WO2023025169A1 - 一种抗Taq DNA聚合酶的抗体及其应用 - Google Patents

一种抗Taq DNA聚合酶的抗体及其应用 Download PDF

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WO2023025169A1
WO2023025169A1 PCT/CN2022/114352 CN2022114352W WO2023025169A1 WO 2023025169 A1 WO2023025169 A1 WO 2023025169A1 CN 2022114352 W CN2022114352 W CN 2022114352W WO 2023025169 A1 WO2023025169 A1 WO 2023025169A1
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binding protein
seq
antibody
amino acid
dna polymerase
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French (fr)
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孟媛
钟冬梅
何雯雯
杨浩
熊俊文
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东莞市朋志生物科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/40—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum bacterial
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/64—General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes

Definitions

  • the present disclosure relates to the technical field of immunization, in particular, to a recombinant antibody against Taq DNA polymerase and its application.
  • Taq DNA polymerase also known as Taq enzyme
  • Taq DNA polymerase can withstand high temperatures above 90°C without loss. Live, which is of great significance in PCR reactions that require a high temperature environment. Therefore, Taq DNA polymerase replaces the DNA polymerase in Escherichia coli, which was previously commonly used in PCR reactions.
  • the application of Taq DNA polymerase in the PCR reaction does not require adding enzymes in each cycle, which makes the PCR technology very simple and greatly reduces the cost.
  • the PCR technology can be widely used and gradually applied in clinical practice.
  • Taq DNA polymerase also has certain defects in the use process, that is, it also has certain enzymatic properties at room temperature, which leads to non-specific and primer-dimer amplification during PCR amplification. and long-term stability problems.
  • hot-start enzyme technology can make the enzymatic properties of ordinary Taq DNA polymerase qualitatively improved.
  • commonly used methods include antibody-modified hot-start enzymes and chemically modified hot-start enzymes, among which antibody-modified hot-start enzymes are more commonly used.
  • Antibody modification hot start enzyme requires the use of a monoclonal antibody specific to Taq DNA polymerase.
  • the monoclonal antibody specific to Taq DNA polymerase binds to Taq DNA polymerase to form a complex, which can effectively block Taq DNA at room temperature
  • the activity of polymerase prevents it from exerting polymerase activity under low temperature conditions. At high temperature, this complex will dissociate to release active Taq DNA polymerase, and then carry out PCR amplification reaction, which can effectively Avoid the formation of primer dimers, reduce the amplification of non-specific products, and prolong the long-term stability of Taq DNA polymerase.
  • said antigen binding domain comprises at least one complementarity determining region selected from the following amino acid sequences:
  • Complementarity determining region CDR-VH1 which comprises the amino acid sequence shown in SEQ ID NO: 1 or consists of it;
  • Complementarity determining region CDR-VH2 which comprises the amino acid sequence shown in SEQ ID NO: 2 or consists of it;
  • Complementarity determining region CDR-VH3 which comprises the amino acid sequence shown in SEQ ID NO: 3 or consists of it;
  • Complementarity determining region CDR-VL1 which comprises the amino acid sequence shown in SEQ ID NO: 4 or consists of it;
  • Complementarity determining region CDR-VL2 which comprises the amino acid sequence shown in SEQ ID NO: 5 or consists of it;
  • Complementarity determining region CDR-VL3 which comprises or consists of the amino acid sequence shown in SEQ ID NO: 6.
  • the beneficial technical effect of the present disclosure is that the binding protein has strong activity, has a high affinity with Taq DNA polymerase, and can effectively block the activity of Taq DNA polymerase at room temperature.
  • Fig. 1 is the electrophoresis image of the monoclonal antibody against Taq DNA polymerase in Example 1 of the present disclosure.
  • isolated binding protein is a protein which, by virtue of its origin or source of derivation, is not associated with the naturally associated component with which it is associated in its native state; substantially free of other proteins; expressed by cells from a different species; or not found in nature.
  • a protein that is chemically synthesized or synthesized in a cellular system different from the cell of its natural origin will be “isolated” from the components with which it is naturally associated. Proteins can also be rendered substantially free of naturally associated components by isolation, using protein purification techniques well known in the art.
  • antibody includes naturally occurring antibodies as well as non-naturally occurring antibodies including, for example, chimeric, bifunctional, and humanized antibodies, and related synthetic isomeric forms (isoforms).
  • antibody is used interchangeably with "immunoglobulin”.
  • Fab fragment generally refers to a monovalent fragment composed of VL, VH , CL and CH1 domains.
  • F(ab') 2 fragment generally refers to a bivalent fragment comprising two Fab fragments linked by a disulfide bond at the hinge region.
  • Fd fragment generally refers to a fragment composed of VH and CH1 domains.
  • VH variable domain of the heavy chain of an antibody
  • VL variable domain of a light chain
  • CDRs complementarity determining regions
  • the framework regions and the extent of the CDRs have been precisely defined, for example in Kabat (see Sequences of Proteins of Immunological Interest), E. Kabat et al., U.S. Department of Health and Human Services Human Services), (1983)) and Chothia.
  • the framework regions of an antibody, the combined framework regions of the constituent light and heavy chains, serve to position and align the CDRs that are primarily responsible for binding to the antigen.
  • variable region or “variable domain” of an antibody refers to the amino-terminal domain of the heavy or light chain of an antibody.
  • the variable domain of the heavy chain may be referred to as “VH.”
  • the variable domain of the light chain may be referred to as “VL.” These domains are usually the most variable part of an antibody and contain the antigen binding site.
  • the light or heavy chain variable region consists of framework regions interrupted by three hypervariable regions called “complementarity determining regions" or "CDRs.”
  • CDRs complementarity determining regions
  • backbone As used herein, "backbone”, “framework” or “FR” regions mean regions of an antibody variable domain excluding those regions defined as CDRs. Each antibody variable domain framework can be further subdivided into contiguous regions (FR1, FR2, FR3 and FR4) separated by CDRs.
  • variable region VL/VH of the heavy chain and the light chain can be obtained by connecting the CDRs and FRs numbered as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • CDR notation methods commonly used in the field include: Kabat numbering scheme, IMGT, Chothia and Lesk numbering schemes, and a new standardized numbering system introduced by Lefranc et al. in 1997 for all protein sequences of the immunoglobulin superfamily.
  • Kabat et al. were the first to propose a standardized numbering scheme for immunoglobulin variable regions.
  • the accumulation of sequences over the past few decades has led to the creation of the KABATMAN database, and the Kabat numbering scheme is generally considered the widely adopted standard for numbering antibody residues.
  • the present disclosure adopts the Kabat annotation standard to mark the CDR region, but the CDR region marked by other methods also belongs to the protection scope of the present disclosure.
  • Isolated polypeptides include such polypeptides as they are naturally produced contained in cell lysates, in purified or partially purified form, recombinant polypeptides, expressed or secreted by cells, and in heterologous host cells or cultures. of the polypeptide.
  • the term isolated or purified indicates, for example, that the nucleic acid is not in its natural genomic context (eg, in a vector, as an expression cassette, linked to a promoter, or artificially introduced into a heterologous host cell).
  • affinity means the equilibrium constant for the reversible binding of two reagents and is expressed as KD.
  • the affinity of a binding protein for a ligand can be, for example, from about 100 nanomolar (nM) to about 0.1 nM, from about 100 nM to about 1 picomolar (pM), or from about 100 nM to about 1 ⁇ M Mole (fM).
  • the term "avidity” as used herein means the resistance of a complex of two or more agents to dissociation upon dilution. Apparent affinity can be determined by methods such as enzyme-linked immunosorbent assay (ELISA) or any other technique familiar to those skilled in the art.
  • the term “about” generally means within plus or minus 10% or plus or minus 5% of the indicated value.
  • “about 10%” can indicate a range of 9% to 11%, and “about 1” can mean from 0.9 to 1.1.
  • Other meanings of "about” may be apparent from the context, such as rounding, so for example "about 1” could also mean from 0.5 to 1.4.
  • homology refers to sequence similarity between two peptides or between two nucleic acid molecules.
  • identity refers to sequence similarity between two peptides or between two nucleic acid molecules.
  • identity can be determined by comparing positions in each sequence that are aligned for comparison purposes. When equivalent positions in the compared sequences are occupied by the same base or amino acid, then the molecules are identical at that position; When it is occupied, then the molecules can be said to be homologous (similar) at this position.
  • a percentage expression of homology/similarity or identity refers to a function of the number of identical or similar amino acids at positions shared by the compared sequences.
  • Complementarity determining region CDR-VH1 which comprises the amino acid sequence shown in SEQ ID NO: 1 or consists of it;
  • Complementarity determining region CDR-VH2 which comprises the amino acid sequence shown in SEQ ID NO: 2 or consists of it;
  • Complementarity determining region CDR-VH3 which comprises the amino acid sequence shown in SEQ ID NO: 3 or consists of it;
  • Complementarity determining region CDR-VL1 which comprises the amino acid sequence shown in SEQ ID NO: 4 or consists of it;
  • Complementarity determining region CDR-VL2 which comprises the amino acid sequence shown in SEQ ID NO: 5 or consists of it;
  • Complementarity determining region CDR-VL3 which comprises or consists of the amino acid sequence shown in SEQ ID NO: 6.
  • the binding protein comprises at least 3 CDRs (eg, 3 light chain CDRs or 3 heavy chain CDRs).
  • the binding protein comprises at least 6 CDRs.
  • the antigen binding domain has the following 6 CDRs:
  • CDR1 of VH which comprises or consists of the amino acid sequence shown in SEQ ID NO: 1;
  • CDR2 of VH which comprises or consists of the amino acid sequence shown in SEQ ID NO: 2;
  • CDR3 of VH which comprises or consists of the amino acid sequence shown in SEQ ID NO: 3;
  • CDR1 of VL which comprises the amino acid sequence shown in SEQ ID NO: 4 or consists of it;
  • the CDR2 of VL which comprises the amino acid sequence shown in SEQ ID NO: 5 or consists of it;
  • the binding protein is a whole antibody comprising variable and constant regions.
  • the binding protein is one of antibody, F(ab') 2 , Fab', Fab, Fv, scFv, bispecific antibody and antibody minimal recognition unit.
  • the binding protein comprises a sequence as shown in SEQ ID NO: 11-14 or has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, or 99% sequence identity of the light chain framework region FR-L1, FR-L2, FR-L3 and FR-L4, and/or, the sequence is as shown in SEQ ID NO:7-10 or Heavy chain framework regions FR-H1, FR-H2, FR having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith -H3 and FR-H4.
  • the binding protein further comprises antibody constant region sequences.
  • the constant region sequence is selected from any one of the constant region sequences of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD.
  • the light chain constant region sequence is shown in SEQ ID NO: 16;
  • the heavy chain constant region sequence is shown in SEQ ID NO: 15.
  • the binding protein comprises a heavy chain variable region as set forth in SEQ ID NO: 17 and a light chain variable region as set forth in SEQ ID NO: 19.
  • the nucleic acid sequence therein is operably linked to at least one regulatory sequence.
  • "Operably linked” means that a coding sequence is linked to a regulatory sequence in a manner that permits the expression of the coding sequence.
  • Regulatory sequences are selected to direct the expression of the protein of interest in a suitable host cell and include promoters, enhancers and other expression control elements.
  • a vector may refer to a molecule or an agent comprising a nucleic acid of the present disclosure or a fragment thereof, capable of carrying genetic information and delivering the genetic information into a cell.
  • Typical vectors include plasmids, viruses, phage, cosmids and minichromosomes.
  • a vector may be a cloning vector (i.e. a vector used to transfer genetic information into a cell, which can be propagated and selected for the presence or absence of said genetic information) or an expression vector (i.e. a vector containing the necessary genetic elements A vector thereby allowing the genetic information of said vector to be expressed in a cell).
  • cloning vectors may contain a selectable marker and an origin of replication compatible with the cell type for which the cloning vector is intended, while expression vectors may contain the regulatory elements necessary to affect expression in the intended target cell.
  • a nucleic acid of the disclosure or a fragment thereof can be inserted into a suitable vector to form a cloning vector or an expression vector carrying a fragment of the nucleic acid of the disclosure.
  • This new vector is also part of this disclosure.
  • Said vectors may include plasmids, phages, cosmids, minichromosomes or viruses, and also include naked DNA that is only transiently expressed in specific cells.
  • the cloning vectors and expression vectors of the present disclosure are capable of spontaneous replication and thus can provide high copy numbers for high level expression or high level replication purposes for subsequent cloning.
  • the expression vector may include a promoter for driving the expression of the nucleic acid fragment of the present disclosure, an optional nucleic acid sequence encoding a signal peptide for secretion or integration of the peptide expression product into the membrane, the nucleic acid fragment of the present disclosure, and an optional A nucleic acid sequence encoding a terminator.
  • the expression vector When the expression vector is manipulated in a production strain or cell line, the vector may or may not be integrated into the genome of the host cell when introduced into the host cell. Vectors typically carry replication sites, as well as marker sequences that provide phenotypic selection in transformed cells.
  • the present disclosure also provides a host cell transformed with the above-mentioned vector.
  • the expression vectors of the present disclosure are used to transform host cells. Such transformed cells are also part of the present disclosure, and can be cultured cells or cell lines used to propagate the nucleic acid fragments and vectors of the present disclosure, or to recombinantly produce the polypeptides of the present disclosure.
  • Transformed cells of the present disclosure include microorganisms such as bacteria (eg, E. coli, Bacillus, etc.).
  • Host cells also include cells from multicellular organisms such as fungi, insect cells, plant cells or mammalian cells, preferably from mammals, eg CHO cells.
  • the transformed cells are capable of replicating the nucleic acid fragments of the present disclosure.
  • the expression products can be exported into the culture medium or carried on the surface of the transformed cells.
  • the present disclosure also provides a method for producing the above-mentioned binding protein, comprising the following steps:
  • the above-mentioned host cells are cultured in appropriate culture conditions, and the produced binding protein is recovered from the culture medium or from the cultured host cells.
  • the method can be, for example, transfecting a host cell with a nucleic acid vector encoding at least a part of the binding protein, and culturing the host cell under suitable conditions to express the binding protein.
  • Host cells can also be transfected with one or more expression vectors, which alone or in combination contain DNA encoding at least a portion of the binding protein.
  • Bound proteins can be isolated from culture medium or cell lysates using conventional techniques for purification of proteins and peptides, including ammonium sulfate precipitation, chromatography (e.g., ion exchange, gel filtration, affinity chromatography, etc.) and/or Electrophoresis.
  • Suitable vectors containing the coding and regulatory sequences of interest can be performed using standard ligation and restriction techniques well known in the art. Isolated plasmids, DNA sequences or synthetic oligonucleotides are cut, tailed and religated as desired. Mutations may be introduced into the coding sequence by any means, and these mutations may comprise deletions or insertions or substitutions, etc., to produce variants of the present disclosure.
  • the present disclosure also provides the use of the above-mentioned binding protein in PCR.
  • Taq DNA polymerase is used in said PCR and said Taq DNA polymerase is modified by said binding protein.
  • the binding protein is an antibody.
  • the antibody or binding protein-modified Taq enzyme further comprises a Taq enzyme modified by the binding protein.
  • the present disclosure also relates to a composition for DNA sequencing reaction, which comprises the above-mentioned Taq enzyme.
  • restriction endonuclease and Prime Star DNA polymerase were purchased from Takara Company.
  • MagExtractor-RNA extraction kit was purchased from TOYOBO Company.
  • BD SMART TM RACE cDNA Amplification Kit was purchased from Takara Company.
  • the pMD-18T vector was purchased from Takara Company.
  • Plasmid extraction kit was purchased from Tiangen Company. Primer synthesis and gene sequencing were performed by Invitrogen.
  • the VL gene sequence is 333bp, which belongs to the VkII gene family, and there is a 57bp leader peptide sequence in front of it; in the gene fragment amplified by the Heavy Chain primer pair, the VH gene sequence is 366bp, which belongs to the VH1 gene family, and there is 57bp in front of it the leader peptide sequence.
  • the main components of solution A include citric acid + sodium acetate + acetanilide + carbamide peroxide; the main components of solution B include citric acid + EDTA ⁇ 2Na+TMB+ concentrated HCl; the stop solution includes EDTA ⁇ 2Na+ concentrated H 2 SO 4 .
  • the cells were first cultured in a 125ml shaker flask with an inoculation volume of 30ml, the medium was 100% Dynamis medium, and placed in a shaker with a rotation speed of 120r/min, a temperature of 37°C, and 8% carbon dioxide. Cultivate for 72 hours, inoculate and expand the culture at an inoculation density of 500,000 cells/ml. The expansion volume is calculated according to production requirements, and the medium is 100% Dynamis medium. Then every 72h expansion once. When the cell volume meets the production requirements, strictly control the inoculation density to about 500,000 cells/ml for production.
  • Shake flask parameters rotational speed 120r/min, temperature 37°C, carbon dioxide 8%.
  • Fed-batch feeding Feed feeding starts every day when the shake flask is cultured for 72 hours.
  • HyCloneTM Cell BoostTM Feed 7a feeds 3% of the initial culture volume every day, and Feed 7b feeds 1/1000 of the initial culture volume every day. Replenish until the 12th day (feeding on the 12th day).
  • Glucose was supplemented with 3g/L on the sixth day. Receive samples on the 13th day.
  • Affinity purification was performed with a proteinA affinity chromatography column. Take 4 ⁇ g of purified antibody for reducing SDS-PAGE, and 4 ⁇ g of foreign control antibody as a control.
  • the electropherogram is shown in Figure 1. Two bands are shown after reducing SDS-PAGE, one Mr is 50KD (heavy chain), the sequence is as shown in SEQ ID NO:18; the other Mr is 28KD (light chain), the sequence is as shown in SEQ ID NO:20 Show.
  • Example 1 The obtained antibody in Example 1 (having sequences such as the heavy chain variable region and the light chain variable region shown in SEQ ID NO: 17 and SEQ ID NO: 19) was analyzed, and the complementarity determining region of the heavy chain:
  • CDR-VH1 is the amino acid sequence shown in SEQ ID NO: 1;
  • CDR-VH2 is the amino acid sequence shown in SEQ ID NO: 2;
  • CDR-VH3 is the amino acid sequence shown in SEQ ID NO: 3;
  • CDR-VL1 is the amino acid sequence shown in SEQ ID NO: 4;
  • FR-H1, FR-H2, FR-H3 and FR-H4 are shown in SEQ ID NO: 7-10.
  • the purified antibody was diluted to 10ug/ml with PBST, and the Taq enzyme was serially diluted with PBST.
  • Solution A main component citric acid + sodium acetate + acetanilide + carbamide peroxide
  • solution B main component citric acid + EDTA ⁇ 2Na+TMB+concentrated HCl
  • stop solution EDTA ⁇ 2Na+concentrated H 2 SO 4
  • Taq DNA polymerase as the modification object to amplify the template containing DNA, and the template concentrations are 20 copies/ml, 200 copies/ml, 2000 copies/ml and 20000 copies/ml. It can be seen from the results that the amplification effect of Taq DNA polymerase modified with Anti-TAQ 6A7 antibody is significantly better than that of unmodified antibody.
  • NTC no template control, used to monitor the environment, when the negative control without template is not detected, other indicators are meaningful.
  • the disclosure provides an anti-Taq DNA polymerase antibody or binding protein, the anti-Taq DNA polymerase antibody binding protein has strong activity, has a high affinity with Taq DNA polymerase, and can effectively block Taq DNA at room temperature polymerase activity and has excellent stability.

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Abstract

提供了一种包含Taq DNA聚合酶抗原结合结构域的分离的结合蛋白，以及该结合蛋白的制备和应用。所述结合蛋白与Taq DNA聚合酶具有很高的亲和力，可应用于PCR检测领域。

Description

一种抗Taq DNA聚合酶的抗体及其应用
相关申请的交叉引用
本申请要求于2021年8月24日提交中国专利局的申请号为202110973666.5、名称为“一种抗TAQ DNA聚合酶的抗体及其应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本公开涉及免疫技术领域，具体而言，涉及一种抗Taq DNA聚合酶的重组抗体及其应用。
背景技术
1988年,Randall K.Saiki等[1.2]从水生栖热菌(Thermus aquatics)中分离纯化到了Taq DNA聚合酶,Taq DNA聚合酶(也称Taq酶)可以耐受90℃以上的高温而不失活，这在需要高温环境的PCR反应中有着重要意义。因此Taq DNA聚合酶取代了之前常用于PCR反应的大肠埃希菌中的DNA聚合酶。PCR反应中应用Taq DNA聚合酶，不需要每个循环加酶，使PCR技术变得非常简捷，大大降低了成本，PCR技术得以大量应用，并逐步应用于临床。然而Taq DNA聚合酶在使用过程中也存在一定的缺陷，即在常温下，其也具有一定的酶学性质，进而导致在PCR扩增过程中会出现非特异性和引物二聚体的扩增，以及长期稳定性存在问题。
所以，随着PCR技术应用的普及以及对PCR扩增质量要求的提高，不断出现新的方法和技术，其中热启动酶技术的出现得以使普通Taq DNA聚合酶的酶学性质得到质的提升，目前常用的方法有抗体修饰的热启动酶和化学修饰的热启动酶，其中抗体修饰的热启动酶使用较为普遍。
抗体修饰热启动酶是需要用到特异性Taq DNA聚合酶的单克隆抗体，特异性Taq DNA聚合酶的单克隆抗体与Taq DNA聚合酶结合后形成复合物，在室温下可以有效地封闭Taq DNA聚合酶的活性，使其在低温条件下不发挥聚合酶活性，在高温时，这种复合物会解离，释放出具有活性的Taq DNA聚合酶，再进行PCR扩增反应，这样可以有效地避免引物二聚体的形成，降低非特异产物的扩增，同时可以延长Taq DNA聚合酶的长期稳定性。
目前市场上Taq DNA聚合酶的单克隆抗体原料仍存在性能缺陷。
有鉴于此，特提出本公开。
发明内容
本公开涉及一种新颖的包含Taq DNA聚合酶抗原结合结构域的分离的结合蛋白，并对该结合蛋白的制备、应用等方面进行研究。
其中所述抗原结合结构域包括选自下述氨基酸序列的至少一个互补决定区：
互补决定区CDR-VH1，其包含SEQ ID NO：1所示的氨基酸序列或由其组成；
互补决定区CDR-VH2，其包含SEQ ID NO：2所示的氨基酸序列或由其组成；
互补决定区CDR-VH3，其包含SEQ ID NO：3所示的氨基酸序列或由其组成；
互补决定区CDR-VL1，其包含SEQ ID NO：4所示的氨基酸序列或由其组成；
互补决定区CDR-VL2，其包含SEQ ID NO：5所示的氨基酸序列或由其组成；
互补决定区CDR-VL3，其包含SEQ ID NO：6所示的氨基酸序列或由其组成。
本公开的有益技术效果在于，所述结合蛋白活性强，与Taq DNA聚合酶具有很高的亲和力,在室温下可以有效地封闭Taq DNA聚合酶的活性。
附图说明
为了更清楚地说明本公开具体实施方式或现有技术中的技术方案，下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图是本公开的一些实施方式，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为本公开实施例1中抗Taq DNA聚合酶的单克隆抗体电泳图。
具体实施方式
本公开可通过后续对于本公开一些实施方案描述以及其中所包括的实施例的详细内容而更容易被了解。
在进一步叙述本公开之前，应明了本公开不会被局限于所述特定实施方案中，因为这些实施方案必然是多样的。亦应明了本说明书中所使用的用语仅是为了阐述特定实施方案，而非作为限制，因为本公开的范围将会被仅仅界定在所附的权利要求中。
除非本文另有定义，连同本公开使用的科学和技术术语应具有本领域普通技术人员通常理解的含义。术语的含义和范围应当清晰，然而，在任何潜在不明确性的情况下，本文提供的定义优先于任何字典或外来定义。在本申请中，除非另有说明，“或”的使用意味着“和/或”。此外，术语“包括”及其他形式的使用是非限制性的。
一般地，连同本文描述的细胞和组织培养、分子生物学、免疫学、微生物学、遗传学 以及蛋白和核酸化学和杂交使用的命名法和其技术是本领域众所周知和通常使用的那些。除非另有说明，本公开的方法和技术一般根据本领域众所周知，且如各种一般和更具体的参考文献中所述的常规方法来进行，所述参考文献在本说明书自始至终引用和讨论。酶促反应和纯化技术根据制造商的说明书、如本领域通常实现的或如本文所述来进行。连同本文描述的分析化学、合成有机化学以及医学和药物化学使用的命名法、以及其实验室程序和技术是本领域众所周知和通常使用的那些。
为了本公开可以更容易地理解，选择的术语在下文定义。
如本文所用，术语Taq DNA聚合酶也称Taq酶，二者可以互换使用。
术语“氨基酸”表示天然存在或非天然存在的梭基α-氨基酸。术语“氨基酸”用在本申请中可以包括天然存在的氨基酸和非天然存在的氨基酸。天然存在的氨基酸包括丙氨酸(三字母密码：A1a，单字母密码：A)，精氨酸(Arg，R)，天冬酰胺(Asn，N)，天冬氨酸(Asp，D)，半胱氨酸(Cys，c)，谷氨酰胺(G1n，Q)，谷氨酸(G1u，E)，甘氨酸(G1y，G)，组氨酸(His，H)，异亮氨酸(I1e，I)，亮氨酸(Leu，L)，赖氨酸(Lys，K)，甲硫氨酸(Met，M)，苯丙氨酸(Phe，F)，脯氨酸(Pro，P)，丝氨酸(Ser，S)，苏氨酸(Thr，T)，色氨酸(Trp，W)，酪氨酸(Tyr，Y)，和缬氨酸(Va1，V)。非天然存在的氨基酸包括但不限于α-氨基己二酸，氨基丁酸，瓜氨酸，高瓜氨酸，高亮氨酸，高精氨酸，羟基脯氨酸，正亮氨酸，吡啶基丙氨酸，肌氨酸等等。
术语“分离的结合蛋白”是这样的蛋白，其由于衍生起源或来源不与天然结合的组分结合，所述天然结合的组分在其天然状态下与其伴随；基本上不含来自相同物种的其他蛋白；由来自不同物种的细胞表达；或在自然界中不存在。因此，化学合成或在不同于其天然起源的细胞的细胞系统中合成的蛋白将是与其天然结合的组分“分离的”。还可以通过分离，使用本领域众所周知的蛋白纯化技术，使得蛋白基本上不含大然结合的组分。
术语“包括抗原结合结构域的分离的结合蛋白”泛指包含CDR区的一切蛋白/蛋白片段。“抗体”此用语包括多克隆抗体及单克隆抗体以及这些抗体的抗原化合物结合片段，包括Fab、F(ab’) 2、Fd、Fv、scFv、双特异抗体和抗体最小识别单位，以及这些抗体和片段的单链衍生物。抗体的类型可以选择IgG1、IgG2、IgG3、IgG4、IgA、IgM、IgE、IgD。此外，“抗体”此用语包括天然发生的抗体以及非天然发生的抗体，包括例如嵌合型(chimeric)、双功能型(bifunctional)和人源化(humanized)抗体，以及相关的合成异构形式(isoforms)。“抗体”此用语可和“免疫球蛋白”互换使用。
如本文中所使用的，术语Fab片段，通常是指一种由V L、V H、C L和C H1结构域构成的单价片段。术语F(ab’) 2片段通常指一种包含由铰链区处的二硫键连接的两个Fab片段的二价片段。术语Fd片段通常指由V H和C H1结构域构成的片段。
抗体的“可变区”或“可变结构域”是指抗体的重链或轻链的氨基端结构域。重链的可变结构域可以被称为“VH”。轻链的可变结构域可以被称为“VL”。这些结构域通常是抗体的最可变的部分，并含有抗原结合位点。轻链或重链可变区(VL或VH)由被三个称为“互补决定区”或“CDR”的高变区打断的构架区构成。构架区和CDR的范围已被精确定义，例如在Kabat(参见《免疫重要的蛋白质的序列》(Sequences of Proteins of Immunological Interest)，E.Kabat等，美国卫生与人类服务部(U.S.Department of Health and Human Services)，(1983))和Chothia中。抗体的构架区，即构成要件轻链和重链的组合的构架区，起到定位和对齐CDR的作用，所述CDR主要负责与抗原的结合。
尽管Fv片段的2个结构域(VL和VH)由单独的基因编码，使用重组方法，通过合成的连接物可以连接它们，所述连接物使它们能够制成单个蛋白链，其中VL和VH区配对形成单价分子(称作单链Fv(scFv)，“单链Fv”或“sFv”抗体片段包含抗体的VH和VL结构域，在有些实施方案中，所述Fv多肽另外包含在VH和VL结构域之间的多肽连接物，其使sFv能够形成抗原结合所希望的结构。抗体的“可变区”或“可变结构域”是指抗体的重链或轻链的氨基端结构域。重链的可变结构域可以被称为“VH”。轻链的可变结构域可以被称为“VL”。这些结构域通常是抗体的最可变的部分，并含有抗原结合位点。轻链或重链可变区由被三个称为“互补决定区”或“CDR”的高变区打断的构架区构成。抗体的构架区，即构成要件轻链和重链的组合的构架区，起到定位和对齐CDR的作用，所述CDR主要负责与抗原的结合。
当在本文中使用时，“骨架”、“构架”或“FR”区意味着抗体可变结构域的排除被定义为CDR的那些区域之外的区域。每个抗体可变结构域构架可以被进一步细分成被CDR分隔开的毗邻区域(FR1、FR2、FR3和FR4)。
通常情况下，重链和轻链的可变区VL/VH可由以下编号的CDR与FR按如下组合排列连接获得：FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。
本领域常用的CDR标示方法包括：Kabat编号方案、IMGT、Chothia和Lesk编号方案以及1997年Lefranc等人为免疫球蛋白超家族的所有蛋白质序列引入的新的标准化编号系统。Kabat等人是第一个为免疫球蛋白可变区提出标准化编号方案的人。在过去的几十年中，序列的积累导致了KABATMAN数据库的创建，Kabat编号方案通常被认为是编号抗体残基广泛采用的标准。本公开采用Kabat注释标准标示CDR区，但其他方法标示的CDR区也属于本公开的保护范围。
当在本文中使用时，与多肽或核酸相关联的术语“纯化的”或“分离的”是指多肽或核酸不是处于其天然介质中或天然形式下。因此，术语“分离的”包括从其原始环境，例如如果它是天然存在的，从天然环境取出的多肽或核酸。例如，分离的多肽通常不含通常 与其结合或通常与其混合或在溶液中的至少某些蛋白质或其他细胞组分。分离的多肽包括细胞裂解物中包含的天然生产的所述多肽，纯化或部分纯化形式的所述多肽，重组多肽，被细胞表达或分泌的所述多肽，以及在异源宿主细胞或培养物中的所述多肽。与核酸相关联，术语分离的或纯化的指示例如所述核酸不在其天然的基因组背景中(例如在载体中，作为表达盒，连接到启动子，或人工引入到异源宿主细胞中)。
本公开中使用的术语“亲和力”表示2种试剂的可逆结合的平衡常数，并表示为KD。结合蛋白对配体的亲和力(诸如抗体对表位的亲和力)可以是，例如，约100纳摩尔(nM)至约0.1nM、约100nM至约1皮摩尔(pM)或约100nM至约1飞摩尔(fM)。本文使用的术语“亲合力”表示，2种或更多种试剂的复合物在稀释以后对解离的抵抗力。通过诸如酶联免疫吸附测定(ELISA)或本领域技术人员熟悉的任意其它技术等方法，可以测定表观亲和力。
如本文所用，术语“约”通常指的是指示数值的正或负10％或正或负5％之内。例如，“约10％”可以指示9％到11％的范围，并且“约1”可以表示从0.9到1.1。“约”的其他含义可以从上下文显而易见，比如四舍五入，因此，例如“约1”还可以表示从0.5到1.4。
本公开中使用的术语“同源性”或“同一性”或“相似性”是指两个肽之间或两个核酸分子之间的序列相似性。同源性和同一性中的每一个可以通过比较每个序列中的位置来确定，所述序列为了比较目的而进行比对。当所比较的序列中的等同位置被相同的碱基或氨基酸占据时，则所述分子在此位置是相同的；当等同位置被相同或相似氨基酸残基(例如在空间和/或电子性质方面相似的)占据时，则所述分子可称作在此位置是同源的(相似的)。同源性/相似性或同一性的百分比表示是指，在所比较的序列共有的位置处的相同或相似的氨基酸的数目的函数。在比较两个序列中，残基(氨基酸或核酸)的缺失或额外残基的存在，也会降低同一性和同源性/相似性。本公开涉及一种包含抗原结合结构域的分离的结合蛋白，其中所述抗原结合结构域包括选自下述氨基酸序列的至少一个互补决定区：
互补决定区CDR-VH1，其包含SEQ ID NO：1所示的氨基酸序列或由其组成；
互补决定区CDR-VH2，其包含SEQ ID NO：2所示的氨基酸序列或由其组成；
互补决定区CDR-VH3，其包含SEQ ID NO：3所示的氨基酸序列或由其组成；
互补决定区CDR-VL1，其包含SEQ ID NO：4所示的氨基酸序列或由其组成；
互补决定区CDR-VL2，其包含SEQ ID NO：5所示的氨基酸序列或由其组成；
互补决定区CDR-VL3，其包含SEQ ID NO：6所示的氨基酸序列或由其组成。
在一些实施方式中，所述结合蛋白中包含至少3个CDRs(例如3个轻链CDR或3个重链CDR)。
在一些实施方式中，所述结合蛋白包含至少6个CDRs。
在一些实施方式中，所述抗原结合结构域具有以下6个CDRs：
VH的CDR1，其包含SEQ ID NO：1所示的氨基酸序列或由其组成；
VH的CDR2，其包含SEQ ID NO：2所示的氨基酸序列或由其组成；
VH的CDR3，其包含SEQ ID NO：3所示的氨基酸序列或由其组成；
VL的CDR1，其包含SEQ ID NO：4所示的氨基酸序列或由其组成；
VL的CDR2，其包含SEQ ID NO：5所示的氨基酸序列或由其组成；
VL的CDR3，其包含SEQ ID NO：6所示的氨基酸序列或由其组成。
在一些实施方式中，所述结合蛋白为包含可变区和恒定区的完整抗体。
在一些实施方式中，所述结合蛋白为抗体、F(ab’) 2、Fab’、Fab、Fv、scFv、双特异抗体和抗体最小识别单位中的一种。
在一些实施方式中，所述结合蛋白包括序列依次如SEQ ID NO:11-14所示或与其具有至少90％同源性的轻链骨架区FR-L1、FR-L2、FR-L3及FR-L4，和/或，序列依次如SEQ ID NO:7-10所示或与其具有至少90％同源性的重链骨架区FR-H1、FR-H2、FR-H3及FR-H4。
在可选的实施方式中，所述结合蛋白包括序列依次如SEQ ID NO:11-14所示或与其具有90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％的序列同一性的轻链骨架区FR-L1、FR-L2、FR-L3及FR-L4，和/或，序列依次如SEQ ID NO:7-10所示或与其具有90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％的序列同一性的重链骨架区FR-H1、FR-H2、FR-H3及FR-H4。
在一些实施方式中，所述结合蛋白还包含抗体恒定区序列。
在一些实施方式中，所述恒定区序列选自IgG1、IgG2、IgG3、IgG4、IgA、IgM、IgE、IgD任何其中之一恒定区的序列。
在一些实施方式中，所述恒定区的种属来源为牛、马、乳牛、猪、绵羊、山羊、大鼠、小鼠、狗、猫、兔、驴、鹿、貂、鸡、鸭、鹅或人。
在一些实施方式中，所述恒定区来源于小鼠；
轻链恒定区序列如SEQ ID NO：16所示；
重链恒定区序列如SEQ ID NO：15所示。
在一些实施方式中，所述结合蛋白包括如SEQ ID NO:17所示的重链可变区以及如SEQ ID NO:19所示的轻链可变区。
在一些实施方式中，所述结合蛋白包括如SEQ ID NO:18所示的重链以及如SEQ ID NO:20所示的轻链。
另一方面，本公开还提供了一种分离的核酸分子，所述核酸分子为DNA或RNA，其 编码如上所述的结合蛋白。
在本文中，核酸包含其保守置换的变体(例如简并密码子的置换)和互补序列。术语“核酸”和“多核苷酸”是同义的，包含基因、cDNA分子、mRNA分子以及它们的片段例如寡核苷酸。
根据本公开的一方面，本公开还提供了一种载体，所述载体包含上述的核酸分子。
其中的核酸序列与至少一种调节序列可操作连接。“可操作连接”指的是编码序列以允许编码序列的表达的方式与调节序列连接。调节序列选择用来在合适的宿主细胞中指导目的蛋白质的表达，包含启动子、增强子和其它的表达调控元件。
在本文中，载体可以指包含本公开的核酸或其片段的、能够携带遗传信息并且可以将遗传信息递送到细胞中的分子或试剂。典型的载体包括质粒、病毒、噬菌体、黏粒和微型染色体。载体可以是克隆载体(即用于将遗传信息转移到细胞中的载体，可以繁殖所述细胞并且可以选择存在或不存在所述遗传信息的所述细胞)或表达载体(即包含必要的遗传元件从而允许所述载体的遗传信息在细胞中表达的载体)。因此，克隆载体可以包含选择标记，以及与所述克隆载体所指定的细胞类型相匹配的复制起点，而表达载体则包含对于影响指定靶细胞中的表达必要的调节元件。
本公开的核酸或其片段可以插入到合适的载体中以形成携带本公开核酸片段的克隆载体或表达载体。这种新载体也是本公开的一部分。所述载体可以包括质粒、噬菌体、黏粒、微型染色体或病毒，也包括只在特定细胞中瞬时表达的裸DNA。本公开克隆载体和表达载体能够自发的复制，因此能够为用于随后克隆的高水平表达或高水平复制目的提供高拷贝数。表达载体可以包括用于驱动本公开的核酸片段表达的启动子，可选的编码使所述肽表达产物分泌或整合到膜上的信号肽的核酸序列，本公开的核酸片段，以及可选的编码终止子的核酸序列。当在生产菌株或细胞系中操作表达载体时，载体引入到宿主细胞中时可以整合到宿主细胞的基因组中，也可以不被整合到宿主细胞基因组中。载体通常携带复制位点，以及能够在转化细胞中提供表型选择的标记序列。
另一方面，本公开还提供了一种宿主细胞，其被如上所述的载体转化。
本公开的表达载体用于转化宿主细胞。这种转化细胞也是本公开的一部分，可以是用于增殖本公开的核酸片段和载体、或用于重组制备本公开的多肽的培养细胞或细胞系。本公开的转化细胞包括微生物如细菌(如大肠杆菌、芽抱杆菌等)。宿主细胞也包括来自多细胞生物如真菌、昆虫细胞、植物细胞或哺乳动物细胞，优选来自哺乳动物的细胞，例如CHO细胞。所述转化细胞能够复制本公开的核酸片段。当重组制备本公开的肽组合时，所述表达产物可以输出到培养基中或携带在所述转化细胞的表面。
根据本公开的一方面，本公开还提供了一种生产上述结合蛋白的方法，包括如下步骤：
在合适的培养条件中培养上述的宿主细胞，从培养基中或从所培养的宿主细胞中回收产生的结合蛋白。
所述方法可以是例如，用编码至少一部分结合蛋白的核酸载体转染宿主细胞，在合适的条件下培养该宿主细胞使其表达该结合蛋白。宿主细胞也可以用一个或多个表达载体转染，该表达载体可以单独或结合地包含编码至少一部分结合蛋白的DNA。利用常规的纯化蛋白质和肽的技术可从培养基或细胞裂解物中分离结合蛋白，所述技术包括硫酸铵沉淀，层析(如离子交换，凝胶过滤，亲合层析等)和/或电泳。
构建合适的含有目的编码和调控序列的载体可以使用本领域公知的标准连接和限制技术进行。将分离的质粒、DNA序列或合成的寡核苷酸按需要的形式切割、加尾和再连接。可以用任何方法向编码序列中引入突变以产生本公开的变体，这些突变可以包含缺失或插入或置换等。
根据本公开的一方面，本公开还提供了如上所述的结合蛋白在PCR中的应用。
在一些实施方式中，在所述PCR中使用Taq DNA聚合酶，并且所述Taq DNA聚合酶被所述结合蛋白修饰。
根据本公开的一方面，本公开还涉及一种抗体修饰的Taq酶，其包括如上所述的结合蛋白。
根据本公开的一方面，本公开还涉及一种结合蛋白修饰的Taq DNA聚合酶，其包括如上所述的结合蛋白。
在一些实施方式中，所述结合蛋白是抗体。
在一些实施方式中，所述抗体或结合蛋白修饰的Taq酶还包括Taq酶，所述Taq酶被所述结合蛋白修饰。
根据本公开的一方面，本公开还涉及一种用于DNA测序反应的组合物，其包含有如上所述的Taq酶。
根据本公开的一方面，本公开还涉及一种试剂或试剂盒，其包含有如上所述的结合蛋白、如上所述的修饰的Taq酶、或如上所述的组合物。下面将结合实施例对本公开的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本公开，而不应视为限制本公开的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。
实施例1
本实施例提供了一种抗Taq DNA聚合酶的重组抗体的示例性制备方法。
1.表达质粒构建
本实施例中限制性内切酶、Prime Star DNA聚合酶购自Takara公司。MagExtractor-RNA提取试剂盒购自TOYOBO公司。BD SMART TM RACE cDNA Amplification Kit试剂盒购自Takara公司。pMD-18T载体购自Takara公司。质粒提取试剂盒购自天根公司。引物合成和基因测序由Invitrogen公司完成。
1.1、Anti-Taq抗体基因制备
从由发明人所在实验室制备的分泌Anti-Taq单克隆抗体的杂交瘤细胞株中提取mRNA，通过RT-PCR方法获得DNA产物，该产物用rTaq DNA聚合酶进行加A反应后插入到pMD-18T载体中，转化到DH5α感受态细胞中，长出菌落后分别取Heavy Chain及Light Chain基因克隆各4个克隆送基因测序公司进行测序。
1.2、Anti-Taq6A7抗体可变区基因的序列分析
将上述测序得到的基因序列放在Kabat抗体数据库中进行分析，并利用VNTI11.5软件进行分析确定重链和轻链引物对扩增出的基因都是正确的，其中Light Chain扩增出的基因片段中，VL基因序列为333bp，属于VkII基因家族，其前方有57bp的前导肽序列；Heavy Chain引物对扩增出的基因片段中，VH基因序列为366bp，属于VH1基因家族，其前方有57bp的前导肽序列。
1.3、重组抗体表达质粒的构建
pcDNA TM 3.4
Figure PCTCN2022114352-appb-000001
vector为构建的重组抗体真核表达载体，该表达载体已经引入HindIII、BamHI、EcoRI等多克隆酶切位点，并命名为pcDNA3.4A表达载体，后续简称3.4A表达载体；根据上述pMD-18T中抗体可变区基因测序结果，设计Anti-Taq 6A7抗体的VL和VH基因特异性引物，两端分别带有HindIII、EcoRI酶切位点和保护碱基，通过PCR扩增方法扩出0.73KB的Light Chain基因片段和1.43kb的Heavy Chain基因片段。
Heavy Chain和Light Chain基因片段分别采用HindIII/EcoRI双酶切，3.4A载体采用HindIII/EcoRI双酶切，将片段和载体纯化回收后Heavy Chain基因和Light Chain基因分别连接3.4A表达载体中，分别得到Heavy Chain和Light Chain的重组表达质粒。
2.稳定细胞株筛选
2.1重组抗体表达质粒瞬时转染CHO细胞，确定表达质粒活性
质粒用超纯水稀释至40ug/100ul，调节CHO细胞1.43×10 7cells/ml于离心管中，100ul质粒与700ul细胞混合，转入电转杯，电转，第3、5、7天取样计数，第7天收样检测。
包被液(主要成分NaHCO 3)稀释Taq酶至1ug/ml，每孔100uL，4℃过夜；次日，洗涤液(主要成份Na 2HPO 4+NaCl)清洗2次，拍干；加入封闭液(20％BSA+80％PBS)，每孔120uL，37℃，1h，拍干；加入稀释后的细胞上清，100uL/孔，37℃,30min(部分上清1h)；洗涤液清洗5次，拍干；加入羊抗鼠IgG-HRP，每孔100uL，37℃,30min；洗涤液清洗5 次，拍干；加入显色液A液(50uL/孔)，加入显色液B液(50uL/孔)，10min；加入终止液，50uL/孔；酶标仪上450nm(参考630nm)处读OD值。结果显示细胞上清稀释1000倍后反应OD仍大于1.0，未加细胞上清孔反应OD小于0.1，表明质粒瞬转后产生的抗体对Taq酶有活性。
备注：A液主要成份包括柠檬酸+醋酸钠+乙酰苯胺+过氧化脲；B液主要成份包括柠檬酸+EDTA·2Na+TMB+浓HCl；终止液包括EDTA·2Na+浓H 2SO 4。
2.2重组抗体表达质粒线性化
准备下述试剂：Buffer 50ul、DNA 100ug/管、PvuⅠ酶10ul、无菌水补至500ul，37℃水浴酶切过夜；先用等体积酚/氯仿/异戊醇(下层)25:24:1，再用氯仿(水相)依次进行抽提；0.1倍体积(水相)3M醋酸钠和2倍体积乙醇冰上沉淀，70％乙醇漂洗沉淀，去除有机溶剂，待乙醇挥发完全用适量的灭菌水进行复融，最后进行浓度的测定。
2.3重组抗体表达质粒稳定转染，加压筛选稳定细胞株
质粒用超纯水稀释至40ug/100ul，调节CHO细胞1.43×10 7cells/ml于离心管中，100ul质粒与700ul细胞混合，转入电转杯，电转，次日计数；25umol/L MSX 96孔加压培养约25天。
显微镜下观察标记长有细胞的克隆孔,并记录汇合度；取培养上清，送样检测；挑选抗体浓度、相对浓度高的细胞株转24孔，3天左右转6孔；3天后保种批培，调整细胞密度0.5×10 6cells/ml，2.2ml进行批培养，细胞密度0.3×10 6cells/ml，2ml进行保种；7天6孔批培上清送样检测，挑选抗体浓度及细胞直径较小的细胞株转TPP保种传代。3.重组抗体生产
3.1细胞扩培
细胞复苏之后先在125ml规格的摇瓶中培养，接种体积为30ml，培养基为100％Dynamis培养基，放置于转速120r/min，温度为37℃，二氧化碳为8％的摇床中。培养72h，以50万cells/ml接种密度接种扩培，扩培体积根据生产需求进行计算，培养基为100％Dynamis培养基。之后每72h扩培一次。当细胞量满足生产需求时，严格控制接种密度为50万cells/ml左右进行生产。
3.2摇瓶生产及纯化
摇瓶参数：转速120r/min，温度为37℃，二氧化碳为8％。流加补料：在摇瓶中培养至72h时开始每天补料，HyCloneTM Cell BoostTM Feed 7a每天流加初始培养体积的3％，Feed 7b每天流加量为初始培养体积的千分之一，一直补到第12天(第12天补料)。葡萄糖在第六天补加3g/L。第13天收样。用proteinA亲和层析柱进行亲和纯化。取4μg纯化的抗体进行还原性SDS-PAGE，4μg外来对照抗体作为对照，电泳图如图1所示。在还原性 SDS-PAGE后显示两条带，1条Mr为50KD(重链)，序列如SEQ ID NO:18所示；另一条Mr为28KD(轻链)，序列如SEQ ID NO:20所示。
实施例2
抗体亲和力分析及活性鉴定
得到的实施例1中的抗体(具有序列如SEQ ID NO:17以及SEQ ID NO:19所示的重链可变区和轻链可变区)经分析，重链的互补决定区：
CDR-VH1，为SEQ ID NO：1所示的氨基酸序列；
CDR-VH2，为SEQ ID NO：2所示的氨基酸序列；
CDR-VH3，为SEQ ID NO：3所示的氨基酸序列；
轻链的互补决定区：
CDR-VL1，为SEQ ID NO：4所示的氨基酸序列；
CDR-VL2，为SEQ ID NO：5所示的氨基酸序列；
CDR-VL3，为SEQ ID NO：6所示的氨基酸序列。
轻链骨架区FR-L1、FR-L2、FR-L3及FR-L4，序列依次如SEQ ID NO:11-14所示；
重链骨架区FR-H1、FR-H2、FR-H3及FR-H4，序列依次如SEQ ID NO:7-10所示。
1.亲和力分析
利用AMC传感器，纯化出来的抗体用PBST稀释到10ug/ml，Taq酶用PBST进行梯度稀释。
运行流程：缓冲液1(PBST)中平衡60s，抗体溶液中固化抗体300s，缓冲液2(PBST)中孵育180s，抗原溶液中结合420s，缓冲液2中解离1200s，用10mM pH 1.69 GLY溶液及缓冲液3进行传感器再生，输出数据。(KD表示平衡解离常数即亲和力；kon表示结合速率；kdis表示解离速率。PBST主要成分Na 2HPO 4+NaCl+TW-20),表1即为所得亲和力检测数据。
表1亲和力检测数据
样品名称 KD(M) kon(1/Ms) kdis(1/s)
对照 2.03E-09 4.92E+03 1.00E-05
Anti-TAQ 6A7 1.10E-11 1.13E+06 1.24E-05
2.活性鉴定
包被液(主要成分NaHCO 3)稀释Taq酶至1ug/ml，每孔100uL，4℃过夜；次日，洗涤液(主要成份Na 2HPO 4+NaCl)清洗2次，拍干；加入封闭液(20％BSA+80％PBS)，每孔 120uL，37℃，1h，拍干；加入稀释后的纯化抗体和对照抗体，100uL/孔，37℃,30min；洗涤液清洗5次，拍干；加入羊抗鼠IgG-HRP，每孔100uL，37℃,30min；洗涤液清洗5次，拍干；加入显色液A液(50uL/孔)，加入显色液B液(50uL/孔)，10min；加入终止液，50uL/孔；酶标仪上450nm(参考630nm)处读OD值,表2即为所得抗体活性分析数据。
备注：A液(主要成份柠檬酸+醋酸钠+乙酰苯胺+过氧化脲)；B液(主要成份柠檬酸+EDTA·2Na+TMB+浓HCl)；终止液(EDTA·2Na+浓H 2SO 4)
表2抗体活性分析数据
样品浓度(ng/ml) 6.25 1.56 0.78 0.39 0.20 0
对照 2.099 1.367 0.748 0.440 0.255 0.071
Anti-TAQ 6A7 2.191 1.878 1.072 0.640 0.372 0.071
3.稳定性分析
裸抗稳定性考核：
将自产抗体置于4℃(冰箱)、-80℃(冰箱)、37℃(恒温箱)放置21天，取7天、14天、21天样品进行状态观察，并对21天样品进行活性检测，结果显示三种考核条件下抗体放置21天均未见明显蛋白状态变化，活性也未随考核温度的升高呈下降趋势，说明自产抗体稳定。表3为考核21天的酶免活性检测OD结果。
表3抗体稳定性检测数据
样品浓度(ng/ml) 1.56 0.39 0
4℃，21天样品 1.818 0.413 0.063
-80℃，21天样品 1.899 0.419 0.075
37℃，21天样品 1.871 0.440 0.071
4.PCR扩增性能评估
以Taq DNA聚合酶为修饰对象，扩增含有DNA的模板，模板浓度分别为20拷贝/ml、200拷贝/ml、2000拷贝/ml和20000拷贝/ml。由结果可知，Taq DNA聚合酶使用Anti-TAQ 6A7抗体修饰后扩增效果明显优于无修饰抗体。
表4 PCR扩增的CT值
Figure PCTCN2022114352-appb-000002
Figure PCTCN2022114352-appb-000003
5.抗体与酶混合物的稳定性考核
将上述制备的抗体与Taq DNA聚合酶制备成混合物，将该混合物放置于37℃处理7天后，以E.coli的基因组为模板，测定其在不同模板浓度下进行PCR扩增的CT值，结果如下表5所示。结果显示37℃加压考核7天后CT值与0天的相比无明显差异，说明该抗体与酶混合物稳定性良好。在同样的方法下测定对照抗体与酶混合物的稳定性。结果显示Anti-TAQ 6A7与Taq DNA聚合酶的混合物的稳定性明显好于对照抗体与Taq DNA聚合酶的混合物的稳定性。
表5
  0天 37℃-7天
100拷贝/mL 36.12 36.15
100拷贝/mL 36.14 36.02
100拷贝/mL 36.02 36.18
1,000拷贝/mL 32.82 32.88
1,000拷贝/mL 32.66 32.81
1,000拷贝/mL 32.61 32.62
10,000拷贝/mL 29.43 29.34
10,000拷贝/mL 29.45 29.44
10,000拷贝/mL 29.32 29.31
100,000拷贝/mL 25.59 25.40
100,000拷贝/mL 25.43 25.41
100,000拷贝/mL 25.34 25.52
NTC No CT No CT
NTC No CT No CT
NTC No CT No CT
NTC：no template control，用来监测环境，无模板的阴性对照无检出时，其他的指标才有意义。
最后应说明的是：以上各实施例仅用以说明本公开的技术方案，而非对其限制；尽管参照前述各实施例对本公开进行了详细的说明，但本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进 行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本公开各实施例技术方案的范围。
工业实用性
本公开提供一种抗Taq DNA聚合酶的抗体或结合蛋白，所述抗Taq DNA聚合酶的抗体结合蛋白活性强，与Taq DNA聚合酶具有很高的亲和力,在室温下可以有效地封闭Taq DNA聚合酶的活性，并且具有优异的稳定性。

Claims (15)

  1. 一种包含Taq DNA聚合酶抗原结合结构域的分离的结合蛋白，其特征在于，其中所述抗原结合结构域包括选自下述氨基酸序列的至少一个互补决定区：
    互补决定区CDR-VH1，其包含SEQ ID NO：1所示的氨基酸序列或由其组成；
    互补决定区CDR-VH2，其包含SEQ ID NO：2所示的氨基酸序列或由其组成；
    互补决定区CDR-VH3，其包含SEQ ID NO：3所示的氨基酸序列或由其组成；
    互补决定区CDR-VL1，其包含SEQ ID NO：4所示的氨基酸序列或由其组成；
    互补决定区CDR-VL2，其包含SEQ ID NO：5所示的氨基酸序列或由其组成；
    互补决定区CDR-VL3，其包含SEQ ID NO：6所示的氨基酸序列或由其组成。
  2. 如权利要求1所述包含抗原结合结构域的分离的结合蛋白，其特征在于，所述结合蛋白中包括至少3个CDRs；或者，所述结合蛋白包括至少6个CDRs；
    优选的，所述结合蛋白为抗体、F(ab’)2、Fab’、Fab、Fv、scFv、双特异抗体和抗体最小识别单位中的一种；
    优选的，所述结合蛋白包括序列依次如SEQ ID NO:11-14所示或与其具有至少90％同源性的轻链骨架区FR-L1、FR-L2、FR-L3及FR-L4，和/或，序列依次如SEQ ID NO:7-10所示或与其具有至少90％同源性的重链骨架区FR-H1、FR-H2、FR-H3及FR-H4。
  3. 如权利要求1或2所述包含抗原结合结构域的分离的结合蛋白，其特征在于，所述结合蛋白还包含抗体恒定区序列；
    优选的，所述恒定区序列选自IgG1、IgG2、IgG3、IgG4、IgA、IgM、IgE、IgD任何其中之一恒定区的序列；
    优选的，所述恒定区的种属来源为牛、马、乳牛、猪、绵羊、山羊、大鼠、小鼠、狗、猫、兔、驴、鹿、貂、鸡、鸭、鹅或人；
    优选的，所述恒定区来源于小鼠；
    轻链恒定区序列如SEQ ID NO：16所示；
    重链恒定区序列如SEQ ID NO：15所示。
  4. 如权利要求1或2所述包含抗原结合结构域的分离的结合蛋白，其特征在于，所述结合蛋白包括如SEQ ID NO:17所示的重链可变区以及如SEQ ID NO:19所示的轻链可变区。
  5. 如权利要求1-3中任一项所述包含抗原结合结构域的分离的结合蛋白，其特征在于，所述结合蛋白包括如SEQ ID NO:18所示的重链以及如SEQ ID NO:20所示的轻链。
  6. 一种分离的核酸分子，其特征在于，所述核酸分子为DNA或RNA，其编码权利要求1～5任一项所述的结合蛋白。
  7. 一种载体，其包含权利要求6所述的核酸分子。
  8. 一种宿主细胞，其特征在于，所述宿主细胞包括权利要求6所述的核酸或权利要求7所述的载体；
    优选的，所述宿主细胞为哺乳动物细胞。
  9. 一种生产权利要求1～5任一项所述的结合蛋白的方法，其特征在于，包括如下步骤：在合适的培养条件下培养权利要求8所述的宿主细胞，从培养基中或从所培养的宿主细胞中回收产生的结合蛋白。
  10. 权利要求1～5任一项所述的结合蛋白在PCR中的应用。
  11. 权利要求10所述的应用，其中在所述PCR中使用Taq DNA聚合酶，并且所述Taq DNA聚合酶被所述结合蛋白修饰。
  12. 一种抗体修饰的Taq酶，其包括权利要求1～5任一项所述的结合蛋白。
  13. 根据权利要求12所述的抗体修饰的Taq酶，其还包括Taq酶，所述Taq酶被所述结合蛋白修饰。
  14. 一种用于PCR反应的组合物，其包含有如权利要求12或13所述的抗体修饰的Taq酶。
  15. 一种试剂或试剂盒，其包含如权利要求1～5任一项所述的结合蛋白、权利要求12～13任一所述的修饰的Taq酶、或如权利要求14所述的组合物。
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