Glucose-6-phosphatase (G6PC) rabbit monoclonal antibody and application thereof

Abstract

This invention relates to the field of biotechnology and discloses a rabbit anti-human glucose-6-phosphatase (G6PC) monoclonal antibody, OTIR5G10. The antibody is produced by sorting specific B cells, culturing and screening, and molecular clonal recombination. The immunogen of the rabbit monoclonal antibody OTIR5G10 is a synthetic polypeptide ranging from 80 to 120 amino acids. The amino acid sequence of the OTIR5G10 antibody light chain (VL) is shown in SEQ ID NO. 1; the amino acid sequence of the OTIR5G10 antibody heavy chain (VH) is shown in SEQ ID NO. 2. The VL region of the OTIR5G10 antibody includes three antigenic determinants: CDR1, CDR2, and CDR3, whose amino acid sequences are shown in SEQ ID NO. 3-5, respectively. The VH region of the OTIR5G10 antibody also includes three antigenic determinants: CDR1, CDR2, and CDR3, whose amino acid sequences are shown in SEQ ID NO. 6-8, respectively. This invention also relates to the application of anti-human glucose-6-phosphatase (G6PC) rabbit monoclonal antibody in the preparation of immunoassay tools for detecting G6PC protein, including but not limited to its application in immunohistochemical kits and in the preparation of kits for labeling tumors, providing an auxiliary means for the diagnosis of G6PC-related diseases and laying the foundation for the preparation of engineered antibodies in the next step.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and particularly relates to rabbit monoclonal antibodies against human glucose-6-phosphatase (G6PC) and their application in immune detection. Background Technology

[0002] The G6PC gene, located on chromosome 17, encodes the liver catalytic subunit glucose-6-phosphatase. It has five exons, is 12.5 kb in length, and encodes a 35.5 kDa protein composed of 357 amino acids. The G6PC gene is a multi-transmembrane protein expressed in the liver, kidneys, and small intestine, involved in the final step of glycogenolysis and gluconeogenesis. It functions in the endoplasmic reticulum (ER) to break down glucose-6-phosphate.

[0003] Glycogen storage disease type Ia (GSD1a) is a hereditary metabolic disorder caused by a deficiency of glucose-6-phosphatase (G6Pase or G6PC). Glucose-6-phosphatase is a key enzyme for glucose homeostasis. G6Pase catalyzes the final reactions of hepatic and renal glycogenolysis and gluconeogenesis, as well as the hydrolysis of glucose-6-phosphate (G6P) into glucose and inorganic phosphate.

[0004] The glucose-6-phosphatase catalytic subunit (G6PC) catalyzes the breakdown of glucose-6-phosphate into glucose and phosphate. Changes in the expression level and activity of this gene directly affect the dynamic homeostasis of glucose in the body. Overexpression of G6PC is closely related to the occurrence and development of diabetes, and mutations in this gene can lead to type 1a glycogen storage disease. Currently, immunohistochemistry (IHC) is commonly used in clinical practice to detect the expression of proteins in disease-related cells. However, the core of IHC experiments is the use of monoclonal antibodies that specifically bind to the proteins, and their performance directly determines the sensitivity and specificity of the entire detection. Therefore, finding highly specific and sensitive anti-G6PC antibodies is crucial for establishing methods to detect G6PC in cells. A search of patent websites in China, the United States, EPO, and WIPO using G6PC or G6PC+monoclonal antibody or antibody as keywords yielded no patents related to monoclonal antibodies with high affinity and specificity for G6PC. No patents were also found related to immunoassay kits prepared using the anti-G6PC rabbit monoclonal antibody OTIR5G10. Using G6PC or G6PC+monoclonal antibody or antibody as keywords, no similar literature related to the content of this invention was found on PubMed. A search of the National Medical Products Administration website revealed no registered G6PC-related diagnostic reagents. Summary of the Invention

[0005] The purpose of this invention is to provide the anti-G6PC rabbit monoclonal antibody OTIR5G10 with high specificity and affinity and its application in the preparation of an immunoassay tool for detecting G6PC protein, so as to provide auxiliary diagnosis and monitoring for glycogen storage disease type Ia (GSD1a) and also to provide a foundation for the preparation of engineered antibodies in the next step.

[0006] The rabbit monoclonal antibody is rabbit monoclonal antibody OTIR5G10.

[0007] The rabbit monoclonal antibody OTIR5G10 uses a synthetic G6PC polypeptide as an immunogen. It is obtained by immunizing New Zealand white rabbits, collecting peripheral blood mononuclear cells (PMBCs) from the immunized rabbits, sorting and culturing specific B cells, screening, and using molecular clonal recombination technology.

[0008] The rabbit monoclonal antibody OTIR5G10 has a light chain variable region (VL) containing 110 amino acids, and its amino acid sequence is shown in SEQ ID NO. 1; the heavy chain (VH) of the antibody contains 109 amino acids, and its amino acid sequence is shown in SEQ ID NO. 2.

[0009] The rabbit monoclonal antibody OTIR5G10 comprises three antigenic determinants in its VL region: CDR1, CDR2, and CDR3, with corresponding regions of 27aa-34aa, 52aa-54aa, and 91aa-100aa, respectively. Its amino acid sequences are shown in SEQ ID No. 3-5.

[0010] The rabbit monoclonal antibody OTIR5G10 comprises three antigenic determinants in its VH region: CDR1, CDR2, and CDR3, with corresponding regions of 25aa-32aa, 50aa-56aa, and 94aa-98aa, respectively. Its amino acid residue sequences are shown in SEQ ID No. 6-8.

[0011] The rabbit monoclonal antibody OTIR5G10 described above can bind with high specificity to G6PC and can be prepared into various immunoassay kits for detecting G6PC using methods known to those skilled in the art. In particular, it can be used in immunohistochemical kits and tumor labeling kits. Attached Figure Description

[0012] Figure 1 Electrophoresis diagram of the full-length amplification products of the heavy and light chains of rabbit monoclonal antibody OTIR5G10, where M is the DNA molecular weight marker.

[0013] Figure 2Western blot results of rabbit monoclonal antibody OTIR5G10 specifically recognizing the complete G6PC (Full length G6PC, G6PC-FL) protein;

[0014] Figure 3 Immunohistochemical results of formalin-fixed and paraffin-embedded human liver (primary antibody: G6PC monoclonal antibody OTIR5G10);

[0015] Figure 4 Immunohistochemical results of formalin-fixed and paraffin-embedded human kidney (primary antibody: G6PC monoclonal antibody OTIR5G10);

[0016] Figure 5 Immunohistochemical results of formalin-fixed and paraffin-embedded human duodenum (primary antibody: G6PC monoclonal antibody OTIR5G10). Detailed Implementation

[0017] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions, conditions described in a laboratory manual, or conditions recommended by the manufacturer.

[0018] Example 1: Preparation of G6PC rabbit monoclonal antibody

[0019] I. Preparation of G6PC Immunogen

[0020] An immunogenic peptide was designed based on the G6PC 1-357aa sequence, with a sequence range of 80-120aa and a peptide purity of over 90%, meeting the purity requirements for monoclonal antibody preparation. The peptide was synthesized by a third-party company, Zhongtai Biochemical Co., Ltd.

[0021] II. Animal Immunization

[0022] The synthesized G6PC peptide was emulsified with complete Freund's adjuvant and administered subcutaneously to approximately 2 kg New Zealand White rabbits at a dose of 500 μg per rabbit. A second immunization was performed two weeks later, using incomplete Freund's adjuvant emulsification at a dose of 250 μg per rabbit. Tail blood was collected after both immunizations and serum titers were determined using a serially diluted ELISA method. The OD450 at an ELISA titer of 128,000 was considered to be greater than 1.0. Based on the results, it was determined whether to collect PBMCs or continue immunization. Rabbits with the highest antibody titers were selected for PBMC collection.

[0023] III. PBMC Isolation, Specific B Cell Sorting, and Clonal Recombination: Rabbits were fixed supine on the operating table. The fur around the heart was trimmed, and the skin was disinfected with alcohol. A 50ml syringe was used to puncture the area where the heartbeat was most pronounced. Blood flowed into the syringe immediately after the needle pierced the heart. The needle was quickly withdrawn, and the whole blood was transferred to a sterile 50ml tube. This was mixed with an equal volume of PBS and slowly added dropwise onto the lymphocyte separation medium. The mixture was centrifuged at 400×g for 30 minutes at room temperature. After centrifugation, the liquid surface separated into four layers from top to bottom: a yellow plasma layer, a white thin film layer (mononuclear cell layer), a separation medium layer, and a red blood cell layer. The mononuclear cell layer was carefully aspirated and washed with PBS to remove platelets and lymphocyte separation medium, yielding rabbit PBMCs. Antigen-specific B cells were further sorted from the rabbit PBMCs for culture. Positive clones were selected from the cultured B cell supernatant using an antigen-coated ELISA plate. Cells from positive clones were collected, lysed, and RNA was extracted and reverse transcribed into cDNA. The full-length light and heavy chain sequences of naturally paired rabbit monoclonal antibodies were amplified from the cDNA of the corresponding positive clones. A rabbit monoclonal antibody expression vector was constructed using clonal recombination, and the sequence was confirmed by sequencing. The results of the amplified full-length PCR products are shown below. Figure 1 .

[0024] IV. Preparation and Purification of Monoclonal Antibodies To obtain rabbit monoclonal antibodies recognizing human G6PC protein, this invention loads the heavy and light chain genes of rabbit monoclonal antibodies into an expression vector, and transfects the plasmid into HEK293 cells. After 120-144 hours of transfection, the culture supernatant contains recombinant rabbit monoclonal antibodies recognizing human G6PC protein. The cell suspension is collected, the supernatant is obtained by centrifugation, and the antibody is purified by affinity chromatography. The concentration of the purified monoclonal antibody is determined by the BCA method, and then aliquoted and lyophilized.

[0025] Example 2: Identification of anti-G6PC rabbit monoclonal antibody OTIR5G10

[0026] I. Western blot identification of rabbit monoclonal antibody OTIR5G10

[0027] Western blot (WB) was used for detection. Lane 1 contained cell lysis buffer transfected with the pCMV6-Entry control plasmid; lane 2 contained cell lysis buffer transfected with the full-length pCMV6-G6PC expression plasmid; lane 3 contained cell lysis buffer transfected with the full-length pCMV6-G6PC2 expression plasmid; and lane 4 contained cell lysis buffer transfected with the full-length pCMV6-G6PC3 expression plasmid. 5 μg of each lysis buffer was loaded for SDS-PAGE, and Western blot was performed after transfer to a membrane.

[0028] The results showed that the rabbit monoclonal antibody OTIR5G10 specifically recognized only the intact G6PC protein and not the family proteins G6PC2 and G6PC3. Antibody dilution ratio: 1:5000-8000, 1 mg / ml. See attached results. Figure 2 .

[0029] II. Titer of Rabbit Monoclonal Antibodies

[0030] Rabbit monoclonal antibody OTIR5G10 was diluted using a serial dilution method, and the antibody titer was measured by indirect ELISA. The results showed that the titer of the rabbit monoclonal antibody OTIR5G10 involved in this invention was 1.2 × 10⁻⁶. 7 .

[0031] Example 3: Analysis of the variable region gene and amino acid sequence of rabbit monoclonal antibody OTIR5G10

[0032] Using the recombinant plasmid of the OTIR5G10 antibody as a DNA template, sequencing primers for the light chain variable region and heavy chain variable region were designed based on the vector sequences at the 5' ends of the light and heavy chains on the template. Sequencing was performed using an ABI 3730 sequencer. The nucleotide sequences of the light and heavy chain variable regions of the rabbit monoclonal antibody OTIR5G10 were obtained by sequencing.

[0033] Using the internet and the IMGT / V-QUEST analysis software at http: / / www.imgt.org, the nucleotide sequences of the light and heavy chains were sequenced and analyzed. The light chain amino acid sequence of the rabbit monoclonal antibody OTIR5G10 is shown in SEQ ID NO.1, and the heavy chain amino acid sequence is shown in SEQ ID NO.2. The full length of VL is 110 amino acids. The number of amino acids in the four domains of its FR are 26, 17, 36, and 10, respectively, and the number of amino acids in the three domains of CDR are 8, 3, and 10, respectively. The regions of CDR1, CDR2, and CDR3 are 27aa-34aa, 52aa-54aa, and 91aa-100aa, respectively, and their amino acid sequences are QSVYSNNY, RAS, and LGGYDCSSAD, respectively.

[0034] Analysis revealed that the rabbit monoclonal antibody OTIR5G10VH has a full length of 109 amino acids. The number of amino acids in the four domains of its FR is 24, 17, 37 and 11, respectively. The number of amino acids in the three domains of its CDR is 8, 7 and 5, respectively. CDR1, CDR2 and CDR3 are 25aa-32aa, 50aa-56aa and 94aa-98aa, respectively. Their amino acid sequences are GADLSTYG, IVSGGRP and VLGNL, respectively.

[0035] Example 4: Immunohistochemical detection using monoclonal antibody OTIR5G10 as the primary antibody

[0036] I. Experimental Methods:

[0037] 1. Formalin-fixed human liver, kidney, and duodenal tissues were embedded in paraffin and sectioned using a Finesse tissue slicer to a thickness of 6 μm.

[0038] 2. Dewaxing and hydration: Analytical grade xylene for 3×10 min, anhydrous ethanol for 3×10 min, 95% ethanol for 5 min, 85% ethanol for 5 min, 75% ethanol for 5 min, and deionized water for 3 min × 3 times.

[0039] 3. Add antigen retrieval solution (0.01M, pH 6.0 sodium citrate buffer) and autoclave for 3 minutes. When the autoclave temperature drops to approximately 90°C, open the autoclave, remove the specimen, and allow it to cool naturally to room temperature. Immerse in deionized water for 3 minutes each time, repeating 3 times.

[0040] 4. Inactivate endogenous peroxidase in tissues with 3% hydrogen peroxide and let stand at room temperature for 10 minutes. Soak in deionized water for 5 minutes three times.

[0041] 5. Add blocking solution (PBS + 5% skim milk powder + 5% normal goat serum) and incubate at 37°C for 60 minutes.

[0042] 6. Remove the blocking buffer, do not rinse. Add G6PC monoclonal antibody (OTIR5G10) at a dilution ratio of 1:2000, diluted with blocking buffer. Incubate in a humidified chamber at 37°C for 60 min. Wash twice with PBST (0.1% Tween-20), 5 min each time. Wash once with PBST (0.02% Tween-20), 5 min each time.

[0043] 7. Add reagent 1 from Polink-2 kit (Catlog No. D37-15) and incubate at 37°C for 10-20 minutes. Wash three times with PBS, 5 minutes each time. Add reagent 2 from Polink-2 kit (Catlog No. D37-15) and incubate at 37°C for 10-20 minutes. Wash three times with PBS, 5 minutes each time.

[0044] 8. Develop color using DAB solution (Zhongshan Jinqiao ZLI-9019) for 3–10 minutes. Wash with distilled water.

[0045] 9. Counterstain cell nuclei with hematoxylin for 2 min, rinse with distilled water, and differentiate with 1% hydrochloric acid. Rinse 3 times with distilled water and let stand at room temperature for 1 min.

[0046] 10. Dehydration and clearing: 75% ethanol for 5 min, 100% ethanol for 5 min x 3 times, 85% ethanol for 5 min, 95% ethanol for 5 min, 100% ethanol for 3 x 5 min; xylene for 3 x 5 min, neutral resin for mounting.

[0047] 11. Microscopic examination, see Figure 3-5 .

[0048] II. Experimental Results:

[0049] Depend on Figure 3-5 The results showed that even at lower dilutions (higher dilution ratios), the antibody produced distinct cytoplasmic staining in human liver, kidney, and duodenal tissues. This result is consistent with the intracellular localization and tissue expression specificity of G6PC, indicating that the monoclonal antibody OTIR5G10 can be used for immunohistochemical detection of G6PC protein levels. Immunohistochemical staining with OTIR5G10 demonstrates the high sensitivity of the monoclonal antibody OTIR5G10, making it suitable for immunohistochemical detection of G6PC expression in normal human tissues and related tumor diseases. sequence list <110> Wuxi Aorui Dongyuan Biotechnology Co., Ltd. <120> Rabbit monoclonal antibodies against glucose-6-phosphatase (G6PC) and their applications <160> 8 <170> SIPOSequenceListing 1.0 <210> 1 <211> 110 <212> PRT <213> Oryctolagus cuniculus <400> 1 Ala Gln Val Leu Thr Gln Thr Ala Ser Pro Val Ser Ala Ala Val Gly 1 5 10 15 Gly Thr Val Thr Ile Asn Cys Gln Ala Ser Gln Ser Val Tyr Ser Asn 20 25 30 Asn Tyr Leu Ser Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro Lys Gln 35 40 45 Leu Ile Tyr Arg Ala Ser Lys Leu Ala Ser Gly Val Ser Ser Arg Phe 50 55 60 Lys Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Asp Val 65 70 75 80 Gln Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Leu Gly Gly Tyr Asp Cys 85 90 95 Ser Ser Ala Asp Phe Gly Gly Gly Thr Glu Val Val Val Lys 100 105 110 <210> 2 <211> 109 <212> PRT <213> Oryctolagus cuniculus <400> 2 Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro 1 5 10 15 Leu Thr Leu Thr Cys Thr Val Ser Gly Ala Asp Leu Ser Thr Tyr Gly 20 25 30 Val Ile Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Ile Ala 35 40 45 Ala Ile Val Ser Gly Gly Arg Pro Tyr Tyr Arg His Trp Ala Lys Gly 50 55 60 Arg Phe Thr Ile Ser Lys Thr Ser Ser Thr Thr Val Glu Leu Lys Ile 65 70 75 80 Thr Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Val Leu Gly 85 90 95 Asn Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 100 105 <210> 3 <211> 8 <212> PRT <213> Oryctolagus cuniculus <400> 3 Gln Be Val Tyr Be Asn Asn Tyr 1 5 <210> 4 <211> 3 <212> PRT <213> Oryctolagus cuniculus <400> 4 Arg Wing Ser 1 <210> 5 <211> 10 <212> PRT <213> Oryctolagus cuniculus <400> 5 Leu Gly Gly Tyr Asp Cys Ser Ser Ala Asp 1 5 10 <210> 6 <211> 8 <212> PRT <213> Oryctolagus cuniculus <400> 6 Gly Ala Asp Leu Ser Thr Tyr Gly 1 5 <210> 7 <211> 7 <212> PRT <213> Oryctolagus cuniculus <400> 7 Ile Val Ser Gly Gly Arg Pro 1 5 <210> 8 <211> 5 <212> PRT <213> Oryctolagus cuniculus <400> 8 Val Leu Gly Asn Leu 1 5

Claims

1. A rabbit monoclonal antibody against human glucose-6-phosphatase (G6PC), characterized in that, The rabbit monoclonal antibody is rabbit monoclonal antibody OTIR5G10, which includes a light chain variable region and a heavy chain variable region. The light chain variable region includes three antigenic determinants: CDR1, CDR2, and CDR3, and the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID No. 3-5, respectively. The heavy chain variable region includes three antigenic determinants: CDR1, CDR2, and CDR3, and the amino acid residue sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID No. 6-8, respectively.

2. The rabbit monoclonal antibody of claim 1, characterized in that, The light chain variable region contains 110 amino acids, and its amino acid sequence is shown in SEQ ID No.

1. The heavy chain variable region contains 109 amino acids, and its amino acid sequence is shown in SEQ ID No.

2.

3. The use of the anti-human glucose-6-phosphatase (G6PC) rabbit monoclonal antibody according to any one of claims 1-2 in the preparation of a glucose-6-phosphatase (G6PC) immunoassay kit, characterized in that, The kit includes an immunohistochemistry kit.

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