CN114605537B - Anti-gastrin releasing peptide antibody, detection reagent and kit - Google Patents

Anti-gastrin releasing peptide antibody, detection reagent and kit Download PDF

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CN114605537B
CN114605537B CN202011446251.4A CN202011446251A CN114605537B CN 114605537 B CN114605537 B CN 114605537B CN 202011446251 A CN202011446251 A CN 202011446251A CN 114605537 B CN114605537 B CN 114605537B
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antibody
cdr
gastrin
releasing peptide
functional fragment
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CN114605537A (en
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崔鹏
何志强
孟媛
钟冬梅
邓晨曦
陈晓倩
王晨
范凌云
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Dongguan Pengzhi Biotechnology Co Ltd
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Abstract

The invention discloses an antibody, a detection reagent and a kit for resisting gastrin-releasing peptide, and relates to the technical field of antibodies. The anti-gastrin releasing peptide antibody disclosed by the invention comprises a heavy chain complementarity determining region and a light chain complementarity determining region. The antibody disclosed by the invention has better specificity and sensitivity on the gastrin releasing peptide, and can be used for detecting the gastrin releasing peptide and diagnosing or assisting in diagnosing related diseases by taking the gastrin releasing peptide as a marker.

Description

Anti-gastrin releasing peptide antibody, detection reagent and kit
Technical Field
The invention relates to the technical field of antibodies, and particularly relates to an anti-gastrin-releasing peptide antibody, a detection reagent and a kit.
Background
Gastrin-releasing peptide (GRP) is a regulatory molecule that stimulates gastrin secretion from gastric G cells, participates in smooth muscle cell contraction, and promotes cell-cell interactions. ProGRP (pro-gastrin-releasing peptide) is a precursor of gastrin-releasing peptide, and has 3 variants with amino acid sequence lengths of 115aa, 118aa and 125aa, respectively, designated as subtype 1, subtype 2 and subtype 3, respectively, at a ratio of 60: 5: 35. The GRP gene encodes a preproprotein of 148 amino acids in humans, whose 148 preproproteins are further decomposed with dissociation of the signal peptide to form gastrin releasing peptide of 27 amino acids and ProGRP of 68 amino acids. Compared with the very short half-life of GRP in blood, the half-life of ProGRP is moderate, and the level of GRP and the expression of GRP gene can be reflected.
Fetal and neonatal bronchial epithelial endocrine cells GRPs are abundant, and adult cells are present only in brain, gastrointestinal nerve fibers and a small part of lung neuroendocrine cells at low levels. Besides renal insufficiency, pulmonary neuroendocrine tumors and medullary thyroid carcinoma, proGRP is rarely elevated in patients with other malignancies or benign diseases.
ProGRP has high sensitivity and specificity for Small Cell Lung Cancer (SCLC) diagnosis, and its level can reflect the therapeutic effect and judge whether the disease progresses or regresses. ProGRP has a better effect in monitoring the recurrence of SCLC patients. The level of ProGRP has high correlation with prognostic factors in patient prognostic evaluation, so that the prognostic evaluation and the therapeutic effect can be predicted earlier.
Due to economic cost and other factors, low-dose spiral CT is not suitable for large-scale lung cancer population screening. The detection of the tumor marker meets the clinical requirements of rapidness, no wound and economy. Since the detection of the SCLC marker in 1989, enzyme-linked immunosorbent assay (1995), chemiluminescence immunoassay (chemiluminescence immunoassay), electrochemiluminescence assay (2013), time-resolved immunofluorescence assay and other detection methods have been established for decades in ProGRP detection. The different methods have their own advantages and disadvantages, but all require specific antibodies against ProGRP. Good quality antibodies are critical for clinical detection of GRP. At present, monoclonal antibodies aiming at GRP have few sources, and have defects in sensitivity, affinity and specificity.
In view of this, the invention is particularly proposed.
Disclosure of Invention
The invention aims to provide an antibody, a detection reagent and a kit for resisting gastrin-releasing peptide, wherein the antibody has better specificity and sensitivity to the gastrin-releasing peptide, and can be used for detecting the gastrin-releasing peptide and diagnosing or assisting diagnosis of related diseases with the gastrin-releasing peptide as a marker.
The invention is realized by the following steps:
in one aspect, the present invention provides an antibody against gastrin releasing peptide, or a functional fragment thereof, having the following complementarity determining regions:
CDR-VH1: G-Y-X1-F-X2-D-Y-N-M-X3; wherein: x1 is R or K; x2 is S or T; x3 is N or D;
CDR-VH2: D-X1-N-P-X2-S-D-X3-T-X4-Y-N-Q-K-F-K-G; wherein: x1 is L or I; x2 is N or D; x3 is G, A or V; x4 is I or F;
CDR-VH3: X1-T-W-X2-H; wherein: x1 is S or T; x2 is I, V or L;
CDR-VL1: S-A-X1-Q-G-X2-S-N-Y-X3-S; wherein: x1 is S or T; x2 is I, V or L; x3 is I, V or L;
CDR-VL2: X1-T-S-R-X2-Y-S; wherein: x1 is H or Y; x2 is I, V or L;
CDR-VL3: X1-Q-Y-S-K-X2-P-W; wherein: x1 is Q or H; x2 is I, V or L.
The invention provides an antibody or a functional fragment thereof with the complementarity determining region structure, wherein the antibody can specifically bind to gastrin-releasing peptide and has better affinity to gastrin-releasing peptide antigen, and the antibody has better specificity and sensitivity when used for detecting gastrin-releasing peptide. Can be used for detecting gastrin releasing peptide and diagnosing or assisting diagnosis of related diseases using gastrin releasing peptide as a marker. The invention provides a more abundant antibody selection for detecting the gastrin releasing peptide.
In alternative embodiments, in CDR-VH1, X1 is R; in CDR-VH2, X1 is I; in CDR-VH3, X1 is S; in CDR-VL1, X1 is S; in CDR-VL3, X1 is H.
The present inventors have found that when the mutation site in each complementarity determining region is the amino acid residue, the antibody exhibits a better affinity for gastrin releasing peptide.
In an alternative embodiment, in CDR-VH1, X2 is S.
In an alternative embodiment, in CDR-VH1, X2 is T.
In an alternative embodiment, in CDR-VH1, X3 is N.
In an alternative embodiment, in CDR-VH1, X3 is D.
In an alternative embodiment, in CDR-VH2, X2 is N.
In an alternative embodiment, in CDR-VH2, X2 is D.
In an alternative embodiment, in CDR-VH2, X3 is G.
In an alternative embodiment, in CDR-VH2, X3 is a.
In an alternative embodiment, in CDR-VH2, X3 is V.
In an alternative embodiment, in CDR-VH2, X4 is I.
In an alternative embodiment, in CDR-VH2, X4 is F.
In an alternative embodiment, in CDR-VH3, X2 is I.
In an alternative embodiment, in CDR-VH3, X2 is V.
In an alternative embodiment, in CDR-VH3, X2 is L.
In an alternative embodiment, in CDR-VL1, X2 is I.
In an alternative embodiment, in CDR-VL1, X2 is V.
In an alternative embodiment, in CDR-VL1, X2 is L.
In an alternative embodiment, in CDR-VL1, X3 is I.
In an alternative embodiment, in CDR-VL1, X3 is V.
In an alternative embodiment, in CDR-VL1, X3 is L.
In an alternative embodiment, in CDR-VL2, X1 is H.
In an alternative embodiment, in CDR-VL2, X1 is Y.
In an alternative embodiment, in CDR-VL2, X2 is I.
In an alternative embodiment, in CDR-VL2, X2 is V.
In an alternative embodiment, in CDR-VL2, X2 is L.
In an alternative embodiment, in CDR-VL3, X2 is I.
In an alternative embodiment, in CDR-VL3, X2 is V.
In an alternative embodiment, in CDR-VL3, X2 is L.
In alternative embodiments, each complementarity determining region of the antibody or functional fragment thereof is selected from any one of the following combinations of mutations 1-47:
Figure BDA0002824496030000021
Figure BDA0002824496030000031
Figure BDA0002824496030000041
in alternative embodiments, the antibody or functional fragment thereof and gastrin-releasing peptide are represented by K D ≤3×10 - 7 Affinity binding in mol/L.
In an alternative embodiment, K D ≤2×10 -7 mol/L、K D ≤1×10 -7 mol/L、K D ≤9×10 -8 mol/L、K D ≤8×10 -8 mol/L、K D ≤7×10 -8 mol/L、K D ≤6×10 -8 mol/L、K D ≤5×10 -8 mol/L、K D ≤4×10 -8 mol/L、K D ≤3×10 -8 mol/L、K D ≤2×10 -8 mol/L、K D ≤1×10 -8 mol/L、K D ≤9×10 -9 mol/L、K D ≤8×10 - 9 mol/L、K D ≤7×10 -9 mol/L、K D ≤6×10 -9 mol/L、K D ≤5×10 -9 mol/L、K D ≤4×10 -9 mol/L、K D ≤3×10 -9 mol/L、K D ≤2×10 -9 mol/L or K D ≤1×10 -9 mol/L。
In an alternative embodiment, K D ≤7.7×10 -9 mol/L。
In an alternative embodiment, 1.22 × 10 -9 mol/L≤K D ≤7.73×10 -9 mol/L。
K D The detection of (c) is carried out with reference to the method in the example of the present invention.
In an alternative embodiment, in CDR-VH1, X1 is K; in CDR-VH2, X1 is L; in CDR-VH3, X1 is T; in CDR-VL1, X1 is T; in CDR-VL3, X1 is Q.
In alternative embodiments, each complementarity determining region of the antibody, or functional fragment thereof, is selected from any one of the following combinations of mutations 48-53:
Figure BDA0002824496030000042
in alternative embodiments, the antibody comprises the light chain framework regions FR1-L, FR2-L, FR3-L and FR4-L, as shown in sequence in SEQ ID NOS: 1-4, and/or the heavy chain framework regions FR1-H, FR2-H, FR3-H and FR4-H, as shown in sequence in SEQ ID NOS: 5-8.
In general, the variable regions of the heavy chain (VH) and light chain (VL) can be obtained by linking the CDRs and FRs numbered below in a combined arrangement as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
It is noted that in other embodiments, each framework region amino acid sequence of an antibody or functional fragment thereof provided herein can have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology to the corresponding framework region described above (SEQ ID NO:1, 2, 3, 4, 5, 6, 7, or 8).
In alternative embodiments, the antibody further comprises a constant region.
In alternative embodiments, the constant region is selected from the constant regions of any one of IgG1, igG2, igG3, igG4, igA, igM, igE, and IgD.
In alternative embodiments, the species of the constant region is derived from a cow, horse, dairy cow, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, goose, turkey, chicken fountains, or human.
In alternative embodiments, the constant region is derived from a mouse.
In alternative embodiments, the light chain constant region sequence of the constant region is set forth in SEQ ID NO. 9 and the heavy chain constant region sequence of the constant region is set forth in SEQ ID NO. 10.
In alternative embodiments, the functional fragment is selected from any one of VHH, F (ab ') 2, fab', fab, fv and scFv of the antibody.
Functional fragments of the above antibodies typically have the same binding specificity as the antibody from which they are derived. It will be readily understood by those skilled in the art from the disclosure of the present invention that functional fragments of the above antibodies can be obtained by methods such as enzymatic digestion (including pepsin or papain) and/or by chemical reduction cleavage of disulfide bonds. Based on the disclosure of the structure of the intact antibody, the above-described functional fragments are readily available to those skilled in the art.
Functional fragments of the above antibodies can also be obtained by recombinant genetic techniques also known to those skilled in the art or synthesized by, for example, automated peptide synthesizers, such as those sold by Applied BioSystems and the like.
In another aspect, the present invention provides a reagent or kit for detecting gastrin-releasing peptide, comprising the antibody or functional fragment thereof according to any one of the above.
In an alternative embodiment, the antibody or functional fragment thereof in the above reagent or kit is labeled with a detectable label.
Detectable labels are substances having properties, such as luminescence, color development, radioactivity, etc., which can be observed directly by the naked eye or detected by an instrument, by which qualitative or quantitative detection of the respective target substance can be achieved.
In alternative embodiments, the detectable labels include, but are not limited to, fluorescent dyes, enzymes that catalyze the development of a substrate, radioisotopes, chemiluminescent reagents, and nanoparticle-based labels.
In the actual use process, one skilled in the art can select a suitable marker according to the detection condition or actual requirement, and whatever marker is used belongs to the protection scope of the present invention.
In alternative embodiments, the fluorescent dyes include, but are not limited to, fluorescein-based dyes and derivatives thereof (e.g., including, but not limited to, fluorescein Isothiocyanate (FITC) hydroxyphoton (FAM), tetrachlorofluorescein (TET), etc. or analogs thereof), rhodamine-based dyes and derivatives thereof (e.g., including, but not limited to, rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc. or analogs thereof), cy-series dyes and derivatives thereof (e.g., including, but not limited to, cy2, cy3B, cy3.5, cy5, cy5.5, cy3, etc. or analogs thereof), alexa-series dyes and derivatives thereof (e.g., including, but not limited to, alexa fluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, chlorophyll, 700, 750, etc. or analogs thereof), and protein-based dyes and derivatives thereof (e.g., including, but not limited to, phycoerythrin (PE), phycocyanin (PC), allophycocyanin (polymetaxanthin), polymetaxanthin-phycoerythrin-protein (cp), etc.).
In alternative embodiments, the enzyme that catalyzes the development of a substrate includes, but is not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate deoxyenzyme.
In alternative embodimentsThe radioactive isotope includes but is not limited to 212 Bi、 131 I、 111 In、 90 Y、 186 Re、 211 At、 125 I、 188 Re、 153 Sm、 213 Bi、 32 P、 94 mTc、 99 mTc、 203 Pb、 67 Ga、 68 Ga、 43 Sc、 47 Sc、 110 mIn、 97 Ru、 62 Cu、 64 Cu、 67 Cu、 68 Cu、 86 Y、 88 Y、 121 Sn、 161 Tb、 166 Ho、 105 Rh、 177 Lu、 172 Lu and 18 F。
in alternative embodiments, the chemiluminescent reagent includes, but is not limited to, luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, bipyridyl ruthenium and its derivatives, acridinium ester and its derivatives, dioxane and its derivatives, lotrine and its derivatives, and peroxyoxalate and its derivatives.
In alternative embodiments, the nanoparticle-based labels include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.
In alternative embodiments, the colloid includes, but is not limited to, colloidal metals, disperse dyes, dye-labeled microspheres, and latex.
In alternative embodiments, the colloidal metals include, but are not limited to, colloidal gold, colloidal silver, and colloidal selenium.
In another aspect, the present invention provides the use of the antibody or the functional fragment thereof as described above in the preparation of a reagent or a kit for the diagnosis or the auxiliary diagnosis of a disease related to gastrin-releasing peptide as a marker.
The antibody or functional fragment thereof provided by the invention can be used for diagnosis or auxiliary diagnosis of any related diseases (such as renal insufficiency, lung neuroendocrine tumor, medullary thyroid cancer and the like) taking gastrin releasing peptide as a marker.
In another aspect, the present invention provides a nucleic acid molecule encoding the above antibody or a functional fragment thereof.
In another aspect, the invention provides a vector comprising the nucleic acid molecule described above.
In another aspect, the present invention provides a recombinant cell comprising the vector described above.
In another aspect, the present invention provides a method of preparing an antibody or functional fragment thereof, comprising: culturing the recombinant cell as described above, and separating and purifying the antibody or functional fragment thereof from the culture product.
Based on the disclosure of the amino acid sequence of the antibody or its functional fragment, it is easy for those skilled in the art to think that the antibody or its functional fragment can be prepared by genetic engineering techniques or other techniques (chemical synthesis, hybridoma cells), for example, by separating and purifying the antibody or its functional fragment from the culture product of recombinant cells capable of recombinantly expressing the antibody or its functional fragment as described above, and this is within the scope of the present invention, regardless of the technique used to prepare the antibody or its functional fragment.
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In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is a result of reducing SDS-PAGE of the anti-gastrin-releasing peptide antibody of example 1.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are conventional products which are not indicated by manufacturers and are commercially available.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the formulations or unit dosages herein, some are now described. Unless otherwise indicated, the techniques employed or contemplated herein are standard methods. The materials, methods, and examples are illustrative only and not intended to be limiting.
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry and immunology, which are within the skill of the art. Such techniques are well explained in the literature, e.g. "molecular cloning: laboratory Manual (Molecular Cloning: A Laboratory Manual), second edition (Sambrook et al, 1989); oligonucleotide Synthesis (oligo Synthesis) (eds. M.j. Goal, 1984); animal Cell Culture (Animal Cell Culture) (edited by r.i. freshney, 1987); methods in Enzymology (Methods in Enzymology), academic Press, inc. (Academic Press, inc.), "Handbook of Experimental Immunology" ("D.M.Weir and C.C.Black well"), gene Transfer Vectors for Mammalian Cells (J.M.Miller and M.P.Calos.), "Current Protocols in Molecular Biology" (F.M.Ausubel et al., 1987), "PCR, polymerase Chain Reaction (PCR: the Polymerase Chain Reaction) (Mullis et al., 1994), and" Current Protocols in Immunology "(blood), each of which is incorporated herein by reference, cold, 1991.
The features and properties of the present invention are described in further detail below with reference to examples.
Examples restriction enzymes, prime Star DNA polymerase was purchased from Takara. MagExtractor-RNA extraction kit was purchased from TOYOBO. BD SMART TM RACE cDNA Amplification Kit was purchased from Takara. The pMD-18T vector was purchased from Takara. Plasmid extraction kits were purchased from Tiangen corporation. Primer synthesis and gene sequencing were done by Invitrogen.
Example 1
1 construction of recombinant plasmid
(1) Antibody Gene preparation
mRNA is extracted from a hybridoma cell strain secreting an anti-gastrin releasing peptide antibody, a DNA product is obtained by an RT-PCR method, the product is added with A by rTaq DNA polymerase for reaction and then inserted into a pMD-18T vector, the product is transformed into DH5 alpha competent cells, after colonies are grown out, 4 clones of the Heavy Chain and Light Chain gene clones are respectively taken and sent to a gene sequencing company for sequencing.
(2) Sequence analysis of antibody variable region genes
Putting the gene sequence obtained by sequencing in an IMGT antibody database for analysis, and analyzing by using VNTI11.5 software to determine that the genes amplified by the heavy Chain primer pair and the Light Chain primer pair are correct, wherein in the gene fragment amplified by the Light Chain, the VL gene sequence is 324bp, belongs to VkII gene family, and a leader peptide sequence of 57bp is arranged in front of the VL gene sequence; in the gene fragment amplified by the Heavy Chain primer pair, the VH gene sequence is 336bp, belongs to a VH1 gene family, and has a leader peptide sequence of 57bp in front.
(3) Construction of recombinant antibody expression plasmid
pcDNA TM 3.4
Figure BDA0002824496030000071
vector is a constructed recombinant antibody eukaryotic expression vector, and multiple cloning enzyme cutting sites such as HindIII, bamHI, ecoRI and the like are introduced into the expression vector and named as pcDNA3.4A expression vector, and the vector is called as 3.4A expression vector for short in the following; according to the sequencing result of the antibody variable region gene in the pMD-18T, VL and VH gene specific primers of the antibody are designed, two ends of the VL and VH gene specific primers are respectively provided with HindIII and EcoRI enzyme cutting sites and protective bases, and 0.73KB L is amplified by a PCR amplification methodAn ight Chain gene fragment and a 1.43kb Heavy Chain gene fragment.
The gene fragments of the Heavy Chain and the Light Chain are subjected to double enzyme digestion by HindIII/EcoRI respectively, the 3.4A vector is subjected to double enzyme digestion by HindIII/EcoRI, the gene of the Heavy Chain and the gene of the Light Chain are respectively connected into the 3.4A expression vector after the fragments and the vector are purified and recovered, and recombinant expression plasmids of the Heavy Chain and the Light Chain are respectively obtained.
2 Stable cell line selection
(1) Transient transfection of recombinant antibody expression plasmid into CHO cells and determination of expression plasmid activity
Plasmid was diluted to 40 ug/100. Mu.L with ultrapure water and CHO cells were conditioned at 1.43X 10 7 cells/ml are put into a centrifuge tube, 100 mu L of plasmid and 700 mu L of cells are mixed, transferred into an electric rotating cup, electrically rotated, sampled and counted on days 3, 5 and 7, and sampled and detected on day 7.
Diluting GRP antigen to 1 μ g/ml with coating solution, 100 μ L per well, and standing overnight at 4 deg.C; the next day, washing with the washing solution for 2 times, and patting dry; add blocking solution (20% BSA +80% PBS), 120 μ L per well, 37 deg.C, 1h, pat dry; adding diluted cell supernatant at a concentration of 100 μ L/well at 37 deg.C for 30min (partial supernatant for 1 h); washing with the washing solution for 5 times, and drying; adding goat anti-mouse IgG-HRP (goat anti-mouse IgG-HRP) with the concentration of 100 mu L per well at 37 ℃ for 30min; washing with washing solution for 5 times, and drying; adding color development liquid A (50 muL/hole, containing citric acid + sodium acetate + acetanilide + carbamide peroxide), adding color development liquid B (50 muL/hole, containing citric acid + EDTA & 2Na + TMB + concentrated HCl), 10min; adding stop solution (50 μ L/well, EDTA-2 Na + concentrated H) 2 SO 4 ) (ii) a OD readings were taken at 450nm (reference 630 nm) on the microplate reader. The results show that the reaction OD after the cell supernatant is diluted 1000 times is still larger than 1.0, and the reaction OD of the wells without the cell supernatant is smaller than 0.1, which indicates that the antibody generated after the plasmid is transiently transformed has activity to GRP antigen.
(2) Linearization of recombinant antibody expression plasmids
The following reagents were prepared: 50 mu L of Buffer, 100 mu g/tube of DNA, 10 mu L of PuvI enzyme and sterile water are supplemented to 500 mu L, and the mixture is subjected to enzyme digestion in water bath at 37 ℃ overnight; extraction was performed sequentially with equal volumes of phenol/chloroform/isoamyl alcohol (lower layer) 25, followed by chloroform (aqueous phase); precipitating with 0.1 times volume (water phase) of 3M sodium acetate and 2 times volume of ethanol on ice, rinsing with 70% ethanol, removing organic solvent, re-melting with appropriate amount of sterilized water after ethanol is completely volatilized, and finally measuring concentration.
(3) Stable transfection of recombinant antibody expression plasmid, pressurized screening of stable cell lines
Plasmid was diluted to 40 ug/100. Mu.L with ultrapure water and CHO cells were conditioned at 1.43X 10 7 cells/ml are put into a centrifuge tube, 100 mu L of plasmid is mixed with 700 mu L of cells, and the mixture is transferred into an electric rotating cup and is electrically rotated, and the next day is counted; 25umol/L MSX 96-well pressure culture for about 25 days.
Observing the marked clone holes with cells under a microscope, and recording the confluence degree; taking culture supernatant, and carrying out sample detection; selecting cell strains with high antibody concentration and relative concentration, transferring the cell strains into 24 holes, and transferring the cell strains into 6 holes after 3 days; after 3 days, the seeds are preserved and cultured in batches, and the cell density is adjusted to be 0.5 multiplied by 10 6 cells/ml,2.2ml, batch culture, cell density 0.3X 10 6 cells/ml,2ml for seed preservation; and (4) 7 days, carrying out batch culture supernatant sample sending detection in 6 holes, and selecting cell strains with small antibody concentration and cell diameter to transfer TPP for seed preservation and passage.
3 recombinant antibody production
(1) Cell expanding culture
After the cell recovery, the cells were first cultured in 125ml size shake flasks, inoculated with 30ml Dynamis medium at 100% volume, and placed in a shaker at a rotation speed of 120r/min, a temperature of 37 ℃ and a carbon dioxide content of 8%. Culturing for 72h, inoculating and expanding at inoculation density of 50 ten thousand cells/ml, and calculating the expanding volume according to production requirements, wherein the culture medium accounts for 100 percent. Then the culture is expanded every 72 h. When the cell amount meets the production requirement, the production is carried out by strictly controlling the inoculation density to be about 50 ten thousand cells/ml.
(2) Shake flask production and purification
Shake flask parameters: the rotating speed is 120r/min, the temperature is 37 ℃, and the carbon dioxide is 8 percent. Feeding in a flowing manner: daily feeding was started when the culture was carried out for 72h in a shake flask, 3% of the initial culture volume was fed daily to HyCloneTM Cell BoostTM Feed 7a, and one thousandth of the initial culture volume was fed daily to Feed 7b, up to day 12 (day 12 feeding). Glucose was supplemented with 3g/L on the sixth day. Samples were collected on day 13. Affinity purification was performed using a proteinA affinity column. Mu.g of the purified antibody was subjected to reducing SDS-PAGE, and 4. Mu.g of an external control antibody was used as a control, and the electrophoretogram showed two bands, 1 of which Mr was 50KD (heavy chain, SEQ ID NO: 14) and the other Mr was 28KD (light chain, SEQ ID NO: 13), as shown in FIG. 1 below, after the reducing SDS-PAGE.
Example 2
Detection of antibody Performance
(1) Example 1 Activity assay of antibodies and mutants thereof
Analyzing the sequence of the antibody (WT) of example 1, wherein the heavy chain variable region is represented by SEQ ID NO:12, wherein the amino acid sequence of each complementarity determining region in the heavy chain variable region is as follows:
CDR-VH1：G-Y-K(X1)-F-S(X2)-D-Y-N-M-D(X3)；
CDR-VH2：D-L(X1)-N-P-N(X2)-S-D-A(X3)-T-I(X4)-Y-N-Q-K-F-K-G；
CDR-VH3：T(X1)-T-W-I(X2)-H；
the light chain variable region is shown as SEQ ID NO. 11, wherein the amino acid sequences of the complementarity determining regions on the light chain variable region are as follows:
CDR1-VL：S-A-T(X1)-Q-G-V(X2)-S-N-Y-I(X3)-S；
CDR-VL2：H(X1)-T-S-R-V(X2)-Y-S；
CDR-VL3：Q(X1)-Q-Y-S-K-I(X2)-P-W。
based on the anti-gastrin releasing peptide antibody (WT) of example 1, a mutation was made in the complementarity determining region at a site involved in the activity of the antibody, wherein each of X1, X2, X3, and X4 was a mutation site. See table 1 below.
TABLE 1 mutant sites associated with antibody Activity
Figure BDA0002824496030000081
Antibody binding activity assay in table 1:
coating liquid (main component NaHCO) 3 ) Diluting GRP antigen to 1 μ g/ml for coating with 100 μ l/well at 4 deg.C overnight; the next day, washing with the washing solution for 2 times, and patting dry; addition of blocking solution (20% BSA + 80%%) Patting to dry at 37 ℃ for 1h and 120 mu l per hole; adding diluted GRP monoclonal antibody in 100 μ l/hole at 37 deg.c for 30-60 min; washing with washing solution for 5 times, and drying; adding goat anti-mouse IgG-HRP (goat anti-mouse IgG-HRP) with the concentration of 100 mu l per well at 37 ℃ for 30min; washing with washing solution (PBS) for 5 times, and drying; adding color development liquid A (50 μ L/well containing 2.1g/L citric acid, 12.25g/L citric acid, 0.07g/L acetanilide and 0.5g/L carbamide peroxide) and adding color development liquid B (50 μ L/well containing 1.05g/L citric acid, 0.186g/L LEDTA.2Na, 0.45g/L TMB and 0.2ml/L concentrated HCl) for 10min; stop solution (50. Mu.l/well, containing 0.75 g/EDTA-2 Na and 10.2ml/L concentrated H) was added 2 SO 4 ) (ii) a OD readings were taken at 450nm (reference 630 nm) on the microplate reader. The results are shown in Table 2 below.
TABLE 2 Activity data of WT antibodies and mutants thereof
Antibody concentration (ng/ml) 37.50 18.75 9.38 4.69 2.34 0.00
WT 1.311 0.924 0.410 0.220 0.023 0.123
Mutation 1 2.027 1.376 0.791 0.448 0.222 0.019
Mutation 2 1.978 1.326 0.740 0.493 0.199 0.019
Mutation 3 1.951 1.36 0.681 0.479 0.182 0.017
Mutation 4 0.246 0.125 - - - -
Mutation 5 0.283 0.193 - - - -
Mutation 6 0.279 0.182 - - - -
According to the results in Table 2, the mutant 4-mutant 6 antibody had substantially no binding activity, while the WT and mutant 1-mutant 3 antibodies had better binding activity, with the mutant 1 antibody having the highest binding activity.
(2) Affinity detection of antibodies and mutants thereof
(a) Based on mutation 1, other sites were mutated, and the sequence of each mutation is shown in table 3 below.
TABLE 3 mutation sites related to antibody affinity
Figure BDA0002824496030000091
Figure BDA0002824496030000101
Affinity assay
Using AMC sensors, purified antibody was diluted to 10ug/mL with PBST, and GRP antigen (0.699 mg/mL) was diluted with PBST in a gradient: 1.8. Mu.g/mL, 0.9. Mu.g/mL, 0.45. Mu.g/mL, 0.23. Mu.g/mL, 0.11. Mu.g/mL, and 0.056. Mu.g/mL.
The operation flow is as follows: equilibration for 60s in buffer 1 (PBST), immobilization of antibody in antibody solution for 300s, incubation in buffer 2 (PBST) for 180s, antigen solubilizationThe binding time in the solution was 420s, the dissociation time in buffer 2 was 1200s, and the sensor was regenerated using 10mM pH 1.69GLY solution and buffer 3, and the data was output. The results are given in Table 4 below. K D Represents the equilibrium dissociation constant, i.e., affinity; kon denotes the binding rate; kdis denotes the off-rate.
Table 4 affinity assay data
Figure BDA0002824496030000102
Figure BDA0002824496030000111
From the results in table 4, it can be seen that both mutation 1 and its series of mutants have higher affinity for GRP antigen, indicating that the antibodies obtained by mutation in the manner of the mutations in table 3 have higher affinity based on mutation 1.
(b) Based on WT, mutation is carried out on other sites, and the affinity of each mutant is detected, wherein the sequence of each mutation is shown in Table 5, and the corresponding affinity data is shown in Table 6.
TABLE 5 mutations with WT as backbone
Figure BDA0002824496030000121
TABLE 6 affinity assay results for WT antibodies and their mutants
K D (M) kon(1/Ms) kdis(1/s)
WT 1.53E-07 3.60E+04 5.50E-03
WT 1 3.06E-07 1.93E+04 5.91E-03
WT 2 1.57E-07 3.93E+04 6.18E-03
WT 3 1.13E-07 3.56E+04 4.03E-03
WT 4 1.14E-07 3.96E+04 4.52E-03
WT 5 1.18E-07 3.03E+04 3.59E-03
From the results in Table 6, it can be seen that WT and its series of mutants also have good affinity, and it is demonstrated that the antibodies obtained by mutating WT in accordance with the mutation pattern in Table 5 also have good affinity.
(3) Evaluation of stability against naked antibody
Placing the antibody in a temperature range of 4 ℃ (refrigerator), -80 ℃ (refrigerator) and 37 ℃ (thermostat) for 21 days, taking samples in 7 days, 14 days and 21 days for state observation, and performing activity detection on the samples in 21 days, wherein the result shows that under three examination conditions, no obvious protein state change is seen in 21 days of placing the antibody, and the activity does not show a descending trend along with the rise of the examination temperature, which indicates that the antibody is stable. The following table 7 shows the results of the OD detection of the 21-day assessment of the antibody to mutation 1.
TABLE 7
Sample concentration (ng/ml) 30 8 0
Samples at 4 ℃ for 21 days 1.853 0.666 0.026
21 days samples at-80 deg.C 1.783 0.637 0.023
21 day samples at 37 deg.C 1.842 0.562 0.027
(4) Evaluation of application Performance
The antibodies were subjected to a double antibody sandwich pairing assay, detected on a chemiluminescent platform, and paired with another strain of ProGRP antibody (available from the fenpeng organism). The detection linear range of the mutation 1 and the series antibodies thereof is 3-5000pg/mL, the detection correlation under different antigen concentrations can reach more than 0.96, the sensitivity can reach 1.3-1.5pg/mL, and the method has good linearity and sensitivity.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Sequence listing
<110> Dongguan City of Pengzhi Biotech Co., ltd
<120> anti-gastrin releasing peptide antibody, detection reagent and kit
<160> 14
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Asp Ile Gln Met Thr Gln Thr Ala Ser Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Asp Arg Val Thr Ile Ser Cys
20
<210> 2
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<212> PRT
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Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile His
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Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser
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Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys Arg
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Glu Val Leu Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
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Ser Val Lys Ile Ser Cys Lys Ala Ser
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Trp Val Arg Gln Asn His Gly Lys Ser Leu Glu Trp Ile Gly
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Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Glu
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Leu Arg Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys
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Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala
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Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln
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Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr
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Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln
35 40 45
Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr
50 55 60
Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg
65 70 75 80
His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro
85 90 95
Ile Val Lys Ser Phe Asn Arg Asn Glu Cys
100 105
<210> 10
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Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala
1 5 10 15
Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu
50 55 60
Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val
65 70 75 80
Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys
85 90 95
Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro
100 105 110
Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu
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Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser
130 135 140
Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu
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Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr
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Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn
180 185 190
Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro
195 200 205
Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln
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Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val
225 230 235 240
Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val
245 250 255
Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln
260 265 270
Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn
275 280 285
Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val
290 295 300
Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His
305 310 315 320
Ser Pro Gly Lys
<210> 11
<211> 108
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<213> Artificial sequence
<400> 11
Asp Ile Gln Met Thr Gln Thr Ala Ser Ser Leu Ser Ala Ser Leu Gly
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Asp Arg Val Thr Ile Ser Cys Ser Ala Thr Gln Gly Val Ser Asn Tyr
20 25 30
Ile Ser Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile
35 40 45
His His Thr Ser Arg Val Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Pro
65 70 75 80
Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Lys Ile Pro Trp
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys Arg
100 105
<210> 12
<211> 112
<212> PRT
<213> Artificial sequence
<400> 12
Glu Val Leu Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Lys Phe Ser Asp Tyr
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Asn Met Asp Trp Val Arg Gln Asn His Gly Lys Ser Leu Glu Trp Ile
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Gly Asp Leu Asn Pro Asn Ser Asp Ala Thr Ile Tyr Asn Gln Lys Phe
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Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
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Thr Thr Trp Ile His Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala
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<210> 13
<211> 214
<212> PRT
<213> Artificial sequence
<400> 13
Asp Ile Gln Met Thr Gln Thr Ala Ser Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Asp Arg Val Thr Ile Ser Cys Ser Ala Thr Gln Gly Val Ser Asn Tyr
20 25 30
Ile Ser Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile
35 40 45
His His Thr Ser Arg Val Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Pro
65 70 75 80
Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Lys Ile Pro Trp
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys Arg Ala Asp Ala Ala
100 105 110
Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly
115 120 125
Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile
130 135 140
Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu
145 150 155 160
Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser
165 170 175
Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr
180 185 190
Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser
195 200 205
Phe Asn Arg Asn Glu Cys
210
<210> 14
<211> 436
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<213> Artificial sequence
<400> 14
Glu Val Leu Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Lys Phe Ser Asp Tyr
20 25 30
Asn Met Asp Trp Val Arg Gln Asn His Gly Lys Ser Leu Glu Trp Ile
35 40 45
Gly Asp Leu Asn Pro Asn Ser Asp Ala Thr Ile Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Thr Thr Trp Ile His Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala
100 105 110
Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala
115 120 125
Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr
130 135 140
Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser
145 150 155 160
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu
165 170 175
Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val
180 185 190
Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys
195 200 205
Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro
210 215 220
Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu
225 230 235 240
Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser
245 250 255
Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu
260 265 270
Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr
275 280 285
Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn
290 295 300
Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro
305 310 315 320
Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln
325 330 335
Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val
340 345 350
Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val
355 360 365
Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln
370 375 380
Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn
385 390 395 400
Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val
405 410 415
Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His
420 425 430
Ser Pro Gly Lys
435

Claims (26)

1. An antibody or functional fragment thereof against gastrin-releasing peptide, comprising the complementarity determining regions: CDR-VH1: G-Y-X1-F-X2-D-Y-N-M-X3; wherein: x1 is R;
CDR-VH2: D-X1-N-P-X2-S-D-X3-T-X4-Y-N-Q-K-F-K-G; wherein: x1 is I;
CDR-VH3: X1-T-W-X2-H; wherein: x1 is S;
CDR-VL1: S-A-X1-Q-G-X2-S-N-Y-X3-S; wherein: x1 is S;
CDR-VL2：X1-T-S-R-X2-Y-S；
CDR-VL3: X1-Q-Y-S-K-X2-P-W; wherein: x1 is H;
each complementarity determining region of the antibody or functional fragment thereof is selected from any one of the following mutation combinations 1-47:
Figure FDA0004023402980000011
Figure FDA0004023402980000021
2. an antibody or functional fragment thereof against gastrin-releasing peptide, comprising the complementarity determining regions: CDR-VH1: G-Y-X1-F-X2-D-Y-N-M-X3; wherein: x1 is K;
CDR-VH2: D-X1-N-P-X2-S-D-X3-T-X4-Y-N-Q-K-F-K-G; wherein: x1 is L;
CDR-VH3: X1-T-W-X2-H; wherein: x1 is T;
CDR-VL1: S-A-X1-Q-G-X2-S-N-Y-X3-S; wherein: x1 is T;
CDR-VL2：X1-T-S-R-X2-Y-S；
CDR-VL3: X1-Q-Y-S-K-X2-P-W; wherein: x1 is Q;
each complementarity determining region of the antibody or functional fragment thereof is selected from any one of the following combinations of mutations:
Figure FDA0004023402980000022
3. an antibody or functional fragment thereof against gastrin-releasing peptide, comprising the following complementarity determining regions: CDR-VH1: G-Y-X1-F-X2-D-Y-N-M-X3; wherein: x1 is R;
CDR-VH2: D-X1-N-P-X2-S-D-X3-T-X4-Y-N-Q-K-F-K-G; wherein: x1 is I;
CDR-VH3: X1-T-W-X2-H; wherein: x1 is S;
CDR-VL1: S-A-X1-Q-G-X2-S-N-Y-X3-S; wherein: x1 is S;
CDR-VL2：X1-T-S-R-X2-Y-S；
CDR-VL3: X1-Q-Y-S-K-X2-P-W; wherein: x1 is H;
each complementarity determining region of the antibody or functional fragment thereof is selected from any one of the following combinations of mutations 1-47:
Figure FDA0004023402980000031
Figure FDA0004023402980000041
and the antibody comprises light chain framework regions FR1-L, FR2-L, FR3-L and FR4-L with the sequences shown as SEQ ID NO. 1-4 in sequence, and/or heavy chain framework regions FR1-H, FR2-H, FR3-H and FR4-H with the sequences shown as SEQ ID NO. 5-8 in sequence.
4. An antibody or functional fragment thereof against gastrin-releasing peptide, comprising the complementarity determining regions:
CDR-VH1: G-Y-X1-F-X2-D-Y-N-M-X3; wherein: x1 is K;
CDR-VH2: D-X1-N-P-X2-S-D-X3-T-X4-Y-N-Q-K-F-K-G; wherein: x1 is L;
CDR-VH3: X1-T-W-X2-H; wherein: x1 is T;
CDR-VL1: S-A-X1-Q-G-X2-S-N-Y-X3-S; wherein: x1 is T;
CDR-VL2：X1-T-S-R-X2-Y-S；
CDR-VL3: X1-Q-Y-S-K-X2-P-W; wherein: x1 is Q;
each complementarity determining region of the antibody or functional fragment thereof is selected from any one of the following combinations of mutations:
Figure FDA0004023402980000042
and the antibody comprises light chain framework regions FR1-L, FR2-L, FR3-L and FR4-L with the sequences shown as SEQ ID NO. 1-4 in sequence, and/or heavy chain framework regions FR1-H, FR2-H, FR3-H and FR4-H with the sequences shown as SEQ ID NO. 5-8 in sequence.
5. The anti-gastrin-releasing peptide antibody or a functional fragment thereof, according to any one of claims 1 to 4, wherein the antibody further comprises a constant region.
6. The anti-gastrin-releasing peptide antibody or the functional fragment thereof according to claim 5, wherein the constant region is selected from the constant regions of any one of IgG1, igG2, igG3, igG4, igA, igM, igE and IgD.
7. The anti-gastrin-releasing peptide antibody or the functional fragment thereof according to claim 5, wherein the species of the constant region is bovine, horse, pig, sheep, goat, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose or human.
8. The anti-gastrin-releasing peptide antibody or the functional fragment thereof, according to claim 7, wherein the species source of the constant region is a bovine cow.
9. The anti-gastrin-releasing peptide antibody or the functional fragment thereof according to claim 7, wherein the species origin of the constant region is turkey or turkey.
10. The anti-gastrin-releasing peptide antibody or a functional fragment thereof according to claim 7, wherein the constant region is derived from a mouse.
11. The anti-gastrin-releasing peptide antibody or functional fragment thereof, according to claim 10, wherein the constant region has the light chain constant region sequence shown in SEQ ID NO 9 and the constant region has the heavy chain constant region sequence shown in SEQ ID NO 10.
12. The anti-gastrin-releasing peptide antibody or the functional fragment thereof according to any one of claims 1 to 4, wherein the functional fragment is selected from F (ab') 2 Any of Fab', fab, fv and scFvOne kind of the method.
13. A reagent or kit for the detection of gastrin-releasing peptide, comprising the antibody or functional fragment thereof according to any one of claims 1 to 12.
14. The reagent or kit for detecting gastrin-releasing peptide according to claim 13, wherein the antibody or functional fragment thereof is labeled with a detectable label.
15. The reagent or kit for the detection of gastrin-releasing peptide according to claim 14, wherein the detectable label is selected from the group consisting of a fluorescent dye, an enzyme catalyzing the development of a substrate, a radioisotope, a chemiluminescent agent and a nanoparticle-based label.
16. The reagent or the kit for detecting gastrin-releasing peptide according to claim 15, wherein the fluorescent dye is selected from a fluorescein dye and a derivative thereof, a rhodamine dye and a derivative thereof, a Cy series dye and a derivative thereof, an Alexa series dye and a derivative thereof, and a protein dye and a derivative thereof.
17. The reagent or kit for the detection of gastrin-releasing peptide according to claim 15, wherein the enzyme catalyzing the development of the substrate is selected from the group consisting of horseradish peroxidase, alkaline phosphatase, β -galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate deoxyenzyme.
18. The reagent or kit for detecting gastrin-releasing peptide according to claim 15, wherein the radioisotope is selected from the group consisting of 212 Bi、 131 I、 111 In、 90 Y、 186 Re、 211 At、 125 I、 188 Re、 153 Sm、 213 Bi、 32 P、 94 mTc、 99 mTc、 203 Pb、 67 Ga、 68 Ga、 43 Sc、 47 Sc、 110 mIn、 97 Ru、 62 Cu、 64 Cu、 67 Cu、 68 Cu、 86 Y、 88 Y、 121 Sn、 161 Tb、 166 Ho、 105 Rh、 177 Lu、 172 Lu and 18 F。
19. the reagent or kit for the detection of gastrin-releasing peptide according to claim 15, wherein the chemiluminescent reagent is selected from luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridinium esters and their derivatives, dioxane and its derivatives, lopowderine and its derivatives and peroxyoxalate and its derivatives.
20. The reagent or kit for detecting gastrin-releasing peptide according to claim 15, wherein the nanoparticle-based label is selected from a nanoparticle and a colloid.
21. The reagent or kit for detecting gastrin-releasing peptide according to claim 20, wherein the nanoparticle is selected from the group consisting of an organic nanoparticle, a magnetic nanoparticle, a quantum dot nanoparticle, and a rare earth complex nanoparticle.
22. The reagent or kit for the detection of gastrin-releasing peptide according to claim 20, wherein the colloid is selected from the group consisting of colloidal selenium, latex, colloidal metal, disperse dye, dye-labeled microsphere, and latex.
23. The reagent or kit for detecting gastrin-releasing peptide according to claim 22, wherein the colloidal metal is selected from the group consisting of colloidal gold and colloidal silver.
24. A vector comprising a nucleic acid fragment encoding the antibody or functional fragment thereof according to any one of claims 1 to 12.
25. A recombinant cell comprising a vector comprising the vector of claim 24.
26. A method of producing the antibody or functional fragment thereof of any one of claims 1 to 12, comprising: culturing the recombinant cell of claim 25, and isolating and purifying the antibody or functional fragment thereof from the culture product.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5550026A (en) * 1992-06-12 1996-08-27 Tonen Corporation Antibodies to human gastrin-releasing peptide precursor and use thereof
CN1830489A (en) * 2006-04-11 2006-09-13 中国药科大学 Tumour polypeptide vaccine based on gastrin release peptide

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE69637411T2 (en) * 1996-06-03 2009-01-22 Advanced Life Science Institute, Inc., Wako Antibodies to human gastrin releasing peptide precursor and their use
CN105067599A (en) * 2015-08-05 2015-11-18 南京闻智生物科技有限公司 Chemiluminescence immune detection kit for detecting pro-gastrin-releasing peptide

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5550026A (en) * 1992-06-12 1996-08-27 Tonen Corporation Antibodies to human gastrin-releasing peptide precursor and use thereof
CN1830489A (en) * 2006-04-11 2006-09-13 中国药科大学 Tumour polypeptide vaccine based on gastrin release peptide

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
抗胃泌素释放肽单克隆抗体的制备及其在体外对乳腺癌细胞生长的抑制;吴国君等;《药物生物技术》;20070615;第14卷(第3期);173-177 *

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