CN113817743B - IgDR gene, igDR monoclonal antibody, preparation method and application thereof - Google Patents

Abstract

The application discloses an IgDR gene, an IgDR monoclonal antibody, a preparation method and application thereof. Wherein the nucleotide sequence of the IgDR gene is shown as SEQ ID NO.1, and the amino acid sequence is shown as SEQ ID NO. 2; the IgDR monoclonal antibody system is prepared by using the encoding protein of the IgDR gene as antigen and utilizing hybridoma monoclonal antibody technology. The application provides the nucleotide and amino acid sequence of the IgDR gene, and the IgDR monoclonal antibody with high specificity and high affinity is prepared by utilizing the IgDR gene. The IgDR monoclonal antibody is used for preparing the related products for detecting IgDR, can provide means for accurately qualitatively, quantitatively and positionally determining IgDR, can be widely applied to clinical diagnosis and scientific research work, and provides a new thought and solving way for researching IgD and IgDR in the fields of medicine, pharmacy and biology.

Description

IgDR gene, igDR monoclonal antibody, preparation method and application thereof

Technical Field

The application relates to the technical field of monoclonal antibody preparation and immune application, in particular to an IgDR gene, an IgDR monoclonal antibody and application thereof, and a preparation method of the IgDR monoclonal antibody.

The IgDR gene is hereinafter abbreviated as IgDR.

Background

Immunoglobulins (Ig), also known as antibodies, are produced by proliferation and differentiation of B cells into plasma cells under antigen stimulation, are important effector molecules mediating fluid immunity, and are involved in the regulation of the immune system. Depending on the immunogenicity of the Ig heavy chain constant regions, five classes, including IgM, igG, igA, igE and IgD, are currently known, and the delicate balance of individual Igs and effector cells together maintain normal physiology in humans, which may lead to autoimmune disease once the balance is broken. Currently, immunology has been studied extensively for IgM, igG, igA and IgE functions, but the understanding of IgD is still limited. IgD was originally found in the serum of a myeloma patient by Rowe and Fahey in 1965, and mainly includes membrane-bound IgD (IgD) and secretory IgD (IgD). In humans, mice, act as B cell surface receptors in membrane-bound form, with igd involved in B cell development, immune function and tolerance. Igd may function as an antibody. serum concentrations of sIgD vary widely and are secreted in small amounts in human nose, nasopharynx, oral cavity and tears. The igd has been found to be involved in mucosal immune responses and can regulate mucosal homeostasis by colonizing mucosal sites and establishing interactions with co-viable flora. Abnormally elevated sIgD may occur in patients with IgD myeloma, skin allergies, various infectious and autoimmune diseases, and the like.

In an immune response, antibodies such as Ig recognize specific antigens through their Fab fragments, and the Fc portion is required for antibody-mediated biological activity. The Fc segment that determines the class of immunoglobulins can exert diverse biological activities mediated by different classes of immunoglobulins through binding to Fc receptors (fcrs) expressed on the membranes of various cells. In vivo and in vitro studies show that IgDR expressed on different immune cells acts like an "effector" to play an important role in regulating physiological, pathological and other functions of IgD in peripheral circulation. Immunologists have demonstrated specific fcrs for both IgM, igG, igA and IgE by techniques such as molecular biology and have conducted a number of structural and immune studies. Human peripheral blood T cells and non-T cells were first found in 1980 to express IgDR. However, specific FcR (fcδr, igDR) of mystery IgD has not been cloned so far, and its presence has been indirectly demonstrated only by sheep red blood cell rosette assay, radioimmunoassay, flow cytometry, and the like. The amino acid sequence of IgDR is unknown so far, and the bottleneck problem of intensive research on IgD and IgDR physiological function and pathological action is also the problem to be solved in the fields of medicine, pharmacy and biology.

Disclosure of Invention

The technical problem to be solved by the application is to provide an IgDR gene.

Another technical problem to be solved by the present application is to provide an IgDR monoclonal antibody.

The application also solves the technical problem of providing a preparation method of the IgDR monoclonal antibody.

The application also solves the technical problem of providing an in vitro detection/diagnosis kit.

For IgDR gene, the present application adopts the technological scheme that the nucleotide sequence is shown in SEQ ID No.1 and the amino acid sequence of the encoded protein is shown in SEQ ID No. 2.

Preferably, the IgDR gene is an Fc receptor (FcR) that specifically binds to the Fc-segment domain of human IgD.

The application adopts the technical scheme that the IgDR monoclonal antibody is prepared by taking the encoding protein of IgDR gene (IgDR protein is called as IgDR protein in the following) as antigen and utilizing hybridoma monoclonal antibody technology.

The preparation method of the IgDR monoclonal antibody adopts the technical scheme that the preparation method comprises the following steps:

(1) Purifying by using an escherichia coli expression system and adopting three steps of affinity chromatography, an ion exchange column and a molecular sieve to obtain prokaryotic expression purified IgDR protein;

(2) IgDR protein is used as antigen, and the IgDR monoclonal antibody is obtained through mouse immunity, antiserum detection, cell fusion, hybridoma screening, establishment of stable cell strain, ascites preparation and purification of antibody.

For in vitro detection/diagnosis kit, the application adopts the technical scheme that the kit comprises IgDR monoclonal antibody.

The application also includes:

the application of IgDR monoclonal antibody in preparing reagent for flow cytometry detection of human cell surface expression IgDR.

The application of IgDR monoclonal antibody in preparing reagent for the Westernblot detection of IgDR in cell or tissue.

The application of IgDR monoclonal antibody in preparing immunohistochemical detection reagent for IgDR expression in tissue.

The application of IgDR monoclonal antibody in preparing ELISA, chemiluminescent immunoassay or fluorescent immunoassay reagent for IgDR.

The beneficial effects of the application are as follows:

provides the nucleotide and amino acid sequence of the IgDR gene, and the IgDR monoclonal antibody with high specificity and high affinity is prepared by using the IgDR gene.

The IgDR monoclonal antibody is used to prepare relevant IgDR detecting product, and may be used in the accurate qualitative, quantitative and locating means including marking cell surface or intracellular IgDR protein expression via flow cytometry, laser confocal technology to display the intracellular IgDR protein location, western blot to detect the IgDR protein expression in protein mixture, enzyme-linked immunosorbent assay (ELISA) to detect IgDR expression in peripheral blood sample, etc.

Drawings

The application will be described in further detail with reference to the drawings and the detailed description.

FIG. 1 is a 3D diagram of IgDR homology modeling of example 1 of the present application. The circled portions represent IgD-IgDR binding peptide fragments.

FIG. 2 is a molecular modeling docking analysis of the primary docking sites of the Fc domains of IgDR and IgD in example 1 of the present application.

FIG. 3 is a SDS-PAGE Coomassie blue staining of the purified IgDR protein of example 2 of the present application.

FIG. 4 shows CD4 of IgDR monoclonal antibodies of example 3 of the present application labeled healthy controls and RA patients ⁺ Expression of IgDR on T cells.

FIG. 5 shows IgDR expression on IgD-induced Jurkat cells by two methods, igDR monoclonal antibody and biotin-labeled IgD of example 3 of the present application.

Detailed Description

Example 1: determination of the nucleotide and amino acid sequences of IgDR:

1. the experimental method comprises the following steps:

1.1 Mass Spectrometry detection of IgD-IgDR binding peptide fragments:

jurkat cells in log phase after 8h stimulation with PMA were taken (10 ⁷ Per mL), adding protein lysate, centrifuging at 12000 Xg for 20min at 4deg.C, and collecting protein supernatant. Incubating human natural IgD protein and 6FF agarose gel microbeads for 2h at 4 ℃ in a shaking table, centrifugally collecting the microbeads, incubating the collected microbeads and protein supernatant for 2h at 4 ℃ in a shaking table, and centrifugally collecting the microbeads. The protein eluate (containing 300mM imidazole) was washed 3 times at 4℃for 10 min/time, and the eluted protein was collected 3 times, respectively. Adding 3 times of eluted proteins into a 5mL ultrafiltration tube (intercepting 10-30 kD of proteins), centrifugally collecting proteins, adding protein replacement liquid (low-salt protein buffer solution) into the 5mL ultrafiltration tube, centrifugally replacing protein buffer solution to obtain desalted proteins, and carrying out mass spectrum detection on the protein solution.

1.2 screening of Membrane proteins binding to IgD to determine IgDR nucleotide and amino acid sequences:

(1) According to the mass spectrum detection result, searching a protein library through a uniprot website, primarily screening out possible membrane protein receptors, namely IgDR candidate proteins, and primarily detecting the binding capacity of the membrane protein receptors and IgD proteins on Jurkat cells by using an immune coprecipitation method.

Immunoprecipitation method: collecting Jurkat cells by centrifugation, adding co-immunoprecipitation (co-IP) lysate, fully mixing, cracking for 30min on ice, repeatedly freezing and thawing for 3 times, collecting protein supernatant by high-speed centrifugation, immunoprecipitation of cell proteins by using candidate membrane protein receptor antibody, detecting expression of phosphorylated membrane protein receptor protein by using a western blot method by using an anti-protein tyrosine phosphorylating antibody 4G10, and detecting binding condition of IgD and receptor protein by using an anti-IgD antibody.

(2) The three-dimensional structure of the receptor protein was obtained by homology modeling using Discovery studio client software. The structure of the IgD protein was downloaded from the RCSB protein database (http:// www.rcsb.org/PDB) (PDB ID:1 ZVO). And running a Discovery Studio 2020client program to carry out molecular simulation butt joint on receptor protein and ligand IgD protein, and carrying out visualized optimization treatment on an optimal complex structure obtained by running a Z-DOCK module through an R-DOCK module of the Discovery Studio, thereby obtaining information of interaction active sites through screening.

2. Experimental results:

the mixed solution of the PMA continuously induced Jurkat cell lysis protein eluted from the 6FF agarose beads and the human IgD protein was detected by LC-MS mass spectrometry. Label-free quantitative protein analysis (LFQ) was used to determine the unknown peptide stretch between IgD and IgDR binding. A total of 5 most likely candidate receptor proteins were screened.

The homology modeling of the amino acid primary sequence is carried out on the 5 candidate receptor proteins by using Discovery Studio 2020Client molecular simulation docking software, and the homologous model of the 5 candidate receptor proteins is subjected to one-to-one simulation docking with human IgD protein (PDB ID:1 ZVO) by using a ZDOCK module. After the optimization analysis of the RDOCK module, four candidate proteins were excluded because their amino acid sequences that interface with IgD mimics were not included in the sequence of the binding peptide fragments shown by the mass spectrometry results. According to the retrieval result of the uniprot website, the IgDR extracellular binding part protein is finally preliminarily determined to be TR beta V12-4. The nucleotide sequence and the primary amino acid sequence of IgDR are shown as SEQ ID NO.1 and SEQ ID NO.2 respectively, and are specifically as follows:

the nucleotide sequence of the IgDR gene is as follows:

the amino acid sequence of IgDR gene is as follows:

the effectiveness of homology modeling of the nucleotide sequence and the amino acid primary sequence of the IgDR gene is shown in FIG. 1. The main docking sites for IgD-IgDR were further screened by software (FIG. 2).

Example 2: determination of affinity and specificity of IgDR binding to IgD:

1. the experimental method comprises the following steps:

1.1 construction, expression and purification of IgDR proteins using E.coli expression systems:

the target protein fragment was amplified by standard Polymerase Chain Reaction (PCR) according to the amino acid primary sequence of IgDR, purified using PCR purification kit, and digested with Nco I and Xho I restriction enzymes or BamH I and Xho I restriction enzymes. The vector pET-28a (+) and pcDNA3.1 (+) -His were linearized by the same enzyme combination, followed by ligation of the vector with digested IgDR protein fragments using T4 DNA ligase (5U/. Mu.L) to generate plasmid constructs. The forward and reverse primer sequences are shown as SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6 respectively, and are specifically as follows:

IgDR Nco I forward：

5′-aattttgtttaactttaagaaggagatataccatgggtgtgattcagagtccgcgccatgaagtg-3′,

IgDR Xho I Reverse：

5′-agccggatctcagtggtggtggtggtggtgctcgagcagactactggcacaaaaataaactgcac-3′；

IgDR BamH I forward：

5′-cccaagcttatggactcctggaccctctg-3′,

IgDR Xho I reverse：

5′-cgggatccgctaaactgctggcacagaagt-3′。

IgDR was expressed in E.coli system using PET-28a (+) eukaryotic expression vector in Luria-Bertani (LB) medium. The IgDR-expressed protein was then purified using a protein purification device, using a combination of "PBS" buffer +8M urea buffer system (ph=7.4), affinity chromatography column, and anion exchange column. Finally, the protein was extensively dialyzed against 0.5m arg,0.2/0.02GSSH/GSH,2mM DTT,20%GLY PBS buffer (ph=7.4), and the purified protein was frozen at-80 ℃. Meanwhile, purity of the protein was detected by coomassie brilliant blue staining using SDS-PAGE gel (purity > 90%) (fig. 3).

1.2 construction, expression and purification of IgD-Fc fragment proteins:

total RNA is extracted from human peripheral blood PBMC and subjected to reverse transcription to obtain a cDNA library, and the cDNA library is digested with EcoR I and Nhe I restriction enzymes to obtain an IgD-Fc gene fragment. The vectors pGMT-easy and PET28a (+) are linearized by the same enzyme combination, and subsequently ligated with the digested IgD-Fc protein fragments using a T4 DNA ligase to generate the construct. The sequences of the forward and reverse primers are shown as SEQ ID NO.7 and SEQ ID NO.8, and are specifically as follows:

IgD-Fc forward：5'-gctagcatgttgtccgagcc-3'，

IgD-Fc Reverse：5'-tctgagctagttgagcagagtccg-3'。

IgD-Fc was expressed in E.coli systems using a PET-28a (+) eukaryotic expression vector in Luria-Bertani (LB) medium. The IgD-Fc expressed protein is then purified using a protein purification device using "Na" as the source of the protein ₂ HPO ₄ "phosphate buffer system (ph=7.4), affinity chromatography column and anion exchange column combination for protein purification. Finally, the protein was extensively dialyzed against 0.5m arg,0.2/0.02GSSH/GSH,2mM DTT,20%GLY PBS buffer (ph=7.4), and the purified protein was frozen at-80 ℃. Meanwhile, purity (purity) of the protein was checked by electrophoresis on SDS-PAGE gel using coomassie brilliant blue staining>90％)。

1.3 study of binding specificity of IgDR:

(1) The affinity (KD value) of IgDR protein, igD protein and IgD-Fc segment protein obtained by purification and collection was detected by Isothermal Titration Calorimetry (ITC).

ITC method: the IgDR protein, igD protein and IgD-Fc segment protein are diluted to proper loading concentrations respectively by PBS, the IgDR protein reacts with the IgDR protein respectively in a sample cell (loading volume 70 mu L, proper concentration 10 mu M) and the IgD protein and IgD-Fc segment protein are ligand proteins (loading volume 200 mu L, proper concentration 100 mu M), and the binding affinity (KD value) and enthalpy change value (delta H value) are detected.

(2) The binding characteristics of IgDR proteins and other immunoglobulins (IgG, igM, igA and IgE) were examined to demonstrate the specificity of receptor protein binding.

ITC method: igDR protein in the sample cell (loading volume 70 muL, suitable concentration 10 muM), immunoglobulin (IgG, igM, igA and IgE) in the cell are ligand proteins respectively reacted with receptor protein (loading volume 200 muL, suitable concentration 100 muM), after a series of parameters are set, the machine detects the affinity (KD value) and enthalpy change value (delta H value) of the receptor protein and the immunoglobulin (IgG, igM, igA and IgE).

1.4 active site study of IgD-IgDR binding:

protein-protein interactions between IgDR proteins, igDR mutants and human IgD were observed by co-immunoprecipitation (co-IP) and pull-down techniques.

(1) Construction, expression and purification of four-point muteins:

the primary amino acid sequence of IgDR is known, and five IgDR mutant plasmids H29R, T32A, M34A, T39A and H29R/T32A/M34A/T39A are constructed by utilizing a site-directed mutagenesis kit according to the results of mass spectrometry detection and molecular simulation docking. The IgDR H29R/T32A/M34A/T39A protein fragment was then amplified by PCR, purified using a PCR purification kit, and digested with Nco I and Xho I restriction enzymes. The vector pET-28a (+) was linearized by the same enzyme combination, followed by ligation of the vector with the digested IgDR H29R/T32A/M34A/T39A protein fragment using T4 DNA ligase (5U/. Mu.L) to generate a plasmid construct. The forward and reverse primer sequences are shown as SEQ ID NO.9 and SEQ ID NO.10, and are specifically as follows:

IgDR H29R/T32A/M34A/T39A Nco I forward：

5′-ttgtttaactttaagaaggagatataccatgggtgtgattcagagtccgcgccgtgaag-3′

IgDR H29R/T32A/M34A/T39A Xho I Reverse：

5′-ccggatctcagtggtggtggtggtggtgctcgagcagactactggcacaaaaataaact-3′

IgDR muteins were expressed in E.coli systems using a PET-28a (+) eukaryotic expression vector in Luria-Bertani (LB) medium. The IgDR mutein was then purified using a protein purification device, using a combination of "PBS" buffer+8M urea buffer system (ph=7.4), affinity chromatography column and anion exchange column for protein purification. Finally, the protein was extensively dialyzed against 300mM NaCl,10%Glycerol PBS buffer (ph=7.4) and the purified protein was frozen at-80 ℃. Meanwhile, purity of the protein was detected by coomassie brilliant blue staining using SDS-PAGE gel (purity > 90%).

(2) The binding of IgDR muteins to IgD proteins was detected by co-immunoprecipitation and pull-down techniques:

adding co-IP lysate, centrifuging at 12000 Xg for 15 min at 4deg.C to obtain protein supernatant, performing co-immunoprecipitation experiment on protein sample, using IgD as probe, forming protein-protein coupled complex by protein A/G microbeads, and detecting the binding of membrane protein receptor protein and IgD protein by Western blot method.

Respectively transfecting HEK 293T cells with the constructed IgDR plasmid and IgDR mutant plasmid, collecting transfected cells, adding protein lysate, crushing cells by a cell crusher, centrifuging 12000 Xg for 20min at 4 ℃, incubating IgDR with His tag with 6FF agarose gel microbeads for 2h at 4 ℃, centrifuging to collect microbeads, washing protein washing solution (containing 20mM imidazole) for 3 times at 4 ℃,30 min/time, centrifuging to collect microbeads. The human natural IgD protein and the treated microbeads are incubated for 12-16 h at 4 ℃. The microbeads are collected by centrifugation, 20-30 mu L of protein loading buffer solution is added, and the mixture is boiled in boiling water 10min,Western blot to detect the co-expression of IgDR and IgD.

(3) The ITC method detects the combination condition of IgDR mutant protein and IgD protein:

eluting IgDR mutant protein, adding it into sample pool (loading volume 70. Mu.L, proper concentration 10. Mu.M), adding IgD protein as ligand protein into cell pool (loading volume 200. Mu.L, proper concentration 100. Mu.M), setting a series of parameters, and machine detecting the affinity (K) of mutant plasmid protein and IgD protein _D Value) and the enthalpy change value (Δh value).

2. Experimental results:

the TR βV12-4 is IgDR extracellular binding partial protein with a molecular weight of 11.9kDa. The parameters obtained by ITC experiments indicate (see table 1): human IgD protein and IgD-Fc segment eggwhite-IgDR-bound K _D Values are all 10 ^-12 Around M, has high affinity binding characteristics. To further confirm the receptor profile of IgDR, ITC was first used to detect binding affinities between IgDR proteins and various immunoglobulin subtypes (IgD, igA, igE, igM, igG). Observing thermodynamic parameters (K) _D ΔG, ΔH, and-TΔS), the results indicate that: igDR and IgA, igE, igM and IgG binding K _D The larger values indicate that in addition to IgD, the affinity of several other immunoglobulin subtypes to IgDR is low. ITC experimental data show that the Fc domain of IgD can be specifically combined with IgDR, the ligand receptor is combined with higher affinity, and K _D Is 10 ^-12 M. The experimental results further demonstrate that IgDR binds IgD with high affinity and high specificity.

TABLE 1 thermodynamic parameters of affinity experiments

The protein-protein interaction of IgDR mutant protein and human IgD is obviously reduced, wherein the H29R/T32A/M34A/T39A site mutation leads to the most obvious reduction of the protein-protein interaction of IgDR and human IgD.

In view of the above experimental results, the IgDR H29R/T32A/M34A/T39A four-point mutant protein was further constructed, expressed and purified by using E.coli expression system, and compared with the K of the wild-type protein _D Comparing the values, the IgDR H29R/T32A/M34A/T39A four-point mutant protein and IgD combined K _D A value of 10 ^-3 Around M, the affinity was greatly reduced (table 1). The above results demonstrate that the four amino acid positions H29, T32, M34 and T39 of IgDR may be key binding sites for binding to the human IgD Fc domain.

Example 3: preparation of monoclonal antibodies and performance evaluation:

1. the experimental method comprises the following steps:

the purified IgDR protein is used as antigen for preparing IgDR monoclonal antibody, B cell and myeloma cell are fused by cell fusion technology to form hybridoma cell, hybridoma cell with antibody secretion function and unlimited proliferation is selected through screening and cloning, mouse ascites is prepared through intraperitoneal injection of hybridoma into BALB/c mouse, and finally high purity IgDR monoclonal antibody (IgDR mAb) is purified from ascites. And further using flow cytometry to evaluate the effect of IgDR mAb.

1.1 mouse immunization and antiserum detection:

immunization: 5 BALB/c mice (female, 6-8 weeks old) were selected, labeled and numbered, and the tail vein was collected with 30-50. Mu.l blood, and after coagulation, serum before immunization was collected by centrifugation. The required antigen volume was calculated at 200 μg per mouse immunization, antigen emulsification was performed using an equal volume of Freund's complete adjuvant, and immunization was performed by subcutaneous multipoint injection. Two weeks later, a second immunization was performed, the required antigen volume was calculated for 100 μg per mouse, antigen emulsification was performed using an equal volume of Freund's incomplete adjuvant, and immunization was performed by subcutaneous multipoint injection. Two weeks later, a third immunization was performed, and the required antigen volume was calculated for 100 μg per mouse, and antigen emulsification was performed using an equal volume of Freund's incomplete adjuvant, followed by subcutaneous multipoint injection. One week after the three-phase, the tail vein is used for blood collection of 30-50 μl, blood is collected by centrifugation after coagulation, and serum titer is detected by ELISA.

Detection titers: CBS is coated with IgDR antigen, the coating concentration is 1 mug/ml, 100 mug/hole, the blocking is carried out by antibody blocking liquid at 4 ℃ overnight, the plate is washed after blocking for 1h at room temperature, the initial dilution of mouse serum is 1:1000, the dilution is multiplied by the dilution, PBS is taken as negative control, the serum titer is detected by ELISA, and the OD450 reading is measured by an enzyme-labeling instrument.

1.2 cell fusion:

at least one week prior to fusion, mouse myeloma cells SP2/0 were prepared, cell status was adjusted to log phase, mice with highest ELISA titers were selected, and the mice were boosted 3 days prior to fusion, 50 μg/mouse, and injected intraperitoneally.

Fusion: taking spleen of a mouse, mechanically crushing, collecting spleen cells, filtering by a 200-mesh screen, and washing with PBS for 3 times; SP2/0 cells were collected and washed 3 times with PBS; after cell counting, the mixture was mixed at a ratio of SP2/0 to splenocyte=1:5, and after centrifugation, PBS was discarded and cell fusion was performed using PEG. At the end of the fusion, cells were centrifuged and HAT-containing complete medium was added, and the cells were resuspended and plated in 96-well plates.

Liquid replacement: half-changing was performed once 7 days after fusion.

1.3 hybridoma screening

ELISA detection: coating IgDR antigen, coating concentration 1 mug/ml, 00 mug/hole, absorbing cell culture supernatant 7 days after fusion for ELISA detection, recording hole with reading more than 0.5, marking and changing liquid.

ELISA rechecking: the positive wells subjected to liquid exchange were rechecked the next day, and clone numbers with stable titers were recorded.

1.4 establishment of stable cell lines:

first subcloning: from the previous step, 12 strains were selected for subcloning screening, complete medium, plated in 196 well plate according to limiting dilution method. After 7 days, observing under a microscope, marking a monoclonal hole, performing half liquid exchange once, performing ELISA detection on the next day, discarding the clone number converted into negative, and selecting the hole with vigorous growth for performing half liquid exchange once for positive clones.

Secondary subcloning: from the previous step 10 strains were selected for subcloning screening, plated in half 96-well plates at 1 cell per well. After 7 days, observing under a microscope, marking a monoclonal hole, performing half liquid exchange once, performing ELISA detection on the next day, subcloning until the ELISA detection positive rate is 100%, selecting the hole with vigorous growth for expansion culture, freezing and preserving the seed.

1.5 ascites preparation and antibody purification:

sensitization of mice: liquid paraffin sensitized BALB/c mice, preferably mice produced by mice, were injected at a volume of 500 ul/mouse. Ascites can be prepared by injecting hybridoma cells 10 days later.

Injecting cells: hybridoma cells are collected and washed twice by PBS, 100 to 150 ten thousand cells are taken and injected into the abdominal cavity of a mouse, the state of the mouse is inactive after one week, the abdominal cavity of the mouse is enlarged, the mouse is killed after the abdominal cavity of the mouse is obviously swelled after 10 days, ascites is taken out, and the titer of the ascites is detected by ELISA.

Purifying the antibody in ascites: the ascites fluid is affinity purified by Protein A, sampled and SDS-PAGE electrophoresis is used to verify purity, and an ultra-micro spectrophotometer is used to detect the concentration value of the antibody and record. The purified antibody is subjected to liquid exchange and concentration by an ultrafiltration tube, finally the liquid exchange is carried out in PBS buffer solution, and the sterile filtration is carried out by a 0.22 mu M pore-size filter membrane in an ultra-clean bench, and the product is preserved at the temperature of minus 20 ℃ for standby.

Antibody titer detection: CBS coats IgDR protein, the coating concentration is 1 mug/ml, 100 mul/hole, blocking is carried out by blocking liquid after 4 ℃ for night, the plate is washed after blocking for 1h at room temperature, the initial dilution of antibody is 1:2000, the dilution is multiplied by the ratio, PBS is negative control, ELISA detects the antibody titer, and an ELISA reader determines OD450 reading.

Antibody purity detection: SDS-PAGE detects antibody purity.

Concentration measurement: antibody concentration was determined using Nano-500.

1.6IgDR mAb Performance evaluation

(1) Collecting peripheral blood of a Rheumatoid Arthritis (RA) patient and a healthy control patient, obtaining PBMC cells in the peripheral blood of the healthy control patient and the RA patient by a density gradient centrifugation method, incubating the PBMC cells with BV421-CD4 flow antibody and IgDR monoclonal antibody for 50min on ice in the dark, incubating the PBMC cells with Alexa Fluor 488-sheep anti-mouse fluorescent secondary antibody for 30min on ice in the dark, and detecting CD4 of the healthy control patient and the RA patient by a flow cytometer ⁺ Expression of IgDR on T cells.

(2) Jurkat cells were incubated with different concentrations of IgD (0.03,0.1,0.3,1,3,10,30. Mu.g/mL) at 37℃with 5% CO ₂ After 24h of co-incubation, the IgDR expression differences on the two primary antibody-labeled Jurkat cells were detected and compared by flow cytometry using the fluorescent biotin-labeled IgDR and IgDR monoclonal antibodies, respectively, as primary antibody-labeled Jurkat cells.

2. Experimental results

BALB/c mice subcutaneous multipoint immune IgDR antigen, utilizing the spleen cells of mice to fuse with myeloma cells, HAT screening and culturing, coating antigen, ELISA screening positive clone, finally obtaining 5 hybridoma cell strains, injecting mice abdominal cavity through the hybridoma, inducing to generate mouse ascites, purifying the ascites to obtain monoclonal antibodies, determining the concentration of antibodies by Nano-500, determining the purity of antibodies by Coomassie brilliant blue method, detecting the antibody titer by ELISA method, successfully preparing IgDR mAb, the purity is more than 85%, and the concentration is adjusted to 2mg/ml.

Consistent with previous reports, igDR was reported in CD4 in peripheral blood of RA-patients and healthy controls ⁺ T cells were all significantly expressed and IgDR was expressed in RA patients significantly higher than in healthy controls (P<0.05 (fig. 4). Jurkat cells showed significant increases in IgDR expression (P) 24 hours after IgD (0.03, 0.1,0.3,1,3,10, 30. Mu.g/mL) stimulation<0.01). As a specific antibody for accurately characterizing, quantifying and locating IgDR, igDR monoclonal antibodies have higher specificity and sensitivity than biotin-labeled IgD (FIG. 5). The product can be further used for preparing in vitro detection/diagnosis kits, and can be used for accurately and qualitatively determining, quantifying, positioning and the like IgDR proteins, including but not limited to chemiluminescent immunoassay kits, enzyme-linked immunoassay kits, colloidal gold immunoassay kits and fluorescent immunoassay kits. In conclusion, the IgDR monoclonal antibody can be used for detecting IgDR expression on the surface of human cells by flow cytometry, detecting IgDR expression in cells or tissues by Western blot, detecting IgDR expression in tissues by immunohistochemistry, and applying various detection methods such as ELISA, chemiluminescence immunity or fluorescence immunity to the expression detection of IgDR in human samples.

The foregoing is a further detailed description of the application in connection with specific embodiments, and is not intended to limit the practice of the application to such descriptions. It will be apparent to those skilled in the art that several simple deductions or substitutions may be made without departing from the spirit of the application, and these should be considered to be within the scope of the application.

Sequence listing

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<213> Artificial sequence (Artificial Sequence)

<400> 5

cccaagctta tggactcctg gaccctctg 29

<210> 6

<211> 30

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 6

cgggatccgc taaactgctg gcacagaagt 30

<210> 7

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 7

gctagcatgt tgtccgagcc 20

<210> 8

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 8

tctgagctag ttgagcagag tccg 24

<210> 9

<211> 59

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 9

ttgtttaact ttaagaagga gatataccat gggtgtgatt cagagtccgc gccgtgaag 59

<210> 10

<211> 59

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 10

ccggatctca gtggtggtgg tggtggtgct cgagcagact actggcacaa aaataaact 59

Claims (2)

1. IgDR gene, its nucleotide sequence is shown in SEQ ID NO. 1.
2. 2. The use of the IgDR gene of claim 1 for preparing an IgDR monoclonal antibody.