Anti-taq DNA polymerase monoclonal antibody and use thereof

Abstract

The present invention relates to a novel anti-Taq DNA polymerase monoclonal antibody, or an antigen-binding fragment thereof, which exhibit high sensitivity and high specificity in specifically binding Taq DNA polymerase, and can therefore bind Taq DNA polymerase to form a stable and efficiently-blocked hot-start Taq DNA polymerase, thus avoiding non-specific amplification during polymerase chain reactions at low temperatures and improving the specificity and sensitivity of PCR reactions.

Description

Anti-Taq DNA polymerase monoclonal antibody and its application Technical Field

[0001] The present invention relates to the technical field of DNA fragment amplification, and in particular to an antibody of Taq DNA polymerase, an antibody-enzyme complex for amplification, and a method for amplifying DNA fragments using the complex. Background Art

[0002] Polymerase Chain Reaction (PCR) is a method for rapidly amplifying specific DNA fragments in vitro and is widely used in fields such as genetic testing and biochemical testing. Taq DNA polymerase has polymerase activity at the 5'-3' end and good heat resistance. It can withstand the thermal denaturation step of PCR and does not require additional addition during the process. It is a commonly used DNA polymerase. However, because Taq DNA polymerase is also partially active under low temperature conditions, this can lead to sequence mismatches and the formation of primer dimers, resulting in non-specific amplification of DNA fragments. In particular, during the preparation stage and preheating process of the PCR reaction system, Taq DNA polymerase will catalyze the extension of mismatched primers or the formation of primer dimers, which is the main reason affecting the synthesis of the target fragment.

[0003] The use of hot-start Taq DNA polymerase can effectively reduce the non-specific synthesis of DNA fragments. Hot-start Taq DNA polymerase is inactive at low temperatures and will not cause mismatches. It will only recover activity at high temperatures and perform specific amplification, thereby avoiding mismatches and the formation of primer dimers.

[0004] Hot-start DNA polymerases can be antibody-modified DNA polymerases. At low temperatures, Taq DNA polymerase binds to its antibody, blocking its activity. After a period of hot start at a higher temperature, the Taq DNA polymerase antibody denatures at high temperature and detaches from the active center of the Taq DNA polymerase, releasing the activity of the Taq DNA polymerase and allowing the amplification reaction to proceed. By using Taq DNA polymerase antibodies to block the activity of Taq DNA polymerase at room temperature, nonspecific amplification caused by primer mismatches is avoided, improving amplification specificity. Furthermore, antibody-modified Taq DNA polymerases require a short hot start time and can effectively protect the template DNA. However, the binding strength of the antibody to the enzyme is easily affected by the environment, making ineffective blocking a possibility. Furthermore, there are many Taq DNA polymerase mutants, making it difficult to find antibodies that are compatible and can effectively block the enzyme.

[0005] Therefore, an adaptive and stable and efficient blocking antibody is needed to modify the hot-start Taq DNA polymerase, thereby reducing nonspecific amplification and improving the specificity and sensitivity of the PCR reaction.

[0006] Summary of the Invention

[0007] The inventors immunized mice with wild-type Taq DNA polymerase, fused the mouse spleen cells with myeloma cells, screened for hybridoma cell lines that specifically bound to Taq DNA polymerase using ELISA, constructed recombinant antibodies, and obtained recombinant anti-Taq DNA polymerase antibodies. This led to the present invention.

[0008] Therefore, in the first aspect, the present invention provides an anti-Taq DNA polymerase monoclonal antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a heavy chain complementary determining region V H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementarity determining region V L CDR1, V L CDR2 and V L CDR3; of which:

[0009] V H The amino acid sequence of CDR1 is shown in SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 22 or SEQ ID NO: 28;

[0010] V H The amino acid sequence of CDR2 is shown in SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:23, SEQ ID NO:29, SEQ ID NO:32 or SEQ ID NO:37;

[0011] V H The amino acid sequence of CDR3 is shown in SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO: 18, SEQ ID NO: 24, SEQ ID NO: 33 or SEQ ID NO: 38;

[0012] V L The amino acid sequence of CDR1 is shown in SEQ ID NO:4, SEQ ID NO:10, SEQ ID NO:15, SEQ ID NO:19, SEQ ID NO:25, SEQ ID NO:30, SEQ ID NO:34 or SEQ ID NO:39;

[0013] V L The amino acid sequence of CDR2 is shown in SEQ ID NO: 5, SEQ ID NO: 11, SEQ ID NO: 20, SEQ ID NO: 26 or SEQ ID NO: 35; and

[0014] V L The amino acid sequence of CDR3 is shown in SEQ ID NO:6, SEQ ID NO:12, SEQ ID NO:21, SEQ ID NO:27, SEQ ID NO:31, SEQ ID NO:36 or SEQ ID NO:40.

[0015] In a second aspect, the present invention provides a nucleic acid molecule encoding the anti-Taq DNA polymerase monoclonal antibody or antigen-binding fragment thereof of the first aspect.

[0016] In a third aspect, the present invention provides a vector comprising the nucleic acid molecule of the second aspect.

[0017] In a fourth aspect, the present invention provides an expression cell comprising the nucleic acid molecule of the second aspect or the vector of the third aspect.

[0018] In a fifth aspect, the present invention provides a hot-start Taq DNA polymerase, comprising the anti-Taq DNA polymerase monoclonal antibody or antigen-binding fragment thereof according to the first aspect and Taq DNA polymerase.

[0019] In a sixth aspect, the present invention provides a method for amplifying a target DNA sequence by polymerase chain reaction, the method comprising the step of using the hot-start Taq DNA polymerase of the fifth aspect to amplify the target DNA sequence.

[0020] In a seventh aspect, the present invention provides a kit for polymerase chain reaction, comprising: the hot-start Taq DNA polymerase of the fifth aspect; and instructions for use.

[0021] In summary, the present invention provides a novel anti-Taq DNA polymerase monoclonal antibody or antigen-binding fragment thereof, which exhibits high sensitivity and high specificity for specific binding to Taq DNA polymerase, thereby being able to bind to Taq DNA polymerase to form a stable and efficient blocked hot-start Taq DNA polymerase. This hot-start Taq DNA polymerase inhibits the activity of Taq DNA polymerase at low temperatures and restores the activity of Taq DNA polymerase at high temperatures. Thus, when used in polymerase chain reaction (PCR), it can effectively reduce sequence mismatches and primer dimer formation at low temperatures, avoid nonspecific amplification of DNA fragments, and does not affect the specific amplification of DNA at high temperatures, thereby improving the specificity and sensitivity of the PCR reaction. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the specific implementation of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the specific implementation or the description of the prior art.

[0023] FIG1 shows the binding reaction results of the recombinant monoclonal antibodies T1, T3, T5 and T8 of the present invention with three Taq DNA polymerases and Pfu DNA polymerase (as a control).

[0024] FIG2 shows the binding reaction results of the recombinant monoclonal antibodies T11, T12, T15 and T19 of the present invention with three Taq DNA polymerases and Pfu DNA polymerase (as a control).

[0025] Figure 3 shows the enzyme activity results of the antibody-enzyme complex prepared according to one embodiment of the present invention, wherein S1: antibody-Taq WT complex without hot start, S2: antibody-enzyme Taq 22 complex without hot start, S3: antibody-enzyme KlenTaq10 complex without hot start, S4: antibody-enzyme Taq WT complex with hot start, S5: antibody-enzyme Taq 22 complex with hot start, S6: antibody-enzyme KlenTaq10 complex with hot start, S7: single enzyme Taq WT without antibody, S8: single enzyme Taq 22 without antibody, S9: single enzyme KlenTaq10 without antibody, S10: negative control, S11: positive control.

[0026] FIG4 shows the specificity test results of the hot-start Taq DNA polymerase of the recombinant monoclonal antibodies T8, T9 and T11 of the present invention combined with the Taq22 enzyme.

[0027] FIG5 shows the specificity test results of the hot-start Taq DNA polymerase of the recombinant monoclonal antibodies T8, T9 and T11 of the present invention combined with TaqWT enzyme.

[0028] FIG6 shows the specificity test results of the hot-start Taq DNA polymerase combined with the KlenTaq10 enzyme by the recombinant monoclonal antibodies T8, T9 and T11 of the present invention. DETAILED DESCRIPTION

[0029] Hereinafter, the present invention will be described in detail with reference to the accompanying drawings. It should be understood that the following description is merely illustrative of the present invention and is not intended to limit the scope of the present invention. The scope of protection of the present invention shall be subject to the appended claims. Furthermore, those skilled in the art will appreciate that the technical solutions of the present invention may be modified without departing from the spirit and purpose of the present invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter of the present invention belongs. Before describing the present invention in detail, the following definitions are provided for a better understanding of the present invention.

[0031] Where a numerical range is provided, such as a concentration range, a percentage range, or a ratio range, it is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limits of the range and any other stated or intervening values ​​in the stated range are encompassed within the subject matter unless the context clearly dictates otherwise. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and such embodiments are also encompassed within the subject matter, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also encompassed within the subject matter.

[0032] In the context of the present invention, many embodiments use the expressions "comprising", "including" or "consisting essentially / mainly of..." The expressions "comprising", "including" or "consisting essentially / mainly of..." can generally be understood as open-ended expressions, indicating that in addition to the various elements, components, assemblies, method steps, etc. specifically listed after the expression, other elements, components, assemblies, method steps, etc. are also included. In addition, in this document, the expressions "comprising", "including" or "consisting essentially / mainly of..." can also be understood as closed-ended expressions in some cases, indicating that only the various elements, components, assemblies, method steps specifically listed after the expression are included, and no other elements, components, assemblies, method steps are included. In this case, the expression is equivalent to the expression "consisting of..."

[0033] For a better understanding of the present teachings and without limiting the scope of the present teachings, all numbers and other numerical values ​​expressing quantities, percentages or ratios used in the specification and claims should be understood as being modified in all cases by the term "about", unless otherwise indicated. Therefore, unless indicated to the contrary, the numerical parameters set forth in the following specification and the appended claims are approximate values ​​that may vary depending on the desired properties sought to be obtained. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0034] As used herein, the term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains, each pair having one "light" (L) chain and one "heavy" (H) chain. Antibody light chains can be classified as kappa and lambda light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and the isotype of the antibody can be defined accordingly as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are connected by a "J" region (hinge region) of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (V H ) and heavy chain constant region (C H ). The heavy chain constant region consists of three domains (C H1 、C H2 and C H3 Each light chain consists of a light chain variable region (V L ) and the light chain constant region (C L ). The light chain constant region consists of a domain C L The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. H and V L The variable regions can be further subdivided into regions of high variability, called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). For each heavy or light chain, the variable region comprises three CDRs, namely CDR1, CDR2 and CDR3. H and V L It consists of three CDRs and four FRs arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable region (V H and V L ) respectively form the antigen binding site.

[0035] The rules for assigning amino acids to various regions or domains are defined in many literatures: Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda Md (1987 and 1991)); Chothia & Lesk J. Mol. Biol. 1987; 196: 901-917; Chothia et al., Nature 1989; 342: 878-883; Ehrenmann, Francois, Quentin Kaas, and Marie-Paule Lefranc. "IMGT / 3Dstructure-DB and IMGT / DomainGapAlign: a database and a tool for immunoglobulins or antibodies, T cell receptors, MHC, IgSF and MhcSF." Nucleic acids research 2009; 38(suppl_1): D301-D307.

[0036] The exact boundaries of CDRs have been defined differently according to different systems. The Kabat system not only provides a clear residue numbering system that can be applied to any variable region of an antibody, but also provides precise residue boundaries that define the three CDRs. These CDRs are called Kabat CDRs. Chothia discovered that certain sub-portions within the Kabat system CDRs, despite having great diversity at the amino acid sequence level, have almost identical peptide backbone conformations. These sub-portions are called Chothia CDRs, and Chothia CDRs have overlapping boundaries with Kabat CDRs. The above-mentioned overlapping boundaries are further described by Padlan and MacCallum. CDR boundary definitions may not strictly adhere to the above system, such as the AbM definition. In this article, CDRs can be defined according to any of these systems, although the preferred embodiment uses the antibody numbering system of Chothia et al. to define CDRs.

[0037] As used herein, the term "monoclonal antibody" or "monoclonal antibody" refers to an antibody or a fragment of an antibody from a group of highly homologous antibody molecules, that is, a group of identical antibody molecules except for natural mutations that may occur spontaneously. The antibody molecule can be an immunoglobulin, whether it is a natural immunoglobulin or an immunoglobulin obtained partially or entirely by a synthetic method. The antibody molecule can also include all polypeptides or proteins with an antibody binding domain, and antibody fragments with antibody domains are molecules such as Fab, scFv, Fv, dAb, Fd, and bifunctional antibodies. Monoclonal antibodies have high specificity for a single epitope on an antigen. Polyclonal antibodies are relative to monoclonal antibodies, and they usually contain at least two or more different antibodies, and these different antibodies usually recognize different epitopes on the antigen. Monoclonal antibodies can usually be obtained using the hybridoma technology first reported by Kohler et al. ( G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity [J]. Nature, 1975; 256 (5517): 495), but can also be obtained by recombinant DNA technology (see, for example, US Patent 4,816,567). As used herein, the terms "monoclonal antibody" and "monoclonal antibody" have the same meaning and are used interchangeably; the terms "polyclonal antibody" and "polyclonal antibody" have the same meaning and are used interchangeably; the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. In addition, in the present invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by Ala or A. Glycine can be represented by Gly or G, valine can be represented by Val or V, leucine can be represented by Leu or L, isoleucine can be represented by Ile or I, proline can be represented by Pro or P, phenylalanine can be represented by Phe or F, tyrosine can be represented by Tyr or Y, tryptophan can be represented by Trp or W, serine can be represented by Ser or S, threonine can be represented by Thr or T, cysteine ​​can be represented by Cys or C, methionine can be represented by Met or M, asparagine can be represented by Asn or N, glutamine can be represented by Gln or Q, aspartic acid can be represented by Asp or D, glutamic acid can be represented by Glu or E, lysine can be represented by Lys or K, arginine can be represented by Arg or R, and histidine can be represented by His or H.

[0038] As used herein, the term "recombinant antibody" refers to an antibody obtained by cloning an antibody gene into an expression vector and then transfecting the expression vector into a suitable host cell line for expression by molecular biology techniques. The recombinant antibody encoding gene may be identical or non-identical to the antibody encoding gene of natural origin. For example, the complete encoding gene of the antibody obtained by immunizing an animal may be cloned into an expression vector for expression, thereby obtaining an antibody identical to the antibody obtained by the immunizing animal. Alternatively, the gene encoding the variable region (including heavy chain variable region and light chain variable region) of the antibody obtained by immunizing an animal may be cloned into an expression vector together with the gene encoding the constant region of the antibody in another species (such as human) source, thereby obtaining an antibody comprising heavy chain and light chain variable region sequences from one species and constant region sequences from another species, such as an antibody with mouse heavy chain and light chain variable region connected to a human constant region. This antibody is commonly referred to as a "chimeric antibody" in the art.

[0039] As used herein, the term "antigen-binding fragment" refers to fragments and antibody analogs from antibodies that are capable of binding to an antigen, which generally include at least a portion of the antigen-binding region or variable region (e.g., one or more CDRs) of the parental antibody. The antigen-binding fragment retains at least some of the binding activity of the parental antibody. Typically, when the activity is expressed on a molar basis, the antigen-binding fragment retains at least 10% of the parental binding activity. Specifically, the antigen-binding fragment retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the binding affinity of the parental antibody for the target. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, linear antibodies, single-chain antibodies, nanobodies, domain antibodies, and multispecific antibodies. Among them, a "Fab fragment" consists of a light chain, the CH1 of a heavy chain, and the variable region. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule. A "Fab' fragment" contains one light chain and part of one heavy chain (including the VH domain, the CH1 domain, and the region between the CH1 and CH2 domains); thus, an interchain disulfide bond can form between the two heavy chains of the two Fab' fragments to form an F(ab')2 molecule. An "Fv region" contains the variable regions from both the heavy and light chains, but lacks the constant regions.

[0040] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its antigen.

[0041] In the present invention, primers are also used to perform PCR amplification of the nucleotide sequence encoding the antibody. In the primer sequence, some sites only involve a single base, such as any one of adenine (A), guanine (G), cytosine (C), and thymine (T), while some sites involve a combination of two, three, or four bases. In this case, these bases are referred to as degenerate bases of each other, which are mainly determined based on the degeneracy of the codons. Degenerate bases can be represented by the letters R, Y, M, K, S, W, H, B, V, D, and N, where R represents A / G, Y represents C / T, M represents A / C, K represents G / T, S represents C / G, W represents A / T, H represents A / T / C, B represents G / T / C, V represents G / A / C, D represents G / A / T, and N represents A / T / C / G.

[0042] As used herein, the terms sequence "identity," "identity," or "homology" have their art-recognized meanings, and the percentage of sequence identity between two nucleic acid or polypeptide molecules or regions can be calculated using published techniques. Sequence identity can be measured along the entire length of a polynucleotide or polypeptide or along a region of the molecule (see, e.g., Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). While there are many methods for measuring the identity between two polynucleotides or polypeptides, the term "identity" is well known to those skilled in the art to apply conservative amino acid substitutions in peptides or proteins, and generally can be made without altering the biological activity of the resulting molecule. In general, those skilled in the art recognize that single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al., Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub.co., p. 224).

[0043] In this article, the terms "hot start", "heat activation" and "heat shock" can be used interchangeably and have the meaning commonly understood in the art, referring to the phenomenon that DNA polymerase only exerts its polymerization activity when the sample temperature exceeds at least a certain temperature, such as at least 90°C.

[0044] As previously mentioned, the present invention aims to provide an anti-Taq DNA polymerase monoclonal antibody. As previously mentioned, the inventors immunized mice with wild-type Taq DNA polymerase, fused the mouse spleen cells with myeloma cells, and screened for hybridoma cell lines that specifically bind to Taq DNA polymerase using ELISA.

[0045] Therefore, in the first aspect, the present invention provides an anti-Taq DNA polymerase monoclonal antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a heavy chain complementary determining region V H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementarity determining region V L CDR1, V L CDR2 and V L CDR3; of which:

[0046] V H The amino acid sequence of CDR1 is shown in SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 22 or SEQ ID NO: 28;

[0047] V H The amino acid sequence of CDR2 is shown in SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:23, SEQ ID NO:29, SEQ ID NO:32 or SEQ ID NO:37;

[0048] V H The amino acid sequence of CDR3 is shown in SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO: 18, SEQ ID NO: 24, SEQ ID NO: 33 or SEQ ID NO: 38;

[0049] V L The amino acid sequence of CDR1 is shown in SEQ ID NO:4, SEQ ID NO:10, SEQ ID NO:15, SEQ ID NO:19, SEQ ID NO:25, SEQ ID NO:30, SEQ ID NO:34 or SEQ ID NO:39;

[0050] V LThe amino acid sequence of CDR2 is shown in SEQ ID NO: 5, SEQ ID NO: 11, SEQ ID NO: 20, SEQ ID NO: 26 or SEQ ID NO: 35; and

[0051] V L The amino acid sequence of CDR3 is shown in SEQ ID NO:6, SEQ ID NO:12, SEQ ID NO:21, SEQ ID NO:27, SEQ ID NO:31, SEQ ID NO:36 or SEQ ID NO:40.

[0052] In a specific embodiment, the heavy chain variable region includes the heavy chain complementary determining region V represented by the amino acid sequence of SEQ ID NO: 1-3, H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V shown in SEQ ID NO: 4-6, respectively. L CDR1, V L CDR2 and V L CDR3.

[0053] In another specific embodiment, the heavy chain variable region includes the heavy chain complementary determining region V represented by the amino acid sequence of SEQ ID NO: 7-9, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V shown in SEQ ID NO: 10-12 respectively. L CDR1, V L CDR2 and V L CDR3.

[0054] In another specific embodiment, the heavy chain variable region includes the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 3, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 15, SEQ ID NO: 5 and SEQ ID NO: 6, respectively. L CDR1, V L CDR2 and V L CDR3.

[0055] In another specific embodiment, the heavy chain variable region comprises a heavy chain complementary determining region V represented by the amino acid sequence of SEQ ID NO: 16-18, H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V shown in SEQ ID NO: 19-21 respectively. L CDR1, V L CDR2 and V L CDR3.

[0056] In another specific embodiment, the heavy chain variable region includes the heavy chain complementary determining region V represented by the amino acid sequence of SEQ ID NO: 22-24, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 25-27, respectively. L CDR1, V L CDR2 and V L CDR3.

[0057] In another specific embodiment, the heavy chain variable region includes the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 3, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 30, SEQ ID NO: 5 and SEQ ID NO: 31, respectively. L CDR1, V L CDR2 and V L CDR3.

[0058] In another specific embodiment, the heavy chain variable region includes the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 13, SEQ ID NO: 32, and SEQ ID NO: 33, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 34-36, respectively. L CDR1, V LCDR2 and V L CDR3.

[0059] In another specific embodiment, the heavy chain variable region includes the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 13, SEQ ID NO: 37, and SEQ ID NO: 38, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 39, SEQ ID NO: 5 and SEQ ID NO: 40, respectively. L CDR1, V L CDR2 and V L CDR3.

[0060] In another specific embodiment, the heavy chain variable region includes the heavy chain complementary determining region V represented by the amino acid sequence of SEQ ID NO: 1-3, H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 15, SEQ ID NO: 5 and SEQ ID NO: 6, respectively. L CDR1, V L CDR2 and V L CDR3.

[0061] In another specific embodiment, the heavy chain variable region includes the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 18, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 39, SEQ ID NO: 5 and SEQ ID NO: 21, respectively. L CDR1, V L CDR2 and V L CDR3.

[0062] In another specific embodiment, the heavy chain variable region includes the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 22, SEQ ID NO: 32, and SEQ ID NO: 38, respectively. H CDR1, V HCDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 39, SEQ ID NO: 26 and SEQ ID NO: 31, respectively. L CDR1, V L CDR2 and V L CDR3.

[0063] In another specific embodiment, the heavy chain variable region includes the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 13, SEQ ID NO: 37, and SEQ ID NO: 3, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 25, SEQ ID NO: 5 and SEQ ID NO: 40, respectively. L CDR1, V L CDR2 and V L CDR3.

[0064] In another specific embodiment, the heavy chain variable region includes the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 28, SEQ ID NO: 32, and SEQ ID NO: 33, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 12, respectively. L CDR1, V L CDR2 and V L CDR3.

[0065] In another specific embodiment, the heavy chain variable region includes the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 33, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 30, SEQ ID NO: 5 and SEQ ID NO: 6, respectively. L CDR1, V L CDR2 and VL CDR3.

[0066] In another specific embodiment, the heavy chain variable region includes the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 22, SEQ ID NO: 14, and SEQ ID NO: 9, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 15, SEQ ID NO: 5 and SEQ ID NO: 6, respectively. L CDR1, V L CDR2 and V L CDR3.

[0067] In another specific embodiment, the heavy chain variable region includes the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 16, SEQ ID NO: 32, and SEQ ID NO: 38, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 27, respectively. L CDR1, V L CDR2 and V L CDR3.

[0068] In another specific embodiment, the heavy chain variable region includes the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 33, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 35 and SEQ ID NO: 12, respectively. L CDR1, V L CDR2 and V L CDR3.

[0069] In another specific embodiment, the heavy chain variable region includes the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 37, and SEQ ID NO: 38, respectively. HCDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 19, SEQ ID NO: 5 and SEQ ID NO: 6, respectively. L CDR1, V L CDR2 and V L CDR3.

[0070] In another specific embodiment, the heavy chain variable region includes the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 13, SEQ ID NO: 32, and SEQ ID NO: 38, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 40, respectively. L CDR1, V L CDR2 and V L CDR3.

[0071] In another specific embodiment, the heavy chain variable region includes the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 22, SEQ ID NO: 37, and SEQ ID NO: 24, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 15, SEQ ID NO: 5 and SEQ ID NO: 36, respectively. L CDR1, V L CDR2 and V L CDR3.

[0072] In a specific embodiment, the antibody is a complete antibody comprising a variable region and a constant region. For the antibodies of the present invention, any framework region (FR) and any constant region can be used. The amino acid sequence of the FR or constant region used in the antibodies of the present invention can be the amino acid sequence of the original FR or constant region as a source, or it can be a different amino acid sequence obtained by performing one or more amino acid substitutions, deletions, additions and / or insertions on the amino acid sequence of the original FR or constant region. The structure used to support the CDR or CDR group of the present invention generally belongs to an antibody heavy chain or light chain sequence or a major part thereof, wherein the CDR or CDR group is located in a region corresponding to a naturally occurring V sequence encoded by a rearranged immunoglobulin gene. H and V L The CDR or CDR group of the antibody variable domain corresponds to the position of the antibody variable domain.

[0073] In a specific embodiment, the heavy chain variable region further comprises heavy chain framework regions HFR1, HFR2, HFR3 and HFR4, H CDR1, V H CDR2 and V H CDR3 is arranged from amino terminus to carboxyl terminus according to HFR1, V H CDR1、HFR2、V H CDR2、HFR3、V H Sequential arrangement of CDR3 and HFR4.

[0074] In a further specific embodiment, the heavy chain framework regions HFR1, HFR2, HFR3 and HFR4 respectively have the sequences shown in SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43 and SEQ ID NO:44, or sequences having more than 80%, more than 85%, more than 90% or more than 95%, or even 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or more identity with said sequences.

[0075] In another specific embodiment, the light chain variable region further comprises light chain framework regions LFR1, LFR2, LFR3 and LFR4, which are closely related to the V L CDR1, V L CDR2 and V L CDR3 is arranged from amino terminus to carboxyl terminus according to LFR1, V L CDR1, LFR2, V L CDR2, LFR3, V L Sequential arrangement of CDR3 and LFR4.

[0076] In a further specific embodiment, the light chain framework regions LFR1, LFR2, LFR3 and LFR4 respectively have the sequences shown in SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47 and SEQ ID NO:48, or sequences having more than 80%, more than 85%, more than 90% or more than 95%, or even 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or more identity with said sequences.

[0077] In a specific embodiment, the antibody further includes a constant region sequence, such as but not limited to a constant region sequence selected from any one of IgG, IgA, IgM, IgE and IgD. Those skilled in the art can make a selection based on needs, and this is not particularly limited herein.

[0078] In another specific embodiment, the species origin of the constant region sequence can be rat, mouse, rabbit, goat, sheep, horse, dog, cow, pig, chicken, duck, goose or human, but is not limited thereto.

[0079] In a specific embodiment, the constant region of the antibody of the present invention is derived from mouse.

[0080] In some embodiments, the species origin of the constant region sequence is mouse.

[0081] In a second aspect, the present invention provides a nucleic acid molecule encoding the anti-Taq DNA polymerase monoclonal antibody or antigen-binding fragment thereof of the first aspect.

[0082] For those skilled in the art, knowing the amino acid sequence of a protein, such as the anti-Taq DNA polymerase monoclonal antibody of the present invention, it is well within their capabilities to determine the nucleic acid encoding the sequence. Furthermore, to obtain a monoclonal antibody via recombinant means, the nucleic acid molecule can be cloned into a vector, and the vector can be further introduced into an expression cell, which can then express the antibody protein.

[0083] In a third aspect, the present invention provides a vector comprising the nucleic acid molecule of the second aspect of the present invention.

[0084] In a preferred embodiment, the vector may be a plasmid vector, such as pEE12, pCAGGS, pTOPO, pcDNA, pTT, pTT3, pEFBOS, pBV, pJV and pBJ.

[0085] In a specific embodiment, the vector is a pTOPO vector.

[0086] In another specific embodiment, the vector is a eukaryotic expression vector.

[0087] In a preferred embodiment, the pcDNA vector may be a pCDNA3.1 vector.

[0088] In a fourth aspect, the present invention provides an expression cell comprising the nucleic acid molecule of the second aspect or the vector of the third aspect.

[0089] The expression cell is prepared by introducing the above-mentioned nucleic acid molecule or the above-mentioned vector into a host cell by molecular biological methods well known to those skilled in the art.

[0090] As described above, the present inventors immunized mice with wild-type Taq DNA polymerase, fused the mouse spleen cells with myeloma cells, and screened for hybridoma cell lines that specifically bind to Taq DNA polymerase by ELISA. After screening for a monoclonal cell line that secretes the target antibody, heavy and light chain variable region cDNAs can be recovered from the cell line by reverse transcription-PCR, and a suitable immunoglobulin constant region (e.g., a human constant region) can be selected. The heavy and light chain variable region cDNAs and constant region cDNAs are then transferred into host cells such as COS or CHO cells, thereby obtaining cells expressing the target antibody of the present invention.

[0091] The above-mentioned monoclonal antibodies and recombinant DNA techniques can be used to produce other antibodies or chimeric molecules that retain the specificity of the original antibody. These techniques may include introducing DNA encoding the variable region or complementarity determining region (CDR) of the antibody immunoglobulin into a eukaryotic expression vector comprising the constant region or the constant region plus a framework region of a different immunoglobulin, or introducing both into a suitable eukaryotic expression vector, and then introducing the eukaryotic expression vector into an expression cell, such as a CHO host cell, to thereby obtain each recombinant anti-Taq DNA polymerase antibody.

[0092] In a specific embodiment, the expressing cells can be mammalian cells, such as Chinese hamster ovary cells, baby hamster kidney cells, monkey kidney cells, mouse thymoma cells, and human embryonic kidney cells. In a more specific embodiment, the expressing cells can be, for example, monkey kidney cells transformed with SV40 (COS-7, ATCC CRL1651), human embryonic kidney cells (HEK293 or HEK293 cells subcloned for growth in suspension culture, Graham et al., 1977, J. Gen Virol. 36:59), baby hamster kidney cells (BHK, ATCC CCL10), Chinese hamster ovary cells / -DHFR1 (CHO, Urlaub et al., 1980, Proc. Natl. Acad. Sci. USA 77:4216; e.g., DG44), mouse thymoma cells (NSO), mouse Sertoli cells (TM4, Mather, 1980, Biol. Reprod. 23:243-251), monkey kidney cells (CV-1, ATCC CCL70), African green monkey kidney cells (VERO-76, ATCC CRL-1587), human cervical cancer cells (HELA, ATCC CCL2), canine kidney cells (MDCK, ATCC CCL34), buffalo rat liver cells (BRL3A, ATCC CRL1442), human lung cells (W138, ATCC CCL75), human liver cells (HepG2, HB8065), mouse mammary tumor (MMT060562, ATCC CCL51), TR1 cells (Mather et al., 1982, Annals NY Acad. Sci. 383:44-68), MRC5 cells, FS4 cells, etc., but are not limited thereto.

[0093] In a fifth aspect, the present invention provides a hot-start Taq DNA polymerase, comprising the anti-Taq DNA polymerase monoclonal antibody or antigen-binding fragment thereof according to the first aspect and Taq DNA polymerase.

[0094] In a specific embodiment, the weight ratio of the anti-Taq DNA polymerase monoclonal antibody or antigen-binding fragment thereof to Taq DNA polymerase is 5:1 to 1:5.

[0095] In a preferred embodiment, the weight ratio of the anti-Taq DNA polymerase monoclonal antibody or antigen-binding fragment thereof to Taq DNA polymerase is 2:1 to 1:2.

[0096] In a more preferred embodiment, the weight ratio of the anti-Taq DNA polymerase monoclonal antibody or antigen-binding fragment thereof to Taq DNA polymerase is 1:1.

[0097] In another specific embodiment, the Taq DNA polymerase includes but is not limited to wild-type Taq DNA polymerase or a mutant thereof, such as KlenTaq10 DNA polymerase, Taq22 DNA polymerase, Klentaq1 DNA polymerase, Stoffel fragment, recombinant Taq-omni, KT-C3 DNA polymerase, S-Taq, S-Taq (Δ289). Wherein, "wild-type Taq DNA polymerase" (abbreviated as Taq WT) is a full-length Taq DNA polymerase, comprising 832 amino acids. "KlenTaq10 DNA polymerase" is a mutant lacking 287 amino acids at the N-terminus, and its inhibitor resistance is improved by 10-100 times compared to the full-length Taq DNA polymerase. "Taq22 enzyme" is obtained by mutating three sites (E626K, I170L and E708Q) of the wild-type Taq DNA polymerase, and has improved inhibition resistance.

[0098] As previously described, the anti-Taq DNA polymerase monoclonal antibody of the present invention can specifically bind to Taq DNA polymerase to form an antibody-enzyme complex, thereby obtaining a hot-start Taq DNA polymerase. At low temperatures, Taq DNA polymerase remains bound to the anti-Taq DNA polymerase antibody, blocking the activity of the Taq DNA polymerase. After a period of hot start at a higher temperature (e.g., a 2-minute heat shock treatment at 95°C), the anti-Taq DNA polymerase antibody denatures at high temperature and detaches from the active center of the Taq DNA polymerase, releasing the activity of the Taq DNA polymerase and allowing the amplification reaction to proceed. The hot-start Taq DNA polymerase of the present invention is inactive at low temperatures and does not cause mismatches or primer-dimer formation. Only at high temperatures does the Taq DNA polymerase regain activity, allowing specific amplification to proceed.

[0099] In a sixth aspect, the present invention provides a method for amplifying a target DNA sequence by polymerase chain reaction, the method comprising the step of amplifying the target DNA sequence using the hot-start Taq DNA polymerase of the fifth aspect.

[0100] In a specific embodiment, the polymerase chain reaction includes quantitative real-time PCR (qPCR), reverse transcription-PCR (RT-PCR), reverse transcription-quantitative PCR (RT-qPCR), digital PCR (dPCR), digital titration PCR (ddPCR), microfluidic PCR, etc., but is not limited thereto.

[0101] In another specific embodiment, the hot start Taq DNA polymerase is activated by hot start before being used in polymerase chain reaction. The purpose of hot start is to dissociate the antibody and enzyme in the hot start Taq DNA polymerase (i.e., antibody-enzyme complex) to release active single enzyme.

[0102] In a further specific embodiment, the hot start employs a temperature of at least 90°C, such as a temperature of 95°C.

[0103] In a seventh aspect, the present invention provides a kit for polymerase chain reaction, comprising: the hot-start Taq DNA polymerase of the fifth aspect; and instructions for use.

[0104] Example

[0105] The following examples illustrate the preparation methods and characterization of the relevant properties of the antibodies of the present invention. Unless otherwise specified, the experimental methods used are all conventional methods, and unless otherwise specified, the experimental materials used in the following examples are all purchased from conventional reagent stores. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0106] It should be noted that the terms used in the description of the present invention are intended only to describe specific embodiments and are not intended to limit the present invention. The above summary of the invention and the detailed description below are intended only to illustrate the present invention and are not intended to limit the present invention in any way. Without departing from the spirit and purpose of the present invention, the scope of the present invention is determined by the appended claims.

[0107] Example 1: Preparation of anti-Taq DNA polymerase monoclonal antibodies

[0108] Immunization: Highly purified Taq DNA polymerase (WT sequence, SEQ ID NO: 49) was used as an immunogen to immunize mice. Female BALB / c mice aged 6-8 weeks were selected. Mice were immunized four times, with a 2-week interval between immunizations, and the immunization dose was 100 μg / mouse. For the first immunization, Taq DNA polymerase was mixed with equal volumes of Freund's complete adjuvant (Sigma-Aldrich) and injected subcutaneously at multiple points on the back. For the next three immunizations, Taq DNA polymerase was mixed with equal volumes of Freund's incomplete adjuvant (Sigma-Aldrich) and injected intraperitoneally. Seven days after the fourth immunization, mice were bled by tail clipping, and serum was isolated. Antibody titers of antisera from immunized mice were measured using an indirect ELISA to assess the immune response. Mice with serum antibody titers exceeding 1:10,000 were selected for cell fusion experiments. Three days before the cell fusion experiments, a booster immunization (100 μg / mouse) of unadjuvanted Taq DNA polymerase was administered intraperitoneally.

[0109] The sequence of SEQ ID NO:49 is as follows: MRGMLPLFEPKGRVLLVDGHHLAYRTFHALKGLTTSRGEPVQAVYGFAKSLLKALKEDGDAVIVVFDAKAPSFRHEAYGGYKAGRAPTPEDFPRQLALIKELVDLLGLARLEVPGYEADDVLASLAKKAEKEGYEVRILTADKDLYQLLSDRIHVLHPEGYLITPAWLWEKYGLRPDQWADYRALTGDESDNLPGVKGIGEKTARKLLEEWGSLEALLKNLDRLKPAIREKILAHMDDLKLSWDLAKVRTDLPLEVDFAKRREPDRERLRAFLERLEFGSLLHEFGLLESPKALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALRDLKEARGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSERLFANLWGRLEGEERLLWLYREVERPLSAVLAHMEATGVRLDVAYLRALSLEVAEEIARLEAEVFRLAGHPFNLNSRDQLERVLFDELGLPAIGKTEKTGKRSTSAAVLEALREAHPIVEKILQYRELTKLKSTYIDPLPDLIHPRTGRLHTRFNQTATATGRLCCCDPNLQNIPVRTPLGQRIRRGFIAEEGWLLVALDYSQIELRVLAHLSGDENLIRVFQEGRDIHTETASWMFGVPREAVDPLMRRAAKTINFGVLYGMSAHRLSQELAIPYEEAQAFIERYFQSFPKVRAWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKSVREAAERMAFNMPVQGTAADLMKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGIGEDWLSAKE

[0110] Establishment of hybridoma cells: On the day of fusion, the spleen of the immunized mouse was removed under sterile conditions and the organ was made into a single cell suspension. Mouse myeloma cells (SP2 / 0) were fused with the above-mentioned immunized BALB / c mouse spleen cells at a ratio of 1:5, mixed thoroughly, and washed twice before fusion with PEG. Preheated PEG1500 was added, gently shaken, the cells were washed with preheated serum-free RPMI-1640 medium, and then resuspended with HAT selective medium. The cell suspension was plated into a 96-well culture plate at 200 μL / well and the cells were cultured at 37°C and 5% CO2. After culturing for 4 to 7 days, HT medium was used for culture. When the fused cells grew to 1 / 10-1 / 5 of the bottom area of ​​the wells of the 96-well plate, the supernatant was taken for antibody detection.

[0111] Screening of positive hybridoma cells: Taq DNA polymerase was diluted with coating buffer (0.05 mol / L, pH 9.6, PBS) to a final concentration of 1 μg / mL, and 100 μL / well was added to a 96-well plate and coated at 4°C overnight; the coating solution was discarded, and the plates were washed three times with phosphate buffered saline (PBST) and patted dry; the plates were blocked with PBST containing 2% BSA, 150 μL / well, incubated at 37°C for 2 h, washed three times with PBST, and patted dry; the supernatant of the fused cells, 1:1000 diluted positive serum of immune mice (as positive control) and 1:1000 diluted negative serum of mice (as negative control) were added to the plates. 100 μL / well was added to the corresponding wells, incubated at 37°C for 1 h, washed three times with PBST, and patted dry; 1:5000 diluted horseradish peroxidase (HRP)-labeled goat anti-mouse IgG (purchased from Sigma) was added, 100 μL / well, incubated at 37°C for 1 h, washed three times with PBST, and patted dry; tetramethylbenzidine (3,3',5,5'-Tetramethylbenzidine, TMB) substrate was added, 100 μL / well, and color was developed at room temperature in the dark for 10 min; 50 μL of 2 mol / L sulfuric acid was added to each well to terminate the reaction.

[0112] The OD of all wells was measured at 450 nm using a microplate reader. 450nm When the OD of negative serum 450nm ≤0.1, to measure the absorbance value OD of the well 450nm The value is the OD of the negative well 450nm The positive value was 2.1 times or more, and the positive hybridoma cells were selected for the next cloning step.

[0113] Cloning of positive cell lines: After sampling and counting the positive cell wells that secrete antibodies, dilute them to 100 cells / 10mL culture medium, plate the diluted cell suspension at 100μL / well to a 96-well cell culture plate, and culture in a 37°C, 5% CO2 cell culture incubator. After 6-7 days, the formation of cloned cells can be observed under a microscope. Mark the single clone growth well, remove the cell supernatant, perform ELISA test (the same as the above fusion test), and select positive monoclonal cells. Perform limiting dilution on the positive well cells, measure the ELISA value 5-6 days after each limiting dilution, and pick the OD value obtained by ELISA test. 450nm Limiting dilutions were performed on wells containing monoclonal clones with high positive values ​​until the entire 96-well plate was positive by ELISA. Stable monoclonal lines with high positive values ​​were selected. Finally, a series of cell lines that stably secreted anti-Taq DNA polymerase antibodies were obtained and named Taq1, Taq2, Taq3, Taq4, Taq5, Taq6, Taq7, and Taq8.

[0114] Example 2: Cloning and sequencing of antibody variable region sequences

[0115] Total RNA is isolated from the hybridoma cell line, and cDNA is prepared by reverse transcription to clone immunoglobulin sequences from the hybridoma cell line, and the variable region sequences of the antibodies of the hybridoma cell line are determined.

[0116] a. RNA extraction: Total RNA was extracted from the hybridoma cell lines according to the instructions of the M5 total RNA extraction kit (purchased from Beijing PolyBiotech Co., Ltd.).

[0117] b. RNA was reverse transcribed into cDNA: The total RNA extracted in the previous step was reverse transcribed using the M5 First Strand cDNA Synthesis Kit (purchased from Beijing Polymer Biotechnology Co., Ltd.) to produce cDNA, which was then frozen at -20°C for later use.

[0118] c. PCR amplification and recovery of variable region sequences: The cDNA obtained in the previous step was used as a template and the universal heavy chain primer Mu Ig V H 5′-A and Mu IgG V H 3′-2, immunoglobulin heavy chain (IgH) cDNA was amplified by PCR; similarly, light chain primers Mu Igκ V L 5′-A and Mu Igκ V L 3′-1, immunoglobulin light chain (IgK) cDNA was amplified by PCR, and the PCR product was recovered; the thermostable Pfu DNA polymerase was used throughout the PCR reaction.

[0119] d. Cloning and sequencing of variable region sequences: According to the instructions of the cloning vector pTOPO-Blunt Cloning kit (purchased from Beijing Polymer Biotechnology Co., Ltd.), the heavy and light chain variable region genes were ligated to the pTOPO vector, transformed into Escherichia coli DH5α, and positive clones were selected and sequenced by Beijing Ruibo Xingke Biotechnology Co., Ltd.

[0120] The antibody heavy chain variable region gene sequences and light chain variable region gene sequences of the hybridoma cell lines obtained by sequencing were analyzed. The heavy chain complementary determining region sequences and light chain complementary determining region sequences are shown in Table 1 below (based on the Chothia numbering system).

[0121] Table 1: Complementarity determining region sequences of heavy and light chains

[0122] Example 3: Preparation and purification of recombinant antibodies

[0123] Construct recombinant antibodies, prepare cell lines that stably express antibodies through eukaryotic expression, and culture and purify them on a large scale.

[0124] Specifically, for the V L and V H The sequenced CDRs were sequentially linked to the framework region (FR) sequences shown in Table 2 below to obtain the heavy and light chain variable regions. Subsequently, a recombinant antibody eukaryotic expression plasmid was constructed using molecular cloning methods. This eukaryotic expression plasmid was electroporated into CHO host cells and cultured in pressure selection medium (50 μM MSX) for 20 days. The supernatant was then assayed by ELISA (using HRP-labeled goat anti-mouse IgG as a secondary antibody, as described above) to identify cell lines stably expressing the recombinant antibody.

[0125] Table 2: Framework region sequences of recombinant antibodies

[0126] The selected stable cell lines were cultured on a large scale using roller bottle culture technology for the preparation of recombinant antibodies. The cells were cultured in Vega CHO medium at a rate of (0.2-0.3) × 10 6cells / mL are inoculated into roller bottles. A 1L roller bottle contains 300mL of culture medium (Vega CHO). The number of inoculation bottles is determined according to production needs. The roller bottles inoculated with cells are placed in a cell roller bottle machine and cultured in a cell culture incubator. The culture conditions are 900 rpm, 37°C, and 5% carbon dioxide. After 7-9 days of culture, samples are taken and observed under a microscope. When the cell viability is less than 50%, the samples are collected by centrifugation. The samples are affinity purified using a protein A affinity chromatography column to obtain antibodies, which are recombinant monoclonal antibodies T1-T8. Note: The heavy chain and light chain CDRs of the recombinant monoclonal antibodies T1-T8 correspond to the heavy chain and light chain CDRs of the monoclonal antibodies Taq1-8 produced by hybridomas in the previous article.

[0127] The concentration of each monoclonal antibody was measured using a microspectrophotometer, and the results showed that the concentrations of monoclonal antibodies T1-T8 were all greater than 4 mg / mL. Each monoclonal antibody was aliquoted and stored at 4°C-8°C.

[0128] The monoclonal antibody was identified by SDS-PAGE electrophoresis. The antibody had an antibody heavy chain band of about 51 KD and an antibody light chain band of about 26 KD.

[0129] Purity testing: Monoclonal antibodies were analyzed by size exclusion chromatography (SEC-HPLC). When all components in the sample were detected, the purity percentage of the main peak was calculated using the peak area normalization method. The purity was greater than 98%.

[0130] Example 4: Binding ability of antibodies to Taq DNA polymerase

[0131] Dilute Taq DNA polymerase to a concentration of 1 μg / mL in 0.05 M carbonate buffer, pH 9.6. Add 100 μL / well to a 96-well microtiter plate. Coat overnight at 4°C, wash three times with PBST on an automated plate washer, and pat dry. Block with PBST containing 2% BSA (150 μL / well), incubate at 37°C for 2 h, wash three times with PBST, and pat dry. Prepare a serial dilution series of monoclonal antibody samples against Taq DNA polymerase using 0.02 M PBS buffer, pH 7.4, starting at a concentration of 5 μg / mL. Repeat the three-fold serial dilution series to generate a series of monoclonal antibody samples at varying concentrations. Add 100 μL / well of the diluted monoclonal antibody sample to the microtiter plate, incubate at 37°C for 1 h, wash three times, and pat dry. Add 100 μL / well of horseradish peroxidase (HRP)-labeled goat anti-mouse IgG (Sigma) at a dilution of 1:15,000 and incubate at 37°C for 1 hour. Wash three times with PBST and pat dry. Add 100 μL / well of TMB substrate and develop for 10 minutes at room temperature in the dark. Terminate the reaction by adding 50 μL / well of 2 mol / L sulfuric acid. Measure the OD value using a microplate reader. 450nm The values ​​were plotted against the antibody concentrations, as shown in Figure 1. As shown in Figure 1, the recombinant monoclonal antibodies T1, T3, T5, and T8 were able to bind to the three Taq DNA polymerases, but not to the Pfu DNA polymerase (as a control), indicating that the antibodies of the present invention have excellent specificity. The EC values ​​of the recombinant monoclonal antibodies were obtained by analyzing the ELISA results using software. 50 The results are shown in Table 3 below.

[0132] Table 3: EC values ​​of each recombinant monoclonal antibody binding to each Taq DNA polymerase 50 value

[0133] In addition, the CDRs of the eight monoclonal antibodies described above were recombined using genetic recombination technology, and another series of recombinant antibodies were constructed using the framework region sequences given in Table 2. The heavy and light chain gene expression plasmids of the antibodies were similarly electroporated into CHO host cells, and the recombinant antibodies were screened. A series of recombinant antibodies T9-T20 that could bind to Taq DNA polymerase were screened. The sequences of the heavy chain CDRs and light chain CDRs of each recombinant antibody are shown in Table 4 below.

[0134] Table 4: Recombinant Antibody Sequence CDR Sequence

[0135] The binding ability of the recombinant antibodies to the three Taq DNA polymerases was tested according to the aforementioned method. The binding reaction results of some recombinant antibodies (T11, T12, T15, and T19) with the three Taq DNA polymerases are shown in Figure 2. As shown in Figure 2, the recombinant antibodies also have strong binding ability to the three Taq DNA polymerases and do not bind to the control (Pfu DNA polymerase).

[0136] OD was measured by microplate reader 450nm The above ELISA results were analyzed by software to obtain the EC value of each recombinant antibody. 50 The values ​​(unit: nM) are shown in Table 5 below.

[0137] Table 5: EC values ​​of various recombinant monoclonal antibodies and Taq DNA polymerase 50 value

[0138] Example 5: Detection of the blocking effectiveness of the antibody of the present invention on Taq DNA polymerase and the effect of restoring the enzyme activity of Taq DNA polymerase

[0139] The T1 antibody was combined with three Taq DNA polymerases in a 2:1 ratio to generate three antibody-enzyme complexes. Hot-start Taq DNA polymerase was then incubated at 37°C for 1 hour. The activity of these hot-start antibody-enzyme complexes and those without hot-start was tested. The group without enzyme served as a negative control, and the positive control was the enzyme alone without antibody.

[0140] Hot start of the antibody-enzyme complex: heat shock treatment at 95°C for 2 minutes. The purpose of the hot start is to dissociate the antibody and enzyme in the antibody-enzyme complex and release active single enzyme.

[0141] The reaction was carried out according to the following system:

[0142] After the reaction system was treated at 94°C for 20 seconds and 55°C for 30 seconds, 0.5 μL DNA polymerase was added to each experimental group and reacted at 37°C for 1 hour. The enzyme activity was detected at this temperature.

[0143] The experimental groups include S1: antibody-Taq WT complex without hot start; S2: antibody-enzyme Taq22 complex without hot start; S3: antibody-enzyme KlenTaq10 complex without hot start; S4: antibody-enzyme Taq WT complex with hot start; S5: antibody-enzyme Taq 22 complex with hot start; S6: antibody-enzyme KlenTaq10 complex with hot start; S7: single enzyme Taq WT without antibody; S8: single enzyme Taq 22 without antibody; S9: single enzyme KlenTaq10 without antibody; S10: negative control; S11: positive control.

[0144] After the reaction was completed, 5 μL of the reaction product was taken from each experimental group, added into 6× loading buffer and mixed, and then subjected to 1% agarose gel electrophoresis at 180 V for 30 min. The gel plate was removed and placed on an observer to observe the experimental results and take photos for preservation.

[0145] The experimental results are shown in Figure 3. As can be seen from Figure 3, the amplification results of the antibody-enzyme complexes without hot start (experimental groups S1-S3) are basically consistent with the negative control (experimental group S10), indicating that the three antibody-enzyme complexes of the present invention can almost completely inhibit the activity of Taq DNA polymerase at 37°C, that is, the antibodies of the present invention can successfully block these three Taq DNA polymerases. The amplification results using the antibody-enzyme complexes of the present invention with hot start (experimental groups S4-S6) are basically consistent with the positive control (experimental group S11) and the single enzyme (S7-S10), indicating that the three antibody-enzyme complexes of the present invention can almost completely restore enzyme activity after 95°C heat shock.

[0146] Example 6: Specificity test of hot-start Taq DNA polymerase

[0147] Hot-start Taq DNA polymerases were prepared by combining Taq DNA polymerase monoclonal antibodies T8 / T9 / T11 with three different Taq DNA polymerases (Taq WT, Taq 22, and KlenTaq10). Nine antibody-enzyme complexes were prepared by combining the T8, T9, and T11 antibodies with each of the three enzymes at a 1:1 mass ratio. The activity and specificity of these nine antibody-enzyme complexes were tested and compared with the three individual enzymes.

[0148] Prepare the reaction system in a PCR tube according to the following system:

[0149] Place the PCR tubes in a fluorescent PCR instrument and perform the following steps:

[0150] The results are shown in Figures 4-6. According to the results in Figures 4-6, the antibody-enzyme complexes prepared by combining three types of Taq DNA polymerase with T8, T9 and T11 antibodies, respectively, can significantly reduce the occurrence of primer dimers compared with the single enzyme that has not been modified with antibodies.

Claims

1. An anti-Taq DNA polymerase monoclonal antibody or an antigen-binding fragment thereof, the antibody or the antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising a heavy chain complementary determining region V H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementarity determining region V L CDR1, V L CDR2 and V L CDR3; of which: V H The amino acid sequence of CDR1 is shown by SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 22 or SEQ ID NO: 28; V H The amino acid sequence of CDR2 is shown by SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:23, SEQ ID NO:29, SEQ ID NO:32 or SEQ ID NO:37; V H The amino acid sequence of CDR3 is shown in SEQ ID NO:3, SEQ ID NO:9, SEQ ID NO:18, SEQ ID NO:24, SEQ ID NO:33 or SEQ ID NO:38; V L The amino acid sequence of CDR1 is shown by SEQ ID NO:4, SEQ ID NO:10, SEQ ID NO:15, SEQ ID NO:19, SEQ ID NO:25, SEQ ID NO:30, SEQ ID NO:34 or SEQ ID NO:39; V L The amino acid sequence of CDR2 is shown by SEQ ID NO:5, SEQ ID NO:11, SEQ ID NO:20, SEQ ID NO:26 or SEQ ID NO:35; and V L The amino acid sequence of CDR3 is shown in SEQ ID NO:6, SEQ ID NO:12, SEQ ID NO:21, SEQ ID NO:27, SEQ ID NO:31, SEQ ID NO:36 or SEQ ID NO:

40.

2. The anti-Taq DNA polymerase monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein 1) The heavy chain variable region includes the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 1-3, H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V shown in SEQ ID NO: 4-6 respectively L CDR1, V L CDR2 and V L CDR3; 2) The heavy chain variable region includes the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 7-9, H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V shown in SEQ ID NO: 10-12 respectively. L CDR1, V L CDR2 and V L CDR3; 3) The heavy chain variable region includes the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 3, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 15, SEQ ID NO: 5 and SEQ ID NO: 6, respectively. L CDR1, V L CDR2 and V L CDR3; 4) The heavy chain variable region includes the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 16-18, H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V shown in SEQ ID NO: 19-21 respectively. L CDR1, V L CDR2 and V L CDR3; 5) The heavy chain variable region includes the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 22-24, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V shown in SEQ ID NO: 25-27 respectively. L CDR1, V L CDR2 and V L CDR3; 6) The heavy chain variable region includes the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 3, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 30, SEQ ID NO: 5 and SEQ ID NO: 31, respectively. L CDR1, V L CDR2 and V L CDR3; 7) The heavy chain variable region includes the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 13, SEQ ID NO: 32 and SEQ ID NO: 33, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V shown in SEQ ID NO: 34-36 respectively. L CDR1, V L CDR2 and V L CDR3; 8) The heavy chain variable region includes the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 13, SEQ ID NO: 37 and SEQ ID NO: 38, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 39, SEQ ID NO: 5 and SEQ ID NO: 40, respectively. L CDR1, V L CDR2 and V L CDR3; 9) The heavy chain variable region includes the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 1-3, H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 15, SEQ ID NO: 5 and SEQ ID NO: 6, respectively. L CDR1, V L CDR2 and V L CDR3; 10) The heavy chain variable region includes the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 18, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 39, SEQ ID NO: 5 and SEQ ID NO: 21, respectively. L CDR1, V L CDR2 and V L CDR3; 11) The heavy chain variable region includes the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 22, SEQ ID NO: 32 and SEQ ID NO: 38, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 39, SEQ ID NO: 26 and SEQ ID NO: 31, respectively. L CDR1, V L CDR2 and V L CDR3; 12) The heavy chain variable region comprises a heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 13, SEQ ID NO: 37 and SEQ ID NO: 3, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 25, SEQ ID NO: 5 and SEQ ID NO: 40, respectively. L CDR1, V L CDR2 and V L CDR3; 13) The heavy chain variable region includes the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 28, SEQ ID NO: 32 and SEQ ID NO: 33, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 12, respectively. L CDR1, V L CDR2 and V L CDR3; 14) The heavy chain variable region includes the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 33, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 30, SEQ ID NO: 5 and SEQ ID NO: 6, respectively. L CDR1, V L CDR2 and V L CDR3; 15) The heavy chain variable region includes the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 22, SEQ ID NO: 14 and SEQ ID NO: 9, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 15, SEQ ID NO: 5 and SEQ ID NO: 6, respectively. L CDR1, V L CDR2 and V L CDR3; 16) The heavy chain variable region includes the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 16, SEQ ID NO: 32 and SEQ ID NO: 38, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 27, respectively. L CDR1, V L CDR2 and V L CDR3 17) The heavy chain variable region includes the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 33, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 35 and SEQ ID NO: 12, respectively. L CDR1, V L CDR2 and V L CDR3; 18) The heavy chain variable region includes the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 37 and SEQ ID NO: 38, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 19, SEQ ID NO: 5 and SEQ ID NO: 6, respectively. L CDR1, V L CDR2 and V L CDR3; 19) The heavy chain variable region comprises a heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 13, SEQ ID NO: 32 and SEQ ID NO: 38, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 40, respectively. L CDR1, V L CDR2 and V L CDR3; or 20) The heavy chain variable region includes the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 22, SEQ ID NO: 37 and SEQ ID NO: 24, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 15, SEQ ID NO: 5 and SEQ ID NO: 36, respectively. L CDR1, V L CDR2 and V L CDR3; Preferably, the heavy chain variable region further comprises framework regions HFR1, HFR2, HFR3 and HFR4, which are closely related to V H CDR1, V H CDR2 and V H CDR3 is arranged from the amino terminus to the carboxyl terminus according to HFR1, V H CDR1、HFR2、V H CDR2、HFR3、V H The light chain variable region further comprises framework regions LFR1, LFR2, LFR3 and LFR4, wherein the HFR1, HFR2, HFR3 and HFR4 have the sequences shown in SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43 and SEQ ID NO:44, respectively, or sequences having 80% or more, 85% or more, 90% or more identity with the sequences, and the light chain variable region further comprises framework regions LFR1, LFR2, LFR3 and LFR4, which are arranged in the order of CDR3 and HFR4, wherein the HFR1, HFR2, HFR3 and HFR4 have the sequences shown in SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43 and SEQ ID NO:44, respectively, or sequences having 80% or more, 85% or more, 90% or more identity with the sequences, L CDR1, V L CDR2 and V L CDR3 is arranged from amino terminus to carboxyl terminus according to LFR1, V L CDR1, LFR2, V L CDR2, LFR3, V L The invention relates to a method for treating a pluripotent stem cell comprising: a first peptide bonded to ...

3. A nucleic acid molecule encoding the anti-Taq DNA polymerase monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2.

4. A vector comprising the nucleic acid molecule according to claim 3; preferably, the vector is a plasmid vector, such as any one of pEE12, pCAGGS, pTOPO, pcDNA such as pCDNA3.1, pTT, pTT3, pEFBOS, pBV, pJV and pBJ.

5. An expression cell comprising the nucleic acid molecule according to claim 3 or the vector according to claim 4, preferably, the expression cell is a mammalian cell, for example, selected from Chinese hamster ovary cells, mini-hamster kidney cells, monkey kidney cells, mouse thymoma cells, and human embryonic kidney cells. 6 . A hot-start Taq DNA polymerase, comprising the anti-Taq DNA polymerase monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2 and Taq DNA polymerase.

7. The hot-start Taq DNA polymerase according to claim 6, wherein the weight ratio of the anti-Taq DNA polymerase monoclonal antibody or antigen-binding fragment thereof to the Taq DNA polymerase is 5:1 to 1:5, preferably 2:1 to 1:2, preferably 1:

1.

8. The hot-start Taq DNA polymerase according to claim 6 or 7, wherein The Taq DNA polymerase includes wild-type Taq DNA polymerase or mutants thereof, such as KlenTaq10 DNA polymerase, Taq22 DNA polymerase, Klentaq1 DNA polymerase, Stoffel fragment, recombinant Taq-omni, KT-C3 DNA polymerase, S-Taq, S-Taq (Δ289).

9. A method for amplifying a target DNA sequence by polymerase chain reaction, the method comprising the step of amplifying the target DNA sequence using the hot-start Taq DNA polymerase according to any one of claims 6 to 8.

10. A kit for polymerase chain reaction, comprising: The hot-start Taq DNA polymerase according to any one of claims 6 to 8; And instructions for use.

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