WO2017005048A1 - 人源降钙素原的单克隆抗体其制备方法和用途 - Google Patents

人源降钙素原的单克隆抗体其制备方法和用途 Download PDF

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WO2017005048A1
WO2017005048A1 PCT/CN2016/081545 CN2016081545W WO2017005048A1 WO 2017005048 A1 WO2017005048 A1 WO 2017005048A1 CN 2016081545 W CN2016081545 W CN 2016081545W WO 2017005048 A1 WO2017005048 A1 WO 2017005048A1
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procalcitonin
antibody
pct
cells
amino acid
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French (fr)
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叶森
唐勇
何仕钊
汪金凤
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NANJING NORMAN BIOLOGICAL TECHNOLOGY Co Ltd
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NANJING NORMAN BIOLOGICAL TECHNOLOGY Co Ltd
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Priority to EP16820699.3A priority Critical patent/EP3321284B1/en
Priority to EP20167035.3A priority patent/EP3702370B1/en
Publication of WO2017005048A1 publication Critical patent/WO2017005048A1/zh
Priority to US15/728,480 priority patent/US10882900B2/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/26—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against hormones ; against hormone releasing or inhibiting factors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/575—Hormones
    • G01N2333/585—Calcitonins

Definitions

  • the invention relates to the field of medical diagnosis.
  • the present invention relates to a hybridoma cell line which can produce a monoclonal antibody specific for human procalcitonin and a process for the preparation thereof.
  • the monoclonal antibody prepared by the hybridoma can be applied to the diagnosis of diseases or related fields.
  • Procalcitonin is a specific marker of bacterial and fungal infections discovered in the 1990s. It is a hormone-free calcitonin propeptide consisting of 116 amino acids. It is about 13KD. It can be gradually lysed into 57 amino acid aminoprocalcitonin (N-PCT), 32 amino acid calcitonin (CT) and 21 amino acid calcitonin under the action of enzyme. Procalcitonin is the expression product of the calcitonin I gene (CALC-I) on chromosome 11.
  • Procalcitonin has a total of 116 amino acids, which can be divided into three segments depending on the domain and function.
  • 1-57 is its N segment.
  • the N-terminus begins to be cleaved at position 57 to produce amino acids 60-116 containing calcitonin.
  • it is also cleaved by protease at position 91 to form calcitonin and procalcitonin (katacalcin of amino acids 96-116). Therefore, there is a procalcitonin whole and a plurality of its sheared fragments in human serum, and these fragments exert a physiological function different from procalcitonin in the human body.
  • the polyclonal antibody against the 1-57 fragment was selected for the determination of procalcitonin.
  • a monoclonal antibody against the 1-57 fragment and a monoclonal antibody against the 60-116 fragment were selected for sandwich assay.
  • the monoclonal antibody against the N-stage and calcitonin-binding region and the monoclonal antibody against the 60-116 fragment were selected for sandwich assay.
  • the present invention aims to provide a monoclonal antibody which specifically binds to human procalcitonin protein. And its preparation method.
  • the inventors recombinantly expressed the mutated protein in vitro by mutating individual amino acids of 1-57 amino acids, mainly for individual amino acids at positions 19-40.
  • the inventors have found that the above treatment can improve the stability of procalcitonin protein, and it is not easily degraded when injected into an animal after co-emulsification with an adjuvant, thereby producing a more efficient antibody.
  • the inventors found that the final epitope analysis identified amino acids at positions 20-35.
  • the present invention provides an anti-procalcitonin antibody which specifically binds to human procalcitonin, wherein the anti-procalcitonin antibody and the hybridoma cell line deposited under the accession number CGMCC No. 10417 produce calcium-reducing
  • the primogen monoclonal antibody specifically binds to the same procalcitonin epitope.
  • the anti-pro-procalcitonin antibody comprises a humanized sequence of a procalcitonin monoclonal antibody produced by a hybridoma cell line deposited under the number CGMCC No. 10417.
  • the anti-procalcitonin antibody is a monoclonal antibody.
  • the anti-procalcitonin antibody specifically binds to an epitope having the amino acid sequence RLLLAALVQDYV (SEQ ID NO: 1).
  • the anti-procalcitonin antibody is obtained from a hybridoma cell line with accession number CGMCC No. 10417.
  • the present invention also provides an immunodetection reagent comprising the above anti-pro-procalcitonin antibody for detecting or quantifying procalcitonin or a fragment thereof.
  • the present invention further provides the use of the above anti-pro-calcitonin antibody for the preparation of an immunodetection reagent for detecting or quantifying procalcitonin or a fragment thereof.
  • the present invention also provides a kit comprising the above anti-pro-procalcitonin antibody for detecting or quantifying procalcitonin or a fragment thereof.
  • the kit of the present invention comprises at least the above anti-pro-procalcitonin antibody, and an antibody against an epitope in the amino acid sequence of the procalcitonin 50-116; preferably, the The antibody against the epitope in the amino acid sequence of the procalcitonin 50-116 may be an antibody against the epitope in the amino acid sequence of the procalcitonin 50-116 disclosed in the prior art.
  • the detection or quantification of procalcitonin or a fragment thereof according to the present invention is carried out by an immunoassay.
  • the immunoassay comprises methods conventional in the art, such as chemical methods, fluorescent immunization, and enzyme-linked immunosorbent assay. , immunochromatography, etc., the specific detection steps of the above detection methods can be obtained by the prior art.
  • the invention further provides the use of the above-described anti-procalcitonin antibody for the preparation of a kit for detecting or quantifying procalcitonin or a fragment thereof.
  • the present invention provides a hybridoma cell line producing a procalcitonin monoclonal antibody having the accession number CGMCC No. 10417.
  • the present invention provides a method of preparing an anti-pro-pro-protinogen antibody comprising the steps of:
  • the mutation is directed to amino acid residues 1 to 57 of procalcitonin.
  • the mutated procalcitonin amino acid sequence is SEQ ID NOs: 2-4.
  • an antibody prepared by the above method is also provided.
  • the antibody is a monoclonal antibody.
  • the beneficial effects of the invention are that after the mutant modification of human procalcitonin, the stability is higher than that of the common antigen, and it can be used as a calibrator for the quantitative detection kit of human procalcitonin, which is not easy to be degraded; A purified recombinant protein that can be used for immunogens and screens for antibody preparation.
  • the antibody produced by the human procalcitonin modified by the mutation can not only specifically bind to human procalcitonin, but also can obtain better efficacy than the existing human procalcitonin antibody. Valence, higher specific binding.
  • Figure 1 shows the results of agarose gel electrophoresis identification of three mutants.
  • TB1 Mutant 1
  • M DL2000 DNA Marker
  • TB2 Mutant 2
  • TB3 Mutant 3 where the arrows indicate the positions of 500 bp and 300 bp Marker, respectively.
  • Figure 2 shows the results of agarose gel electrophoresis identification of pET34 double digestion.
  • Figure 3 shows the results of agarose gel electrophoresis identification of bacterial liquid PCR.
  • TB1 Mutant 1
  • TB2 Mutant 2
  • TB3 Mutant 3 where the arrows indicate the positions of the 500 bp and 300 bp Marker, respectively.
  • Figure 4 shows the results of agarose gel electrophoresis for the extraction of pET34-PCT-TB1, pET34-PCT-TB2, and pET34-PCT-TB3 plasmids.
  • Figure 5 shows the results of SDS-PAGE electrophoresis for purification of pET34-PCT-TB1, pET34-PCT-TB2, and pET34-PCT-TB3 proteins.
  • the arrow refers to the 15kd protein band.
  • L loading
  • M Protein Marker
  • P Leakage
  • 80 80 mM imidazole
  • 250 250 mM imidazole.
  • the Balb/c mouse hybridoma cells obtained by the present invention were deposited with the General Microbiology Center of the China Microbial Culture Collection Management Committee on April 1, 2015 under the deposit number CGMCC No. 10417.
  • the address of the deposit center is Beichen, Chaoyang District, Beijing. No. 3, West Road No. 1, Institute of Microbiology, Chinese Academy of Sciences, Post Code: 100101.
  • sequences involved in the present invention including nucleotide sequences and amino acid sequences, have been compiled into sequence listings, which are appended to the specification, and the inventors simultaneously filed a computer readable form of the sequence listing.
  • antibody refers to any immunoglobulin or intact molecule that binds to a particular epitope, as well as fragments thereof.
  • Such antibodies include, but are not limited to, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, single chain antibodies, and fragments and/or portions of intact antibodies, so long as such fragments or portions retain the antigen binding ability of the parent antibody.
  • anti-pro-procalcitonin antibody refers to a monoclonal antibody, a polyclonal antibody, a single-chain antibody, and a monoclonal antibody, a polyclonal antibody, or a functional fragment thereof, which specifically binds procalcitonin protein, or a functional variant or functional fragment thereof Immunologically active fragments or parts.
  • terms such as “procalcitonin antibody”, “anti-procalcitonin antibody”, and “antibody against procalcitonin” are used interchangeably.
  • binding refers to the use of a purified wild-type antigen in an in vitro assay, preferably in a plasmon resonance assay (BIAcore, GE-Healthcare Uppsala, Sweden). The combination of epitopes. Binding affinity is defined by the terms ka (binding rate constant for antibodies from antibody/antigen complexes), kD (dissociation constant) and KD (kD/ka). Binding or specific binding means a binding affinity (KD) of 10 -8 mol/l or less, preferably 10 -9 to 10 -13 mol/l.
  • the procalcitonin antibody according to the present invention specifically binds to the antigen with a binding affinity (Kd) of 10 -8 mol/l or less, preferably 10 -9 to 10 -13 mol/l.
  • human monoclonal antibody refers to an antibody that exhibits a single binding specificity with variable and constant regions derived from human germline immunoglobulin sequences.
  • the human monoclonal antibody is produced by a hybridoma comprising a transgenic non-human animal having a genome comprising a human heavy chain transgene and a human light chain transgene fused to an immortalized cell, eg, a small transgene B cells obtained from mice.
  • the agarose gel DNA recovery kit was purchased from Tiangen Bio; the plasmid extraction kit was purchased from Axygen; Tryptone and Yeast extract were OXOID products, IPTG and Protein Marker were purchased from Shengxing Company, Ni Column gum (Ni-Sepharose 6 FF) was purchased from GE.
  • Buffer A 20 mM Tris, 300 mM NaCl, 20 mM Imi, pH 7.4
  • Buffer B 20 mM Tris, 300 mM NaCl, 80 mM Imi, pH 7.4
  • Buffer C 20 mM Tris, 300 mM NaCl, 250 mM Imi, pH 7.4
  • the 16 primers were ligated by overlap extension PCR to obtain the full length of the three mutants.
  • the agarose gel was identified (see Figure 1) and the PCR product was purified using an agarose gel DNA recovery kit.
  • the above reaction solution was separately mixed, reacted at 37 ° C for 30 minutes, and identified by electrophoresis (see Fig. 2). After digestion, the DNA fragment purification kit was used for purification.
  • the PCR product and the vector DNA were reacted at a molar ratio of 9:1, and were ligated at 22 ° C for 30 minutes under the action of T4-DNA ligase.
  • PCR conditions 95 ° C, 3 minutes. 95 ° C, 30 seconds, 58 ° C, 30 seconds, 72 ° C, 30 seconds. 30 cycles. 8 ⁇ l of the sample was verified by agarose gel electrophoresis (see Figure 3).
  • Plasmid extraction of pET34-PCT-TB1, pET34-PCT-TB2, and pET34-PCT-TB3 was performed according to the kit instructions, and 2 ⁇ l of the sample was confirmed by agarose gel electrophoresis (see Figure 4).
  • the extracted pET34-PCT-TB1, pET34-PCT-TB2, pET34-PCT-TB3 plasmids were sent to Kingsley for sequencing. The sequencing results are consistent with the design sequence.
  • the correctly sequenced recombinant pET34-PCT-TB1, pET34-PCT-TB2, pET34-PCT-TB3 plasmids were transformed into BL21(DE3) competent state, respectively.
  • the cells were collected by centrifugation at 12000 rpm, and the cells were resuspended in 20 ml Buffer A, sonicated in an ice bath for 20 minutes, centrifuged at 12000 rpm for 20 minutes, and the supernatant was taken, purified by Ni column, pre-washed with Buffer A, Buffer B, and eluted with Buffer C. Dialysis into PBS, 20 ⁇ l of the sample after dialysis was identified by SDS-PAGE electrophoresis (see Figure 5).
  • mice Six Balb/c mice, 6 weeks old, were emulsified with Freund's adjuvant and antigen, then injected into the back of the mice, immunized three times, every two weeks. After the end of the immunization, the mice were tested for blood titer. The mice with the same titer were injected intraperitoneally with the antigen, and the immunization was boosted for three days.
  • DMEM is not completely cultured. 10% fetal calf serum and 1% double antibody are added to prepare DMEM complete medium. 15ml complete medium is used in T75 cell culture flask to culture SP2/0 myeloma cells, and changed every two days. In the medium, when the cell growth reaches a logarithmic phase, when the cell volume is covered with 70% of the bottom of the bottle, the medium is replaced and the next day is reserved.
  • the spare mouse spleen was ground in a 200 mesh sterile sieve, washed with DMEM incomplete medium, and the suspension was transferred to a 50 ml sterile centrifuge tube and mixed for counting; the SP2/0 culture supernatant was discarded, and DMEM was not used. Complete medium, the cells were blown off, transferred to a 50 ml sterile centrifuge tube and mixed for counting; the number of spleen cells and myeloma cells was mixed at a ratio of 5:1, centrifuged at 1500 rpm for 3 minutes, the supernatant was discarded, and the mixture was beaten and beaten.
  • the fusion wells were replaced with HT medium (DMEM complete medium + HT solution) for half a volume. After three days, the supernatant was taken for detection, 60 ⁇ l/well, and the OD 450 value of the supernatant was determined by indirect ELISA. The measured value was greater than that of the control 2.5.
  • the doubled holes are positive holes and the markers are reserved.
  • the labeled positive wells were blotted with a Pasteur pipette, mixed and counted, and 200-500 cells were diluted with DMEM complete medium and plated into a 96-well cell culture plate containing feeder cells at a volume of 200 ⁇ l. /well, the first round of subcloning is completed; after 5-7 days, the supernatant is taken for detection, and the monoclonal cell heap is selected for the second round of subcloning, and the cycle is repeated until the selected hybridoma cells are successively cultured twice. If the cells are positive and the monoclonal cells are subcloned, the subcloning is stopped, and the finally obtained monoclonal cells are picked out and cultured in a 24-well cell culture plate for expansion and cryopreservation.
  • the hybridoma cells in the 24 wells were covered with 80% of the bottom of the wells, and the pasteurized tubes were mixed and transferred to a cell culture flask. After they were covered with 80% of the bottom of the flask, the cells were frozen; the cells in the flask were washed off and transferred to In a 50 ml sterile centrifuge tube, take a small number of counts, centrifuge the remaining cells at 1500 rpm, centrifuge for 3 minutes, discard the supernatant, pat the mixture and add the frozen solution (90% fetal bovine serum + 10% DMSO) to make 1 ⁇ 10 6 cells/ml cells. The suspension, 1 ml/tube was placed in a 2 ml cryotube, placed in a cell cryopreservation box, and stored at -70 ° C overnight, and transferred to liquid nitrogen for storage the next day.
  • the desired hybridoma cells were taken out from the liquid nitrogen, and the frozen tube was quickly placed in a 37 ° C water bath, which was quickly thawed by shaking back and forth, completely thawed in 1 minute, centrifuged at 1500 rpm for 3 minutes, the supernatant was discarded, and DMEM complete medium was added. The cells were resuspended and transferred to a cell culture flask for resuscitation. The resuscitation was successful if the cells were observed under the microscope, otherwise resuscitation was required.
  • the fresh medium was replaced the next day, and then changed every two days.
  • the cells were covered with 70% of the bottom of the bottle, the cells were blown down in DMEM complete medium, half removed to a new flask, and freshly cultured.
  • the cells in the culture flask are washed with a sterile PBS solution, collected into a 50 ml sterile centrifuge tube, centrifuged at 1500 rpm for 3 minutes, the supernatant is discarded, and the cells are resuspended in PBS, mixed again. After centrifugation, the supernatant was discarded, and resuspended again with PBS to prepare a cell suspension of 2 ⁇ 10 6 /ml, and set aside.
  • mice were intraperitoneally injected with the above cell suspension, 0.5 ml/mouse.
  • mice produced ascites After 5 days, the mice produced ascites, inserted into the abdominal cavity of the mouse with a 20 ml syringe needle, and collected ascites with a centrifuge tube at the end of the needle. After the abdomen was not dripped, the needle was pulled out, the mouse was returned, and the next day, again, until repeated. The mouse died.
  • Preservatives were added to the ascites at 4 ° C. After the ascites collection was completed, the ascites antibody was purified.
  • the mouse ascites was taken, an equal volume of binding buffer was added, mixed, and allowed to stand at 4 ° C for 1 hour, centrifuged (4 ° C, 12000 rotation, 20 minutes), and the supernatant was taken, and the 0.45 ⁇ m membrane was filtered to remove the particulate inclusions.
  • the ascites sample after suction filtration was subjected to a protein A affinity chromatography column previously equilibrated with a binding buffer, and the flow rate was controlled at 2 mL/min.
  • Elution was carried out with a pH 3.0 eluate, and the fraction with high protein content was collected in a centrifuge tube previously added with a neutralization buffer to purify the monoclonal antibody; after dialysis with PBS, the immunological activity of the antibody was determined, and a preservative was added for storage. .
  • the column was regenerated with 3 column volumes of regeneration buffer, washed with 20% ethanol, and stored in a refrigerator at 4 °C.
  • Binding buffer 0.1 M Gly, pH 9.0, containing 3 M NaCl;
  • Elution buffer 0.1 M Gly, pH 3.0, containing 0.15 M NaCl;
  • Regeneration buffer 0.1 M Gly, pH 2.0, containing 0.15 M NaCl;
  • Neutralization buffer 1M Tris-HCl, pH 8.0, containing 0.15 M NaCl
  • Antibody titers were determined by indirect ELISA. The purified antibody was taken and the dilution gradient was from 10 -4 to 10 -7 . Set 3 duplicate holes.
  • Antigen coating Dilute the corresponding antigen to 2 ug/ml with a coating solution (carbonate buffer pH 9.6, 0.05 mol/L), add to a 96-well microtiter plate, 100 ⁇ l/well, and overnight at 4 ° C (or Incubate at 37 ° C for 1 h), wash 3 times, and pat dry.
  • a coating solution carbonate buffer pH 9.6, 0.05 mol/L
  • Blocking Block with 1% BSA, 200 ⁇ l/well, incubate for 1 hour at 37 ° C, wash 3 times, and pat dry.
  • the diluted antibody was added to the well, 100 ⁇ l/well, incubated at 37 ° C for 1 h (or overnight at 4 ° C), washed 3 times, and patted dry.
  • the enzyme-labeled secondary antibody was added: goat anti-mouse IgG-HRP was added, and the suitable dilution concentration was determined by a checkerboard titration method, 100 ⁇ l/well, incubated at 37 ° C for 1 h, washed 3 times, and patted dry.
  • TMB coloring solution was placed in a 37 ° C incubator at a temperature of 100 ⁇ l/well and incubated at 37 ° C for 10 to 15 minutes.
  • the reaction was terminated: 50 ⁇ l of 2 mol/L sulfuric acid was added to each well to terminate the reaction.
  • the OD value was measured at 450 nm: the OD value was measured by a microplate reader, and the blank hole was adjusted to zero. If the OD value of the hole to be tested exceeded 0.2 and was greater than 2.1 times of the negative control well, the determination was positive, and the antibody dilution factor corresponding to the minimum determination value was used. For the antibody titer, the antibody release is optimal for antibody dilution when the OD value is close to 1.0.

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Abstract

本发明提供了一种与人源降钙素原蛋白特异性结合的单克隆抗体及其用途。本发明还提供了分泌该单克隆抗体的保藏号为CGMCC No.10417的杂交瘤细胞系以及一种以降钙素原突变抗原为免疫原制备抗降钙素原抗体的方法。

Description

人源降钙素原的单克隆抗体其制备方法和用途 技术领域
本发明涉及医学诊断领域。具体地，本发明涉及一种可以产生特异针对人源降钙素原的单克隆抗体的杂交瘤细胞系及其制备方法。该杂交瘤制备的单克隆抗体可以应用于疾病的诊断或者相关领域。
背景技术
降钙素原(procalcitonin，PCT)是二十世纪九十年代才发现的细菌、真菌性感染的特异性标志物，是一种无激素活性的降钙素前肽，由116个氨基酸组成，分子量大约为13KD。它可以在酶的作用下逐步裂解成57个氨基酸的氨基降钙素原(N-PCT)、32个氨基酸的降钙素(calcitonin，CT)和21个氨基酸的降钙蛋白。降钙素原是11号染色体上降钙素I基因(CALC-I)的表达产物。在不存在感染的情况下，甲状腺外CALC-I表达被抑制，而主要局限于甲状腺和肺的神经内分泌细胞一定程度的表达；细菌感染诱导全身各种组织多种细胞类型的CALC-I表达和降钙素原连续性释放。近年来研究表明在严重全身性细菌、真菌、寄生虫、急性疟疾感染、全身性炎症反应综合征(SIRS)、多器官功能衰竭综合征(MODS)的诊断方面，降钙素原是一个具有高灵敏度、特异性的新指标。
降钙素原一共116个氨基酸，其根据结构域和功能可以分为三段。1-57位为其N段。在体内N端会在57位开始被剪切，生成含有降钙素的60-116位氨基酸。同时，在91位也会被蛋白酶剪切，形成降钙素和抗钙素原(96-116位氨基酸的katacalcin)。因此在人血清中存在着降钙素原整体以及其多种剪切后的片段，而这些片段在人体内发挥着与降钙素原不一样的生理功能。
为了特异地、准确地诊断识别出完整的降钙素原的片段，目前有三种方案来测定降钙素原的含量。第一种，选择抗1-57片段的多抗来进行降钙素原的测定。第二种，选择抗1-57片段的单抗和抗60-116片段的单抗进行夹心测定。第三种，选择抗N段和降钙素结合的区域的单抗和抗60-116片段的单抗进行夹心测定。
由于降钙素原的N末端不稳定，容易降解，因而以纯人源降钙素原抗原进行免疫时，不稳定的抗原会产生较弱的免疫应答，达不到高效抗体的制备条件。因此，现有技术中存在解决纯人源降钙素原抗原进行免疫时由于不稳定的抗原会产生较弱的免疫应答的问题的需要。
发明内容
为了解决纯人源降钙素原抗原进行免疫时由于不稳定的抗原会产生较弱的免疫应答的问题，本发明旨在提供在制备与人源降钙素原蛋白特异性结合的单克隆抗体及其制备方法。发明人通过突变1-57个氨基酸中的个别氨基酸，主要是针对19-40位的个别氨基酸，并体外重组表达这种突变的蛋白。发明人发现上述处理可以改善降钙素原蛋白的稳定性，使其在与佐剂共同乳化后注射入动物体内不容易降解，从而产生更加高效的抗体。发明人发现最终表位分析确定为20-35位的氨基酸。
本发明提供了一种与人源降钙素原特异性结合的抗降钙素原抗体，其中所述抗降钙素原抗体与保藏号为CGMCC No.10417的杂交瘤细胞系产生的降钙素原单克隆抗体特异性结合相同的降钙素原表位。
在本发明的一个方面，所述抗降钙素原抗体包含保藏号为CGMCC No.10417的杂交瘤细胞系产生的降钙素原单克隆抗体的人源化序列。
在本发明的另一个方面，所述抗降钙素原抗体是单克隆抗体。
在本发明的又一个方面，所述抗降钙素原抗体特异性结合具有氨基酸序列为RLLLAALVQDYV(SEQ ID NO:1)的表位。
在本发明的一个实施方案中，所述抗降钙素原抗体获得自保藏号为CGMCC No.10417的杂交瘤细胞系。
本发明还提供了含有上述的抗降钙素原抗体的免疫检测试剂，其用于检测或者定量降钙素原或其片段。
本发明又提供了上述抗降钙素原抗体用于制备检测或者定量降钙素原或其片段的免疫检测试剂的用途。
本发明还提供了含有上述抗降钙素原抗体的试剂盒，其用于检测或者定量降钙素原或其片段。在本发明的实施方案中，本发明所述的试剂盒，至少包含上述抗降钙素原抗体，以及针对降钙素原50-116位氨基酸序列中的表位的抗体；优选的，所述的针对降钙素原50-116位氨基酸序列中的表位的抗体可以为现有技术中所公开的针对降钙素原50-116位氨基酸序列中的表位的抗体。更优选的，本发明所述的检测或者定量降钙素原或其片段采用免疫测定法，进一步的，所述的免疫测定法包含本领域常规的方法，如化学方法，荧光免疫，酶联免疫，免疫层析等，上述检测方法具体测定步骤均可由现有技术获得。
本发明又提供了上述抗降钙素原抗体用于制备检测或者定量降钙素原或其片段的试剂盒的用途。
本发明提供了一种产生降钙素原单克隆抗体的杂交瘤细胞系，其保藏号为CGMCC No.10417。
此外，本发明提供了一种制备抗降钙素原抗体的方法，包括以下步骤：
(1)对降钙素原N端氨基酸残基进行突变，获得与突变后氨基酸残基序列相对应的核苷酸序列；
(2)将所述核苷酸序列插入到原核表达载体中，进行诱导表达以及蛋白纯化；
(3)将纯化蛋白作为抗原免疫动物，取其脾细胞与小鼠骨髓瘤细胞融合制备杂交瘤细胞，经筛选获得可稳定分泌抗降钙素原抗体的杂交瘤细胞，并获得相应单克隆抗体。
在本发明的一方面，所述突变是针对降钙素原第1至57位氨基酸残基进行的。
在本发明的一个实施方案中，突变后的降钙素原氨基酸序列为SEQ ID NOs:2-4。
在本发明中，还提供了由上述的方法制备的抗体。
在本发明的一个实施方案中，所述抗体是单克隆抗体。
本发明的有益效果在于对人源降钙素原进行突变修饰后，比普通抗原稳定性更高，可作为人降钙素原定量检测试剂盒时的校准品，不易降解；同时是一种高纯度的重组蛋白，可用于抗体制备的免疫原和筛选原。此外，由该突变修饰后的人源降钙素原所产生的抗体不仅能够特异性结合到人源降钙素原，并且能够得到与现有人源降钙素原抗体相比的更好的效价、更高的特异性结合。
下面结合附图对本发明进行更详细的说明。从下文的详细描述中，本发明的上述方面和本发明的其他方面将是明显的。
附图说明
图1为三种突变体的琼脂糖凝胶电泳鉴定结果。TB1：突变体1，M：DL2000 DNA Marker，TB2：突变体2，TB3：突变体3，箭头所指分别为500bp和300bp Marker所在位置。
图2为pET34双酶切的琼脂糖凝胶电泳鉴定结果。
图3为菌液PCR的琼脂糖凝胶电泳鉴定结果。TB1：突变体1，TB2：突变体2，TB3：突变体3，箭头所指分别为500bp和300bp Marker所在位置。
图4为pET34-PCT-TB1、pET34-PCT-TB2、pET34-PCT-TB3质粒提取的琼脂糖凝胶电泳鉴定结果。
图5为pET34-PCT-TB1、pET34-PCT-TB2、pET34-PCT-TB3蛋白纯化的SDS-PAGE电泳鉴定结果。箭头所指为15kd蛋白条带。L：上样，M：蛋白Marker，P：漏过，80：80mM咪唑，250：250mM咪唑。
保藏说明
本发明所获得的Balb/c小鼠杂交瘤细胞已于2015年4月1日以保藏号CGMCC No.10417保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏中心地址：北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所，邮政编码：100101。
序列说明
本发明涉及的序列，包括核苷酸序列和氨基酸序列，已经整理成序列表，附在说明书的后面，发明人同时提交了序列表的计算机可读形式文件。
具体实施方式
本发明并不限于本文所描述的特定方法学、方案、抗体或细胞系，因为它们可以有所变化。另外，本文所用的术语只用于描述特定实施方式的目的，而不是用于限定本发明的范围。
除非有不同的定义，所有的技术和科学术语以及任何的缩写在此都具有与本发明领域普通技术人员所通常理解的含义相同的含义。尽管在本发明的实践中可以使用与那些在本文所描述的方法和 材料相似或等价的方法和材料，在此描述了例证性的方法、设备和材料。
除非特别说明，本发明的术语具有本领域通常使用的含义。
本发明所用的术语“抗体”，是指结合特定表位的任何免疫球蛋白或完整分子以及其片段。所述抗体包括但不限于多克隆抗体、单克隆抗体、嵌合抗体、人源化抗体、单链抗体以及完整抗体的片段和/或部分，只要这些片段或部分保留亲本抗体的抗原结合能力。例如，本发明中，“抗降钙素原抗体”是指能特异性地结合降钙素原蛋白，或其功能变体或功能片段的单克隆抗体、多克隆抗体、单链抗体和其具有免疫活性的片段或部分。本发明中，诸如“降钙素原抗体”、“抗降钙素原抗体”以及“针对降钙素原的抗体”这样的术语均可以互换使用。
本发明所用的术语“结合”或“特异性结合”指用纯化的野生型抗原在体外测定法中，优选地在等离振子共振测定法(BIAcore，GE-Healthcare Uppsala，Sweden)中抗体对抗原的表位的结合。结合亲和力通过术语ka(来自抗体/抗原复合物的抗体的结合速率常数)、kD(解离常数)和KD(kD/ka)限定。结合或特异性结合意指10-8mol/l或更小，优选地10-9至10-13mol/l的结合亲和力(KD)。如此，依照本发明的降钙素原抗体以10-8mol/l或更小，优选地10-9至10-13mol/l的结合亲和力(Kd)特异性结合抗原。
本发明所用的术语“人单克隆抗体”指具有自人种系免疫球蛋白序列衍生的可变和恒定区的展现出单一结合特异性的抗体。在一个实施方案中，人单克隆抗体是由杂交瘤生成的，所述杂交瘤包含与永生化细胞融合的具有包含人重链转基因和人轻链转基因的基因组的转基因非人动物，例如转基因小鼠获得的B细胞。
实施例
实施例1.降钙素原突变体质粒构建及重组蛋白的表达与纯化
1.1实验材料
1.1.1菌株和质粒
大肠杆菌Top10、BL21(DE3)
质粒pET34
1.1.2引物
突变方案：
突变1：
Figure PCTCN2016081545-appb-000001
突变2：
Figure PCTCN2016081545-appb-000002
突变3：
Figure PCTCN2016081545-appb-000003
全基因合成：
突变1：
Figure PCTCN2016081545-appb-000004
突变2：
Figure PCTCN2016081545-appb-000005
突变3：
Figure PCTCN2016081545-appb-000006
设计引物：
突变1：
PCT-TB1-1：
Figure PCTCN2016081545-appb-000007
PCT-TB1-2：
Figure PCTCN2016081545-appb-000008
PCT-TB1-3：
Figure PCTCN2016081545-appb-000009
PCT-TB1-4：
Figure PCTCN2016081545-appb-000010
PCT-TB1-5：
Figure PCTCN2016081545-appb-000011
PCT-TB1-6：
Figure PCTCN2016081545-appb-000012
PCT-TB1-7：
Figure PCTCN2016081545-appb-000013
PCT-TB1-8：
Figure PCTCN2016081545-appb-000014
PCT-TB1-9：
Figure PCTCN2016081545-appb-000015
PCT-TB1-10：
Figure PCTCN2016081545-appb-000016
PCT-TB1-11：
Figure PCTCN2016081545-appb-000017
PCT-TB1-12：
Figure PCTCN2016081545-appb-000018
PCT-TB1-13：
Figure PCTCN2016081545-appb-000019
PCT-TB1-14：
Figure PCTCN2016081545-appb-000020
PCT-TB1-15：
Figure PCTCN2016081545-appb-000021
PCT-TB1-16：
Figure PCTCN2016081545-appb-000022
突变2：
PCT-TB2-1：
Figure PCTCN2016081545-appb-000023
PCT-TB2-2：
Figure PCTCN2016081545-appb-000024
PCT-TB2-3：
Figure PCTCN2016081545-appb-000025
PCT-TB2-4：
Figure PCTCN2016081545-appb-000026
PCT-TB2-5：
Figure PCTCN2016081545-appb-000027
PCT-TB2-6：
Figure PCTCN2016081545-appb-000028
PCT-TB2-7：
Figure PCTCN2016081545-appb-000029
PCT-TB2-8：
Figure PCTCN2016081545-appb-000030
PCT-TB2-9：
Figure PCTCN2016081545-appb-000031
PCT-TB2-10：
Figure PCTCN2016081545-appb-000032
PCT-TB2-11：
Figure PCTCN2016081545-appb-000033
PCT-TB2-12：
Figure PCTCN2016081545-appb-000034
PCT-TB2-13：
Figure PCTCN2016081545-appb-000035
PCT-TB2-14：
Figure PCTCN2016081545-appb-000036
PCT-TB2-15：
Figure PCTCN2016081545-appb-000037
PCT-TB2-16：
Figure PCTCN2016081545-appb-000038
突变3：
PCT-TB3-1：
Figure PCTCN2016081545-appb-000039
PCT-TB3-2：
Figure PCTCN2016081545-appb-000040
PCT-TB3-3：
Figure PCTCN2016081545-appb-000041
PCT-TB3-4：
Figure PCTCN2016081545-appb-000042
PCT-TB3-5：
Figure PCTCN2016081545-appb-000043
PCT-TB3-6：
Figure PCTCN2016081545-appb-000044
PCT-TB3-7：
Figure PCTCN2016081545-appb-000045
PCT-TB3-8：
Figure PCTCN2016081545-appb-000046
PCT-TB3-9：
Figure PCTCN2016081545-appb-000047
PCT-TB3-10：
Figure PCTCN2016081545-appb-000048
PCT-TB3-11：
Figure PCTCN2016081545-appb-000049
PCT-TB3-12：
Figure PCTCN2016081545-appb-000050
PCT-TB3-13：
Figure PCTCN2016081545-appb-000051
PCT-TB3-14：
Figure PCTCN2016081545-appb-000052
PCT-TB3-15：
Figure PCTCN2016081545-appb-000053
PCT-TB3-16：
Figure PCTCN2016081545-appb-000054
1.1.3主要试剂及配制
限制性内切酶BamH I、Xho I，Fast pfu DNA聚合酶，Taq酶购自Takara公司；T4-DNA连接酶购自Promega公司；氨苄抗生素购自上海生工；DL2000 DNA Marker购自金斯瑞公司。琼脂糖凝胶DNA回收试剂盒购自天根生物；质粒提取试剂盒购自Axygen公司；胰蛋白胨(Tryptone)、酵母提取物(Yeast extract)为OXOID产品，IPTG、蛋白Marker购自生兴公司，Ni柱胶(Ni-Sepharose 6 FF)购自GE公司。
Buffer A：20mM Tris，300mM NaCl，20mM Imi，pH 7.4
Buffer B：20mM Tris，300mM NaCl，80mM Imi，pH 7.4
Buffer C：20mM Tris，300mM NaCl，250mM Imi，pH 7.4
1.1.4主要实验仪器
1)低温离心机
2)台式离心机
3)PCR仪
4)核酸电泳仪
5)蛋白电泳仪
6)凝胶成像分析仪
7)超声破碎仪
8)恒温培养震荡器
1.2实验方法
1.2.1目的基因的获取
通过重叠延伸PCR，分别将16条引物连接起来，得到三种突变体的全长。琼脂糖凝胶鉴定(见图1)，用琼脂糖凝胶DNA回收试剂盒对PCR产物进行纯化。
1.2.2质粒及PCR产物的双酶切
1)三种PCR产物酶切
Figure PCTCN2016081545-appb-000055
2)质粒pET34酶切
Figure PCTCN2016081545-appb-000056
分别混匀上述反应液，37℃反应30分钟，电泳鉴定(见图2)，酶切完全后，按照DNA片段纯化试剂盒进行纯化。
1.2.3连接反应
运用DNA连接反应计算器计算，PCR产物与载体DNA按照摩尔比为9:1进行反应，在T4-DNA连接酶的作用下，22℃连接30分钟。
1.2.4转化
将10μl连接产物加于100μl Top10感受态细胞中。用枪头吹打几下，冰浴30分钟，42℃热击90秒，再冰浴2分钟，加入800μl LB培养基。37℃摇床孵育1小时，6000rpm离心30秒，去除约800μl上清，重悬菌体，全部涂氨苄LB平板。
1.2.5菌液PCR
次日长出菌体后，分别挑单菌落
突变1：
Figure PCTCN2016081545-appb-000057
Figure PCTCN2016081545-appb-000058
突变2：
Figure PCTCN2016081545-appb-000059
突变3：
Figure PCTCN2016081545-appb-000060
PCR条件：95℃，3分钟。95℃，30秒、58℃，30秒、72℃，30秒。30个循环。取8μl样品通过琼脂糖凝胶电泳验证(见图3)。
1.2.6质粒提取
pET34-PCT-TB1、pET34-PCT-TB2、pET34-PCT-TB3质粒提取按照试剂盒说明进行，取2μl样品通过琼脂糖凝胶电泳验证(见图4)。
1.2.7测序
将抽提的pET34-PCT-TB1、pET34-PCT-TB2、pET34-PCT-TB3质粒送至金斯瑞公司测序。测序结果与设计序列一致。
1.2.8重转
将测序正确的重组pET34-PCT-TB1、pET34-PCT-TB2、pET34-PCT-TB3质粒分别转化BL21(DE3)感受态。
1.2.9表达
1)活化：挑取含pET34-PCT-TB1、pET34-PCT-TB2、pET34-PCT-TB3的单菌落于3ml LB培养基，培养过夜。
2)扩大培养及诱导表达：向100ml LB液体培养基中，加入上述活化菌液1ml扩大培养，37℃，200rpm震摇2-3小时后，测定细菌OD600吸光度值，待A600nm为0.4～0.6时，加入1mol/L IPTG溶液，使其终浓度分别为1mmol/L，诱导表达4小时，收菌。
1.2.10蛋白纯化
12000rpm离心收集菌体，用20ml Buffer A重悬菌体，冰浴超声20分钟，12000rpm离心20分钟，取上清，过Ni柱纯化，用Buffer A、Buffer B预洗，Buffer C洗脱目的蛋白，透析到PBS中，取透析后样品20μl通过SDS-PAGE电泳鉴定(见图5)。
实施例2.杂交瘤细胞株的制备
2.1免疫小鼠
6周龄Balb/c小鼠3只，用弗氏佐剂和抗原混合乳化后，分别注射小鼠的背部皮下，免疫三次，每次间隔两周，免疫结束后采小鼠眼眶血测效价，效价达到要求的小鼠直接腹腔注射抗原，加强免疫三天后备用。
2.2 SP2/0骨髓瘤细胞培养
DMEM不完全培养内基加入10％的胎牛血清和1％的双抗配制DMEM完全培养基，取15ml完全培养基于T75的细胞培养瓶内，培养SP2/0骨髓瘤细胞，每两天更换一次培养基，待细胞生长达到对数期，细胞量铺满瓶底70％时，更换培养基，第二天备用。
2.3细胞融合
取备用小鼠脾脏于200目无菌筛网中研磨，用DMEM不完全培养基吹洗，将悬液移至50ml无菌离心管内混匀后计数；弃SP2/0培养上清，用DMEM不完全培养基将细胞吹下，移至50ml无菌离心管内混匀后计数；脾细胞和骨髓瘤细胞数目以5:1的比例混合，1500rpm，离心3分钟，弃上清，拍打混匀后37℃水浴加入1ml 50％PEG 1500，1分钟内加完，静止1.5分钟，加DMEM不完全培养基40ml终止反应，1000rpm，10分钟，弃上清，拍匀后同上再清洗一次并离心，弃上清，用HAT培养基(DMEM完全培养基+HAT溶液)重悬，混匀后铺到96孔细胞培养板内，100μl/孔。
实施例3.杂交瘤细胞株的筛选
3.1培养上清检测
融合第7天，融合孔用HT培养基(DMEM完全培养基+HT溶液)半量换液，三天后取上清检测，60μl/孔，ELISA间接法测定上清OD450值，测定值大于对照2.5倍的孔则为阳性孔，标记备用。
3.2细胞亚克隆
用巴氏吸管吹打标记的阳性孔，混匀后取少量计数，取200-500个细胞用DMEM完全培养基进行有限稀释，铺到含有饲养层细胞的96孔细胞培养板内，装液量200μl/孔，第一轮亚克隆完成；5-7天后，再取上清检测，选单克隆细胞堆进行第二轮亚克隆，如此循环，直至筛选到的杂交瘤细胞连续两次整块培养板(有细胞的)为阳性且是单克隆细胞亚克隆所得，则停止亚克隆，同时挑出最后获得的单克隆细胞，于24孔细胞培养板内培养，用于扩培冻存。
3.3冻存细胞
24孔内的杂交瘤细胞铺满80％孔底，巴氏吸管吹匀后转移至细胞培养瓶内，待其铺满80％瓶底准备冻存细胞；将瓶内细胞吹洗下来并转移至50ml无菌离心管内，取少量计数，剩余细胞1500rpm，离心3分钟，弃上清，拍匀后加冻存液(90％胎牛血清+10％DMSO)配制成1×106个/ml细胞悬液，1ml/管分装于2ml冻存管内，放入细胞冻存盒，于-70℃过夜，第二天转移至液氮内保存。
实施例4.单克隆抗体的制备
4.1杂交瘤细胞复苏及培养
从液氮中取出所需的杂交瘤细胞，迅速将冻存管放入37℃水浴，来回震荡促其快速解冻，1分钟完全融化，1500rpm，离心3分钟，弃上清，加入DMEM完全培养基将细胞重悬并转移至细胞培养瓶内培养，复苏完成，显微镜下观察为活细胞则复苏成功，否则需重新复苏。
第二天更换新鲜培养基，之后每隔两天换液，待细胞铺满70％瓶底，则用DMEM完全培养基将细胞吹下，移走一半至新的培养瓶内，补加新鲜培养基培养，原瓶则补液继续培养，如此扩培至所需数量。
4.2制备腹水抗体
待细胞铺满80％瓶底时，用无菌PBS溶液吹洗培养瓶内细胞，收集至50ml无菌离心管内，1500rpm，离心3分钟，弃上清，用PBS重悬细胞，混匀后再次离心，弃上清，用PBS再次重悬，制成2×106个/ml的细胞悬液，备用。
收集细胞前三天，用无菌石蜡油腹腔注射6-8周龄Balb/c雄鼠，0.5ml/只，备用。
用上述细胞悬液腹腔注射备用小鼠，0.5ml/只。
5天后，小鼠产生腹水，用20ml注射器针头插入小鼠腹腔，在针头尾部用离心管收集腹水，无腹水滴出后拔出针头，放回小鼠，隔天再取，如此反复，至小鼠死亡。
所收腹水添加防腐剂于4℃保存，待腹水收集完成后混匀，开始纯化腹水抗体。
4.3腹水抗体纯化
取小鼠腹水，加入等体积的结合缓冲液，混匀，4℃静置1小时，离心(4℃，12000转速，20分钟)，取上清，0.45μm膜抽滤除去颗粒装杂物。
抽滤后的腹水上样于预先用结合缓冲液平衡好的protein A亲和层析柱，流速控制在2mL/分钟。
上样结束后，继续用结合缓冲液进行流洗，至无蛋白流出。
用pH 3.0洗脱液进行洗脱，用预先加入中和缓冲液的离心管收集蛋白含量高的部分，即为纯化单抗；用PBS进行透析后，测定抗体免疫活性，加入防腐剂进行保存备用。
接收完抗体，层析柱用3倍柱体积再生缓冲液进行再生后，20％乙醇清洗，保存于4℃冰箱。
结合缓冲液：0.1M Gly，pH9.0，含3M NaCl；
洗脱缓冲液：0.1M Gly，pH3.0，含0.15M NaCl；
再生缓冲液：0.1M Gly，pH2.0，含0.15M NaCl；
中和缓冲液：1M Tris-HCl，pH8.0，含0.15M NaCl
4.4单克隆抗体的效价
采用间接ELISA法测定抗体效价。取已纯化的抗体，稀释梯度从10-4至10-7。设置复孔3个。抗原包被：用包被液(碳酸盐缓冲液pH9.6，0.05mol/L)将对应的抗原稀释到2ug/ml，加到96孔酶标板，100μl/孔，4℃过夜(或者37℃温育1h)，洗涤3次，拍干。
封闭：用1％BSA进行封闭，200μl/孔，37℃温育1小时，洗涤3次，拍干。
加入待测抗体：稀释好的抗体加到板孔，100μl/孔，37℃温育1h(或4℃过夜)，洗涤3次，拍干。
加入酶标二抗：加入羊抗鼠IgG-HRP，适宜稀释浓度用棋盘滴定法测定，100μl/孔，37℃温育1h，洗涤3次，拍干。
显色：TMB显色液避光放入37℃恒温培养箱调温，100μl/孔，37℃温育10～15分钟。
终止反应：每孔加入50μl 2mol/L的硫酸终止反应。
450nm测定OD值：用酶标仪测出OD值，以空白孔调零，若待测孔OD值超过0.2且大于阴性对照孔的2.1倍，判定为阳性，以最小判定值对应的抗体稀释倍数为抗体的效价，OD值接近1.0时抗体释度最佳抗体稀释度。
效价测定结果如下：
表1
Figure PCTCN2016081545-appb-000061
本领域的技术人员应当明了，尽管为了举例说明的目的，本文描述了本发明的具体实施方式，但可以对其进行各种修改而不偏离本发明的精神和范围。因此，本发明的具体实施方式和实施例不应当视为限制本发明的范围。本发明仅受所附权利要求书的限制。
Figure PCTCN2016081545-appb-000062
Figure PCTCN2016081545-appb-000063
Figure PCTCN2016081545-appb-000064
Figure PCTCN2016081545-appb-000065
Figure PCTCN2016081545-appb-000066
Figure PCTCN2016081545-appb-000067
Figure PCTCN2016081545-appb-000068
Figure PCTCN2016081545-appb-000069
Figure PCTCN2016081545-appb-000070
Figure PCTCN2016081545-appb-000071

Claims (14)

  1. 一种与人源降钙素原特异性结合的抗降钙素原抗体，其中所述抗降钙素原抗体与保藏号为CGMCC No.10417的杂交瘤细胞系产生的降钙素原单克隆抗体特异性结合相同的降钙素原表位。
  2. 根据权利要求1所述的抗降钙素原抗体，其包含保藏号为CGMCC No.10417的杂交瘤细胞系产生的降钙素原单克隆抗体的人源化序列。
  3. 根据权利要求1所述的抗降钙素原抗体，其中所述抗降钙素原抗体是单克隆抗体。
  4. 根据权利要求1所述的抗降钙素原抗体，其中所述抗降钙素原抗体特异性结合具有氨基酸序列为RLLLAALVQDYV(SEQ ID NO:1)的表位。
  5. 根据权利要求1所述的抗降钙素原抗体，其中所述抗降钙素原抗体获得自保藏号为CGMCC No.10417的杂交瘤细胞系。
  6. 含有权利要求1-5任一项所述的抗降钙素原抗体的免疫检测试剂，其用于检测或者定量降钙素原或其片段。
  7. 根据权利要求1-5任一项所述的抗降钙素原抗体的用途，其用于制备检测或者定量降钙素原或其片段的免疫检测试剂的用途。
  8. 根据权利要求1-5任一项所述的抗降钙素原抗体的用途，其用于制备检测或者定量降钙素原或其片段的试剂盒的用途。
  9. 含有权利要求1-5任一项所述的抗降钙素原抗体的试剂盒，其用于检测或者定量降钙素原或其片段。
  10. 一种产生降钙素原单克隆抗体的杂交瘤细胞系，其保藏号为CGMCC No.10417。
  11. 一种制备抗降钙素原抗体的方法，包括以下步骤：
    (1)对降钙素原N端氨基酸残基进行突变，获得与突变后氨基酸残基序列相对应的核苷酸序列；
    (2)将所述核苷酸序列插入到原核表达载体中，进行诱导表达以及蛋白纯化；
    (3)将纯化蛋白作为抗原免疫动物，取其脾细胞与小鼠骨髓瘤细胞融合制备杂交瘤细胞，经筛选获得可稳定分泌抗降钙素原抗体的杂交瘤细胞，并获得相应单克隆抗体。
  12. 根据权利要求11所述的方法，其中所述突变是针对降钙素原第1至57位氨基酸残基进行的。
  13. 根据权利要求11-12任一项所述的方法，其中突变后的降钙素原氨基酸序列为SEQ ID NOs:2-4。
  14. 根据权利要求11-13任一项所述的方法制备的抗体。
PCT/CN2016/081545 2015-07-09 2016-05-10 人源降钙素原的单克隆抗体其制备方法和用途 Ceased WO2017005048A1 (zh)

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