Use of a recombinant protein in the preparation of a product for diagnosing or assisting in the diagnosis of IgA nephropathy

Abstract

The present invention discloses the use of a recombinant protein in the preparation of a product for diagnosing or assisting in the diagnosis of IgA nephropathy. Specifically, it discloses the use of a protein with an amino acid sequence of SEQ ID No.1 in the preparation of a product for diagnosing or assisting in the diagnosis of IgA nephropathy. The present invention uses a recombinant baculovirus containing the gene encoding human glycosyltransferase B3GNT6(43-384) to infect insect cells, obtaining a secreted form of glycosyltransferase B3GNT6(43-384), which can efficiently and specifically recognize the Tn antigen in the hinge region of Gd-IgA1. Based on this, the present invention has developed a novel chemical enzyme method technology for serological diagnosis of IgA nephropathy. This technology can effectively distinguish IgA nephropathy patients, healthy controls, and other nephropathy controls, with high stability, good repeatability, and the characteristics of non-invasive, rapid, specific, and sensitive, having a very broad clinical application prospect and important significance.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to the application of a recombinant protein in the preparation of a product for diagnosing or assisting in the diagnosis of IgA nephropathy. Background Art

[0002] IgA nephropathy (Immunoglobulin A nephropathy, IgAN) is the most common primary glomerular disease in the world and an important cause of end-stage renal disease (ESRD). Patients with IgA nephropathy often onset in young and middle-aged adults. More than 30% of the patients develop end-stage renal disease (uremia) 10 years after the onset, eventually leading to renal failure. The clinical manifestations of IgA nephropathy are as follows: the content of IgA antibodies in the circulatory system of patients increases, with polyuria, accompanied by proteinuria, hematuria, and at the same time, symptoms such as hypertension and edema will appear. The specific molecular diagnosis is that IgA deposition mainly occurs in the glomerular mesangial region, with or without the deposition of other immunoglobulins such as IgG and IgM, and complement protein C3 (complement 3) etc. in the glomerular mesangial region. Light microscopy commonly shows hyperplasia of glomerular mesangial matrix, proliferation of mesangial cells, and often accompanied by focal segmental fibrinoid necrosis of the glomerulus and the formation of glomerular crescents. Therefore, the current gold standard for the diagnosis of IgA nephropathy is only kidney pathological tissue biopsy, and immunopathological examination to check whether there is IgA deposition around the glomerulus, accompanied by varying degrees of histopathological damage. However, the method of tissue biopsy is invasive, causing great pain to patients, not suitable for repeated use, and unable to evaluate the degree of disease progression through the initial renal biopsy results.

[0003] At present, the pathogenesis of IgA nephropathy is not clear, but it is generally believed that the mechanism is related to the inducing factors associated with mucosal immunity. At the same time, there is also an accepted "four-hit" theory in this field, which mainly includes: First, there is a phenomenon of galactose deficiency in the O-glycosylation of the hinge region of IgA1 molecules in the blood circulation system of IgA nephropathy patients (human galactose-deficient IgA1, Gd-IgA1). This glycosylation-deficient IgA1 molecule is prone to spontaneous aggregation in the blood and generates polycyclic immune complexes. Second, anti-glycosylation antibodies against Gd-IgA1 are produced in the blood circulation, such as the production of IgG. Then, the anti-glycosylation antibody takes Gd-IgA1 as its own antigen and participates in the formation of immune complexes such as IgG-IgA1 in the circulatory system or in the glomerular mesangial area. Finally, this circulating immune complex containing IgA is locally deposited in the glomerular mesangial area, thereby activating the complement system and inducing an inflammatory response, leading to kidney tissue damage. As the most important initiating link of the "four-hit" theory, many domestic and foreign studies have deeply explored its clinical application value. Many studies have proved that the formation of Gd-IgA1 plays a key role in the pathogenesis of IgAN. The Novak J laboratory confirmed that the level of Gd-IgA1 in the circulation of IgA nephropathy patients is significantly higher than that of healthy people, and its specificity and sensitivity for diagnosing IgA nephropathy are as high as 94% and 76.5% respectively, and thus proposed the non-invasive diagnostic value of Gd-IgA1. Some studies have shown that Gd-IgA1 is more likely to bind to mesangial cells and induce mesangial cell proliferation, and the higher the content of Gd-IgA1 in the circulatory system of IgA nephropathy patients, the worse the prognosis and the shorter the survival time of the kidneys. Compared with other diseases, such as IgA vasculitis, there is no galactose deficiency in IgA1 in its blood, which only causes skin damage and produces a purpura reaction, but Gd-IgA1 can also cause severe glomerular damage.

[0004] Although the clinical value of Gd-IgA1 has been confirmed by multiple studies, the measurement protocol of Gd-IgA1 is still limited to the laboratory level and has not been clinically promoted. The obstacle to the clinical promotion of Gd-IgA1 lies in the lack of a stable, reliable and large-scale detection method. Previous studies mainly used ELISA methods based on lectins HAA (Helix aspersa agglutinin), HPA (Helix pomatia agglutinin) and VVL (Vicia villosa lectin) to measure Gd-IgA1. The disadvantages of this method are that the lectin has a weak binding ability to O-glycans, the effective enrichment concentration is as high as several hundred micrograms, and the substrate specificity is poor, making it difficult to construct a stable Gd-IgA1 measurement system. And relevant reports point out that in addition to recognizing the GalNAc modification of Gd-IgA1, VVL can also recognize the N-glycosylation on IgA2 without O-glycosylation modification. In addition to the lectin ELISA method, Japanese scholars immunized mice with the hinge region of Gd-IgA1 to obtain a specific antibody against Gd-IgA1 - KM55, and established a new method for measuring Gd-IgA1 using KM55. However, this antibody is expensive and difficult to be applied clinically for large-scale sample measurement. And this antibody has some disadvantages. Although a hapten carrying 5 Tn's was used in the preparation process, in the experiment of binding with glycopeptides, KM55 only had obvious binding to the first peptide segment with Tn modification, and did not bind to other Tn antigens modified in the hinge region.

[0005] The dilemmas faced by the Gd-IgA1 detection method limit its clinical application and hinder the progress of non-invasive diagnosis and targeted treatment of IgA nephropathy. Therefore, it is very necessary to explore new, non-invasive, rapid, specific and sensitive serological diagnostic technologies for IgA nephropathy, which have broad clinical application prospects and important significance. Summary of the Invention

[0006] The technical problem to be solved by the present invention is how to conveniently, rapidly and specifically achieve the labeling and / or detection of Gd-IgA1 based on chemical enzymatic technology, and / or, how to provide a product for non-invasive, rapid, specific and sensitive serological diagnosis of IgA nephropathy that can be applied clinically. The technical problems to be solved are not limited to the described technical themes, and those skilled in the art can clearly understand other technical themes not mentioned herein through the following description.

[0007] To solve the above technical problems, the present invention first provides the application of a protein, and the application can be any one of the following:

[0008] B1) The application of the protein in the preparation of a product for diagnosing or assisting in diagnosing IgA nephropathy;

[0009] B2) Use of the protein in the preparation of a product for predicting the prognosis or efficacy of IgA nephropathy;

[0010] B3) Use of the protein in labeling Gd-IgA1;

[0011] B4) Use of the protein in the preparation of a product for labeling Gd-IgA1;

[0012] B5) Use of the protein in the preparation of a product for detecting Gd-IgA1;

[0013] B6) Use of the protein in the preparation of a product for differential diagnosis between IgA nephropathy and other kidney diseases;

[0014] The name of the protein is B3GNT6 (43 - 384), and it can be any of the following:

[0015] A1) A protein with the amino acid sequence of SEQ ID No.1;

[0016] A2) A protein obtained by substituting and / or deleting and / or adding amino acid residues to the amino acid sequence of SEQ ID No.1, having more than 80% identity with the protein shown in A1) and having the same function;

[0017] A3) A fusion protein with the same function obtained by connecting a tag to the N-terminus and / or C-terminus of A1) or A2).

[0018] The product for diagnosing or assisting in the diagnosis of IgA nephropathy described in B1) includes products for early diagnosis or early auxiliary diagnosis of IgA nephropathy.

[0019] The use described in B3) can be an application based on the chemical enzyme method.

[0020] In the above applications, the product can be a reagent or a kit, and the kit can be an enzyme-linked immunosorbent assay kit.

[0021] The protein B3GNT6 (43 - 384) is a truncated B3GNT6 protein. Compared with the B3GNT6 protein, the N-terminus is truncated by 42 amino acids; the truncated B3GNT6 protein can act as a glycosyltransferase and can specifically recognize the Tn antigen.

[0022] The protein B3GNT6 (43 - 384) can be human-derived.

[0023] Proteins that are homologous but from different families and proteins with the same function but different origins as the protein B3GNT6 (43 - 384) are all within the scope of protection of the present invention.

[0024] In this article, the terms "protein B3GNT6 (43-384)", "glycosyltransferase B3GNT6 (43-384)", "B3GNT6 (43-384)", and "B3GNT6 (43-384) truncation" can be used interchangeably.

[0025] In order to facilitate the purification or detection of the protein in A1), a tag protein can be linked to the amino terminus or carboxyl terminus of the protein consisting of the amino acid sequence shown in SEQ ID No.1 in the sequence listing.

[0026] The tag proteins include, but are not limited to: GST (glutathione S-transferase) tag protein, His6 tag protein (His-tag), MBP (maltose-binding protein) tag protein, Flag tag protein, SUMO tag protein, HA tag protein, Myc tag protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow-green fluorescent protein), mCherry (monomeric red fluorescent protein), or AviTag tag protein.

[0027] Those of ordinary skill in the art can easily mutate the nucleotide sequence encoding protein B3GNT6 (43-384) of the present invention using known methods, such as directed evolution or site-directed mutagenesis. Nucleotides that have been artificially modified and have 75% or more identity with the nucleotide sequence of protein B3GNT6 (43-384) isolated from the present invention, as long as they encode protein B3GNT6 (43-384) and have the function of protein B3GNT6 (43-384), are all derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention.

[0028] The above 75% or more identity can be 80%, 85%, 90%, or 95% or more identity.

[0029] In this article, identity refers to the identity of amino acid sequences or nucleotide sequences. The identity of amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST web page on the NCBI homepage website. For example, in Advanced BLAST 2.1, by using blastp as the program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as the Matrix, setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing a search to calculate the identity of a pair of amino acid sequences, and then the identity value (%) can be obtained.

[0030] In this text, the identity of more than 80% may be an identity of at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

[0031] The present invention also provides the use of a biomaterial related to the protein B3GNT6(43 - 384), and the use may be any one of the following:

[0032] C1) The use of a biomaterial related to the protein B3GNT6(43 - 384) in the preparation of a product for diagnosing or assisting in diagnosing IgA nephropathy;

[0033] C2) The use of a biomaterial related to the protein B3GNT6(43 - 384) in the preparation of a product for prognosis or efficacy prediction of IgA nephropathy;

[0034] C3) The use of a biomaterial related to the protein B3GNT6(43 - 384) in labeling Gd - IgA1;

[0035] C4) The use of a biomaterial related to the protein B3GNT6(43 - 384) in the preparation of a product for labeling Gd - IgA1;

[0036] C5) The use of a biomaterial related to the protein B3GNT6(43 - 384) in the preparation of a product for detecting Gd - IgA1;

[0037] C6) The use of a biomaterial related to the protein B3GNT6(43 - 384) in the preparation of the protein B3GNT6(43 - 384);

[0038] C7) The use of a biomaterial related to the protein B3GNT6(43 - 384) in the preparation of a product for differential diagnosis between IgA nephropathy and other kidney diseases;

[0039] The biomaterial may be any one of the following D1) to D5):

[0040] D1) A nucleic acid molecule encoding the protein B3GNT6(43 - 384);

[0041] D2) An expression cassette containing the nucleic acid molecule described in D1);

[0042] D3) A recombinant vector containing the nucleic acid molecule described in D1), or a recombinant vector containing the expression cassette described in D2);

[0043] D4) A recombinant microorganism containing the nucleic acid molecule described in D1), or a recombinant microorganism containing the expression cassette described in D2), or a recombinant microorganism containing the recombinant vector described in D3);

[0044] D5) A cell line containing the nucleic acid molecule described in D1), or a cell line containing the expression cassette described in D2), or a cell line containing the recombinant vector described in D3).

[0045] The product for diagnosing or assisting in the diagnosis of IgA nephropathy described in C1) includes products for early diagnosis or early auxiliary diagnosis of IgA nephropathy.

[0046] The application described in C3) can be an application based on a chemical enzyme method.

[0047] In the above applications, the product can be a reagent or a kit, and the kit can be an enzyme-linked immunosorbent assay kit.

[0048] In the above applications, the nucleic acid molecule described in D1) can be any of the following:

[0049] E1) A cDNA molecule whose coding sequence is SEQ ID No. 2;

[0050] E2) A DNA molecule whose nucleotide sequence is SEQ ID No. 2.

[0051] The amino acid sequence encoded by the DNA molecule shown in SEQ ID No. 2 is the protein B3GNT6 (43 - 384) of SEQ ID No. 1.

[0052] The nucleic acid molecule described herein can be DNA, such as cDNA, genomic DNA, or recombinant DNA.

[0053] The vectors described herein are well-known to those skilled in the art and include, but are not limited to: plasmids, phages (such as λ phage or M13 filamentous phage, etc.), cosmids (i.e., cosmid plasmids), artificial chromosomes (such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), P1 artificial chromosomes (PAC), or Ti plasmid artificial chromosomes (TAC), etc.), viral vectors (such as retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, or herpesviruses (such as herpes simplex virus), etc.). In one embodiment of the present invention, the vector can specifically be the vector pI-secSUMOstar.

[0054] The microorganisms described in this article can be yeast, bacteria, algae or fungi. Among them, the bacteria can be from the genus Escherichia, Erwinia, Agrobacterium, Flavobacterium, Alcaligenes, Pseudomonas, Bacillus, etc. In one embodiment of the present invention, the microorganism can specifically be Escherichia coli DH10Bac.

[0055] The cells (host cells) described in this article refer to cells that can be used to introduce vectors, including but not limited to: eukaryotic cells (such as yeast cells, Aspergillus), animal cells (such as mammalian cells, insect cells), plant cells or prokaryotic cells. In one embodiment of the present invention, the cell can specifically be sf21 cells.

[0056] The recombinant vector can specifically be recombinant vector pI-secSUMOstar-B3GNT6(43-384). The recombinant vector pI-secSUMOstar-B3GNT6(43-384) is obtained by replacing the fragment (small fragment) between the BamHI and EcoRI recognition sites of the pI-secSUMOstar vector with a DNA fragment whose nucleotide sequence is SEQ ID NO.2 in the sequence listing, while keeping other sequences of the pI-secSUMOstar vector unchanged. The recombinant vector pI-secSUMOstar-B3GNT6(43-384) expresses a fusion protein with a His6-Sumo tag at the N-terminus. There is a TEV cleavage site between the His6-Sumo tag protein and the protein B3GNT6(43-384), which facilitates the subsequent excision of the His6-Sumo tag at the N-terminus.

[0057] The present invention also provides a method for labeling Gd-IgA1, which includes the step of treating Gd-IgA1 in a sample to be tested with sialidase to obtain desialylated Gd-IgA1.

[0058] The method for labeling Gd-IgA1 can be achieved by labeling the Tn antigen in the hinge region of the IgA1 antibody.

[0059] The sialidase, also known as neuraminidase (NA), is widely distributed in organisms and participates in the metabolism of sialic acid. It can act on the terminal sialic acid residue, excise it and thus expose the galactose residue.

[0060] After the Gd-IgA1 in the sample to be tested is treated with sialidase, a large number of Tn antigens present in the hinge region of Gd-IgA1 are exposed, that is, the galactose residue GalNAc linked to Ser or Thr is exposed. The exposed Tn antigen can be specifically recognized by the glycosyltransferase B3GNT6 (43-384), and GlcNAl containing a bioorthogonal group is transferred to the Tn antigen through an enzymatic reaction. Then, using a bioorthogonal reaction (such as a click reaction), a labeling group with a complementary orthogonal reaction group is transferred to the Tn antigen for labeling or detection.

[0061] In the above method, the method further includes the following steps:

[0062] (1) Using the protein B3GNT6 (43-384) as a glycosyltransferase and using a glycosyltransferase donor substrate containing an alkyne as an enzymatic reaction substrate, performing an enzymatic reaction on the sample to be tested;

[0063] (2) The sample treated in step (1) is subjected to a click reaction with a detection reagent containing an azide group under the action of a click reaction catalyst to obtain Gd-IgA1 labeled with the detection reagent containing an azide group.

[0064] In the above method, the glycosyltransferase donor substrate containing an alkyne can be UDP-GlcNAl.

[0065] In the above method, the enzymatic reaction can use metal ions as a catalyst, and the metal ions can be manganese ions, cobalt ions, and / or zinc ions, specifically manganese ions.

[0066] The optimal pH value of the enzymatic reaction can be 7-8. Specifically, the optimal pH value of the enzymatic reaction can be 7.5.

[0067] In the above method, the detection reagent containing an azide group can be any one of the following:

[0068] F1) A small molecule label modified with an azide group;

[0069] F2) A dye modified with an azide group;

[0070] F3) A hapten modified with an azide group;

[0071] F4) N3-Cy5.

[0072] In the above method, the sample can be a blood sample.

[0073] Furthermore, the blood sample can be serum, plasma, or whole blood.

[0074] Further, the click reaction may be a copper-catalyzed click reaction (copper-catalyzed azide-alkyne cycloaddition reaction), but is not limited thereto.

[0075] Further, the click reaction catalyst may be cuprous ion (Cu + ) compounds, such as cuprous bromide, cuprous iodide, etc.

[0076] In one embodiment of the present invention, the click reaction catalyst is copper sulfate (CuSO4) and sodium ascorbate (VcNa), and sodium ascorbate (VcNa) reduces Cu 2+ to Cu + and catalyzes the click reaction of azide and alkyne (copper-catalyzed azide-alkyne Husigen cycloaddition reaction).

[0077] Further, in one embodiment of the present invention, the copper-catalyzed click reaction is carried out in the presence of a copper ion ligand.

[0078] The copper ion ligands include BTTAA, BTTES, TBTA, BTTP, THPTA, but are not limited thereto. The presence of copper ion ligands can stabilize the monovalent copper salt, protect the monovalent copper ion from oxidation or disproportionation, enhance the catalytic effect on the click reaction, and reduce the toxicity of monovalent copper.

[0079] In one embodiment of the present invention, the copper ion ligand is BTTAA. BTTAA is an accelerating ligand for the copper salt-catalyzed "azide-alkyne" bioorthogonal reaction and is also a ligand with ultra-low toxicity and biocompatibility.

[0080] Further, in one embodiment of the present invention, the labeling method of Gd-IgA1 is as follows:

[0081] (1) Perform desialylation treatment with sialidase, use the protein B3GNT6 (43-384) as a glycosyltransferase, and use a glycosyltransferase donor substrate containing alkyne (such as UDP-GlcNAl) as an enzymatic reaction substrate to perform an enzymatic reaction on a sample to be tested (plasma sample);

[0082] (2) The sample after enzymatic reaction treatment (Gd-IgA1 alkyne derivative) and a detection reagent containing an azide group are subjected to a click reaction under the action of a click reaction catalyst to obtain Gd-IgA1 labeled with the detection reagent containing an azide group.

[0083] In the above labeling method of Gd-IgA1, the sample to be tested may be a plasma sample or a sample obtained by enriching Gd-IgA1 antibody from a plasma sample.

[0084] In the above labeling method of Gd-IgA1, the reaction system and reaction conditions of the enzymatic reaction in step (1) can be: 100 μg Gd-IgA1, pH = 8.0 HEPES (50 mM), MnCl2 (2 mM), MgCl2 (5 mM), UDP-GlcNAl (100 μM), sialidase (0.5 mg / mL), B3GNT6(43-384) (0.2 mg / mL), made up to 50 μL with deionized water, and reacted for 60 minutes under the condition of 37 °C water bath.

[0085] In the above labeling method of Gd-IgA1, the reaction system and reaction conditions of the click reaction in step (2) are: the sample after enzymatic reaction treatment (Gd-IgA1 alkyne derivative), 100 μM azide-modified Cy5 (N3-Cy5), 500 μM copper sulfate (CuSO4), 1 mM ligand BTTAA, 10 mM VcNa, and reacted for 60 minutes under the condition of 37 °C.

[0086] The present invention also provides a reagent or kit for diagnosing or assisting in the diagnosis of IgA nephropathy, and the reagent or kit includes any one of the following:

[0087] G1) The reagent or kit includes the protein B3GNT6(43-384);

[0088] G2) The reagent or kit includes the protein B3GNT6(43-384) and IgA antibody;

[0089] G3) The reagent or kit includes the protein B3GNT6(43-384), IgA antibody, glycosyltransferase donor substrate containing alkyne, detection reagent containing azide group, and click reaction catalyst.

[0090] G4) The reagent or kit includes the protein B3GNT6(43-384), sialidase, IgA antibody, glycosyltransferase donor substrate containing alkyne, detection reagent containing azide group, and click reaction catalyst.

[0091] The present invention also provides a Gd-IgA1 detection reagent or kit, and the reagent or kit includes the protein B3GNT6(43-384).

[0092] The kit can be an enzyme-linked immunosorbent assay kit.

[0093] Specifically, in an embodiment of the present invention, the enzyme-linked immunosorbent assay kit for diagnosing or assisting in the diagnosis of IgA nephropathy contains an enzyme-labeled plate coated with anti-IgA antibody.

[0094] Further, the kit further comprises an enzymatic reaction catalyst, and the enzymatic reaction catalyst can be a metal ion-containing compound. The metal ion can be manganese ion, cobalt ion, and / or zinc ion.

[0095] Specifically, in one embodiment of the present invention, the enzymatic reaction catalyst is MnCl2.

[0096] Further, the kit further comprises a copper ion ligand (catalyst stabilizer). The copper ion ligand can be BTTAA, BTTES, TBTA, BTTP, THPTA, phosphoramidite, or carboxylic acid.

[0097] Specifically, in one embodiment of the present invention, the copper ion ligand is BTTAA.

[0098] In the above kit, the alkyne-containing glycosyltransferase donor substrate can be UDP-GlcNAl.

[0099] In the above kit, the azide group-containing detection reagent (i.e., a labeled molecule containing an azide group) can be a small molecule label modified with an azide group, and the small molecule label can be a dye or a hapten.

[0100] Specifically, in one embodiment of the present invention, in the above kit, the azide group-containing detection reagent is azide-modified biotin (Az-biotin).

[0101] In the above kit, the click reaction catalyst can be a cuprous ion (Cu + ) compound, such as cuprous bromide or cuprous iodide, but not limited thereto.

[0102] Specifically, in one embodiment of the present invention, the click reaction catalyst is copper sulfate (CuSO4) and sodium ascorbate (VcNa).

[0103] Further, the kit further comprises a solvent, and the solvent can be water, tetrahydrofuran, DMSO, acetonitrile, or a mixture of tetrahydrofuran and water, or a mixture of DMSO and water, or a mixture of the above compounds.

[0104] Further, the present invention also provides the use of the kit in the differential diagnosis of IgA nephropathy and non-IgA nephropathy.

[0105] Those skilled in the art should understand that the click reaction described herein refers to the chemical synthesis of various molecules through the splicing of small units. The representative reaction of the click reaction is the copper-catalyzed azide–alkyne cycloaddition, and its basic reaction principle is that Cu + catalyzes the click reaction between the azide group and the alkyne group, causing the azide and alkyne to form a covalent bond, thereby forming a five-membered triazole ring as the cycloaddition product. Therefore, when using the click reaction to label Gd-IgA1, the azide and alkyne moieties are interchangeable, that is, the labeled molecule containing an azide group (N3-Cy5) and the glycosyltransferase donor substrate containing an alkyne group (UDP-GlcNAl) described in the present invention can also be replaced with a labeled molecule containing an alkyne group (such as an alkyne-modified fluorescent group or hapten) and a glycosyltransferase donor substrate containing an azide group (such as UDP-GlcNAz), and the object of the present invention can also be achieved without departing from the protection purpose of the present invention.

[0106] The objects of the applications and methods described in the present invention can be for disease diagnosis, disease prognosis, and / or disease treatment purposes, and they can also be for non-disease diagnosis, non-disease prognosis, and non-disease treatment purposes; their direct objects can be to obtain information on intermediate results of disease diagnosis results, disease prognosis results, and / or disease treatment results, and their direct objects can be non-disease diagnosis, non-disease prognosis, and / or non-disease treatment purposes.

[0107] The method for diagnosing or assisting in the diagnosis of IgA nephropathy described in the present invention is as follows: Take the sera of IgA patients to be tested, sera of other nephropathy controls, and sera of healthy controls, and perform ELISA detection using any of the above-described kits, and determine whether the patient to be tested has IgA nephropathy or differentially diagnose IgA nephropathy from other nephropathies according to the detection results.

[0108] The present invention uses a recombinant baculovirus containing the gene encoding human glycosyltransferase B3GNT6 (43-384) to infect insect cells sf21, obtaining a secreted form of glycosyltransferase B3GNT6 (43-384), which can efficiently and specifically recognize the Tn antigen in the hinge region of Gd-IgA1. Based on this, the present invention combines this glycosyltransferase tool with bioorthogonal reactions to develop a novel chemoenzymatic technique for serological diagnosis of IgA nephropathy. Since the hinge region of Gd-IgA1 in the blood circulation system of IgA nephropathy patients contains abundant Tn antigens, the chemoenzymatic technique described in the present invention can be used to achieve in vitro recognition and labeling of the glycosylation of the hinge region of Gd-IgA1 in human blood samples (such as plasma samples), so as to achieve the purpose of quantitatively detecting galactose-deficient IgA1 (Gd-IgA1). Further, the level of galactose-deficient Gd-IgA1 in the blood is closely related to IgA nephropathy. The detection method of the chemoenzymatic method of the present invention can effectively distinguish IgA nephropathy patients, healthy controls and other nephropathy controls, and this method has high stability and good repeatability, and is a choice to overcome the defects of weak lectin and antibody affinity, poor specificity, large dosage and long operation time in the prior art. The technique for diagnosing IgA nephropathy based on the chemoenzymatic method described in the present invention has the characteristics of non-invasive, rapid, specific and sensitive, integrates bioorthogonal reaction groups, shows superior biocompatibility advantages, can provide effective help for the early diagnosis and treatment of IgA nephropathy, and has very broad clinical application prospects and important significance. Brief Description of the Drawings

[0109] Figure 1 It is a purification result diagram of recombinant protein B3GNT6 (43-384).

[0110] Figure 2 It is a schematic diagram of chemoenzymatic labeling of galactose-deficient IgA1 (Gd-IgA1).

[0111] Figure 3 It is a fluorescence scanning result of chemoenzymatic labeling of Gd-IgA1 in healthy control and IgA nephropathy control samples. "+" indicates the addition of this substance, and "-" indicates the non-addition of this substance.

[0112] Figure 4 It is a statistical analysis diagram of Gd-IgA1 in samples of IgA nephropathy patients, healthy patients and other nephropathy control patients with non-IgA nephropathy. Detailed Description of the Invention

[0113] The present invention will be further described in detail below in conjunction with specific embodiments. The provided embodiments are only for clarifying the present invention, rather than limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way.

[0114] The experimental methods in the following embodiments are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0115] The vector pI-secSUMOstar in the following embodiments is a product of lifesensors company;

[0116] The following embodiments use ANOVA statistical software to process the data. The experimental results are expressed as mean ± standard deviation, and the Brown-Forsythe test is used. P < 0.05 (****) indicates extremely significant differences.

[0117] Example 1 Preparation of Recombinant Protein B3GNT6(43 - 384)

[0118] Recombinant protein B3GNT6(43 - 384) (i.e., the protein B3GNT6(43 - 384) of the present invention) is a truncated B3GNT6 protein. Compared with the B3GNT6 protein, the N-terminus is truncated by 42 amino acids; the truncated B3GNT6 protein can act as a glycosyltransferase and can specifically recognize the Tn antigen.

[0119] The amino acid sequence of recombinant protein B3GNT6(43 - 384) is shown in SEQ ID No.1 (consisting of 342 amino acid residues); the nucleotide sequence of the gene encoding recombinant protein B3GNT6(43 - 384) is shown in SEQ ID No.2 (consisting of 1026 nucleotides); the DNA molecule shown in SEQ ID No.2 encodes the protein B3GNT6(43 - 384) of SEQ ID No.1.

[0120] I. Construction and Expression of Recombinant Expression Vector

[0121] 1. The gene encoding the codon-optimized recombinant protein B3GNT6 (43-384) (SEQ ID No.2) was constructed into the pI-secSUMOstar plasmid, and the successfully constructed recombinant vector was named pI-secSUMOstar-B3GNT6 (43-384). The recombinant vector pI-secSUMOstar-B3GNT6 (43-384) was obtained by replacing the fragment between the BamHI and EcoRI recognition sites (small fragment) of the pI-secSUMOstar vector with the DNA fragment whose nucleotide sequence is SEQ ID NO.2 in the sequence listing, while keeping other sequences of the pI-secSUMOstar vector unchanged, thus obtaining a recombinant expression vector.

[0122] The recombinant vector pI-secSUMOstar-B3GNT6 (43-384) expresses a fusion protein with a His6-Sumo tag at the N-terminus. There is a TEV cleavage site between the His6-Sumo tag protein and the protein B3GNT6 (43-384), which is convenient for subsequent excision of the N-terminal His6-Sumo tag. After successful sequencing identification of the recombinant vector pI-secSUMOstar-B3GNT6 (43-384), it indicates that the recombinant vector pI-secSUMOstar-B3GNT6 (43-384) has been successfully constructed.

[0123] The successfully constructed recombinant vector pI-secSUMOstar-B3GNT6 (43-384) was transformed into MAX Efficiency DH10Bac (10361-012, Invitrogen) competent cells, cultured in LB medium at 37 °C with a rotation speed of 220 for 4 hours, and then blue-white screening was used to select white colonies for the extraction of bacmid (recombinant baculovirus shuttle plasmid).

[0124] 2. Transfect sf21 cells by liposome transfection method: Take sf21 cells in the logarithmic growth phase and seed them in a 6-well plate (2×10 6Cells / well), let them adhere at room temperature. Transfect sf21 cells in a 6-well plate with 2 μg of baculovirus plasmid (transfection reagent: Roche X-tremeGENE 9 DNA transfect ref 06365787001). Set up a control group and a transfection group. The culture environment is 27 °C without carbon dioxide, and the culture time is 72 hours. The specific transfection operation is as follows: Prepare a mixture of plasmid and transfection reagent: Dilute with antibiotic-free SIM SF medium (Sino Biological). Dilute 2 μg of baculovirus plasmid with 100 μL of medium, and dilute 6 μL of transfection reagent with 100 μL of medium. After pipetting evenly, add the liposome component (transfection reagent) dropwise to the baculovirus plasmid DNA component, mix gently, and let it stand for 30 minutes to obtain the transfection mixture. Add 200 μL of the mixture to the cells without changing the medium in the middle, and seal it with a sealing film to prevent the solution from drying out.

[0125] 3. Harvest the virus: Four days after the sf21 cells are infected, harvest the supernatant to obtain the first-generation recombinant baculovirus (P1 virus). The specific operation steps are as follows: First, centrifuge at a low speed of 800×g to remove the cell pellet, transfer the supernatant to a new centrifuge tube, then centrifuge at a high speed of 2000×g for 5 minutes to remove cell debris, take the supernatant, and then add serum to a concentration of 5% to obtain the first-generation recombinant baculovirus (P1 virus), and store it at 4 °C.

[0126] 4. Infect sf21 cells with P1 virus: Take 30 ml of sf21 cells with a cell concentration of 2×10 6 cells / mL, and infect the virus at a ratio of 1:100, that is, add 300 μL of P1 virus to the sf21 cells and mix well; Culture at 110 rpm, 27 °C without carbon dioxide, and harvest the supernatant after 4 days. The treatment method is as described in step 3.

[0127] 5. Expand the culture system to 1 L and expand the number of virus infections in the same proportion. Other operations are as described in step 3.

[0128] II. Expression and purification of recombinant protein B3GNT6 (43-384)

[0129] After culturing and infecting sf21 cells according to step 1, recombinant protein B3GNT6 (43-384) is produced. The produced recombinant protein B3GNT6 (43-384) has a His6-Sumo tag at the N-terminus and can be further used for purification.

[0130] 1. Collect the supernatant after culturing infected sf21 cells in Step 1, and perform ultrafiltration or dialysis concentration; then enrich the protein using a nickel column. Specifically, first perform the column binding process, and then sequentially elute the nickel column with 10 mmol / L, 30 mmol / L, and 50 mmol / L imidazole solutions for about 5 column volumes to remove the impurity proteins. Finally, elute the nickel column with 250 mmol / L imidazole solution for about 3 column volumes, and collect the eluted solution, which is the target protein solution.

[0131] 2. Detect the protein using 12% SDS-PAGE. After collecting the concentrated protein solutions and mixing them, perform BCA quantification after ultrafiltration using a 30 K MiliQ ultrafiltration membrane, and store them at -80 °C using 20 mM HEPES (pH = 7.5) containing 10% glycerol. The specific results of protein purification are shown in Figure 1 , and the recombinant protein B3GNT6 (43 - 384) containing the His6-Sumo tag showed clear bands at the corresponding positions (molecular weight approximately 70 KDa).

[0132] Example 2 Functional Assay of Recombinant Protein B3GNT6 (43 - 384)

[0133] In this part of the experiment, Tn-modified glycopeptides were used as the receptor substrates for recombinant protein B3GNT6 (43 - 384) to explore its activity. The specific implementation is as follows:

[0134] 1. Exploration of the donor substrate of B3GNT6 (43 - 384)

[0135] First, the Tn-modified glycopeptide sequence, PIMAAAT(α-GalNAc)PAPAAK, was designed in the experimental protocol and synthesized by Shanghai Gil Biochemical Technology Co., Ltd. The enzyme activity differences of the recombinant protein B3GNT6(43-384) prepared in Example 1 were compared using the donor substrates uridine 5'-diphospho-N-acetylglucosamine (UDP-GlcNAc) (CAS: 91183-98-1, Sigma-Aldrich (Shanghai) Trading Co., Ltd.) and its azide derivative (UDP-GlcNAz) (CAS: 1611490-64-2, Qingdao Shuge Biotechnology Co., Ltd.) and alkyne derivative (UDP-GlcNAl) (Qingdao Shuge Biotechnology Co., Ltd.), respectively. Then, the reaction system was designed. The enzymatic reaction system was 30 μl, containing HEPES (100 mM, pH = 8.0), MnCl2 (2 mM), glycopeptide (1 mM), UDP-GlcNAc or UDP-GlcNAz or UDP-GlcNAl (2 mM), B3GNT6(43-384) (0.2 mg / ml), and made up with deionized water. The reaction was carried out in a water bath at 37 °C for 30 minutes and terminated by heating at 95 °C for 2 minutes. The reaction solution was analyzed by HPLC. In the HPLC detection, an Agilent ZORBAX C18 analytical column was used in the HPLC analysis, the column temperature was 40 °C, the injection volume was 20 μl, the mobile phase was a liquid composed of acetonitrile and water containing one-thousandth trifluoroacetic acid, the gradient of acetonitrile was 15%-45% (20 minutes), the flow rate was 1 ml / min, and the separation map was automatically formed by detecting at a wavelength of 220 nm using a UV detector.

[0136] The detection and analysis results are shown in Table 1:

[0137] Table 1 Yield of the enzymatic reaction

[0138]

[0139] The results showed that the recombinant protein B3GNT6 (43-384) could transfer the GlcNAc group of UDP-GlcNAc to the glycopeptide, and the yield of the product GlcNAc-GalNAc-peptide was 100%; the recombinant protein B3GNT6 (43-384) could transfer the GlcNAz group of UDP-GlcNAz to the glycopeptide, and the product GlcNAz-GalNAc-peptide was obtained with a yield of 11.3%; the recombinant protein B3GNT6 (43-384) could transfer the GlcNAl group of UDP-GlcNAl to the glycopeptide, and the product GlcNAl-GalNAc-peptide was obtained with a yield of 99.6%. It was shown that the recombinant protein B3GNT6 (43-384) could efficiently utilize natural nucleoside sugars and their analogs, had high biocatalytic activity, and the activity of using UDP-GlcNAl was higher than that of UDP-GlcNAz. Therefore, UDP-GlcNAl was used as the donor substrate of the recombinant protein B3GNT6 (43-384) in the subsequent labeling process.

[0140] Example 3 Chemical-enzymatic Labeling of the Hinge Region Tn Antigen of Gd-IgA1

[0141] I. Chemical-enzymatic Labeling of Plasma-derived Monomeric Gd-IgA1

[0142] This chemical-enzymatic labeling technique involves three steps of operation, aiming to convert two sugar-deficient forms of Gd-IgA1 into one form, namely Tn modification, and then be labeled, as Figure 2 shown: First, Gd-IgA1 is treated with sialidase, then B3GNT6 (43-384) is used to recognize the Tn antigen and transfer GlcNAl to the Tn antigen, and the last step is a bioorthogonal reaction to introduce a labeling group (such as a fluorescent group) for observation. The specific operation steps are as follows:

[0143] 1. The control plasma of IgA nephropathy patients and other nephropathy patients was from patients diagnosed with IgA nephropathy or other nephropathy patients with non-IgA nephropathy who had signed informed consent in the Department of Nephrology of Peking University First Hospital. The control plasma of healthy people was from healthy blood donors who had signed informed consent. The Gd-IgA1 in the plasma samples of IgA nephropathy patients, other nephropathy patients or healthy people was enriched using a Jacalin agarose column, eluted with melibiose, and then the Gd-IgA1 monomer was enriched using a molecular sieve to obtain the Gd-IgA1 sample of IgA nephropathy patients, the Gd-IgA1 sample of other nephropathy patients or the Gd-IgA1 sample of healthy people.

[0144] 2. Enzymatic labeling: Measure the concentration of the Gd-IgA1 samples from IgA nephropathy patients, other nephropathy patients, or healthy individuals obtained in step 1 using A280 of nanodrop. Then, take 25 μg of the Gd-IgA1 samples from IgA nephropathy patients, other nephropathy patients, or healthy individuals respectively for the labeling reaction, and record them as three experimental groups: the IgA nephropathy patient group, the other nephropathy control group (abbreviated as the nephropathy control group), and the healthy control group. Perform the following 4 treatments on the above 3 groups of Gd-IgA1 samples respectively to obtain the samples after enzymatic reaction treatment:

[0145] 1) B3GNT6(43 - 384) + UDP-GlcNAl+

[0146] Use the protein B3GNT6(43 - 384) as the glycosyltransferase, and use the glycosyltransferase donor substrate containing alkyne (such as UDP-GlcNAlK (Qingdao Shuge Biotechnology Co., Ltd.)) as the enzymatic reaction substrate to perform the enzymatic reaction on the sample to be tested. The reaction system and reaction conditions are: 100 ug Gd-IgA1, pH = 8.0 HEPES (50 mM), MnCl2 (2 mM), MgCl2 (5 mM), UDP-GlcNAl (100 μM), sialidase (0.5 mg / mL), B3GNT6(43 - 384) (0.2 mg / mL), make up to 50 μL with deionized water, and react for 60 minutes under the condition of 37 °C water bath.

[0147] 2) B3GNT6(43 - 384) - UDP-GlcNAl-

[0148] Compared with 1), the difference is only that B3GNT6(43 - 384) and UDP-GlcNAl are replaced with the same volume of HEPES with pH = 8.0.

[0149] 3) B3GNT6(43 - 384) + UDP-GlcNAl-

[0150] Compared with 1), the difference is only that UDP-GlcNAl is replaced with the same volume of HEPES with pH = 8.0.

[0151] 4) B3GNT6(43 - 384) - UDP-GlcNAl+

[0152] Compared with 1), the difference is only that B3GNT6(43 - 384) is replaced with the same volume of HEPES with pH = 8.0.

[0153] 3. Bioorthogonal reaction: The sample treated by the enzymatic reaction (Gd-IgA1 alkyne derivative) and the detection reagent containing an azide group undergo a click reaction under the action of a click reaction catalyst to obtain Gd-IgA1 labeled by the detection reagent containing an azide group. The reaction system and reaction conditions are as follows: The sample (Gd-IgA1 alkyne derivative) treated by the B3GNT6 enzymatic reaction, 100 μM azide-modified Cy5 (N3-Cy5), 500 μM copper sulfate (CuSO4), 1 mM ligand BTTAA, 10 mM VcNa, and react at 37 °C for 60 minutes.

[0154] 4. Add 5× reducing loading buffer, heat at 95 °C, then load 10 μL and perform SDS-PAGE (12%) gel electrophoresis for observation.

[0155] 5. After the gel electrophoresis is completed, use a Tythoon in-gel fluorescence scanner to scan the Cy5 fluorescence signal, and the scanning voltage is 800V.

[0156] 6. After the scanning is completed, stain and observe the protein gel with Coomassie Brilliant Blue R250.

[0157] 7. After completing steps 5 and 6, perform relative quantitative analysis on it using ImageJ.

[0158] 8. The results show that this chemoenzymatic strategy can effectively label the Tn antigen on Gd-IgA1, and the labeling signal of Gd-IgA1 in IgA nephropathy patients is stronger than that of healthy controls. For specific results, see Figure 3 。

[0159] 9. During the experiment, plasma from 43 IgA nephropathy patients, 26 healthy patients, and 28 other nephropathy control patients with non-IgA nephropathy was collected. According to steps 1-7, the content of Gd-IgA1 was measured.

[0160] 10. From the statistical results, this method can better distinguish IgA nephropathy patients from the other two control groups. For details, see Figure 4 。

[0161] The above has described the present invention in detail. For those skilled in the art, without departing from the spirit and scope of the present invention and without the need for unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations, and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to cover any modifications, uses, or improvements to the present invention, including those that depart from the scope disclosed in this application but are made by using conventional techniques known in the art. Some basic features can be applied within the scope of the following appended claims.

Claims

1. Application of a protein, characterized in that, The application is any one of the following: B1) The application of the protein in the preparation of a product for diagnosing or assisting in the diagnosis of IgA nephropathy; B2) The application of the protein in labeling Gd-IgA1; B3) The application of the protein in the preparation of a product for labeling Gd-IgA1; B4) The application of the protein in the preparation of a product for detecting Gd-IgA1; The protein is any one of the following: A1) A protein with an amino acid sequence of SEQ ID No.1; A2) A fusion protein with the same function obtained by connecting a tag to the N-terminus and / or C-terminus of A1).

2. Use of a biological material related to the protein described in claim 1, characterized in that, The application is any one of the following: C1) The application of the biological material related to the protein described in claim 1 in the preparation of a product for diagnosing or assisting in the diagnosis of IgA nephropathy; C2) The application of the biological material related to the protein described in claim 1 in labeling Gd-IgA1; C3) The application of the biological material related to the protein described in claim 1 in the preparation of a product for labeling Gd-IgA1; C4) The application of the biological material related to the protein described in claim 1 in the preparation of a product for detecting Gd-IgA1; C5) The application of the biological material related to the protein described in claim 1 in the preparation of the protein described in claim 1; The biological material is any one of the following D1) to D5): D1) A nucleic acid molecule encoding the protein described in claim 1; D2) An expression cassette containing the nucleic acid molecule described in D1); D3) A recombinant vector containing the nucleic acid molecule described in D1), or a recombinant vector containing the expression cassette described in D2); D4) A recombinant microorganism containing the nucleic acid molecule described in D1), or a recombinant microorganism containing the expression cassette described in D2), or a recombinant microorganism containing the recombinant vector described in D3); D5) A cell line containing the nucleic acid molecule described in D1), or a cell line containing the expression cassette described in D2), or a cell line containing the recombinant vector described in D3).

3. The application according to claim 2, characterized in that, The nucleotide sequence of the nucleic acid molecule described in D1) is as shown in SEQ ID No.

2.

4. A method for labeling Gd-IgA1, characterized in that, The method includes subjecting Gd-IgA1 in a sample to be tested to neuraminidase treatment to obtain desialylated Gd-IgA1, then using the protein described in claim 1 to recognize the Tn antigen on the desialylated Gd-IgA1, transferring GlcNAl to the Tn antigen, performing a bioorthogonal reaction, and introducing a labeling group for detection.

5. The method according to claim 4, wherein The method further includes the following steps: (1) Using the protein described in claim 1 as a glycosyltransferase, and using a glycosyltransferase donor substrate containing an alkyne as an enzymatic reaction substrate to perform an enzymatic reaction on the sample to be tested; (2) Reacting the sample treated in step (1) with a detection reagent containing an azide group under the action of a click reaction catalyst to obtain Gd-IgA1 labeled with the detection reagent containing an azide group.

6. The method according to claim 5, wherein The glycosyltransferase donor substrate containing an alkyne is UDP-GlcNAl.

7. The method according to claim 5 or 6, characterized in that, The detection reagent containing an azide group is any one of the following: F1) A small molecule label modified with an azide group; F2) A dye modified with an azide group. F3) Hapten modified with an azide group; F4) N3-Cy5.

8. The method according to any one of claims 4-6, characterized in that The sample is a blood sample.

9. A reagent or kit for diagnosing or assisting in the diagnosis of IgA nephropathy, characterized in that, The reagent or kit includes the protein, neuraminidase, IgA antibody, alkyne-containing glycosyltransferase donor substrate, azide group-containing detection reagent, and click reaction catalyst described in claim 1.

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