 4 degrees.
[0030] (2) Resuspend cells in PBS and filter them through a 100-mesh filter before processing. Five minutes before processing, add 2% PI for staining to remove dead cells. Use a control to adjust the voltage and fluorescence compensation settings. Use FSC-A / SSC-A to select lymphocytes and FSC-W / FSC-A to remove adherent cells. Select IgM-negative, antigen polypeptide-BSA-positive, and PI-negative B cells.
[0031] (3) By flow cytometry, add one B cell to each well of a 96-well plate containing 100 μl of complete 1640 medium (containing 10% FBS, 1% penicillin-streptomycin, 1% glutamine, and 2 μg / mL human IL2), centrifuge the 96-well plate at 300 g for 5 min, and incubate at 37℃ with 5% CO2 for 7 days.
[0032] IV. ELISA screening for positive clones
[0033] (1) Plate packing: Slc7a10a polypeptide-BSA antigen, 5 μg / ml, 100 μl / well, overnight at 4℃.
[0034] (2) Blocking: 3% BSA-PBS, 300 μl / well, 37℃, 1.5 h.
[0035] (3) Washing: PBST, 300 μl / well, twice.
[0036] (4) Sample addition: Take 50 μl of culture supernatant from a 96-well plate, add 50 μl of PBS at a ratio of 1:1 for dilution, 100 μl / well, 37℃, 1h.
[0037] (5) Washing: PBST, 300 μl / well, 3 times.
[0038] (6) Add Goat-anti Rabbit IgG secondary antibody: 100 μl / well, 37℃, 30 min.
[0039] (7) Washing: PBST, 300 μl / well, 3 times.
[0040] (8) Add TMB Substrate Solution, 100 μl / well, 37℃, 5-10 min.
[0041] (9) Termination: 2 M HCl, 50 μl / well.
[0042] (10) Reading: OD 450 -OD 630 .
[0043] V. Sequencing and Expression of Specific Rabbit Monoclonal Antibodies
[0044] B cells corresponding to ELISA-positive antibodies were collected, and RNA was extracted and reverse transcribed into cDNA using conventional methods.
[0045] The primer sequences for the heavy chain variable region gene are as follows.
[0046] Forward primer: 5'-AAGCTTGCCAACATGCAGACTGGGCTGCGCTGGCTTC-3', as shown in SEQ ID NO.12.
[0047] Reverse primer: 5'-CCATTGGTGAGCATGCCCGAG-3', as shown in SEQ ID NO.13.
[0048] The primer sequences for light chain variable region gene amplification are as follows.
[0049] Forward primer: 5'-AAGCTTGCTACCATGCACAGAGGGCCCCCACTC-3', as shown in SEQ ID NO.14.
[0050] Reverse primer: 5'-CAGTGTCTGCTGAGGATGTAGGTAC-3', as shown in SEQ ID NO.15.
[0051] The variable region gene of the heavy and light chains was amplified using cDNA as a template. The PCR reaction system was as follows: 95℃ for 3 min; 95℃ for 1 min, 58℃ for 30 s, 72℃ for 1 min, for a total of 25 cycles; and finally 72℃ for 5 min. The PCR product was recovered and purified.
[0052] The heavy and light chain variable region genes of the rabbit recombinant monoclonal antibody were ligated into a T vector for transformation and plated.
[0053] Finally, colony PCR was performed to verify positive clones, and gene sequencing was performed on the positive clones to obtain the variable region gene sequence of the rabbit monoclonal antibody.
[0054] The heavy chain variable region gene of the correctly sequenced rabbit monoclonal antibody was subcloned into PATX1-Rabbit H, and the light chain variable region gene was subcloned into PATX1-Rabbit L. Both plasmids were co-transfected into Xten CHO cells. After 72 h of transfection, the cells were removed by centrifugation, and the culture supernatant was purified using Protein A resin. The QC of the purified antibody is detailed below. Figure 3 The antibody size was normal; the rabbit antibody was 150 kDa on a non-reducing gel and had two bands on a reducing gel, one for the heavy chain and one for the light chain (50 kDa and 24.82 kDa respectively). The purity was over 90%.
[0055] The heavy chain amino acid sequence of the high-affinity rabbit monoclonal antibody obtained in this purification step is as follows:
[0056] QSLEESGGRLVTPGGSLTLTCTVSGIDLSSYAMDWVRQAPGKGLEYIGFIGSTGNTYYASWAKGRFTISKTSSTTVDLKMTSLTASDTATYFCARGFGIWGPGTLVTVS SGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPE LLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQ PLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK, as SEQ Shown as ID NO.5.
[0057] The light chain amino acid sequence is as follows:
[0058] AQVLTQTASSVSAAVGGTVTISCQSSQSVYDNNWLAWYQQKPGQPPKRLIYRASTLESGVPSRFSGSGSGTQFTLTISGVQCDDAATYYCLGGFSGNIYTFGGGTEVVVK GDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC, such as SEQID Shown in NO.10.
[0059] VI. Affinity Identification of Recombinant Antibody
[0060] The recombinant antibody was analyzed for affinity using an ELISA method. The ELISA method was the same as in step four, and the antibody detection ranges were: 10 μg / ml, 3.33 μg / ml, 1.11 μg / ml, 0.37 μg / ml, 0.123 μg / ml, 0.041 μg / ml, 0.014 μg / ml, and 0 μg / ml.
[0061] VII. Zebrafish frozen sections and immunofluorescence
[0062] 1. Preparation of frozen sections
[0063] Three-day-old zebrafish were anesthetized with tricaine (100 mg / L), fixed with 4% paraformaldehyde (PFA), dehydrated with a (20%-30%) gradient of sucrose, embedded in OCT, and cut into 20 μm thick adhering brain tissue sections using a Leica cryostat.
[0064] 2. Immunofluorescence staining
[0065] After washing with PBS, the slides were permeabilized with PBST containing 0.3% Triton X-100 and then blocked with 10% goat serum. They were incubated overnight at 4°C with slc7a10a primary antibody, washed with PBST, and then Alexa Fluor® 647-labeled goat anti-rabbit IgG (H+L) secondary antibody was added. Nuclear staining was performed simultaneously with DAPI and incubated at room temperature for 2 h. After washing with PBST, the slides were mounted with anti-fluorescence quencher and finally images were acquired using a Nikon laser confocal microscope.
[0066] The effectiveness of the 13D9 antibody was verified using immunofluorescence on frozen sections of zebrafish brains, and the results are as follows: Figure 2 As shown, a positive signal (magenta) of slc7a10a was detected in the brain of zebrafish.
[0067] The above detailed embodiments describe the implementation of the present invention; however, the present invention is not limited to the specific details described in the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.
Claims
1. A high affinity rabbit monoclonal antibody against zebrafish slc7a10a, characterized in that, The amino acid sequences of the heavy chain complementarity-determining regions CDR1, CDR2, and CDR3 of the high-affinity rabbit monoclonal antibody are shown in SEQ ID NO. 1-3, respectively; the amino acid sequences of the light chain complementarity-determining regions CDR1 and CDR3 are shown in SEQ ID NO. 7 and 8, respectively; and the amino acid sequence of the light chain complementarity-determining region CDR2 is RAS.
2. The high affinity rabbit monoclonal antibody against zebrafish slc7a10a of claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the high-affinity rabbit monoclonal antibody is shown in SEQ ID NO.4, and the amino acid sequence of the light chain variable region of the high-affinity rabbit monoclonal antibody is shown in SEQ ID NO.
9.
3. The high affinity rabbit monoclonal antibody against zebrafish slc7a10a of claim 2, characterized in that, The amino acid sequence of the heavy chain of the high-affinity rabbit monoclonal antibody is shown in SEQ ID NO.5, and the amino acid sequence of the light chain of the high-affinity rabbit monoclonal antibody is shown in SEQ ID NO.
10.
4. The high affinity rabbit monoclonal antibody against zebrafish slc7a10a of claim 1, characterized in that, The nucleotide sequence of the heavy chain encoding the high-affinity rabbit monoclonal antibody is shown in SEQ ID NO.6, and the nucleotide sequence of the light chain of the high-affinity rabbit monoclonal antibody is shown in SEQ ID NO.
11.
5. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the high-affinity rabbit monoclonal antibody against zebrafish slc7a10a as described in any one of claims 1-4.
6. A genetically engineered cell or strain of recombinant organism, wherein the genetically engineered cell or strain of recombinant organism is used to express the high-affinity rabbit monoclonal antibody against zebrafish slc7a10a as described in any one of claims 1-4.
7. The use of the high-affinity rabbit monoclonal antibody according to any one of claims 1-4 in the preparation of a product for detecting Slc7a10a protein not for the purpose of disease diagnosis and treatment.
8. A product for detecting the amino acid transporter Slc7a10a not for the purpose of disease diagnosis and treatment, characterized in that, Including the high-affinity rabbit monoclonal antibody against zebrafish slc7a10a as described in any one of claims 1-4.
9. The use of the high-affinity rabbit monoclonal antibody according to any one of claims 1-4 in the preparation of a product for treating diseases, characterized in that, The diseases mentioned are amino acid metabolism disorders, nervous system disorders, and tumors; the amino acid metabolism disorders include, but are not limited to, nonketotic hyperglycemia, hyperprolineemia, and urea cycle disorders; the nervous system disorders include, but are not limited to, epilepsy, schizophrenia, and neuropathic pain; and the tumors include, but are not limited to, glioma, liver cancer, and colorectal cancer.
10. A product for treating a disease, characterized in that, The invention includes a high-affinity rabbit monoclonal antibody against zebrafish slc7a10a as described in any one of claims 1-4; the disease is an amino acid metabolism disorder, a nervous system disorder, or a tumor; the amino acid metabolism disorder includes, but is not limited to, nonketotic hyperglycinemia, hyperprolineemia, and urea cycle disorder; the nervous system disorder includes, but is not limited to, epilepsy, schizophrenia, and neuropathic pain; and the tumor includes, but is not limited to, glioma, liver cancer, and colorectal cancer.