CN116082517A

CN116082517A - Fusion proteins and uses thereof

Info

Publication number: CN116082517A
Application number: CN202210869039.1A
Authority: CN
Inventors: 赖兵; 何玲; 张知明; 龚石林
Original assignee: Chengdu Diao Pharmaceutical Group Co Ltd
Current assignee: Chengdu Diao Pharmaceutical Group Co Ltd
Priority date: 2022-05-26
Filing date: 2022-07-22
Publication date: 2023-05-09

Abstract

The invention provides a fusion protein and a method for detecting PLA2R antibody. The fusion protein comprises: a PLA2R epitope peptide, albumin or fragment thereof, said PLA2R epitope peptide being linked to said albumin or fragment thereof; the method comprises the following steps: and detecting the PLA2R antibody in the sample to be detected by adopting the fusion protein, and determining the content of the PLA2R antibody in the sample to be detected based on a detection result. The fusion protein can be specifically combined with PLA2R antibody, and has the advantages of strong combining ability, high sensitivity and the like; the method provided by the invention has higher sensitivity and accuracy for detecting PLA2R antibodies.

Description

Fusion proteins and uses thereof

Technical Field

The invention belongs to the technical field of biomedical engineering, and particularly relates to a fusion protein and application thereof, and more particularly relates to a fusion protein, a nucleic acid molecule, an expression vector, a recombinant cell, a kit and application thereof, and a method for detecting a PLA2R antibody.

Background

Primary membranous nephropathy is also called idiopathic membranous nephropathy, spontaneous membranous nephropathy, which is one of the important causes of renal failure and is also the primary cause of nephrotic syndrome. Although no more than 30% of primary membranous nephropathy may spontaneously alleviate, 30% of patients 'kidney function slowly progresses to renal failure over about 10 years, causing serious threat to people's life health. Basic research in recent years has demonstrated that primary membranous nephropathy is a kidney-specific autoimmune disease, and that autoimmune antigens of the body activate the immune system under disease conditions, producing autoimmune antibodies. Autoimmune antibodies bind to autoimmune antigens on glomerular podocytes, resulting in the immune system's attack on podocytes and surrounding tissues, causing inflammation and glomerular injury.

At present, the autoimmune antigen of primary membranous nephropathy is studied clearly with PLA2R and THSD7A, and can be classified into PLA2R type and THSD7A type according to the type of antigen. Wherein about 70% -80% of the primary membranous nephropathy is PLA2R type, less than 10% of the primary membranous nephropathy is THSD7A type. anti-PLA 2R antibodies (PLA 2R-abs) play an important role in the development and progression of PLA 2R-type membranous nephropathy. The level of PLA2R-Ab in serum can serve as a predictive marker for PLA 2R-type membranous nephropathy, with higher PLA2R-Ab titers implying lower spontaneous remission rates. PLA2R-Ab titer reduction is often accompanied by spontaneous remission and is as low as undetectable in patients with complete remission. Immunosuppressive treatment can reduce PLA2R-Ab titers.

Current diagnostic criteria for PLA 2R-type membranous nephropathy are immunofluorescence or immunohistochemistry of kidney puncture samples. This method is a minor surgery and has some drawbacks such as: 1) The pain of the patient is great; 2) Some cases cannot be performed; 3) Cannot be repeatedly performed, and cannot provide reference for the treatment effect of the diseases.

Thus, there is a need for a diagnostic reagent or method that is non-invasive, accurate, and cost effective for patients.

Disclosure of Invention

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

The present invention has been completed based on the following findings by the inventors:

PLA2R is a membrane protein highly expressed on the surface of glomerular podocyte, and comprises a CysR domain, a fibractin type-II (FnII for short) domain, 8C-type lectin domains with very high sequence similarity (namely CTLD domains), a transmembrane domain and an intracellular domain. Because it contains a transmembrane hydrophobic structure, inclusion bodies are easy to form during recombinant expression, and the yield is very low.

Currently, PLA2R-Ab is usually detected using a diagnostic kit of germany, which uses human recombinant PLA2R as capture molecule, wherein the complete human recombinant PLA2R refers to the extracellular region of the PLA2R protein, which contains more than 1300 amino acid residues, and has a large molecular weight of about 185kD. The molecule has the advantages of high expression difficulty, easy aggregation of products, low yield and difficult purification. Whether the full-length protein or the extracellular region protein fragment of PLA2R is expressed, the expression quantity is low in the preparation process, a large amount of required protein is difficult to obtain, the development of PLA2R type membranous nephropathy diagnostic reagents is hindered, and the production cost is increased. In addition, in the clinical application process, the inventor finds that the diagnosis kit of German European Mongolian possibly has false negative phenomenon on the diagnosis of PLA2R type membranous nephropathy, and influences the accuracy of the diagnosis.

However, in a first aspect of the invention, the invention proposes a fusion protein. According to an embodiment of the invention, the fusion protein comprises: PLA2R epitope peptide; albumin or a fragment thereof, to which the PLA2R epitope peptide is linked. The fusion protein disclosed by the embodiment of the invention has the advantages of high expression quantity, easiness in acquisition, capability of being specifically combined with the PLA2R antibody, high combining capability, high sensitivity and the like, can be used for detecting the content of the PLA2R antibody and diagnosing PLA2R type membranous nephropathy, and has high accuracy.

In a second aspect of the invention, the invention provides a nucleic acid molecule. According to an embodiment of the invention, the nucleic acid molecule encodes the fusion protein of the first aspect. The nucleic acid molecules of the invention may be effective in expressing the fusion proteins of the first aspect.

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

In a fourth aspect of the invention, the invention provides a recombinant cell. According to an embodiment of the invention, the recombinant cell comprises: carrying the nucleic acid molecule of the second aspect or the expression vector of the third aspect; alternatively, the fusion protein of the first aspect is expressed. The recombinant cells of the embodiments described herein may be used for in vitro expression and bulk acquisition of the fusion proteins described in the first aspect.

In a fifth aspect of the invention, the invention provides a kit. According to an embodiment of the invention, the kit comprises: 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. The kit disclosed by the embodiment of the invention can be used for effectively detecting the content of the PLA2R antibody and also can be used for diagnosing related diseases caused by the PLA2R antibody or evaluating the stage or prognosis of the related diseases caused by the PLA2R antibody, such as PLA2R antibody positive membranous nephropathy.

In a sixth aspect of the invention, the invention provides the use of a fusion protein according to the first aspect, a nucleic acid molecule according to the second aspect, an expression vector according to the third aspect or a recombinant cell according to the fourth aspect for the preparation of a kit for detecting a PLA2R antibody, diagnosing a disease associated with excessive PLA2R antibody, assessing the prognosis of a disease associated with PLA2R antibody or assessing the staging of a disease associated with PLA2R antibody.

In a seventh aspect of the invention, the invention provides a method of detecting a PLA2R antibody. According to an embodiment of the invention, the method comprises: detecting PLA2R antibodies in a sample to be tested using the fusion protein of the first aspect, the nucleic acid molecule of the second aspect, the expression vector of the third aspect, the recombinant cell of the fourth aspect or the kit of the fifth aspect; and determining the content of PLA2R antibody in the sample to be detected based on the detection result. The method provided by the embodiment of the invention can effectively detect the content of the PLA2R antibody, and has the advantages of high detection sensitivity, high detection accuracy and the like.

Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.

Drawings

The foregoing and/or additional aspects and advantages of the invention will become apparent and may be better understood from the following description of embodiments taken in conjunction with the accompanying drawings in which:

FIG. 1 is a schematic diagram of a PLA2R structure in accordance with an embodiment of the present invention;

FIG. 2 is a schematic view showing the structure of an expression frame in embodiment 1 of the present invention;

FIG. 3 is a graph showing the result of electrophoresis of pcDNA3.4 of example 1 of the present invention;

FIG. 4 is a graph showing the electrophoresis results of RC21-RC24 in example 1 of the present invention;

FIG. 5 is a schematic diagram showing the structure of the RC21-RC24 protein of example 1 of the present invention;

FIG. 6 shows the purification effect of the RC22 and RC24 proteins detected by SDS-PAGE in example 1 of the present invention;

FIG. 7 is a graph showing determination of the concentration of RC22 in example 3 of the present invention;

FIG. 8 is a graph of determination of the concentration of RC24 coated plates in example 3 of the present invention;

FIG. 9 is a graph of plasma dilution determinations of RC22 in example 3 of the present invention;

FIG. 10 is a graph of plasma dilution determination of RC24 in example 3 of the present invention;

FIG. 11 is a standard curve of the European-Mongolian kit, RC22, RC24 in example 4 of the present invention;

FIG. 12 is a plot of RC24 versus RC22 for example 4 of the present invention;

FIG. 13 is a graph showing the results of the detection of the European style kit in example 4 of the present invention with respect to the results of the detection of RC 22;

FIG. 14 is a graph showing the results of the detection of the European style kit in example 4 of the present invention with respect to the results of the detection of RC 24;

FIG. 15 is a graph showing the results of the detection of the European style kit after the sample size is enlarged in example 5 of the present invention with respect to the results of the detection of RC 22;

FIG. 16 shows the purification effect of the RC3 protein detected by SDS-PAGE in example 6 of the present invention;

FIG. 17 is a graph showing the result of neutralization test conducted by the European-Mongolian kit in example 6 of the present invention;

FIG. 18 is a graph showing the results of neutralization test conducted in accordance with the RC22 test of example 6 of the present invention.

Detailed Description

Embodiments of the present invention are described in detail below. The following examples are illustrative only and are not to be construed as limiting the invention.

It should be noted that the terms "first," "second," and "second" are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implying a number of technical features being indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include one or more such feature. Further, in the description of the present invention, unless otherwise indicated, the meaning of "a plurality" is two or more.

The endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and are understood to encompass values approaching those ranges or values. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.

In this document, the terms "comprise" or "include" are used in an open-ended fashion, i.e., to include what is indicated by the present invention, but not to exclude other aspects.

In this document, the terms "optionally," "optional," or "optionally" generally refer to the subsequently described event or condition may, but need not, occur, and the description includes instances in which the event or condition occurs, as well as instances in which the event or condition does not.

In this context, the terms "identity", "homology" or "similarity" are used to describe the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences when compared to the amino acid sequence or nucleic acid sequence of a reference sequence, using conventional methods, e.g., see Ausubel et al, et al (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: support.3 (National Biomedical Research Foundation, washington, D.C.), there are many algorithms for alignment and determination of sequence identity, including homology alignment algorithms of needle et al (1970) J.mol.biol.48:443, computer programs using these algorithms are also available and include, but are not limited to, ALIGN or Megalign (DNASTAR) software, or the programs of Pearson et al (1988) Proc.Natl.Acad.Sci.85:2444, the Smith-Waterman algorithm (Meth.mol.70:173-187 (1997), and BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al (1990) J.Mol.biol.215:403-410), and include but are also available in the programs of ALIGN or Megalign (DNASTAR), or the programs of BLAST-2, and the programs of Abelson.G.35:266, and the programs of Abelson.35:266, respectively.

In this context, the term "nucleotide" generally refers to a modified form that may be a ribonucleotide, a deoxynucleotide, or any type of nucleotide, as well as combinations thereof.

As used herein, the term "at least 90% similarity" refers to a similarity of at least 90%, and may be 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% with each reference sequence.

Herein, the term "PLA2R epitope peptide" refers to a polypeptide whose amino acid sequence is derived from PLA2R, which "PLA2R epitope peptide" comprises an epitope that can specifically bind to a PLA2R antibody, e.g., a PLA2R epitope peptide comprises CysR, fnII, and PLA2R binding region 1; wherein the term "CysR" refers to the CysR domain in PLA2R, and the term "PLA2R binding region 1" refers to CTLD domain 1 in PLA 2R; the term "PLA2R binding region 2" refers to CTLD domain 2 in PLA2R (see fig. 1).

In this context, the term "expression vector" generally refers to a nucleic acid molecule capable of insertion into a suitable host for self-replication, which transfers the inserted nucleic acid molecule into and/or between host cells. The expression vector may include a vector mainly used for inserting DNA or RNA into cells, a vector mainly used for replicating DNA or RNA, and a vector mainly used for expression of transcription and/or translation of DNA or RNA. The expression vector also includes vectors having a plurality of the above functions. The expression vector may be a polynucleotide capable of transcription and translation into a polypeptide when introduced into a suitable host cell. Typically, the expression vector will produce the desired expression product by culturing a suitable host cell containing the expression vector.

As used herein, the term "recombinant cell" generally refers to a cell that has been modified or recombined with genetic material of a host cell using genetic engineering techniques or cell fusion techniques to obtain a unique trait that is stably inherited. Wherein the term "host cell" refers to a prokaryotic or eukaryotic cell into which a recombinant expression vector may be introduced. The term "transformed" or "transfected" as used herein refers to the introduction of a nucleic acid (e.g., vector) into a cell by various techniques known in the art. Suitable host cells can be transformed or transfected with the DNA sequences of the invention and can be used for 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 (human amniotic fluid derived cells), insect cells, per.c6 cells and CoS cells.

In this context, the term "fusion protein" refers to a novel protein in which at least two proteins or polypeptides are fused, and such fusion operations are usually achieved by techniques such as genetic engineering, for example, by recombinant expression products of two genes obtained by DNA recombination techniques. By PLA2R is meant herein in a fused state with serum albumin.

The term "pharmaceutical composition" as used herein generally refers to unit dosage forms and may be prepared by any of the methods well known in the pharmaceutical arts. 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 intimately bringing into association the active compound with liquid carriers, finely divided solid carriers or both.

As used herein, the term "pharmaceutically acceptable" means that the substance or composition must be chemically and/or toxicologically compatible with the other ingredients comprising the formulation and/or the mammal being treated therewith. Preferably, the term "pharmaceutically acceptable" as used herein refers to use in animals, particularly humans, approved by the federal regulatory agency or a state government or listed in the U.S. pharmacopeia or other generally recognized pharmacopeia.

As used herein, the term "pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier" may include any solvent, solid excipient, diluent or other liquid excipient, and the like, as appropriate for the particular dosage form of interest. In addition to the extent to which any conventional adjuvant is incompatible with the compounds of the present invention, such as any adverse biological effects produced or interactions with any other component of the pharmaceutically acceptable composition in a deleterious manner, their use is also contemplated by the present invention.

As used herein, the term "administering" refers to introducing a predetermined amount of a substance into a patient by some suitable means. The fusion protein or pharmaceutical composition of the invention may be administered by any common route, provided that it reaches the desired tissue. Various modes of administration are contemplated, including peritoneal, intravenous, intramuscular, subcutaneous, etc., but the invention is not limited to these illustrated modes of administration. Preferably, the compositions of the present invention are administered by intravenous or subcutaneous injection.

Herein, the term "PLA2R antibody-associated disease" generally refers to a disease caused by an abnormality of a PLA2R antibody, such as a disease caused by an increase in a PLA2R antibody, including, but not limited to, PLA2R antibody positive membranous nephropathy. Among them, PLA2R is a membrane protein expressed in glomerular podocytes, and becomes an antigen for unknown reasons, resulting in the production of corresponding autoimmune antibodies (PLA 2R antibodies) by the body. PLA2R antibodies bind to PLA2R and deposit under glomerular basement membrane, triggering downstream effects such as C3 deposition, resulting in PLA2R antibody positive membranous nephropathy.

The terms "anti-PLA 2R antibody", "PLA2R antibody" and "PLA2R-Ab" are used interchangeably herein.

In this context, the term "treatment" is intended to mean obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of completely or partially preventing the disease or symptoms thereof, and/or may be therapeutic in terms of partially or completely curing the disease and/or adverse effects caused by the disease. As used herein, "treating" encompasses diseases in mammals, particularly humans, including: (a) Preventing the occurrence of a disease or disorder in an individual susceptible to the disease but not yet diagnosed with the disease; (b) inhibiting disease, e.g., arresting disease progression; or (c) alleviating a disease, e.g., alleviating symptoms associated with a disease. As used herein, "treating" or "treatment" encompasses any administration of a drug or compound to an individual to treat, cure, alleviate, ameliorate, reduce or inhibit a disease in the individual, including, but not limited to, administration of a drug comprising a compound described herein to an individual in need thereof.

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 a PLA2R antibody, a method for diagnosing a disease associated with the PLA2R antibody, a method for assessing the stage of a disease associated with the PLA2R antibody, and a method for assessing the prognosis of a disease associated with the PLA2R antibody, which will be described in detail below, respectively.

Fusion proteins

In a first aspect of the invention, the invention provides a fusion protein. According to an embodiment of the invention, the fusion protein comprises: PLA2R epitope peptide; albumin or a fragment thereof, to which the PLA2R epitope peptide is linked. The fusion protein disclosed by the embodiment of the invention has the advantages of high expression quantity, easiness in acquisition, capability of being specifically combined with the PLA2R antibody, high combining capability, high sensitivity and the like, can be used for detecting the content of the PLA2R antibody and diagnosing PLA2R type membranous nephropathy, and has high accuracy.

According to one implementation of the embodiments of the invention, the C-terminus of the PLA2R epitope peptide is linked to the N-terminus of the albumin or fragment thereof.

According to one implementation of the embodiments of the invention, the N-terminus of the PLA2R epitope peptide is linked to the C-terminus of the albumin or fragment thereof.

According to one implementation of the examples of the invention, the PLA2R epitope peptide includes CysR, fnII, and PLA2R binding region 1 and optionally PLA2R binding region 2. The structure of PLA2R is shown in FIG. 1. The inventor finds through experiments that the binding capacity of PLA2R epitope and PLA2R-Ab can be maintained by adopting a CysR domain, an FNII domain and a part of CTLD domain (PLA 2R binding region 1 or PLA2R binding region 1 and PLA2R binding region 2) of PLA2R as PLA2R epitope peptide and fusing the PLA2R epitope peptide with albumin or fragments thereof to obtain fusion protein. The fusion protein prepared from the PLA2R epitope peptide has strong binding capacity with PLA2R-Ab, has the advantages of high sensitivity, high accuracy and the like, and has high expression quantity.

According to one implementation of the embodiment of the invention, the C-terminal of the CysR is connected to the N-terminal of the FNII, which is connected to the N-terminal of the PLA2R binding region 1.

According to one implementation of the embodiment of the invention, the C-terminal of CysR is connected to the N-terminal of FnII, the C-terminal of FnII is connected to the N-terminal of PLA2R binding region 1, and the C-terminal of PLA2R binding region 1 is connected to the N-terminal of PLA2R binding region 2.

According to one embodiment of the examples of the invention, the PLA2R 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.

EGVAAALTPERLLEWQDKGIFVIQSESLKKCIQAGKSVLTLENCKQANKHMLWKWVSNHGLFNIGGSGCLGLNFSAPEQPLSLYECDSTLVSLRWRCNRKMITGPLQYSVQVAHDNTVVASRKYIHKWISYGSGGGDICEYLHKDLHTIKGNTHGMPCMFPFQYNHQWHHECTREGREDDLLWCATTSRYERDEKWGFCPDPTSAEVGCDTIWEKDLNSHICYQFNLLSSLSWSEAHSSCQMQGGTLLSITDETEENFIREHMSSKTVEVWMGLNQLDEHAGWQWSDGTPLNYLNWSPEVNFEPFVEDHCGTFSSFMPSAWRSRDCESTLPYICKKYLNHIDHEIVE(SEQ ID NO:1)。

EGVAAALTPERLLEWQDKGIFVIQSESLKKCIQAGKSVLTLENCKQANKHMLWKWVSNHGLFNIGGSGCLGLNFSAPEQPLSLYECDSTLVSLRWRCNRKMITGPLQYSVQVAHDNTVVASRKYIHKWISYGSGGGDICEYLHKDLHTIKGNTHGMPCMFPFQYNHQWHHECTREGREDDLLWCATTSRYERDEKWGFCPDPTSAEVGCDTIWEKDLNSHICYQFNLLSSLSWSEAHSSCQMQGGTLLSITDETEENFIREHMSSKTVEVWMGLNQLDEHAGWQWSDGTPLNYLNWSPEVNFEPFVEDHCGTFSSFMPSAWRSRDCESTLPYICKKYLNHIDHEIVEKDAWKYYATHCEPGWNPYNRNCYKLQKEEKTWHEALRSCQADNSALIDITSLAEVEFLVTLLGDENASETWIGLSSNKIPVSFEWSNDSSVIFTNWHTLEPHIFPNRSQLCVSAEQSEGHWKVKNCEERLFYICKKAGHVLSD(SEQ ID NO:2)。

According to one implementation of the embodiment of the invention, the albumin or fragment thereof is serum albumin or a fragment thereof, preferably human serum albumin.

According to one embodiment of the examples of the invention, the albumin or 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.

DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL(SEQ ID NO:3)。

According to an embodiment of the invention, the fusion protein further comprises a connecting peptide.

According to one embodiment of the examples of the present invention, the N-terminus of the linker peptide is linked to the C-terminus of the PLA2R epitope peptide, and the C-terminus of the linker peptide is linked to the N-terminus of the albumin or fragment thereof.

According to one embodiment of the examples of the present invention, the N-terminus of the linker peptide is linked to the C-terminus of the albumin or fragment thereof, and the C-terminus of the linker peptide is linked to the N-terminus of the PLA2R epitope peptide.

According to one embodiment of the embodiments 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.

According to one embodiment of the examples of the present invention, the connecting peptide comprises the amino acid sequence shown in SEQ ID NO. 4.

GGGGSGGGGS(SEQ ID NO:4)。

According to an embodiment of the invention, the fusion protein further comprises a tag.

According to one implementation of the embodiments of the invention, the tag is attached to the end of the PLA2R epitope peptide or albumin or fragment thereof.

According to one implementation of the 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 subjected to adsorption purification for subsequent detection of the PLA2R antibody.

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

HHHHHH(SEQ ID NO:5)。

According to an embodiment of the invention, the N-terminus of the HIS tag is linked to the C-terminus of the albumin or fragment thereof; or the C end of the HIS label is connected with the N end of the PLA2R epitope peptide. The inventors found that the expression level of the fusion protein was higher when the N-terminus of the HIS tag was linked to the C-terminus of the albumin or fragment thereof.

According to an embodiment of the invention, the N-terminus of the HIS tag is linked to the C-terminus of the albumin or fragment thereof, further comprising a TG sequence. Furthermore, the expression of the fusion protein can be further improved.

According to an embodiment of the invention, the N-terminus of the TG sequence is linked to the C-terminus of the albumin or fragment thereof and the C-terminus of the TG sequence is linked to the N-terminus of the HIS tag.

According to one embodiment of the examples of the present invention, the fusion protein has an amino acid sequence as shown in any one of SEQ ID NOS.6 to 9.

Preferably, the fusion protein has the amino acid sequence shown in SEQ ID NO. 6. The inventor finds that compared with other fusion proteins, the expression quantity of the fusion protein is obviously improved.

EGVAAALTPERLLEWQDKGIFVIQSESLKKCIQAGKSVLTLENCKQANKHMLWKWVSNHGLFNIGGSGCLGLNFSAPEQPLSLYECDSTLVSLRWRCNRKMITGPLQYSVQVAHDNTVVASRKYIHKWISYGSGGGDICEYLHKDLHTIKGNTHGMPCMFPFQYNHQWHHECTREGREDDLLWCATTSRYERDEKWGFCPDPTSAEVGCDTIWEKDLNSHICYQFNLLSSLSWSEAHSSCQMQGGTLLSITDETEENFIREHMSSKTVEVWMGLNQLDEHAGWQWSDGTPLNYLNWSPEVNFEPFVEDHCGTFSSFMPSAWRSRDCESTLPYICKKYLNHIDHEIVEKDAWKYYATHCEPGWNPYNRNCYKLQKEEKTWHEALRSCQADNSALIDITSLAEVEFLVTLLGDENASETWIGLSSNKIPVSFEWSNDSSVIFTNWHTLEPHIFPNRSQLCVSAEQSEGHWKVKNCEERLFYICKKAGHVLSDGGGGSGGGGSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLTGHHHHHH(SEQ ID NO:6)。

HHHHHHEGVAAALTPERLLEWQDKGIFVIQSESLKKCIQAGKSVLTLENCKQANKHMLWKWVSNHGLFNIGGSGCLGLNFSAPEQPLSLYECDSTLVSLRWRCNRKMITGPLQYSVQVAHDNTVVASRKYIHKWISYGSGGGDICEYLHKDLHTIKGNTHGMPCMFPFQYNHQWHHECTREGREDDLLWCATTSRYERDEKWGFCPDPTSAEVGCDTIWEKDLNSHICYQFNLLSSLSWSEAHSSCQMQGGTLLSITDETEENFIREHMSSKTVEVWMGLNQLDEHAGWQWSDGTPLNYLNWSPEVNFEPFVEDHCGTFSSFMPSAWRSRDCESTLPYICKKYLNHIDHEIVEKDAWKYYATHCEPGWNPYNRNCYKLQKEEKTWHEALRSCQADNSALIDITSLAEVEFLVTLLGDENASETWIGLSSNKIPVSFEWSNDSSVIFTNWHTLEPHIFPNRSQLCVSAEQSEGHWKVKNCEERLFYICKKAGHVLSDGGGGSGGGGSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL(SEQ ID NO:7)。

EGVAAALTPERLLEWQDKGIFVIQSESLKKCIQAGKSVLTLENCKQANKHMLWKWVSNHGLFNIGGSGCLGLNFSAPEQPLSLYECDSTLVSLRWRCNRKMITGPLQYSVQVAHDNTVVASRKYIHKWISYGSGGGDICEYLHKDLHTIKGNTHGMPCMFPFQYNHQWHHECTREGREDDLLWCATTSRYERDEKWGFCPDPTSAEVGCDTIWEKDLNSHICYQFNLLSSLSWSEAHSSCQMQGGTLLSITDETEENFIREHMSSKTVEVWMGLNQLDEHAGWQWSDGTPLNYLNWSPEVNFEPFVEDHCGTFSSFMPSAWRSRDCESTLPYICKKYLNHIDHEIVEGGGGSGGGGSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLTGHHHHHH(SEQ ID NO:8)。

HHHHHHEGVAAALTPERLLEWQDKGIFVIQSESLKKCIQAGKSVLTLENCKQANKHMLWKWVSNHGLFNIGGSGCLGLNFSAPEQPLSLYECDSTLVSLRWRCNRKMITGPLQYSVQVAHDNTVVASRKYIHKWISYGSGGGDICEYLHKDLHTIKGNTHGMPCMFPFQYNHQWHHECTREGREDDLLWCATTSRYERDEKWGFCPDPTSAEVGCDTIWEKDLNSHICYQFNLLSSLSWSEAHSSCQMQGGTLLSITDETEENFIREHMSSKTVEVWMGLNQLDEHAGWQWSDGTPLNYLNWSPEVNFEPFVEDHCGTFSSFMPSAWRSRDCESTLPYICKKYLNHIDHEIVEGGGGSGGGGSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL(SEQ ID NO:9)。

Nucleic acid molecules, expression vectors and recombinant cells

In preparing or obtaining these fusion proteins, nucleic acid molecules expressing these fusion proteins may be used, linked to different vectors, and then expressed in different cells to obtain the corresponding fusion proteins.

In a second aspect of the invention, the invention provides a nucleic acid molecule. According to an embodiment of the invention, the nucleic acid molecule encodes the fusion protein of the first aspect. The nucleic acid molecules can be used to express the fusion proteins of the first aspect, especially in prokaryotes or lower eukaryotes.

According to an embodiment of the invention, the nucleic acid molecule is DNA.

The nucleotide sequence shown as SEQ ID NO. 10 is used for encoding SEQ ID NO. 6.

The nucleotide sequence shown as SEQ ID NO. 11 is used for encoding SEQ ID NO. 7.

The nucleotide sequence shown as SEQ ID NO. 12 is used for encoding SEQ ID NO. 8.

The nucleotide sequence shown as SEQ ID NO. 13 is used for encoding SEQ ID NO. 9.

GAGGGTGTGGCGGCGGCGCTTACCCCCGAGCGGCTCCTGGAGTGGCAGGATAAAGGAATATTTGTTATCCAAAGTGAGAGTCTCAAGAAATGCATTCAAGCAGGTAAATCGGTTCTGACCCTGGAGAACTGCAAGCAAGCAAACAAGCACATGCTGTGGAAATGGGTTTCAAACCATGGCCTCTTTAACATAGGAGGCAGTGGTTGCCTGGGCCTGAATTTCTCCGCCCCAGAGCAGCCATTAAGCTTATATGAATGTGACTCCACCCTCGTTTCCTTACGGTGGCGCTGTAACAGGAAGATGATCACAGGCCCGCTGCAGTACTCTGTCCAGGTGGCGCATGACAACACAGTGGTGGCCTCACGGAAGTATATTCATAAGTGGATTTCTTATGGGTCAGGTGGTGGAGACATTTGTGAATATCTACACAAAGATTTGCATACAATCAAAGGGAACACCCACGGGATGCCGTGTATGTTTCCCTTCCAGTATAACCATCAGTGGCATCATGAATGTACCCGTGAAGGTCGGGAAGATGACTTACTGTGGTGTGCCACGACAAGCCGTTATGAAAGAGATGAAAAGTGGGGATTTTGCCCTGATCCCACCTCTGCAGAAGTAGGTTGTGATACTATTTGGGAGAAGGACCTCAATTCACACATTTGCTACCAGTTCAACCTGCTTTCATCTCTCTCTTGGAGTGAGGCACATTCTTCATGCCAGATGCAAGGAGGTACGCTGTTAAGTATTACAGATGAAACTGAAGAAAATTTCATAAGGGAGCACATGAGCAGTAAAACAGTGGAGGTGTGGATGGGCCTCAATCAGCTGGATGAACACGCTGGCTGGCAGTGGTCTGATGGAACGCCGCTCAACTATCTGAATTGGAGCCCAGAGGTAAATTTTGAGCCATTTGTTGAAGATCACTGTGGAACATTTAGTTCATTTATGCCAAGTGCCTGGAGGAGTCGGGATTGTGAGTCCACCTTGCCATATATATGTAAAAAATATCTAAACCACATTGATCATGAAATAGTTGAAAAAGATGCGTGGAAATATTATGCTACCCACTGTGAGCCTGGCTGGAATCCCTACAATCGTAATTGCTACAAACTTCAGAAAGAAGAAAAGACCTGGCATGAGGCTCTGCGTTCTTGTCAGGCTGATAACAGTGCATTAATAGACATAACCTCATTAGCAGAGGTGGAGTTTCTTGTAACCCTCCTTGGAGATGAAAATGCATCAGAAACATGGATTGGTTTGAGCAGCAATAAAATTCCAGTTTCCTTTGAATGGTCTAATGACTCTTCAGTCATCTTTACTAATTGGCACACACTTGAGCCCCACATTTTTCCAAATAGAAGCCAGCTGTGTGTCTCAGCAGAGCAGTCTGAGGGACACTGGAAAGTCAAAAATTGTGAAGAAAGACTTTTTTACATTTGTAAAAAAGCAGGCCATGTCCTCTCTGATGGCGGTGGAGGCTCCGGCGGAGGTGGAAGCGATGCACACAAGAGTGAGGTTGCTCATCGGTTTAAAGATTTGGGAGAAGAAAATTTCAAAGCCTTGGTGTTGATTGCCTTTGCTCAGTATCTTCAGCAGTGTCCATTTGAAGATCATGTAAAATTAGTGAATGAAGTAACTGAATTTGCAAAAACATGTGTTGCTGATGAGTCAGCTGAAAATTGTGACAAATCACTTCATACCCTTTTTGGAGACAAATTATGCACAGTTGCAACTCTTCGTGAAACCTATGGTGAAATGGCTGACTGCTGTGCAAAACAAGAACCTGAGAGAAATGAATGCTTCTTGCAACACAAAGATGACAACCCAAACCTCCCCCGATTGGTGAGACCAGAGGTTGATGTGATGTGCACTGCTTTTCATGACAATGAAGAGACATTTTTGAAAAAATACTTATATGAAATTGCCAGAAGACATCCTTACTTTTATGCCCCGGAACTCCTTTTCTTTGCTAAAAGGTATAAAGCTGCTTTTACAGAATGTTGCCAAGCTGCTGATAAAGCTGCCTGCCTGTTGCCAAAGCTCGATGAACTTCGGGATGAAGGGAAGGCTTCGTCTGCCAAACAGAGACTCAAGTGTGCCAGTCTCCAAAAATTTGGAGAAAGAGCTTTCAAAGCATGGGCAGTAGCTCGCCTGAGCCAGAGATTTCCCAAAGCTGAGTTTGCAGAAGTTTCCAAGTTAGTGACAGATCTTACCAAAGTCCACACGGAATGCTGCCATGGAGATCTGCTTGAATGTGCTGATGACAGGGCGGACCTTGCCAAGTATATCTGTGAAAATCAAGATTCGATCTCCAGTAAACTGAAGGAATGCTGTGAAAAACCTCTGTTGGAAAAATCCCACTGCATTGCCGAAGTGGAAAATGATGAGATGCCTGCTGACTTGCCTTCATTAGCTGCTGATTTTGTTGAAAGTAAGGATGTTTGCAAAAACTATGCTGAGGCAAAGGATGTCTTCCTGGGCATGTTTTTGTATGAATATGCAAGAAGGCATCCTGATTACTCTGTCGTGCTGCTGCTGAGACTTGCCAAGACATATGAAACCACTCTAGAGAAGTGCTGTGCCGCTGCAGATCCTCATGAATGCTATGCCAAAGTGTTCGATGAATTTAAACCTCTTGTGGAAGAGCCTCAGAATTTAATCAAACAAAATTGTGAGCTTTTTGAGCAGCTTGGAGAGTACAAATTCCAGAATGCGCTATTAGTTCGTTACACCAAGAAAGTACCCCAAGTGTCAACTCCAACTCTTGTAGAGGTCTCAAGAAACCTAGGAAAAGTGGGCAGCAAATGTTGTAAACATCCTGAAGCAAAAAGAATGCCCTGTGCAGAAGACTATCTATCCGTGGTCCTGAACCAGTTATGTGTGTTGCATGAGAAAACGCCAGTAAGTGACAGAGTCACCAAATGCTGCACAGAATCCTTGGTGAACAGGCGACCATGCTTTTCAGCTCTGGAAGTCGATGAAACATACGTTCCCAAAGAGTTTAATGCTGAAACATTCACCTTCCATGCAGATATATGCACACTTTCTGAGAAGGAGAGACAAATCAAGAAACAAACTGCACTTGTTGAGCTTGTGAAACACAAGCCCAAGGCAACAAAAGAGCAACTGAAAGCTGTTATGGATGATTTCGCAGCTTTTGTAGAGAAGTGCTGCAAGGCTGACGATAAGGAGACCTGCTTTGCCGAGGAGGGTAAAAAACTTGTTGCTGCAAGTCAAGCTGCCTTAGGCTTAACCGGTCATCATCACCATCACCAT(SEQ ID NO:10)。

CATCATCACCATCACCATGAGGGTGTGGCGGCGGCGCTTACCCCCGAGCGGCTCCTGGAGTGGCAGGATAAAGGAATATTTGTTATCCAAAGTGAGAGTCTCAAGAAATGCATTCAAGCAGGTAAATCGGTTCTGACCCTGGAGAACTGCAAGCAAGCAAACAAGCACATGCTGTGGAAATGGGTTTCAAACCATGGCCTCTTTAACATAGGAGGCAGTGGTTGCCTGGGCCTGAATTTCTCCGCCCCAGAGCAGCCATTAAGCTTATATGAATGTGACTCCACCCTCGTTTCCTTACGGTGGCGCTGTAACAGGAAGATGATCACAGGCCCGCTGCAGTACTCTGTCCAGGTGGCGCATGACAACACAGTGGTGGCCTCACGGAAGTATATTCATAAGTGGATTTCTTATGGGTCAGGTGGTGGAGACATTTGTGAATATCTACACAAAGATTTGCATACAATCAAAGGGAACACCCACGGGATGCCGTGTATGTTTCCCTTCCAGTATAACCATCAGTGGCATCATGAATGTACCCGTGAAGGTCGGGAAGATGACTTACTGTGGTGTGCCACGACAAGCCGTTATGAAAGAGATGAAAAGTGGGGATTTTGCCCTGATCCCACCTCTGCAGAAGTAGGTTGTGATACTATTTGGGAGAAGGACCTCAATTCACACATTTGCTACCAGTTCAACCTGCTTTCATCTCTCTCTTGGAGTGAGGCACATTCTTCATGCCAGATGCAAGGAGGTACGCTGTTAAGTATTACAGATGAAACTGAAGAAAATTTCATAAGGGAGCACATGAGCAGTAAAACAGTGGAGGTGTGGATGGGCCTCAATCAGCTGGATGAACACGCTGGCTGGCAGTGGTCTGATGGAACGCCGCTCAACTATCTGAATTGGAGCCCAGAGGTAAATTTTGAGCCATTTGTTGAAGATCACTGTGGAACATTTAGTTCATTTATGCCAAGTGCCTGGAGGAGTCGGGATTGTGAGTCCACCTTGCCATATATATGTAAAAAATATCTAAACCACATTGATCATGAAATAGTTGAAAAAGATGCGTGGAAATATTATGCTACCCACTGTGAGCCTGGCTGGAATCCCTACAATCGTAATTGCTACAAACTTCAGAAAGAAGAAAAGACCTGGCATGAGGCTCTGCGTTCTTGTCAGGCTGATAACAGTGCATTAATAGACATAACCTCATTAGCAGAGGTGGAGTTTCTTGTAACCCTCCTTGGAGATGAAAATGCATCAGAAACATGGATTGGTTTGAGCAGCAATAAAATTCCAGTTTCCTTTGAATGGTCTAATGACTCTTCAGTCATCTTTACTAATTGGCACACACTTGAGCCCCACATTTTTCCAAATAGAAGCCAGCTGTGTGTCTCAGCAGAGCAGTCTGAGGGACACTGGAAAGTCAAAAATTGTGAAGAAAGACTTTTTTACATTTGTAAAAAAGCAGGCCATGTCCTCTCTGATGGCGGTGGAGGCTCCGGCGGAGGTGGAAGCGATGCACACAAGAGTGAGGTTGCTCATCGGTTTAAAGATTTGGGAGAAGAAAATTTCAAAGCCTTGGTGTTGATTGCCTTTGCTCAGTATCTTCAGCAGTGTCCATTTGAAGATCATGTAAAATTAGTGAATGAAGTAACTGAATTTGCAAAAACATGTGTTGCTGATGAGTCAGCTGAAAATTGTGACAAATCACTTCATACCCTTTTTGGAGACAAATTATGCACAGTTGCAACTCTTCGTGAAACCTATGGTGAAATGGCTGACTGCTGTGCAAAACAAGAACCTGAGAGAAATGAATGCTTCTTGCAACACAAAGATGACAACCCAAACCTCCCCCGATTGGTGAGACCAGAGGTTGATGTGATGTGCACTGCTTTTCATGACAATGAAGAGACATTTTTGAAAAAATACTTATATGAAATTGCCAGAAGACATCCTTACTTTTATGCCCCGGAACTCCTTTTCTTTGCTAAAAGGTATAAAGCTGCTTTTACAGAATGTTGCCAAGCTGCTGATAAAGCTGCCTGCCTGTTGCCAAAGCTCGATGAACTTCGGGATGAAGGGAAGGCTTCGTCTGCCAAACAGAGACTCAAGTGTGCCAGTCTCCAAAAATTTGGAGAAAGAGCTTTCAAAGCATGGGCAGTAGCTCGCCTGAGCCAGAGATTTCCCAAAGCTGAGTTTGCAGAAGTTTCCAAGTTAGTGACAGATCTTACCAAAGTCCACACGGAATGCTGCCATGGAGATCTGCTTGAATGTGCTGATGACAGGGCGGACCTTGCCAAGTATATCTGTGAAAATCAAGATTCGATCTCCAGTAAACTGAAGGAATGCTGTGAAAAACCTCTGTTGGAAAAATCCCACTGCATTGCCGAAGTGGAAAATGATGAGATGCCTGCTGACTTGCCTTCATTAGCTGCTGATTTTGTTGAAAGTAAGGATGTTTGCAAAAACTATGCTGAGGCAAAGGATGTCTTCCTGGGCATGTTTTTGTATGAATATGCAAGAAGGCATCCTGATTACTCTGTCGTGCTGCTGCTGAGACTTGCCAAGACATATGAAACCACTCTAGAGAAGTGCTGTGCCGCTGCAGATCCTCATGAATGCTATGCCAAAGTGTTCGATGAATTTAAACCTCTTGTGGAAGAGCCTCAGAATTTAATCAAACAAAATTGTGAGCTTTTTGAGCAGCTTGGAGAGTACAAATTCCAGAATGCGCTATTAGTTCGTTACACCAAGAAAGTACCCCAAGTGTCAACTCCAACTCTTGTAGAGGTCTCAAGAAACCTAGGAAAAGTGGGCAGCAAATGTTGTAAACATCCTGAAGCAAAAAGAATGCCCTGTGCAGAAGACTATCTATCCGTGGTCCTGAACCAGTTATGTGTGTTGCATGAGAAAACGCCAGTAAGTGACAGAGTCACCAAATGCTGCACAGAATCCTTGGTGAACAGGCGACCATGCTTTTCAGCTCTGGAAGTCGATGAAACATACGTTCCCAAAGAGTTTAATGCTGAAACATTCACCTTCCATGCAGATATATGCACACTTTCTGAGAAGGAGAGACAAATCAAGAAACAAACTGCACTTGTTGAGCTTGTGAAACACAAGCCCAAGGCAACAAAAGAGCAACTGAAAGCTGTTATGGATGATTTCGCAGCTTTTGTAGAGAAGTGCTGCAAGGCTGACGATAAGGAGACCTGCTTTGCCGAGGAGGGTAAAAAACTTGTTGCTGCAAGTCAAGCTGCCTTAGGCTTA(SEQ ID NO:11)。

GAGGGTGTGGCGGCGGCGCTTACCCCCGAGCGGCTCCTGGAGTGGCAGGATAAAGGAATATTTGTTATCCAAAGTGAGAGTCTCAAGAAATGCATTCAAGCAGGTAAATCGGTTCTGACCCTGGAGAACTGCAAGCAAGCAAACAAGCACATGCTGTGGAAATGGGTTTCAAACCATGGCCTCTTTAACATAGGAGGCAGTGGTTGCCTGGGCCTGAATTTCTCCGCCCCAGAGCAGCCATTAAGCTTATATGAATGTGACTCCACCCTCGTTTCCTTACGGTGGCGCTGTAACAGGAAGATGATCACAGGCCCGCTGCAGTACTCTGTCCAGGTGGCGCATGACAACACAGTGGTGGCCTCACGGAAGTATATTCATAAGTGGATTTCTTATGGGTCAGGTGGTGGAGACATTTGTGAATATCTACACAAAGATTTGCATACAATCAAAGGGAACACCCACGGGATGCCGTGTATGTTTCCCTTCCAGTATAACCATCAGTGGCATCATGAATGTACCCGTGAAGGTCGGGAAGATGACTTACTGTGGTGTGCCACGACAAGCCGTTATGAAAGAGATGAAAAGTGGGGATTTTGCCCTGATCCCACCTCTGCAGAAGTAGGTTGTGATACTATTTGGGAGAAGGACCTCAATTCACACATTTGCTACCAGTTCAACCTGCTTTCATCTCTCTCTTGGAGTGAGGCACATTCTTCATGCCAGATGCAAGGAGGTACGCTGTTAAGTATTACAGATGAAACTGAAGAAAATTTCATAAGGGAGCACATGAGCAGTAAAACAGTGGAGGTGTGGATGGGCCTCAATCAGCTGGATGAACACGCTGGCTGGCAGTGGTCTGATGGAACGCCGCTCAACTATCTGAATTGGAGCCCAGAGGTAAATTTTGAGCCATTTGTTGAAGATCACTGTGGAACATTTAGTTCATTTATGCCAAGTGCCTGGAGGAGTCGGGATTGTGAGTCCACCTTGCCATATATATGTAAAAAATATCTAAACCACATTGATCATGAAATAGTTGAAGGCGGTGGAGGCTCCGGCGGAGGTGGAAGCGATGCACACAAGAGTGAGGTTGCTCATCGGTTTAAAGATTTGGGAGAAGAAAATTTCAAAGCCTTGGTGTTGATTGCCTTTGCTCAGTATCTTCAGCAGTGTCCATTTGAAGATCATGTAAAATTAGTGAATGAAGTAACTGAATTTGCAAAAACATGTGTTGCTGATGAGTCAGCTGAAAATTGTGACAAATCACTTCATACCCTTTTTGGAGACAAATTATGCACAGTTGCAACTCTTCGTGAAACCTATGGTGAAATGGCTGACTGCTGTGCAAAACAAGAACCTGAGAGAAATGAATGCTTCTTGCAACACAAAGATGACAACCCAAACCTCCCCCGATTGGTGAGACCAGAGGTTGATGTGATGTGCACTGCTTTTCATGACAATGAAGAGACATTTTTGAAAAAATACTTATATGAAATTGCCAGAAGACATCCTTACTTTTATGCCCCGGAACTCCTTTTCTTTGCTAAAAGGTATAAAGCTGCTTTTACAGAATGTTGCCAAGCTGCTGATAAAGCTGCCTGCCTGTTGCCAAAGCTCGATGAACTTCGGGATGAAGGGAAGGCTTCGTCTGCCAAACAGAGACTCAAGTGTGCCAGTCTCCAAAAATTTGGAGAAAGAGCTTTCAAAGCATGGGCAGTAGCTCGCCTGAGCCAGAGATTTCCCAAAGCTGAGTTTGCAGAAGTTTCCAAGTTAGTGACAGATCTTACCAAAGTCCACACGGAATGCTGCCATGGAGATCTGCTTGAATGTGCTGATGACAGGGCGGACCTTGCCAAGTATATCTGTGAAAATCAAGATTCGATCTCCAGTAAACTGAAGGAATGCTGTGAAAAACCTCTGTTGGAAAAATCCCACTGCATTGCCGAAGTGGAAAATGATGAGATGCCTGCTGACTTGCCTTCATTAGCTGCTGATTTTGTTGAAAGTAAGGATGTTTGCAAAAACTATGCTGAGGCAAAGGATGTCTTCCTGGGCATGTTTTTGTATGAATATGCAAGAAGGCATCCTGATTACTCTGTCGTGCTGCTGCTGAGACTTGCCAAGACATATGAAACCACTCTAGAGAAGTGCTGTGCCGCTGCAGATCCTCATGAATGCTATGCCAAAGTGTTCGATGAATTTAAACCTCTTGTGGAAGAGCCTCAGAATTTAATCAAACAAAATTGTGAGCTTTTTGAGCAGCTTGGAGAGTACAAATTCCAGAATGCGCTATTAGTTCGTTACACCAAGAAAGTACCCCAAGTGTCAACTCCAACTCTTGTAGAGGTCTCAAGAAACCTAGGAAAAGTGGGCAGCAAATGTTGTAAACATCCTGAAGCAAAAAGAATGCCCTGTGCAGAAGACTATCTATCCGTGGTCCTGAACCAGTTATGTGTGTTGCATGAGAAAACGCCAGTAAGTGACAGAGTCACCAAATGCTGCACAGAATCCTTGGTGAACAGGCGACCATGCTTTTCAGCTCTGGAAGTCGATGAAACATACGTTCCCAAAGAGTTTAATGCTGAAACATTCACCTTCCATGCAGATATATGCACACTTTCTGAGAAGGAGAGACAAATCAAGAAACAAACTGCACTTGTTGAGCTTGTGAAACACAAGCCCAAGGCAACAAAAGAGCAACTGAAAGCTGTTATGGATGATTTCGCAGCTTTTGTAGAGAAGTGCTGCAAGGCTGACGATAAGGAGACCTGCTTTGCCGAGGAGGGTAAAAAACTTGTTGCTGCAAGTCAAGCTGCCTTAGGCTTAACCGGTCATCATCACCATCACCAT(SEQ ID NO:12)。

CATCATCACCATCACCATGAGGGTGTGGCGGCGGCGCTTACCCCCGAGCGGCTCCTGGAGTGGCAGGATAAAGGAATATTTGTTATCCAAAGTGAGAGTCTCAAGAAATGCATTCAAGCAGGTAAATCGGTTCTGACCCTGGAGAACTGCAAGCAAGCAAACAAGCACATGCTGTGGAAATGGGTTTCAAACCATGGCCTCTTTAACATAGGAGGCAGTGGTTGCCTGGGCCTGAATTTCTCCGCCCCAGAGCAGCCATTAAGCTTATATGAATGTGACTCCACCCTCGTTTCCTTACGGTGGCGCTGTAACAGGAAGATGATCACAGGCCCGCTGCAGTACTCTGTCCAGGTGGCGCATGACAACACAGTGGTGGCCTCACGGAAGTATATTCATAAGTGGATTTCTTATGGGTCAGGTGGTGGAGACATTTGTGAATATCTACACAAAGATTTGCATACAATCAAAGGGAACACCCACGGGATGCCGTGTATGTTTCCCTTCCAGTATAACCATCAGTGGCATCATGAATGTACCCGTGAAGGTCGGGAAGATGACTTACTGTGGTGTGCCACGACAAGCCGTTATGAAAGAGATGAAAAGTGGGGATTTTGCCCTGATCCCACCTCTGCAGAAGTAGGTTGTGATACTATTTGGGAGAAGGACCTCAATTCACACATTTGCTACCAGTTCAACCTGCTTTCATCTCTCTCTTGGAGTGAGGCACATTCTTCATGCCAGATGCAAGGAGGTACGCTGTTAAGTATTACAGATGAAACTGAAGAAAATTTCATAAGGGAGCACATGAGCAGTAAAACAGTGGAGGTGTGGATGGGCCTCAATCAGCTGGATGAACACGCTGGCTGGCAGTGGTCTGATGGAACGCCGCTCAACTATCTGAATTGGAGCCCAGAGGTAAATTTTGAGCCATTTGTTGAAGATCACTGTGGAACATTTAGTTCATTTATGCCAAGTGCCTGGAGGAGTCGGGATTGTGAGTCCACCTTGCCATATATATGTAAAAAATATCTAAACCACATTGATCATGAAATAGTTGAAGGCGGTGGAGGCTCCGGCGGAGGTGGAAGCGATGCACACAAGAGTGAGGTTGCTCATCGGTTTAAAGATTTGGGAGAAGAAAATTTCAAAGCCTTGGTGTTGATTGCCTTTGCTCAGTATCTTCAGCAGTGTCCATTTGAAGATCATGTAAAATTAGTGAATGAAGTAACTGAATTTGCAAAAACATGTGTTGCTGATGAGTCAGCTGAAAATTGTGACAAATCACTTCATACCCTTTTTGGAGACAAATTATGCACAGTTGCAACTCTTCGTGAAACCTATGGTGAAATGGCTGACTGCTGTGCAAAACAAGAACCTGAGAGAAATGAATGCTTCTTGCAACACAAAGATGACAACCCAAACCTCCCCCGATTGGTGAGACCAGAGGTTGATGTGATGTGCACTGCTTTTCATGACAATGAAGAGACATTTTTGAAAAAATACTTATATGAAATTGCCAGAAGACATCCTTACTTTTATGCCCCGGAACTCCTTTTCTTTGCTAAAAGGTATAAAGCTGCTTTTACAGAATGTTGCCAAGCTGCTGATAAAGCTGCCTGCCTGTTGCCAAAGCTCGATGAACTTCGGGATGAAGGGAAGGCTTCGTCTGCCAAACAGAGACTCAAGTGTGCCAGTCTCCAAAAATTTGGAGAAAGAGCTTTCAAAGCATGGGCAGTAGCTCGCCTGAGCCAGAGATTTCCCAAAGCTGAGTTTGCAGAAGTTTCCAAGTTAGTGACAGATCTTACCAAAGTCCACACGGAATGCTGCCATGGAGATCTGCTTGAATGTGCTGATGACAGGGCGGACCTTGCCAAGTATATCTGTGAAAATCAAGATTCGATCTCCAGTAAACTGAAGGAATGCTGTGAAAAACCTCTGTTGGAAAAATCCCACTGCATTGCCGAAGTGGAAAATGATGAGATGCCTGCTGACTTGCCTTCATTAGCTGCTGATTTTGTTGAAAGTAAGGATGTTTGCAAAAACTATGCTGAGGCAAAGGATGTCTTCCTGGGCATGTTTTTGTATGAATATGCAAGAAGGCATCCTGATTACTCTGTCGTGCTGCTGCTGAGACTTGCCAAGACATATGAAACCACTCTAGAGAAGTGCTGTGCCGCTGCAGATCCTCATGAATGCTATGCCAAAGTGTTCGATGAATTTAAACCTCTTGTGGAAGAGCCTCAGAATTTAATCAAACAAAATTGTGAGCTTTTTGAGCAGCTTGGAGAGTACAAATTCCAGAATGCGCTATTAGTTCGTTACACCAAGAAAGTACCCCAAGTGTCAACTCCAACTCTTGTAGAGGTCTCAAGAAACCTAGGAAAAGTGGGCAGCAAATGTTGTAAACATCCTGAAGCAAAAAGAATGCCCTGTGCAGAAGACTATCTATCCGTGGTCCTGAACCAGTTATGTGTGTTGCATGAGAAAACGCCAGTAAGTGACAGAGTCACCAAATGCTGCACAGAATCCTTGGTGAACAGGCGACCATGCTTTTCAGCTCTGGAAGTCGATGAAACATACGTTCCCAAAGAGTTTAATGCTGAAACATTCACCTTCCATGCAGATATATGCACACTTTCTGAGAAGGAGAGACAAATCAAGAAACAAACTGCACTTGTTGAGCTTGTGAAACACAAGCCCAAGGCAACAAAAGAGCAACTGAAAGCTGTTATGGATGATTTCGCAGCTTTTGTAGAGAAGTGCTGCAAGGCTGACGATAAGGAGACCTGCTTTGCCGAGGAGGGTAAAAAACTTGTTGCTGCAAGTCAAGCTGCCTTAGGCTTA(SEQ ID NO:13)。

In a third aspect of the invention, the invention provides an expression vector. According to an embodiment of the invention, the expression vector carries a nucleic acid molecule according to the second aspect. The expression vector can be effectively used for expressing the fusion protein of the first aspect, and particularly can be effectively expressed in a prokaryotic or lower eukaryote expression system.

According to one implementation of the embodiments of the invention, the expression vector is a eukaryotic expression vector.

According to one embodiment of the embodiments of the invention, the expression vector is a lentiviral vector.

In a fourth aspect of the invention, the invention provides a recombinant cell. According to an embodiment of the invention, the recombinant cell comprises: carrying the nucleic acid molecule of the second aspect or the expression vector of the third aspect; alternatively, the fusion protein of the first aspect is expressed. The recombinant cells according to embodiments of the present invention may be used for in vitro expression and mass production of the fusion protein according to the first aspect.

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

According to one implementation of the embodiments of the invention, the recombinant cell is a eukaryotic cell.

According to one implementation of the embodiments of the invention, the recombinant cell is a mammalian cell.

Those skilled in the art will appreciate that the features and advantages described above for fusion proteins are equally applicable to such nucleic acid molecules, expression vectors and recombinant cells and will not be described in detail herein.

Kit for detecting a substance in a sample

In a fifth aspect of the invention, the invention provides a kit. According to an embodiment of the invention, the kit comprises: 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. The kit disclosed by the embodiment of the invention can be used for effectively detecting the content of the PLA2R antibody, and can also be used for diagnosing related diseases caused by the PLA2R antibody or evaluating the stage or prognosis of the related diseases caused by the PLA2R antibody, such as PLA2R antibody positive membranous nephropathy.

According to one embodiment of the examples of the present invention, the working concentration of the fusion protein is 0.01-50. Mu.g/mL.

According to one embodiment of the examples of the present invention, the working concentration of the fusion protein is 0.05-40. Mu.g/mL.

According to one embodiment of the examples of the present invention, the working concentration of the fusion protein is 0.1-30. Mu.g/mL.

According to one implementation of the embodiments of the invention, the working concentration of the fusion protein is 1-20. Mu.g/mL, and is exemplified by 10. Mu.g/mL.

Those skilled in the art will appreciate that the features and advantages described above for fusion proteins, nucleic acid molecules, expression vectors and recombinant cells are equally applicable to the kit and will not be described in detail herein.

Use of the same

According to an embodiment of the invention, the disease associated with excessive PLA2R antibodies is selected from PLA2R antibody positive membranous nephropathy.

Those skilled in the art will appreciate that the features and advantages described above for fusion proteins, nucleic acid molecules, expression vectors, recombinant cells and kits are equally applicable for this purpose and will not be described in detail herein.

Method

In a seventh aspect of the invention, the invention provides a method of detecting a PLA2R antibody. According to an embodiment of the invention, the method comprises: detecting PLA2R antibodies in a sample to be tested using the fusion protein of the first aspect, the nucleic acid molecule of the second aspect, the expression vector of the third aspect, the recombinant cell of the fourth aspect or the kit of the fifth aspect; and determining the content of PLA2R antibody in the sample to be detected based on the detection result. The method provided by the embodiment of the invention can be used for effectively detecting the content of the PLA2R antibody in the sample to be detected, and has the advantages of high detection sensitivity, high detection accuracy and the like if being used for subsequent scientific researches.

In an eighth aspect of the invention, the invention provides a method of diagnosing a disease associated with a PLA2R antibody. According to an embodiment of the invention, the method comprises: detecting PLA2R antibodies in a sample to be tested using the fusion protein of the first aspect, the nucleic acid molecule of the second aspect, the expression vector of the third aspect, the recombinant cell of the fourth aspect or the kit of the fifth aspect; and determining the content of PLA2R antibody in the sample to be detected based on the detection result. The method provided by the embodiment of the invention can effectively diagnose the related diseases caused by the PLA2R antibody, and has the advantages of high diagnosis accuracy and the like.

According to an embodiment of the invention, the content of PLA2R antibodies in the sample to be tested is not lower than a predetermined threshold value, which is an indication that the sample to be tested is derived from a patient suffering from a disease related to PLA2R antibodies. For example, the predetermined threshold may be obtained by determining the PLA2R antibody content in plasma samples of individuals known to have PLA2R antibody positive membranous nephropathy and normal individuals multiple times.

In a ninth aspect of the invention, the invention provides a method of assessing the stage of a disease associated with a PLA2R antibody. According to an embodiment of the invention, the method comprises: detecting PLA2R antibodies in a sample to be tested, which is derived from a patient to be tested, using the fusion protein of the first aspect, the nucleic acid molecule of the second aspect, the expression vector of the third aspect, the recombinant cell of the fourth aspect or the kit of the fifth aspect; and determining the content of PLA2R antibody in the sample to be detected based on the detection result. The method provided by the embodiment of the invention can effectively evaluate the stage of the related diseases caused by the PLA2R antibody, and has the advantages of high accuracy of evaluation results and the like.

According to an embodiment of the present invention, the stage of the patient to be tested suffering from the related disease caused by the PLA2R antibody is determined based on the content of the PLA2R antibody in the sample to be tested.

In a tenth aspect of the invention, the invention provides a prognostic method for assessing a disease associated with a PLA2R antibody. According to an embodiment of the invention, the method comprises: detecting PLA2R antibodies in a sample to be tested, which is derived from a patient to be tested, using the fusion protein of the first aspect, the nucleic acid molecule of the second aspect, the expression vector of the third aspect, the recombinant cell of the fourth aspect or the kit of the fifth aspect; and determining the content of PLA2R antibody in the sample to be detected based on the detection result. The method provided by the embodiment of the invention can effectively evaluate the prognosis of the related diseases caused by the PLA2R antibody, and has the advantages of high accuracy of evaluation results and the like.

According to one implementation of the embodiments of the invention, the sample to be tested is derived from a patient suffering from a PLA2R antibody-induced related disease before or after treatment.

According to one implementation of the examples of the present invention, the prognostic effect of a PLA2R antibody-induced disease is determined based on the content of PLA2R antibody in a test sample of a patient suffering from a PLA2R antibody-induced disease before or after treatment.

According to one implementation of the examples of the invention, a decrease in the content of PLA2R antibodies in a test sample of a patient suffering from a disease associated with PLA2R antibodies after treatment is an indication that the prognosis of the patient is good.

In an eleventh aspect of the invention, the invention provides a method of using the kit of the fifth aspect. According to an embodiment of the invention, the method comprises: detecting the PLA2R antibody in the sample to be detected by adopting the kit in the fifth aspect; and determining the content of PLA2R antibody in the sample to be detected based on the detection result. The method provided by the embodiment of the invention can be used for effectively detecting the content of the PLA2R antibody, and has the advantages of high detection sensitivity, high detection accuracy and the like.

According to an embodiment of the embodiments of the present invention, the method according to the seventh, eighth, ninth, tenth and eleventh aspects may further include at least one of the following technical features:

according to an embodiment of the invention, the detecting comprises: carrying out first contact treatment on the sample to be detected and the fusion protein; subjecting the first contact treatment product to a second contact treatment with a detection antibody; and determining the content of PLA2R antibody in the sample to be detected based on the detection value of the second contact treatment product. In the detection process, the sample to be detected and the fusion protein are subjected to first contact, so that the fusion protein can specifically bind to the PLA2R antibody in the sample to be detected, and then the fusion protein bound with the PLA2R antibody is obtained. And then, the fusion protein combined with the PLA2R antibody is subjected to secondary contact with a detection antibody, the detection antibody can be combined with the PLA2R antibody, and the signal of the detection antibody is detected to obtain a detection value, so that the content of the PLA2R antibody in the sample to be detected is determined.

According to one implementation of the embodiments of the invention, the fusion protein is subjected to a wrapper in advance.

According to one embodiment of the present invention, the fusion protein is present in the wrapper sheet process at a concentration of 0.01 to 50 μg/mL.

According to one implementation of the embodiment of the invention, the concentration of the fusion protein in the wrapper sheet treatment is 0.05-40 μg/mL.

According to one implementation of the embodiment of the invention, the concentration of the fusion protein in the wrapper sheet treatment is 0.1-30 μg/mL.

According to one implementation of the embodiment of the invention, the concentration of the fusion protein in the wrapper sheet treatment is 1-20. Mu.g/mL, and is exemplified by 10. Mu.g/mL.

According to an embodiment of the present invention, the sample to be measured is blood.

According to an embodiment of the present invention, the blood is subjected to a dilution treatment in advance before the detection, so as to obtain a test sample, the dilution treatment being 10 to 500 times, preferably 50 to 200 times, and exemplified by 100 times.

According to one embodiment of the examples of the present invention, the fusion protein is subjected to a purification treatment in advance before the second contacting. Thus, the fusion protein combined with the PLA2R antibody can be purified so as to obtain the PLA2R antibody, thereby facilitating the subsequent detection of the PLA2R antibody.

According to one implementation of the embodiments of the invention, the detection antibody is a mouse anti-human IgG monoclonal antibody.

According to one implementation of the examples of the present invention, the detection antibody is an HRP-conjugated detection antibody.

According to one implementation of the embodiment of the invention, the detection value is a absorbance value, such as absorbance values in the wavelength range of 450-490 nm.

According to an embodiment of the present invention, the determining the content of PLA2R antibody in the sample to be tested 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 PLA2R standard antibody concentration absorbance curve; determining the content of PLA2R antibodies in the test sample based on the comparison; and determining the content of the PLA2R antibody in the sample to be tested based on the product of the content of the PLA2R antibody in the sample to be tested and the dilution multiple of the sample to be tested. Therefore, the content of PLA2R antibody in the sample to be detected can be accurately detected.

According to an embodiment of the invention, the PLA2R standard antibody is an antibody solution of known predetermined concentration. For example, PLA2R standard antibodies in the ommond kit can be used.

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/x 0)/(p), wherein A1, A2, x0 and p are constants determined by Logistic regression analysis independently, respectively; x represents the content of PLA2R antibodies in the test sample; y represents absorbance.

According to one implementation of the embodiment of the invention, R of the standard curve ² Above 0.9994, optionally R ² 0.9996 or 0.9999.

According to one implementation of the example of the present invention, the standard curve corresponds to the formula y=a2+ (a1—a2)/(1+ (x/x 0)/(p)), where A1 is 0.01 to 0.05, A2 is 2 to 2.1, x0 is 112 to 124, p is 0.87 to 0.96, x represents the content of PLA2R antibody in the test sample, and y represents absorbance; or the standard curve corresponds to the formula of y=A2+ (A1-A2)/(1+ (x/x 0)/(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 absorbance.

According to an embodiment of the invention, the PLA2R antibody-causing related disease is selected from PLA2R antibody positive membranous nephropathy.

Those skilled in the art will appreciate that the features and advantages described above for fusion proteins, nucleic acid molecules, expression vectors, recombinant cells and kits apply equally to the method and are not described in detail herein.

The scheme of the present invention will be explained below with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

Example 1: construction of fusion protein expression plasmid

1. The plasmid vector adopts pcDNA3.4 transient expression vector, the vector comprises a native full-length CMV promoter and WPRE elements downstream of cloning sites, the vector based on the CMV promoter generally has good expression level for a CHO cell transient expression system, the WPRE elements are positioned downstream of multiple cloning sites, the transcription and the expression of genes can be effectively improved, and the vector has ampicillin resistance genes in escherichia coli. Then, the PCR technology is utilized to amplify the gene sequence of the vector pcDNA3.4 connected with the target gene (gene of interest) and the signal peptide (signal peptide), the PCR template is obtained through gene synthesis, the pcDNA3.4 vector fragment and the target gene fragment are connected through a homologous recombination kit, DH5 alpha competence is transformed to obtain monoclonal, the monoclonal is sequenced after amplified, and after sequencing confirmation, plasmids are extracted to obtain target gene recombination expression plasmids, namely pcDNA3.4-RC21, pcDNA3.4-RC22, pcDNA3.4-RC23 and pcDNA3.4-RC24 respectively. Wherein, the target gene coding DNA and the coding DNA of the secretory expression signal peptide are cloned between CMV promoter and WPRE gene of plasmid pcDNA3.4 after gene synthesis (short for synthetic gene), see specifically FIG. 2.

1.1pcDNA3.4 vector and synthetic Gene composition and primer and template information therefor

The synthetic genes are specifically as follows: carrying out gene synthesis on RC21 target genes (the amino acid sequence is SEQ ID NO:8, the nucleotide sequence is SEQ ID NO: 12) and signal peptide, wherein RC21 is short; carrying out gene synthesis on RC22 target genes (the amino acid sequence is SEQ ID NO:6, the nucleotide sequence is SEQ ID NO: 10) and signal peptide, wherein the gene synthesis is abbreviated as RC22; carrying out gene synthesis on RC23 target genes (the amino acid sequence is SEQ ID NO:9, the nucleotide sequence is SEQ ID NO: 13) and signal peptide, wherein RC23 is short; the gene synthesis of RC24 destination gene (SEQ ID NO:7, SEQ ID NO: 11) and signal peptide, abbreviated as RC24, is carried out. The pcDNA3.4 vector and primers and templates for the synthetic genes are shown in Table 1.

Wherein the amino acid sequence of the signal peptide is:

MLLSPSLLLLLLLGAPRGCA(SEQ ID NO：14)；

the nucleotide sequence of the signal peptide is:

ATGCTGCTGTCGCCGTCGCTGCTGCTGCTGCTGCTGCTGGGGGCGCCGCGGGGCTGCGCC(SEQ ID NO：15)。

table 1: information on synthetic genes

1.2PCR amplification of fragments of interest

The KOD reaction system (Cat. KFX-101), reaction conditions and reagents of the PCR reaction using TAKARA are shown in tables 2 to 4.

Table 2: KOD reaction System of target Gene or pcDNA3.4

Reagent(s)	Concentration of	Final concentration	Volume (mu L)
				2*PCR buffer for KOD	2*	1*	25
dNTP (dATP, dTTP, dCTP and dGTP, four kinds)	2 mM/each	0.4 mM/each	10
				5' primer	10μM	0.5μM	2.5
3' primer	10μM	0.5μM	2.5
				Stencil (target gene or pcDNA3.4)	10ng/μL	0.2ng/μL	1
ddH ₂ O		N/A	8
				KOD FX	1U/μL	20mU/μL	1
Totals to			50

Table 3: PCR reaction conditions of vector pcDNA3.4

Table 4: PCR reaction conditions of the target Gene

Procedure	Temperature (temperature)	Time	Conditions (conditions)
				1	94℃	2min
2	98℃	10s
				3	60℃	30s
4	68℃	2-6min	Returning to procedure 2, a total of 30 cycles from procedures 2 to 4 were performed
				5	10℃	Permanent set

1.3 gel electrophoresis detection of PCR results

After the PCR was completed, the sample from step 1.2 was subjected to 1.5% agarose gel electrophoresis in which 1 x tae 300ml was added to the electrophoresis tank and run at 100V for 30 min. The results showed that the agarose gel electrophoresis band size was consistent with the theoretical size. And (3) recovering the electrophoresis result by using a Omega Gel Extraction Kit gel recovery kit (Cat.D2500-01), and eluting with 20 mu L of eluent to obtain a purified PCR product. The electrophoresis results are shown in FIG. 3-4, wherein the leftmost lane in FIG. 3 is DL5000 marker, and the other 4 lanes are pcDNA3.4 skeleton. In FIG. 4, the leftmost lane is DL5000 marker, and the rest lanes are RC21, RC22, RC23, RC24, and RC24 in this order from left to right.

1.4 obtaining the plasmid of interest

The purified PCR bands obtained in 1.3 were subjected to homologous recombination, and spliced according to the instructions using a ready-to-use seamless cloning kit (B632219) from Shanghai Co., ltd. Adding the spliced product into competent cells E.coli DH5 alpha at a ratio of 1:10, placing the mixture on ice for 10min, then incubating the mixture at a metal bath at 42 ℃ for 45s, placing the mixture on ice for 10min, finally coating the solution on LB solid medium containing ampicillin (Amp, final concentration of 50 mug/mL), culturing the mixture overnight at 37 ℃, picking up the single clone in 2mL LB liquid medium (final concentration of 50 mug/mL Amp) the next day, shaking the bacterium at 37 ℃ and 200rpm, and sending the obtained bacterial liquid to sequencing to confirm that all the sequence is consistent with the original sequence. 100 mu L of the monoclonal bacterial liquid is added into 50mL of LB liquid medium containing ampicillin (final concentration of 50 mu g/mL), the culture is carried out at 37 ℃ overnight at 200rpm, the strain is preserved, and plasmids are extracted, thus obtaining the target gene expression plasmids.

2. Transient expression of RC21-RC24

1) About 24 hours before transfection, freeStyle was taken ^TM

Cells (cell density 5-6×10) ⁵ /mL from Gibco corporation of America, freeStyle ^TM MAX CHO expression system, cat: k900020 At 37℃with 5% CO ₂ And culturing at 120-135 rpm/min.

2) On the day of transfection, cells were diluted to a cell density of 1X 10 ⁶ Per mL, 10mL of cells (cell viability above 95%) were added to each flask.

3) Gently mix freeform ^TM MAX Reagent several times, note that vortex oscillation is not possible.

4) The pcDNA3.4-RC21, pcDNA3.4-RC22, pcDNA3.4-RC23 and pcDNA3.4-RC24 were taken at 12.5. Mu.g each, and OptiPRO was added ^TM SFM to 0.2mL total volume and gently mixed.

5) Taking 25. Mu.L of FreeStyle ^TM MAX Reagent, add Opti-Pro ^TM SFM to a total volume of 0.4mL, gently mix. After mixing uniformly, the mixture was added to the plasmid mixture on average, and gently mixed to obtain 2 branches of 0.4mL DNA-FreeStyle ^TM MAX mixture and incubated at room temperature for 10 min to allow complex formation.

6) Slowly add the 2-branch 0.4mL DNA-FreeStyle ^TM MAX complex was added to the cells while the shake flask was slowly rotated.

7) At 37℃with 5% CO ₂ The cells are cultured at 120-135rpm/min without changing or supplementing the culture medium, and the cell sap is collected after 7 d.

3. Purification and identification of RC21-RC24

The RC21-RC24 cell culture supernatants obtained in step 2 were centrifuged at 7000rpm at 4℃for 15min, and then filtered and sterilized with a 0.22um needle filter. And loading on a Ni sepharose 6FF column with the concentration of 2mL/min. The plates were washed with 20mM phosphate buffer, 500mM NaCl and 20mM imidazole at pH 6.8. 5 column volumes of 0-100%20mM phosphate buffer, 1M NaCl and 500mM imidazole, eluting at pH 6.8. The 2 mL/tube was partially collected. The elution peak was ultrafiltered 3 times in a 30kD ultrafiltration centrifuge tube and the solution was replaced with 20mM phosphate buffer pH 6.8. Quantification was performed using the Bradford method.

10-well SDS-PAGE pre-gels were selected and run at 150V for 50min. The sample was heated at 90℃for 5min and 10. Mu.g per well was loaded. SDS-PAGE detects the purification effect of the RC21-RC24 protein, and the result shows that the RC21-RC24 fusion protein is successfully purified. The structural diagram of RC21-RC24 is shown in FIG. 5, wherein CFC1 protein has the amino acid sequence shown in SEQ ID NO. 1, and CFC2 protein has the amino acid sequence shown in SEQ ID NO. 2.

Wherein, the purifying effect of RC22 and RC24 is shown in FIG. 6, wherein lanes from left to right are the purifying RC22 non-reduction, purifying RC22 reduction, RC22 culture medium non-reduction, RC22 culture medium reduction, purifying RC24 non-reduction, purifying RC24 reduction, and protein molecular weight reference in sequence; the expression level of RC22 was 66mg/L and the expression level of RC24 was 22mg/L, which revealed that the expression level of RC22 was 3 times the expression level of RC 24. Subsequent experiments were performed using the RC22 and RC24 fusion proteins.

Example 2: plasma preparation and PLA2R-Ab detection for patients with membranous nephropathy

23 patients with membranous nephropathy were anticoagulated with EDTA in an amount of 2mL, and plasma was isolated and 100-fold diluted. PLA2R-Ab concentration was measured using an European Mongolian kit, and the average was taken 2 times, and specific procedures were referred to the European Mongolian kit instructions. The results of plasma tests for patients with membranous nephropathy are shown in Table 5.

Table 5: plasma detection result of membranous nephropathy patient

According to the kit instructions, the detection value >20RU/mL is a positive sample and <14RU/mL is a negative sample. Thus, samples pMN-2, pMN-4, pMN-7, pMN-18, pMN-19, pMN-21 were positive samples.

Example 3: determination of plate concentration and dilution of plasma samples

1. Determination of the concentration of the wrapper

The 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. Mu.g/mL, respectively) obtained in example 1, and 100-fold diluted plasma samples pMN1 or pMN2 were added to detect color development after incubation of the antibodies.

As shown in FIGS. 7 and 8, the abscissa in FIGS. 7 to 8 is the concentration (concentration), and it can be seen that the developed intensity reaches substantially the plateau at 1. Mu.g/mL or more. Subsequent experiments selected a 10. Mu.g/mL concentration of coated plate.

2. Determining the dilution of a plasma sample

Detecting plasma samples subjected to gradient dilution, wherein the dilution factors are 1, 10, 100, 1000 and 10 respectively ⁴ 、10 ⁵ 、10 ⁶ And 10 ⁷ To determine optimal dilution conditions. The samples were pMN-1, pMN-2, pMN-3, pMN-4, pMN-5, pMN-6, pMN-7, pMN-18, RTX (rituximab). Wherein RTX is a negative reference, pMN-1, pMN-3, pMN-5, PMN-6 are negative samples, and the rest are positive samples.

As shown in fig. 9 and 10, the abscissa in fig. 9 to 10 is the dilution ratio (dilution ratio), and it can be seen that the distinction between the positive sample and the negative sample is good. The dilution is 50-500 times, the color background is low, and the distinction between positive samples and negative samples is high. Subsequent experiments used 100-fold dilutions to detect samples.

Example 4: RC22 and RC24 as capture molecules for detecting plasma samples and correlation analysis of results

1. Drawing concentration standard curve

The standard of the European Mongolian kit is taken as PLA2R-Ab standard solution, RC22 and RC24 obtained in the example 1 are taken as capture molecules, an HRP-coupled mouse anti-human IgG monoclonal antibody is taken as a detection antibody, and a concentration standard curve is established by ELISA technology, and the concentration is shown in FIG. 11, and the abscissa in FIG. 11 is concentration.

From the standard curve of FIG. 11, R of RC22 ² R of 0.9999, RC24 ² 0.9996, all higher than 0.9994 (R ² ) Indicating a better fit. RC22 and RC24 have higher absorbance at 100RU/mL than the European Meng Shiji box, and X0 in the fitting parameters is 118RU/mL and 92RU/mL, respectively, significantly lower than 8×10 of the European kit ⁶ RU/mL. A lower X0 means that a 50% detection signal value occurs at a lower concentration, i.e., the global fit curve moves toward a lower concentration, indicating that RC22 and RC24 are more sensitive than the omnipotent kit.

2. Plasma sample detection and correlation analysis

Detecting PLA2R-Ab concentrations of pMN-1, pMN-2, pMN-3, pMN-4, pMN-5, pMN-6, pMN-7 and pMN-18 by taking RC22 and RC24 as capture molecules, comparing the detected results with the standard curve to obtain PLA2R-Ab concentration, and then performing correlation analysis, wherein the specific analysis results are as follows:

the RC24 test results are plotted against the RC22 results, the fitting equation is shown in Table 6, and FIG. 12 shows that the linear correlation is excellent. In FIG. 12, the abscissa indicates the PLA2R-Ab titer (PLA 2R-Ab titre by RC 22) detected by RC22, and the ordinate indicates the PLA2R-Ab titer by RC24 detected by RC 24.

Table 6: fitting equation

The results of the European Mongolian kit are plotted against the RC22 detection results, the fitting equation is shown in Table 7, and FIG. 13 shows that the linear correlation is better. In FIG. 13, the abscissa indicates the PLA2R-Ab titer (PLA 2R-Ab titre by RC 22) detected by RC22, and the ordinate indicates the PLA2R-Ab titer (standard PLA2R-Ab titre) detected by the European kit.

Table 7: fitting equation

The results of the European Mongolian kit are plotted against the RC24 detection results, the fitting equation is shown in Table 8, and FIG. 14 shows that the linear correlation is better. In FIG. 14, the abscissa indicates the PLA2R-Ab titer (PLA 2R-Ab titre by RC 24) detected by RC24, and the ordinate indicates the PLA2R-Ab titer (standard PLA2R-Ab titre) detected by the European kit.

Table 8: fitting equation

The results show that the concentration of PLA2R-Ab detected by RC22 or RC24 is consistent with the detection result of the European-Mongolian kit. The detection results of RC22 and RC24 are highly consistent.

Example 5: RC22 as capture molecule for detecting plasma sample and result correlation analysis

1. Plasma sample detection

The RC22 and European-Mongolian kit obtained in example 1 were used to amplify the plasma sample amounts and to detect the samples of 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, respectively. According to the European standard, the detection value >20RU/mL is a positive sample, and <20RU/mL is a negative sample.

As a result of detection, samples positive for the European-Mongolian kit were pMN-2 (44.795 RU/mL), pMN-4 (124.905 RU/mL), pMN-7 (58.505 RU/mL), pMN-18 (133.560 RU/mL), pMN-19 (39.51 RU/mL) and pMN-21 (141.735 RU/mL), and for the above samples, RC22 was detected as pMN-2 (176.8826 RU/mL), pMN-4 (302.5741 RU/mL), pMN-7 (158.8119 RU/mL), pMN-18 (722.1047 RU/mL), pMN-19 (96.01469 RU/mL) and pMN-21 (440.9976 RU/mL). Clearly, all samples positive for the European style kit were tested, with higher RC22 detection.

For pMN-9, pMN-12 and pMN-20 samples, the European kit test was negative, while RC22 test was positive. Specifically, in the European Mongolian kit detection, pMN-9 is 7.625RU/mL, pMN-12 is 6.772RU/mL and pMN-20 is 5.400RU/mL; in the RC22 assay, pMN-9 was 81.11847RU/mL, pMN-12 was 98.68588RU/mL, and pMN-20 was 28.40804RU/mL. The appearance of the OuMonte kit results were negative, while the RC22 results were positive, indicating that RC22 did detect more sensitively than the OuMonte kit.

2. Correlation analysis

The results of the European Mongolian kit are plotted against the RC22 detection results, the fitting equation is shown in Table 9, and FIG. 15 shows that the linear correlation is better. In FIG. 15, the abscissa represents the PLA2R-Ab titer (PLA 2R-Ab titre by RC 22) detected by RC22, and the ordinate represents the PLA2R-Ab titer (standard PLA2R-Ab titre) detected by the European kit.

Table 9: fitting equation

3. Statistical correlation analysis

SPSS was analyzed for bivariate linear correlations and the results are shown in Table 10.

Table 10: correlation analysis

Note that: * *. is significantly correlated at the.01 level (bilateral).

The RC22 detection result has very good correlation with the result of the European kit, the Pearson correlation is 0.913, and the confidence is 0.000.

Example 6: plasma neutralization experiments verify diagnostic reagent accuracy

To verify that the European Mongolian kit detected negative, RC22 detected positive plasma was indeed positive, but not false positive, neutralization experiments were performed using RC3 (amino acid sequence SEQ ID NO:21; nucleotide sequence SEQ ID NO: 22).

1. RC3 is an Fc fusion protein developed by the inventors of the present application for neutralizing PLA2R-Ab, and RC3 contains PLA2R epitope peptide. RC3 was prepared by referring to the preparation method of RC22 fusion protein in example 1 of the present application, wherein the PCR was performed using the synthetic gene "SEQ ID NO:15+SEQ ID NO:22" as a template, and the primers were 6-f (SEQ ID NO: 23) and 6-r (SEQ ID NO: 24). Then, RC3 protein was purified and identified, see specifically the method for purifying and identifying RC22 fusion protein in specific example 1 of the present application, and the purified RC3 was used in the following neutralization experiment, where the purification effect is as shown in fig. 16, lane 1 is RC3 eluting non-reducing, lane 2 is RC3 eluting reducing, and lane M is marker. The detection was then performed using the only current European Mongolian kit (Germany, ELISA method for quantitative detection of PLA2R-Ab concentration in blood samples) approved by the FDA of the United states for diagnosis of PLA2R antibody positive membranous nephropathy, all steps were performed according to the instructions, and the dosage of RC3 protein was 5. Mu.g/mL. The plasma numbers of 3 PLA2R antibody-positive patients with membranous nephropathy were 1, 2 and 3, respectively. The neutralization effect of RC3 on PLA2R-Ab in the plasma of the 3 patients with positive membranous nephropathy was detected, and the neutralization rate shown in Table 11 was obtained, and the result shows that RC3 has higher neutralization rate on PLA 2R-Ab. Thus, RC3 can act as a competitive inhibitor of PLA 2R.

Wherein, the amino acid sequence of RC 3:

EGVAAALTPERLLEWQDKGIFVIQSESLKKCIQAGKSVLTLENCKQANKHMLWKWVSNHGLFNIGGSGCLGLNFSAPEQPLSLYECDSTLVSLRWRCNRKMITGPLQYSVQVAHDNTVVASRKYIHKWISYGSGGGDICEYLHKDLHTIKGNTHGMPCMFPFQYNHQWHHECTREGREDDLLWCATTSRYERDEKWGFCPDPTSAEVGCDTIWEKDLNSHICYQFNLLSSLSWSEAHSSCQMQGGTLLSITDETEENFIREHMSSKTVEVWMGLNQLDEHAGWQWSDGTPLNYLNWSPEVNFEPFVEDHCGTFSSFMPSAWRSRDCESTLPYICKKYLNHIDHEIVEGGGGSGGGGSTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:21)。

nucleotide sequence of RC 3:

GAGGGTGTGGCGGCGGCGCTTACCCCCGAGCGGCTCCTGGAGTGGCAGGATAAAGGAATATTTGTTATCCAAAGTGAGAGTCTCAAGAAATGCATTCAAGCAGGTAAATCGGTTCTGACCCTGGAGAACTGCAAGCAAGCAAACAAGCACATGCTGTGGAAATGGGTTTCAAACCATGGCCTCTTTAACATAGGAGGCAGTGGTTGCCTGGGCCTGAATTTCTCCGCCCCAGAGCAGCCATTAAGCTTATATGAATGTGACTCCACCCTCGTTTCCTTACGGTGGCGCTGTAACAGGAAGATGATCACAGGCCCGCTGCAGTACTCTGTCCAGGTGGCGCATGACAACACAGTGGTGGCCTCACGGAAGTATATTCATAAGTGGATTTCTTATGGGTCAGGTGGTGGAGACATTTGTGAATATCTACACAAAGATTTGCATACAATCAAAGGGAACACCCACGGGATGCCGTGTATGTTTCCCTTCCAGTATAACCATCAGTGGCATCATGAATGTACCCGTGAAGGTCGGGAAGATGACTTACTGTGGTGTGCCACGACAAGCCGTTATGAAAGAGATGAAAAGTGGGGATTTTGCCCTGATCCCACCTCTGCAGAAGTAGGTTGTGATACTATTTGGGAGAAGGACCTCAATTCACACATTTGCTACCAGTTCAACCTGCTTTCATCTCTCTCTTGGAGTGAGGCACATTCTTCATGCCAGATGCAAGGAGGTACGCTGTTAAGTATTACAGATGAAACTGAAGAAAATTTCATAAGGGAGCACATGAGCAGTAAAACAGTGGAGGTGTGGATGGGCCTCAATCAGCTGGATGAACACGCTGGCTGGCAGTGGTCTGATGGAACGCCGCTCAACTATCTGAATTGGAGCCCAGAGGTAAATTTTGAGCCATTTGTTGAAGATCACTGTGGAACATTTAGTTCATTTATGCCAAGTGCCTGGAGGAGTCGGGATTGTGAGTCCACCTTGCCATATATATGTAAAAAATATCTAAACCACATTGATCATGAAATAGTTGAAGGCGGTGGAGGCTCCGGCGGAGGTGGAAGCACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA(SEQ ID NO:22)。

the nucleotide sequence of 6-f is:

GCCTCCGGACTCTAGAGGGTACGGGCCAGATATACGCGT(SEQ ID NO:23)。

the nucleotide sequence of 6-r is:

CATTACTAACCGGTAGGGTCATTTAGCTCTGTGTGGTAAGCAGGT(SEQ ID NO:24)。

table 11: neutralization rate of PLA2R-Ab in plasma of the 3 patients with positive membranous nephropathy by RC3

Neutralization rate	1	2	3
				RC3	95.76％	82.03％	89.26％

2. The inventors selected pMN-9, pMN-12, pMN-20 and pMN-14 blood samples for neutralization experiments, wherein among the detection results of the pMN-9, pMN-12 and pMN-20 samples in example 5, the detection result of the European kit is negative, and the detection result of the RC22 is positive; pMN-14 samples were negative for both the European kit test and the RC22 test. PLA2R-Ab concentrations in blood samples of pMN-9, pMN-12, pMN-20 and pMN-14 before and after addition of RC3 were measured using the European Mongolian kit and RC22 obtained in example 1, respectively. If the plasma is indeed positive, a significantly higher neutralization effect should be exhibited compared to the negative sample. Wherein, the neutralization detection of PLA2R-Ab in plasma sample by RC3 is described in step 1 of the present example. RC22 detection see example 4.

Prior to testing, samples of pMN-9, pMN-12, pMN-20 and pMN-14 were diluted 100-fold, the results of this example are shown in Table 12 and Table 13 and in FIGS. 17 and 18 (drug concentration =5. Mu.g/mL in FIGS. 17-18 means that the amount of RC3 protein administered was 5. Mu.g/mL; FIGS. 17-18, on the ordinate, represent the concentration of PLA2R-ab (PLA 2Rab control)), and the neutralization rate (neutralization ratio) after addition of RC3 was significantly higher for samples (pMN-9, pMN-12 and pMN-20) that were negative for the European test kit, and for those samples (pMN-14) that were negative for both the European test kit and RC 22. That is, in pMN-9, pMN-12 and pMN-20, there was more PLA2R-Ab before adding RC3, so that after adding RC3, neutralization rate was shown to be significantly higher than that of pMN-14. Therefore, it can be said that, for a sample in which the European kit detects negative and the RC22 detects positive, the positive result of the RC22 detection is not false positive, and the RC22 detection sensitivity and accuracy are higher than those of the European kit.

Table 12: neutralization test results of European Mongolian kit detection

Sample of	Detection value RU/mL	RC3 neutralization assay 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％

Table 13: neutralization assay results for RC22 detection

Sample of	Detection value RU/mL	RC3 neutralization assay 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％

In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.

While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.

Claims

1. A fusion protein comprising:

PLA2R epitope peptide;

albumin or a fragment thereof, to which the PLA2R epitope peptide is linked.

2. The fusion protein of claim 1, wherein the C-terminus of the PLA2R epitope peptide is linked to the N-terminus of the albumin or fragment thereof;

optionally, the N-terminus of the PLA2R epitope peptide is linked to the C-terminus of the albumin or fragment thereof;

optionally, the PLA2R epitope peptide includes CysR, fnII, PLA2R binding region 1 and optionally PLA2R binding region 2;

optionally, the C-terminus of CysR is linked to the N-terminus of FnII, which is linked to the N-terminus of PLA2R binding region 1;

optionally, the C-terminus of CysR is linked to the N-terminus of FnII, which is linked to the N-terminus of PLA2R binding region 1, which is linked to the N-terminus of PLA2R binding region 2;

optionally, the PLA2R 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;

optionally, the albumin or fragment thereof is serum albumin or a fragment thereof, preferably human serum albumin;

Optionally, the albumin or 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.

3. The fusion protein of claim 1 or 2, further comprising a linker peptide;

optionally, the N-terminus of the linker peptide is linked to the C-terminus of the PLA2R epitope peptide, the C-terminus of the linker peptide being linked to the N-terminus of the albumin or fragment thereof;

optionally, the N-terminus of the linker peptide is linked to the C-terminus of the albumin or fragment thereof, and the C-terminus of the linker peptide is linked to the N-terminus of the PLA2R epitope peptide;

optionally, 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;

optionally, the linker peptide comprises the amino acid sequence shown in SEQ ID NO. 4;

optionally, the fusion protein further comprises a tag;

optionally, the tag is attached to the terminus of the PLA2R epitope peptide or albumin or fragment thereof;

optionally, the tag is at least one of a HIS tag, FLAG tag, HA tag, GST tag, strep II tag, and MBP tag;

Optionally, the HIS tag has an amino acid sequence as shown in SEQ ID NO. 5;

optionally, the N-terminus of the HIS tag is linked to the C-terminus of the albumin or fragment thereof; or (b)

The C end of the HIS tag is connected with the N end of the PLA2R epitope peptide;

preferably, the N-terminus of the HIS tag is linked to the C-terminus of the albumin or fragment thereof, further comprising a TG sequence;

optionally, the N-terminus of the TG sequence is linked to the C-terminus of the albumin or fragment thereof, and the C-terminus of the TG sequence is linked to the N-terminus of the HIS tag;

optionally, the fusion protein has an amino acid sequence as shown in any one of SEQ ID NOs 6 to 9.

4. A nucleic acid molecule encoding the fusion protein of any one of claims 1-3;

optionally, the nucleic acid molecule is DNA.

5. An expression vector carrying the nucleic acid molecule of claim 4;

optionally, the expression vector is a eukaryotic expression vector, preferably the expression vector is a lentiviral vector.

6. A recombinant cell comprising:

carrying the nucleic acid molecule of claim 4 or the expression vector of claim 5; or alternatively, the first and second heat exchangers may be,

Expressing the fusion protein of any one of claims 1-3;

optionally, the recombinant cell is obtained by introducing the expression vector of claim 5 into a host cell;

optionally, the recombinant cell is a eukaryotic cell;

preferably, the recombinant cell is a mammalian cell.

7. A kit, comprising: the fusion protein of any one of claims 1-3, the nucleic acid molecule of claim 4, the expression vector of claim 5, or the recombinant cell of claim 6;

optionally, the fusion protein has a working concentration of 0.01-50. Mu.g/mL, 0.05-40. Mu.g/mL, 0.1-30. Mu.g/mL, 1-20. Mu.g/mL, or 10. Mu.g/mL.

8. Use of the fusion protein of any one of claims 1-3, the nucleic acid molecule of claim 4, the expression vector of claim 5 or the recombinant cell of claim 6 in the preparation of a kit for detecting a PLA2R antibody, diagnosing a disease associated with excessive PLA2R antibody, assessing the prognosis of a disease associated with PLA2R antibody or assessing the staging of a disease associated with PLA2R antibody;

optionally, the disease associated with excessive PLA2R antibodies is selected from PLA2R antibody positive membranous nephropathy.

9. A method of detecting PLA2R antibodies, comprising:

detecting PLA2R antibodies in a sample to be tested using the fusion protein of any one of claims 1 to 3, the nucleic acid molecule of claim 4, the expression vector of claim 5 or the recombinant cell of claim 6 or the kit of claim 7;

and determining the content of PLA2R antibody in the sample to be detected based on the detection result.

10. The method of claim 9, wherein the detecting comprises:

carrying out first contact treatment on the sample to be detected and the fusion protein;

subjecting the first contact treatment product to a second contact treatment with a detection antibody;

determining the content of PLA2R antibodies in the sample to be tested based on the detection value of the second contact treatment product;

optionally, the fusion protein is subjected to a plating treatment in advance;

preferably, the concentration of the fusion protein in the wrapper sheet treatment is 0.01-50 μg/mL, 0.05-40 μg/mL, 0.1-30 μg/mL, 1-20 μg/mL or 10 μg/mL;

optionally, the sample to be tested is blood;

preferably, the blood is subjected to a dilution treatment in advance before the detection, so as to obtain a test sample, the dilution treatment being performed by a factor of 10 to 500, preferably 50 to 200;

Optionally, prior to the second contacting, the fusion protein is subjected to a purification treatment in advance;

optionally, the detection antibody is a mouse anti-human IgG monoclonal antibody;

optionally, the detection antibody is an HRP-conjugated detection antibody;

optionally, the detection value is an absorbance value in the wavelength range of 450-490 nm;

optionally, the determining the content of PLA2R antibodies in the sample to be tested 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 PLA2R standard antibody concentration absorbance curve;

determining the content of PLA2R antibodies in the test sample based on the comparison;

determining the content of PLA2R antibody in the sample to be tested based on the product of the content of PLA2R antibody in the sample to be tested and the dilution multiple of the sample to be tested;

optionally, the PLA2R standard antibody is an antibody solution of known predetermined concentration;

optionally, 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 determined independently from each other by Logistic regression analysis;

x represents the content of PLA2R antibodies in the test sample;

y represents absorbance;

optionally, the PLA2R antibody-causing related disease is selected from PLA2R antibody positive membranous nephropathy.