A polyclonal antibody against phospholipase A2 receptor, its preparation method and application

Abstract

This invention relates to an anti-phospholipase A2 receptor polyclonal antibody, its preparation method, and its application. The preparation method includes immunizing animals with phospholipase A2 receptor protein as an antigen and collecting serum; separating and purifying the serum to obtain the anti-phospholipase A2 receptor polyclonal antibody; the amino acid sequence of the phospholipase A2 receptor protein includes the sequence shown in SEQ ID NO.1. This invention designs specific antigens for the preparation of polyclonal antibodies, enabling efficient preparation of polyclonal antibodies that specifically recognize the phospholipase A2 receptor. It has also been found that polyclonal antibodies prepared using eukaryotically expressed antigens, compared to those prepared using prokaryotically expressed or peptide-derived antigens, can more stably and efficiently construct an anti-PLA2R-associated membranous nephropathy (PLA2R MN) animal model.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to an anti-phospholipase A2 receptor polyclonal antibody, its preparation method, and its application. Background Technology

[0002] Phospholipase A2 (PLA2) can be divided into secretory and cytoplasmic types. The secretory phospholipase A2 (sPLA2) receptor acts as the receptor for sPLA2-IB and PLA2G1B, but not for sPLA2-IIA and PLA2G2A. The binding of sPLA2 to its receptor is involved in the positive and negative regulation of sPLA2 function and its clearance. The binding of sPLA2-IB / PLA2G1B induces various effects depending on cell type, such as activating the mitogen-activated protein kinase (MAPK) cascade to induce cell proliferation, lipid mediator production, and selective release of arachidonic acid from bone marrow-derived mast cells. In neutrophils, the binding of sPLA2-IB / PLA2G1B can activate p38 MAPK to stimulate elastase release and cell adhesion. It may be involved in the production of pro-inflammatory cytokines during endotoxic shock. It also possesses endocytic properties and rapidly internalizes sPLA2 ligands, which is particularly important for clearing extracellular sPLA2 to protect its effective enzymatic activity. In podocytes, the binding of sPLA2-IB / PLA2G1B can regulate podocyte survival and glomerular homeostasis.

[0003] Studies have reported the use of CRISPR Cas9 technology to construct mouse models, generating transgenic mouse strains that express the full-length mouse phospholipase A2 receptor in podocytes. By injecting anti-phospholipase A2 receptor antibodies, a passively immunized anti-PLA2R-associated membranous nephropathy (PLA2R MN) mouse model was successfully constructed, providing an important experimental animal platform for understanding the mechanism and effects of podocyte injury.

[0004] However, there are few reports on the preparation and model construction of antiphospholipase A2 receptor antibodies. It is unclear which source of antiphospholipase A2 receptor antibody should be selected to stably and efficiently construct the PLA2R MN animal model. Therefore, in-depth research on the preparation process of antiphospholipase A2 receptor antibodies and the subsequent animal model construction process is of great significance for achieving stable and efficient preparation of PLA2R MN animal models. Summary of the Invention

[0005] To address the shortcomings of existing technologies and practical needs, this invention provides an anti-phospholipase A2 receptor polyclonal antibody, its preparation method, and its application, aiming to achieve stable and efficient preparation of PLA2R MN animal models.

[0006] To achieve this objective, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a method for preparing an anti-phospholipase A2 receptor polyclonal antibody, the method comprising:

[0008] Animals were immunized with phospholipase A2 receptor protein (M-phospholipase A2 receptor, PLA2R) as an antigen, and serum was collected. The serum was then subjected to antibody separation and purification, and the antibody was collected to obtain the anti-phospholipase A2 receptor polyclonal antibody. The amino acid sequence of the phospholipase A2 receptor protein includes the sequence shown in SEQ ID NO.1.

[0009] In this invention, a specific antigen is designed and used to prepare polyclonal antibodies, which can efficiently prepare polyclonal antibodies that specifically recognize the phospholipase A2 receptor.

[0010] SEQ ID NO.1:

[0011] HIQEPSLEWRDKGIFIIQSESLKTCIQAGKSVLTLENCKQPNEHMLWKWVSDDHLFNVGGSGCLGLNISALEQPLKLYECDSTLISLRWHCDRKMIEGPLQYKVQVKSDNTVVARKQIHRWIAYTSSGGDICEHPSRDLYTLKGNAHGMPCVFPFQFKGHWHHDCI REGQKEHLLWCATTSRYEEDEKWGFCPDPTSMKVFCDATWQRNGSSRICYQFNLLSSLSWNQAHSSCLMQGGALLSIADEDEEDFIRKHLSKVVKEVWIGLNQLDEKAGWQWSDGTPLSYLNWSQEITPGPFVEHHCGTLEVVSAAWRSRDCESTLPYICKRDLN.

[0012] Preferably, the method for preparing the phospholipase A2 receptor protein includes:

[0013] The nucleic acid sequence encoding the phospholipase A2 receptor protein is inserted into an expression vector to obtain a recombinant vector. The recombinant vector is introduced into eukaryotic cells, cultured, and then the phospholipase A2 receptor protein is isolated and purified to collect the phospholipase A2 receptor protein.

[0014] In this invention, the use of eukaryotic cell expression to prepare phospholipase A2 receptor protein as an antigen enables more efficient preparation of polyclonal antibodies and is effectively used to construct the PLA2R MN animal model.

[0015] Preferably, the nucleic acid sequence encoding the phospholipase A2 receptor protein includes the sequence shown in SEQ ID NO.2.

[0016] SEQ ID NO.2:

[0017] CACATCCAGGAACCGTCCCTCGAGTGGCGTGATAAAGGAATTTTCATTATCCAGAGCGAGAGCCTGAAGACATGCATTCAGGCAGGTAAATCTGTACTGACCCTGGAGAACTGCAAGCAGCCAAACGAGCACATGCTGTGGAAATGGGTTTCCGACGACCACCTCTTTAATGTGGGAGGCAGTGGCTGCCTAGGCCTGAACATATCTGCTCTGGAGCAACCATTAAAGCTCTATGAATGTGATTCCACCCTCATTTCCCTGAGATGGCACTGTGACAGGAAGATGATCGAGGGCCCACTCCAGTACAAGGTCCAGGTGAAGTCTGACAACACAGTGGTGGCAAGAAAACAGATCCATAGGTGGATCGCCTACACGTCCAGTGGTGGGGACATTTGTGAGCATCCTAGCAGAGATTTATATACGTTAAAAGGGAATGCCCATGGGATGCCTTGCGTGTTTCCTTTCCAGTTCAAAGGTCACTGGCACCATGACTGTATCCGAGAAGGGCAGAAAGAGCACCTGCTGTGGTGTGCCACCACAAGCCGATACGAAGAAGACGAGAAGTGGGGATTCTGCCCAGATCCTACCTCTATGAAGGTGTTCTGTGATGCTACGTGGCAAAGGAATGGCAGTTCACGCATTTGCTACCAGTTCAACCTGCTTTCGTCTCTGTCTTGGAACCAGGCCCATTCTTCATGCCTGATGCAAGGAGGTGCTTTGCTAAGTATTGCAGATGAAGATGAAGAAGATTTCATAAGGAAGCACTTGAGCAAGGTAGTCAAGGAAGTGTGGATTGGTTTGAACCAGCTGGATGAGAAGGCTGGCTGGCAGTGGTCTGATGGAACACCACTCAGCTACCTGAACTGGAGCCAAGAGATAACTCCTGGGCCATTTGTTGAGCATCACTGTGGAACCCTGGAGGTTGTTTCAGCTGCCTGGAGGAGCAGGGACTGCGAGTCCACCTTGCCTTATATATGCAAACGGGATCTAAAC。

[0018] Preferably, the expression vector includes a plasmid vector.

[0019] It is understood that commonly used eukaryotic expression plasmid vectors in this field are applicable to this invention without special limitations, such as pcDNA vector. Besides pcDNA vector, there are several other common eukaryotic expression plasmids, each with unique characteristics and applications: 1. pEGFP-N1: This is an enhanced green fluorescent protein (EGFP) expression vector, commonly used for fusion protein expression and intracellular localization studies; 2. pCMVp-NEO-BAN: This vector contains a cytomegalovirus (CMV) promoter, suitable for high-level gene expression, and carries a neomycin resistance gene for screening; 3. pSV2: This is a commonly used eukaryotic expression vector that provides basic expression elements and is suitable for transfection of various cell types; 4. CMV4: This vector constructs an expression framework containing a CMV promoter for efficient gene expression; 5. Pichia expression vector: A plasmid used in yeast expression systems, such as the plasmid used in the Pichia pastoris system, containing the yeast methanol alcohol oxidase gene promoter (PAXOI) for exogenous gene expression, especially when methanol is the sole carbon source; 6. Baculovirus expression. Vector: A baculovirus expression vector used in insect cell expression systems to achieve efficient exogenous gene expression using nuclear polyhedrosis gene promoters. Each vector has its specific applications and advantages, and the appropriate expression vector can be selected according to the experimental purpose and cell type. For example, the pcDNA3.1(+) vector contains a CMV enhancer and promoter, making it suitable for achieving high-level gene expression in various mammalian cell lines, and it contains multiple cloning sites to facilitate the insertion of the target gene.

[0020] It is understood that eukaryotic cells commonly used in the art for protein expression and preparation are applicable to this invention, such as HEK293 and CHO cells. In addition to HEK293 and CHO cells, the following common eukaryotic cell lines are also included: 1. COS cells: a cell line derived from African green monkey kidney cells, commonly used for transient transfection experiments to produce high levels of protein expression; 2. HeLa cells: a widely used human cervical cancer cell line, widely used due to its rapid growth and ease of transfection; 3. NIH / 3T3 cells: a mouse embryonic fibroblast cell line, commonly used for gene expression, cell signaling, and cancer research; 4. Jurkat cells: a human leukemia T cell line, commonly used for immunological and T cell biology research; 5. BHK-21 cells: a cell line derived from Syrian hamster kidneys, commonly used for virology research and protein production; 6. Sf9 and Sf21 cells: these two insect cell lines are derived from the fall armyworm (Spodoptera exigua). 7. *Frugiperda*: A type of baculovirus expression system, commonly used for efficient expression of exogenous genes; 8. *Pichia pastoris*: A type of methanolycidal yeast, commonly used for protein expression, especially in industrial-scale production; 9. *Xenopus oocytes*: Oocytes of the African clawed frog, commonly used to study the function of ion channels and other membrane proteins; 10. MDCK cells: Canine kidney epithelial cells, commonly used to study cell polarity and virology; 11. K562 cells: A human chronic myeloid leukemia cell line, commonly used to study cell cycle and gene regulation. The appropriate cell line can be selected based on the specific circumstances.

[0021] In this invention, any culture method capable of culturing eukaryotic cells to express phospholipase A2 receptor protein is applicable, as are any methods for isolating and purifying phospholipase A2 receptor protein, without any special limitations. For example, a method for isolating and purifying phospholipase A2 receptor protein could be: obtaining the purified target protein through the specific binding of a His affinity purification tag on the protein and a nickel column.

[0022] Preferably, the animal includes any one of rabbit, guinea pig, rat, mouse, or sheep.

[0023] Preferably, the rabbit includes the New Zealand rabbit, etc.

[0024] Preferably, the immunization specifically includes:

[0025] The phospholipase A2 receptor protein was emulsified with Freund's complete adjuvant and used for the first immunization of animals. Ten to eighteen days after the first immunization, the phospholipase A2 receptor protein was emulsified with Freund's incomplete adjuvant for the second immunization. Five to nine days after the second immunization, the phospholipase A2 receptor protein was emulsified with Freund's incomplete adjuvant for the third immunization. Five to nine days after the third immunization, the phospholipase A2 receptor protein was emulsified with Freund's incomplete adjuvant for the fourth immunization.

[0026] It is understandable that the amount of antigen used during immunization can be adjusted according to the actual situation without special restrictions. For example, if the concentration of antigen for the first immunization is 1 mg / mL, the amount of antigen used for immunizing rabbits is 0.5 mL / rabbit, and the amount of antigen used for the second to fourth immunizations is halved.

[0027] Preferably, the antibody separation and purification includes:

[0028] Clean the affinity chromatography column, filter the serum to be purified, pass the filtered serum into the affinity chromatography column, clean the affinity chromatography column and elute.

[0029] Preferably, the immunization process further includes determining the serum titer against the phospholipase A2 receptor and whether it can specifically recognize the phospholipase A2 receptor using an ELISA assay.

[0030] As a preferred technical solution, the preparation method of the anti-phospholipase A2 receptor polyclonal antibody includes the following steps:

[0031] (1) Insert the nucleic acid sequence encoding phospholipase A2 protein into an expression vector to obtain a recombinant vector. Introduce the recombinant vector into eukaryotic cells, culture them, and then isolate and purify the phospholipase A2 receptor protein. Collect the phospholipase A2 receptor protein. The amino acid sequence of the phospholipase A2 receptor protein includes the sequence shown in SEQ ID NO.1.

[0032] (2) The phospholipase A2 receptor protein was emulsified with Freund's complete adjuvant and the animals were immunized for the first time; 10 to 18 days after the first immunization, the phospholipase A2 receptor protein was emulsified with Freund's incomplete adjuvant and a second immunization was performed; 5 to 9 days after the second immunization, the phospholipase A2 receptor protein was emulsified with Freund's incomplete adjuvant and a third immunization was performed; 5 to 9 days after the third immunization, the phospholipase A2 receptor protein was emulsified with Freund's incomplete adjuvant and a fourth immunization was performed; 5 to 9 days after the fourth immunization, serum was collected.

[0033] (3) Clean the affinity chromatography column, filter the serum to be purified, pass the filtered serum into the affinity chromatography column, clean the affinity chromatography column and elute, collect the antibody, and obtain the antiphospholipase A2 receptor polyclonal antibody.

[0034] In a second aspect, the present invention provides an antiphospholipase A2 receptor polyclonal antibody, which is prepared by the method for preparing the antiphospholipase A2 receptor polyclonal antibody described in the first aspect.

[0035] Thirdly, the present invention provides the application of the anti-phospholipase A2 receptor polyclonal antibody described in the second aspect in detecting phospholipase A2 receptor or constructing an animal model of anti-PLA2R-related membranous nephropathy.

[0036] Fourthly, the present invention provides a method for constructing an animal model of anti-PLA2R-related membranous nephropathy, the method comprising:

[0037] The CAG-LSL-Pla2r1-polyA gene fragment was inserted at the Rosa26 site in the genome of an experimental animal to obtain a transgenic experimental animal. The transgenic experimental animal was then injected with the anti-phospholipase A2 receptor polyclonal antibody as described in claim 7 to induce a passive immune response and obtain an animal model of anti-PLA2R-related membranous nephropathy.

[0038] Preferably, the CAG-LSL-PLA2R1-polyA gene fragment refers to the CAG promoter-loxp-stop-loxp-PLA2R1 CDS-polyA terminator. This involves a specific gene expression system that utilizes a LoxP-Stop-LoxP (LSL) expression cassette to achieve Cre-mediated conditional gene activation expression in mammalian cells and animals. The LSL expression cassette contains an SV40 polyA sequence with LoxP sites at both ends. The selected promoter is located upstream of the LSL expression cassette, while the target gene is located downstream. In the absence of Cre recombinase, theoretically, this expression cassette blocks normal expression of the target gene; when Cre is introduced into cells carrying this vector, the LSL expression cassette upstream of the target gene is deleted, allowing the target gene to be permanently activated and expressed under the selected promoter. This vector system is suitable for constructing transgenic animal models.

[0039] In the initial mouse model of this invention, CAG was used as a broad-spectrum promoter, approximately 1.7 kb in length. The mouse PLA2R1 gene has six transcripts, one of which (PLA2R1-201) has a defined CDS. The CDS of the PLA2R1-201 transcript (ENSMUST00000112525.4) was selected for model creation; it is 4464 nt long and encodes 1487 aa. Rosa26, located on mouse chromosome 6, serves as a safe site for exogenous gene insertion. Exogenous genes integrated into this site can be expressed stably and efficiently without disrupting the function of endogenous genes. Combined with the Cre-loxP system, this can be used to construct a versatile conditional gene knock-in mouse model. In this protocol, before Cre is introduced, the expression of the transformed PLA2R1 is turned off.

[0040] Preferably, the experimental animals include mice or rats.

[0041] Preferably, the injection method includes intraperitoneal injection.

[0042] Preferably, the injection dose of the antiphospholipase A2 receptor polyclonal antibody is 3-10 mg / animal, the injection frequency is 1-2 times / day, and the cycle is 1-3 days.

[0043] In a specific embodiment of this invention, the CAG-LSL-Pla2r1-polyA gene fragment can be inserted into the Rosa26 site of mice using CRISPR / Cas9 technology. The simplified process is as follows: sgRNA is transcribed in vitro, a donor vector is constructed, and Cas9, the donor, and sgRNA are microinjected into the fertilized eggs of C57BL / 6J mice to obtain F0 generation mice. F0 generation positive mice, verified by PCR, sequencing, and Southern PCR, are then mated with C57BL / 6J mice to obtain a stably heritable F1 generation positive mouse model. Transgenic mice were bred using 13-14 week old mice. Twelve PLA2R1 transgenic mice (CAG-LSL-PLA2R1-polyA-KI(Rosa26) / NPHS2-CreT, abbreviated as KI mice) and six PLA2R control mice (CAG-LSL-PLA2R1-polyA-KI(Rosa26) / NPHS2-CreW, abbreviated as WT mice) were randomly divided into four groups: KI mice + anti-mouse PLA2R polyclonal antibody group (KI+PLA2R), KI mice + unimmunized rabbit total IgG group (KI+IgG), and WT mice + PBS solution group (WT+PBS). On the first day of the experiment, mice in the KI+PLA2R group were intraperitoneally injected with anti-mouse PLA2R polyclonal antibody (3 mg / mouse), and on the second day, they were injected intraperitoneally again with anti-mouse PLA2R polyclonal antibody (2 mg / mouse). In the KI+IgG group, mice were intraperitoneally injected with unimmunized rabbit total IgG (3 mg / mouse) on the first day, and on the second day, they were injected intraperitoneally again with unimmunized rabbit total IgG (2 mg / mouse). In the WT+PBS group, mice were intraperitoneally injected with the same volume of PBS solution on the first day, and on the second day, they were injected intraperitoneally again with the same volume of PBS solution. Urine was collected from each group on days 1, 3, 5, and 7 (24 hours) and urine volume was assessed. The urine albumin-to-creatinine ratio was measured. On day 8, mice were sacrificed, and serum was collected for biochemical analysis: serum albumin and serum cholesterol. Bilateral kidneys were collected for pathological analysis: kidney pathology (paraffin sections stained with PAS and PASM and examined under electron microscopy), and immunopathology (PLA2R, IgG, and complement C3 immunofluorescence).

[0044] This invention aims to construct a PLA2R MN mouse model by repeatedly passively immunizing PLA2R1 transgenic mice with an anti-phospholipase A2 receptor (PLA2R) antibody. This rapidly induces nephrotic syndrome in PLA2R1 transgenic mice and activates the complement system, suggesting that the antibody can rapidly induce podocyte immune damage. This model exhibits similar characteristics to human PLA2R-related membranous nephropathy in both biochemical indicators and pathological features, providing an important experimental animal platform for further research on PLA2R MN. In this invention, in-depth analysis of the construction of the PLA2R MN animal model revealed that using a specific sequence of phospholipase A2 receptor prepared by eukaryotic cell expression as an antigen to prepare polyclonal antibodies can stably and efficiently construct the PLA2R MN animal model.

[0045] Compared with the prior art, the present invention has the following beneficial effects:

[0046] In this invention, specific antigens were designed and used to prepare polyclonal antibodies, which can efficiently prepare polyclonal antibodies that specifically recognize the phospholipase A2 receptor. It was also found that polyclonal antibodies prepared using eukaryotically expressed antigens can more stably and efficiently construct animal models compared to polyclonal antibodies prepared using prokaryotically expressed antigens or antigens derived from peptide routes. The first animal model of anti-phospholipase A2 receptor antibody-related membranous nephropathy in China was successfully constructed through passive immunization, providing an important experimental animal platform for understanding the mechanism and action of phospholipase A2 receptor in situ immune complexes in causing podocyte damage. Attached Figure Description

[0047] Figure 1 The image shows the identification results of the phospholipase A2 receptor protein.

[0048] Figure 2 A schematic diagram illustrating the principle of site-specific insertion of the CAG-LSL-PLA2R1-polyA gene fragment into the Rosa26 site in mice;

[0049] Figure 3 Figures show the results of urine and biochemical indicators in mice in the WT+PBS group, KI+IgG group, and KI+PLA2R group. Figure A shows the ratio of urinary albumin to creatinine in each group, Figure B shows the serum albumin level in each group, Figure C shows the changes in 24-hour urine volume on days 1, 3, 5, and 7 in each group, and Figure D shows the serum cholesterol level in each group. *P<0.0001;

[0050] Figure 4 Figures showing the results of PAS, PASM and electron microscopic pathological index detection in mice in the WT+PBS group, KI+IgG group and KI+PLA2R group;

[0051] Figure 5Figure 400 shows the results of immunopathological marker detection of PLA2R, C3, and IgG in mice in the WT+PBS group, KI+IgG group, and KI+PLA2R group. Detailed Implementation

[0052] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention is determined by the claims.

[0053] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased from legitimate channels.

[0054] Example 1

[0055] In this embodiment, the phospholipase A2 receptor protein was prepared and identified.

[0056] 1. Preparation of phospholipase A2 receptor protein by eukaryotic expression

[0057] The process of preparing antigens through eukaryotic expression involves several key steps, including plasmid construction, transduction, cell culture, and antigen isolation and purification.

[0058] 1. Plasmid construction

[0059] Step 1: Select a carrier

[0060] Choose a suitable eukaryotic expression vector (such as pcDNA vector) based on the characteristics of the target antigen gene (SEQ ID NO.2). Commonly used vectors include plasmids and viral vectors. These vectors typically contain basic elements such as promoters, selection markers, and terminators, as well as possible regulatory elements such as enhancers.

[0061] Step Two: Gene Cloning

[0062] The target antigen gene sequence is determined, and appropriate restriction enzyme sites are selected. The target DNA fragment is obtained through PCR amplification or gene synthesis. The vector and target DNA fragment are double-digested using restriction endonucleases to ensure correct orientation and location.

[0063] Step 3: DNA Ligation and Transformation

[0064] The cut vector and insert fragment are mixed, and a ligase (such as T4 DNA ligase) is added to carry out the ligation reaction. The ligation product is transformed into host cells (such as E. coli), and positive clones are screened by plate culture.

[0065] Step 4: Plasmid Extraction and Validation

[0066] Plasmid DNA was extracted from positive clones and sequenced to verify the correctness of the inserted fragment sequence.

[0067] 2. Transduction

[0068] Step 1: Cell Preparation

[0069] Choose a suitable eukaryotic cell line (such as mammalian cell HEK293, insect cells, etc.) to ensure that the cells are in good condition.

[0070] Step 2: Selection of transduction methods

[0071] Choose the appropriate transduction method based on experimental needs and cell type, such as plasmid transfection (e.g., heat shock transfection, electroporation transfection, chemical transfection, etc.) or viral infection.

[0072] Taking plasmid transfection as an example, the constructed expression plasmid is introduced into eukaryotic cells. The specific steps include mixing the plasmid with the transfection reagent, adding it to the cell culture medium, gently mixing it, and then placing it under appropriate culture conditions.

[0073] 3. Cell Culture

[0074] Step 1: Optimization of cultivation conditions

[0075] Adjust culture conditions such as culture medium composition, temperature, and CO2 concentration to promote cell growth and antigen expression.

[0076] Step 2: Inducing expression (if applicable)

[0077] For certain genes that require induced expression, an inducer (such as IPTG, tetracycline, etc.) is added at a specific time point to stimulate cells to express the target antigen.

[0078] Step 3: Cell Growth Monitoring

[0079] Regularly monitor cell growth to ensure cell health and achieve the desired degree of fusion.

[0080] 4. Separation and purification

[0081] Step 1: Cell Harvesting

[0082] Once the cells have expressed a sufficient amount of antigen, they are harvested using methods such as centrifugation.

[0083] Step 2: Cell lysis

[0084] Cells are disrupted using an appropriate lysis buffer to release intracellular proteins. Lysis methods may include mechanical disruption, sonication, and chemical lysis.

[0085] Step 3: Antigen purification

[0086] Choose an appropriate purification method based on the characteristics of the antigen, such as affinity chromatography, ion exchange chromatography, or gel filtration chromatography. These methods can be used alone or in combination to improve purity and yield.

[0087] During the purification process, multiple washing and elution steps may be required to remove impurities and collect the target antigen.

[0088] Step 4: Antigen Detection and Identification

[0089] The specificity and activity of the antigen were further identified using enzyme-linked immunosorbent assay (ELISA). High-purity, high-activity antigen proteins were successfully prepared using a eukaryotic expression system.

[0090] 1) Sample preparation: Take a certain volume of sample, add the corresponding volume of 5× loading buffer, heat in boiling water at 100℃ for 10 min, centrifuge at 12000 rpm for 5 min, and wait for loading.

[0091] 2) Sample loading: Calculate the loading volume based on the protein concentration. Generally, 1-5 μg of sample is loaded per well.

[0092] 3) Electrophoresis: Run the stacking gel at 80V, then run the separating gel at 120V. Stop electrophoresis when the leading edge of the bromophenol blue indicator in the protein sample reaches the bottom of the glass plate.

[0093] 4) Staining: Remove the gel from the electrophoresis tank after electrophoresis, carefully remove the gel from the glass plate, add Coomassie Brilliant Blue staining solution, and shake on a horizontal shaker for 60 minutes.

[0094] 5) Add decolorizing solution, changing it several times until the background is clear (to save time, the number of times the decolorizing solution is changed can be increased).

[0095] 6) After the background is clear, pour off the decolorizing solution, wash the glue several times with deionized water, seal the glue with plastic film and scan (avoid air bubbles when sealing the glue), scan, edit and save the record with Photoshop.

[0096] Antigen identification results as follows Figure 1 As shown, the protein was obtained to the expected size.

[0097] Example 2

[0098] In this embodiment, the phospholipase A2 receptor protein prepared in Example 1 is used as the antigen for the preparation of polyclonal antibodies.

[0099] 1. Rabbit immunization program

[0100] 1.1 Animal Selection: New Zealand White rabbits, weighing approximately 2.5 kg, were selected. Animals should have glossy fur and be healthy with free movement. Selected animals were pre-fed for about two weeks. The purpose was to weed out any unsuitable animals and ensure the smooth progress of later experiments.

[0101] 1.2 Preparations before the experiment: Mark the rabbits.

[0102] 1.3 Antigen preparation:

[0103] 1.3.1 Remove the antigen from the -20°C freezer and thaw it at room temperature, avoiding repeated freeze-thaw cycles. Label the syringe with the project number and animal number.

[0104] 1.3.2 Extract the antigen (the antigen should be completely mixed). The concentration of the antigen for the first immunization is 1 mg / mL, and for rabbits it is 0.5 mL / rabbit. The amount of antigen for the second to fourth immunizations is halved.

[0105] 1.3.3 Adjuvant should be drawn at a 1:1 volume ratio to antigen. Complete adjuvant is used for the first immunization, while incomplete adjuvant is used for the second to fourth immunizations. The adjuvant must be thoroughly mixed before being drawn into the syringe.

[0106] 1.3.4 After connecting the two syringes with the syringe connecting tube, complete emulsification is performed. The emulsification standard is: the emulsified immunogen is qualified if it does not disperse when dropped into 37°C water.

[0107] 1.4 Immunization: Rabbits were administered multiple subcutaneous injections, with 0.2 mL at each point.

[0108] Immunization schedule: The second immunization is given 14 days after the first immunization, and the interval between the second and third immunizations is 7 days. On the 7th day after the third immunization, a small serum sample is taken from the middle ear artery of the rabbit for testing. If the test is qualified, a booster immunization is given 7 days later, and whole blood can be collected 7 days after the booster immunization.

[0109] 1.4.1 Procedures for collecting small serum samples

[0110] The rabbit was restrained in a frame, and its ears were gently tapped to dilate the central auricular artery. The area was disinfected with 75% alcohol. The rabbit's ear was held still with the left hand, and the syringe was held in the right hand. The needle was inserted into the central auricular artery at a point one-third of the way down from the distal end, parallel to the artery and directed towards the heart. 8 mL of blood was collected at a time, and pressure was applied with a cotton ball to stop the bleeding.

[0111] 1.4.2 Whole blood collection procedure

[0112] 1.4.2.1 Capture the rabbits to be immunized, check the ear tag number, weigh them, and anesthetize them by intravenous injection of 1 ml of 3% sodium pentobarbital per kilogram.

[0113] 1.4.2.2 After anesthesia, the immunized rabbits were placed with their abdomen facing upwards, and their limbs were restrained on a stainless steel mesh frame. Blood was drawn from the heart using the cardiac blood sampling method.

[0114] 1.4.2.3 After blood collection, place the centrifuge tube containing the blood in a 37°C water bath for 20 minutes, then remove and cool it before placing it in a 4°C refrigerator. Wait for the blood to separate automatically, then transfer the supernatant to a clean 50mL centrifuge tube.

[0115] 1.4.2.4 Centrifuge at 12000 rpm for 2 min, transfer the supernatant to a clean centrifuge tube, add 100 μL of 10% sodium thimerosal solution (final concentration 0.02%) to 50 mL of supernatant, mix well, and store at -20℃.

[0116] 2. ELISA detection (indirect method)

[0117] 2.1 Plate coating: Dilute the known antigen to 1 μg / mL with coating buffer (Na2CO3 and NaHCO3 buffer), add 50 μL to each reaction well of the polystyrene plate, incubate overnight at 4°C, and the next day, discard the solution in the wells and wash once with 180 μL of 1×PBST washing buffer per well.

[0118] 2.2 Blocking: Add 150 μL of 1% BSA (prepared with PBST) to each well for blocking, and incubate at 37°C for 1 h. Then discard the blocking solution.

[0119] 2.3 Sample addition: Add 50 μL of the diluted test sample (dilute the test sample according to a certain ratio) to the above-mentioned sealed reaction wells. Also, set up negative control wells (1% BSA). Incubate at 37°C for 30 min, then wash three times with 150 μL of 1×PBST washing buffer per well.

[0120] 2.4 Add enzyme-labeled antibody: Add freshly diluted secondary antibody-HRP (diluted with 1% BSA) at 50 μL / well to the wells of the ELISA plate, incubate at 37°C for 45 min, and wash 3 times with 1×PBST buffer at 150 μL / well.

[0121] 2.5 Adding substrate solution for color development: Add 50 μL of the temporarily prepared TMB substrate solution to each reaction well and incubate at 37 °C for 5 min.

[0122] 2.6 Termination of reaction: Add 50 μL of 1M sulfuric acid to each reaction well.

[0123] 2.7 Plate reading: Place the ELISA plate in a preheated ELISA reader (450nm) to read the data, save the data, and perform analysis.

[0124] 3. Antibody purification

[0125] 3.1 The affinity chromatography column was thoroughly washed with 20 mL of pure water and 1×PBS (pH 7.4) at a flow rate of 70 mL / h.

[0126] 3.2 Take 10 mL of the serum to be purified into a 50 mL centrifuge tube and filter it using a microporous membrane with a pore size of 0.45 μm and a diameter of 25 mm.

[0127] 3.3 Load the filtered serum sample at a flow rate of 40 mL / h, and repeat once.

[0128] 3.4 Wash the column with 20 mL of 1×PBS (pH 7.4) at a flow rate of 70 mL / h. After 10 min, connect the protein analyzer. During the washing process, adjust the instrument transmittance (T setting) to 100.

[0129] 3.5 When the absorbance reading of the protein detector (1A range) is 0, turn on the HD-A computer acquisition device on the computer desktop and adjust the full-screen range to 5. Elute the antibody with glycine solution (pH 2.7, 0.2M) at a rate of 40mL / h. Press the green elution record button to start elution. When the instrument reading starts to rise, start collecting the antibody.

[0130] 3.6 During antibody collection, the pH of the antibody was adjusted to around 7 with 1M sodium bicarbonate in a timely manner, and the highest peak value of the elution peak was recorded.

[0131] 3.7 After the antibody collection is complete, adjust the pH value to about 7 and record the volume of eluted antibody. Then rinse the rubber tubing connected to the collector with purified water.

[0132] 3.8 Wash the affinity chromatography column sequentially with 20 mL of 1×PBS and pure water at a rate of 70 mL / h, then add 20% ethanol, seal the column, and store at 4°C.

[0133] 3.9 The purified antibodies are sent for testing according to different requirements.

[0134] 3.10 After purifying enough antibodies to meet the delivery requirements and ensuring the titer of the semi-finished product is qualified, mix all the antibodies and concentrate them using an ultrafiltration concentrator to achieve a certain concentration and volume.

[0135] 3.11 Place the concentrated antibody in 1L of 0.01M PBS (pH 7.4) and dialyze at room temperature, changing the medium every 3 hours for a total of 3 times (for overnight dialyze, place in a 4°C refrigerator).

[0136] 3.12 Take the dialyzed antibody into a clean centrifuge tube, filter the antibody in a clean bench using a 0.22μm disposable low-adsorption filter, take a small sample for testing, and take another 5μL to test the concentration.

[0137] 3.13 Concentration detection using a Protein A280 on a micro-volume spectrophotometer (denovix DS-11).

[0138] The results of serum ELISA are shown in Table 1. The results show that when the serum is coated with 50 ng / well of antigen and diluted 4 times starting from 250, the serum titer can reach more than 1,024,000.

[0139] Table 1

[0140]

[0141] The ELISA results of the purified antibody are shown in Table 2. The results show that when coated with 50 ng / well of antigen and serially diluted from 250 to 4-fold, the purified antibody titer can reach over 1,024,000.

[0142] Table 2

[0143]

[0144]

[0145] Example 3

[0146] This embodiment utilizes the anti-phospholipase A2 receptor polyclonal antibody prepared in Example 2 to construct a PLA2R MN animal model and to validate the model.

[0147] Both “CAG-LSL-PLA2R1-polyA-KI(Rosa26) / NPHS2-CreT” and “CAG-LSL-PLA2R1-polyA-KI(Rosa26) / NPHS2-CreW” are gene editing strategies. “CAG-LSL-PLA2R1-polyA” is a construct, and “KI(Rosa26)” indicates that the construct was knocked into the Rosa26 site in the mouse genome.

[0148] CAG is a potent, comprehensive promoter (Cytomegalovirus enhancer / chicken β-actin promoter) that drives high levels of gene expression across multiple cell types.

[0149] LSL (LoxP-Stop-LoxP): This is a conditional expression system containing two LoxP sites and a termination sequence. In the absence of Cre recombinase, the termination sequence prevents the expression of downstream genes. When Cre recombinase is present, it removes the termination sequence, thereby activating the expression of downstream genes.

[0150] PLA2R1: Pla2r1 is the gene encoding the phosphatidylinositol-anchored protein PLA2R1, an antigen associated with idiopathic membranous nephropathy (a kidney disease). Knocking the Pla2r1 gene into the Rosa26 site allows for the regulation of PLA2R1 expression in mice, which is helpful for studying the role of PLA2R1 in the disease.

[0151] polyA: is a polyadenylate signal sequence, usually located at the 3' end of a gene, which helps in the stability of mRNA and translation efficiency.

[0152] KI (Knock-In): refers to cas9-ki, a gene knock-in technology that uses homologous recombination to precisely insert foreign genes or gene fragments into specific locations in the genome.

[0153] (Rosa26): Rosa26 is a "safe harbor" site in the mouse genome, often used for gene knock-in. Inserting a foreign gene at this site usually does not affect the normal development and physiological function of the mouse.

[0154] NPHS2-CreT / W: "NPHS2" encodes the podocyte protein podocin, which is associated with various kidney diseases. It refers to a Cre recombinase driven by the NPHS2 promoter, which removes the STOP sequence to activate the expression of the PLA2R1 gene.

[0155] CAG promoter-loxp-stop-loxp-Pla2r1 CDS-polyA terminator mice, after crossbreeding with NPHS2-Cre mice, produce double-positive mice. Ultimately, the region between the two loxp sites is deleted, resulting in the "CAG-LSL-PLA2R1-polyA-KI(Rosa26) / NPHS2-CreT" mice (KI mice). These are genetically engineered mice used to study the role of PLA2R1 in kidney disease. A specific gene construct is knocked into the Rosa26 site, and conditional expression of the PLA2R1 gene is achieved using the Cre recombinase driven by the NPHS2 promoter. The "CAG-LSL-PLA2R1-polyA-KI(Rosa26) / NPHS2-CreW" mice (WT mice), serving as control mice (wild-type mice), are Cre-negative and do not ultimately have the region between the two loxp sites deleted. This typically means that recombination events between loxP sites do not occur in the absence of Cre enzyme. In other words, without Cre enzyme activity, the STOP box in the LSL sequence will not be deleted, and the PLA2R1 gene will not be expressed. This design allows researchers to control the on / off state of genes by introducing or removing the Cre enzyme, thereby studying the role of specific genes in specific cells or tissues. This model can be used as a control group to study the biological effects in the absence of specific gene expression.

[0156] Transgenic mice were bred, and 12 PLA2R1 transgenic mice aged 13-14 weeks were selected: CAG-LSL-PLA2R1-polyA-KI(Rosa26) / NPHS2-CreT (KI mice for short). A schematic diagram illustrating the principle of CAG-LSL-Pla2r1-polyA gene fragment insertion into the Rosa26 site in mice is shown below. Figure 2(As shown); 6 PLA2R control mice: CAG-LSL-PLA2R1-polyA-KI(Rosa26) / NPHS2-CreW (WT mice), randomly divided into KI mice + anti-mouse PLA2R polyclonal antibody group (KI+PLA2R), KI mice + unimmunized rabbit total IgG group (KI+IgG), and WT mice + PBS solution group (WT+PBS). On the first day of the experiment, the KI+PLA2R group was injected intraperitoneally with the anti-mouse PLA2R polyclonal antibody prepared in Example 2 (3 mg / mouse), and on the second day, it was injected intraperitoneally again with the anti-mouse PLA2R polyclonal antibody (2 mg / mouse); on the first day of the experiment, the KI+IgG group was injected intraperitoneally with unimmunized rabbit serum IgG (3 mg / mouse), and on the second day, it was injected intraperitoneally again with unimmunized rabbit total IgG (2 mg / mouse); on the first day of the experiment, the WT+PBS group was injected intraperitoneally with the same volume of PBS solution, and on the second day, it was injected intraperitoneally with the same volume of PBS solution. Urine samples were collected from mice in each group on days 1, 3, 5, and 7 for 24 hours, and urine volume was assessed. The urine albumin-to-creatinine ratio was measured. Mice in each group were sacrificed on day 8, and serum samples were collected for biochemical analysis: serum albumin and serum cholesterol. Bilateral kidney samples were collected from each group for pathological analysis: kidney pathology (paraffin sections stained with PAS and PASM and examined under an electron microscope) and immunopathology (immunofluorescence of PLA2R, IgG, and complement C3).

[0157] The results of urine and biochemical index tests in mice in the WT+PBS group, KI+IgG group, and KI+PLA2R group are as follows: Figure 3As shown, compared with the KI+IgG group and the WT+PBS group, mice in the KI+PLA2R group showed a decrease in 24-hour urine output on days 1, 3, 5, and 7 (P<0.05); the urine albumin-to-creatinine ratio was significantly increased (P<0.05), peaking on day 5 (KI+PLA2R group: 946.36±304.33 μg / mL vs. KI+IgG group: 32.34±3.24 μg / mL vs. WT+PBS group: 43.46±25.41 μg / mL, P<0.001), and slightly decreasing on day 7 (KI+PLA2R group: 578.31±161.75 μg / mL). The serum albumin levels were significantly lower in the KI+IgG group (29.33±5.35 μg / mL vs. WT+PBS group (36.48±12.15 μg / mL, P<0.001) and significantly higher in the WT+PBS group (28.00±1.02 g / L vs. WT+PBS group (28.49±1.03 g / L, P<0.001)). Serum cholesterol levels were also significantly elevated on day 8 (23.33±1.95 mmol / L vs. KI+IgG group (7.21±2.54 mmol / L vs. WT+PBS group (8.48±2.46 mmol / L, P<0.001)). Electron microscopy pathological markers were also observed. Figure 4 As shown, PAS (periodic acid-Schiff staining) and PASM (periodic acid-hexamethylenetetramine silver staining) staining in each group of mice showed normal morphology of glomeruli and renal tubular interstitium. PASM staining in the KI+PLA2R group indicated mild thickening of the basement membrane, with obvious foot process fusion visible under electron microscopy. Electron microscopy also revealed a small amount of electron-dense material in the subepithelial region and within the basement membrane. Immunopathological marker detection results are as follows: Figure 5 As shown, immunopathology in the KI+PLA2R group revealed granular positivity for IgG and complement C3 in the glomerular basement membrane region of mice, as well as granular positivity for PLA2R antigen expression. In the KI+IgG group, IgG expression was weakly positive, and no significant changes were observed in the renal pathology of the remaining mice.

[0158] Conclusion: A stable PLA2R MN mouse model was successfully established through repeated passive immunization with anti-PLA2R antibodies. This demonstrates that continuous stimulation with anti-PLA2R antibodies can rapidly induce nephrotic syndrome in PLA2R1 transgenic mice and activate the complement system, suggesting that this antibody can rapidly induce podocyte immune damage. This model exhibits similar biochemical and pathological characteristics to human PLA2R-related membranous nephropathy, providing an important experimental animal platform for further research on PLA2R MN.

[0159] In summary, this invention designs specific antigens for the preparation of polyclonal antibodies, enabling the efficient preparation of polyclonal antibodies that specifically recognize the phospholipase A2 receptor. Furthermore, it was found that polyclonal antibodies prepared using eukaryotically expressed antigens, compared to those prepared using prokaryotically expressed antigens or antigens derived via peptide routes, can more stably and efficiently construct animal models. The invention successfully established China's first animal model of anti-phospholipase A2 receptor antibody-related membranous nephropathy through passive immunization, providing an important experimental animal platform for understanding the mechanism and action of phospholipase A2 receptor in situ immune complexes in causing podocyte damage.

[0160] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A method for preparing an anti-phospholipase A2 receptor polyclonal antibody, characterized in that, The preparation method includes: Animals were immunized with phospholipase A2 receptor protein as an antigen, and serum was collected. The serum was subjected to antibody separation and purification, and the antibodies were collected to obtain the antiphospholipase A2 receptor polyclonal antibody; The amino acid sequence of the phospholipase A2 receptor protein is shown in SEQ ID NO.

1.

2. The method for preparing the anti-phospholipase A2 receptor polyclonal antibody according to claim 1, characterized in that, The method for preparing the phospholipase A2 protein includes: The nucleic acid sequence encoding the phospholipase A2 receptor protein is inserted into an expression vector to obtain a recombinant vector. The recombinant vector is introduced into eukaryotic cells, cultured, and then the phospholipase A2 receptor protein is isolated and purified to collect the phospholipase A2 receptor protein.

3. The method for preparing the anti-phospholipase A2 receptor polyclonal antibody according to claim 2, characterized in that, The nucleic acid sequence encoding the phospholipase A2 receptor protein is shown in SEQ ID NO.

2.

4. The method for preparing the anti-phospholipase A2 receptor polyclonal antibody according to claim 2, characterized in that, The expression vector includes a plasmid vector.

5. The method for preparing the anti-phospholipase A2 receptor polyclonal antibody according to claim 1, characterized in that, The animals include any one of rabbits, guinea pigs, rats, mice, or sheep.

6. The method for preparing the antiphospholipase A2 receptor polyclonal antibody according to claim 5, characterized in that, The rabbits mentioned include New Zealand rabbits.

7. The method for preparing the antiphospholipase A2 receptor polyclonal antibody according to claim 1, characterized in that, The immunity specifically includes: The phospholipase A2 receptor protein was emulsified with Freund's complete adjuvant and the animals were immunized for the first time. Ten to eighteen days after the first immunization, the phospholipase A2 receptor protein is emulsified with Freund's incomplete adjuvant for a second immunization. Five to nine days after the second immunization, the phospholipase A2 receptor protein was emulsified with Freund's incomplete adjuvant and a third immunization was performed. Five to nine days after the third immunization, the phospholipase A2 receptor protein was emulsified with Freund's incomplete adjuvant and then immunized four times.

8. The method for preparing the antiphospholipase A2 receptor polyclonal antibody according to claim 1, characterized in that, The antibody separation and purification includes: Clean the affinity chromatography column, filter the serum to be purified, pass the filtered serum into the affinity chromatography column, clean the affinity chromatography column and elute.

9. The method for preparing the antiphospholipase A2 receptor polyclonal antibody according to any one of claims 1-8, characterized in that, The preparation method includes the following steps: (1) Insert the nucleic acid sequence encoding the phospholipase A2 receptor protein into the expression vector to obtain a recombinant vector. Introduce the recombinant vector into eukaryotic cells, culture them, and then isolate and purify the phospholipase A2 receptor protein. Collect the phospholipase A2 receptor protein. The amino acid sequence of the phospholipase A2 receptor protein is shown in SEQ ID NO.

1. (2) The phospholipase A2 receptor protein was emulsified with Freund's complete adjuvant and the animals were immunized for the first time; 10-18 days after the first immunization, the phospholipase A2 receptor protein was emulsified with Freund's incomplete adjuvant and a second immunization was performed; 5-9 days after the second immunization, the phospholipase A2 receptor protein was emulsified with Freund's incomplete adjuvant and a third immunization was performed; 5-9 days after the third immunization, the phospholipase receptor A2 protein was emulsified with Freund's incomplete adjuvant and a fourth immunization was performed; 5-9 days after the fourth immunization, serum was collected. (3) Clean the affinity chromatography column, filter the serum to be purified, pass the filtered serum into the affinity chromatography column, clean the affinity chromatography column and elute, collect the antibody, and obtain the antiphospholipase A2 receptor polyclonal antibody.

10. A polyclonal antibody against phospholipase A2 receptor, characterized in that, The polyclonal antibody is prepared by the method for preparing the antiphospholipase A2 receptor polyclonal antibody according to any one of claims 1-9.

11. The use of the antiphospholipase A2 receptor polyclonal antibody according to claim 10 in constructing an animal model of anti-PLA2R-related membranous nephropathy.

12. A method for constructing an animal model of anti-PLA2R-related membranous nephropathy, characterized in that, The method for constructing an animal model of anti-PLA2R-related membranous nephropathy includes: The CAG-LSL-PLA2R1-polyA gene fragment was inserted at the Rosa26 site in the genome of an experimental animal to obtain a transgenic experimental animal. The transgenic experimental animal was then injected with the anti-phospholipase A2 receptor polyclonal antibody as described in claim 10 to induce a passive immune response and obtain an anti-PLA2R-related membranous nephropathy animal model.

13. The method for constructing an animal model of anti-PLA2R-related membranous nephropathy according to claim 12, characterized in that, The experimental animals include mice or rats.

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