CN111153996B - Antibody of G protein coupled receptor, preparation method thereof and G protein coupled receptor kit - Google Patents
Antibody of G protein coupled receptor, preparation method thereof and G protein coupled receptor kit Download PDFInfo
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- CN111153996B CN111153996B CN202010027428.0A CN202010027428A CN111153996B CN 111153996 B CN111153996 B CN 111153996B CN 202010027428 A CN202010027428 A CN 202010027428A CN 111153996 B CN111153996 B CN 111153996B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2869—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against hormone receptors
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones
- G01N2333/726—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH
Abstract
The invention relates to the technical field of biological detection reagents, in particular to an antibody of a G protein-coupled receptor, a preparation method thereof and a G protein-coupled receptor kit. An antibody of a G protein coupled receptor is prepared by coupling antigenic determinant polypeptide and hemocyanin to form a whole antigen and then preparing the whole antigen by a polyclonal antibody technology; the antigenic determinant polypeptide comprises a plurality of polypeptide immunogens which are modified by cysteine and arginine respectively. The antibody of the G protein coupled receptor provided by the invention can simultaneously identify G protein coupled receptors with similar homology, and a G protein coupled receptor kit produced by using the antibody can rapidly detect a cell-level G protein coupled receptor signal.
Description
Technical Field
The invention relates to the technical field of biological detection reagents, in particular to an antibody of a G protein-coupled receptor, a preparation method thereof and a G protein-coupled receptor kit.
Background
G protein-coupled receptors (GPCRs) are the most well known family of membrane receptors, which play important biological roles in physiopathological processes and are the major superfamily of drug targets. The main function is to mediate extracellular signals to enter the cell interior and participate in autocrine, paracrine and endocrine system signals which are a series of important reactions in the cell interior. Approximately 50% of small molecule therapeutics are targeted to GPCRs, whose ligands comprise a wide range of proteins, polypeptides, carbohydrates, lipids, and the like. For most GPCRs, external ligand binding to receptors within the membrane causes structural changes that activate several or hundreds of guanylate trimers within the membrane to bind G proteins, effecting signaling in a molecular conformational sense. At present, people have limited understanding on the structure and function of GPCRs, signal pathways, regulation mechanisms of the GPCRs and the like, more than half of GPCRs are orphan receptors with unknown ligands, and the biological functions of the GPCRs are not clear, so that the production of antibodies capable of detecting the GPCRs on a molecular level has wide scientific research value and economic prospect. The existing methods for detecting GPCRs generally adopt an antibody production process using polypeptides as immunogens, and aiming at the content change detection of a specific GPCR, the detection result has limitation, and downstream experiments can be carried out only by purchasing a large amount of antibodies or kits to detect the change of the GPCR. The kit adopts a high-degree homologous sequence in GPCR protein, synthesizes a plurality of polypeptide sequences for testing by predicting a plurality of antigenic determinants in order to ensure specific application, and finally adopts a polyclonal antibody preparation kit of mixed immunogen, so that the change of a corresponding signal path can be rapidly identified, and a convenient and rapid tool is provided for drug screening and target point research.
Disclosure of Invention
In order to overcome the defects of the technical defects, the invention designs an antibody of a G protein coupled receptor, a preparation method thereof and a G protein coupled receptor kit.
In order to achieve the above object, the present invention provides an antibody for a G protein-coupled receptor, which is characterized in that: the antigenic determinant polypeptide is coupled with hemocyanin to form a holoantigen, and then the holoantigen is prepared by a polyclonal antibody technology;
the antigenic determinant polypeptide comprises a plurality of polypeptide immunogens, and the polypeptide immunogens are respectively modified by cysteine and arginine.
Preferably, the amino acid sequence of the epitope polypeptide is:
FZD8/FZD5 CNHDTQDEAGLEVHQFWR；
GPR109/HCAR2 CLQRKMTGEPDNNRSTR；
OR10C1/OR10C1 CHLLTGRRHISRSR；
OR2A5/2A14 CKSRHPEEQQKVLR；
OR2AG1/2 CADFLRRENTISFR；
OR2T2/2T35 CHRMNSAEGRRKAFR；
OR2T2/2T35 CLQDLLSKDKTISFR；
OR4F4/4F5/4F17 CDFFSQRKVISFKR；
OR5AU1/OR5AU1 CLKMSSAQGRFKAFR；
OR1S1/1S2 CPSSTHPEDTDKIGR；
OR2A4/7 CVGPRYGNPKEQKR；
OR2AG1/2 CLADFLRRENTISFR；
OR2T2/35 CQDLLSKDKTISFR；
OR2T3/34 CLRSMMQSRMNQEKR；
OR4A4/47 CYTLRNSEMTSAMKKLR；
OR8B2/B3 CHIKSTQGRSKAFR；
OR11H1/11H2/11H12 CSLQNKEIKAALRKR；
OR13C2/13C9 CMKPKSKETLNSDR；
OR2T5/2T29 VNKVSAPECGMQMR；
OR10H3/10H4 CYLKPKGLHSMYSDR；
OR10K1/10K2 CDLLSQKKTISFLR；
OR1D4/5 CQMPSASKKYKTFR；
OR1D4/5 CLQMPSASKKYKTFSR；
OR3A2/3 CRLLSHKSTISYDR；
OR5M1/5M10 CMYVRPPSEKSVER；
OR10X1/OR10R3P CSAEGKQKAFR。
the key point of the preparation method of the antibody of the G protein coupled receptor is that the method comprises the following steps:
step one, selecting a plurality of polypeptide immunogens as antigen polypeptide groups of two or more G protein coupled receptors with similar homology, respectively modifying each polypeptide immunogen by adopting cysteine and arginine, and purifying to obtain antigenic determinant polypeptides;
step two, mixing the raw materials in a mass ratio of (3-8): (3-8): 1, hemocyanin and Sulfo-SMCC, purifying, adding Freund's adjuvant, emulsifying to form complete antigen;
thirdly, preparing antiserum from the whole antigen by using a polyclonal antibody technology;
and step four, pretreating the antiserum, mixing the antiserum with the antigenic determinant polypeptide and the coupling resin, loading the mixture into a chromatographic column, taking a glycine buffer solution with the pH value of 3.0 as an eluent, and collecting the eluent to obtain the antibody.
Preferably, the antiserum pretreatment in the fourth step is specifically as follows: mixing the serum to be processed in each batch, sequentially adding 1mol/L Tris.HCl buffer solution with pH7.5, 0.2 mol/L sodium orthovanadate solution and 0.1 mol/L sodium fluoride solution, uniformly mixing at room temperature, standing, and filtering for later use.
Preferably, the coupling resin in the fourth step is Sulfolink Gel, and the mass ratio of the antigenic determinant polypeptide to the coupling resin is 0.5: 1.
The key point of the G protein coupled receptor kit is that: antibodies and antibody dilutions comprising any of the above G protein-coupled receptors.
The invention has the beneficial effects that: the antibody of the G protein coupled receptor provided by the invention can simultaneously identify G protein coupled receptors with similar homology, and a G protein coupled receptor kit produced by using the antibody can rapidly detect a cell-level G protein coupled receptor signal.
Drawings
FIG. 1 is a graph showing the trend of GPCR signal changes produced by Hela cells stimulated by UV irradiation;
FIG. 2 is a graph showing the trend of the GPCR signals produced by Hela cells stimulated by TNF;
detailed description of the invention
In order to make the technical solutions of the present invention better understood, the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.
EXAMPLE 1 preparation of antibody I to G protein-coupled receptor
Step one, selecting the following polypeptide immunogens as antigen polypeptide groups of two or more G protein coupled receptors with similar homology, respectively modifying each polypeptide immunogen by adopting cysteine and arginine, and purifying to obtain antigenic determinant polypeptides;
the amino acid sequence of the antigenic determinant polypeptide is as follows:
FZD8/FZD5 CNHDTQDEAGLEVHQFWR；
GPR109/HCAR2 CLQRKMTGEPDNNRSTR；
OR10C1/OR10C1 CHLLTGRRHISRSR；
OR2A5/2A14 CKSRHPEEQQKVLR；
OR2AG1/2 CADFLRRENTISFR；
OR2T2/2T35 CHRMNSAEGRRKAFR；
OR2T2/2T35 CLQDLLSKDKTISFR；
OR4F4/4F5/4F17 CDFFSQRKVISFKR；
OR5AU1/OR5AU1 CLKMSSAQGRFKAFR；
OR1S1/1S2 CPSSTHPEDTDKIGR；
OR2A4/7 CVGPRYGNPKEQKR；
OR2AG1/2 CLADFLRRENTISFR；
OR2T2/35 CQDLLSKDKTISFR；
OR2T3/34 CLRSMMQSRMNQEKR。
coupling the antigenic determinant polypeptide, the hemocyanin and the Sulfo-SMCC, purifying, and adding Freund's adjuvant for emulsification to form a complete antigen;
thirdly, preparing antiserum from the whole antigen by using a polyclonal antibody technology;
the polyclonal antibody technique comprises the following steps: 1. rabbits were immunized and the basal immunization was injected subcutaneously with 8-10 sites, each of 0.1-0.2ml of antigen. And performing booster immunization according to a plan; the strengthening immunity time is the first strengthening after the 21 st day of basic immunity, the first strengthening immunity is carried out by shearing hairs on the right side of the back of the rabbit, the strengthening immunity is additionally injected with two muscle sites except for 5 points of subcutaneous immunity of the back, and each part is injected with 0.2-0.3 ml of antigen. Boosting for the second time at intervals of 21 days, boosting for the third time at intervals of 21 days, boosting for the fourth time at intervals of 21 days, and boosting for the fifth time at intervals of 35 days;
2. the positive serum is generally collected nine times, wherein the collection time is about 7 days after the third booster immunization (C), about 7 days after the fourth booster immunization (DI), about 14 days after the DI blood collection (DII), about 7 days after the fifth booster immunization (EI), about 14 days after the EI blood collection (EII), about 7 days after the sixth booster immunization (FI), 14 days after the FI blood collection (FII), about 7 days after the sixth booster immunization (GI), and if the positive serum is the venous immunization, the fourth day after the immunization and 14 days after the GI blood collection (GII);
3. marking the collected blood with a batch number, placing the blood at room temperature for one hour, centrifuging the blood at 3000rpm for 15min, transferring the upper serum into another clean centrifugal tube, labeling the blood, adding 2 wt% of sodium azide solution into the serum to be purified, diluting the blood to the concentration of 0.02 wt%, and placing the blood in a refrigerator at 4 ℃ for storage;
and step four, mixing the serum to be processed in each batch, sequentially adding 1mol/L Tris.HCl buffer solution with pH7.5, 0.2 mol/L sodium orthovanadate solution and 0.1 mol/L sodium fluoride solution, uniformly mixing and standing at room temperature, filtering, mixing with the epitope polypeptide and Sulfolink Gel in a mass ratio of 0.5:1, loading into a chromatographic column, and collecting eluent by using glycine buffer solution with pH3.0 as eluent to obtain the antibody.
EXAMPLE 2 preparation of antibody II of G protein-coupled receptor
The preparation method is the same as example 1, except that the amino acid sequence of the epitope polypeptide prepared in step one is:
OR2T2/2T35 CHRMNSAEGRRKAFR；
OR2T2/2T35 CLQDLLSKDKTISFR；
OR4F4/4F5/4F17 CDFFSQRKVISFKR；
OR5AU1/OR5AU1 CLKMSSAQGRFKAFR；
OR1S1/1S2 CPSSTHPEDTDKIGR；
OR2A4/7 CVGPRYGNPKEQKR；
OR2AG1/2 CLADFLRRENTISFR；
OR2T2/35 CQDLLSKDKTISFR；
OR2T3/34 CLRSMMQSRMNQEKR；
OR4A4/47 CYTLRNSEMTSAMKKLR；
OR8B2/B3 CHIKSTQGRSKAFR；
OR11H1/11H2/11H12 CSLQNKEIKAALRKR；
OR13C2/13C9 CMKPKSKETLNSDR；
OR2T5/2T29 VNKVSAPECGMQMR；
OR10H3/10H4 CYLKPKGLHSMYSDR；
OR10K1/10K2 CDLLSQKKTISFLR；
OR1D4/5 CQMPSASKKYKTFR；
OR1D4/5 CLQMPSASKKYKTFSR；
OR3A2/3 CRLLSHKSTISYDR；
OR5M1/5M10 CMYVRPPSEKSVER；
OR10X1/OR10R3P CSAEGKQKAFR。
in the second step, the mass ratio of the antigen determinant polypeptide to the hemocyanin to the Sulfo-SMCC is 8: 8: 1.
EXAMPLE 3 preparation of antibody III for G protein-coupled receptor
The preparation method is the same as example 1, except that the amino acid sequence of the epitope polypeptide prepared in step one is:
OR2A5/2A14 CKSRHPEEQQKVLR；
OR2AG1/2 CADFLRRENTISFR；
OR2T2/2T35 CHRMNSAEGRRKAFR；
OR2T2/2T35 CLQDLLSKDKTISFR；
OR4F4/4F5/4F17 CDFFSQRKVISFKR；
OR5AU1/OR5AU1 CLKMSSAQGRFKAFR；
OR1S1/1S2 CPSSTHPEDTDKIGR；
OR2A4/7 CVGPRYGNPKEQKR；
OR2AG1/2 CLADFLRRENTISFR；
OR2T2/35 CQDLLSKDKTISFR；
OR2T3/34 CLRSMMQSRMNQEKR；
OR4A4/47 CYTLRNSEMTSAMKKLR；
OR8B2/B3 CHIKSTQGRSKAFR；
OR11H1/11H2/11H12 CSLQNKEIKAALRKR；
OR13C2/13C9 CMKPKSKETLNSDR；
OR2T5/2T29 VNKVSAPECGMQMR；
OR10H3/10H4 CYLKPKGLHSMYSDR；
OR10K1/10K2 CDLLSQKKTISFLR；
OR1D4/5 CQMPSASKKYKTFR；
OR1D4/5 CLQMPSASKKYKTFSR；
OR3A2/3 CRLLSHKSTISYDR；
OR5M1/5M10 CMYVRPPSEKSVER；
OR10X1/OR10R3P CSAEGKQKAFR。
in the second step, the mass ratio of the antigen determinant polypeptide, the hemocyanin and the Sulfo-SMCC is 5: 5: 1.
EXAMPLE 4 preparation of antibody IV for G protein-coupled receptor
The preparation method is the same as example 1, except that the amino acid sequence of the epitope polypeptide prepared in step one is:
FZD8/FZD5 CNHDTQDEAGLEVHQFWR；
GPR109/HCAR2 CLQRKMTGEPDNNRSTR；
OR10C1/OR10C1 CHLLTGRRHISRSR；
OR2A5/2A14 CKSRHPEEQQKVLR；
OR2AG1/2 CADFLRRENTISFR；
OR2T2/2T35 CHRMNSAEGRRKAFR；
OR2T2/2T35 CLQDLLSKDKTISFR；
OR4F4/4F5/4F17 CDFFSQRKVISFKR；
OR5AU1/OR5AU1 CLKMSSAQGRFKAFR；
OR1S1/1S2 CPSSTHPEDTDKIGR；
OR2A4/7 CVGPRYGNPKEQKR；
OR2AG1/2 CLADFLRRENTISFR；
OR2T2/35 CQDLLSKDKTISFR；
OR2T3/34 CLRSMMQSRMNQEKR；
OR4A4/47 CYTLRNSEMTSAMKKLR；
OR8B2/B3 CHIKSTQGRSKAFR；
OR11H1/11H2/11H12 CSLQNKEIKAALRKR；
OR13C2/13C9 CMKPKSKETLNSDR；
OR2T5/2T29 VNKVSAPECGMQMR；
OR10H3/10H4 CYLKPKGLHSMYSDR；
OR10K1/10K2 CDLLSQKKTISFLR；
OR1D4/5 CQMPSASKKYKTFR；
OR1D4/5 CLQMPSASKKYKTFSR；
OR3A2/3 CRLLSHKSTISYDR；
OR5M1/5M10 CMYVRPPSEKSVER；
OR10X1/OR10R3P CSAEGKQKAFR。
in the second step, the mass ratio of the antigen determinant polypeptide to the hemocyanin to the Sulfo-SMCC is 4: 4: 1.
example 5A G protein-coupled receptor kit I
The kit comprises an antibody I of a G protein coupled receptor, an antibody diluent I, a cleaning solution, a quenching solution, a fixing solution, a membrane penetrating solution and a confining solution, wherein the antibody diluent comprises the following components in parts by mass: 0.5% Bovine Serum Albumin (BSA), 0.1% Triton X-100, 0.05% sodium azide solution and 99.35% PBS.
The formula of the cleaning solution is as follows: 8g of NaCl, 0.2g of KCL, 0.2g of KH2PO40.2g of Na2PO4.12H2O 2.85g of NaCl, and H2O 1L of NaCl;
the formula of the quenching liquid is as follows: 1mL of 30% H202, 29mL of PBS;
the formula of the stationary liquid is as follows: 10ug/mL PBS;
the formula of the membrane penetrating liquid is as follows: 4% wt formaldehyde, 96% wtPSS, suitable for adherent cells; 8% wt formaldehyde, 92% wtPSS, suitable for suspension cells;
the formula of the sealing liquid is as follows: 2% BSA, 96% wtPSS.
Example 6A G protein-coupled receptor kit II
The same as in example 5, except for including antibody II of G protein-coupled receptor and antibody diluent II, the antibody diluent II comprises the following components in mass fraction: 2% Bovine Serum Albumin (BSA), 1% Triton X-100, 0.2% sodium azide solution and 96.8% PBS.
Example 7A G protein-coupled receptor kit III
Same as example 5, except that antibody III of a G protein-coupled receptor and antibody diluent III were included, the antibody diluent III having the following composition in terms of mass fraction: 1.5% Bovine Serum Albumin (BSA), 1% Triton X-100, 0.1% sodium azide solution and 97.4% PBS.
Example 8A G protein-coupled receptor kit IV
Same as example 5, except that antibody IV of G protein-coupled receptor and antibody diluent IV were included, the antibody diluent IV was composed of the following components in mass fraction: 1% Bovine Serum Albumin (BSA), 0.5% Triton X-100, 0.05% sodium azide solution and 98.45% PBS.
The product performance of the G protein coupled receptor kit prepared by the invention is tested, taking the G protein coupled receptor kit IV as an example:
(1) detecting the change trend of GPCR signals generated by Hela cells stimulated by ultraviolet irradiation
And (3) performing conventional culture on the Hela cells, irradiating the cell culture holes by ultraviolet, inoculating the cells for culture every ten minutes, and inoculating the cells for 7 times to obtain the cell culture holes irradiated by the ultraviolet for 0-70 minutes. The medium was discarded and 200ul of wash solution was used to wash the cell culture wells three times for 5 minutes each time. 100ul of quench liquid per well was added for 20 minutes at room temperature. The cell culture wells were washed three times, 5 minutes each time, using 200ul of wash solution per well without quench. 100ul of fixative was added per well for 1.5 hours at room temperature. The fixative was discarded and the cell culture wells were washed three times 5 minutes each with 200ul of wash solution per well. 100ul of membrane-penetrating fluid was added to each well, and the reaction was carried out at room temperature for 20 minutes. Cell culture wells were washed three times for 5 minutes each time, with 200ul of fixative wash discarded per well. The mixture was added to a closed cell and the reaction was warmed for 1.5 hours. Blocking solution was discarded and 1:500 diluted GPCR antibody was added four degrees overnight. The GPCR antibody was discarded and 200ul of wash solution was used to wash the cell culture wells three times for 5 minutes each. Adding HRP labeled goat anti-rabbit secondary antibody for reaction at room temperature for 45 minutes. HRP-labeled secondary goat anti-rabbit antibodies were discarded and 200ul of wash solution was used to wash the cell culture wells three times for 5 minutes each time. 100ul of secondary substrate developing solution is added into each hole for 15 minutes at room temperature, 50ul of stopping solution is added to stop the reaction, and the result is shown in figure 1 after the reading of an enzyme-linked immunosorbent assay.
As can be seen in FIG. 1, the signal was activated in a large amount after the Hela cells were irradiated with UV light for 70 minutes.
(2) Using the kit to detect the change trend of GPCR signals generated by stimulating Hela cells by tumor necrosis factor
HeLa cells were routinely cultured by adding TNF 20ng/ml cell culture wells every ten minutes and seeding 7 times to give TNF-stimulated cells for 0-70 min. The medium was discarded and 200ul of wash solution was used to wash the cell culture wells three times for 5 minutes each time. 100ul of quench liquid per well was added for 20 minutes at room temperature. The cell culture wells were washed three times, 5 minutes each time, using 200ul of wash solution per well without quench. 100ul of fixative was added per well for 1.5 hours at room temperature. The fixative was discarded and the cell culture wells were washed three times 5 minutes each with 200ul of wash solution per well. 100ul of membrane-penetrating fluid was added to each well, and the reaction was carried out at room temperature for 20 minutes. Cell culture wells were washed three times for 5 minutes each time, with 200ul of fixative wash discarded per well. The mixture was added to a closed cell and the reaction was warmed for 1.5 hours. Blocking solution was discarded and 1:500 diluted GPCR antibody was added four degrees overnight. The GPCR antibody was discarded and 200ul of wash solution was used to wash the cell culture wells three times for 5 minutes each. Adding HRP labeled goat anti-rabbit secondary antibody for reaction at room temperature for 45 minutes. HRP-labeled secondary goat anti-rabbit antibodies were discarded and 200ul of wash solution was used to wash the cell culture wells three times for 5 minutes each time. 100ul of secondary substrate developing solution is added into each hole for 15 minutes at room temperature, 50ul of stopping solution is added to stop the reaction, and the result is shown in figure 2 after the reading of an enzyme-linked immunosorbent assay.
In FIG. 2, it can be seen that the signal started to activate after 30 minutes when the tumor necrosis factor stimulated Hela cells, and reached a peak after 40 minutes, after which the signal gradually decreased.
Finally, it should be noted that the above-mentioned description is only a preferred embodiment of the present invention, and those skilled in the art can make various similar representations without departing from the spirit and scope of the present invention.
Sequence listing
<110> Rui Ying Biotechnology Limited, Suzhou
<120> antibody of G protein-coupled receptor, preparation method thereof and G protein-coupled receptor kit
<140> 2020100274280
<141> 2020-01-10
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<400> 17
Cys Ser Leu Gln Asn Lys Glu Ile Lys Ala Ala Leu Arg Lys Arg
1 5 10 15
<210> 18
<211> 14
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 18
Cys Met Lys Pro Lys Ser Lys Glu Thr Leu Asn Ser Asp Arg
1 5 10
<210> 19
<211> 14
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 19
Val Asn Lys Val Ser Ala Pro Glu Cys Gly Met Gln Met Arg
1 5 10
<210> 20
<211> 15
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 20
Cys Tyr Leu Lys Pro Lys Gly Leu His Ser Met Tyr Ser Asp Arg
1 5 10 15
<210> 21
<211> 14
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 21
Cys Asp Leu Leu Ser Gln Lys Lys Thr Ile Ser Phe Leu Arg
1 5 10
<210> 22
<211> 14
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 22
Cys Gln Met Pro Ser Ala Ser Lys Lys Tyr Lys Thr Phe Arg
1 5 10
<210> 23
<211> 16
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 23
Cys Leu Gln Met Pro Ser Ala Ser Lys Lys Tyr Lys Thr Phe Ser Arg
1 5 10 15
<210> 24
<211> 14
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 24
Cys Arg Leu Leu Ser His Lys Ser Thr Ile Ser Tyr Asp Arg
1 5 10
<210> 25
<211> 14
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 25
Cys Met Tyr Val Arg Pro Pro Ser Glu Lys Ser Val Glu Arg
1 5 10
<210> 26
<211> 11
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 26
Cys Ser Ala Glu Gly Lys Gln Lys Ala Phe Arg
1 5 10
Claims (5)
1. A method for preparing an antibody of a G protein-coupled receptor, comprising the steps of:
step one, selecting a plurality of polypeptide immunogens as antigen polypeptide groups of two or more G protein coupled receptors with similar homology, modifying each polypeptide immunogen, and purifying to obtain antigenic determinant polypeptides;
the amino acid sequence of the antigenic determinant polypeptide is as follows:
FZD8/FZD5 CNHDTQDEAGLEVHQFWR；
GPR109/HCAR2 CLQRKMTGEPDNNRSTR；
OR10C1/OR10C1 CHLLTGRRHISRSR；
OR2A5/2A14 CKSRHPEEQQKVLR；
OR2AG1/2 CADFLRRENTISFR；
OR2T2/2T35 CHRMNSAEGRRKAFR；
OR2T2/2T35 CLQDLLSKDKTISFR；
OR4F4/4F5/4F17 CDFFSQRKVISFKR；
OR5AU1/OR5AU1 CLKMSSAQGRFKAFR；
OR1S1/1S2 CPSSTHPEDTDKIGR；
OR2A4/7 CVGPRYGNPKEQKR；
OR2AG1/2 CLADFLRRENTISFR；
OR2T2/35 CQDLLSKDKTISFR；
OR2T3/34 CLRSMMQSRMNQEKR；
OR4A4/47 CYTLRNSEMTSAMKKLR；
OR8B2/B3 CHIKSTQGRSKAFR；
OR11H1/11H2/11H12 CSLQNKEIKAALRKR；
OR13C2/13C9 CMKPKSKETLNSDR；
OR2T5/2T29 VNKVSAPECGMQMR；
OR10H3/10H4 CYLKPKGLHSMYSDR；
OR10K1/10K2 CDLLSQKKTISFLR；
OR1D4/5 CQMPSASKKYKTFR；
OR1D4/5 CLQMPSASKKYKTFSR；
OR3A2/3 CRLLSHKSTISYDR；
OR5M1/5M10 CMYVRPPSEKSVER；
OR10X1/OR10R3P CSAEGKQKAFR；
step two, mixing the raw materials in a mass ratio of (3-8): (3-8): 1, hemocyanin and Sulfo-SMCC, purifying, adding Freund's adjuvant, emulsifying to form complete antigen; the mass ratio of the antigenic determinant polypeptide to the hemocyanin is 1: 1;
thirdly, preparing antiserum from the whole antigen by using a polyclonal antibody technology;
and step four, pretreating the antiserum, mixing the antiserum with the antigenic determinant polypeptide and the coupling resin, loading the mixture into a chromatographic column, taking a glycine buffer solution with the pH value of 3.0 as an eluent, and collecting the eluent to obtain the antibody.
2. The method for preparing an antibody against a G protein-coupled receptor according to claim 1, wherein the antiserum pretreatment in the fourth step comprises: mixing the serum to be processed in each batch, sequentially adding 1mol/L Tris.HCl buffer solution with pH7.5, 0.2 mol/L sodium orthovanadate solution and 0.1 mol/L sodium fluoride solution, uniformly mixing at room temperature, standing, and filtering for later use.
3. The method for producing an antibody against a G protein-coupled receptor according to claim 2, wherein the coupling resin in step four is Sulfolink Gel, and the mass ratio of the epitope polypeptide to the coupling resin is 0.5: 1.
4. A G protein-coupled receptor kit, characterized in that: an antibody comprising a G protein-coupled receptor prepared according to any one of claims 1 to 3 and an antibody dilution.
5. The G protein-coupled receptor kit of claim 4, wherein: the antibody diluent comprises the following components in parts by mass: 0.5-2% BSA, 0.1-1% Triton X-100, 0.05-0.2% sodium azide solution and 96.8-99.35% PBS.
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