CN117088984B - Anti-benzodiazepine antibody, and kit and reagent for detecting benzodiazepine - Google Patents

Anti-benzodiazepine antibody, and kit and reagent for detecting benzodiazepine Download PDF

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CN117088984B
CN117088984B CN202210510664.7A CN202210510664A CN117088984B CN 117088984 B CN117088984 B CN 117088984B CN 202210510664 A CN202210510664 A CN 202210510664A CN 117088984 B CN117088984 B CN 117088984B
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孟媛
钟冬梅
覃文新
游辉
曹慧方
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Dongguan Pengzhi Biotechnology Co Ltd
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Abstract

The invention discloses an anti-benzodiazepine antibody, a benzodiazepine detection reagent and a benzodiazepine detection kit, and relates to the technical field of antibodies. The anti-benzodiazepine antibodies disclosed herein comprise a heavy chain complementarity determining region and a light chain complementarity determining region. The antibody provides an important raw material source for detecting benzodiazepine, and has improved affinity and activity.

Description

Anti-benzodiazepine antibody, and kit and reagent for detecting benzodiazepine
Technical Field
The invention relates to the technical field of antibodies, in particular to an anti-benzodiazepine antibody, a benzodiazepine detection reagent and a benzodiazepine detection kit.
Background
Benzodiazepine (Benzodiazepines, BZO) drugs are widely used in clinic, are mostly derivatives of 1, 4-benzodiazepine, mainly comprise diazepam (diazepam), fluoxazepam (fludiazepam), chlordiazepoxide, oxazepam, triazolam and the like, and are sedatives acting on the central nervous system. The benzodiazepine is a derivative of 1, 4-benzodiazepine, and the basic structure of the benzodiazepine is composed of two benzene rings and a heterocycle containing 7 atoms. Has sedative, hypnotic, anxiolytic, anticonvulsant, and muscle relaxing effects, and has low toxicity. However, large doses of benzodiazepines cause ataxia, excessive acute poisoning causes coma and respiratory depression, prolonged administration causes physical and mental dependencies, and the risk of dependency increases with increasing doses and prolonged treatment periods. Once physical dependence occurs, withdrawal symptoms, including headache, muscle pain, extreme anxiety tension, agitation, excitement, and delirium, appear upon immediate withdrawal. Insomnia, disturbance of consciousness, loss of intelligence, hyperacusis, numbness, tingling of limbs, hallucination and epileptic seizure occur in severe cases.
In view of their serious harmfulness, benzodiazepines have been administered by national antipsychotics. The metabolites of benzodiazepines are slowly eliminated, the elimination time is long, and the benzodiazepines can be continuously detected in urine even in the weeks or even months after stopping the administration of the benzodiazepines for individuals who use the benzodiazepines for months or years.
At present, the detection method of benzodiazepine mainly comprises a colloidal gold immunochromatography, has the advantages of rapidness, simplicity in operation and the like, is an immunological detection method based on the specific reaction of antibodies and antigens, and simultaneously utilizes colloidal gold to amplify and display detected signals, and the acquisition of the anti-benzodiazepine antibodies is a key for developing colloidal gold immunochromatography test paper. Thus, there is a strong need in the art for antibodies that are effective and bind to and detect benzodiazepines.
In view of this, the present invention has been made.
Disclosure of Invention
The application provides an anti-benzodiazepine Zhuo Kangti with improved affinity and/or activity to improve the detection of benzodiazepine, and provides an important raw material source for the detection of benzodiazepine.
In order to achieve the above object, according to one aspect of the present invention, there is provided an antibody or a functional fragment thereof comprising HCDR1 having an amino acid sequence shown in SEQ ID NO. 1, HCDR2 having an amino acid sequence shown in SEQ ID NO. 2, HCDR3 having an amino acid sequence shown in SEQ ID NO. 3 or 15, and LCDR1 having an amino acid sequence shown in SEQ ID NO. 4 or 16, LCDR2 having an amino acid sequence shown in SEQ ID NO. 5, LCDR3 having an amino acid sequence shown in SEQ ID NO. 6.
In order to achieve the above object, according to a second aspect of the present invention, there is provided an anti-benzodiazepine antibody or a functional fragment thereof, comprising a heavy chain variable region comprising the sequence structure of HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4 and/or a light chain variable region comprising the sequence structure of LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4, wherein the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 are the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 described above.
In order to achieve the above object, according to a third aspect of the present invention, there is provided an anti-benzodiazepine antibody comprising a heavy chain comprising the heavy chain variable region described above and/or a light chain comprising the light chain variable region described above.
In order to achieve the above object, according to a fourth aspect of the present invention, there is provided an antibody conjugate comprising the above antibody or a functional fragment thereof.
In order to achieve the above object, according to a fifth aspect of the present invention, there is provided a reagent or kit for detecting benzodiazepine, the reagent or kit comprising the above-mentioned antibody or a functional fragment thereof or the above-mentioned antibody conjugate.
In order to achieve the above object, the present invention also provides a vector, a cell and a method for preparing the above antibody or a functional fragment thereof.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 shows the results of reducing SDS-PAGE of Anti-BZO-9C238mut1 to Anti-BZO-9C238mut 5.
Detailed Description
The invention provides an antibody or a functional fragment thereof, which comprises HCDR1 with an amino acid sequence shown as SEQ ID NO. 1, HCDR2 with an amino acid sequence shown as SEQ ID NO.2, HCDR3 with an amino acid sequence shown as SEQ ID NO. 3 or 15, and LCDR1 with an amino acid sequence shown as SEQ ID NO. 4 or 16, LCDR2 with an amino acid sequence shown as SEQ ID NO. 5 and LCDR3 with an amino acid sequence shown as SEQ ID NO. 6. The antibodies have improved affinity and/or activity.
In the present invention, the term "antibody" is used in the broadest sense and may include full length monoclonal antibodies, bispecific or multispecific antibodies, and chimeric antibodies so long as they exhibit the desired biological activity.
In the present invention, the terms "complementarity determining regions", "CDRs" or "CDRs" refer to the highly variable regions of the heavy and light chains of immunoglobulins, and refer to regions comprising one or more or even all of the major amino acid residues responsible for the binding of an antibody or antigen-binding fragment to the antigen or epitope recognized by it. In a specific embodiment of the invention, CDRs refer to the highly variable regions of the heavy and light chains of the antibody.
In the present invention, the heavy chain complementarity determining region is represented by HCDR, which includes HCDR1, HCDR2 and HCDR3; the light chain complementarity determining regions are denoted by LCDR and include LCDR1, LCDR2 and LCDR3. CDR labeling methods commonly used in the art include: the Kabat numbering scheme, the IMGT numbering scheme, the Chothia and Lesk numbering schemes, 1997 Lefranc et al, all protein sequences of the immunoglobulin superfamily. Kabat et al were the first to propose a standardized numbering scheme for immunoglobulin variable regions. Over the past few decades, the accumulation of sequences has led to the creation of KABATMAN databases, and the Kabat numbering scheme is generally considered as a widely adopted standard for numbering antibody residues. The invention adopts Kabat annotation standard to mark CDR regions, but other methods to mark CDR regions also belong to the protection scope of the invention.
In the present invention, a "framework region" or "FR" region includes a heavy chain framework region and a light chain framework region, and refers to regions other than CDRs in an antibody heavy chain variable region and a light chain variable region; wherein the heavy chain framework regions can be further subdivided into contiguous regions separated by CDRs comprising HFR1, HFR2, HFR3 and HFR4 framework regions; the light chain framework regions may be further subdivided into contiguous regions separated by CDRs comprising LFR1, LFR2, LFR3 and LFR4 framework regions.
In the present invention, the heavy chain variable region is obtained by connecting the following numbered CDRs with FRs in the following combination arrangement: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is obtained by ligating the following numbered CDRs with the FR in the following combination arrangement: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.
In alternative embodiments, the antibody or functional fragment thereof further comprises HFR1, HFR2, HFR3, HFR4 having amino acid sequences shown in SEQ ID NO. 7 through SEQ ID NO. 10, and LFR1, LFR2, LFR3 and LFR4 having amino acid sequences shown in SEQ ID NO. 11 through SEQ ID NO. 14.
In other embodiments, each of the framework region amino acid sequences of the antibodies or functional fragments thereof provided herein may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the corresponding framework region (SEQ ID NO:7, 8, 9, 10, 11, 12, 13 or 14) described above.
In an alternative embodiment, the antibody or functional fragment thereof binds benzodiazepine with an affinity of KD.ltoreq.10 10 -7M、KD≤10-8M、KD≤10- 9M、KD≤10-10M、KD≤10-11 M.
In an alternative embodiment, the antibody or functional fragment thereof binds to benzodiazepine with an affinity of KD.ltoreq.8.43X10 -10 M.
In an alternative embodiment, the antibody or functional fragment thereof binds to benzodiazepine with an affinity of KD.ltoreq.4.43X10 -11 M.
In an alternative embodiment, the amino acid sequence of HFR1 of the antibody or functional fragment thereof is set forth in SEQ ID NO. 17.
In an alternative embodiment, the amino acid sequence of HFR3 of the antibody or functional fragment thereof is set forth in SEQ ID NO. 18.
In another aspect, embodiments of the present invention provide an anti-benzodiazepine antibody or a functional fragment thereof, the antibody or the functional fragment thereof comprises a heavy chain variable region and/or a light chain variable region, the heavy chain variable region comprises a sequence structure of HFR1-HCDR 2-HFR3-HCDR3-HFR4, the light chain variable region comprises a sequence structure of LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4, and the amino acid sequence of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 is the amino acid sequence of HCDR1, HCDR2, HCDR3, LCDR1, LCDR3, HFR1, HFR2, HFR3, HFR4, LFR3, r4 is the amino acid sequence of HFR1, HFR2, HFR3, lcr 4, LFR2, LFR 4.
In an alternative embodiment, the heavy chain variable region amino acid sequence is set forth in any one of SEQ ID NOs 21 to 28.
In an alternative embodiment, the light chain variable region amino acid sequence is as set forth in any one of SEQ ID NOs 29 to 30.
In alternative embodiments, the antibody further comprises a constant region.
In alternative embodiments, the constant region comprises a heavy chain constant region and/or a light chain constant region.
In alternative embodiments, the heavy chain constant region is selected from the group consisting of an IgG1, igG2, igG3, igG4, igA, igM, igE, or IgD heavy chain constant region, and the light chain constant region is selected from the group consisting of kappa-type or lambda-type light chain constant regions.
In alternative embodiments, the constant region is of a species derived from a cow, horse, cow, pig, sheep, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose, turkey, chicken, or human.
In an alternative embodiment, the constant region is of murine species origin.
In an alternative embodiment, the heavy chain constant region sequence (CH) is shown in SEQ ID NO. 19 and the light chain constant region (CL) sequence is shown in SEQ ID NO. 20.
In other embodiments, the constant region sequence may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the constant region (SEQ ID NO:19 or 20) described above.
In alternative embodiments, the functional fragment is selected from any one of F (ab ') 2, fab', fab, fv, and scFv of the antibody.
The functional fragments of the above antibodies generally have the same binding specificity as the antibody from which they were derived. It will be readily appreciated by those skilled in the art from the disclosure herein that functional fragments of the above antibodies may be obtained by methods such as enzymatic digestion (including pepsin or papain) and/or by methods of chemical reduction cleavage of disulfide bonds. The above functional fragments are readily available to those skilled in the art based on the disclosure of the structure of the intact antibodies.
Functional fragments of the above antibodies may also be synthesized by recombinant genetic techniques also known to those skilled in the art or by, for example, automated peptide synthesizers such as those sold by Applied BioSystems and the like.
In another aspect, the invention provides an anti-benzodiazepine antibody comprising a heavy chain comprising the heavy chain variable region described above and the heavy chain constant region described above and/or a light chain; the light chain comprises the light chain variable region described above and the light chain constant region described above.
In an alternative embodiment, the amino acid sequence of the heavy chain is as shown in any one of SEQ ID NOs 31 to 38.
In an alternative embodiment, the amino acid sequence of the light chain is as shown in any one of SEQ ID NOS: 39 to 40.
In another aspect, the invention provides an antibody conjugate comprising an antibody or functional fragment thereof as described above.
In an alternative embodiment, the antibody or functional fragment thereof in the above antibody conjugate is labeled with a label.
In an alternative embodiment, the above-mentioned marker refers to a substance having a property such as luminescence, color development, radioactivity, etc., which can be directly observed by naked eyes or detected by an instrument, by which qualitative or quantitative detection of the corresponding target can be achieved.
In alternative embodiments, the labels include, but are not limited to, fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents, and nanoparticle-based labels.
In the actual use process, a person skilled in the art can select a suitable marker according to the detection conditions or actual needs, and no matter what marker is used, the marker belongs to the protection scope of the invention.
In alternative embodiments, the fluorescent dyes include, but are not limited to, fluorescein-based dyes and derivatives thereof (including, but not limited to, fluorescein Isothiocyanate (FITC) hydroxy-light (FAM), tetrachlorolight (TET), and the like, or analogs thereof), rhodamine-based dyes and derivatives thereof (including, but not limited to, red Rhodamine (RBITC), tetramethyl rhodamine (TAMRA), rhodamine B (TRITC), and the like, or analogs thereof), cy-based dyes and derivatives thereof (including, but not limited to, cy2, cy3B, cy3.5, cy5, cy5.5, cy3, and the like, or analogs thereof), alexa-based dyes and derivatives thereof (including, but not limited to, alexa fluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, and the like, or analogs thereof), and protein-based dyes and derivatives thereof (including, but not limited to, for example, phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polyazosin (preCP), and the like).
In alternative embodiments, the enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose 6-phosphate deoxygenase.
In alternative embodiments, the radioisotope includes, but is not limited to 212Bi、131I、111In、90Y、186Re、211At、125I、188Re、153Sm、213Bi、32P、94mTc、99mTc、203Pb、67Ga、68Ga、43Sc、47Sc、110mIn、97Ru、62Cu、64Cu、67Cu、68Cu、86Y、88Y、121Sn、161Tb、166Ho、105Rh、177Lu、172Lu and 18 F.
In alternative embodiments, the chemiluminescent reagents include, but are not limited to, luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridinium esters and its derivatives, dioxane and its derivatives, lomustine 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 colloids include, but are 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 an alternative embodiment, the antibody or functional fragment thereof in the above antibody conjugate is coated onto a solid phase.
In alternative embodiments, the solid phase is selected from the group consisting of microspheres, plates, and membranes.
In alternative embodiments, the solid phase includes, but is not limited to, magnetic microspheres, plastic microparticles, microplates, glass, capillaries, nylon, and nitrocellulose membranes.
In an alternative embodiment, the solid phase is a nitrocellulose membrane.
In another aspect, the invention provides a reagent or kit for detecting benzodiazepines, the reagent or kit comprising an antibody or functional fragment thereof as described above or an antibody conjugate as described above.
In another aspect, the invention provides the use of an antibody as described above or a functional fragment thereof, an antibody conjugate or a reagent or kit as described above in the detection of benzodiazepines.
In another aspect, the invention provides a nucleic acid molecule encoding an antibody or functional fragment thereof as described above.
In another aspect, the invention provides a vector comprising the nucleic acid molecule described above.
In another aspect, the invention provides a cell comprising the vector described above.
In another aspect, the invention provides a method of making an antibody or functional fragment thereof comprising: the cells as described above were cultured.
On the basis of the present invention, which discloses the amino acid sequence of an antibody or a functional fragment thereof, it is easy for a person skilled in the art to prepare the antibody or the functional fragment thereof by genetic engineering techniques or other techniques (chemical synthesis, recombinant expression), for example, by separating and purifying the antibody or the functional fragment thereof from a culture product of recombinant cells capable of recombinantly expressing the antibody or the functional fragment thereof according to any one of the above, and on the basis of this, it is within the scope of the present invention to prepare the antibody or the functional fragment thereof by any technique.
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
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 formulations or unit doses herein, some methods and materials are now described. Unless otherwise indicated, techniques employed or contemplated herein are standard methods. The materials, methods, and examples are illustrative only and not intended to be limiting.
Unless otherwise indicated, practice of the present invention will employ conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry and immunology, which are within the ability of a person skilled in the art. This technique is well explained in the literature, as is the case for molecular cloning: laboratory Manual (Molecular Cloning: A Laboratory Manual), second edition (Sambrook et al, 1989); oligonucleotide Synthesis (Oligonucleotide Synthesis) (M.J.Gait, eds., 1984); animal cell Culture (ANIMAL CELL Culture) (r.i. freshney, 1987); methods of enzymology (Methods in Enzymology) (academic Press Co., ltd. (ACADEMIC PRESS, inc.)), experimental immunology handbook (Handbook of Experimental Immunology) (D.M.Weir and C.C. Blackwell, inc.), gene transfer Vectors for mammalian cells (GENE TRANSFER vector for MAMMALIAN CELLS) (J.M.Miller and M.P.Calos, inc., 1987), methods of contemporary molecular biology (Current Protocols in Molecular Biology) (F.M.Ausubel et al, 1987), polymerase chain reaction (PCR: the Polymerase Chain Reaction) (Mullis et al, 1994), and methods of contemporary immunology (Current Protocols in Immunology) (J.E.Coligan et al, 1991), each of which are expressly incorporated herein by reference.
The features and capabilities of the present invention are described in further detail below in connection with the examples.
Example 1 preparation of Anti-BZO-9C238 monoclonal antibody
Restriction enzymes, PRIME STAR DNA polymerase in this example were purchased from Takara. MagExtractor-RNA extraction kit was purchased from TOYOBO company. BD SMART TM RACE cDNA Amplification Kit kit was purchased from Takara. pMD-18T vector was purchased from Takara. Plasmid extraction kits were purchased from Tiangen. Primer synthesis and gene sequencing were accomplished by Invitrogen corporation.
1 Construction of recombinant plasmid
(1) Antibody Gene production
MRNA is extracted from hybridoma cell strains secreting monoclonal antibodies against benzodiazepine, a DNA product is obtained through an RT-PCR method, the product is inserted into a pMD-18T vector after an A adding reaction by rTaq DNA polymerase, the product is transformed into DH5 alpha competent cells, HEAVY CHAIN and LIGHT CHAIN gene clones are respectively taken after colony growth, and each 4 clones are sent to a gene sequencing company for sequencing.
(2) Sequence analysis of Anti-BZO-9C238 antibody variable region Gene
The gene sequence obtained by sequencing is placed in a Kabat antibody database for analysis, and VNTI 11.5.5 software is utilized for analysis to determine that the amplified genes of the heavy chain primer pair and the light chain primer pair are correct, wherein in the LIGHT CHAIN amplified gene fragment, the VL gene sequence is 336bp, and the front side of the VL gene fragment is 57bp leader peptide sequence; the gene fragment amplified by HEAVY CHAIN primer pair has 354bp VH gene sequence, belonging to VH1 gene family, and the front of the gene fragment has 57bp leader peptide sequence.
(3) Construction of recombinant antibody expression plasmids
pcDNATM3.4Vector is a constructed eukaryotic expression vector of the recombinant antibody, and the expression vector is introduced with HindIII, bamHI, ecoRI and other polyclonal enzyme cutting sites and named pcDNA3.4A expression vector, and is hereinafter abbreviated as 3.4A expression vector; according to the result of the gene sequencing of the antibody variable region in pMD-18T, VL and VH gene specific primers of the antibody are designed, and the two ends of the VL and VH gene specific primers are respectively provided with HindIII, ecoRI digestion sites and protective bases, and a LIGHT CHAIN gene fragment of 0.74kb and a HEAVY CHAIN gene fragment of 1.40kb are amplified by a PCR amplification method.
The HEAVY CHAIN and LIGHT CHAIN gene fragments are respectively cut by HindIII/EcoRI double enzyme, the 3.4A vector is cut by HindIII/EcoRI double enzyme, the HEAVY CHAIN gene and LIGHT CHAIN gene after the fragments and the vector are purified and recovered are respectively connected with the 3.4A expression vector, and recombinant expression plasmids of HEAVY CHAIN and LIGHT CHAIN are respectively obtained.
2 Stable cell line selection
(1) Recombinant antibody expression plasmid transient transfection CHO cells, determination of expression plasmid activity
The plasmid was diluted to 40ug/100ul with ultrapure water, CHO cells were adjusted to 1.43X10 7 cells/ml in centrifuge tubes, 100. Mu.L of plasmid was mixed with 700. Mu.L of cells, transferred to an electrotransfer cup, electrotransferred, sampled and counted on days 3, 5, 7, and collected on day 7.
The coating solution (main component NaHCO 3) was diluted to 2ug/ml with BZO-BSA (Handson, product number HDS-A4020, qingdao) 100. Mu.L per well overnight at 4 ℃; the next day, washing with washing liquid for 2 times, and drying; blocking solution (20% BSA+80% PBS) was added, 120. Mu.L per well, 37℃for 1h, and the mixture was dried by shaking; adding diluted cell supernatant at 100. Mu.L/well, 37℃for 30min (1 h for part of supernatant); washing with washing liquid for 5 times, and drying; adding goat anti-mouse IgG-HRP, 100 mu L of each hole, and 30min at 37 ℃; washing with washing liquid for 5 times, and drying; adding a color development solution A (50 mu L/hole, containing citric acid, sodium acetate, acetanilide and carbamide peroxide), and adding a color development solution B (50 mu L/hole, containing citric acid, EDTA, 2Na+TMB and concentrated HCL) for 10min; adding stop solution (50 mu L/hole, EDTA.2Na+ concentrated H 2SO4); OD was read on the microplate reader at 450nm (reference 630 nm). The results showed that the reaction OD after 1000-fold dilution of the cell supernatant was still greater than 1.0, and that the reaction OD without cell supernatant addition was less than 0.1, indicating that the antibodies produced after transient plasmid transformation were active on BZO-BSA.
The reactivity of the cell supernatant with BSA was examined by the same method as described above, and the result showed that the cell supernatant was diluted 1000 times and the reaction OD was less than 0.1, and that the cell supernatant was not added and the reaction OD was less than 0.1, indicating that the antibody produced after transient transformation of the plasmid was inactive against BSA, and further proving that the cell supernatant was active against BZO (benzodiazepine).
(2) Linearization of recombinant antibody expression plasmids
The following reagents were prepared: buffer 50 mu L, DNA mu g/tube, puv I enzyme 10 mu L, sterile water to 500 mu L, water bath at 37 ℃ for enzyme digestion overnight; firstly, extracting with equal volume of phenol/chloroform/isoamyl alcohol (lower layer) 25:24:1, and then sequentially extracting with chloroform (water phase); precipitating 0.1 times volume (water phase) of 3M sodium acetate and 2 times volume of ethanol on ice, rinsing the precipitate with 70% ethanol, removing organic solvent, completely volatilizing ethanol, re-thawing with appropriate amount of sterilized water, and measuring concentration.
(3) Stable transfection of recombinant antibody expression plasmid and pressure screening of stable cell strain
Diluting the plasmid to 40ug/100ul with ultrapure water, regulating CHO cells to 1.43X10 7 cells/ml in a centrifuge tube, mixing 100 mu L of plasmid with 700 mu L of cells, transferring into an electrorotating cup, electrorotating, and counting the next day; 25umol/L MSX 96-well pressure culture for about 25 days.
Observing the clone holes with the cells under a microscope, and recording the confluency; taking culture supernatant, and carrying out sample feeding detection; selecting cell strains with high antibody concentration and relative concentration, turning 24 holes, and turning 6 holes about 3 days; seed preservation batch culture is carried out after 3 days, cell density is adjusted to be 0.5X10 6 cells/ml, batch culture is carried out by 2.2ml, and seed preservation is carried out by 2ml, wherein the cell density is 0.3X10 6 cells/ml; and (3) carrying out sample feeding detection on the culture supernatant of the 6-hole batch culture for 7 days, and selecting cell strains with smaller antibody concentration and smaller cell diameter to transfer TPP for seed preservation and passage.
3 Recombinant antibody production
(1) Cell expansion culture
After cell recovery, the cells were first cultured in 125ml shake flasks with an inoculation volume of 30ml and a medium of 100% dynamis and placed in a shaker at a speed of 120r/min at 37℃and with 8% carbon dioxide. Culturing for 72h, inoculating and expanding culture at 50 ten thousand cells/ml inoculating density, and calculating the expanding culture volume according to production requirements, wherein the culture medium is 100% Dynamis culture medium. After that, the culture was spread every 72 hours. When the cell quantity meets the production requirement, the inoculation density is strictly controlled to be about 50 ten thousand cells/ml for production.
(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%. Feeding: feeding is started every day when the culture is carried out in a shake flask for 72 hours, hyCloneTM Cell BoostTM Feed a is fed with 3% of the initial culture volume every day, and the feeding amount of Feed 7b is one thousandth of the initial culture volume every day until the 12 th day (feeding on 12 th day). Glucose was fed at 3g/L on day six. Samples were collected on day 13. Affinity purification was performed using a proteona affinity column. 3 μg of purified antibody was subjected to reducing SDS-PAGE, and the electrophoretogram shows two bands after reducing SDS-PAGE, 1 Mr at 50KD and the other Mr at 28KD.
Example 2 affinity and Activity optimization
Although the Anti-BZO-9C238 monoclonal antibody obtained in example 1 had the ability to bind benzodiazepine, the affinity and antibody activity were not ideal, and thus the applicant performed directed mutation on the light chain CDR and the heavy chain CDR of the antibody. The method comprises the steps of performing structural simulation of an antibody variable region, structural simulation of an antigen-antibody variable region acting complex, analysis of key amino acids of an antibody and mutation design by using a computer, designing and synthesizing a two-way primer covering a mutation site according to a mutation scheme, synthesizing primers at two ends of target DNA, performing high-fidelity PCR reaction, cloning a PCR product to a vector, and preparing the mutant antibody according to the method described in the example 1. Monoclonal antibodies with remarkably improved affinity and antibody activity are obtained through screening and are named as: anti-BZO-9C238mut1 to Anti-BZO-9C238mut5. The amino acid sequences of the respective monoclonal antibodies are shown in the following table:
TABLE 1 antibody sequences
Example 3 detection of Performance of antibodies
1 Affinity assay
Using the AMC sensor, the purified antibody was diluted to 10ug/ml with PBST, and BZO-BSA (Handson, product No. HDS-A4020) was gradient diluted with PBST:
The operation flow is as follows: equilibration for 60s in buffer 1 (PBST), antibody 300s in antibody solution, incubation for 180s in buffer 2 (PBST), binding for 420s in antigen solution, dissociation for 1200s in buffer 2, sensor regeneration with 10mM pH 1.69GLY solution and buffer 3 (PBST), output data. (KD represents equilibrium dissociation constant, i.e., affinity; kon represents binding rate; kdis represents dissociation rate. PBST major component Na2 HPO4+NaCl+TW-20).
Table 2 affinity data
Sample name KD(M) kon(1/Ms) kdis(1/s)
Control 8.43E-10 4.52E+05 3.81E-04
Anti-BZO-9C238mut1 1.30E-11 4.00E+04 5.20E-07
Anti-BZO-9C238mut2 4.43E-11 2.30E+04 1.02E-06
Anti-BZO-9C238mut3 3.05E-11 1.10E+04 3.36E-07
Anti-BZO-9C238mut4 1.75E-11 1.30E+04 2.27E-07
Anti-BZO-9C238mut5 1.35E-11 3.96E+04 5.33E-07
2 Activity assay
The coating solution (main component NaHCO 3) was diluted to 2ug/ml with BZO-BSA (Handson, product number HDS-A4020, qingdao) 100. Mu.L per well overnight at 4 ℃; the next day, washing with washing liquid for 2 times, and drying; blocking solution (20% BSA+80% PBS) was added, 120. Mu.l per well, 37℃for 1h, and the mixture was dried by pipetting; adding the diluted monoclonal antibody into the mixture at the temperature of 37 ℃ for 30-60 min at the concentration of 100 mu l/hole; washing with washing liquid for 5 times, and drying; adding goat anti-mouse IgG-HRP, 100 μl per well, 37deg.C, 30min; washing with washing solution (PBS) for 5 times, and drying; adding a color development solution A (50. Mu.l/hole, containing 2.1g/L citric acid, 12.25g/L citric acid, 0.07g/L acetanilide and 0.5g/L carbamide peroxide), and adding a color development solution B (50. Mu.l/hole, containing 1.05g/L citric acid, 0.186 g/LEDTA.2Na, 0.45g/L TMB and 0.2ml/L concentrated HCl) for 10min; stop solution (50. Mu.l/well, 0.75 g/EDTA.2Na and 10.2ml/L concentrated H 2SO4) was added; OD was read on the microplate reader at 450nm (reference 630 nm). The results are shown in the following table.
TABLE 3 Activity data
3 Stability assessment
Placing the antibody at 4 ℃ (refrigerator), 80 ℃ (refrigerator) and 37 ℃ (incubator) for 21 days, taking 7 days, 14 days and 21 days samples for state observation, and detecting the activity of the 21 days samples, wherein the result shows that no obvious protein state change is seen for the antibody placed for 21 days under three examination conditions, and the activity is not in a descending trend along with the increase of the examination temperature, thus indicating that the antibody is stable. Table 4 below shows the results of the detection of OD after 21 days of antibody examination.
Table 4 stability data
Sample concentration (ng/ml) 1.953 0.488 0
4 ℃,21 Days sample 1.771 0.754 0.054
Sample at-80℃for 21 days 1.798 0.787 0.039
37 ℃ And 21 days of sample 1.768 0.734 0.035
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
The partial amino acid sequence related to the application is as follows:
Sequence numbering Sequence fragments
SEQ ID NO:1 SDYAWV
SEQ ID NO:2 YISYSGGTNYNPSLKS
SEQ ID NO:3 WGWDEAL
SEQ ID NO:4 RSSQNIVHSNGNTYLE
SEQ ID NO:5 KVSNRFS
SEQ ID NO:6 FQTSHVP
SEQ ID NO:15 WGWDEAI
SEQ ID NO:16 RSSQNLVHSNGNTYLE
SEQUENCE LISTING
<110> Dongguan City, pengzhi biotechnology Co., ltd
<120> Anti-benzodiazepine antibody, kit for detecting benzodiazepine, and kit for detecting benzodiazepine
<130> P2022053CN01
<160> 40
<170> PatentIn version 3.5
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Ser Asp Tyr Ala Trp Val
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Tyr Ile Ser Tyr Ser Gly Gly Thr Asn Tyr Asn Pro Ser Leu Lys Ser
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Trp Gly Trp Asp Glu Ala Leu
1 5
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Arg Ser Ser Gln Asn Ile Val His Ser Asn Gly Asn Thr Tyr Leu Glu
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Lys Val Ser Asn Arg Phe Ser
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Phe Gln Thr Ser His Val Pro
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Asp Val His Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
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Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr
20 25 30
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Trp Ile Arg Gln Phe Pro Gln Asn Lys Leu Glu Trp Met Gly
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Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe Leu Leu
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Leu Asn Ser Met Thr Thr Lys Asp Thr Ala Thr Tyr Tyr Cys Ala Arg
20 25 30
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Ala Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser
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Asn Val Leu Met Thr Gln Ser Pro Leu Ser Leu Pro Val Ser Leu Gly
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Asp Gln Ala Ser Ile Ser Cys
20
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Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr
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Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr
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Leu Lys Ile Asn Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys
20 25 30
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Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
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Trp Gly Trp Asp Glu Ala Ile
1 5
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Arg Ser Ser Gln Asn Leu Val His Ser Asn Gly Asn Thr Tyr Leu Glu
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Asp Val His Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 15
Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Leu Thr
20 25 30
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Arg Leu Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe Leu Leu
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Leu Asn Ser Met Thr Thr Lys Asp Thr Ala Thr Tyr Tyr Cys Ala Arg
20 25 30
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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
115 120 125
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
145 150 155 160
Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr
165 170 175
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
210 215 220
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
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Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu
1 5 10 15
Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe
20 25 30
Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg
35 40 45
Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser
50 55 60
Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu
65 70 75 80
Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser
85 90 95
Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys
100 105
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Asp Val His Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 15
Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Asp
20 25 30
Tyr Ala Trp Val Trp Ile Arg Gln Phe Pro Gln Asn Lys Leu Glu Trp
35 40 45
Met Gly Tyr Ile Ser Tyr Ser Gly Gly Thr Asn Tyr Asn Pro Ser Leu
50 55 60
Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe
65 70 75 80
Leu Leu Leu Asn Ser Met Thr Thr Lys Asp Thr Ala Thr Tyr Tyr Cys
85 90 95
Ala Arg Trp Gly Trp Asp Glu Ala Leu Ala Tyr Trp Gly Gln Gly Thr
100 105 110
Ser Val Thr Val Ser Ser
115
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Asp Val His Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 15
Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Leu Thr Ser Asp
20 25 30
Tyr Ala Trp Val Trp Ile Arg Gln Phe Pro Gln Asn Lys Leu Glu Trp
35 40 45
Met Gly Tyr Ile Ser Tyr Ser Gly Gly Thr Asn Tyr Asn Pro Ser Leu
50 55 60
Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe
65 70 75 80
Leu Leu Leu Asn Ser Met Thr Thr Lys Asp Thr Ala Thr Tyr Tyr Cys
85 90 95
Ala Arg Trp Gly Trp Asp Glu Ala Leu Ala Tyr Trp Gly Gln Gly Thr
100 105 110
Ser Val Thr Val Ser Ser
115
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Asp Val His Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 15
Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Asp
20 25 30
Tyr Ala Trp Val Trp Ile Arg Gln Phe Pro Gln Asn Lys Leu Glu Trp
35 40 45
Met Gly Tyr Ile Ser Tyr Ser Gly Gly Thr Asn Tyr Asn Pro Ser Leu
50 55 60
Lys Ser Arg Leu Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe
65 70 75 80
Leu Leu Leu Asn Ser Met Thr Thr Lys Asp Thr Ala Thr Tyr Tyr Cys
85 90 95
Ala Arg Trp Gly Trp Asp Glu Ala Leu Ala Tyr Trp Gly Gln Gly Thr
100 105 110
Ser Val Thr Val Ser Ser
115
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Asp Val His Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 15
Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Asp
20 25 30
Tyr Ala Trp Val Trp Ile Arg Gln Phe Pro Gln Asn Lys Leu Glu Trp
35 40 45
Met Gly Tyr Ile Ser Tyr Ser Gly Gly Thr Asn Tyr Asn Pro Ser Leu
50 55 60
Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe
65 70 75 80
Leu Leu Leu Asn Ser Met Thr Thr Lys Asp Thr Ala Thr Tyr Tyr Cys
85 90 95
Ala Arg Trp Gly Trp Asp Glu Ala Ile Ala Tyr Trp Gly Gln Gly Thr
100 105 110
Ser Val Thr Val Ser Ser
115
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Asp Val His Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 15
Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Leu Thr Ser Asp
20 25 30
Tyr Ala Trp Val Trp Ile Arg Gln Phe Pro Gln Asn Lys Leu Glu Trp
35 40 45
Met Gly Tyr Ile Ser Tyr Ser Gly Gly Thr Asn Tyr Asn Pro Ser Leu
50 55 60
Lys Ser Arg Leu Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe
65 70 75 80
Leu Leu Leu Asn Ser Met Thr Thr Lys Asp Thr Ala Thr Tyr Tyr Cys
85 90 95
Ala Arg Trp Gly Trp Asp Glu Ala Leu Ala Tyr Trp Gly Gln Gly Thr
100 105 110
Ser Val Thr Val Ser Ser
115
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Asp Val His Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 15
Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Asp
20 25 30
Tyr Ala Trp Val Trp Ile Arg Gln Phe Pro Gln Asn Lys Leu Glu Trp
35 40 45
Met Gly Tyr Ile Ser Tyr Ser Gly Gly Thr Asn Tyr Asn Pro Ser Leu
50 55 60
Lys Ser Arg Leu Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe
65 70 75 80
Leu Leu Leu Asn Ser Met Thr Thr Lys Asp Thr Ala Thr Tyr Tyr Cys
85 90 95
Ala Arg Trp Gly Trp Asp Glu Ala Ile Ala Tyr Trp Gly Gln Gly Thr
100 105 110
Ser Val Thr Val Ser Ser
115
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Asp Val His Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 15
Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Leu Thr Ser Asp
20 25 30
Tyr Ala Trp Val Trp Ile Arg Gln Phe Pro Gln Asn Lys Leu Glu Trp
35 40 45
Met Gly Tyr Ile Ser Tyr Ser Gly Gly Thr Asn Tyr Asn Pro Ser Leu
50 55 60
Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe
65 70 75 80
Leu Leu Leu Asn Ser Met Thr Thr Lys Asp Thr Ala Thr Tyr Tyr Cys
85 90 95
Ala Arg Trp Gly Trp Asp Glu Ala Ile Ala Tyr Trp Gly Gln Gly Thr
100 105 110
Ser Val Thr Val Ser Ser
115
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Asp Val His Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 15
Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Leu Thr Ser Asp
20 25 30
Tyr Ala Trp Val Trp Ile Arg Gln Phe Pro Gln Asn Lys Leu Glu Trp
35 40 45
Met Gly Tyr Ile Ser Tyr Ser Gly Gly Thr Asn Tyr Asn Pro Ser Leu
50 55 60
Lys Ser Arg Leu Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe
65 70 75 80
Leu Leu Leu Asn Ser Met Thr Thr Lys Asp Thr Ala Thr Tyr Tyr Cys
85 90 95
Ala Arg Trp Gly Trp Asp Glu Ala Ile Ala Tyr Trp Gly Gln Gly Thr
100 105 110
Ser Val Thr Val Ser Ser
115
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Asn Val Leu Met Thr Gln Ser Pro Leu Ser Leu Pro Val Ser Leu Gly
1 5 10 15
Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Asn Ile Val His Ser
20 25 30
Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Lys Ile
65 70 75 80
Asn Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Thr
85 90 95
Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 110
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Asn Val Leu Met Thr Gln Ser Pro Leu Ser Leu Pro Val Ser Leu Gly
1 5 10 15
Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Asn Leu Val His Ser
20 25 30
Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Lys Ile
65 70 75 80
Asn Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Thr
85 90 95
Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 110
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Asp Val His Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 15
Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Asp
20 25 30
Tyr Ala Trp Val Trp Ile Arg Gln Phe Pro Gln Asn Lys Leu Glu Trp
35 40 45
Met Gly Tyr Ile Ser Tyr Ser Gly Gly Thr Asn Tyr Asn Pro Ser Leu
50 55 60
Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe
65 70 75 80
Leu Leu Leu Asn Ser Met Thr Thr Lys Asp Thr Ala Thr Tyr Tyr Cys
85 90 95
Ala Arg Trp Gly Trp Asp Glu Ala Leu Ala Tyr Trp Gly Gln Gly Thr
100 105 110
Ser Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro
115 120 125
Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu Gly
130 135 140
Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn
145 150 155 160
Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175
Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser Thr
180 185 190
Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser Ser
195 200 205
Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys Pro
210 215 220
Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro
225 230 235 240
Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys
245 250 255
Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp
260 265 270
Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu
275 280 285
Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met
290 295 300
His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser
305 310 315 320
Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly
325 330 335
Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln
340 345 350
Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe Phe
355 360 365
Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu
370 375 380
Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe
385 390 395 400
Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn
405 410 415
Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His Thr
420 425 430
Glu Lys Ser Leu Ser His Ser Pro Gly
435 440
<210> 32
<211> 441
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 32
Asp Val His Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 15
Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Leu Thr Ser Asp
20 25 30
Tyr Ala Trp Val Trp Ile Arg Gln Phe Pro Gln Asn Lys Leu Glu Trp
35 40 45
Met Gly Tyr Ile Ser Tyr Ser Gly Gly Thr Asn Tyr Asn Pro Ser Leu
50 55 60
Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe
65 70 75 80
Leu Leu Leu Asn Ser Met Thr Thr Lys Asp Thr Ala Thr Tyr Tyr Cys
85 90 95
Ala Arg Trp Gly Trp Asp Glu Ala Leu Ala Tyr Trp Gly Gln Gly Thr
100 105 110
Ser Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro
115 120 125
Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu Gly
130 135 140
Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn
145 150 155 160
Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175
Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser Thr
180 185 190
Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser Ser
195 200 205
Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys Pro
210 215 220
Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro
225 230 235 240
Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys
245 250 255
Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp
260 265 270
Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu
275 280 285
Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met
290 295 300
His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser
305 310 315 320
Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly
325 330 335
Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln
340 345 350
Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe Phe
355 360 365
Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu
370 375 380
Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe
385 390 395 400
Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn
405 410 415
Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His Thr
420 425 430
Glu Lys Ser Leu Ser His Ser Pro Gly
435 440
<210> 33
<211> 441
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 33
Asp Val His Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 15
Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Asp
20 25 30
Tyr Ala Trp Val Trp Ile Arg Gln Phe Pro Gln Asn Lys Leu Glu Trp
35 40 45
Met Gly Tyr Ile Ser Tyr Ser Gly Gly Thr Asn Tyr Asn Pro Ser Leu
50 55 60
Lys Ser Arg Leu Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe
65 70 75 80
Leu Leu Leu Asn Ser Met Thr Thr Lys Asp Thr Ala Thr Tyr Tyr Cys
85 90 95
Ala Arg Trp Gly Trp Asp Glu Ala Leu Ala Tyr Trp Gly Gln Gly Thr
100 105 110
Ser Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro
115 120 125
Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu Gly
130 135 140
Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn
145 150 155 160
Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175
Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser Thr
180 185 190
Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser Ser
195 200 205
Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys Pro
210 215 220
Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro
225 230 235 240
Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys
245 250 255
Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp
260 265 270
Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu
275 280 285
Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met
290 295 300
His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser
305 310 315 320
Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly
325 330 335
Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln
340 345 350
Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe Phe
355 360 365
Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu
370 375 380
Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe
385 390 395 400
Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn
405 410 415
Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His Thr
420 425 430
Glu Lys Ser Leu Ser His Ser Pro Gly
435 440
<210> 34
<211> 441
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 34
Asp Val His Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 15
Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Asp
20 25 30
Tyr Ala Trp Val Trp Ile Arg Gln Phe Pro Gln Asn Lys Leu Glu Trp
35 40 45
Met Gly Tyr Ile Ser Tyr Ser Gly Gly Thr Asn Tyr Asn Pro Ser Leu
50 55 60
Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe
65 70 75 80
Leu Leu Leu Asn Ser Met Thr Thr Lys Asp Thr Ala Thr Tyr Tyr Cys
85 90 95
Ala Arg Trp Gly Trp Asp Glu Ala Ile Ala Tyr Trp Gly Gln Gly Thr
100 105 110
Ser Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro
115 120 125
Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu Gly
130 135 140
Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn
145 150 155 160
Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175
Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser Thr
180 185 190
Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser Ser
195 200 205
Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys Pro
210 215 220
Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro
225 230 235 240
Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys
245 250 255
Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp
260 265 270
Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu
275 280 285
Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met
290 295 300
His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser
305 310 315 320
Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly
325 330 335
Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln
340 345 350
Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe Phe
355 360 365
Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu
370 375 380
Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe
385 390 395 400
Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn
405 410 415
Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His Thr
420 425 430
Glu Lys Ser Leu Ser His Ser Pro Gly
435 440
<210> 35
<211> 441
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 35
Asp Val His Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 15
Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Leu Thr Ser Asp
20 25 30
Tyr Ala Trp Val Trp Ile Arg Gln Phe Pro Gln Asn Lys Leu Glu Trp
35 40 45
Met Gly Tyr Ile Ser Tyr Ser Gly Gly Thr Asn Tyr Asn Pro Ser Leu
50 55 60
Lys Ser Arg Leu Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe
65 70 75 80
Leu Leu Leu Asn Ser Met Thr Thr Lys Asp Thr Ala Thr Tyr Tyr Cys
85 90 95
Ala Arg Trp Gly Trp Asp Glu Ala Leu Ala Tyr Trp Gly Gln Gly Thr
100 105 110
Ser Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro
115 120 125
Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu Gly
130 135 140
Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn
145 150 155 160
Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175
Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser Thr
180 185 190
Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser Ser
195 200 205
Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys Pro
210 215 220
Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro
225 230 235 240
Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys
245 250 255
Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp
260 265 270
Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu
275 280 285
Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met
290 295 300
His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser
305 310 315 320
Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly
325 330 335
Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln
340 345 350
Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe Phe
355 360 365
Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu
370 375 380
Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe
385 390 395 400
Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn
405 410 415
Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His Thr
420 425 430
Glu Lys Ser Leu Ser His Ser Pro Gly
435 440
<210> 36
<211> 441
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 36
Asp Val His Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 15
Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Asp
20 25 30
Tyr Ala Trp Val Trp Ile Arg Gln Phe Pro Gln Asn Lys Leu Glu Trp
35 40 45
Met Gly Tyr Ile Ser Tyr Ser Gly Gly Thr Asn Tyr Asn Pro Ser Leu
50 55 60
Lys Ser Arg Leu Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe
65 70 75 80
Leu Leu Leu Asn Ser Met Thr Thr Lys Asp Thr Ala Thr Tyr Tyr Cys
85 90 95
Ala Arg Trp Gly Trp Asp Glu Ala Ile Ala Tyr Trp Gly Gln Gly Thr
100 105 110
Ser Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro
115 120 125
Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu Gly
130 135 140
Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn
145 150 155 160
Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175
Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser Thr
180 185 190
Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser Ser
195 200 205
Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys Pro
210 215 220
Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro
225 230 235 240
Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys
245 250 255
Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp
260 265 270
Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu
275 280 285
Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met
290 295 300
His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser
305 310 315 320
Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly
325 330 335
Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln
340 345 350
Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe Phe
355 360 365
Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu
370 375 380
Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe
385 390 395 400
Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn
405 410 415
Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His Thr
420 425 430
Glu Lys Ser Leu Ser His Ser Pro Gly
435 440
<210> 37
<211> 441
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 37
Asp Val His Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 15
Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Leu Thr Ser Asp
20 25 30
Tyr Ala Trp Val Trp Ile Arg Gln Phe Pro Gln Asn Lys Leu Glu Trp
35 40 45
Met Gly Tyr Ile Ser Tyr Ser Gly Gly Thr Asn Tyr Asn Pro Ser Leu
50 55 60
Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe
65 70 75 80
Leu Leu Leu Asn Ser Met Thr Thr Lys Asp Thr Ala Thr Tyr Tyr Cys
85 90 95
Ala Arg Trp Gly Trp Asp Glu Ala Ile Ala Tyr Trp Gly Gln Gly Thr
100 105 110
Ser Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro
115 120 125
Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu Gly
130 135 140
Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn
145 150 155 160
Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175
Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser Thr
180 185 190
Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser Ser
195 200 205
Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys Pro
210 215 220
Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro
225 230 235 240
Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys
245 250 255
Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp
260 265 270
Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu
275 280 285
Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met
290 295 300
His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser
305 310 315 320
Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly
325 330 335
Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln
340 345 350
Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe Phe
355 360 365
Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu
370 375 380
Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe
385 390 395 400
Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn
405 410 415
Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His Thr
420 425 430
Glu Lys Ser Leu Ser His Ser Pro Gly
435 440
<210> 38
<211> 441
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 38
Asp Val His Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 15
Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Leu Thr Ser Asp
20 25 30
Tyr Ala Trp Val Trp Ile Arg Gln Phe Pro Gln Asn Lys Leu Glu Trp
35 40 45
Met Gly Tyr Ile Ser Tyr Ser Gly Gly Thr Asn Tyr Asn Pro Ser Leu
50 55 60
Lys Ser Arg Leu Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe
65 70 75 80
Leu Leu Leu Asn Ser Met Thr Thr Lys Asp Thr Ala Thr Tyr Tyr Cys
85 90 95
Ala Arg Trp Gly Trp Asp Glu Ala Ile Ala Tyr Trp Gly Gln Gly Thr
100 105 110
Ser Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro
115 120 125
Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu Gly
130 135 140
Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn
145 150 155 160
Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175
Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser Thr
180 185 190
Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser Ser
195 200 205
Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys Pro
210 215 220
Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro
225 230 235 240
Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys
245 250 255
Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp
260 265 270
Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu
275 280 285
Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met
290 295 300
His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser
305 310 315 320
Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly
325 330 335
Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln
340 345 350
Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe Phe
355 360 365
Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu
370 375 380
Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe
385 390 395 400
Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn
405 410 415
Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His Thr
420 425 430
Glu Lys Ser Leu Ser His Ser Pro Gly
435 440
<210> 39
<211> 219
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 39
Asn Val Leu Met Thr Gln Ser Pro Leu Ser Leu Pro Val Ser Leu Gly
1 5 10 15
Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Asn Ile Val His Ser
20 25 30
Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Lys Ile
65 70 75 80
Asn Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Thr
85 90 95
Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 110
Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu
115 120 125
Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe
130 135 140
Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg
145 150 155 160
Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser
165 170 175
Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu
180 185 190
Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser
195 200 205
Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys
210 215
<210> 40
<211> 219
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 40
Asn Val Leu Met Thr Gln Ser Pro Leu Ser Leu Pro Val Ser Leu Gly
1 5 10 15
Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Asn Leu Val His Ser
20 25 30
Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Lys Ile
65 70 75 80
Asn Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Thr
85 90 95
Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 110
Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu
115 120 125
Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe
130 135 140
Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg
145 150 155 160
Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser
165 170 175
Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu
180 185 190
Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser
195 200 205
Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys
210 215

Claims (35)

1. An anti-benzodiazepine antibody or antigen-binding fragment thereof, comprising:
HCDR1 with the amino acid sequence shown as SEQ ID NO. 1, HCDR2 with the amino acid sequence shown as SEQ ID NO.2, HCDR3 with the amino acid sequence shown as SEQ ID NO. 3, and LCDR1 with the amino acid sequence shown as SEQ ID NO. 4, LCDR2 with the amino acid sequence shown as SEQ ID NO. 5, LCDR3 with the amino acid sequence shown as SEQ ID NO. 6; or alternatively, the first and second heat exchangers may be,
HCDR1 with the amino acid sequence shown as SEQ ID NO. 1, HCDR2 with the amino acid sequence shown as SEQ ID NO. 2, HCDR3 with the amino acid sequence shown as SEQ ID NO. 3, and LCDR1 with the amino acid sequence shown as SEQ ID NO. 16, LCDR2 with the amino acid sequence shown as SEQ ID NO. 5, LCDR3 with the amino acid sequence shown as SEQ ID NO. 6; or alternatively, the first and second heat exchangers may be,
The amino acid sequences are HCDR1 shown in SEQ ID NO.1, HCDR2 shown in SEQ ID NO.2, HCDR3 shown in SEQ ID NO. 15, and LCDR1 shown in SEQ ID NO. 4, LCDR2 shown in SEQ ID NO. 5, LCDR3 shown in SEQ ID NO. 6.
2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof further comprises HFR1, HFR2, HFR3, HFR4 having the amino acid sequences shown in SEQ ID No. 7 to SEQ ID No. 10 and LFR1, LFR2, LFR3 and LFR4 having the amino acid sequences shown in SEQ ID No. 11 to SEQ ID No. 14, or an amino acid sequence having at least 80% identity to each of said sequences.
3. The antibody or antigen-binding fragment thereof according to claim 2, wherein the antibody or antigen-binding fragment thereof binds benzodiazepine with an affinity of kd.ltoreq.10 -9 M.
4. The antibody or antigen-binding fragment thereof according to claim 2, wherein the amino acid sequence of HFR1 of the antibody or antigen-binding fragment thereof is shown in SEQ ID No. 17.
5. The antibody or antigen-binding fragment thereof according to claim 2, wherein the amino acid sequence of HFR3 of the antibody or antigen-binding fragment thereof is shown in SEQ ID No. 18.
6. An anti-benzodiazepine antibody or antigen-binding fragment thereof, comprising a heavy chain variable region comprising the sequence structure of HFR1-HCDR 2-HFR3-HCDR3-HFR4 and a light chain variable region comprising the sequence structure of LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4, wherein the amino acid sequence of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 is the amino acid sequence of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 as defined in claim 1, the amino acid sequence of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, LFR4 is the amino acid sequence of HFR1, HFR2, LCDR3, LFR4 as defined in any one of claims 2 to 4.
7. An anti-benzodiazepine antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region;
The amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 21, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 29; or (b)
The amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 22, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 29; or (b)
The amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 23, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 29; or (b)
The amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 24, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 29; or (b)
The amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 21, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 30.
8. The antibody or antigen-binding fragment thereof of any one of claims 1 to 7, wherein the antibody or antigen-binding fragment thereof further comprises a constant region.
9. The antibody or antigen-binding fragment thereof of claim 8, wherein the constant regions comprise a heavy chain constant region and a light chain constant region.
10. The antibody or antigen-binding fragment thereof of claim 9, wherein the heavy chain constant region is selected from the group consisting of the heavy chain constant region of IgG1, igG2, igG3, igG4, igA, igM, igE, or IgD; the light chain constant region is selected from kappa-type or lambda-type light chain constant regions.
11. The antibody or antigen-binding fragment thereof of claim 9, wherein the constant region is of a species origin of bovine, equine, porcine, ovine, caprine, rat, mouse, canine, feline, rabbit, donkey, deer, mink, chicken, duck, goose, or human.
12. The antibody or antigen-binding fragment thereof of claim 11, wherein the constant region is of murine species origin.
13. The antibody or antigen-binding fragment thereof of claim 9, wherein the heavy chain constant region sequence is as shown in SEQ ID No. 19 or has at least 80% identity thereto.
14. The antibody or antigen-binding fragment thereof of claim 9, wherein the light chain constant region sequence is as set forth in SEQ ID No. 20 or has at least 80% identity thereto.
15. The antibody or antigen-binding fragment thereof of any one of claims 1 to 7, wherein the antigen-binding fragment is selected from any one of F (ab ') 2, fab', fab, fv, and scFv of the antibody.
16. An anti-benzodiazepine antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the heavy chain variable region of any one of claims 6 to 7 and the heavy chain constant region of any one of claims 9, 10 or 13; the light chain comprises the light chain variable region of any one of claims 6 to 7 and the light chain constant region of any one of claims 9, 10 or 14.
17. An anti-benzodiazepine antibody comprises a heavy chain and a light chain, and is characterized in that the amino acid sequence of the heavy chain is shown as SEQ ID NO. 31, and the amino acid sequence of the light chain is shown as SEQ ID NO. 39; or alternatively, the first and second heat exchangers may be,
The amino acid sequence of the heavy chain is shown as SEQ ID NO. 32, and the amino acid sequence of the light chain is shown as SEQ ID NO. 39; or alternatively, the first and second heat exchangers may be,
The amino acid sequence of the heavy chain is shown as SEQ ID NO. 33, and the amino acid sequence of the light chain is shown as SEQ ID NO. 39; or alternatively, the first and second heat exchangers may be,
The amino acid sequence of the heavy chain is shown as SEQ ID NO. 34, and the amino acid sequence of the light chain is shown as SEQ ID NO. 39; or alternatively, the first and second heat exchangers may be,
The amino acid sequence of the heavy chain is shown as SEQ ID NO. 31, and the amino acid sequence of the light chain is shown as SEQ ID NO. 40.
18. An antibody conjugate comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 15 or the anti-benzodiazepine Zhuo Kangti of claim 16 or 17, wherein the antibody or antigen-binding fragment thereof is labeled with a label.
19. The antibody conjugate of claim 18, wherein the label is selected from the group consisting of a fluorescent dye, an enzyme, a radioisotope, a chemiluminescent reagent, and a nanoparticle-based label.
20. The antibody conjugate of claim 19, wherein the fluorescent dye is selected from the group consisting of fluorescein-based dyes, rhodamine-based dyes, cy-based dyes, alexa-based dyes, and protein-based dyes.
21. The antibody conjugate of claim 19, wherein the enzyme is selected from horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and 6-phosphoglucose deoxygenase.
22. The antibody conjugate of claim 19, wherein the radioisotope is selected from the group consisting of 212Bi、131I、111In、90Y、186Re、211At、125I、188Re、153Sm、213Bi、32P、94mTc、99mTc、203Pb、67Ga、68Ga、43Sc、47Sc、110mIn、97Ru、62Cu、64Cu、67Cu、68Cu、86Y、88Y、121Sn、161Tb、166Ho、105Rh、177Lu、172Lu and 18 F.
23. The antibody conjugate of claim 19, wherein the chemiluminescent reagent is selected from the group consisting of luminol, luciferin, crustacean fluorescein, ruthenium bipyridine, acridinium esters, dioxane, lomustine and peroxyoxalate.
24. The antibody conjugate of claim 19, wherein the nanoparticle-based label is selected from the group consisting of a nanoparticle and a colloid.
25. The antibody conjugate of claim 24, wherein the nanoparticle-based label is selected from the group consisting of an organic nanoparticle, a magnetic nanoparticle, a quantum dot nanoparticle, and a rare earth complex nanoparticle.
26. The antibody conjugate of claim 24, wherein the colloid is selected from the group consisting of colloidal selenium, latex, colloidal metal, disperse dye, and dye-labeled microsphere.
27. The antibody conjugate of claim 26, wherein the colloidal metal is selected from the group consisting of colloidal gold and colloidal silver.
28. The antibody conjugate of claim 18, wherein the antibody or antigen binding fragment thereof is coated onto a solid phase.
29. The antibody conjugate of claim 28, wherein the solid phase is selected from the group consisting of a microsphere, a plate, and a membrane.
30. The antibody conjugate of claim 29, wherein the solid phase is selected from the group consisting of magnetic microspheres, plastic microparticles, microwell plates, glass, capillaries, nylon, and nitrocellulose membranes.
31. A reagent or kit for detecting benzodiazepines, comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 15 or the anti-benzodiazepine antibody of claim 16 or 17 or the antibody conjugate of any one of claims 18 to 30.
32. A nucleic acid encoding the antibody or antigen-binding fragment thereof of any one of claims 1 to 15 or the anti-benzodiazepine Zhuo Kangti of claim 16 or 17.
33. A vector comprising the nucleic acid of claim 32.
34. A cell comprising the nucleic acid of claim 32 or the vector of claim 33.
35. A method of preparing the antibody or antigen-binding fragment thereof of any one of claims 1 to 15 or the anti-benzodiazepine antibody of claim 16 or 17, comprising: culturing the cell of claim 34.
CN202210510664.7A 2022-05-11 2022-05-11 Anti-benzodiazepine antibody, and kit and reagent for detecting benzodiazepine Active CN117088984B (en)

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