Anti-phospholipase A2 receptor polyclonal antibody as well as preparation method and application thereof

Abstract

The invention relates to an anti-phospholipase A2 receptor polyclonal antibody as well as a preparation method and application thereof. The preparation method comprises the following steps: immunizing animals by taking phospholipase A2 receptor protein as an antigen, and collecting serum; carrying out antibody separation and purification on the serum, and collecting an antibody to obtain the anti-phospholipase A2 receptor polyclonal antibody, the amino acid sequence of the phospholipase A2 receptor protein comprises a sequence as shown in SEQ ID NO. 1. The specific antigen is designed and used for preparing the polyclonal antibody, the polyclonal antibody capable of specifically recognizing the phospholipase A2 receptor can be efficiently prepared, and the polyclonal antibody prepared by using the eukaryotic expression antigen is found to have the advantages that compared with the polyclonal antibody prepared by using the prokaryotic expression or polypeptide route source antigen, the polyclonal antibody prepared by using the eukaryotic expression antigen has the advantages that the specificity is high; and the anti-PLA2R related membranous nephropathy (called PLA2R MN for short) animal model can be more stably and efficiently constructed.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to an anti-phospholipase A2 receptor polyclonal antibody and a preparation method and application thereof. Background Art

[0002] Phospholipase A2 (PLA2) can be divided into two types: secretory and cytoplasmic. The secretory phospholipase A2 (sPLA2) receptor acts as a receptor for the phospholipases 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 the clearance of sPLA2. The binding of sPLA2-IB / PLA2G1B induces various effects depending on the cell type, such as activation of the mitogen-activated protein kinase (MAPK) cascade to induce cell proliferation, the production of lipid mediators, and the selective release of arachidonic acid in 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 response to proinflammatory cytokine production during endotoxic shock. It also has 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] A study reported the use of CRISPR Cas9 technology to construct a mouse model, generating a transgenic mouse strain that expresses the full-length mouse phospholipase A2 receptor in podocytes, and successfully constructed a passive immune anti-PLA2R-associated membranous nephropathy (PLA2R MN) mouse model by injecting anti-phospholipase A2 receptor antibodies, providing an important experimental animal platform for the mode and mechanism of podocyte injury.

[0004] However, there are few reports on the preparation of anti-phospholipase A2 receptor antibodies and the construction of models, and it is not clear what specific source of anti-phospholipase A2 receptor antibodies should be selected to stably and efficiently construct the PLA2R MN animal model. Therefore, in-depth research on the preparation process of anti-phospholipase 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 and other fields. Summary of the invention

[0005] In view of the deficiencies of the prior art and actual needs, the present invention provides an anti-phospholipase A2 receptor polyclonal antibody and a preparation method and application thereof, in order to achieve stable and efficient preparation of a PLA2R MN animal model.

[0006] To achieve this object, the present invention adopts the following technical solutions:

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

[0008] Animals are immunized with phospholipase A2 receptor protein (M-phospholipase A2 receptor, PLA2R) as an antigen, and serum is collected; the serum is subjected to antibody separation and purification, and antibodies are 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 the present invention, a specific antigen is designed and used to prepare a polyclonal antibody, so that a polyclonal antibody that specifically recognizes the phospholipase A2 receptor can be efficiently prepared.

[0010] SEQ ID NO.1:

[0011] HIQEPSLEWRDKGIFIIQSESLKTCIQAGKSVLTLENCKQPNEHMLWKWVSDDHLFNVGGSGCLGLNISALEQPLKLYECDSTLISLRWHCDRKMIEGPLQYKVQVKSDNTVVARKQIHRWIAYTSSGGDICEHPSRDLYTLKGNAHGMPCVFPFQFKGHWHHDCI REGQKEHLLWCATTSRYEEDEKWGFCPDPTSMKVFCDATWQRNGSSRICYQFNLLSSLSWNQAHSSCLMQGGALLSIADEDEEDFIRKHLSKVVKEVWIGLNQLDEKAGWQWSDGTPLSYLNWSQEITPGPFVEHHCGTLEVVSAAWRSRDCESTLPYICKRDLN.

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

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

[0014] In the present invention, the phospholipase A2 receptor protein is prepared by expressing eukaryotic cells as an antigen, which can more effectively prepare polyclonal antibodies and be effectively used to construct a 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 comprises a plasmid vector.

[0019] It can be understood that the commonly used eukaryotic expression plasmid vectors in the art are applicable to the present invention without special restrictions, such as pcDNA vectors. In addition to pcDNA vectors, there are several other common eukaryotic expression plasmids, each of which has unique characteristics and application ranges: 1. pEGFP-N1: This is an enhanced green fluorescent protein (EGFP) expression vector, commonly used for the expression of fusion proteins and intracellular localization studies; 2. pCMVp-NEO-BAN: This vector contains a cytomegalovirus (CMV) promoter, which is 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 a yeast expression system, such as the plasmid used in the Pichia pastoris system, which contains the methanol yeast alcohol oxidase gene promoter (PAXOI) for the expression of exogenous genes, especially when methanol is the sole carbon source; 6. Baculovirus expression vector: a plasmid used in a yeast expression system, such as a plasmid used in a Pichia pastoris system, which contains the methanol yeast alcohol oxidase gene promoter (PAXOI) for the expression of exogenous genes, especially when methanol is used as the sole carbon source; Vector: Baculovirus expression vector used in insect cell expression system, using nuclear polyhedron gene promoter for efficient exogenous gene expression. 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, pcDNA3.1(+) vector contains CMV enhancer and promoter, which is suitable for achieving high-level gene expression in a variety of mammalian cell lines, and contains multiple cloning sites for the insertion of target genes.

[0020] It can be understood that the eukaryotic cells commonly used in the art for expressing and preparing proteins are suitable for the present invention, such as HEK293 and CHO. 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 in transient transfection experiments to produce high levels of protein expression; 2. HeLa cells: a widely used human cervical cancer cell line, which is widely used due to its rapid growth and easy transfection characteristics; 3. NIH / 3T3 cells: a mouse embryonic fibroblast cell line, commonly used in gene expression, cell signaling and cancer research; 4. Jurkat cells: a human leukemia T cell line, commonly used in immunology research and T cell biology research; 5. BHK-21 cells: a cell line derived from Syrian hamster kidney, commonly used in virology research and protein production; 6. Sf9 and Sf21 cells: these two insect cell lines are derived from Spodoptera frugiperda (Spodoptera frugiperda), commonly used in baculovirus expression systems for efficient expression of foreign genes; 7. Pichia pastoris: a methanol yeast, commonly used for protein expression, especially in industrial-scale production; 8. Xenopus oocytes: oocytes of African clawed frogs, often used to study the functions of ion channels and other membrane proteins; 9. MDCK cells: canine kidney epithelial cells, often used to study cell polarity and virology; 10. K562 cells: a human chronic myeloid leukemia cell line, often used to study cell cycle and gene regulation. You can choose according to the actual situation.

[0021] In the present invention, any culture method that can culture eukaryotic cells to express phospholipase A2 receptor protein is applicable to the present invention, and any method for separating and purifying phospholipase A2 receptor protein is applicable to the present invention without special restrictions. For example, the method for separating and purifying phospholipase A2 receptor protein can be: through the his affinity purification tag on the protein, through specific binding with a nickel column, to obtain a purified target protein.

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

[0023] Preferably, the rabbit includes New Zealand rabbit and the like.

[0024] Preferably, the immunization specifically comprises:

[0025] Freund's complete adjuvant is used to emulsify the phospholipase A2 receptor protein and immunize animals for the first time; 10 to 18 days after the first immunization, the phospholipase A2 receptor protein is emulsified with Freund's incomplete adjuvant for a second immunization; 5 to 9 days after the second immunization, the phospholipase A2 receptor protein is emulsified with Freund's incomplete adjuvant for a third immunization; 5 to 9 days after the third immunization, the phospholipase A2 receptor protein is emulsified with Freund's incomplete adjuvant for a fourth immunization.

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

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

[0028] The affinity chromatography column is cleaned, the serum to be purified is filtered, the filtered serum is passed through the affinity chromatography column, the affinity chromatography column is cleaned and eluted.

[0029] Preferably, after the immunization, the method further comprises determining the titer of the serum against the phospholipase A2 receptor and whether the serum can specifically recognize the phospholipase A2 receptor by an ELISA experiment.

[0030] As a preferred technical solution, the method for preparing the anti-phospholipase A2 receptor polyclonal antibody comprises the following steps:

[0031] (1) inserting a nucleic acid sequence encoding a phospholipase A2 protein into an expression vector to obtain a recombinant vector, introducing the recombinant vector into eukaryotic cells, culturing the cells, separating and purifying the phospholipase A2 receptor protein, and collecting the phospholipase A2 receptor protein, wherein the amino acid sequence of the phospholipase A2 receptor protein includes the sequence shown in SEQ ID NO.1;

[0032] (2) using Freund's complete adjuvant to emulsify the phospholipase A2 receptor protein and immunize the animal for the first time; 10 to 18 days after the first immunization, using Freund's incomplete adjuvant to emulsify the phospholipase A2 receptor protein and perform a second immunization; 5 to 9 days after the second immunization, using Freund's incomplete adjuvant to emulsify the phospholipase A2 receptor protein and perform a third immunization; 5 to 9 days after the third immunization, using Freund's incomplete adjuvant to emulsify the phospholipase A2 receptor protein and perform a fourth immunization; 5 to 9 days after the fourth immunization, collecting serum;

[0033] (3) cleaning the affinity chromatography column, filtering the serum to be purified, passing the filtered serum into the affinity chromatography column, cleaning the affinity chromatography column and eluting it, collecting the antibody, and obtaining the anti-phospholipase A2 receptor polyclonal antibody.

[0034] In a second aspect, the present invention provides an anti-phospholipase A2 receptor polyclonal antibody, wherein the polyclonal antibody is prepared by the method for preparing the anti-phospholipase A2 receptor polyclonal antibody according to the first aspect.

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

[0036] In a fourth aspect, the present invention provides a method for constructing an anti-PLA2R-associated membranous nephropathy animal model, the method for constructing an anti-PLA2R-associated membranous nephropathy animal model comprising:

[0037] The CAG-LSL-Pla2r1-polyA gene fragment is site-specifically inserted into the Rosa26 site of the experimental animal genome to obtain a transgenic experimental animal, and the anti-phospholipase A2 receptor polyclonal antibody according to claim 7 is injected into the transgenic experimental animal to induce the transgenic animal to passively produce an immune response, thereby obtaining an anti-PLA2R-related membranous nephropathy animal model.

[0038] Preferably, the CAG-LSL-PLA2R1-polyA gene fragment refers to the CAG promoter-loxp-stop-loxp-PLA2R1 CDS-polyA termination element. It involves a specific gene expression system, which uses the 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, and the target gene is located downstream thereof. In the absence of Cre recombinase, the expression cassette theoretically blocks the normal expression of the target gene; when Cre is introduced into cells carrying the vector, the LSL expression cassette upstream of the target gene will be deleted, so that the target gene can be permanently activated for expression under the selected promoter. This vector system is suitable for the construction of transgenic animal models.

[0039] In the early stage, the mouse model of the present invention uses CAG as a broad-spectrum promoter with a length of about 1.7kb; the mouse PLA2R1 gene has 6 transcripts, one of which (PLA2R1-201) has a determined CCDS, and the CDS of the Pla2r1-201 (ENSMUST00000112525.4) transcript is selected for model making, with a length of 4464nt and encoding 1487aa; Rosa26 is located on mouse chromosome 6 as a safe site for exogenous gene insertion, and the exogenous gene integrated into this site can be stably and efficiently expressed without destroying the function of the endogenous gene. Combined with the use of the Cre-loxP system, it can be used to construct a multi-purpose conditional gene knock-in mouse model. Before the Cre is matched, the expression of the transferred PLA2R1 is in a closed state.

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

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

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

[0043] In a specific embodiment of the present invention, the CRISPR / Cas9 technology can be used to insert the CAG-LSL-Pla2r1-polyA gene fragment into the Rosa26 site of the mouse. The brief process is as follows: sgRNA is transcribed in vitro, a donor vector is constructed, and Cas9, donor and sgRNA are microinjected into the fertilized eggs of C57BL / 6J mice to obtain F0 generation mice. The F0 generation positive mice that are correct by PCR, sequencing and southern verification are mated with C57BL / 6J mice to obtain a stable and heritable F1 generation positive mouse model. To breed transgenic mice, mice aged 13-14 weeks were selected, including 12 PLA2R1 transgenic mice: CAG-LSL-PLA2R1-polyA-KI(Rosa26) / NPHS2-CreT (referred to as KI mice); 6 PLA2R control group mice: CAG-LSL-PLA2R1-polyA-KI(Rosa26) / NPHS2-CreW (referred to as WT mice), and they were randomly divided into KI mice + anti-mouse PLA2R polyclonal antibody group (referred to as KI+PLA2R), KI mice + non-immunized rabbit total IgG group (referred to as KI+IgG); WT mice + PBS solution group (referred to as WT+PBS). In the KI+PLA2R group, anti-mouse PLA2R polyclonal antibody (3 mg / mouse) was injected intraperitoneally on the first day of the experiment, and anti-mouse PLA2R polyclonal antibody (2 mg / mouse) was injected intraperitoneally again on the second day; in the KI+IgG group, unimmunized rabbit total IgG (3 mg / mouse) was injected intraperitoneally on the first day of the experiment, and unimmunized rabbit total IgG (2 mg / mouse) was injected intraperitoneally again on the second day; in the WT+PBS group, an equal volume of PBS solution was injected intraperitoneally on the first day of the experiment, and an equal volume of PBS solution was injected intraperitoneally again on the second day. The 24-hour urine of mice in each group was collected on the 1st, 3rd, 5th, and 7th day, and the urine volume was evaluated, and the urine albumin-creatinine ratio was detected. After the 8th day, the mice in each group were killed, and the serum of mice in each group was collected for biochemical index detection: serum albumin, serum cholesterol; the bilateral kidneys of mice in each group were collected for pathological index detection: renal pathology (paraffin sections were subjected to PAS, PASM staining and electron microscopy), immunopathology (PLA2R, IgG, complement C3 immunofluorescence).

[0044] The present invention aims to induce the construction of a PLA2R MN mouse model by giving PLA2R1 transgenic mice multiple passive immunizations with anti-phospholipase A2 receptor (Anti-Phospholipase A2 Receptor, PLA2R) antibodies, which can quickly induce nephrotic syndrome in PLA2R1 transgenic mice and activate the complement system, indicating that the antibody can quickly induce immune damage to podocytes. The model shows similar characteristics to human PLA2R-related membranous nephropathy in biochemical indicators and pathological characteristics, providing an important experimental animal platform for further research on PLA2R MN. In the present invention, an in-depth analysis of the construction of the PLA2R MN animal model was conducted, and it was found that the polyclonal antibody prepared by using the phospholipase A2 receptor of a specific sequence expressed in eukaryotic cells as an antigen can be used to prepare the polyclonal antibody, and the obtained antibody 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 the present invention, specific antigens are designed and used to prepare polyclonal antibodies, which can efficiently prepare polyclonal antibodies that specifically recognize the phospholipase A2 receptor, and it is found that polyclonal antibodies prepared using eukaryotic expressed antigens can more stably and efficiently construct animal models than polyclonal antibodies prepared using prokaryotic expressed or polypeptide-derived antigens. The first animal model of anti-phospholipase A2 receptor antibody-related membranous nephropathy in China was successfully constructed by passive immunization, providing an important experimental animal platform for understanding the mode and mechanism of action of phospholipase A2 receptor in situ immune complex-induced podocyte injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 This is a diagram of the identification results of phospholipase A2 receptor protein.

[0048] Figure 2 Schematic diagram of the principle of site-directed insertion of the CAG-LSL-PLA2R1-polyA gene fragment into the Rosa26 locus of mice;

[0049] Figure 3 The results of urine and biochemical index detection of mice in WT+PBS group, KI+IgG group and KI+PLA2R group, among which, Figure A is the urine albumin-creatinine ratio of mice in each group, Figure B is the serum albumin level of mice in each group, Figure C is the change of 24h urine volume of mice in each group on the 1st, 3rd, 5th and 7th day, Figure D is the serum cholesterol level of mice in each group, *P<0.0001;

[0050] Figure 4 The results of PAS, PASM and electron microscopic pathological indexes of mice in the WT+PBS group, KI+IgG group and KI+PLA2R group are shown in the figure;

[0051] Figure 5Figure 2 shows the results of detection of PLA2R, C3 and IgG immunopathological indexes in mice in the WT+PBS group, KI+IgG group and KI+PLA2R group (×400). DETAILED DESCRIPTION

[0052] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and through specific implementation methods. However, the following examples are only 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 shall be subject to the claims.

[0053] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0054] Example 1

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

[0056] 1. Eukaryotic expression of phospholipase A2 receptor protein

[0057] The process of preparing antigens by 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] According to the characteristics of the target antigen gene (SEQ ID NO.2), a suitable eukaryotic expression vector (such as pcDNA vector) is selected. Commonly used vectors include plasmids and viral vectors. These vectors usually contain basic elements such as promoters, selection markers, terminators, and possible regulatory elements such as enhancers.

[0061] Step 2: Gene cloning

[0062] Determine the target antigen gene sequence, select the appropriate restriction enzyme cutting site, and obtain the target DNA fragment through PCR amplification or gene synthesis. Use restriction endonucleases to double-cut the vector and target DNA fragment to ensure the correct direction and position.

[0063] Step 3: DNA ligation and transformation

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

[0065] Step 4: Plasmid extraction and verification

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

[0067] 2. Transduction

[0068] Step 1: Cell preparation

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

[0070] Step 2: Transduction method selection

[0071] Select an appropriate transduction method according to experimental requirements and cell types, such as plasmid transfection (such as heat shock transfection, electroporation transfection, chemical transfection, etc.), viral infection, etc.

[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 placing it under appropriate culture conditions.

[0073] 3. Cell Culture

[0074] Step 1: Optimization of culture conditions

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

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

[0077] For some genes that need to be induced to express, inducers (such as IPTG, tetracycline, etc.) are added at specific time points to stimulate cells to express the target antigen.

[0078] Step 3: Cell growth monitoring

[0079] Observe the cell growth status regularly to ensure that the cells are healthy and have reached the expected confluence.

[0080] 4. Separation and purification

[0081] Step 1: Cell harvesting

[0082] When the cells express a sufficient amount of antigen, the cells are harvested by centrifugation or the like.

[0083] Step 2: Cell lysis

[0084] Use an appropriate lysis buffer to disrupt cells and release intracellular proteins. Lysis methods may include mechanical disruption, ultrasonic disruption, chemical lysis, etc.

[0085] Step 3: Antigen purification

[0086] According to the characteristics of the antigen, a suitable purification method is selected, such as affinity chromatography, ion exchange chromatography, gel filtration chromatography, etc. 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 by enzyme-linked immunosorbent assay (ELISA). The eukaryotic expression system was successfully used to prepare high-purity and high-activity antigen proteins.

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

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

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

[0093] 4) Staining: Take out the gel after electrophoresis from the electrophoresis tank, carefully remove the gel from the glass plate, add Coomassie Brilliant Blue staining solution, and shake it horizontally for 60 minutes.

[0094] 5) Add destaining solution and change it several times until the background is clear (in order to save time, the number of changes of destaining solution can be increased).

[0095] 6) After the background is clear, pour out the decolorizing solution, wash the glue several times with deionized water, seal the glue with plastic film and scan (no bubbles when sealing the glue), scan, edit and save the record with Photoshop.

[0096] Antigen identification results Figure 1 As shown, a protein of the expected size was obtained.

[0097] Example 2

[0098] In this example, the phospholipase A2 receptor protein prepared in Example 1 was used as an antigen to prepare polyclonal antibodies.

[0099] 1. Rabbit Immunization Protocol

[0100] 1.1 Animal selection: New Zealand white rabbits are used, weighing about 2.5 kg, young and strong. The animals should be healthy animals with shiny fur and free movement. The selected animals should be pre-raised for about 2 weeks. The purpose is to eliminate some unqualified animals so that the later experiments can proceed smoothly.

[0101] 1.2 Preparation before the experiment: The rabbits were marked.

[0102] 1.3 Antigen preparation:

[0103] 1.3.1 Take the antigen out of the -20℃ freezer and dissolve it at room temperature to avoid repeated freezing and thawing. Mark the syringe with the project number and animal number.

[0104] 1.3.2 Extract antigen (mix antigen completely), the antigen concentration for the first immunization is 1 mg / mL, 0.5 mL / rabbit, and the antigen amount for the second to fourth immunizations is halved.

[0105] 1.3.3 Extract the adjuvant in a 1:1 volume ratio of adjuvant to antigen. Use complete adjuvant for the first immunization and incomplete adjuvant for the second to fourth immunizations. When extracting, mix the adjuvant thoroughly before drawing it into the syringe.

[0106] 1.3.4 After the two syringes are connected with a 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 given multiple subcutaneous injections, with 0.2 mL at each point.

[0108] Immunization time: The second vaccination is carried out on the 14th day after the first vaccination, and the interval between the second and third vaccinations is 7 days. On the 7th day after the third vaccination, a small sample of serum is collected from the middle ear artery of the rabbit for testing. If the test is qualified, an additional vaccination will be carried out 7 days later, and whole blood can be collected 7 days after the additional vaccination.

[0109] 1.4.1 Steps for collecting small serum samples

[0110] The experimental rabbit was fixed in a fixed frame, and the ears were patted by hand to dilate the central artery of the ear. Disinfected with 75% alcohol, the rabbit's ears were fixed with the left hand, and the syringe was held in the right hand. The needle was inserted into the central artery of the ear 1 / 3 of the distance from the end, parallel to the artery, and inserted into the artery in the centripetal direction. 8mL of blood was collected at a time, and cotton balls were used to press and stop bleeding after blood collection.

[0111] 1.4.2 Whole blood collection steps

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

[0113] 1.4.2.2 After anesthesia, the immunized rabbit was placed with its abdomen facing upwards, and its limbs were fixed on a stainless steel mesh frame, and the cardiac blood sampling method was used.

[0114] 1.4.2.3 After blood collection, place the centrifuge tube containing blood in a 37℃ water bath for 20 minutes, take it out, cool it down, and place it in a 4℃ refrigerator. Wait for the blood to automatically separate into layers, and take out the supernatant into a clean 50mL centrifuge tube.

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

[0116] 2.ELISA test (indirect method)

[0117] 2.1 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, and incubate at 4°C overnight. The next day, discard the solution in the wells and wash once with 1×PBST washing buffer (180 μL per well).

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

[0119] 2.3 Sample addition: Add a certain dilution of the sample to be tested (dilute the sample to be tested according to a certain ratio), 50 μL to the above-mentioned blocked reaction wells. At the same time, set up a negative control well (1% BSA). Incubate at 37°C for 30 minutes, and wash 3 times with 1×PBST washing buffer, 150 μL per well.

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

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

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

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

[0124] 3. Antibody Purification

[0125] 3.1 The affinity chromatography column was thoroughly cleaned 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 serum to be purified and place it in a 50 mL centrifuge tube. Filter it using a microporous filter 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 1×PBS (pH 7.4) at a flow rate of 70 mL / h. Connect the column to the protein detector after 10 minutes. During the washing process, adjust the instrument transmittance (T gear) to 100.

[0129] 3.5 Adjust the protein detector absorbance (1A gear) to 0. At this time, turn on the HD-A computer collector on the computer desktop and adjust the full-screen range to 5. Use glycine solution (pH 2.7, 0.2M) at a rate of 40mL / h to elute the antibody. At this time, press the green elution record button to start elution. When the instrument reading begins to rise, start collecting the antibody.

[0130] 3.6 During the antibody collection process, adjust the pH value of the antibody to about 7 with 1M sodium bicarbonate and record the highest peak of the elution peak.

[0131] 3.7 After collecting the antibodies, adjust the pH value to about 7 and record the volume of eluted antibodies. Then rinse the rubber tube connected to the collector with pure water.

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

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

[0134] 3.10 After the antibody quantity for delivery is purified and the semi-finished product has passed the titer test, all antibodies are mixed and concentrated using ultrafiltration concentrators to reach a certain concentration and volume.

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

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

[0137] 3.13 The concentration was measured using the Protein A280 application on a denovix DS-11 ultra-micro spectrophotometer.

[0138] The test results of serum ELISA are shown in Table 1. The results show that when the amount of antigen coated is 50 ng / well, the serum is diluted from 250 and diluted 4 times, the serum titer can reach more than 1024000.

[0139] Table 1

[0140]

[0141] The ELISA test results of the purified antibody are shown in Table 2. The results show that when the antigen amount coated is 50 ng / well, the purified antibody is diluted from 250 and diluted 4 times, the titer of the purified antibody can reach more than 1024000.

[0142] Table 2

[0143]

[0144]

[0145] Example 3

[0146] In this example, the anti-phospholipase A2 receptor polyclonal antibody prepared in Example 2 was used to construct a PLA2R MN animal model and to perform model verification.

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

[0148] CAG: is a powerful comprehensive promoter (Cytomegalovirus enhancer / chickenβ-actinpromoter) that can drive genes to achieve high levels of expression in a variety of cell types.

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

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

[0151] PolyA: is a polyadenylation signal sequence, usually located at the 3' end of the gene, which contributes to the stability and translation efficiency of mRNA.

[0152] KI (Knock-In): refers to cas9-ki, which is a gene knock-in technology that precisely inserts exogenous genes or gene fragments into a specific location in the genome through homologous recombination.

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

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

[0155] CAG promoter-loxp-stop-loxp-Pla2r1 CDS-polyA stop original mice were bred with NPHS2-Cre to obtain double positive mice, which eventually deleted the two loxp middle regions. This is the "CAG-LSL-PLA2R1-polyA-KI(Rosa26) / NPHS2-CreT" mouse (referred to as KI mouse), a genetically engineered mouse used to study the role of PLA2R1 in kidney disease. The conditional expression of the PLA2R1 gene was achieved by knocking in a specific gene construct at the Rosa26 site and using the Cre recombinase driven by the NPHS2 promoter. The "CAG-LSL-PLA2R1-polyA-KI(Rosa26) / NPHS2-CreW" mouse (referred to as WT mouse) is a control group mouse (wild-type mouse), which is cre-negative and will not eventually delete the region between the two loxp sites, which usually means that they will not undergo recombination events between loxP sites in the absence of Cre enzyme. In other words, without the activity of the Cre enzyme, 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 whether the gene is turned on or off by introducing the Cre enzyme, thereby studying the role of a specific gene in a specific cell or tissue. This model can be used as a control group to study the biological effects in the absence of specific gene expression.

[0156] For breeding transgenic mice, 13-14 week-old mice were selected, and 12 PLA2R1 transgenic mice were selected: CAG-LSL-PLA2R1-polyA-KI(Rosa26) / NPHS2-CreT (referred to as KI mice, the CAG-LSL-Pla2r1-polyA gene fragment was inserted into the Rosa26 site of mice. The schematic diagram is as follows Figure 2); 6 PLA2R control group mice: CAG-LSL-PLA2R1-polyA-KI(Rosa26) / NPHS2-CreW (referred to as WT mice), were randomly divided into KI mice + anti-mouse PLA2R polyclonal antibody group (referred to as KI+PLA2R), KI mice + non-immunized rabbit total IgG group (referred to as KI+IgG); WT mice + PBS solution group (referred to as WT+PBS). The KI+PLA2R group was intraperitoneally injected with anti-mouse PLA2R polyclonal antibody (3 mg / mouse) prepared in Example 2 on the first day of the experiment, and the anti-mouse PLA2R polyclonal antibody (2 mg / mouse) was intraperitoneally injected again on the second day; the KI+IgG group was intraperitoneally injected with non-immunized rabbit serum IgG (3 mg / mouse) on the first day of the experiment, and non-immunized rabbit total IgG (2 mg / mouse) was intraperitoneally injected again on the second day; the WT+PBS group was intraperitoneally injected with an equal volume of PBS solution on the first day of the experiment, and the same volume of PBS solution was intraperitoneally injected again on the second day. The 24-hour urine of each group of mice was collected on the 1st, 3rd, 5th and 7th day to evaluate the urine volume and detect the urine albumin-creatinine ratio. The mice in each group were killed on the 8th day, and the serum of each group of mice was collected for biochemical index detection: serum albumin, serum cholesterol; the bilateral kidneys of each group of mice were collected for pathological index detection: renal pathology (paraffin sections for PAS, PASM staining and electron microscopy), immunopathology (PLA2R, IgG, complement C3 immunofluorescence).

[0157] The results of urine and biochemical indexes of 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, the 24-h urine volume of mice in the KI+PLA2R group decreased on days 1, 3, 5, and 7 (P<0.05). The urine albumin-creatinine ratio increased significantly (P<0.05), reaching a peak on day 5 (KI+PLA2R group: 946.36±304.33μg / mL vs.KI+IgG group: 32.34±3.24μg / mL vs.WT+PBS group: 43.46±25.41μg / mL, P<0.001), and slightly decreased on day 7 (KI+PLA2R group: 578.31±161.75μg / mL vs.KI+IgG group: 29.33±5.35μg / mL vs.WT+PBS group: 36.48±12.15μg / mL, P<0.001); serum albumin decreased significantly (experimental day 8: KI+PLA2R group: 11.68±1.24g / L vs.KI+IgG group: 28.00±1.02g / L vs.WT+PBS group: 28.49±1.03g / L, P<0.001); serum cholesterol increased significantly (experimental day 8: KI+PLA2R group: 23.33±1.95mmol / L vs.KI+IgG group: 7.21±2.54mmol / L vs.WT+PBS group: 8.48±2.46mmol / L, P<0.001). The results of electron microscopy pathological index detection are as follows Figure 4 As shown in the figure, PAS (periodic acid-Schiff staining) and PASM (periodic acid hexamethylenetetramine silver staining) staining of mice in each group showed normal morphology of glomeruli and tubulointerstitium; PASM staining of KI+PLA2R group showed mild thickening of basement membrane, obvious foot process fusion was observed under electron microscopy, and a small amount of electron-dense material was observed under the epithelium and in the basement membrane. The results of immunopathological index detection are shown in the figure. Figure 5 As shown in the figure, the immunopathology of the KI+PLA2R group showed that the IgG and complement C3 in the glomerular basement membrane area of ​​the mice were granular positive, and the PLA2R antigen expression was granular positive. The IgG expression of the mice in the KI+IgG group was weakly positive, and there was no obvious change in the renal pathology of the other mice.

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

[0159] In summary, the present invention designs specific antigens and uses them to prepare polyclonal antibodies, which can efficiently prepare polyclonal antibodies that specifically recognize phospholipase A2 receptors, and finds that polyclonal antibodies prepared using eukaryotic expressed antigens can more stably and efficiently construct animal models than polyclonal antibodies prepared using prokaryotic expressed or polypeptide-derived antigens. The first animal model of anti-phospholipase A2 receptor antibody-related membranous nephropathy in China was successfully constructed by passive immunization, providing an important experimental animal platform for understanding the mode and mechanism of action of phospholipase A2 receptor in situ immune complex-induced podocyte injury.

[0160] The applicant declares that the above is only a specific implementation mode 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 thought of by those skilled in the art within the technical scope disclosed by the present invention are within the protection scope 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 comprises: Animals were immunized with phospholipase A2 receptor protein as antigen and serum was collected; Separating and purifying the serum for antibodies, collecting antibodies, and obtaining 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.

2. The method for preparing anti-phospholipase A2 receptor polyclonal antibodies according to claim 1, characterized in that: The preparation method of the phospholipase A2 protein comprises: The nucleic acid sequence encoding the phospholipase A2 receptor protein is inserted into an expression vector to obtain a recombinant vector, and the recombinant vector is introduced into eukaryotic cells. After culturing, the phospholipase A2 receptor protein is separated and purified to collect the phospholipase A2 receptor protein.

3. The method for preparing anti-phospholipase A2 receptor polyclonal antibodies according to claim 2, characterized in that: The nucleic acid sequence encoding the phospholipase A2 receptor protein includes the sequence shown in SEQ ID NO.2; Preferably, the expression vector comprises a plasmid vector.

4. The method for preparing an anti-phospholipase A2 receptor polyclonal antibody according to any one of claims 1 to 3, characterized in that: The animal includes any one of rabbit, guinea pig, rat, mouse or sheep; Preferably, the rabbit comprises a New Zealand rabbit; Preferably, the immunization specifically comprises: emulsifying the phospholipase A2 receptor protein with Freund's complete adjuvant and immunizing animals for the first time; 10 to 18 days after the first immunization, the phospholipase A2 receptor protein is emulsified with Freund's incomplete adjuvant for secondary immunization; 5 to 9 days after the second immunization, the phospholipase A2 receptor protein is emulsified with Freund's incomplete adjuvant and immunized for the third time; 5 to 9 days after the third immunization, the phospholipase A2 receptor protein was emulsified with Freund's incomplete adjuvant and immunized four times.

5. The method for preparing an anti-phospholipase A2 receptor polyclonal antibody according to any one of claims 1 to 4, characterized in that: The antibody separation and purification comprises: The affinity chromatography column is cleaned, the serum to be purified is filtered, the filtered serum is passed through the affinity chromatography column, the affinity chromatography column is cleaned and eluted.

6. The method for preparing an anti-phospholipase A2 receptor polyclonal antibody according to any one of claims 1 to 5, characterized in that: The preparation method comprises the following steps: (1) inserting a nucleic acid sequence encoding a phospholipase A2 receptor protein into an expression vector to obtain a recombinant vector, introducing the recombinant vector into eukaryotic cells, culturing the cells, separating and purifying the phospholipase A2 receptor protein, and collecting the phospholipase A2 receptor protein, wherein the amino acid sequence of the phospholipase A2 receptor protein includes the sequence shown in SEQ ID NO.1; (2) using Freund's complete adjuvant to emulsify the phospholipase A2 receptor protein and immunize the animal for the first time; 10 to 18 days after the first immunization, using Freund's incomplete adjuvant to emulsify the phospholipase A2 receptor protein and perform a second immunization; 5 to 9 days after the second immunization, using Freund's incomplete adjuvant to emulsify the phospholipase A2 receptor protein and perform a third immunization; 5 to 9 days after the third immunization, using Freund's incomplete adjuvant to emulsify the phospholipase A2 receptor protein and perform a fourth immunization; 5 to 9 days after the fourth immunization, collecting serum; (3) cleaning the affinity chromatography column, filtering the serum to be purified, passing the filtered serum into the affinity chromatography column, cleaning the affinity chromatography column and eluting it, collecting the antibody, and obtaining the anti-phospholipase A2 receptor polyclonal antibody.

7. An anti-phospholipase A2 receptor polyclonal antibody, characterized in that: The polyclonal antibody is prepared by the method for preparing the anti-phospholipase A2 receptor polyclonal antibody according to any one of claims 1 to 6.

8. Use of the anti-phospholipase A2 receptor polyclonal antibody according to claim 7 in detecting phospholipase A2 receptor or constructing an anti-PLA2R-related membranous nephropathy animal model.

9. A method for constructing an anti-PLA2R-related membranous nephropathy animal model, characterized in that: The method for constructing an anti-PLA2R-related membranous nephropathy animal model comprises: The CAG-LSL-PLA2R1-polyA gene fragment is site-specifically inserted into the Rosa26 site of the experimental animal genome to obtain a transgenic experimental animal, and the anti-phospholipase A2 receptor polyclonal antibody according to claim 7 is injected into the transgenic experimental animal to induce the transgenic animal to passively produce an immune response, thereby obtaining an anti-PLA2R-related membranous nephropathy animal model.

10. The method for constructing an anti-PLA2R-related membranous nephropathy animal model according to claim 9, characterized in that: The experimental animals include mice or rats.

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