Fusion protein and its use

Abstract

The present invention provides a fusion protein and a method for detecting PLA2R antibodies. The fusion protein comprises a PLA2R antigen epitope peptide, albumin, or a fragment thereof, wherein the PLA2R antigen epitope peptide is linked to the albumin or a fragment thereof. The method comprises: using the fusion protein to detect PLA2R antibodies in a test sample, and determining the PLA2R antibody content in the test sample based on the detection results. The fusion protein of the present invention can specifically bind to PLA2R antibodies, exhibiting advantages such as strong binding ability and high sensitivity. The method of the present invention has high sensitivity and accuracy in detecting PLA2R antibodies.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical engineering technology. Specifically, the present invention relates to a fusion protein and its use. More specifically, the present invention relates to a fusion protein, a nucleic acid molecule, an expression vector, a recombinant cell, a kit and its use, and a method for detecting PLA2R antibodies. Background Art

[0002] Primary membranous nephropathy, also known as idiopathic membranous nephropathy or spontaneous membranous nephropathy, is a major cause of renal failure and the leading cause of nephrotic syndrome. While no more than 30% of cases of primary membranous nephropathy may resolve spontaneously, another 30% of patients experience a slow progression of renal failure over approximately 10 years, posing a serious threat to people's health and life. Recent research has confirmed that primary membranous nephropathy is a kidney-specific autoimmune disease. Under disease conditions, the body's autoimmune antigens activate the immune system, producing autoimmune antibodies. These autoimmune antibodies bind to autoimmune antigens on glomerular podocytes, triggering an immune attack on the podocytes and surrounding tissues, causing inflammation and glomerular damage.

[0003] Currently, the most well-researched autoimmune antigens for primary MN are PLA2R and THSD7A. Based on the antigen type, MN can be divided into PLA2R-type and THSD7A-type. Approximately 70%-80% of primary MN cases are PLA2R-type, while less than 10% are THSD7A-type. Anti-PLA2R antibodies (PLA2R-Ab) play a key role in the development and progression of PLA2R-type MN. Serum levels of PLA2R-Ab can serve as a predictive marker for PLA2R-type MN; higher PLA2R-Ab titers indicate a lower chance of spontaneous remission. Decreased PLA2R-Ab titers often accompany spontaneous remission and can be as low as undetectable in patients with complete remission. Immunosuppressive therapy can reduce PLA2R-Ab titers.

[0004] The current diagnostic standard for PLA2R-type membranous nephropathy is immunofluorescence or immunohistochemistry of renal biopsy specimens. This procedure is a minor surgical procedure with several drawbacks, including: 1) significant pain for the patient; 2) impracticality in some cases; and 3) inability to repeat the procedure, which provides no information on treatment efficacy.

[0005] Therefore, there is an urgent need for a diagnostic reagent or method that is non-invasive, highly accurate, and more cost-effective for patients. Summary of the Invention

[0006] The present invention aims to solve at least one of the technical problems existing in the prior art to a certain extent. To this end, the present invention provides a fusion protein for specifically binding to PLA2R-Ab. The fusion protein of the present invention can specifically bind to PLA2R-Ab.

[0007] The present invention is accomplished based on the following findings of the inventors:

[0008] PLA2R is a membrane protein highly expressed on the surface of glomerular podocytes. It consists of a CysR domain, a Fibronectin type-II (FnII) domain, eight highly similar C-type lectin domains (CTLDs), a transmembrane domain, and an intracellular domain. Due to its hydrophobic transmembrane structure, it is prone to forming inclusion bodies during recombinant expression, resulting in very low yields.

[0009] Currently, the diagnostic kit from Germany's Euromonenter is commonly used to detect PLA2R-Ab. This kit uses recombinant human PLA2R as the capture molecule. The term "intact human recombinant PLA2R" refers to the extracellular domain of the PLA2R protein, which contains over 1,300 amino acid residues and has a large molecular weight of approximately 185 kD. This molecule is challenging to express, with the product prone to aggregation, low yield, and difficulty in purification. Whether expressing the full-length PLA2R protein or extracellular domain protein fragments, the low expression levels during the production process make it difficult to obtain large quantities of the required protein, hindering the development of diagnostic reagents for PLA2R-type membranous nephropathy and increasing production costs. Furthermore, during clinical application, the inventors discovered that the Euromonenter diagnostic kit may produce false negative results for PLA2R-type membranous nephropathy, affecting diagnostic accuracy.

[0010] However, in its first aspect, the present invention provides a fusion protein. According to an embodiment of the present invention, the fusion protein comprises: a PLA2R antigen epitope peptide; and albumin or a fragment thereof, wherein the PLA2R antigen epitope peptide is linked to the albumin or a fragment thereof. The fusion protein according to the embodiment of the present invention has high expression levels, is easily accessible, and can specifically bind to PLA2R antibodies, exhibiting advantages such as strong binding ability and high sensitivity. It can be used to accurately detect PLA2R antibody levels and diagnose PLA2R-type membranous nephropathy.

[0011] In a second aspect of the present invention, the present invention provides a nucleic acid molecule. According to an embodiment of the present invention, the nucleic acid molecule encodes the fusion protein described in the first aspect. The nucleic acid molecule of the present invention can effectively express the fusion protein described in the first aspect.

[0012] In a third aspect, the present invention provides an expression vector. According to an embodiment of the present invention, the expression vector carries the nucleic acid molecule described in the second aspect. The expression vector of the present invention can effectively express the fusion protein described in the first aspect.

[0013] In its fourth aspect, the present invention provides a recombinant cell. According to embodiments of the present invention, the recombinant cell comprises: a nucleic acid molecule as described in the second aspect or an expression vector as described in the third aspect; or expresses the fusion protein as described in the first aspect. The recombinant cell of the embodiments of the present invention can be used for in vitro expression and large-scale production of the fusion protein as described in the first aspect.

[0014] In a fifth aspect, the present invention provides a kit. According to embodiments of the present invention, the kit comprises: the fusion protein described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, or the recombinant cell described in the fourth aspect. The kit described in embodiments of the present invention can effectively detect PLA2R antibody levels and can also be used to diagnose diseases caused by PLA2R antibodies or assess the staging or prognosis of diseases caused by PLA2R antibodies, such as PLA2R antibody-positive membranous nephropathy.

[0015] In a sixth aspect, the present invention provides a use of the fusion protein of the first aspect, the nucleic acid molecule of the second aspect, the expression vector of the third aspect, or the recombinant cell of the fourth aspect in preparing a kit for detecting PLA2R antibodies, diagnosing diseases related to excessive PLA2R antibodies, evaluating the prognosis of diseases related to PLA2R antibodies, or evaluating the staging of diseases related to PLA2R antibodies.

[0016] In its seventh aspect, the present invention provides a method for detecting PLA2R antibodies. According to embodiments of the present invention, the method comprises: detecting PLA2R antibodies in a test sample using the fusion protein described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, the recombinant cell described in the fourth aspect, or the kit described in the fifth aspect; and determining the PLA2R antibody content in the test sample based on the detection results. The methods described in the embodiments of the present invention can effectively detect PLA2R antibody content and have advantages such as high detection sensitivity and accuracy.

[0017] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the following description of the embodiments with reference to the accompanying drawings, in which:

[0019] Figure 1 Schematic diagram of the structure of PLA2R in an embodiment of the present invention;

[0020] Figure 2 Schematic diagram of the structure of the expression framework in Example 1 of the present invention;

[0021] Figure 3 This is the electrophoresis result of pcDNA3.4 in Example 1 of the present invention;

[0022] Figure 4 This is the electrophoresis result of RC21-RC24 in Example 1 of the present invention;

[0023] Figure 5 Schematic diagram of the structure of RC21-RC24 protein in Example 1 of the present invention;

[0024] Figure 6 The purification effect of RC22 and RC24 proteins was detected by SDS-PAGE in Example 1 of the present invention;

[0025] Figure 7 This is a diagram for determining the coating concentration of RC22 in Example 3 of the present invention;

[0026] Figure 8 This is a diagram for determining the RC24 plate concentration in Example 3 of the present invention;

[0027] Figure 9 This is a graph showing the determination of the plasma dilution of RC22 in Example 3 of the present invention;

[0028] Figure 10 This is a graph showing the determination of the plasma dilution of RC24 in Example 3 of the present invention;

[0029] Figure 11 The standard curves of the Ou Meng test kit, RC22, and RC24 in Example 4 of the present invention are shown below:

[0030] Figure 12 The RC24 test results in Example 4 of the present invention are plotted against the RC22 test results;

[0031] Figure 13 The results of the RC22 test were plotted against the results of the Oumeng test kit in Example 4 of the present invention;

[0032] Figure 14 The results of the RC24 test were plotted against the results of the Oumeng test kit in Example 4 of the present invention;

[0033] Figure 15The results of the RC22 test were plotted against the results of the Euromon test kit after increasing the sample size in Example 5 of the present invention;

[0034] Figure 16 The purification effect of RC3 protein was detected by SDS-PAGE in Example 6 of the present invention;

[0035] Figure 17 This is a graph showing the results of a neutralization experiment performed by the Ou Meng test kit according to Example 6 of the present invention;

[0036] Figure 18 This is a graph showing the neutralization test results of RC22 detected in Example 6 of the present invention. DETAILED DESCRIPTION

[0037] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0038] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, features defined as "first" or "second" may explicitly or implicitly include one or more of such features. Furthermore, in the description of the present invention, unless otherwise specified, "plurality" means two or more.

[0039] The endpoints of the ranges and any values ​​disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0040] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention, but not excluding other contents.

[0041] As used herein, the terms "optionally," "optional," or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0042] As used herein, the terms "identity," "homology," or "similarity" are used to describe an amino acid sequence or nucleic acid sequence relative to a reference sequence, and the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences is determined by conventional methods, for example, see Ausubel et al., eds. (1995), Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN program (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research Foundation, Washington, DC). There are many algorithms for aligning sequences and determining sequence identity, including the homology alignment algorithm of Needleman et al. (1970) J. Mol. Biol. 48:443; the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the similarity search method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 85:2444; the Smith-Waterman algorithm (Meth. Mol. Biol. 10:106); the similarity search method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 10:116; the similarity search method of Pearson et al. (1990) Proc. Natl. Acad. Sci. 10:117; the similarity search method of Pearson et al. (1990 ... .70:173-187 (1997); and BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J. Mol. Biol. 215:403-410). Computer programs that utilize these algorithms are also available and include, but are not limited to, ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul et al., Meth. Enzym., 266:460-480 (1996)); or GAP, BESTFIT, BLAST Altschul et al., supra, FASTA, and TFASTA, available in the Genetics Computing Group (GCG) package, Version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0043] As used herein, the term "nucleotide" generally refers to a nucleotide that can be a ribonucleotide, a deoxynucleotide, or a modified form of either type of nucleotide, and combinations thereof.

[0044] As used herein, the term "at least 90% similarity" refers to at least 90%, and may be 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% similarity to the respective reference sequence.

[0045] Herein, the term "PLA2R antigen epitope peptide" refers to a polypeptide having an amino acid sequence derived from PLA2R. The "PLA2R antigen epitope peptide" comprises an antigen epitope that can specifically bind to a PLA2R antibody. For example, the PLA2R antigen epitope peptide comprises CysR, FnII, and PLA2R binding region 1; wherein the term "CysR" refers to the CysR domain in PLA2R, the term "PLA2R binding region 1" refers to the CTLD domain 1 in PLA2R, and the term "PLA2R binding region 2" refers to the CTLD domain 2 in PLA2R (refer to Figure 1 ).

[0046] In this article, the term "expression vector" generally refers to a nucleic acid molecule that can be inserted into a suitable host and replicates itself, and transfers the inserted nucleic acid molecule into and / or between host cells. The expression vector may include a vector primarily used to insert DNA or RNA into a cell, a vector primarily used to replicate DNA or RNA, and a vector primarily used for expression of the transcription and / or translation of DNA or RNA. The expression vector also includes vectors with multiple of the above functions. The expression vector can be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, the expression vector can produce a desired expression product by culturing a suitable host cell containing the expression vector.

[0047] In this article, the term "recombinant cell" generally refers to a cell in which the genetic material of a host cell is modified or reorganized using genetic engineering techniques or cell fusion techniques to obtain a cell with a unique trait of stable inheritance. Among them, the term "host cell" refers to a prokaryotic cell or eukaryotic cell into which a recombinant expression vector can be introduced. The terms "transformed" or "transfected" as used herein refer to the introduction of a nucleic acid (e.g., a vector) into a cell by various techniques known in the art. Suitable host cells can be transformed or transfected with the DNA sequence of the present invention and can be used for the expression and / or secretion of a target protein. Examples of suitable host cells that can be used in the present invention include immortalized hybridoma cells, NS / 0 myeloma cells, 293 cells, Chinese hamster ovary (CHO) cells, HeLa cells, Cap cells (cells derived from human amniotic fluid), insect cells, PER.C6 cells, and CoS cells.

[0048] As used herein, the term "fusion protein" refers to a novel protein formed by the fusion of at least two proteins or polypeptides. This fusion can typically be achieved through genetic engineering techniques, for example, by recombinant DNA technology, yielding the expression product of two genes recombined. In this context, it refers to a fusion of PLA2R and serum albumin.

[0049] As used herein, the term "pharmaceutical composition" generally refers to unit dosage form and can be prepared by any of the methods well known in the pharmaceutical art. All methods include the step of bringing the active ingredient into association with the carrier which constitutes one or more accessory ingredients. Generally, the compositions are prepared by uniformly and thoroughly combining the active compound with liquid carriers, finely divided solid carriers, or both.

[0050] As used herein, the term "pharmaceutically acceptable" means that the substance or composition must be chemically and / or toxicologically compatible with the other ingredients of the formulation and / or the mammal to be treated therewith. Preferably, the term "pharmaceutically acceptable" as used herein means approved by federal regulatory agencies or national governments or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopeia for use in animals, particularly humans.

[0051] As used herein, the term "pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier" may include any solvent, solid excipient, diluent or other liquid excipient, etc., suitable for the particular target dosage form. Except to the extent that any conventional excipient is incompatible with the compounds of the present invention, such as by producing any adverse biological effect or interacting in a deleterious manner with any other component of the pharmaceutically acceptable composition, their use is also contemplated by the present invention.

[0052] In this article, the term "administration" refers to introducing a predetermined amount of material into a patient by a certain suitable mode. Fusion protein or pharmaceutical composition of the present invention can be administered by any common approach, as long as it can reach the expected tissue. The various modes of administration are expected to include peritoneal, intravenous, intramuscular, subcutaneous injections, etc., but the present invention is not limited to these exemplified modes of administration. Preferably, compositions of the present invention are administered by intravenous or subcutaneous injection.

[0053] As used herein, the term "PLA2R antibody-induced diseases" generally refers to diseases caused by abnormal PLA2R antibodies, such as those caused by increased PLA2R antibodies, including but not limited to PLA2R antibody-positive membranous nephropathy. PLA2R is a membrane protein expressed in glomerular podocytes. For unknown reasons, it becomes an antigen, leading to the production of corresponding autoimmune antibodies (PLA2R antibodies). PLA2R antibodies bind to PLA2R and deposit beneath the glomerular basement membrane, triggering downstream effects such as C3 deposition, leading to PLA2R antibody-positive membranous nephropathy.

[0054] Herein, the terms "anti-PLA2R antibody," "PLA2R antibody," and "PLA2R-Ab" are used interchangeably.

[0055] As used herein, the term "treatment" refers to any process used to obtain a desired pharmacological and / or physiological effect. The effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and / or therapeutic in terms of partially or completely curing a disease and / or the adverse effects caused by the disease. "Treatment" as used herein covers diseases in mammals, particularly humans, and includes: (a) preventing the occurrence of a disease or condition in individuals who are susceptible to the disease but have not yet been diagnosed with the disease; (b) inhibiting the disease, such as arresting the progression of the disease; or (c) alleviating the disease, such as alleviating the symptoms associated with the disease. "Treatment" as used herein covers any medication that administers a drug or compound to an individual to treat, cure, alleviate, improve, reduce or inhibit the individual's disease, including but not limited to administering a drug containing a compound described herein to an individual in need.

[0056] The present invention provides a fusion protein, a nucleic acid molecule, an expression vector, a recombinant cell, a kit and uses thereof, a method for detecting PLA2R antibodies, a method for diagnosing diseases related to PLA2R antibodies, a method for evaluating the staging of diseases related to PLA2R antibodies, and a method for evaluating the prognosis of diseases related to PLA2R antibodies, each of which will be described in detail below.

[0057] Fusion protein

[0058] In its first aspect, the present invention provides a fusion protein. According to an embodiment of the present invention, the fusion protein comprises: a PLA2R antigen epitope peptide; and albumin or a fragment thereof, wherein the PLA2R antigen epitope peptide is linked to the albumin or a fragment thereof. The fusion protein according to an embodiment of the present invention has high expression levels, is easily accessible, and can specifically bind to PLA2R antibodies, exhibiting advantages such as strong binding ability and high sensitivity. It can be used to accurately detect PLA2R antibody levels and diagnose PLA2R-type membranous nephropathy.

[0059] According to one embodiment of the present invention, the C-terminus of the PLA2R antigen epitope peptide is connected to the N-terminus of the albumin or a fragment thereof.

[0060] According to one embodiment of the present invention, the N-terminus of the PLA2R antigen epitope peptide is connected to the C-terminus of the albumin or a fragment thereof.

[0061] According to one embodiment of the present invention, the PLA2R antigen epitope peptide comprises CysR, FnII, PLA2R binding region 1 and optionally PLA2R binding region 2. The structure of PLA2R is as follows: Figure 1 As shown. The inventors have experimentally discovered that using the PLA2R CysR domain, FNII domain, and part of the CTLD domain (PLA2R binding region 1, or PLA2R binding region 1 and PLA2R binding region 2) as PLA2R antigen epitope peptides and fusing them with albumin or a fragment thereof to produce a fusion protein can maintain the binding ability of the PLA2R antigen epitope to the PLA2R-Ab. Fusion proteins prepared using these PLA2R antigen epitope peptides have strong binding ability to the PLA2R-Ab, high sensitivity, and high accuracy, and are also expressed in high levels.

[0062] According to one embodiment of the present invention, the C-terminus of the CysR is connected to the N-terminus of the FnII, and the C-terminus of the FnII is connected to the N-terminus of the PLA2R binding region 1.

[0063] According to one embodiment of the present invention, the C-terminus of the CysR is connected to the N-terminus of the FnII, the C-terminus of the FnII is connected to the N-terminus of the PLA2R binding region 1, and the C-terminus of the PLA2R binding region 1 is connected to the N-terminus of the PLA2R binding region 2.

[0064] According to one embodiment of the present invention, the PLA2R antigen epitope peptide has an amino acid sequence as shown in SEQ ID NO: 1 or 2, or an amino acid sequence having at least 95% similarity to SEQ ID NO: 1 or 2.

[0065] EGVAAALTPERLLEWQDKGIFVIQSESLKKCIQAGKSVLTLENCKQANKHMLWKWVSNHGLFNIGGSGCLGLNFSAPEQPLSLYECDSTLVSLRWRCNRKMITGPLQYSVQVAHDNTVVASRKYIHKWISYGSGGGDICEYLHKDLHTIKGNTHGMPCMFPFQYNHQWHHECTRE GREDDLLWCATTSRYERDEKWGFCPDPTSAEVGCDTIWEKDLNSHICYQFNLLSSLSWSEAHSSCQMQGGTLLSITDETEENFIREHMSSKTVEVWMGLNQLDEHAGWQWSDGTPLNYLNWSPEVNFEPFVEDHCGTFSSFMPSAWRSRDCESTLPYICKKYLNHIDHEIVE(SEQ ID NO:1).

[0066] EGVAAALTPERLLEWQDKGIFVIQSESLKKCIQAGKSVLTLENCKQANKHMLWKWVSNHGLFNIGGSGCLGLNFSAPEQPLSLYECDSTLVSLRWRCNRKMITGPLQYSVQVAHDNTVVASRK YIHKWISYGSGGGDICEYLHKDLHTIKGNTHGMPCMFPFQYNHQWHHECTREGREDDLLWCATTSRYERDEKWGFCPDPTSAEVGCDTIWEKDLNSHICYQFNLLSSLSWSEAHSSCQMQGGTL LSITDETEENFIREHMSSKTVEVWMGLNQLDEHAGWQWSDGTPLNYLNWSPEVNFEPFVEDHCGTFSSFMPSAWRSRDCESTLPYICKKYLNHIDHEIVEKDAWKYYATHCEPGWNPYNRNCY KLQKEEKTWHEALRSCQADNSALIDITSLAEVEFLVTLLGDENASETWIGLSSNKIPVSFEWSNDSSVIFTNWHTLEPHIFPNRSQLCVSAEQSEGHWKVKNCEERLFYICKKAGHVLSD(SEQ ID NO:2).

[0067] According to one embodiment of the present invention, the albumin or a fragment thereof is serum albumin or a fragment thereof, preferably human serum albumin.

[0068] According to one embodiment of the present invention, the albumin or a fragment thereof has an amino acid sequence as shown in SEQ ID NO: 3 or an amino acid sequence having at least 90% similarity to SEQ ID NO: 3.

[0069] (SEQ ID NO: 3).

[0070] According to an embodiment of the present invention, the fusion protein further includes a connecting peptide.

[0071] According to one embodiment of the present invention, the N-terminus of the connecting peptide is connected to the C-terminus of the PLA2R antigen epitope peptide, and the C-terminus of the connecting peptide is connected to the N-terminus of the albumin or a fragment thereof.

[0072] According to one embodiment of the present invention, the N-terminus of the connecting peptide is connected to the C-terminus of the albumin or its fragment, and the C-terminus of the connecting peptide is connected to the N-terminus of the PLA2R antigen epitope peptide.

[0073] According to one embodiment of the present invention, the amino acid sequence of the connecting peptide is (GGGGS)n, wherein n is an integer greater than or equal to 1, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, more preferably 1, 2 or 3.

[0074] According to one embodiment of the present invention, the connecting peptide includes the amino acid sequence shown in SEQ ID NO:4.

[0075] GGGGSGGGGS (SEQ ID NO: 4).

[0076] According to an embodiment of the present invention, the fusion protein further includes a tag.

[0077] According to one embodiment of the present invention, the tag is connected to the end of the PLA2R antigen epitope peptide or albumin or a fragment thereof.

[0078] According to one embodiment of the present invention, the tag is at least one of a HIS tag, a FLAG tag, an HA tag, a GST tag, a Strep II tag, and an MBP tag. Thus, during the purification of the fusion protein, the fusion protein can be purified by adsorption to facilitate subsequent detection of the PLA2R antibody.

[0079] According to one embodiment of the present invention, the HIS tag has an amino acid sequence as shown in SEQ ID NO:5.

[0080] HHHHHH (SEQ ID NO: 5).

[0081] According to an embodiment of the present invention, the N-terminus of the HIS tag is linked to the C-terminus of the albumin or its fragment; or the C-terminus of the HIS tag is linked to the N-terminus of the PLA2R antigen epitope peptide. The inventors have discovered that when the N-terminus of the HIS tag is linked to the C-terminus of the albumin or its fragment, the expression level of the fusion protein is higher.

[0082] According to an embodiment of the present invention, the N-terminus of the HIS tag is connected to the C-terminus of the albumin or its fragment, and further comprises a TG sequence, thereby further improving the expression of the fusion protein.

[0083] According to an embodiment of the present invention, the N-terminus of the TG sequence is connected to the C-terminus of the albumin or a fragment thereof, and the C-terminus of the TG sequence is connected to the N-terminus of the HIS tag.

[0084] According to one embodiment of the present invention, the fusion protein has an amino acid sequence as shown in any one of SEQ ID NOs: 6 to 9.

[0085] Preferably, the fusion protein has an amino acid sequence as shown in SEQ ID NO: 6. The inventors have found through experiments that the expression level of this fusion protein is significantly increased compared with other fusion proteins.

[0086]

[0087]

[0088] EGVAAALTPERLLEWQDKGIFVIQSESLKKCIQAGKSVLTLENCKQANKHMLWKWVSNHGLFNIGGSGCLGLNFSAPEQPLSLYECDSTLVSLRWRCNRKMITGPLQYSVQVAHDNTVVASRKYIHKWISYGSGGGDICEYLHKDLHTIKGNTHGMPCMFPFQYNHQWHHECTREGREDDLLWCATTSRYERDEKWGFCPDPTSAEVGCDTIWEKDLNSHICYQFNLLSSLSWSEAHSSCQMQGGTLLSITDETEENFIREHMSSKTVEVWMGLNQLDEHAGWQWSDGTPLNYLNWSPEVNFEPFVEDHCGTFSSFMPSAWRSRDCESTLPYICKKYLNHIDHEIVEGGGGSGGGGSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLTGHHHHHH(SEQ ID NO:8)。

[0089] HHHHHHEGVAAALTPERLLEWQDKGIFVIQSESLKKCIQAGKSVLTLENCKQANKHMLWKWVSNHGLFNIGGSGCLGLNFSAPEQPLSLYECDSTLVSLRWRCNRKMITGPLQYSVQVAHDNTVVASRKYIHKWISYGSGGGDICEYLHKDLHTIKGNTHGMPCMFPFQYNHQWHHECTREGREDDLLWCATTSRYERDEKWGFCPDPTSAEVGCDTIWEKDLNSHICYQFNLLSSLSWSEAHSSCQMQGGTLLSITDETEENFIREHMSSKTVEVWMGLNQLDEHAGWQWSDGTPLNYLNWSPEVNFEPFVEDHCGTFSSFMPSAWRSRDCESTLPYICKKYLNHIDHEIVEGGGGSGGGGSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL(SEQ ID NO:9)。

[0090] Nucleic acid molecules, expression vectors and recombinant cells

[0091] In the process of preparing or obtaining these fusion proteins, nucleic acid molecules expressing these fusion proteins can be connected to different vectors and then expressed in different cells to obtain the corresponding fusion proteins.

[0092] In a second aspect, the present invention provides a nucleic acid molecule. According to an embodiment of the present invention, the nucleic acid molecule encodes the fusion protein described in the first aspect. This nucleic acid molecule can be effectively used to express the fusion protein of the first aspect, particularly in a prokaryotic or lower eukaryotic expression system.

[0093] According to an embodiment of the present invention, the nucleic acid molecule is DNA.

[0094] The nucleotide sequence shown in SEQ ID NO:10 is used to encode SEQ ID NO:6.

[0095] The nucleotide sequence shown in SEQ ID NO:11 is used to encode SEQ ID NO:7.

[0096] The nucleotide sequence shown in SEQ ID NO:12 is used to encode SEQ ID NO:8.

[0097] The nucleotide sequence shown in SEQ ID NO:13 is used to encode SEQ ID NO:9.

[0098]

[0099]

[0100]

[0101]

[0102] In a third aspect, the present invention provides an expression vector. According to an embodiment of the present invention, the expression vector carries the nucleic acid molecule described in the second aspect. This expression vector can be effectively used to express the fusion protein of the first aspect, particularly in a prokaryotic or lower eukaryotic expression system.

[0103] According to one embodiment of the present invention, the expression vector is a eukaryotic expression vector.

[0104] According to one embodiment of the present invention, the expression vector is a lentiviral vector.

[0105] In its fourth aspect, the present invention provides a recombinant cell. According to embodiments of the present invention, the recombinant cell comprises: carrying the nucleic acid molecule described in the second aspect or the expression vector described in the third aspect; or expressing the fusion protein described in the first aspect. The recombinant cell described in the embodiments of the present invention can be used for in vitro expression and large-scale production of the fusion protein described in the first aspect.

[0106] According to an embodiment of the present invention, the recombinant cell is obtained by introducing the aforementioned expression vector into a host cell.

[0107] According to one embodiment of the present invention, the recombinant cell is a eukaryotic cell.

[0108] According to one embodiment of the present invention, the recombinant cell is a mammalian cell.

[0109] Those skilled in the art will appreciate that the features and advantages described above for the fusion protein are also applicable to the nucleic acid molecule, expression vector and recombinant cell, and will not be described in detail here.

[0110] Reagent test kit

[0111] In a fifth aspect, the present invention provides a kit. According to embodiments of the present invention, the kit comprises: the fusion protein described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, or the recombinant cell described in the fourth aspect. The kit described in embodiments of the present invention can effectively detect the level of PLA2R antibodies and can also be used to diagnose diseases associated with PLA2R antibodies or assess the staging or prognosis of diseases associated with PLA2R antibodies, such as PLA2R antibody-positive membranous nephropathy.

[0112] According to one embodiment of the present invention, the working concentration of the fusion protein is 0.01 to 50 μg / mL.

[0113] According to one embodiment of the present invention, the working concentration of the fusion protein is 0.05-40 μg / mL.

[0114] According to one embodiment of the present invention, the working concentration of the fusion protein is 0.1 to 30 μg / mL.

[0115] According to one embodiment of the present invention, the working concentration of the fusion protein is 1 to 20 μg / mL, exemplified by 10 μg / mL.

[0116] Those skilled in the art will appreciate that the features and advantages described above for the fusion protein, nucleic acid molecule, expression vector, and recombinant cell are also applicable to the kit and will not be described in detail here.

[0117] use

[0118] In a sixth aspect, the present invention provides a use of the fusion protein of the first aspect, the nucleic acid molecule of the second aspect, the expression vector of the third aspect, or the recombinant cell of the fourth aspect in preparing a kit for detecting PLA2R antibodies, diagnosing diseases related to excessive PLA2R antibodies, evaluating the prognosis of diseases related to PLA2R antibodies, or evaluating the staging of diseases related to PLA2R antibodies.

[0119] According to an embodiment of the present invention, the related disease caused by excessive PLA2R antibodies is selected from PLA2R antibody-positive membranous nephropathy.

[0120] Those skilled in the art will appreciate that the features and advantages described above for the fusion protein, nucleic acid molecule, expression vector, recombinant cell and kit are also applicable to this use and will not be described in detail here.

[0121] method

[0122] In its seventh aspect, the present invention provides a method for detecting PLA2R antibodies. According to embodiments of the present invention, the method comprises: detecting PLA2R antibodies in a test sample using the fusion protein described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, the recombinant cell described in the fourth aspect, or the kit described in the fifth aspect; and determining the content of PLA2R antibodies in the test sample based on the detection results. The method according to embodiments of the present invention can effectively detect the content of PLA2R antibodies in a test sample and, for subsequent scientific research, has advantages such as high detection sensitivity and accuracy.

[0123] In its eighth aspect, the present invention provides a method for diagnosing diseases caused by PLA2R antibodies. According to embodiments of the present invention, the method comprises: detecting PLA2R antibodies in a test sample using the fusion protein described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, the recombinant cell described in the fourth aspect, or the kit described in the fifth aspect; and determining the PLA2R antibody content in the test sample based on the detection results. The method according to embodiments of the present invention can effectively diagnose diseases caused by PLA2R antibodies and has advantages such as high diagnostic accuracy.

[0124] According to an embodiment of the present invention, the presence of a PLA2R antibody content in the test sample that is not less than a predetermined threshold value indicates that the test sample is derived from a patient suffering from a disease associated with PLA2R antibodies. For example, the predetermined threshold value can be obtained by repeatedly measuring the PLA2R antibody content in plasma samples from individuals with known PLA2R antibody-positive membranous nephropathy and normal individuals.

[0125] In its ninth aspect, the present invention provides a method for assessing the staging of a disease caused by PLA2R antibodies. According to an embodiment of the present invention, the method comprises: detecting PLA2R antibodies in a test sample derived from a patient using the fusion protein described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, the recombinant cell described in the fourth aspect, or the kit described in the fifth aspect; and determining the PLA2R antibody content in the test sample based on the detection results. The method according to an embodiment of the present invention can effectively assess the staging of a disease caused by PLA2R antibodies and has the advantages of high accuracy of the assessment results.

[0126] According to an embodiment of the present invention, the stage of a disease caused by PLA2R antibodies in a patient to be tested is determined based on the content of PLA2R antibodies in the sample to be tested.

[0127] In its tenth aspect, the present invention provides a method for assessing the prognosis of diseases associated with PLA2R antibodies. According to embodiments of the present invention, the method comprises: detecting PLA2R antibodies in a test sample derived from a patient using the fusion protein described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, the recombinant cell described in the fourth aspect, or the kit described in the fifth aspect; and determining the PLA2R antibody content in the test sample based on the detection results. The method according to embodiments of the present invention can effectively assess the prognosis of diseases associated with PLA2R antibodies and has the advantages of high accuracy of the assessment results.

[0128] According to one embodiment of the present invention, the sample to be tested is derived from a patient suffering from a disease related to PLA2R antibodies before or after treatment.

[0129] According to one embodiment of the present invention, the prognosis of a disease caused by a PLA2R antibody is determined based on the PLA2R antibody content in a test sample from a patient suffering from the disease caused by a PLA2R antibody before or after treatment.

[0130] According to one embodiment of the present invention, a decrease in the content of PLA2R antibodies in a test sample of a patient suffering from a disease caused by PLA2R antibodies after treatment is an indication of a good prognosis for the patient.

[0131] In its eleventh aspect, the present invention provides a method for using the kit described in the fifth aspect. According to an embodiment of the present invention, the method comprises: detecting PLA2R antibodies in a test sample using the kit described in the fifth aspect; and determining the content of PLA2R antibodies in the test sample based on the detection results. The method according to the embodiment of the present invention can effectively detect the content of PLA2R antibodies and has the advantages of high detection sensitivity and accuracy.

[0132] According to an implementation manner of the embodiments of the present invention, the methods described in the seventh aspect, the eighth aspect, the ninth aspect, the tenth aspect, and the eleventh aspect may further include at least one of the following technical features:

[0133] According to an embodiment of the present invention, the detection comprises: a first contacting treatment of the test sample with the fusion protein; a second contacting treatment of the product of the first contacting treatment with a detection antibody; and determining the content of the PLA2R antibody in the test sample based on the detection value of the second contacting treatment product. During the detection process, the first contacting treatment of the test sample and the fusion protein allows the fusion protein to specifically bind to the PLA2R antibody in the test sample, thereby obtaining the fusion protein bound to the PLA2R antibody. The fusion protein bound to the PLA2R antibody is then brought into a second contacting treatment with the detection antibody, which allows the detection antibody to bind to the PLA2R antibody. The detection antibody signal is detected to obtain a detection value, thereby determining the content of the PLA2R antibody in the test sample.

[0134] According to one embodiment of the present invention, the fusion protein is pre-coated.

[0135] According to one embodiment of the present invention, the concentration of the fusion protein in the plate coating process is 0.01 to 50 μg / mL.

[0136] According to one embodiment of the present invention, the concentration of the fusion protein in the plate coating process is 0.05 to 40 μg / mL.

[0137] According to one embodiment of the present invention, the concentration of the fusion protein in the plate coating process is 0.1 to 30 μg / mL.

[0138] According to one embodiment of the present invention, the concentration of the fusion protein in the coating process is 1 to 20 μg / mL, exemplified by 10 μg / mL.

[0139] According to an embodiment of the present invention, the sample to be tested is blood.

[0140] According to an embodiment of the present invention, the blood is diluted before the test to obtain a test sample, and the dilution ratio is 10 to 500 times, preferably 50 to 200 times, and exemplarily 100 times.

[0141] According to one embodiment of the present invention, the fusion protein is purified before the second contacting, thereby purifying the fusion protein bound to the PLA2R antibody to obtain the PLA2R antibody, facilitating subsequent detection of the PLA2R antibody.

[0142] According to one embodiment of the present invention, the detection antibody is a mouse anti-human IgG monoclonal antibody.

[0143] According to one embodiment of the present invention, the detection antibody is a detection antibody coupled to HRP.

[0144] According to an implementation manner of the embodiment of the present invention, the detection value is an absorbance value, such as an absorbance value within a wavelength range of 450 to 490 nm.

[0145] According to an embodiment of the present invention, determining the content of the PLA2R antibody in the test sample based on the absorbance of the second contact treatment product is achieved by: comparing the absorbance of the second contact treatment product with a standard curve, wherein the standard curve is a curve of absorbance values ​​of PLA2R standard antibody concentration; determining the content of the PLA2R antibody in the test sample based on the comparison result; and determining the content of the PLA2R antibody in the test sample based on the product of the PLA2R antibody content in the test sample and the dilution factor of the test sample. Thus, the content of the PLA2R antibody in the test sample can be accurately detected.

[0146] According to an embodiment of the present invention, the PLA2R standard antibody is an antibody solution with a known predetermined concentration. For example, the PLA2R standard antibody in the Euromon test kit can be used.

[0147] According to an embodiment of the present invention, the content of PLA2R antibodies in the test sample can be obtained by the following formula: y=A2+(A1-A2) / (1+(x / x0)^p), wherein A1, A2, x0 and p are constants independently determined by logistic regression analysis; x represents the content of PLA2R antibodies in the test sample; and y represents absorbance.

[0148] According to one embodiment of the present invention, the R 2 Above 0.9994, optionally, R 2 is 0.9996 or 0.9999.

[0149] According to one implementation manner of an embodiment of the present invention, the formula corresponding to the standard curve is y=A2+(A1-A2) / (1+(x / x0)^p), wherein A1 is 0.01-0.05, A2 is 2-2.1, x0 is 112-124, p is 0.87-0.96, x represents the content of PLA2R antibody in the test sample, and y represents the absorbance; or the formula corresponding to the standard curve is y=A2+(A1-A2) / (1+(x / x0)^p), wherein A1 is 0.003-0.075, A2 is 1.65-1.8, x0 is 83-101, p is 0.94-1.16, x represents the content of PLA2R antibody in the test sample, and y represents the absorbance.

[0150] According to an embodiment of the present invention, the PLA2R antibody-induced related disease is selected from PLA2R antibody-positive membranous nephropathy.

[0151] Those skilled in the art will appreciate that the features and advantages described above for the fusion protein, nucleic acid molecule, expression vector, recombinant cell, and kit are also applicable to this method and will not be described in detail here.

[0152] Below, the scheme of the present invention will be explained in conjunction with embodiment.It will be understood by those skilled in the art that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.In the embodiment, if specific technology or conditions are not indicated, the technology or conditions described in the literature in this area or the product instructions are used.The reagents or instruments used are not indicated by the manufacturer, and are all conventional products that can be obtained by commercial purchase.

[0153] Example 1: Construction of fusion protein expression plasmid

[0154] 1. The plasmid vector used was the pcDNA3.4 transient expression vector, which contains the native full-length CMV promoter and the WPRE element downstream of the cloning site. CMV promoter-based vectors typically exhibit good expression levels in CHO cell transient expression systems. The WPRE element, located downstream of the multiple cloning site, effectively enhances gene transcription and expression. In E. coli, it also contains an ampicillin resistance gene. PCR was then used to amplify the pcDNA3.4 vector gene and the gene sequence linked to the gene of interest and signal peptide. The PCR template was synthesized, and the pcDNA3.4 vector fragment and the gene of interest fragment were ligated using a homologous recombination kit. Single clones were generated by DH5α competent cell transformation, amplified, and sequenced. After sequencing confirmation, the plasmids were extracted to obtain the target gene recombinant expression plasmids, designated pcDNA3.4-RC21, pcDNA3.4-RC22, pcDNA3.4-RC23, and pcDNA3.4-RC24, respectively. The target gene encoding DNA plus the secretory expression signal peptide encoding DNA were synthesized (referred to as synthetic gene) and cloned between the CMV promoter and WPRE gene of plasmid pcDNA3.4. Figure 2 .

[0155] 1.1 pcDNA3.4 vector and synthetic gene composition and primer and template information

[0156] Specifically, the synthetic genes were: RC21 (amino acid sequence: SEQ ID NO: 8, nucleotide sequence: SEQ ID NO: 12) and a signal peptide were synthesized, referred to as RC21; RC22 (amino acid sequence: SEQ ID NO: 6, nucleotide sequence: SEQ ID NO: 10) and a signal peptide were synthesized, referred to as RC22; RC23 (amino acid sequence: SEQ ID NO: 9, nucleotide sequence: SEQ ID NO: 13) and a signal peptide were synthesized, referred to as RC23; and RC24 (amino acid sequence: SEQ ID NO: 7, nucleotide sequence: SEQ ID NO: 11) and a signal peptide were synthesized, referred to as RC24. The pcDNA3.4 vector and primers and templates for the synthetic genes are shown in Table 1.

[0157] Among them, the amino acid sequence of the signal peptide is:

[0158] MLLSPSLLLLLLLGAPRGCA (SEQ ID NO: 14);

[0159] The nucleotide sequence of the signal peptide is:

[0160] ATGCTGCTGTCGCCGTCGCTGCTGCTGCTGCTGCTGCTGGGGGCGCCGGGGCTGCGCC (SEQ ID NO: 15).

[0161] Table 1: Information of synthetic genes

[0162]

[0163] 1.2 PCR amplification of target fragments

[0164] The PCR reaction used TAKARA's KOD reaction system (Cat. KFX-101). The reaction conditions and reagents are shown in Tables 2 to 4.

[0165] Table 2: KOD reaction system for target gene or pcDNA3.4

[0166] Reagents concentration Final concentration Volume (μL) 2*PCR buffer for KOD 2* 1* 25 dNTP (dATP, dTTP, dCTP, and dGTP, four in total) 2mM / each 0.4mM / each 10 5' primer 10 μM 0.5μM 2.5 3' primer 10 μM 0.5μM 2.5 Template (target gene or pcDNA3.4) 10 ng / μL 0.2 ng / μL 1 <![CDATA[ddH2O]]> N / A 8 KOD FX 1U / μL 20mU / μL 1 total 50

[0167] Table 3: PCR reaction conditions for vector pcDNA3.4

[0168]

[0169]

[0170] Table 4: PCR reaction conditions for target genes

[0171] program temperature time condition 1 94℃ 2min 2 98℃ 10s 3 60℃ 30s 4 68℃ 2-6 minutes Return to program 2 and perform 30 cycles from program 2 to 4 5 10℃ permanent

[0172] 1.3 Gel electrophoresis detection of PCR results

[0173] After the PCR is completed, the sample in step 1.2 is subjected to 1.5% agarose gel electrophoresis. In the electrophoresis tank, 1*TAE300mL is added and the gel is run at 100V for 30min. The results show that the size of the agarose gel electrophoresis band is consistent with the theoretical size. The electrophoresis results are recovered using the Omega Gel Extraction Kit (Cat.D2500-01) and eluted with 20μL eluent to obtain the purified PCR product. The electrophoresis results are shown in Figure 2. Figure 3-4 As shown, Figure 3 The leftmost lane is the DL5000 marker, and the remaining four lanes are the pcDNA3.4 backbone. Figure 4 The leftmost lane is the DL5000 marker, and the remaining lanes from left to right are RC21, RC21, RC22, RC22, RC23, RC23, RC24, and RC24.

[0174] 1.4 Obtaining the target plasmid

[0175] The gel-cleaved and purified PCR bands obtained in 1.3 were homologously recombined and spliced ​​using the ready-to-use seamless cloning kit (B632219) from Sangon Biotech (Shanghai) Co., Ltd. according to the manufacturer's instructions. The spliced ​​product was added to competent E. coli DH5α cells at a ratio of 1:10, placed on ice for 10 minutes, then incubated in a metal bath at 42°C for 45 seconds, and then placed on ice for another 10 minutes. Finally, the solution was spread on LB solid medium containing ampicillin (Amp, final concentration 50 μg / mL) and cultured at 37°C overnight. The next day, a single colony was picked and placed in 2 ml of LB liquid medium (final concentration 50 μg / mL Amp) and shaken at 37°C at 200 rpm. The resulting bacterial solution was sent for sequencing to confirm that all sequenced sequences were consistent with the original sequence. 100 μL of the monoclonal bacterial solution was added to 50 mL of LB liquid culture medium containing ampicillin (final concentration 50 μg / mL), cultured overnight at 37°C and 200 rpm, the strain was preserved, and the plasmid was extracted to obtain the target gene expression plasmid.

[0176] 2. Transient expression of RC21-RC24

[0177] 1) About 24 hours before transfection, take FreeStyle TM Cells (cell density 5-6×10 5 / mL, from FreeStyle, Gibco, USA TM MAX CHO Expression System, Catalog No.: K900020), cultured at 37°C, 5% CO2, and 120-135 rpm / min.

[0178] 2) On the day of transfection, dilute the cells to a cell density of 1×10 6 / mL, add 10mL of cells to each shaking flask (cell survival rate is above 95%).

[0179] 3) Gently blend FreeStyle TM MAX Reagent several times, being careful not to vortex.

[0180] 4) Take 12.5 μg each of pcDNA3.4-RC21, pcDNA3.4-RC22, pcDNA3.4-RC23 and pcDNA3.4-RC24 and add OptiPRO TM SFM to a total volume of 0.2 mL each and mix gently.

[0181] 5) Take 25 μL of FreeStyle TM MAX Reagent, added Opti-ProTM Add SFM to a total volume of 0.4 mL and mix gently. Mix evenly and add evenly to the plasmid mixture and mix gently to obtain two 0.4 mL DNA-FreeStyle TM MAX mixture and incubate at room temperature for 10 minutes to allow complex formation.

[0182] 6) Slowly add the two 0.4mL DNA-FreeStyle TM MAX complex was added to the cells while slowly rotating the flask.

[0183] 7) Culture the cells at 37°C, 5% CO2, and 120-135 rpm / min. No replacement or supplementation of the culture medium is required. Collect the cell fluid after 7 days.

[0184] 3. Purification and identification of RC21-RC24

[0185] The RC21-RC24 cell culture supernatant obtained in step 2 was centrifuged at 7000 rpm for 15 minutes at 4°C, then sterilized by filtration through a 0.22 μm syringe filter. The supernatant was applied to a Ni sepharose 6FF column at 2 mL / min. The column was washed with 20 mM phosphate buffer, 500 mM NaCl, and 20 mM imidazole at pH 6.8. The column was eluted with 0-100% 20 mM phosphate buffer, 1 M NaCl, and 500 mM imidazole at pH 6.8 over 5 column volumes. A 2 mL fraction was collected per tube. The eluted peak was ultrafiltered three times through a 30 kD ultrafiltration centrifuge tube, and the solution was replaced with 20 mM phosphate buffer at pH 6.8. Quantification was performed using the Bradford method.

[0186] Select a 10-well SDS-PAGE precast gel and run it at 150V for 50 minutes. Heat the sample at 90℃ for 5 minutes and load 10μg of sample into each well. The purification effect of RC21-RC24 protein was detected by SDS-PAGE. The results showed that the RC21-RC24 fusion protein was successfully purified. The schematic diagram of the RC21-RC24 structure is shown in the figure. Figure 5 As shown, the CFC1 protein has the amino acid sequence shown in SEQ ID NO: 1, and the CFC2 protein has the amino acid sequence shown in SEQ ID NO: 2.

[0187] Among them, the purification effects of RC22 and RC24 are as follows Figure 6As shown, the lanes from left to right are purified RC22 (non-reduced), purified RC22 (reduced), RC22 culture medium (non-reduced), RC22 culture medium (reduced), purified RC24 (non-reduced), purified RC24 (reduced), and protein molecular weight reference. The expression level of RC22 was 66 mg / L, and that of RC24 was 22 mg / L, indicating that the expression level of RC22 was three times that of RC24. RC22 and RC24 fusion proteins were used in subsequent experiments.

[0188] Example 2: Preparation of plasma from patients with membranous nephropathy and detection of PLA2R-Ab

[0189] 2 mL of EDTA-anticoagulated blood was collected from 23 patients with membranous nephropathy. Plasma was separated and diluted 100-fold. PLA2R-Ab concentrations were measured using the Euromon assay kit, with two measurements taken as the average. For detailed procedures, refer to the Euromon assay kit instructions. The results of the plasma analysis of patients with membranous nephropathy are shown in Table 5.

[0190] Table 5: Plasma test results of patients with membranous nephropathy

[0191]

[0192]

[0193] According to the kit instructions, a value >20 RU / mL is considered a positive sample, and <14 RU / mL is considered a negative sample. Therefore, samples pMN-2, pMN-4, pMN-7, pMN-18, pMN-19, and pMN-21 are positive samples.

[0194] Example 3: Determination of plate concentration and dilution of plasma samples

[0195] 1. Determination of the board concentration

[0196] Blank ELISA plates were coated with RC22 or RC24 at different concentrations (0.000001, 0.00001, 0.0001, 0.001, 0.01, 0.1, 1, 10, and 100 μg / mL, respectively) obtained in Example 1, and 100-fold diluted plasma samples pMN1 or pMN2 were added. The plates were incubated with detection antibodies and color was developed.

[0197] like Figure 7 and Figure 8 As shown, Figure 7-8 The horizontal axis in the figure represents concentration. It can be seen that the color intensity reaches a plateau above 1 μg / mL. The 10 μg / mL concentration was selected for the subsequent experiments.

[0198] 2. Determine the dilution of plasma samples

[0199] The plasma samples were tested after gradient dilution, with dilution multiples of 1, 10, 100, 1000, 10 4 , 10 5 , 10 6 and 10 7 , to determine the optimal dilution conditions. The samples were pMN-1, pMN-2, pMN-3, pMN-4, pMN-5, pMN-6, pMN-7, pMN-18, and RTX (rituximab). RTX was a negative reference, pMN-1, pMN-3, pMN-5, and PMN-6 were negative samples, and the rest were positive samples.

[0200] like Figure 9 and Figure 10 As shown, Figure 9-10 The horizontal axis represents the dilution ratio, demonstrating excellent discrimination between positive and negative samples. Dilutions of 50-500x all exhibited low background color development, resulting in high discrimination between positive and negative samples. Subsequent experiments used a 100-fold dilution.

[0201] Example 4: Detection of plasma samples using RC22 and RC24 as capture molecules and correlation analysis of the results

[0202] 1. Draw a concentration standard curve

[0203] The standard product of the Ou Meng kit was used as the PLA2R-Ab standard solution, RC22 and RC24 obtained in Example 1 were used as capture molecules, and HRP-coupled mouse anti-human IgG monoclonal antibody was used as detection antibody. A concentration standard curve was established by ELISA technology. Figure 11 , Figure 11 The horizontal axis is the concentration.

[0204] from Figure 11 According to the standard curve, the R 2 The R of RC24 is 0.9999. 2 The R 2 ), indicating a good fit. The absorbance values ​​of RC22 and RC24 at 100RU / mL were higher than that of the Euromon test kit, and the X0 in the fitting parameters were 118RU / mL and 92RU / mL, respectively, which were significantly lower than the 8×10 6 RU / mL. A lower X0 means that 50% of the detection signal value appears at a relatively low concentration, that is, the overall fitting curve shifts toward lower concentrations, which means that RC22 and RC24 are more sensitive than the Euromon test kit.

[0205] 2. Plasma sample testing and correlation analysis

[0206] RC22 and RC24 were used as capture molecules to detect the PLA2R-Ab concentrations of pMN-1, pMN-2, pMN-3, pMN-4, pMN-5, pMN-6, pMN-7, and pMN-18. The measured results were compared with the above standard curve to obtain the PLA2R-Ab concentrations, and then correlation analysis was performed. The specific analysis results are as follows:

[0207] The RC24 test results were plotted against the RC22 results, and the fitting equation is shown in Table 6. Figure 12 It shows excellent linear correlation. Figure 12 The horizontal axis is the PLA2R-Ab titer detected by RC22 (PLA2R-Ab titre by RC22), and the vertical axis is the PLA2R-Ab titer detected by RC24 (PLA2R-Ab titre by RC24).

[0208] Table 6: Fitting equations

[0209]

[0210] The results of the RC22 test were plotted using the results of the Omon kit, and the fitting equation is shown in Table 7. Figure 13 It shows good linear correlation. Figure 13 The horizontal axis is the PLA2R-Ab titre detected by RC22 (PLA2R-Ab titre by RC22), and the vertical axis is the PLA2R-Ab titre detected by the Euromon test kit (standard PLA2R-Ab titre).

[0211] Table 7: Fitting equations

[0212]

[0213] The results of the RC24 test were plotted against the results of the Ou Meng test kit, and the fitting equation is shown in Table 8. Figure 14 It shows good linear correlation. Figure 14 The horizontal axis is the PLA2R-Ab titre detected by RC24 (PLA2R-Ab titre by RC24), and the vertical axis is the PLA2R-Ab titre detected by the Euromon test kit (standard PLA2R-Ab titre).

[0214] Table 8: Fitting equations

[0215]

[0216] The above results show that the concentration of PLA2R-Ab detected by RC22 or RC24 is consistent with the detection results of the EUMO kit. The detection results of RC22 and RC24 are highly consistent.

[0217] Example 5: Detection of plasma samples using RC22 as a capture molecule and correlation analysis of the results

[0218] 1. Plasma sample testing

[0219] The plasma sample size was increased, and the RC22 assay and the OMN assay kit obtained in Example 1 were used to detect pMN-1, pMN-2, pMN-3, pMN-4, pMN-5, pMN-6, pMN-7, pMN-8, pMN-9, pMN-10, pMN-11, pMN-12, pMN-13, pMN-14, pMN-15, pMN-16, pMN-17, pMN-18, pMN-19, pMN-20, pMN-21, pMN-22, and pMN-23. According to OMN assay standards, a value >20 RU / mL was considered positive, and <20 RU / mL was considered negative.

[0220] The test results showed that the samples that tested positive with the Euromon test kit included pMN-2 (44.795RU / mL), pMN-4 (124.905RU / mL), pMN-7 (58.505RU / mL), pMN-18 (133.560RU / mL), pMN-19 (39.51RU / mL) and pMN-21 (141.735RU / mL). The RC22 test results for the above samples were pMN-2 (176.8826RU / mL), pMN-4 (302.5741RU / mL), pMN-7 (158.8119RU / mL), pMN-18 (722.1047RU / mL), pMN-19 (96.01469RU / mL) and pMN-21 (440.9976RU / mL). Obviously, the RC22 test values ​​were higher for all samples that tested positive with the Euromon test kit.

[0221] For the pMN-9, pMN-12, and pMN-20 samples, the Euromon test kit tested negative, while the RC22 test tested positive. Specifically, in the Euromon test kit, pMN-9 was 7.625RU / mL, pMN-12 was 6.772RU / mL, and pMN-20 was 5.400RU / mL; in the RC22 test, pMN-9 was 81.11847RU / mL, pMN-12 was 98.68588RU / mL, and pMN-20 was 28.40804RU / mL. The negative result of the Euromon test kit and the positive result of the RC22 indicate that the RC22 test is indeed more sensitive than the Euromon test kit.

[0222] 2. Correlation analysis

[0223] The results of the RC22 test were plotted using the results of the Omon kit, and the fitting equation is shown in Table 9. Figure 15 It shows good linear correlation. Figure 15 The horizontal axis is the PLA2R-Ab titre detected by RC22 (PLA2R-Ab titre by RC22), and the vertical axis is the PLA2R-Ab titre detected by the Euromon test kit (standard PLA2R-Ab titre).

[0224] Table 9: Fitting equations

[0225]

[0226] 3. Statistical correlation analysis

[0227] SPSS was used to perform bivariate linear correlation analysis, and the results are shown in Table 10.

[0228] Table 10: Correlation analysis

[0229]

[0230] Note: **. Significant correlation at the .01 level (two-sided).

[0231] The RC22 test results correlated very well with those of the Euromon test kit, with a Pearson correlation of 0.913 and a confidence level of 0.000.

[0232] Example 6: Plasma neutralization test to verify the accuracy of diagnostic reagents

[0233] To verify that the plasma tested negative by the Euromon test kit and that the positive plasma tested by RC22 was indeed positive and not a false positive, a neutralization experiment was performed using RC3 (amino acid sequence: SEQ ID NO: 21; nucleotide sequence: SEQ ID NO: 22).

[0234] 1. RC3 is an Fc fusion protein developed by the inventors of the present application for neutralizing PLA2R-Ab. RC3 contains a PLA2R antigen epitope peptide. The preparation method of RC3 refers to the preparation method of the RC22 fusion protein in Example 1 of the present application, wherein the PCR uses the synthetic gene "SEQ ID NO:15+SEQ ID NO:22" as a template and the primers are 6-f (nucleotide sequence is SEQ ID NO:23) and 6-r (nucleotide sequence is SEQ ID NO:24). The RC3 protein is then purified and identified, as shown in the purification and identification method of the RC22 fusion protein in Example 1 of the present application. The purified RC3 is used in the following neutralization experiment, wherein the purification effect is as follows: Figure 16 Lane 1 represents the non-reduced elution of RC3, lane 2 represents the reduced elution of RC3, and lane M represents a marker. The assay was then performed using the Euromon kit (Germany, an ELISA method for quantitatively detecting PLA2R-Ab concentrations in blood samples), the only FDA-approved test for the diagnosis of PLA2R antibody-positive MN. All steps were performed according to the manufacturer's instructions, and the RC3 protein dosage was 5 μg / mL. Plasma samples from three patients with PLA2R antibody-positive MN were numbered 1, 2, and 3. The neutralization effect of RC3 on the PLA2R-Ab in the plasma of these three patients with MN was tested, and the neutralization rates were obtained as shown in Table 11. The results show that RC3 has a high neutralization rate against PLA2R-Ab. Therefore, RC3 can act as a competitive inhibitor of PLA2R.

[0235] Among them, the amino acid sequence of RC3 is:

[0236] EGVAAALTPERLLEWQDKGIFVIQSESLKKCIQAGKSVLTLENCKQANKHMLWKWVSNHGLFNIGGSGCLGLNFSAPEQPLSLYECDSTLVSLRWRCNRKMITGPLQYSVQVAHDNTVVASRKYIHKWISYGSGGGDICEYLHKDLHTIKGNTHGMPCMFPFQYNHQWHHECTREGREDDLLWCATTSRYERDEKWGFCPDPTSAEVGCDTIWEKDLNSHICYQFNLLSSLSWSEAHSSCQMQGGTLLSITDETEENFIREHMSSKTVEVWMGLNQLDEHAGWQWSDGTPLNYLNWSPEVNFEPFVEDHCGTFSSFMPSAWRSRDCESTLPYICKKYLNHIDHEIVEGGGGSGGGGSTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:21).

[0237] Nucleotide sequence of RC3:

[0238]

[0239] The nucleotide sequence of 6-f is:

[0240] GCCTCCGGACTCTAGAGGGTACGGGCCAGATATACGCGT (SEQ ID NO: 23).

[0241] The nucleotide sequence of 6-r is:

[0242] CATTACTAACCGGTAGGGTCATTTAGCTCTGTGTGGTAAGCAGGT (SEQ ID NO: 24).

[0243] Table 11: Neutralization rate of RC3 against PLA2R-Ab in the plasma of these three membranous nephropathy-positive patients

[0244] Neutralization rate 1 2 3 RC3 95.76％ 82.03％ 89.26％

[0245] 2. The inventors selected pMN-9, pMN-12, pMN-20, and pMN-14 blood samples for neutralization experiments. In Example 5, the pMN-9, pMN-12, and pMN-20 samples tested negative using the Omen kit and positive using RC22. For the pMN-14 sample, both the Omen kit and RC22 tests were negative. PLA2R-Ab concentrations in the pMN-9, pMN-12, pMN-20, and pMN-14 blood samples were measured using the Omen kit and RC22, respectively, before and after the addition of RC3. If the plasma samples were indeed positive, they should exhibit significantly higher neutralization efficacy compared to negative samples. For the neutralization test of RC3 against PLA2R-Ab in plasma samples, refer to Step 1 of this Example. For the RC22 test, refer to Example 4.

[0246] Before testing, the blood samples of pMN-9, pMN-12, pMN-20 and pMN-14 were diluted 100 times. The results of this example are shown in Tables 12 and 13. Figure 17 and Figure 18 As shown ( Figure 17-18 The drug concentration = 5 μg / mL here means that the dosage of RC3 protein is 5 μg / mL; Figure 17-18The vertical axis represents the concentration of PLA2R-Ab (PLA2Rab conc.). It can be seen that the neutralization ratios after adding RC3 for samples that tested negative with the Euromon test kit but positive with RC22 (pMN-9, pMN-12, and pMN-20) were significantly higher than the neutralization ratios after adding RC3 for a sample that tested negative with both the Euromon test kit and RC22 (pMN-14). This indicates that pMN-9, pMN-12, and pMN-20 possessed a higher concentration of PLA2R-Ab before adding RC3, resulting in significantly higher neutralization ratios than pMN-14 after adding RC3. Therefore, it can be said that for samples that tested negative with the Euromon test kit but positive with RC22, the positive results detected by RC22 were not false positives, and that RC22 demonstrated higher sensitivity and accuracy than the Euromon test kit.

[0247] Table 12: Neutralization test results of the Euromon test kit

[0248] sample Detection value RU / mL RC3 neutralization test value RU / mL Neutralization rate pMN-9 5.90075 1.651845 72.04％ pMN-12 7.619725 1.495215 80.43％ pMN-20 5.89942 2.788775 52.81％ pMN-14 1.62506 1.470055 9.26％

[0249] Table 13: Neutralization test results of RC22

[0250] sample Detection value RU / mL RC3 neutralization test value RU / mL Neutralization rate pMN-9 41.40243 1.42355 96.58％ pMN-12 49.64485 1.199255 97.59％ pMN-20 17.246 1.7908 89.62％ pMN-14 0.95912 0.68177 28.43％

[0251] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0252] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A fusion protein, characterized in that Composed of a PLA2R antigen epitope peptide and albumin, optionally a linker peptide, and optionally a tag; The C-terminus of the PLA2R antigen epitope peptide is connected to the N-terminus of the albumin, or the N-terminus of the PLA2R antigen epitope peptide is connected to the C-terminus of the albumin; The amino acid sequence of the PLA2R antigen epitope peptide is shown in SEQ ID NO: 2; The amino acid sequence of the albumin is shown in SEQ ID NO:

3.

2. The fusion protein according to claim 1, characterized in that The N-terminus of the connecting peptide is connected to the C-terminus of the PLA2R antigen epitope peptide, and the C-terminus of the connecting peptide is connected to the N-terminus of the albumin; or The N-terminus of the connecting peptide is connected to the C-terminus of the albumin, and the C-terminus of the connecting peptide is connected to the N-terminus of the PLA2R antigen epitope peptide.

3. The fusion protein according to claim 2, characterized in that The amino acid sequence of the connecting peptide is (GGGGS) n , where n is an integer greater than or equal to 1.

4. The fusion protein according to claim 3, characterized in that n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

5. The fusion protein according to claim 3, characterized in that n is 1, 2 or 3.

6. The fusion protein according to claim 1, characterized in that The connecting peptide includes the amino acid sequence shown in SEQ ID NO:

4.

7. The fusion protein according to claim 1, characterized in that The tag is connected to the end of the PLA2R antigen epitope peptide or albumin.

8. The fusion protein according to claim 7, characterized in that The tag is at least one of a HIS tag, a FLAG tag, a HA tag, a GST tag, a Strep II tag and a MBP tag.

9. The fusion protein according to claim 8, characterized in that The HIS tag has the amino acid sequence shown in SEQ ID NO:

5.

10. The fusion protein according to claim 8, characterized in that The N-terminus of the HIS tag is connected to the C-terminus of the albumin; or The C-terminus of the HIS tag is connected to the N-terminus of the PLA2R antigen epitope peptide.

11. The fusion protein according to claim 9, characterized in that The N-terminus of the HIS tag is connected to the C-terminus of the albumin, and the HIS tag further includes a TG sequence; The C-terminus of the TG sequence is connected to the N-terminus of the amino acid sequence shown in SEQ ID NO:

5.

12. The fusion protein according to claim 1, characterized in that The amino acid sequence of the fusion protein is shown in any one of SEQ ID NOs: 6 to 7.

13. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the fusion protein according to any one of claims 1 to 12.

14. The nucleic acid molecule according to claim 13, characterized in that The nucleic acid molecule is DNA.

15. An expression vector, characterized in that: Carrying the nucleic acid molecule according to any one of claims 13 to 14.

16. The expression vector according to claim 15, characterized in that The expression vector is a eukaryotic expression vector.

17. The expression vector according to claim 15, characterized in that The expression vector is a lentiviral vector.

18. A recombinant cell, characterized in that include: Carrying the nucleic acid molecule according to any one of claims 13 to 14 or the expression vector according to any one of claims 15 to 17; or, Expressing the fusion protein according to any one of claims 1 to 12.

19. The recombinant cell according to claim 18, characterized in that The recombinant cell is obtained by introducing the expression vector according to any one of claims 15 to 17 into a host cell.

20. The recombinant cell according to claim 18, wherein The recombinant cell is a eukaryotic cell.

21. The recombinant cell according to claim 18, wherein The recombinant cell is a mammalian cell.

22. A kit, characterized in that include: The fusion protein according to any one of claims 1 to 12, the nucleic acid molecule according to any one of claims 13 to 14, the expression vector according to any one of claims 15 to 17, or the recombinant cell according to any one of claims 18 to 21.

23. The kit according to claim 22, wherein The working concentration of the fusion protein is 0.01-50 μg / mL.

24. The kit according to claim 22, wherein The working concentration of the fusion protein is 0.05-40 μg / mL.

25. The kit according to claim 22, wherein The working concentration of the fusion protein is 0.1-30 μg / mL.

26. The kit according to claim 22, wherein The working concentration of the fusion protein is 1-20 μg / mL.

27. The kit according to claim 22, wherein The working concentration of the fusion protein was 10 μg / mL.

28. Use of the fusion protein of any one of claims 1 to 12, the nucleic acid molecule of any one of claims 13 to 14, the expression vector of any one of claims 15 to 17, or the recombinant cell of any one of claims 18 to 21 in preparing a kit for detecting PLA2R antibodies, diagnosing a disease caused by excessive PLA2R antibodies, assessing the prognosis of a disease caused by PLA2R antibodies, or assessing the staging of a disease caused by PLA2R antibodies.

29. The use according to claim 28, characterized in that The related diseases caused by excessive PLA2R antibodies are selected from PLA2R antibody-positive membranous nephropathy.

30. A method for detecting PLA2R antibodies for purposes other than disease diagnosis and treatment, characterized in that: include: Detecting PLA2R antibodies in a sample using the fusion protein of any one of claims 1 to 12, the nucleic acid molecule of any one of claims 13 to 14, the expression vector of any one of claims 15 to 17, the recombinant cell of any one of claims 18 to 21, or the kit of any one of claims 22 to 27; Based on the test results, the content of PLA2R antibodies in the test sample is determined.

31. The method according to claim 30, wherein The detection includes: performing a first contact treatment between the sample to be tested and the fusion protein; performing a second contact treatment on the product of the first contact treatment and a detection antibody; Based on the detection value of the second contact treatment product, the content of the PLA2R antibody in the test sample is determined.

32. The method according to claim 31, characterized in that The fusion protein is pre-coated.

33. The method according to claim 32, wherein The concentration of the fusion protein in the plate coating process is 0.01-50 μg / mL.

34. The method according to claim 32, wherein The concentration of the fusion protein in the plate coating process is 0.05-40 μg / mL.

35. The method according to claim 32, wherein The concentration of the fusion protein in the plate coating process is 0.1-30 μg / mL.

36. The method according to claim 32, wherein The concentration of the fusion protein in the plate coating process is 1-20 μg / mL.

37. The method according to claim 32, wherein The concentration of the fusion protein in the coating treatment was 10 μg / mL.

38. The method according to claim 31, wherein Before the second contact, the fusion protein is purified in advance.

39. The method according to claim 31, wherein The detection antibody is a mouse anti-human IgG monoclonal antibody.

40. The method according to claim 31, wherein The detection antibody is a detection antibody coupled with HRP.

41. The method according to claim 31, wherein The detection value is the absorbance value within the wavelength range of 450~490nm.

42. The method according to claim 31, wherein The determination of the content of the PLA2R antibody in the test sample based on the detection value of the second contact treatment product is achieved by the following method: Comparing the absorbance value of the second contact treatment product with a standard curve, wherein the standard curve is a PLA2R standard antibody concentration absorbance value curve; Based on the comparison results, determining the content of the PLA2R antibody in the test sample; The content of the PLA2R antibody in the test sample is determined based on the product of the content of the PLA2R antibody in the test sample and the dilution factor of the test sample.

43. The method according to claim 42, characterized in that The PLA2R standard antibody is an antibody solution with a known predetermined concentration.

44. The method according to claim 42, wherein The content of PLA2R antibody in the test sample can be obtained by the following formula: y=A2+(A1-A2) / (1+(x / x0)^p), Where A1, A2, x0, and p are constants independently determined by logistic regression analysis; A1 is 0.01-0.05, A2 is 2-2.1, x0 is 112-124, and p is 0.87-0.96; or A1 is 0.003-0.075, A2 is 1.65-1.8, x0 is 83-101, and p is 0.94-1.16; x represents the content of PLA2R antibody in the test sample; y represents absorbance.

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