WO2020125135A1 - 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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WO2020125135A1
WO2020125135A1 PCT/CN2019/109791 CN2019109791W WO2020125135A1 WO 2020125135 A1 WO2020125135 A1 WO 2020125135A1 CN 2019109791 W CN2019109791 W CN 2019109791W WO 2020125135 A1 WO2020125135 A1 WO 2020125135A1
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complementarity determining
determining region
region cdr
cdr
binding protein
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PCT/CN2019/109791
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French (fr)
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崔鹏
何志强
孟媛
钟冬梅
杨浩
梁碧
游辉
马秋燕
李蔚芝
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东莞市朋志生物科技有限公司
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Priority to JP2021526467A priority Critical patent/JP2022511718A/ja
Priority to EP19898854.5A priority patent/EP3865508A4/en
Priority to US17/293,045 priority patent/US20210403601A1/en
Priority to KR1020217014548A priority patent/KR20210119374A/ko
Publication of WO2020125135A1 publication Critical patent/WO2020125135A1/zh

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    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
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    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
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    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
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    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)
    • G01N2333/922—Ribonucleases (RNAses); Deoxyribonucleases (DNAses)

Definitions

  • the present disclosure relates to the fields of biotechnology and medical technology, in particular to an antibody against Taq DNA polymerase and its application.
  • Taq DNA polymerase also has certain defects during use, that is, at room temperature, it also has certain enzymatic properties, which in turn leads to non-specific amplification and primer dimer formation during PCR amplification , And problems with long-term stability.
  • hot-start enzyme technology can qualitatively improve the enzymatic properties of ordinary Taq DNA polymerase.
  • Currently commonly used methods are antibody-modified hot-start enzymes and chemically-modified hot-start enzymes, of which antibody-modified hot-start enzymes are more commonly used.
  • Antibody-modified hot-start enzyme is a monoclonal antibody that requires specific Taq enzyme.
  • the monoclonal antibody specific for Taq enzyme combines with Taq DNA polymerase to form an antigen-antibody complex, which can effectively block Taq DNA polymerase at room temperature.
  • the activity of the polymerase does not exert polymerase activity under low temperature conditions. At high temperatures, this complex will dissociate, releasing active Taq DNA polymerase, and then performing PCR amplification reaction, which can effectively avoid primers
  • the formation of dimers reduces the amplification of non-specific products and at the same time improves the long-term stability of Taq DNA polymerase.
  • Antibodies to modify the specific Taq enzyme used in hot-start enzymes need to be further developed.
  • the present disclosure relates to a novel isolated binding protein containing the Taq DNA polymerase antigen binding domain, and studies on the preparation and application of the binding protein.
  • the antigen binding domain includes at least one complementarity determining region selected from the following amino acid sequence, or has at least 80% sequence identity with the complementarity determining region of the following amino acid sequence and has K D ⁇ 8.568 ⁇ with Taq DNA polymerase 10 -9 mol/L affinity;
  • the complementarity determining region CDR-VH1 is S-V-X1-T-F-X2-T-Y-Y-X3-Y, where,
  • X1 is D, E or N, X2 is S or T, X3 is I or L;
  • the complementarity determining region CDR-VH2 is G-X1-N-P-T-S-X2-P-V-F-X3-E-K, where,
  • X1 is I, V or L, X2 is N or GG, X3 is D, E or N;
  • the complementarity determining region CDR-VH3 is T-R-S-X1-X2-R-R-G-Y-Y-X3-D-Y, where,
  • X1 is I, V or L
  • X2 is I, V or L
  • X3 is F or P
  • the complementarity determining region CDR-VL1 is R-X1-S-Q-D-I-X2-N-Y-X3-N, where,
  • X1 is A or G
  • X2 is N or Q
  • X3 is I, V or L;
  • the complementarity determining region CDR-VL2 is I-Y-X1-T-S-R-L-X2-S-G-X3-P, where,
  • X1 is Y or F
  • X2 is Q
  • H or N
  • X3 is I, V or L;
  • the complementarity determining region CDR-VL3 is Q-D-D-T-X1-P-X2-T-X3-G, where,
  • X1 is I, V or L
  • X2 is I, V or L
  • X3 is W or F.
  • binding protein has strong activity and has a high affinity with Taq DNA polymerase.
  • X3 is F
  • X1 is A
  • X1 is Y
  • X3 is F.
  • X1 is D and X2 is S.
  • X1 is E and X2 is S.
  • X1 is N and X2 is S.
  • X1 is D and X2 is T.
  • X1 is E and X2 is T.
  • X1 is N and X2 is T.
  • X1 is I and X2 is N.
  • X1 is I and X2 is GG.
  • X1 is V and X2 is N.
  • X1 is V and X2 is GG.
  • X1 is L and X2 is N.
  • X1 is L and X2 is GG.
  • X1 is I and X2 is I.
  • X1 is I and X2 is V.
  • X1 is I and X2 is L.
  • X1 is V and X2 is I.
  • X1 is V and X2 is V.
  • X1 is V and X2 is L.
  • X1 is L and X2 is I.
  • X1 is L and X2 is V.
  • X1 is L and X2 is L.
  • X2 is N and X3 is I.
  • X2 is N and X3 is V.
  • X2 is N and X3 is L.
  • X2 is Q and X3 is I.
  • X2 is Q and X3 is V.
  • X2 is Q and X3 is L.
  • X2 is Q and X3 is I.
  • X2 is Q and X3 is V.
  • X2 is Q and X3 is L.
  • X2 is H and X3 is I.
  • X2 is H and X3 is V.
  • X2 is H and X3 is L.
  • X2 is N and X3 is I.
  • X2 is N and X3 is V.
  • X2 is N and X3 is L.
  • X1 is I and X2 is I.
  • X1 is I and X2 is V.
  • X1 is I and X2 is L.
  • X1 is V and X2 is I.
  • X1 is V and X2 is V.
  • X1 is V and X2 is L.
  • X1 is L and X2 is I.
  • X1 is L and X2 is V.
  • X1 is L and X2 is L.
  • the mutation site of each complementarity determining region is selected from any one of the following mutation combinations:
  • the binding protein includes at least 3 CDRs; alternatively, the binding protein includes at least 6 CDRs.
  • the binding protein is a complete antibody comprising variable and constant regions.
  • the binding protein is one of Nanobody, F(ab')2, Fab', Fab, Fv, scFv, bispecific antibody, and antibody minimum recognition unit.
  • the binding protein includes light chain framework regions FR-L1, FR-L2, FR-L3, and FR-L4 whose sequence is as shown in SEQ ID NO: 1-4, and/or Or, the sequence is as shown in SEQ ID NO: 5-8 heavy chain framework regions FR-H1, FR-H2, FR-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 species source of the constant region is cattle, horse, dairy cow, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink , Chicken, duck, goose, turkey, cockfight or human.
  • the constant region is derived from mice
  • the sequence of the light chain constant region is shown in SEQ ID NO: 9;
  • the sequence of the heavy chain constant region is shown in SEQ ID NO: 10.
  • the present disclosure also provides an isolated nucleic acid encoding the aforementioned binding protein.
  • the present disclosure also provides a vector including the above-mentioned nucleic acid.
  • the expression vector of the present disclosure is 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 prepare the polypeptides of the present disclosure.
  • the present disclosure also provides a method for producing the above binding protein, including the following steps:
  • the above-mentioned host cells are cultured in a culture medium, and the produced binding protein is recovered from the culture medium or from the cultured host cells.
  • the present disclosure also provides the use of the aforementioned binding protein in PCR.
  • kits characterized in that the kit contains one or more of the aforementioned binding protein, the aforementioned isolated nucleic acid or the aforementioned carrier.
  • the present disclosure also provides a composition, wherein the composition includes the binding protein and Taq DNA polymerase described in the present disclosure.
  • the composition further includes 4 deoxynucleoside triphosphates.
  • the composition further includes primers and/or probes.
  • the composition further includes MgCl 2 .
  • the composition further includes nucleic acid as a template.
  • the present disclosure also provides a method for amplifying nucleic acids, which includes performing hot-start PCR using the binding protein described in the present disclosure or the composition described in the present disclosure.
  • the hot-start PCR is selected from the group consisting of multiplex PCR, real-time PCR, and real-time quantitative PCR.
  • the present disclosure also provides a method for detecting Taq DNA polymerase in a test sample, which includes:
  • the immune complex further includes a second antibody, and the second antibody binds to the binding protein
  • the immune complex in step a), further includes a second antibody, and the second antibody binds to the Taq DNA polymerase.
  • the present disclosure also provides the binding proteins described herein for use in amplifying nucleic acids
  • FIG. 1 is a monoclonal antibody electrophoresis diagram of the recombinant antibody against Taq DNA polymerase of the present disclosure.
  • Figure 2 shows the PCR electrophoresis of Taq DNA polymerase.
  • amino acid means a naturally occurring or non-naturally occurring carboxy alpha-amino acid.
  • amino acid as used in this application may include naturally occurring amino acids and non-naturally occurring amino acids.
  • Naturally occurring amino acids include alanine (three letter code: A1a, single letter code: A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), Cysteine (Cys, c), Glutamine (G1n, Q), Glutamic acid (G1u, E), Glycine (G1y, G), Histidine (His, H), Isoleucine (I1e , I), Leucine (Leu, L), Lysine (Lys, K), Methionine (Met, M), Phenylalanine (Phe, F), Proline (Pro, P) , Serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W),
  • Non-naturally occurring amino acids include but are not limited to ⁇ -aminoadipate, aminobutyric acid, citrulline, homocitrulline, homoleucine, homoarginine, hydroxyproline, norleucine, pyridine Alanine, sarcosine and so on.
  • isolated binding protein is a protein that does not bind to naturally bound components due to derived origin or source, which naturally accompanies it in its natural state; is substantially free of those from the same species Other proteins; expressed by cells from different species; or not found in nature. Therefore, a protein that is chemically synthesized or synthesized in a cell system different from that of its natural origin will be “isolated” from its naturally associated components.
  • the protein can also be substantially free of naturally bound components by isolation, for example, using protein purification techniques well known in the art.
  • isolated binding protein including an antigen binding domain generally refers to any protein/protein fragment that includes a CDR region.
  • antibody includes polyclonal antibodies and monoclonal antibodies and antigen compound binding fragments of these antibodies, including Fab, F(ab')2, Fd, Fv, scFv, bispecific antibodies and antibody minimum recognition units, and these antibodies And fragments of single-chain derivatives.
  • the type of antibody can be IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, IgD.
  • 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”.
  • variable region refers to the amino-terminal domain of the heavy or light chain of the antibody.
  • the variable domain of the heavy chain may be referred to as "VH”.
  • 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 antigen binding sites.
  • the light or heavy chain variable region is composed of three so-called “complementarity determining regions” or “CDRs” and a framework region that separates them.
  • CDRs complementarity determining regions
  • the framework region of the antibody that is, the framework region constituting the combination of the light chain and the heavy chain of the requirements, plays a role of locating and aligning CDRs, which are mainly responsible for binding to the antigen.
  • backbone region means a region other than those regions of the antibody variable domain that are defined as CDRs.
  • Each antibody variable domain framework region can be further subdivided into adjacent regions (FR1, FR2, FR3, and FR4) separated by CDRs.
  • variable regions VL/VH of the heavy and light chains can be obtained by connecting the following numbered CDRs and FRs in the following combinations: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • the term “purified” or “isolated” in association with a polypeptide or nucleic acid means that the polypeptide or nucleic acid is not in its natural medium or in its natural form. Therefore, the term “isolated” includes polypeptides or nucleic acids taken from the original environment, for example, if it is naturally occurring. For example, an isolated polypeptide generally does not contain at least some proteins or other cellular components that it is usually bound to or usually mixed with or in solution.
  • Isolated polypeptides include the naturally produced polypeptides contained in cell lysates, the polypeptides in purified or partially purified form, recombinant polypeptides, the polypeptides expressed or secreted by cells, and in heterologous host cells or cultures Of the polypeptide.
  • the term isolated or purified indicates that the nucleic acid is not in its natural genomic background (eg, in a vector, as an expression cassette, linked to a promoter, or artificially introduced into a heterologous host cell).
  • bispecific antibody or “bifunctional antibody” refers to an artificial hybrid binding protein with two different heavy/light chain pairs and two different binding sites. Bispecific binding proteins can be produced by a variety of methods, including fusion of hybridomas or attachment of Fab' fragments.
  • sequence identity refers to the similarity between at least two different sequences. This percentage identity can be determined by standard algorithms, such as Basic Local Alignment Search Tool (BLAST); Needleman's algorithm; or Meyers' algorithm. In one or more embodiments, a set of parameters may be a Blosum 62 scoring matrix and a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5. In one or more embodiments, the percent identity between two amino acid or nucleotide sequences can also be determined using the algorithm of Meyers and Miller ((1989) CABIOS 4:11-17), which has been incorporated Into the ALIGN program (version 2.0), use the PAM120 weight residue table, gap length penalty of 12, and gap penalty of 4. Percent identity is usually calculated by comparing sequences of similar length.
  • BLAST Basic Local Alignment Search Tool
  • Needleman's algorithm or Meyers' algorithm.
  • a set of parameters may be a Blosum 62 scoring matrix and a gap penalty of 12, a gap extension penalty of 4, and a frameshift
  • affinity refers to the strength of binding of an antigen-binding domain of a binding protein or antibody to an antigen or antigen epitope. Affinity can be measured by KD value, the smaller the KD value, the greater the affinity.
  • the present disclosure provides an isolated binding protein comprising an antigen binding domain, the antigen binding domain comprising at least one complementarity determining region selected from the following amino acid sequence, or having at least 80 % Sequence identity and affinity with Taq DNA polymerase K D ⁇ 8.568 ⁇ 10 -9 mol/L;
  • the complementarity determining region CDR-VH1 is S-V-X1-T-F-X2-T-Y-Y-X3-Y, where,
  • X1 is D, E or N, X2 is S or T, X3 is I or L;
  • the complementarity determining region CDR-VH2 is G-X1-N-P-T-S-X2-P-V-F-X3-E-K, where,
  • X1 is I, V or L, X2 is N or GG, X3 is D, E or N;
  • the complementarity determining region CDR-VH3 is T-R-S-X1-X2-R-R-G-Y-Y-X3-D-Y, where,
  • X1 is I, V or L
  • X2 is I, V or L
  • X3 is F or P
  • the complementarity determining region CDR-VL1 is R-X1-S-Q-D-I-X2-N-Y-X3-N, where,
  • X1 is A or G
  • X2 is N or Q
  • X3 is I, V or L;
  • the complementarity determining region CDR-VL2 is I-Y-X1-T-S-R-L-X2-S-G-X3-P, where,
  • X1 is Y or F
  • X2 is Q
  • H or N
  • X3 is I, V or L;
  • the complementarity determining region CDR-VL3 is Q-D-D-T-X1-P-X2-T-X3-G, where,
  • X1 is I, V or L
  • X2 is I, V or L
  • X3 is W or F.
  • the binding specificity and affinity of antibodies are mainly determined by CDR sequences. According to mature and well-known existing technologies, the amino acid sequence of non-CDR regions can be easily changed to obtain changes with similar biological activities. body. Therefore, the present disclosure also includes "functional derivatives" of the binding protein. “Functional derivative” refers to a variant of amino acid substitution. A functional derivative retains detectable binding protein activity, preferably the activity of an antibody that binds Taq DNA polymerase. "Functional derivatives" may include "variants” and “fragments” because they have the same CDR sequences as the binding proteins described in this disclosure, and therefore have similar biological activities.
  • the antigen binding domain and the complementarity determining region of the amino acid sequence described below have at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70%, Or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, Or at least 97%, or at least 98%, or at least 99% sequence identity and having a KD ⁇ 8.568 ⁇ 10 -9 mol/L with Taq DNA polymerase Taq DNA polymerase, for example, 8.568 ⁇ 10 -9 mol/L, 5.126 ⁇ 10 -9 mol / L, 3.018 ⁇ 10 -9 mol / L, 2.196 ⁇ 10 -10 mol / L, 3.839 ⁇ 10 -10 mol / L, 4.075 ⁇ 10 -10 mol / L, 6.772 ⁇ 10 -10
  • the affinity is measured according to the method in the present specification.
  • X3 is F
  • X1 is A
  • X1 is Y
  • X3 is F.
  • X1 is D and X2 is S.
  • X1 is E and X2 is S.
  • X1 is N and X2 is S.
  • X1 is D and X2 is T.
  • X1 is E and X2 is T.
  • X1 is N and X2 is T.
  • X1 is I and X2 is N.
  • X1 is I and X2 is GG.
  • X1 is V and X2 is N.
  • X1 is V and X2 is GG.
  • X1 is L and X2 is N.
  • X1 is L and X2 is GG.
  • X1 is I and X2 is I.
  • X1 is I and X2 is V.
  • X1 is I and X2 is L.
  • X1 is V and X2 is I.
  • X1 is V and X2 is V.
  • X1 is V and X2 is L.
  • X1 is L and X2 is I.
  • X1 is L and X2 is V.
  • X1 is L and X2 is L.
  • X2 is N and X3 is I.
  • X2 is N and X3 is V.
  • X2 is N and X3 is L.
  • X2 is Q and X3 is I.
  • X2 is Q and X3 is V.
  • X2 is Q and X3 is L.
  • X2 is Q and X3 is I.
  • X2 is Q and X3 is V.
  • X2 is Q and X3 is L.
  • X2 is H and X3 is I.
  • X2 is H and X3 is V.
  • X2 is H and X3 is L.
  • X2 is N and X3 is I.
  • X2 is N and X3 is V.
  • X2 is N and X3 is L.
  • X1 is I and X2 is I.
  • X1 is I and X2 is V.
  • X1 is I and X2 is L.
  • X1 is V and X2 is I.
  • X1 is V and X2 is V.
  • X1 is V and X2 is L.
  • X1 is L and X2 is I.
  • X1 is L and X2 is V.
  • X1 is L and X2 is L.
  • the mutation site of each complementarity determining region is selected from any one of the following mutation combinations:
  • X1 appearing in the six CDR regions of the binding protein described in the present disclosure each independently of each other represent the amino acids defined in the present disclosure; appearing in the six of the binding protein described in the present disclosure X2 in each CDR region independently represents the amino acid defined in the present disclosure; X3 appearing in the six CDR regions of the binding protein described in the present disclosure each independently represents the amino acid defined in the present disclosure.
  • the binding protein includes at least 3 CDRs; alternatively, the binding protein includes at least 6 CDRs.
  • the binding protein is a complete antibody comprising variable and constant regions.
  • the binding protein is one of Nanobody, F(ab')2, Fab', Fab, Fv, scFv, bispecific antibody, and antibody minimum recognition unit.
  • the binding protein includes light chain framework regions FR-L1, FR-L2, FR-L3, and FR-L4 whose sequence is as shown in SEQ ID NO: 1-4, and/or Or, the sequence is as shown in SEQ ID NO: 5-8 heavy chain framework regions FR-H1, FR-H2, FR-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 species source of the constant region is cattle, horse, dairy cow, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink , Chicken, duck, goose, turkey, cockfight or human.
  • the constant region is derived from mice
  • the sequence of the light chain constant region is shown in SEQ ID NO: 9;
  • the sequence of the heavy chain constant region is shown in SEQ ID NO: 10.
  • the present disclosure also provides an isolated nucleic acid encoding the aforementioned binding protein.
  • nucleic acid contains variants of its conservative substitutions (eg substitution of degenerate codons) and complementary sequences.
  • nucleic acid and polynucleotide are synonymous and include genes, cDNA molecules, mRNA molecules, and fragments thereof such as oligonucleotides.
  • the present disclosure also provides a vector including the above-mentioned nucleic acid.
  • the nucleic acid sequence is operably linked to at least one regulatory sequence.
  • "Operably linked” means that the coding sequence is linked to the regulatory sequence in a manner that allows 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 agent that contains the nucleic acid of the present disclosure or a fragment thereof, is capable of carrying genetic information, and can deliver genetic information to cells.
  • Typical vectors include plasmids, viruses, bacteriophages, cosmids, and mini-chromosomes.
  • the vector can be a cloning vector (ie a vector used to transfer genetic information into a cell, the cell can be propagated and the cell can be selected with or without the genetic information) or an expression vector (ie contains the necessary genetic elements Thereby allowing the genetic information of the vector to be expressed in the cell).
  • a cloning vector can contain a selection marker and an origin of replication that matches the cell type specified by the cloning vector, while an expression vector contains the regulatory elements necessary to affect expression in the designated target cell.
  • the nucleic acid or fragment thereof of the present disclosure can be inserted into a suitable vector to form a cloning vector or expression vector carrying the nucleic acid fragment of the present disclosure.
  • This new carrier is also part of this disclosure.
  • the vector may include plasmids, bacteriophages, cosmids, mini-chromosomes or viruses, as well as naked DNA that is only transiently expressed in specific cells.
  • the cloning vectors and expression vectors of the present disclosure can replicate spontaneously, and therefore can provide a high copy number for high-level expression or high-level replication purposes for subsequent cloning.
  • the expression vector may include a promoter for driving expression of the nucleic acid fragment of the present disclosure, optionally a nucleic acid sequence encoding a signal peptide that secretes or integrates the peptide expression product on the membrane, a nucleic acid fragment of the present disclosure, and optionally Nucleic acid sequence encoding a terminator.
  • a promoter for driving expression of the nucleic acid fragment of the present disclosure optionally a nucleic acid sequence encoding a signal peptide that secretes or integrates the peptide expression product on the membrane, a nucleic acid fragment of the present disclosure, and optionally Nucleic acid sequence encoding a terminator.
  • the vector When an 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.
  • the vector usually carries a replication site and a marker sequence that can provide phenotypic selection in the transformed cell.
  • the expression vector of the present disclosure is 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 prepare the polypeptides of the present disclosure.
  • the 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 cells from mammals, such as CHO cells.
  • the transformed cell can replicate the nucleic acid fragment of the present disclosure.
  • the expression product may be exported to the culture medium or carried on the surface of the transformed cell.
  • the present disclosure also provides a method for producing the above binding protein, including the following steps:
  • the above-mentioned host cells are cultured in a culture medium, and the produced binding protein is recovered from the culture medium or from the cultured host cells.
  • the method may 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.
  • the host cell can also be transfected with one or more expression vectors, which can contain DNA encoding at least a portion of the binding protein, alone or in combination.
  • the binding protein can be separated from the culture medium or cell lysate using conventional techniques for protein and peptide purification, which include ammonium sulfate precipitation, chromatography (eg ion exchange, gel filtration, affinity chromatography, etc.) and/or Electrophoresis.
  • Suitable vectors containing coding and regulatory sequences of interest can be performed using standard ligation and restriction techniques known in the art.
  • the isolated plasmid, DNA sequence, or synthetic oligonucleotide is cut, tailed, and religated as desired. Any method may be used to introduce mutations into the coding sequence to produce the variants of the present disclosure, and these mutations may include deletions or insertions or substitutions and the like.
  • the present disclosure also provides antibodies that can react with the epitope of Taq DNA polymerase, including monoclonal and polyclonal antibodies.
  • the antibody may contain the entire binding protein, or a fragment or derivative thereof.
  • Preferred antibodies contain all or part of the binding protein.
  • the present disclosure also provides the use of the aforementioned binding protein in PCR.
  • kits characterized in that the kit contains one or more of the aforementioned binding protein, the aforementioned isolated nucleic acid or the aforementioned carrier.
  • the present disclosure also provides a composition, wherein the composition includes the binding protein and Taq DNA polymerase described in the present disclosure.
  • the composition further includes 4 deoxynucleoside triphosphates.
  • the composition further includes primers and/or probes.
  • the composition further includes MgCl 2 .
  • the composition further includes nucleic acid as a template.
  • the present disclosure also provides a method for amplifying nucleic acids, which includes performing hot-start PCR using the binding protein described in the present disclosure or the composition described in the present disclosure.
  • the hot-start PCR is selected from the group consisting of multiplex PCR, real-time PCR, and real-time quantitative PCR.
  • hot start PCR refers to PCR that allows Taq DNA polymerase to function only when the sample temperature exceeds at least a certain temperature, thereby improving the specificity of the reaction and avoiding non-specific amplification of nucleic acids .
  • the present disclosure also provides a method for detecting Taq DNA polymerase in a test sample, which includes:
  • the immune complex further includes a second antibody, and the second antibody binds to the binding protein
  • the immune complex in step a), further includes a second antibody, and the second antibody binds to the Taq DNA polymerase.
  • the present disclosure also provides the use of the binding proteins described herein in amplifying nucleic acids.
  • the restriction enzyme and Prime DNA polymerase were purchased from Takara.
  • MagExtractor-RNA extraction kit was purchased from TOYOBO.
  • SMARTERTM RACE cDNA Amplification Kit kit was purchased from Takara.
  • the pMD-18T vector was purchased from Takara Corporation.
  • the plasmid extraction kit was purchased from Tiangen Company. Primer synthesis and gene sequencing were completed by Invitrogen.
  • the hybridoma cell line secreting the Anti-TAQ 2C7 monoclonal antibody is an existing hybridoma cell line, and it is ready for recovery.
  • NUP Nested Universal Primer A
  • mIg-HR 5'-TCATTTACCAGGAGAGTGGGAGAGGC-3'.
  • Light chain genes are amplified with Universal Primer A Mixture (UPM), Nested Universal Primer A (NUP) and mKR Primers, and Heavy Chain Genes with Universal Primer A Mixture (UPM), Nested Universal Primer A (NUP) and mHR Primers Perform amplification.
  • the primer pair of the light chain amplifies the target band of about 0.7KB
  • the primer pair of the heavy chain amplifies the target band of about 1.4KB.
  • Purified and recovered by agarose gel electrophoresis the product was added with rTaq DNA polymerase and then inserted into pMD-18T vector, transformed into DH5 ⁇ competent cells, after growing bacteria, heavy chain and light chain genes were cloned respectively Four clones were sent to Invitrogen for sequencing.
  • VL gene sequence 378bp, which belongs to the VkII gene family, with a 57bp leader peptide sequence in front
  • VH gene sequence 417bp, which belongs to the VH1 gene family, which has 57bp in front Leader sequence.
  • the vector is a constructed recombinant antibody eukaryotic expression vector.
  • the expression vector has been introduced into polyclonal cleavage sites such as HindIII, BamHI, EcoRI, etc., and is named pcDNA 3.4A expression vector, subsequently referred to as 3.4A expression vector; according to the above pMD-18T
  • anti-TAQ 2C7 antibody VL and VH gene specific primers were designed with HindIII, EcoRI cleavage sites and protective bases at both ends.
  • the primers are as follows:
  • Anti-Taq 2C7-HF 5’-CCCAAGCTTGCCACCATGGGATGGAGCTATATCATCCTC-3’;
  • the 0.75KB light chain gene fragment and 1.42KB heavy chain gene fragment were expanded by PCR amplification method.
  • the heavy chain and light chain gene fragments were digested with HindIII/EcoRI double digestion, and the 3.4A vector was digested with HindIII/EcoRI double digestion. After the fragments and vector were purified and recovered, the heavy chain gene and light chain gene were connected to the 3.4A expression vector, respectively. Recombinant expression plasmids for heavy and light chains were obtained.
  • Recombinant antibody expression plasmid was transiently transfected into CHO cells to determine the activity of the expression plasmid
  • reaction OD was still greater than 1.0 after the cell supernatant was diluted 1,000 times, and the reaction OD was less than 0.1 without the cell supernatant, indicating that the antibody produced after the plasmid transient was active against Taq enzyme.
  • Recombinant antibody expression plasmid is stably transfected, and stable cell lines are screened under pressure
  • the cells were recovered, they were first cultivated in 125ml shake flasks, the inoculation volume was 30ml, the medium was 100% Dynamis medium, and they were placed in a shaker with a rotation speed of 120r/min, a temperature of 37°C, and carbon dioxide of 8%. Incubate for 72h, inoculate expansion 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. After every 72h expansion. When the cell volume meets the production requirements, strictly control the seeding density to be around 500,000 cells/ml for production.
  • Shake flask parameters speed 120r/min, temperature 37°C, carbon dioxide 8%.
  • Feeding feed feeding in the shake flask until 72h starts feeding every day, HyCloneTM Cell Booster Feed 7a feeds 3% of the initial culture volume every day, Feed 7b feeds the volume daily as 1/1000th of the initial culture volume.
  • Make up to the 12th day feeding on the 12th day).
  • Glucose was supplemented with 3g/L on the sixth day.
  • the antibody of sample 1 obtained in Example 1 has the ability to bind Taq DNA polymerase, but the affinity and antibody activity are not ideal, so the application Humans mutate the light chain CDR and heavy chain CDR of this antibody.
  • the complementary determination region (WT) of the heavy chain After analysis, the complementary determination region (WT) of the heavy chain:
  • CDR-VH1 is S-V-D(X1)-T-F-S(X2)-T-Y-Y-L(X3)-Y;
  • CDR-VH2 is G-V(X1)-N-P-T-S-N(X2)-P-V-F-D(X3)-E-K;
  • CDR-VH3 is T-R-S-I(X1)-L(X2)-R-R-G-Y-Y-P(X3)-D-Y;
  • CDR-VL1 is R-G(X1)-S-Q-D-I-Q(X2)-N-Y-V(X3)-N;
  • CDR-VL2 is I-Y-F(X1)-T-S-R-L-Q(X2)-S-G-I(X3)-P;
  • CDR-VL3 is Q-D-D-T-I(X1)-P-V(X2)-T-W(X3)-G;
  • X1, X2, X3 are mutation sites.
  • the coating solution diluted Taq DNA polymerase to the specified concentration, 100 ⁇ L per well, overnight at 4°C; the next day, the washing solution was washed twice and patted dry; adding blocking solution (20% BSA+80) %PBS), 120 ⁇ L per well, 37°C, 1h, pat dry; add diluted Taq monoclonal antibody, 100 ⁇ L/well, 37°C, 30min (partial supernatant 1h); wash solution 5 times, pat dry; add Goat anti-mouse IgG-HRP, 100 ⁇ L per well, 37° C., 30 min; wash solution 5 times, pat dry; add solution A (50 ⁇ L/well), add solution B (50 ⁇ L/well), 10 min ; Add stop solution, 50 ⁇ L/well; read the OD value at 450nm (reference 630nm) on the microplate reader.
  • the above antibody was diluted with PBST to 10 ⁇ g/ml, and Taq DNA polymerase was diluted with PBST: 1000 nmol/ml, 500 nmol/ml, 250 nmol/ml, 125 nmol/ml, 62.5 nmol/ml, 31.3 nmol/ml , 15.6nmol/ml, 0nmol/ml.
  • mutation 1 has the best activity effect and affinity, so mutation 1 is used as the backbone sequence to screen for mutation sites with better titers (to ensure that the activity of the screened antibody is similar to mutation 1, and the antibody activity is ⁇ 10 %), some results are as follows.
  • the activity of the enzyme can be blocked below 70°C, and can be dissociated above 70°C, and can be completely dissociated at 95°C in only 1-3 minutes to release the active components of the enzyme.
  • the disclosure also tests the stability of the combination of antibodies and Taq DNA polymerase, rapid activation, and pH compatibility of the mutation combinations in Table 4 and all have excellent effects, indicating that the antibodies provided by the disclosure against Taq DNA polymerase are It has good applications in molecular detection.
  • the binding protein provided by the present disclosure can specifically bind Taq enzyme to form an abzyme complex, so it can effectively block the activity of Taq DNA polymerase at room temperature; while at high temperature, this complex will dissociate and release Active Taq DNA polymerase to perform PCR amplification reaction. This can effectively avoid the formation of primer dimers, reduce the amplification of non-specific products, and improve the long-term stability of Taq DNA polymerase.
  • the binding protein of the present disclosure can be widely applied to various hot-start PCR.

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Abstract

提供一种包含Taq DNA聚合酶抗原结合结构域的分离的结合蛋白，该抗原结合结构域包括选自CDR-VH1-3、 CDR-VL1-3的至少一个互补决定区，或与所述互补决定区具有至少80％的序列同一性且与Taq DNA聚合酶具有K D≤8.568×10 -9mol/L的亲和力。所述结合蛋白可用于分子检测领域。

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2018年12月20日提交中国专利局的申请号为201811566184.2、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
本公开涉及生物技术和医学技术领域，尤其是涉及一种抗Taq DNA聚合酶的抗体及其应用。
背景技术
1988年，Randall K.Saiki等从水生栖热菌(Thermus aquaticus)中分离纯化到了Taq DNA聚合酶，Taq聚合酶可以耐受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聚合酶取代了之前常用于PCR反应的大肠埃希菌(Escherichia coli)中的DNA聚合酶。在PCR反应中应用Taq聚合酶，不需要每个循环加酶，这使PCR技术变得非常简捷，大大降低了成本，PCR技术得以广泛应用，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扩增和引物二聚体的形成，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酶的单克隆抗体，特异性Taq酶的单克隆抗体与Taq DNA聚合酶结合后形成抗原抗体复合物，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，同时可以提高Taq DNA聚合酶的长期稳定性。
抗体修饰热启动酶用到的特异性Taq酶的抗体还有待进一步开发。
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。
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，或与下述氨基酸序列的互补决定区具有至少80％的序列同一性且与Taq DNA聚合酶具有K D≤8.568×10 -9mol/L的亲和力；
互补决定区CDR-VH1为S-V-X1-T-F-X2-T-Y-Y-X3-Y，其中，
X1是D、E或N，X2是S或T，X3是I或L；
互补决定区CDR-VH2为G-X1-N-P-T-S-X2-P-V-F-X3-E-K，其中，
X1是I、V或L，X2是N或GG，X3是D、E或N；
互补决定区CDR-VH3为T-R-S-X1-X2-R-R-G-Y-Y-X3-D-Y，其中，
X1是I、V或L，X2是I、V或L，X3是F或P；
互补决定区CDR-VL1为R-X1-S-Q-D-I-X2-N-Y-X3-N，其中，
X1是A或G，X2是N或Q，X3是I、V或L；
互补决定区CDR-VL2为I-Y-X1-T-S-R-L-X2-S-G-X3-P，其中，
X1是Y或F，X2是Q、H或N，X3是I、V或L；
互补决定区CDR-VL3为Q-D-D-T-X1-P-X2-T-X3-G，其中，
X1是I、V或L，X2是I、V或L，X3是W或F。
一个重要优点在于，所述结合蛋白活性强，与Taq DNA聚合酶具有很高的亲和力。
在一种或多种实施方式中，
所述互补决定区CDR-VH1中，X3是I；
所述互补决定区CDR-VH2中，X3是N；
所述互补决定区CDR-VH3中，X3是F；
所述互补决定区CDR-VL1中，X1是A；
所述互补决定区CDR-VL2中，X1是Y；
所述互补决定区CDR-VL3中，X3是F。
在一种或多种实施方式中，所述互补决定区CDR-VH1中，X1是D，X2是S。
在一种或多种实施方式中，所述互补决定区CDR-VH1中，X1是E，X2是S。
在一种或多种实施方式中，所述互补决定区CDR-VH1中，X1是N，X2是S。
在一种或多种实施方式中，所述互补决定区CDR-VH1中，X1是D，X2是T。
在一种或多种实施方式中，所述互补决定区CDR-VH1中，X1是E，X2是T。
在一种或多种实施方式中，所述互补决定区CDR-VH1中，X1是N，X2是T。
在一种或多种实施方式中，所述互补决定区CDR-VH2中，X1是I，X2是N。
在一种或多种实施方式中，所述互补决定区CDR-VH2中，X1是I，X2是GG。
在一种或多种实施方式中，所述互补决定区CDR-VH2中，X1是V，X2是N。
在一种或多种实施方式中，所述互补决定区CDR-VH2中，X1是V，X2是GG。
在一种或多种实施方式中，所述互补决定区CDR-VH2中，X1是L，X2是N。
在一种或多种实施方式中，所述互补决定区CDR-VH2中，X1是L，X2是GG。
在一种或多种实施方式中，所述互补决定区CDR-VH3中，X1是I，X2是I。
在一种或多种实施方式中，所述互补决定区CDR-VH3中，X1是I，X2是V。
在一种或多种实施方式中，所述互补决定区CDR-VH3中，X1是I，X2是L。
在一种或多种实施方式中，所述互补决定区CDR-VH3中，X1是V，X2是I。
在一种或多种实施方式中，所述互补决定区CDR-VH3中，X1是V，X2是V。
在一种或多种实施方式中，所述互补决定区CDR-VH3中，X1是V，X2是L。
在一种或多种实施方式中，所述互补决定区CDR-VH3中，X1是L，X2是I。
在一种或多种实施方式中，所述互补决定区CDR-VH3中，X1是L，X2是V。
在一种或多种实施方式中，所述互补决定区CDR-VH3中，X1是L，X2是L。
在一种或多种实施方式中，所述互补决定区CDR-VL1中，X2是N，X3是I。
在一种或多种实施方式中，所述互补决定区CDR-VL1中，X2是N，X3是V。
在一种或多种实施方式中，所述互补决定区CDR-VL1中，X2是N，X3是L。
在一种或多种实施方式中，所述互补决定区CDR-VL1中，X2是Q，X3是I。
在一种或多种实施方式中，所述互补决定区CDR-VL1中，X2是Q，X3是V。
在一种或多种实施方式中，所述互补决定区CDR-VL1中，X2是Q，X3是L。
在一种或多种实施方式中，所述互补决定区CDR-VL2中，X2是Q，X3是I。
在一种或多种实施方式中，所述互补决定区CDR-VL2中，X2是Q，X3是V。
在一种或多种实施方式中，所述互补决定区CDR-VL2中，X2是Q，X3是L。
在一种或多种实施方式中，所述互补决定区CDR-VL2中，X2是H，X3是I。
在一种或多种实施方式中，所述互补决定区CDR-VL2中，X2是H，X3是V。
在一种或多种实施方式中，所述互补决定区CDR-VL2中，X2是H，X3是L。
在一种或多种实施方式中，所述互补决定区CDR-VL2中，X2是N，X3是I。
在一种或多种实施方式中，所述互补决定区CDR-VL2中，X2是N，X3是V。
在一种或多种实施方式中，所述互补决定区CDR-VL2中，X2是N，X3是L。
在一种或多种实施方式中，所述互补决定区CDR-VL3中，X1是I，X2是I。
在一种或多种实施方式中，所述互补决定区CDR-VL3中，X1是I，X2是V。
在一种或多种实施方式中，所述互补决定区CDR-VL3中，X1是I，X2是L。
在一种或多种实施方式中，所述互补决定区CDR-VL3中，X1是V，X2是I。
在一种或多种实施方式中，所述互补决定区CDR-VL3中，X1是V，X2是V。
在一种或多种实施方式中，所述互补决定区CDR-VL3中，X1是V，X2是L。
在一种或多种实施方式中，所述互补决定区CDR-VL3中，X1是L，X2是I。
在一种或多种实施方式中，所述互补决定区CDR-VL3中，X1是L，X2是V。
在一种或多种实施方式中，所述互补决定区CDR-VL3中，X1是L，X2是L。
在一种或多种实施方式中，各互补决定区的突变位点选自下述突变组合中的任一种：
Figure PCTCN2019109791-appb-000001
Figure PCTCN2019109791-appb-000002
在一种或多种实施方式中，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。
在一种或多种实施方式中，所述结合蛋白为包含可变区和恒定区的完整抗体。
在一种或多种实施方式中，所述结合蛋白为纳米抗体、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。
在一种或多种实施方式中，所述结合蛋白包括序列依次如SEQ ID NO:1-4所示的轻链骨架区FR-L1、FR-L2、FR-L3及FR-L4，和/或，序列依次如SEQ ID NO:5-8所示的重链骨架区FR-H1、FR-H2、FR-H3及FR-H4。
在一种或多种实施方式中，所述结合蛋白还包含抗体恒定区序列。
在一种或多种实施方式中，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。
在一种或多种实施方式中，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、骆驼、驴、鹿、貂、鸡、鸭、鹅、火鸡、斗鸡或人。
在一种或多种实施方式中，所述恒定区来源于小鼠；
轻链恒定区序列如SEQ ID NO:9所示；
重链恒定区序列如SEQ ID NO:10所示。
本公开还提供了一种分离的核酸，所述核酸编码上述的结合蛋白。
本公开还提供了一种载体，所述载体包含上述的核酸。
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。
本公开还提供了一种生产上述结合蛋白的方法，包括如下步骤：
在培养基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。
本公开还提供了上述的结合蛋白在PCR中的应用。
本公开还提供了一种试剂盒，其特征在于，所述试剂盒上述的结合蛋白、上述的分离的核酸或上述的载体中的一种或多种。
本公开还提供了一种组合物，其中所述组合物包括本公开所述的结合蛋白和Taq DNA聚合酶。
在一种或多种实施方式中，所述组合物还包括4种脱氧核苷三磷酸。
在一种或多种实施方式中，所述组合物还包括引物和/或探针。
在一种或多种实施方式中，所述组合物还包括MgCl 2。
在一种或多种实施方式中，所述组合物还包括作为模板的核酸。
本公开还提供了一种用于扩增核酸的方法，包括使用本公开所述的结合蛋白或本公开所述的组合物进行热启动PCR。
在一种或多种实施方式中，热启动PCR选自由多重PCR、实时PCR和实时定量PCR组成组。
本公开还提供一种检测测试样品中的Taq DNA聚合酶的方法，其包括：
a)在足以发生抗体/抗原结合反应的条件下，使所述测试样品中的Taq DNA聚合酶与本公开的结合蛋白接触以形成免疫复合物；和
b)检测所述免疫复合物的存在，所述复合物的存在指示所述测试样品中所述Taq DNA聚合酶的存在。
在一种或多种实施方式中，所述免疫复合物中还包括第二抗体，所述第二抗体与所述结合蛋白结合；
在一种或多种实施方式中，在步骤a)中，所述免疫复合物中还包括第二抗体，所述第二抗体与所述Taq DNA聚合酶结合。
本公开还提供本文所述的结合蛋白，在扩增核酸中的用途
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为本公开抗Taq DNA聚合酶的重组抗体的单克隆抗体电泳图。图2为Taq DNA聚合酶的PCR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。
术语“氨基酸”表示天然存在或非天然存在的羧基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，例如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天然结合的组分。
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”此用语包括天然存在的抗体以及非天然存在的抗体，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。
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三个称为“互补决定区”或“CDR”以及将其分隔开的骨架区(framework region)构成。抗体的骨架区，即构成要件轻链和重链的组合的骨架区，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，“骨架区”、“构架区”或“FR”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区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。
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)。
如本文所用，术语“双特异性抗体”或“双功能性抗体”指具有两对不同重/轻链和两个不同结合位点的人造杂合结合蛋白。双特异性结合蛋白可通过多种方法来生成，包括融合杂交瘤或连接Fab’片段。
如本文所用，术语“序列同一性”指至少两种不同序列之间的相似性。此百分比同一性可通过标准算法来确定，例如基本局部比对搜索工具(Basic Local Alignment Search Tool，BLAST)；Needleman等的算法；或Meyers等的算法。在一种或多种实施方式中，一组参数可以是Blosum 62评分矩阵及缺口罚分12、缺口延伸罚分4、和移码缺口罚分5。在一种或多种实施方式中，两种氨基酸或核苷酸序列之间的百分比同一性还可以使用Meyers和Miller((1989)CABIOS 4：11-17)的算法来确定，该算法已经掺入ALIGN程序(2.0版)，使用PAM120权重残基表、缺口长度罚分12、和缺口罚分4。百分比同一性通常通过比较相似长度的序列来计算。
如本文所用，术语“亲和力”是指结合蛋白或抗体的抗原结合结构域与抗原或抗原表位的结合强度。亲和力可以用KD值来衡量，KD值越小代表亲和力越大。
本公开提供的一种包含抗原结合结构域的分离的结合蛋白，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，或与下述氨基酸序列的互补决定区具有至少80％的序列同一性且与Taq DNA聚合酶具有K D≤8.568×10 -9mol/L的亲和力；
互补决定区CDR-VH1为S-V-X1-T-F-X2-T-Y-Y-X3-Y，其中，
X1是D、E或N，X2是S或T，X3是I或L；
互补决定区CDR-VH2为G-X1-N-P-T-S-X2-P-V-F-X3-E-K，其中，
X1是I、V或L，X2是N或GG，X3是D、E或N；
互补决定区CDR-VH3为T-R-S-X1-X2-R-R-G-Y-Y-X3-D-Y，其中，
X1是I、V或L，X2是I、V或L，X3是F或P；
互补决定区CDR-VL1为R-X1-S-Q-D-I-X2-N-Y-X3-N，其中，
X1是A或G，X2是N或Q，X3是I、V或L；
互补决定区CDR-VL2为I-Y-X1-T-S-R-L-X2-S-G-X3-P，其中，
X1是Y或F，X2是Q、H或N，X3是I、V或L；
互补决定区CDR-VL3为Q-D-D-T-X1-P-X2-T-X3-G，其中，
X1是I、V或L，X2是I、V或L，X3是W或F。
本领域公知，抗体的结合特异性及亲合力均主要由CDR序列决定，根据成熟、公知的现有各项技术可轻易地将非CDR区域的氨基酸序列改变而获得具有相类似的生物活性的变体。因此，本公开也包括该结合蛋白的“功能性衍生物”。“功能性衍生物”是指氨基酸替换的变体，一个功能性衍生物保留有可检测的结合蛋白活性，优选为能结合Taq DNA聚合酶的抗体的活性。“功能性衍生物”可以包含“变体”和“片段”，因其具有与本公开所述的结合蛋白完全相同的CDR序列，因此具有相类似的生物活性。
在一种或多种实施方式中，所述抗原结合结构域与下述氨基酸序列的互补决定区具有至少50％，或至少55％，或至少60％，或至少65％，或至少70％，或至少75％，或至少80％，或至少85％，或至少90％，或至少91％，或至少92％，或至少93％，或至少94％，或至少95％，或至少96％，或至少97％，或至少98％，或至少99％的序列同一性且与Taq DNA聚合酶Taq DNA聚合酶具有KD≤8.568×10 -9mol/L，例如8.568×10 -9mol/L、5.126×10 -9mol/L、3.018×10 -9mol/L、2.196×10 -10mol/L、3.839×10 -10mol/L、4.075×10 -10mol/L、6.772×10 -10mol/L、8.499×10 -10mol/L、9.870×10 -10mol/L、3.145×10 -11mol/L、5.067×10 -11mol/L、6.643×10 -11mol/L，或者1.328×10 -11mol/L≤KD≤8.568×10 -9mol/L，或者1.328×10 -11mol/L≤KD≤9.870×10 -10mol/L；或KD小于或等于5.126×10 -9mol/L、3.018×10 -9mol/L、2.196×10 -10mol/L、3.839×10 -10mol/L、4.075×10 -10mol/L、6.772×10 -10mol/L、8.499×10 -10mol/L、9.870×10 -10mol/L、3.145×10 -11mol/L、5.067×10 -11mol/L或6.643×10 -11mol/L。
其中，亲和力按照本公开说明书中的方法测定。
在一种或多种实施方式中，
所述互补决定区CDR-VH1中，X3是I；
所述互补决定区CDR-VH2中，X3是N；
所述互补决定区CDR-VH3中，X3是F；
所述互补决定区CDR-VL1中，X1是A；
所述互补决定区CDR-VL2中，X1是Y；
所述互补决定区CDR-VL3中，X3是F。
在一种或多种实施方式中，所述互补决定区CDR-VH1中，X1是D，X2是S。
在一种或多种实施方式中，所述互补决定区CDR-VH1中，X1是E，X2是S。
在一种或多种实施方式中，所述互补决定区CDR-VH1中，X1是N，X2是S。
在一种或多种实施方式中，所述互补决定区CDR-VH1中，X1是D，X2是T。
在一种或多种实施方式中，所述互补决定区CDR-VH1中，X1是E，X2是T。
在一种或多种实施方式中，所述互补决定区CDR-VH1中，X1是N，X2是T。
在一种或多种实施方式中，所述互补决定区CDR-VH2中，X1是I，X2是N。
在一种或多种实施方式中，所述互补决定区CDR-VH2中，X1是I，X2是GG。
在一种或多种实施方式中，所述互补决定区CDR-VH2中，X1是V，X2是N。
在一种或多种实施方式中，所述互补决定区CDR-VH2中，X1是V，X2是GG。
在一种或多种实施方式中，所述互补决定区CDR-VH2中，X1是L，X2是N。
在一种或多种实施方式中，所述互补决定区CDR-VH2中，X1是L，X2是GG。
在一种或多种实施方式中，所述互补决定区CDR-VH3中，X1是I，X2是I。
在一种或多种实施方式中，所述互补决定区CDR-VH3中，X1是I，X2是V。
在一种或多种实施方式中，所述互补决定区CDR-VH3中，X1是I，X2是L。
在一种或多种实施方式中，所述互补决定区CDR-VH3中，X1是V，X2是I。
在一种或多种实施方式中，所述互补决定区CDR-VH3中，X1是V，X2是V。
在一种或多种实施方式中，所述互补决定区CDR-VH3中，X1是V，X2是L。
在一种或多种实施方式中，所述互补决定区CDR-VH3中，X1是L，X2是I。
在一种或多种实施方式中，所述互补决定区CDR-VH3中，X1是L，X2是V。
在一种或多种实施方式中，所述互补决定区CDR-VH3中，X1是L，X2是L。
在一种或多种实施方式中，所述互补决定区CDR-VL1中，X2是N，X3是I。
在一种或多种实施方式中，所述互补决定区CDR-VL1中，X2是N，X3是V。
在一种或多种实施方式中，所述互补决定区CDR-VL1中，X2是N，X3是L。
在一种或多种实施方式中，所述互补决定区CDR-VL1中，X2是Q，X3是I。
在一种或多种实施方式中，所述互补决定区CDR-VL1中，X2是Q，X3是V。
在一种或多种实施方式中，所述互补决定区CDR-VL1中，X2是Q，X3是L。
在一种或多种实施方式中，所述互补决定区CDR-VL2中，X2是Q，X3是I。
在一种或多种实施方式中，所述互补决定区CDR-VL2中，X2是Q，X3是V。
在一种或多种实施方式中，所述互补决定区CDR-VL2中，X2是Q，X3是L。
在一种或多种实施方式中，所述互补决定区CDR-VL2中，X2是H，X3是I。
在一种或多种实施方式中，所述互补决定区CDR-VL2中，X2是H，X3是V。
在一种或多种实施方式中，所述互补决定区CDR-VL2中，X2是H，X3是L。
在一种或多种实施方式中，所述互补决定区CDR-VL2中，X2是N，X3是I。
在一种或多种实施方式中，所述互补决定区CDR-VL2中，X2是N，X3是V。
在一种或多种实施方式中，所述互补决定区CDR-VL2中，X2是N，X3是L。
在一种或多种实施方式中，所述互补决定区CDR-VL3中，X1是I，X2是I。
在一种或多种实施方式中，所述互补决定区CDR-VL3中，X1是I，X2是V。
在一种或多种实施方式中，所述互补决定区CDR-VL3中，X1是I，X2是L。
在一种或多种实施方式中，所述互补决定区CDR-VL3中，X1是V，X2是I。
在一种或多种实施方式中，所述互补决定区CDR-VL3中，X1是V，X2是V。
在一种或多种实施方式中，所述互补决定区CDR-VL3中，X1是V，X2是L。
在一种或多种实施方式中，所述互补决定区CDR-VL3中，X1是L，X2是I。
在一种或多种实施方式中，所述互补决定区CDR-VL3中，X1是L，X2是V。
在一种或多种实施方式中，所述互补决定区CDR-VL3中，X1是L，X2是L。
在一种或多种实施方式中，各互补决定区的突变位点选自下述突变组合中的任一种：
Figure PCTCN2019109791-appb-000003
Figure PCTCN2019109791-appb-000004
在一种或多种实施方式中，出现在本公开所述的结合蛋白的六个CDR区中的X1各自彼此独立地代表本公开所限定的氨基酸；出现在本公开所述的结合蛋白的六个CDR区中的X2各自彼此独立地代表本公开所限定的氨基酸；出现在本公开所述的结合蛋白的六个CDR区中的X3各自彼此独立地代表本公开 所限定的氨基酸。
在一种或多种实施方式中，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。
在一种或多种实施方式中，所述结合蛋白为包含可变区和恒定区的完整抗体。
在一种或多种实施方式中，所述结合蛋白为纳米抗体、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。
在一种或多种实施方式中，所述结合蛋白包括序列依次如SEQ ID NO:1-4所示的轻链骨架区FR-L1、FR-L2、FR-L3及FR-L4，和/或，序列依次如SEQ ID NO:5-8所示的重链骨架区FR-H1、FR-H2、FR-H3及FR-H4。
在一种或多种实施方式中，所述结合蛋白还包含抗体恒定区序列。
在一种或多种实施方式中，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。
在一种或多种实施方式中，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、骆驼、驴、鹿、貂、鸡、鸭、鹅、火鸡、斗鸡或人。
在一种或多种实施方式中，所述恒定区来源于小鼠；
轻链恒定区序列如SEQ ID NO:9所示；
重链恒定区序列如SEQ ID NO:10所示。
本公开还提供了一种分离的核酸，所述核酸编码上述的结合蛋白。
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。
本公开还提供了一种载体，所述载体包含上述的核酸。
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，也可以不能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。
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。
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：
在培养基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。
本公开也提供抗体，能与Taq DNA聚合酶的表位发生反应，包含单克隆的和多克隆的抗体。该抗体可以含有完整的结合蛋白，或其片段或衍生物。优选的抗体含有全部或部分的结合蛋白。
本公开还提供了上述的结合蛋白在PCR中的应用。
本公开还提供了一种试剂盒，其特征在于，所述试剂盒上述的结合蛋白、上述的分离的核酸或上述的载体中的一种或多种。
本公开还提供了一种组合物，其中所述组合物包括本公开所述的结合蛋白和Taq DNA聚合酶。
在一种或多种实施方式中，所述组合物还包括4种脱氧核苷三磷酸。
在一种或多种实施方式中，所述组合物还包括引物和/或探针。
在一种或多种实施方式中，所述组合物还包括MgCl 2。
在一种或多种实施方式中，所述组合物还包括作为模板的核酸。
本公开还提供了一种用于扩增核酸的方法，包括使用本公开所述的结合蛋白或本公开所述的组合物进行热启动PCR。
在一种或多种实施方式中，热启动PCR选自由多重PCR、实时PCR和实时定量PCR组成组。
如本文所用，术语“热启动PCR(hot start PCR)”是指使Taq DNA聚合酶只有在样品温度至少超过一定温度时才发挥作用的PCR，从而提高反应的特异性，避免核酸的非特异性扩增。
本公开还提供一种检测测试样品中的Taq DNA聚合酶的方法，其包括：
a)在足以发生抗体/抗原结合反应的条件下，使所述测试样品中的Taq DNA聚合酶与本公开的结合蛋白接触以形成免疫复合物；和
b)检测所述免疫复合物的存在，所述复合物的存在指示所述测试样品中所述Taq DNA聚合酶的存在。
在一种或多种实施方式中，所述免疫复合物中还包括第二抗体，所述第二抗体与所述结合蛋白结合；
在一种或多种实施方式中，在步骤a)中，所述免疫复合物中还包括第二抗体，所述第二抗体与所述Taq DNA聚合酶结合。
本公开还提供本文所述的结合蛋白，在扩增核酸中的用途。
下文提供了一些实例用于示例性说明本公开，而不是限制本公开的范围。
实施例1
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。SMARTERTM 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。分泌Anti-TAQ 2C7单克隆抗体的杂交瘤细胞株为已有的杂交瘤细胞株，复苏备用。
1、引物
扩增重链和轻链5’RACE引物：
SMARTER II A寡核苷酸：
5’-AAGCAGTGGTATCAACGCAGAGTACXXXXX-3’；
5'-RACE CDS Primer(5'-CDS)：5'-(T) 25VN-3’(N＝A,C,G,orT；V＝A,G,orC)；
通用引物A混合物(UPM)：5’-CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT-3’；
巢式通用引物A(NUP)：5’-AAGCAGTGGTATCAACGCAGAGT-3’；
mIg-KR：5’-CTAACACTCATTCCTGTTGAAGCTCTTGACAAT-3’；
mIg-HR：5’-TCATTTACCAGGAGAGTGGGAGAGGC-3’。
2、抗体可变区基因克隆及测序
从分泌Anti-Taq 2C7克隆抗体的杂交瘤细胞株中提取中RNA，用SMARTER TM RACE cDNA Amplification Kit试剂盒及试剂盒中的SMARTER II A寡核苷酸和5'-CDS引物进行第一链cDNA合成，获得的第一链cDNA产物作为PCR扩增模板。轻链基因以通用引物A混合物(UPM)、巢式通用引物A(NUP)和mKR引物进行扩增，重链基因以通用引物A混合物(UPM)、巢式通用引物A(NUP)和mHR引物进行扩增。其中轻链的引物对扩增出0.7KB左右的目的条带，重链的引物对扩增出1.4KB左右的目的条带。用琼脂糖凝胶电泳纯化回收，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，长出菌落后分别取重链及轻链基因克隆各4个克隆送Invitrogen公司进行测序。
3、Anti-Taq 2C7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IMGT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，其中轻链扩增出的基因片段中，VL基因序列为378bp，属于VkII基因家族，其前方有57bp的前导肽序列；重链引物对扩增出的基因片段中，VH基因序列为417bp，属于VH1基因家族，其前方有57bp的前导肽序列。
4、重组抗体表达质粒的构建
pcDNA TM 3.4
Figure PCTCN2019109791-appb-000005
载体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pcDNA 3.4A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 2C7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，引物如下：
Anti-Taq 2C7-HF：5’-CCCAAGCTTGCCACCATGGGATGGAGCTATATCATCCTC-3’；
Anti-Taq 2C7-HR:
5’-CCCGAATTCTCATTATTTACCAGGAGAGTGGGAGAGGCTCTTCTC-3’；
Anti-Taq 2C7-LF:5’-CCCAAGCTTGCCACCATGTCCTCTGCTCAGTTCCTTGGTCTC-3’；
Anti-Taq 2C7-LR:
5’-CCCGAATTCTCATTAACACTCATTCCTGTTGAAGCTCTTGACAA-3’。
通过PCR扩增方法扩出0.75KB的轻链基因片段和1.42KB的重链基因片段。重链和轻链基因片段分别采用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重链基因和轻链基因分别连接3.4A表达载体中，分别得到重链和轻链的重组表达质粒。
5、筛选稳定细胞株
5.1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400ng/ml，调节CHO细胞1.43×10 7cells/ml于离心管中，100μl质粒与700μl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。
包被液稀释Taq酶到指定浓度，每孔100μL，4℃过夜；次日，洗涤液清洗2次，拍干；加入封闭液(20％BSA+80％PBS)，每孔120μL，37℃，1h，拍干；加入稀释后的细胞上清，100μL/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100μL，37℃,30min；洗涤液清洗5次，拍干；加入显色液A液(50μL/孔)，加入显色液B液(50μL/孔)，10min；加入终止液，50μL/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。
5.2重组抗体表达质粒线性化
准备下述试剂：Buffer 50μl、DNA 100μg/管、PuvⅠ酶10μl、无菌水补至500μl，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。
5.3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400ng/ml，调节CHO细胞1.43×10 7cells/ml于离心管中，100μl质粒与700μl细胞混合，转入电转杯，电转，次日计数；25μmol/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。
6、生产重组抗体
6.1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。
6.2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:11所示；另一条Mr为28KD(轻链)，序列如SEQ ID NO:12所示。
实施例2
实施例1得到的样品1的抗体(具有序列如SEQ ID NO:11以及12所示的重链和轻链)虽然具备结合Taq DNA聚合酶的能力，但亲和力和抗体活性均不够理想，因而申请人通过对该抗体的轻链CDR及重链CDR进行突变。
经分析，重链的互补决定区(WT)：
CDR-VH1为S-V-D(X1)-T-F-S(X2)-T-Y-Y-L(X3)-Y；
CDR-VH2为G-V(X1)-N-P-T-S-N(X2)-P-V-F-D(X3)-E-K；
CDR-VH3为T-R-S-I(X1)-L(X2)-R-R-G-Y-Y-P(X3)-D-Y；
轻链的互补决定区：
CDR-VL1为R-G(X1)-S-Q-D-I-Q(X2)-N-Y-V(X3)-N；
CDR-VL2为I-Y-F(X1)-T-S-R-L-Q(X2)-S-G-I(X3)-P；
CDR-VL3为Q-D-D-T-I(X1)-P-V(X2)-T-W(X3)-G；
其中，X1、X2、X3均为突变位点。
表1与抗体活性有关的突变位点
Figure PCTCN2019109791-appb-000006
在突变后对抗体活性进行检测，包被液稀释Taq DNA聚合酶到指定浓度，每孔100μL，4℃过夜；次日，洗涤液清洗2次，拍干；加入封闭液(20％BSA+80％PBS)，每孔120μL，37℃，1h，拍干；加入稀释后的Taq单克隆抗体，100μL/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100μL，37℃，30min；洗涤液清洗5次，拍干；加入显色液A液(50μL/孔)，加入显色液B液(50μL/孔)，10min；加入终止液，50μL/孔；酶标仪上450nm(参考630nm)处读OD值。
部分结果如下：
表2抗体活性分析数据
浓度(ng/ml) WT 突变1 突变2 突变3 突变4 突变5
37.04 2.298 2.382 2.054 1.784 1.378 1.405
12.35 2.101 2.139 1.841 0.874 0.679 0.701
4.12 1.578 1.697 1.005 0.450 0.354 0.362
1.37 0.798 0.887 0.514 0.067 0.057 0.054
0.46 0.399 0.442 0.059 - - -
0 0.035 0.047 0.050 - - -
“-”代表无活性。
亲和力分析
利用AMC传感器，对上述抗体用PBST稀释到10μg/ml，Taq DNA聚合酶用PBST进行梯度稀释：1000nmol/ml、500nmol/ml、250nmol/ml、125nmol/ml、62.5nmol/ml、31.3nmol/ml、15.6nmol/ml、0nmol/ml。
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表示平衡解亲常数即亲和力；Kon表示结合速率；Kdis表示解离速率。
表3亲和力分析数据
不同突变 KD(M) Kon(1/Ms) Kdis(1/S)
WT 2.457E-09 5.904E+04 1.451E-04
突变1 3.839E-10 9.832E+04 3.775E-05
突变2 9.900E-10 5.924E+04 5.865E-05
突变3 2.066E-08 6.550E+04 1.353E-03
突变4 - - -
突变5 - - -
“-”表示未检测。
从表2和表3可知，突变1的活性效果和亲和力最佳，因而以突变1作为骨架序列筛选效价较好的突变位点(保证筛选得到的抗体活性与突变1相近，抗体活性±10％)，部分结果如下。
表4与抗体亲和力有关的突变位点
Figure PCTCN2019109791-appb-000007
Figure PCTCN2019109791-appb-000008
亲和力分析，方法同上，结果如表5所示。表5亲和力分析数据
不同突变 KD(M) Kon(1/Ms) Kdis(1/S)
突变1 3.839E-10 9.832E+04 3.775E-05
突变1-1 4.914E-10 3.217E+04 1.581E-05
突变1-2 2.850E-10 4.971E+04 1.417E-05
突变1-3 3.675E-10 4.196E+04 1.542E-05
突变1-4 7.192E-10 3.821E+04 2.748E-05
突变1-5 8.734E-10 8.910E+04 7.782E-05
突变1-6 8.085E-10 4.213E+04 3.406E-05
突变1-7 5.986E-10 4.100E+04 2.454E-05
突变1-8 3.435E-11 4.763E+04 1.636E-06
突变1-9 9.261E-10 7.865E+04 7.284E-05
突变1-10 3.839E-10 9.801E+04 3.764E-05
突变1-11 6.126E-10 4.999E+04 3.062E-05
突变1-12 2.196E-11 9.338E+04 2.051E-06
突变1-13 4.075E-10 6.174E+04 2.516E-05
突变1-14 2.286E-10 8.407E+04 1.922E-05
突变1-15 1.201E-10 4.760E+04 5.717E-06
突变1-16 2.227E-10 6.514E+04 1.451E-05
突变1-17 3.980E-10 3.405E+04 1.355E-05
突变1-18 6.772E-10 6.640E+04 4.497E-05
突变1-19 2.913E-10 6.333E+04 1.845E-05
突变1-20 7.281E-10 8.832E+04 6.430E-05
突变1-21 6.643E-11 8.978E+04 5.964E-06
突变1-22 3.839E-10 9.721E+04 3.698E-05
突变1-23 9.131E-10 7.022E+04 6.411E-05
突变1-24 1.989E-10 5.645E+04 1.123E-05
突变1-25 6.784E-10 6.733E+04 4.568E-05
突变1-26 8.518E-10 3.397E+04 2.894E-05
突变1-27 2.805E-11 7.242E+04 2.031E-06
突变1-28 4.018E-10 6.498E+04 2.611E-05
突变1-29 3.145E-11 3.609E+04 1.135E-06
突变1-30 9.113E-10 4.927E+04 4.490E-05
突变1-31 8.499E-10 6.329E+04 5.379E-05
突变1-32 2.810E-10 3.132E+04 8.800E-06
突变1-33 9.259E-10 4.261E+04 3.945E-05
突变1-34 9.870E-10 6.255E+04 6.174E-05
突变1-35 7.055E-10 5.244E+04 3.700E-05
突变1-36 7.678E-10 5.018E+04 3.853E-05
突变1-37 3.820E-10 7.021E+04 2.682E-05
突变1-38 1.060E-10 3.136E+04 3.324E-06
突变1-39 4.574E-10 4.814E+04 2.202E-05
突变1-40 6.154E-10 7.645E+04 4.705E-05
突变1-41 2.968E-10 3.308E+04 9.819E-06
突变1-42 8.030E-10 4.673E+04 3.752E-05
突变1-43 9.713E-10 7.125E+04 6.920E-05
突变1-44 1.328E-11 3.690E+05 4.899E-06
突变1-45 5.824E-11 5.265E+04 3.066E-06
突变1-46 6.547E-10 4.377E+04 2.866E-05
突变1-47 7.394E-10 6.320E+04 4.673E-05
突变1-48 3.839E-10 9.792E+04 3.721E-05
突变1-49 1.983E-10 8.610E+04 1.708E-05
突变1-50 5.929E-10 5.085E+04 3.015E-05
突变1-51 3.836E-10 9.830E+04 3.771E-05
突变1-52 6.981E-10 3.398E+04 2.372E-05
突变1-53 3.839E-10 9.897E+04 3.801E-05
突变1-54 2.727E-10 3.723E+04 1.015E-05
突变1-55 1.734E-10 6.444E+04 1.117E-05
突变1-56 9.136E-10 4.148E+04 3.790E-05
突变1-57 3.616E-10 6.897E+04 2.494E-05
突变1-58 1.785E-10 7.498E+04 1.338E-05
突变1-59 5.067E-11 6.043E+04 3.062E-06
突变1-60 3.140E-11 8.461E+04 2.657E-06
从表5可以看出，表4中列出的突变位点对抗体的亲和力影响不大。
为验证上述结果，以WT作为骨架序列重复上述实验，进行突变位点的亲和力验证，部分结果如下。
表6以WT为骨架进行的突变
Figure PCTCN2019109791-appb-000009
表7亲和力分析数据
不同突变 KD(M) Kon(1/Ms) Kdis(1/S)
WT 2.457E-09 5.904E+04 1.451E-04
WT 1-1 4.652E-09 5.879E+04 2.735E-04
WT 1-2 3.414E-09 4.849E+04 1.655E-04
WT 1-3 2.296E-09 4.802E+04 1.103E-04
WT 1-4 4.100E-09 8.842E+04 3.625E-04
WT 1-5 5.126E-09 6.125E+04 3.139E-04
WT 1-6 6.652E-09 7.610E+04 5.062E-04
WT 1-7 3.418E-09 8.668E+04 2.962E-04
WT 1-8 3.018E-09 3.265E+04 9.855E-05
WT 1-9 8.568E-09 5.054E+04 4.330E-04
WT 1-10 8.300E-09 8.413E+04 6.983E-04
从表6和表7分析，表6中列出的突变位点对抗体的亲和力影响也不大。
本公开的抗体与Taq DNA聚合酶修饰后，70℃以下可以封闭酶的活性，70℃以上即可解离，在95℃时只要1-3min即可完全解离，使酶的活性成分释放。
在同等条件下对加了抗体和没加抗体修饰的Taq DNA聚合酶进行特异性检测，结果如附图2中的PCR电泳图显示，其中1为Taq DNA聚合酶(没有抗体修饰)，2和4为由突变1抗体修饰的Taq DNA聚合酶，3和5为由WT抗体修饰的Taq DNA聚合酶。结果分析表明，经抗体修饰的TAQ酶具有明显的提高扩增特异性。分析各泳道中出现的非特异性条带情况，可以看出经突变1抗体修饰的Taq DNA聚合酶的特异性会稍优于经WT抗体修饰的Taq DNA聚合酶的特异性。本公开还对表4中的突变组合进行抗体与Taq DNA聚合酶结合稳定性、快速激活以及pH的兼容性进行检测，均有优良的效果，说明本公开提供的抗Taq DNA聚合酶的抗体在分子检测中具有良好的应用。
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，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
本公开提供的结合蛋白能够特异性结合Taq酶以形成抗体酶复合物，因此在室温下可以有效地封闭Taq DNA聚合酶的活性；而在高温时，这种复合物会解离，释放出具有活性的Taq DNA聚合酶，从而进行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，同时可以提高Taq DNA聚合酶的长期稳定性。本公开的结合蛋白可以广泛地应用于各种热启动PCR。

Claims (18)

  1. 一种包含抗原结合结构域的分离的结合蛋白，其特征在于，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，或与下述氨基酸序列的互补决定区具有至少80％的序列同一性且与Taq DNA聚合酶具有K D≤8.568×10 -9mol/L的亲和力；
    互补决定区CDR-VH1为S-V-X1-T-F-X2-T-Y-Y-X3-Y，其中，
    X1是D、E或N，X2是S或T，X3是I或L；
    互补决定区CDR-VH2为G-X1-N-P-T-S-X2-P-V-F-X3-E-K，其中，
    X1是I、V或L，X2是N或GG，X3是D、E或N；
    互补决定区CDR-VH3为T-R-S-X1-X2-R-R-G-Y-Y-X3-D-Y，其中，
    X1是I、V或L，X2是I、V或L，X3是F或P；
    互补决定区CDR-VL1为R-X1-S-Q-D-I-X2-N-Y-X3-N，其中，
    X1是A或G，X2是N或Q，X3是I、V或L；
    互补决定区CDR-VL2为I-Y-X1-T-S-R-L-X2-S-G-X3-P，其中，
    X1是Y或F，X2是Q、H或N，X3是I、V或L；
    互补决定区CDR-VL3为Q-D-D-T-X1-P-X2-T-X3-G，其中，
    X1是I、V或L，X2是I、V或L，X3是W或F。
  2. 根据权利要求1所述的结合蛋白，其特征在于，
    所述互补决定区CDR-VH1中，X3是I；
    所述互补决定区CDR-VH2中，X3是N；
    所述互补决定区CDR-VH3中，X3是F；
    所述互补决定区CDR-VL1中，X1是A；
    所述互补决定区CDR-VL2中，X1是Y；
    所述互补决定区CDR-VL3中，X3是F；
    进一步地，所述互补决定区CDR-VH1中，X1是D，X2是S；
    进一步地，所述互补决定区CDR-VH1中，X1是E，X2是S；
    进一步地，所述互补决定区CDR-VH1中，X1是N，X2是S；
    进一步地，所述互补决定区CDR-VH1中，X1是D，X2是T；
    进一步地，所述互补决定区CDR-VH1中，X1是E，X2是T；
    进一步地，所述互补决定区CDR-VH1中，X1是N，X2是T；
    进一步地，所述互补决定区CDR-VH2中，X1是I，X2是N；
    进一步地，所述互补决定区CDR-VH2中，X1是I，X2是GG；
    进一步地，所述互补决定区CDR-VH2中，X1是V，X2是N；
    进一步地，所述互补决定区CDR-VH2中，X1是V，X2是GG；
    进一步地，所述互补决定区CDR-VH2中，X1是L，X2是N；
    进一步地，所述互补决定区CDR-VH2中，X1是L，X2是GG；
    进一步地，所述互补决定区CDR-VH3中，X1是I，X2是I；
    进一步地，所述互补决定区CDR-VH3中，X1是I，X2是V；
    进一步地，所述互补决定区CDR-VH3中，X1是I，X2是L；
    进一步地，所述互补决定区CDR-VH3中，X1是V，X2是I；
    进一步地，所述互补决定区CDR-VH3中，X1是V，X2是V；
    进一步地，所述互补决定区CDR-VH3中，X1是V，X2是L；
    进一步地，所述互补决定区CDR-VH3中，X1是L，X2是I；
    进一步地，所述互补决定区CDR-VH3中，X1是L，X2是V；
    进一步地，所述互补决定区CDR-VH3中，X1是L，X2是L；
    进一步地，所述互补决定区CDR-VL1中，X2是N，X3是I；
    进一步地，所述互补决定区CDR-VL1中，X2是N，X3是V；
    进一步地，所述互补决定区CDR-VL1中，X2是N，X3是L；
    进一步地，所述互补决定区CDR-VL1中，X2是Q，X3是I；
    进一步地，所述互补决定区CDR-VL1中，X2是Q，X3是V；
    进一步地，所述互补决定区CDR-VL1中，X2是Q，X3是L；
    进一步地，所述互补决定区CDR-VL2中，X2是Q，X3是I；
    进一步地，所述互补决定区CDR-VL2中，X2是Q，X3是V；
    进一步地，所述互补决定区CDR-VL2中，X2是Q，X3是L；
    进一步地，所述互补决定区CDR-VL2中，X2是H，X3是I；
    进一步地，所述互补决定区CDR-VL2中，X2是H，X3是V；
    进一步地，所述互补决定区CDR-VL2中，X2是H，X3是L；
    进一步地，所述互补决定区CDR-VL2中，X2是N，X3是I；
    进一步地，所述互补决定区CDR-VL2中，X2是N，X3是V；
    进一步地，所述互补决定区CDR-VL2中，X2是N，X3是L；
    进一步地，所述互补决定区CDR-VL3中，X1是I，X2是I；
    进一步地，所述互补决定区CDR-VL3中，X1是I，X2是V；
    进一步地，所述互补决定区CDR-VL3中，X1是I，X2是L；
    进一步地，所述互补决定区CDR-VL3中，X1是V，X2是I；
    进一步地，所述互补决定区CDR-VL3中，X1是V，X2是V；
    进一步地，所述互补决定区CDR-VL3中，X1是V，X2是L；
    进一步地，所述互补决定区CDR-VL3中，X1是L，X2是I；
    进一步地，所述互补决定区CDR-VL3中，X1是L，X2是V；
    进一步地，所述互补决定区CDR-VL3中，X1是L，X2是L；
    优选地，各互补决定区的突变位点选自下述突变组合中的任一种：
    Figure PCTCN2019109791-appb-100001
    Figure PCTCN2019109791-appb-100002
  3. 根据权利要求1-2任一项所述的结合蛋白，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；
    进一步地，所述结合蛋白为纳米抗体、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。
  4. 根据权利要求1-2任一项所述的结合蛋白，其特征在于，所述结合蛋白包括序列依次如SEQ ID NO:1-4所示的轻链骨架区FR-L1、FR-L2、FR-L3及FR-L4，和/或，序列依次如SEQ ID NO:5-8所示的重链骨架区FR-H1、FR-H2、FR-H3及FR-H4；
    进一步地，所述结合蛋白还包含抗体恒定区序列；
    进一步地，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；
    进一步地，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、骆驼、驴、鹿、貂、鸡、鸭、鹅、火鸡、斗鸡或人；
    进一步地，所述恒定区来源于小鼠；
    轻链恒定区序列如SEQ ID NO:9所示；
    重链恒定区序列如SEQ ID NO:10所示。
  5. 一种分离的核酸，其特征在于，所述核酸编码权利要求1-4任一项所述的结合蛋白。
  6. 一种载体，其特征在于，其包含权利要求5所述的核酸。
  7. 一种宿主细胞，其特征在于，所述宿主细胞包括权利要求5所述的核酸或权利要求6所述的载体。
  8. 一种生产权利要求1-4任一项所述的结合蛋白的方法，其特征在于，包括如下步骤：
    在培养基中培养权利要求7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。
  9. 权利要求1-4任一项所述的结合蛋白在PCR中的应用。
  10. 一种试剂盒，其特征在于，所述试剂盒包括权利要求1-4任一项所述的结合蛋白、权利要求5所述的分离的核酸或权利要求6所述的载体中的一种或多种。
  11. 一种组合物，其中所述组合物包括根据权利要求1-4任一项所述的结合蛋白和Taq DNA聚合酶。
  12. 根据权利要求11所述的组合物，其中所述组合物还包括4种脱氧核苷三磷酸。
  13. 根据权利要求11或12所述的组合物，其中所述组合物还包括引物和/或探针。
  14. 根据权利要求11-13任一项所述的组合物，其中所述组合物还包括MgCl 2。
  15. 根据权利要求11-14任一项所述的组合物，其中所述组合物还包括作为模板的核酸。
  16. 一种用于扩增核酸的方法，包括使用根据权利要求1-4任一项所述的结合蛋白或根据权利要求11-15任一项所述的组合物进行热启动PCR。
  17. 根据权利要求16所述的方法，所述热启动PCR选自由多重PCR、实时PCR和实时定量PCR组成组。
  18. 根据权利要求1-4任一项所述的结合蛋白，在扩增核酸中的用途。
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113321733A (zh) * 2021-05-19 2021-08-31 厦门同仁心生物技术有限公司 一种TaqDNA聚合酶单克隆抗体及应用，包含该抗体的聚合酶反应体系及应用
CN115785276A (zh) * 2021-08-24 2023-03-14 东莞市朋志生物科技有限公司 一种抗Taq DNA聚合酶的抗体及其应用
WO2023035967A1 (zh) * 2021-09-07 2023-03-16 东莞市朋志生物科技有限公司 一种抗Taq DNA聚合酶的抗体及其应用

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111560073B (zh) * 2020-07-16 2020-10-09 翌圣生物科技（上海）有限公司 Taq酶5’-3’聚合酶活性封闭单克隆抗体及其应用
CN116731184B (zh) * 2022-11-25 2024-06-14 厦门康基生物科技有限公司 一种特异性结合Taq酶的单克隆抗体F6H12及其应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107828755A (zh) * 2017-11-29 2018-03-23 东北制药集团辽宁生物医药有限公司 热启动TaqDNA聚合酶及其制备方法与应用
CN108546749A (zh) * 2018-03-27 2018-09-18 深圳市艾伟迪生物科技有限公司 混合型热启动dna聚合酶组合物、pcr扩增试剂盒及其应用

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9951378B2 (en) * 2012-06-14 2018-04-24 Life Technologies Corporation Compositions, methods and kits for real time polymerase chain reaction (PCR)
CN107502600A (zh) * 2017-07-26 2017-12-22 李桂秋 一种含有纳米抗体的双热启动dna聚合酶及pcr扩增检测方法
CN110256566A (zh) * 2019-05-10 2019-09-20 江苏苏博生物医学科技南京有限公司 Taq DNA聚合酶单链免疫球蛋白IgG抗体、制备方法及其在基因分型检测中的应用

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107828755A (zh) * 2017-11-29 2018-03-23 东北制药集团辽宁生物医药有限公司 热启动TaqDNA聚合酶及其制备方法与应用
CN108546749A (zh) * 2018-03-27 2018-09-18 深圳市艾伟迪生物科技有限公司 混合型热启动dna聚合酶组合物、pcr扩增试剂盒及其应用

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
LIN QING , XU MU-YU , LI QING-GE: "Using Antibodies Against Taq-FS Polymerase for Hot-start PCR", CHINESE JOURNAL OF BIOCHEMISTRY AND MOLECULAR BIOLOGY, vol. 24, no. 5, 20 May 2008 (2008-05-20), pages 475 - 480, XP055806227, ISSN: 1007-7626, DOI: 10.13865/j.cnki.cjbmb.2008.05.011 *
MEYERSMILLER, CABIOS, vol. 4, 1989, pages 11 - 17
See also references of EP3865508A4

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113321733A (zh) * 2021-05-19 2021-08-31 厦门同仁心生物技术有限公司 一种TaqDNA聚合酶单克隆抗体及应用，包含该抗体的聚合酶反应体系及应用
CN115785276A (zh) * 2021-08-24 2023-03-14 东莞市朋志生物科技有限公司 一种抗Taq DNA聚合酶的抗体及其应用
CN115785276B (zh) * 2021-08-24 2023-10-03 东莞市朋志生物科技有限公司 一种抗Taq DNA聚合酶的抗体及其应用
WO2023035967A1 (zh) * 2021-09-07 2023-03-16 东莞市朋志生物科技有限公司 一种抗Taq DNA聚合酶的抗体及其应用

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