Anti-Taq DNA polymerase antibody and application thereof

Abstract

The invention discloses an antibody for resisting Taq DNA polymerase and application thereof, researches on preparation, application and the like of the antibody, and relates to the field of antibodies. The anti-Taq DNA polymerase antibody disclosed by the invention comprises a heavy chain complementarity determining region and a light chain complementarity determining region, has excellent affinity or activity, and can be widely applied to the field of PCR (Polymerase Chain Reaction) detection.

Description

[0001] Cross - reference to related applications

[0002] This disclosure claims the priority of the Chinese patent application with the application number 202311810686.6 and the title "Anti - Taq DNA Polymerase Antibody and Its Use" filed on December 26, 2023, the entire content of which is incorporated herein by reference. Technical field

[0003] The present invention belongs to the technical field of antibodies. Specifically, the present invention relates to a Taq DNA polymerase antibody and its use. More specifically, the present invention relates to an antibody against Taq DNA polymerase, a nucleic acid molecule, a vector, a cell or a host, an antibody - modified Taq DNA polymerase and a composition, a method for preparing an antibody, a reagent or a kit and its use, a method for screening a Taq DNA polymerase antibody, and a mutant library. Background art

[0004] In 1988, Randall K. Saiki et al. [1, 2] isolated and purified Taq DNA polymerase from Thermus aquatics. Taq DNA polymerase (also known as Taq enzyme) can tolerate high temperatures above 90°C without inactivation, which is of great significance in PCR reactions that require a high - temperature environment. Therefore, Taq DNA polymerase replaced the DNA polymerase in Escherichia coli that was previously commonly used in PCR reactions. When Taq DNA polymerase is used in PCR reactions, there is no need to add enzyme in each cycle, making the PCR technology very simple and greatly reducing the cost. The PCR technology has been widely applied and gradually applied to the clinic. However, Taq DNA polymerase also has certain defects during use. That is, at room temperature, it also has certain enzymatic properties, which leads to non - specific and primer - dimer amplification during PCR amplification, and there are problems with long - term stability.

[0005] Therefore, with the popularization of the application of PCR technology and the improvement of the requirements for the quality of PCR amplification, new methods and technologies have emerged continuously. Among them, the emergence of hot - start enzyme technology has qualitatively improved the enzymatic properties of ordinary Taq DNA polymerase. Currently, the commonly used methods include antibody - modified hot - start enzymes and chemically modified hot - start enzymes. Among them, antibody - modified hot - start enzymes are more commonly used. Antibody - modified hot - start enzymes require antibodies against Taq DNA polymerase, and currently, the antibody raw materials for Taq DNA polymerase on the market still have performance defects.

[0006] Therefore, there is a strong demand in the art for anti - Taq DNA polymerase antibodies with good performance. Summary of the invention

[0007] The present invention aims to provide an antibody against Taq DNA polymerase and conduct research on the preparation, application, etc. of this binding protein.

[0008] In one aspect of the present invention, an antibody against Taq DNA polymerase is proposed, which includes HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, and the amino acid sequences of HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3 are as follows:

[0009] HCDR 1: TYYIY;

[0010] HCDR 2: GINPTX1GGPVFNEKFKS, where X1 is S or G;

[0011] HCDR 3: SLLRRGYYFDY;

[0012] LCDR 1: RASQX2INNYLN, where X2 is D or Y;

[0013] LCDR 2: YTX3RLHX4, X3 is S, F, I or L, X4 is S, F or Y;

[0014] LCDR 3: QQDDTLPLT.

[0015] In the second aspect of the present invention, an antibody against Taq DNA polymerase is proposed, which includes a heavy chain variable region and / or a light chain variable region; the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 17 or its variant, and compared with the amino acid sequence shown in SEQ ID NO: 17, the variant of SEQ ID NO: 17 includes mutations at the following sites: S55G; the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 18 or its variant, and compared with the amino acid sequence shown in SEQ ID NO: 18, the variant of SEQ ID NO: 18 includes mutations at least one of the following sites: D28Y, S52I / F / L, S56F / Y.

[0016] In the third aspect of the present invention, an antibody against Taq DNA polymerase is proposed, which includes HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, and HCDR1, HCDR2, HCDR3 are HCDR1, HCDR2, HCDR3 of the heavy chain variable region defined by the antibody in the second aspect; LCDR1, LCDR2, LCDR3 are LCDR1, LCDR2, LCDR3 of the light chain variable region defined by the antibody in the second aspect.

[0017] In a fourth aspect of the present invention, the present invention provides a nucleic acid molecule encoding the antibody described in the first, second, or third aspect.

[0018] In a fifth aspect of the present invention, the present invention provides a vector comprising the nucleic acid molecule described in the fourth aspect.

[0019] In a sixth aspect of the present invention, the present invention provides a cell or host comprising: the nucleic acid molecule described in the fourth aspect or the vector described in the fifth aspect; or expressing the antibody described in the first, second, or third aspect.

[0020] In a seventh aspect of the present invention, the present invention provides a method for preparing the antibody described in the first, second, or third aspect, the method comprising culturing the cell or host described in the sixth aspect.

[0021] In an eighth aspect of the present invention, the present invention provides an application of the antibody described in the first, second, or third aspect in PCR.

[0022] In a ninth aspect of the present invention, the present invention provides an antibody-modified Taq DNA polymerase, wherein the Taq DNA polymerase is modified by the antibody described in the first, second, or third aspect.

[0023] In a tenth aspect of the present invention, the present invention provides a composition for PCR reaction, which comprises the antibody described in the first, second, or third aspect and Taq DNA polymerase.

[0024] In an eleventh aspect of the present invention, the present invention provides a reagent or kit, which comprises: the antibody described in the first, second, or third aspect, or the Taq DNA polymerase described in the ninth aspect, or the composition described in the tenth aspect.

[0025] In a twelfth aspect of the present invention, the present invention provides a method for screening Taq DNA polymerase antibodies, the method comprising: a) designing primers for amino acid substitution at the sites of X1, X2, X3, and X4 defined in the antibody described in the first aspect, or at the mutation sites defined in the antibody described in the second aspect; b) using the nucleic acid molecule described in the fourth aspect, the vector described in the fifth aspect, or the cell described in the sixth aspect as a template, and constructing a mutant library with the primers described in a); c) screening Taq DNA polymerase antibodies from the mutant library.

[0026] In a thirteenth aspect of the present invention, the present invention provides a mutant library, which comprises the antibody described in the first, second, or third aspect.

[0027] Additional aspects and advantages of the present invention will be given in part in the following description, become apparent in part from the following description, or be learned through the practice of the present invention. Detailed Description of the Invention

[0028] Embodiments of the present invention will be described in detail below. The embodiments described below are exemplary and are only used to explain the present invention and should not be construed as a limitation of the present invention.

[0029] It should be noted that the terms "first" and "second" are only used for descriptive purposes and should not be construed as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, features defined with "first" and "second" may explicitly or implicitly include one or more of such features. Further, in the description of the present invention, unless otherwise stated, the meaning of "a plurality" is two or more.

[0030] In the ranges disclosed herein, the endpoints and any values are not limited to the exact ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, between the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values, they can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0031] To make the present invention easier to understand, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention pertains. The abbreviations of amino acid residues are the standard three-letter and / or one-letter codes used in the art to refer to one of the 20 common L-amino acids.

[0032] In this document, the term "comprising" or "including" is an open-ended expression, that is, it includes the content specified by the present invention, but does not exclude other aspects.

[0033] In this document, the terms "optionally", "optional", "optionally", "optional" or "option" generally mean that the subsequent events or conditions may or may not occur, and this description includes the cases where the events or conditions occur and the cases where the events or conditions do not occur.

[0034] In this document, the term "antibody" is used in the broadest sense and may include full-length monoclonal antibodies, multispecific antibodies, chimeric antibodies or antigen-binding fragments, and the specific structure is not limited as long as they exhibit the required antigen-binding ability.

[0035] As used herein, the terms "full-length antibody", "full-length monoclonal antibody", or "full-length monoclonal antibody" are each composed of at least two identical light chains and at least two identical heavy chains linked by interchain disulfide bonds, such as immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin D (IgD), or immunoglobulin E (IgE).

[0036] As used herein, the terms "polyclonal antibody" and "multispecific antibody" are synonymous and both refer to antibodies that can recognize multiple antigenic epitopes, such as antibodies that can recognize two antigenic epitopes (bispecific antibodies, abbreviated as bispecific antibodies), antibodies that can recognize three antigenic epitopes, or antibodies that can recognize four antigenic epitopes. This is a broad understanding, and the specific structure is not limited as long as it can recognize multiple antigenic epitopes. In the present invention, at least one of the multiple antigenic epitopes is derived from Taq DNA polymerase.

[0037] As used herein, the term "antigen-binding fragment" is a fragment that contains part or all of an antibody, lacks at least some of the amino acids present in the full-length chain, but still has the functional activity of specifically binding to an antigen. For example, the fragment may contain part or all of the antibody CDR. Such fragments bind to antigens and can compete with other antigen-binding molecules (including intact antibodies) for binding to a given epitope. Such fragments include, but are not limited to, Fab, Fab', F(ab)2, F(ab')2, Fv, scFv, scFv-Fc fusion proteins, scFv-Fv fusion proteins, single-domain antibodies, or minimal recognition units. Such fragments can be produced by recombinant nucleic acid techniques or by enzymatic cleavage or chemical cleavage of antigen-binding molecules (including intact antibodies).

[0038] As used herein, the term "Fab antibody" or "Fab fragment" generally refers to an antibody or fragment that contains only the Fab molecule, which is composed of VH and CH1 of the heavy chain and the intact light chain, and the light chain and the heavy chain are connected by a disulfide bond.

[0039] As used herein, the term "F(ab’)2 antibody" or "F(ab’)2 fragment" has two antigen-binding F(ab’) parts linked together by disulfide bonds.

[0040] As used herein, the term "Fv antibody" or "Fv fragment" generally refers to an antibody or fragment composed only of the variable region of the light chain (VL) and the variable region of the heavy chain (VH) connected by non-covalent bonds, which is the smallest functional fragment of the antibody molecule that retains the complete antigen-binding site.

[0041] As used herein, the terms "single-chain antibody" and "scFv fragment" are antibodies or fragments formed by connecting the variable region of the heavy chain and the variable region of the light chain of an antibody with a short peptide.

[0042] As used herein, the terms "minimal recognition unit" and "MRU" both refer to an antibody or fragment consisting of only one CDR, which has a very small molecular weight, accounting for only about 1% of the complete antibody.

[0043] As used herein, the terms "complementary determining region", "CDR" or "CDRs" refer to the hypervariable regions of the heavy and light chains of an immunoglobulin, and refer to the regions containing one or more or even all of the main amino acid residues that contribute to the binding affinity of the antibody for the antigen or epitope it recognizes.

[0044] As used herein, the heavy chain complementary determining regions (CDRs of the heavy chain variable region) are denoted by "HCDRs" or "HCDR", which include HCDR1 (also known as CDR-H1), HCDR2 (also known as CDR-H2) and HCDR3 (also known as CDR-H3); the light chain complementary determining regions (CDRs of the light chain variable region) are denoted by "LCDRs" or "LCDR", which include LCDR1 (also known as CDR-L1), LCDR2 (also known as CDR-L2) and LCDR3 (also known as CDR-L3). Commonly used CDR definition systems in the art include: Kabat definition, Chothia definition, IMGT definition, Contact definition and AbM definition. As described herein, the "Kabat definition" refers to the definition system described by Kabat et al., U.S. Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). The "Chothia definition" can be found in Chothia et al., J Mol Biol 196: 901-917 (1987). Exemplary defined CDRs are listed in Table 1 below. The definitions in different literatures are slightly different. Given the amino acid sequence of the variable region of an antibody, those skilled in the art can routinely determine which residues contain a specific CDR. It should be noted that the CDRs defined in the present invention include, but are not limited to, the CDRs defined by the methods in Table 1. CDRs defined by other methods disclosed in the art based on the heavy chain variable region and light chain variable region disclosed in this application also fall within the scope of protection of this disclosure.

[0045] Table 1: CDR Definitions 1

[0046] CDR Kabat <![CDATA[AbM 2 > IMGT Chothia HCDR1 <![CDATA[H31~H35 3 > <![CDATA[H26~H35 3 > <![CDATA[H26~H33..35 5 > <![CDATA[H26~H32..34 4 > HCDR2 H50 - H65 H50 - H58 H51 - H57 H52 - H56 HCDR3 H95 - H102 H95 - H102 H93 - H102 H95 - H102 LCDR1 L24 - L34 L24 - L34 L27 - L32 L24 - L34 LCDR2 L50 - L56 L50 - L56 L50 - L51 L50 - L56 LCDR3 L89 - L97 L89 - L97 L89 - L97 L89 - L97

[0047] 1 The numbers of all CDR definitions in Table 1 are based on the Kabat numbering system (see below). The amino acid numbers on the heavy chain are represented by "H + number", and the amino acid numbers on the light chain are represented by "L + number".

[0048] 2 "AbM" as used in Table 1 with a lower case "b" refers to the CDR defined by the "AbM" antibody modeling software of Oxford Molecular.

[0049] 3 When neither H35A nor H35B is present, CDR-H1 ends at position 35; when only H35A is present, CDR-H1 ends at position 35A; when both H35A and H35B are present, CDR-H1 ends at position 35B.

[0050] 4 When neither H35A nor H35B is present, CDR-H1 ends at position 32; when only H35A is present, CDR-H1 ends at position 33; when both H35A and H35B are present, CDR-H1 ends at position 34.

[0051] 5 When neither H35A nor H35B is present, CDR-H1 ends at position 33; when only H35A is present, CDR-H1 ends at position 34; when both H35A and H35B are present, CDR-H1 ends at position 35.

[0052] Kabat et al. also defined a numbering system applicable to the variable region sequences of any antibody. A person of ordinary skill in the art can clearly map the Kabat numbering system to any variable region sequence without relying on any experimental data outside the sequence itself. As described herein, "Kabat numbering" refers to numbering using the numbering system described in "Kabat et al., U.S. Dept. of Health and Human Services, 'Sequence of Proteins of Immunological Interest' (1983)". The HCDRs and LCDRs of the antibodies in this application are numbered using the above numbering system, and the specific numbering results are shown in Table 1. It should be noted that the polypeptide sequences of the present invention are not numbered according to the Kabat numbering system. However, a person of ordinary skill in the art can fully convert the sequence numbers in the sequence listing into Kabat numbers.

[0053] As used herein, the term "framework region" or "FR" region includes the heavy chain framework region and the light chain framework region, and refers to the regions in the heavy chain variable region (which may be designated as VH) and the light chain variable region (which may be designated as VL) of an antibody other than the CDRs; wherein, the heavy chain framework region is designated as "HFR" and may be further subdivided into contiguous regions separated by CDRs, including the HFR1, HFR2, HFR3, and HFR4 framework regions; the light chain framework region is designated as "LFR" and may be further subdivided into contiguous regions separated by CDRs, including the LFR1, LFR2, LFR3, and LFR4 framework regions.

[0054] As used herein, the heavy chain variable region is obtained by connecting the following numbered CDRs and FRs in the following combined arrangement: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is obtained by connecting the following numbered CDRs and FRs in the following combined arrangement: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0055] In this text, when the terms "identity", "homology", or "similarity" are used to describe an amino acid sequence or a nucleic acid sequence relative to a reference sequence, the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences is determined by conventional methods. For example, see Ausubel et al., eds. (1995), Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN program (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research Foundation, Washington, D.C.)). There are many algorithms for aligning sequences and determining sequence identity, including the homology alignment algorithm of Needleman et al. (1970) J. Mol. Biol. 48: 443; the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2: 482; the similarity search method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 85: 2444; the Smith-Waterman algorithm (Meth. Mol. Biol. 70: 173-187 (1997)); and the BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J. Mol. Biol. 215: 403-410). Computer programs utilizing these algorithms are also available and include, but are not limited to: ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul et al., Meth. Enzym., 266: 460-480 (1996)); or GAP, BESTFIT, BLAST Altschul et al., supra, FASTA, and TFASTA, available in the Genetics Computing Group (GCG) package, 8th edition, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0056] Without substantially affecting the antibody activity (retaining at least 90% of the activity), those skilled in the art can substitute, add, and / or delete one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) amino acids in the sequences of the present invention to obtain variants of the antibody sequences. They are all considered to be included within the scope of protection of the present invention. For example, amino acids with similar properties are substituted in the variable region. The variant sequences of the present invention can have at least 90%, 95%, 96%, 97%, 98%, or 99% identity (or homology) with the reference sequences. The sequence identity described in the present invention can be measured using sequence analysis software. For example, using the computer program BLAST with default parameters, especially BLASTP or TBLASTN. The amino acid sequences described in the present invention are all shown in the N-terminal to C-terminal manner.

[0057] It should be noted that the positions or numbers of the mutation sites defined in the description and claims of the present invention also need to be adjusted according to the number and positions of the added and / or deleted amino acids.

[0058] In this article, the term "at least 80% homology" means at least 80% homology with each reference sequence, which can be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% homology. The term "at least 90% homology" means at least 90% homology with each reference sequence, which can be 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% homology.

[0059] In this article, the term "variant" or "mutant" can refer to any naturally occurring or engineered molecule containing one or more nucleotide or amino acid mutations.

[0060] In this article, the term "vector" generally refers to a nucleic acid molecule capable of self-replicating after being inserted into a suitable host, which transfers the inserted nucleic acid molecule into cells or hosts and / or between cells or hosts. The vector can include vectors mainly used for inserting DNA or RNA into cells, vectors mainly used for replicating DNA or RNA, and expression vectors mainly used for transcription and / or translation of DNA or RNA. The vector also includes vectors having multiple above-mentioned functions. The vector can be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable cell or host. Generally, by culturing a suitable cell or host containing the vector, the vector can produce the desired expression product.

[0061] In this text, the term "cell" generally refers to a cell obtained by modifying or recombining the genetic material of a host cell using genetic engineering techniques or cell fusion techniques, and having unique traits with stable inheritance. Among them, the term "host cell" refers to a prokaryotic cell or a eukaryotic cell that can introduce a recombinant vector. The term "transformed" or "transfected" as used herein refers to the introduction of nucleic acid (such as a vector) into a cell by various techniques known in the art. Suitable host cells can be transformed or transfected with the DNA sequence of the present invention and can be used for the expression and / or secretion of a target protein. Examples of suitable host cells that can be used in the present invention include immortalized hybridoma cells, NS / 0 myeloma cells, 293 cells, Chinese hamster ovary (CHO) cells, HeLa cells, Cap cells (cells derived from human amniotic fluid), and CoS cells.

[0062] The present invention provides an anti-Taq DNA polymerase antibody, a nucleic acid molecule, a vector, a cell or a host, a method for preparing the antibody, a conjugate, a reagent or a kit and their uses, a method for detecting Taq DNA polymerase, a method for screening an anti-Taq DNA polymerase antibody, and a mutant library, which will be described in detail below.

[0063] Antibody

[0064] In the first aspect of the present invention, the present invention provides an anti-Taq DNA polymerase antibody. According to an embodiment of the present invention, the antibody includes HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, and the amino acid sequences of HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3 are as follows:

[0065] HCDR 1: TYYIY;

[0066] HCDR 2: GINPTX1GGPVFNEKFKS, where X1 is S or G;

[0067] HCDR 3: SLLRRGYYFDY;

[0068] LCDR 1: RASQX2INNYLN, where X2 is D or Y;

[0069] LCDR 2: YTX3RLHX4, X3 is S, F, I or L, X4 is S, F or Y;

[0070] LCDR 3: QQDDTLPLT.

[0071] According to an embodiment of the present invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Kabat system.

[0072] As can be seen from the aforementioned CDR definition principles, when different systems are used for definition, even for the same heavy chain variable region or the same light chain variable region, the obtained CDRs are different. "HCDR 1: TYYIY" in the present application means that the amino acid sequence of HCDR 1 includes or is TYYIY, and it can also be adjusted according to different systems to obtain an HCDR 1 with a sequence shorter or longer than TYYIY.

[0073] In some alternative embodiments of the present invention, X1 is S;

[0074] In some alternative embodiments of the present invention, X1 is G;

[0075] In some alternative embodiments of the present invention, X2 is D;

[0076] In some alternative embodiments of the present invention, X2 is Y;

[0077] In some alternative embodiments of the present invention, X3 is S;

[0078] In some alternative embodiments of the present invention, X3 is F;

[0079] In some alternative embodiments of the present invention, X3 is I;

[0080] In some alternative embodiments of the present invention, X3 is L;

[0081] In some alternative embodiments of the present invention, X4 is S;

[0082] In some alternative embodiments of the present invention, X4 is F;

[0083] In some alternative embodiments of the present invention, X4 is Y;

[0084] In some alternative embodiments of the present invention, X1, X2, X3, and X4 are selected from any one of the following combinations:

[0085]

[0086] The antibody described in the first aspect of the present invention has excellent activity, affinity, stability, or specificity.

[0087] The antibody described in the first aspect of the present invention has improved activity, affinity, stability, or specificity.

[0088] In a second aspect of the present invention, the present invention provides an anti-Taq DNA polymerase antibody. According to an embodiment of the present invention, the antibody or functional fragment includes: a heavy chain variable region and / or a light chain variable region; the heavy chain variable region includes an amino acid sequence as shown in SEQ ID NO: 17 or a variant thereof, and compared with the amino acid sequence shown in SEQ ID NO: 17, the variant of SEQ ID NO: 17 includes a mutation at the following site: S55G; the light chain variable region includes an amino acid sequence as shown in SEQ ID NO: 18 or a variant thereof, and compared with the amino acid sequence shown in SEQ ID NO: 18, the variant of SEQ ID NO: 18 includes a mutation at at least one of the following sites: D28Y, S52I / F / L, S56F / Y.

[0089] It should be noted that the numbering of the above heavy chain variable region sites is obtained by sequentially numbering the amino acid sequence shown in SEQ ID NO: 17 from the N-terminus to the C-terminus. For example, the 55th position refers to the 55th position of the amino acid sequence shown in SEQ ID NO: 17 starting from the N-terminus; the "S55G" means that the serine at the 55th position of the amino acid sequence shown in SEQ ID NO: 17 is replaced by glycine.

[0090] The numbering of the above light chain variable region sites is obtained by sequentially numbering the amino acid sequence shown in SEQ ID NO: 18 from the N-terminus to the C-terminus. For example, the 28th position refers to the 28th position of the amino acid sequence shown in SEQ ID NO: 18 starting from the N-terminus; the "D28Y" means that the aspartic acid at the 28th position of the amino acid sequence shown in SEQ ID NO: 18 is replaced by tyrosine; the "S52I / F / L" means that the serine at the 52nd position of the amino acid sequence shown in SEQ ID NO: 18 can be replaced by isoleucine or phenylalanine or leucine.

[0091] According to an embodiment of the present invention, the above antibody may further include at least one of the following additional technical features:

[0092] In some alternative embodiments of the present invention, the mutation at the 55th position is S55G;

[0093] In some alternative embodiments of the present invention, the mutation at the 28th position is D28Y;

[0094] In some alternative embodiments of the present invention, the mutation at the 52nd position is S52I;

[0095] In some alternative embodiments of the present invention, the mutation at the 52nd position is S52F;

[0096] In some alternative embodiments of the present invention, the mutation at position 52 is S52L;

[0097] In some alternative embodiments of the present invention, the mutation at position 56 is S56F;

[0098] In some alternative embodiments of the present invention, the mutation at position 56 is S56Y.

[0099] In some alternative embodiments of the present invention, the heavy chain variable region and the light chain variable region are selected from any one of the following combinations:

[0100]

[0101]

[0102] It should be noted that the "," in the above table represents "and", that is, combined mutations. For example, "D28Y, S52F, S56F" represents a combined mutation of three mutations: D28Y, S52F, and S56F.

[0103] In the third aspect of the present invention, the present invention provides an anti-Taq DNA polymerase antibody. According to the embodiments of the present invention, the antibody includes HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3. The HCDR1, HCDR2, and HCDR3 are the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region defined by the antibody in the second aspect; the LCDR1, LCDR2, and LCDR3 are the LCDR1, LCDR2, and LCDR3 of the light chain variable region defined by the antibody in the second aspect.

[0104] It should be noted that the HCDR1, HCDR2, and HCDR3 in the antibody described in the third aspect are the HCDR1, HCDR2, and HCDR3 of the same heavy chain variable region defined by the antibody in the second aspect, and the LCDR1, LCDR2, and LCDR3 are the LCDR1, LCDR2, and LCDR3 of the same light chain variable region defined by the antibody in the second aspect.

[0105] For example, when the heavy chain variable region defined in the antibody described in the second aspect has no mutation compared to SEQ ID NO: 17, the HCDR1, HCDR2, and HCDR3 of this heavy chain variable region are respectively HCDR 1: TYYIY; HCDR 2: GINPTSGGPVFNEKFKS; HCDR 3: SLLRRGYYFDY; then the HCDR1, HCDR2, and HCDR3 contained in the antibody described in the third aspect are also respectively HCDR 1: TYYIY; HCDR 2: GINPTSGGPVFNEKFKS; HCDR 3: VWYSGNGFDFGYFDYSLLRRGYYFDY.

[0106] For example, when the light chain variable region defined in the antibody described in the second aspect has only a mutation at the D28Y site compared to SEQ ID NO: 18, the LCDR1, LCDR2, and LCDR3 of this light chain variable region are respectively LCDR 1: RASQYINNYLN; LCDR2: YTSRLHS; LCDR 3: QQDDTLPLT; then the LCDR1, LCDR2, and LCDR3 contained in the antibody described in the third aspect are also respectively LCDR 1: RASQYINNYLN; LCDR 2: YTSRLHS; LCDR 3: QQDDTLPLT.

[0107] According to an embodiment of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the Kabat system.

[0108] In some alternative embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, or LCDR3 are defined by any one system or a combination of multiple systems among Kabat, Chothia, IMGT, AbM, or Contact.

[0109] According to an embodiment of the present invention, the antibody described in the first aspect or the third aspect further includes at least one of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4.

[0110] In an alternative embodiment of the present invention, at least a part of at least one of the HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4 is derived from at least one of a murine antibody, a human antibody, a primate antibody, a bovine antibody, a horse antibody, a dairy cow antibody, a pig antibody, a sheep antibody, a goat antibody, a dog antibody, a cat antibody, a rabbit antibody, a camel antibody, a donkey antibody, a deer antibody, a mink antibody, a chicken antibody, a duck antibody, a goose antibody, a turkey antibody, a fighting chicken antibody, or a mutant thereof.

[0111] According to an embodiment of the present invention, the HFR1 includes the amino acid sequence shown in SEQ ID NO:7 or an amino acid sequence having at least 80% homology thereto; the HFR2 includes the amino acid sequence shown in SEQ ID NO:8 or an amino acid sequence having at least 80% homology thereto; the HFR3 includes the amino acid sequence shown in SEQ ID NO:9 or an amino acid sequence having at least 80% homology thereto; the HFR4 includes the amino acid sequence shown in SEQ ID NO:10 or an amino acid sequence having at least 80% homology thereto; the LFR1 includes the amino acid sequence shown in SEQ ID NO:11 or an amino acid sequence having at least 80% homology thereto; the LFR2 includes the amino acid sequence shown in SEQ ID NO:12 or an amino acid sequence having at least 80% homology thereto; the LFR3 includes the amino acid sequence shown in SEQ ID NO:13 or an amino acid sequence having at least 80% homology thereto; the LFR4 includes the amino acid sequence shown in SEQ ID NO:14 or an amino acid sequence having at least 80% homology thereto.

[0112] According to an embodiment of the present invention, the antibody described in the first aspect, the second aspect or the third aspect above may further include at least one of the following technical features:

[0113] According to an embodiment of the present invention, the affinity KD of the antibody < 10 -9 M.

[0114] According to an embodiment of the present invention, the affinity KD of the antibody < 10 -10 M.

[0115] According to an embodiment of the present invention, the affinity KD of the antibody < 10 -11 M.

[0116] According to an embodiment of the present invention, the affinity KD of the antibody < 10 -12 M.

[0117] According to an embodiment of the present invention, the affinity KD of the antibody < 10 -13 M.

[0118] According to an embodiment of the present invention, the antibody further includes a constant region; wherein, the constant region includes at least one of a heavy chain constant region and a light chain constant region.

[0119] According to an embodiment of the present invention, at least a part of at least one of the heavy chain constant region and the light chain constant region is derived from at least one of a murine antibody, a human antibody, a primate antibody, a bovine antibody, a horse antibody, a dairy cow antibody, a pig antibody, a sheep antibody, a goat antibody, a dog antibody, a cat antibody, a rabbit antibody, a camel antibody, a donkey antibody, a deer antibody, a mink antibody, a chicken antibody, a duck antibody, a goose antibody, a turkey antibody, a fighting chicken antibody or a mutant thereof.

[0120] According to an embodiment of the present invention, the heavy chain constant region is selected from the heavy chain constant region of any one of IgG, IgA, IgM, IgE, IgD or a combination of multiple constant region segments.

[0121] In an alternative embodiment of the present invention, the heavy chain constant region includes CH1 of IgG, the hinge region of IgG, CH2 of IgM, CH3 of IgM and / or CH4 of IgM.

[0122] In an alternative embodiment of the present invention, the IgG is selected from IgG1, IgG2, IgG3, IgG4.

[0123] According to an embodiment of the present invention, the light chain constant region includes a κ-type or λ-type light chain constant region.

[0124] In an alternative embodiment of the present invention, the heavy chain constant region includes or is the heavy chain constant region shown in SEQ ID NO:15 or an amino acid sequence having at least 80% identity thereto; or the light chain constant region includes or is the light chain constant region shown in SEQ ID NO:16 or an amino acid sequence having at least 80% identity thereto.

[0125] According to an embodiment of the present invention, the N-terminus of the heavy chain constant region is connected to the C-terminus of the heavy chain variable region, and the N-terminus of the light chain constant region is connected to the C-terminus of the light chain variable region.

[0126] In this article, the division of variable region and constant region sequences refers to the IMGT division method. See Lefranc, the international ImMunoGeneTics database. Nucl.Acids Res., 29(1):207-209(2001). DOI:10.1093 / nar / 29.1.207. PMID:11125093. and Martinez-Jean C. and Bosc N. or Ehrenmann, Patrice Duroux, Chantal Ginestoux, Gene table: house mouse (Mus musculus) IGHC, IMGT Repertoire. the international ImMunoGenetics information http: / / www.imgt.org. Created: 16 / 03 / 2011. Version: 17 / 01 / 2020. or Ehrenmann, Patrice Duroux, Chantal Ginestoux, Gene table: house mouse (Mus musculus) IGLC, IMGT Repertoire. the international ImMunoGenetics information http: / / www.imgt.org. Created: 16 / 03 / 2011. Version: 17 / 01 / 2020. There will be some amino acid differences between the variable regions divided by different methods and the C-terminus of the variable region or the N-terminus of the constant region divided by IMGT. The variable regions or constant regions divided by other methods well-known in the art are also within the protection scope of the present invention.

[0127] Those skilled in the art can understand that the features and advantages described for the antibodies in the first aspect also apply to the antibodies in the second aspect or the third aspect, and will not be repeated here.

[0128] Nucleic acid molecule, vector, cell or host, method for preparing antibody

[0129] In the process of preparing or obtaining the antibodies described in the first aspect, the second aspect or the third aspect, nucleic acid molecules expressing these antibodies can be used, connected to different vectors, and then expressed in different cells to obtain the corresponding antibodies.

[0130] In the fourth aspect of the present invention, the present invention provides a nucleic acid molecule. According to an embodiment of the present invention, the nucleic acid molecule encodes the antibody described in the first aspect, the second aspect or the third aspect. The nucleic acid molecule according to the embodiment of the present invention can encode and obtain the above-mentioned antibody.

[0131] According to an embodiment of the present invention, the nucleic acid molecule includes DNA or RNA.

[0132] It should be noted that for the nucleic acid molecules mentioned in this article, those skilled in the art should understand that it actually includes either any one of the complementary double strands or both. For convenience, in this article, although only one strand is given in most cases, the other complementary strand is actually also disclosed. In addition, the molecular sequences in the present invention include DNA or RNA forms. Disclosing one of them means that the other is also disclosed.

[0133] In the fifth aspect of the present invention, the present invention provides a vector. According to the embodiments of the present invention, the vector includes the nucleic acid molecule described in the fourth aspect. When connecting the above-mentioned nucleic acid molecule to the vector, the nucleic acid molecule can be directly or indirectly connected to the control elements on the vector, as long as these control elements can control the translation and expression of the nucleic acid molecule, etc. Of course, these control elements can directly come from the vector itself or be exogenous, that is, not from the vector itself. Of course, the nucleic acid molecule and the control elements can be operably linked. As used herein, "operably linked" means connecting an exogenous gene to a vector such that the control elements within the vector, such as transcriptional control sequences and translational control sequences, etc., can perform their intended functions of regulating the transcription and translation of the exogenous gene. Commonly used vectors can be, for example, plasmids, phages, etc. After introducing the vector according to some specific embodiments of the present invention into a suitable recipient cell, under the mediation of the regulatory system, the expression of the aforementioned antibody can be effectively achieved, and thus a large amount of the antibody can be obtained in vitro.

[0134] In some specific embodiments of the present invention, the vector is a eukaryotic expression vector, a prokaryotic expression vector, a virus or a phage.

[0135] In an alternative embodiment of the present invention, the expression vector is a plasmid expression vector.

[0136] In the sixth aspect of the present invention, the present invention provides a cell or a host. According to the embodiments of the present invention, the cell or the host includes: the nucleic acid molecule described in the fourth aspect or the vector described in the fifth aspect; or expresses the antibody described in the first aspect, the second aspect or the third aspect. Using this cell under suitable conditions, the aforementioned antibody can be effectively expressed intracellularly.

[0137] According to the embodiments of the present invention, the cell is obtained by introducing the vector described in the fifth aspect into the cell.

[0138] It should be noted that the cells of the present invention are not particularly limited and can be prokaryotic cells, eukaryotic cells or phages. The prokaryotic cells can be Escherichia coli, Bacillus subtilis, Streptomyces or Proteus mirabilis, etc. The eukaryotic cells include fungi such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Trichoderma, etc., insect cells such as Spodoptera frugiperda, plant cells such as tobacco, and mammalian cells such as BHK cells, CHO cells, COS cells, myeloma cells, etc.

[0139] In an alternative embodiment of the present invention, the cells are mammalian cells, including BHK cells, CHO cells, NSO cells or COS cells, and do not include animal germ cells, fertilized eggs or embryonic stem cells.

[0140] It should be noted that the "suitable conditions" described in the present invention refer to the conditions suitable for the expression of the antibodies of the present invention. It is easily understood by those skilled in the art that the conditions suitable for the expression of the antibodies include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy cell states, suitable cell densities, suitable cell culture environments, and suitable cell culture times. The "suitable conditions" are not particularly limited, and those skilled in the art can optimize the most suitable conditions for the expression of the antibodies according to the specific environment of the laboratory.

[0141] In a seventh aspect of the present invention, the present invention provides a method for preparing the antibody described in the first aspect, the second aspect or the third aspect. According to an embodiment of the present invention, the method includes culturing the cells or hosts described in the sixth aspect. The methods according to some specific embodiments of the present invention can effectively obtain a large amount of the antibody.

[0142] Based on the amino acid sequence of the antibody of the present disclosure, it is easily conceivable by those skilled in the art to prepare the antibody by using genetic engineering techniques or other techniques (chemical synthesis, recombinant expression), for example, isolating and purifying the antibody from the culture product of recombinant cells capable of recombinantly expressing the antibody described in any one of the above, which is easily achievable for those skilled in the art. Based on this, no matter what technique is used to prepare the antibody of the present disclosure, it falls within the protection scope of the present disclosure.

[0143] Those skilled in the art can understand that the features and advantages described above for the antibody described in the first aspect, the second aspect or the third aspect also apply to the nucleic acid molecule, the vector, the cell or the host, and the method for preparing the antibody, and will not be repeated here.

[0144] Reagents or kits and their uses

[0145] In the eighth aspect of the present invention, the present invention provides an application of the antibody described in the first, second or third aspect in PCR. According to an embodiment of the present invention, Taq DNA polymerase is used in the PCR, and the Taq DNA polymerase is modified by the antibody. According to an embodiment of the present invention, the Taq DNA polymerase modified by the antibody is used to amplify a template containing DNA, and the amplification effect is significantly better than that of the Taq DNA polymerase not modified by the antibody.

[0146] In the ninth aspect of the present invention, the present invention provides a Taq DNA polymerase modified by an antibody, and the Taq DNA polymerase is modified by the antibody described in the first, second or third aspect.

[0147] In the tenth aspect of the present invention, the present invention provides a composition for PCR reaction, which comprises the antibody described in the first, second or third aspect and Taq DNA polymerase, and the composition has good stability. According to an embodiment of the present invention, the composition is placed at 37 °C for 7 days, and the stability is good.

[0148] In the eleventh aspect of the present invention, the present invention provides a reagent or a kit. According to an embodiment of the present invention, the reagent or the kit includes: the antibody described in the first, second or third aspect, the Taq DNA polymerase described in the tenth aspect, or the composition described in the eleventh aspect. As mentioned above, the antibody in some specific embodiments or examples of the present invention can bind to Taq DNA polymerase. Therefore, the reagent or the kit containing the antibody can effectively inhibit the activity of DNA polymerase, thereby preventing non-specific amplification caused by mismatches or primer dimers. As mentioned above, the mutant antibody of the present invention has improved Taq DNA polymerase binding activity, affinity, stability or specificity. Therefore, the reagent or the kit containing the antibody has an improved amplification effect.

[0149] The above-mentioned kit may include any one or more of the following: treatment solution, anti-Taq DNA polymerase antibody, Taq DNA polymerase, PCR composition, instruction manual or literature, etc.

[0150] Method, mutant library

[0151] In the twelfth aspect of the present invention, the present invention provides a method for screening Taq DNA polymerase antibodies. According to an embodiment of the present invention, the method includes: a) designing primers for amino acid substitution at 1, 2, 3, or 4 sites among X1, X2, X3, and X4 defined in the antibody of the first aspect or at the mutation sites defined in the antibody of the second aspect; b) constructing a mutant library with the primers in a) using the nucleic acid molecule of the fourth aspect, the vector of the fifth aspect, or the cell or host of the sixth aspect as a template;

[0152] c) screening Taq DNA polymerase antibodies from the mutant library.

[0153] According to an embodiment of the present invention, the mutant library is a single-site saturation mutant library.

[0154] According to an embodiment of the present invention, the Taq DNA polymerase antibody includes or is the antibody of the first aspect, the second aspect, or the third aspect.

[0155] In the thirteenth aspect of the present invention, the present invention provides a mutant library, which includes the antibody of the first aspect, the second aspect, or the third aspect.

[0156] According to an embodiment of the present invention, the mutant library is obtained by the method of the twelfth aspect.

[0157] The amino acid sequences involved herein are shown in Table 2:

[0158] Table 2: Amino Acid Sequences

[0159]

[0160]

[0161] The solutions of the present invention will be explained below in conjunction with embodiments. Those skilled in the art will understand that the following embodiments are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. For those not specified in the embodiments regarding specific techniques or conditions, they shall be carried out according to the techniques or conditions described in the literature in the art or according to the product specifications. For reagents or instruments not specified as to the manufacturer, they are all conventional products that can be obtained commercially.

[0162] 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 this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the formulations or unit doses herein, some methods and materials are now described. Unless otherwise noted, the techniques employed or contemplated herein are standard methods. The materials, methods, and examples are illustrative only and not limiting.

[0163] Unless otherwise indicated, the practice of this disclosure will employ conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of those of ordinary skill in the art. Such techniques are fully explained in the literature, such as Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989); Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Animal Cell Culture (R.I. Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D.M. Weir and C.C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P. Calos, eds., 1987); Current Protocols in Molecular Biology (F.M. Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction (Mullis et al., eds., 1994); and Current Protocols in Immunology (J.E. Coligan et al., eds., 2011), each of which is hereby expressly incorporated by reference herein.

[0164] In this example, the restriction endonuclease, T4 DNA ligase, and DNA polymerase were purchased from New England Biolabs, the Taq DNA polymerase was purchased from TaKaRa, the gel extraction kit and plasmid extraction kit were commercially available, and primer synthesis and gene sequencing were completed by an outsourcing company. The sequence of the monoclonal antibody against Taq DNA polymerase (hereinafter referred to as the WT antibody) was derived from the sequencing of mouse hybridoma cells.

[0165] Example 1: Construction and screening of the mutant library

[0166] 1. Construction of the template plasmid of the wild-type (WT) Taq DNA polymerase antibody (abbreviated as the WT antibody)

[0167] (1) Synthesis of the WT antibody gene:

[0168] The nucleotide sequences of VH and VL of the WT antibody were optimized for Escherichia coli codons, and then the antibody gene sequence was synthesized. The amino acid sequences of VH and VL of the WT antibody are shown in SEQ ID NO:17 and SEQ ID NO:18 respectively, and the amino acid sequences of the heavy chain and light chain are shown in SEQ ID NO:19 and SEQ ID NO:20 respectively.

[0169] (2) Amplification of the WT antibody gene fragment:

[0170] The nucleotide sequences of the synthesized antibody VH and VL in step (1) were amplified by PCR using DNA polymerase, and then the antibody bands were separated by agarose gel electrophoresis. Then, the antibody gene fragment was purified using a gel extraction kit.

[0171] (3) Enzymatic digestion and ligation of the WT antibody gene fragment:

[0172] The antibody gene fragment obtained in step (2) and the V01 vector plasmid (including the nucleotide sequence of the constant region) were simultaneously digested with a restriction endonuclease, and then the antibody gene fragment and the V01 vector with sticky ends were purified using a gel extraction kit. Then, the antibody gene fragment and the V01 vector were ligated with T4 DNA ligase at 22°C for 4 hours. After that, the ligation reaction product was recovered and purified, and the DNA concentration was measured. Finally, 100 ng of the plasmid was transformed into 100 μl of TG1 Escherichia coli competent cells to obtain a bacterial solution. Then, the bacterial solution was spread evenly on a plate containing ampicillin resistance and cultured overnight at 37°C.

[0173] (4) Extraction and sequencing verification of the WT template plasmid

[0174] Select 10 monoclonal colonies cultured overnight in step (3), perform colony PCR and gel electrophoresis detection using Taq DNA polymerase, select the bacteria with the correct inserted antibody gene sequence for culture and amplification, obtain the WT template plasmid using a plasmid extraction kit, etc., and send it to a sequencing company for gene sequencing verification.

[0175] 2. Construction of single-point mutation library

[0176] In this part of the experiment, single-point saturation mutagenesis was performed on the VH and VL full CDR regions of the WT antibody obtained in step 1 to construct a single-point mutation library.

[0177] (1) Primer design and synthesis

[0178] Using degenerate base codons, design upstream and downstream primer pairs for single-point saturation mutagenesis at amino acid sites in the VH and VL full CDR regions, and hand them over to an outsourcing company for primer synthesis.

[0179] (2) PCR amplification of single-point saturation mutagenesis plasmid

[0180] Using the primers obtained in step (1), perform PCR amplification on the single-point saturation mutagenesis plasmid by the PCR method. Configure the reaction system according to Table 3, and then use the PCR reaction conditions in Table 4 to amplify and prepare the single-point mutation library plasmid. Finally, digest the WT template plasmid obtained in step 1 with a restriction enzyme at 37°C for 1 hour to obtain the plasmid of the mutation library at amino acid sites in the full CDR region.

[0181] Table 3: Reaction system for PCR amplification

[0182] WT template plasmid 50 ng DNA polymerase 1 μl DNA polymerase buffer 10 μl dNTP (2.5 mM) 4 μl Forward primer (10 μM) 1 μl Reverse primer (10 μM) 1 μl ddH2O Volume made up to 50 μl

[0183] Table 4: PCR reaction conditions

[0184] Step1 Step2 Step3 Step4 Step5 Step6 Temperature 95℃ 95℃ 55-60℃ 72℃ 72℃ 4℃ Time 5 min 30s 30s 2 min 5 min ∞

[0185] Note: 22 cycles are performed in Step 2 - Step 4.

[0186] (3) Transformation of single-point saturation mutagenesis plasmid:

[0187] Take the plasmid of the mutation library at amino acid sites in the full CDR region obtained in step (2), take 10 μl of each reaction product and transform it into 100 μl of TG1 Escherichia coli competent cells to obtain a bacterial solution, and then spread all the bacterial solution on a plate containing ampicillin resistance and culture it overnight at 37°C.

[0188] 3. Screening of single-point mutation library

[0189] (1) Expression of mutant library antibodies

[0190] Pre-add 500 μl of culture medium into a 96-well culture plate. For each single-point mutation library, select monoclonal colonies transformed with the single-point saturated mutation plasmids cultured overnight in step 2-(3), and set up WT, negative control (i.e., colonies without insertion of VH\VL genes), and blank control (i.e., only culture medium without colonies). After culturing at 37°C for 5-6 hours, transfer the bacterial solution to a new 96-well culture plate. Then, after culturing at 37°C for 1-2 hours, finally add the induction medium and culture overnight at 37°C to express the antibody, obtaining the antibody expression supernatant of the mutation library at all amino acid sites in the CDR region.

[0191] (2) Screening and sequencing of the mutation library

[0192] Add commercially available Taq DNA polymerase into the ELISA plate at a dose of 0.03 μg / ml and 100 μl / well, and react overnight at 4°C for coating. The next day, block with 1-2% skim milk powder. After diluting the antibody expression supernatant of the mutation library at all amino acid sites in the CDR region obtained in this step (1), add it into the ELISA plate wells at a dose of 100 μl / well, and set up WT, negative control (i.e., colonies without insertion of VH\VL genes), and blank control (i.e., only culture medium without colonies). Incubate at room temperature for 2 hours, and then perform subsequent washing of the plate, color development, and reading using the detection method of conventional ELISA; finally, organize and analyze the data results, send the clones with improved performance for sequencing, and finally analyze the sequencing results to select the mutation sites of 5 unique mutant candidate clones (see Table 5) for constructing the combinatorial mutation library. (The Ratio value in Table 5 represents the degree of affinity improvement. When the Ratio value is equal to 1, it means that the affinity of the mutant clone is the same as that of WT).

[0193] Table 5: Screening results and mutation sites of candidate clones

[0194]

[0195] Note: In this article, WT always represents no mutation relative to the wild-type sequence (the same as in Table 6 and Table 7); the positions of mutant amino acids in this article are obtained by sequentially numbering the amino acid sequences of VH or VL of WT from the N-terminus to the C-terminus.

[0196] 4. Construction of the combinatorial mutation library

[0197] (1) Design and synthesis of library primers:

[0198] According to the mutation sites on VH and VL of the Taq DNA polymerase antibody obtained in step 3-(2), design amplification primers for the combinatorial mutation library and perform primer synthesis.

[0199] (2) Fragment amplification and ligation

[0200] According to the PCR system in Table 3 and the PCR reaction conditions in Table 4, the antibody mutant fragments were amplified, and then the antibody mutant fragments were recovered by gel extraction. The antibody mutant fragments were spliced into complete antibody fragments (heavy chain variable region or light chain variable region) by the method of Overlap PCR.

[0201] Finally, the antibody fragment was inserted into the V01 vector by enzymatic digestion and ligation to form a complete antibody expression plasmid (for the specific steps, see the steps in 1-(3) "Restriction digestion and ligation of WT antibody gene fragment"). 100 ng of the plasmid was transformed into 100 μl of TG1 Escherichia coli competent cells, and all the bacterial solution was spread on a plate containing ampicillin resistance, and cultured overnight at 37 °C.

[0202] 5. Screening of the combinatorial mutant library

[0203] Twenty-one combinatorial mutant antibodies were randomly selected for supernatant expression, ELISA screening detection, and positive clone sequencing analysis. The mutation site information and Ratio values are shown in Table 6.

[0204] Table 6: Information of combinatorial mutant candidate clones

[0205]

[0206] Example 2: Expression of mutant Taq DNA polymerase antibody

[0207] In this example, the mutant Taq DNA polymerase antibody screened in Example 1 was expressed, and the specific experimental operations are as follows:

[0208] 1. Construction of eukaryotic recombinant expression plasmid

[0209] pcDNA TM 3.4 The vector pcDNA3.4A is the constructed recombinant antibody eukaryotic expression vector. Multiple cloning enzyme digestion sites such as HindIII, BamHI, and EcoRI have been introduced into this expression vector, and it is named the pcDNA3.4A expression vector, hereinafter referred to as the 3.4A expression vector for short; according to the variable region gene sequences of the 26 candidate clones screened in Example 1, VL and VH gene-specific amplification primers and constant region overlap primers for the corresponding antibody sequences were designed. The two ends of the primers were respectively provided with HindIII and EcoRI enzyme digestion sites and protection bases, and the light chain gene fragment and the heavy chain gene fragment were amplified by PCR amplification method.

[0210] The heavy and light chain gene fragments were digested with HindIII / EcoRI double enzymes respectively, and the 3.4A vector was digested with HindIII / EcoRI double enzymes. The digested antibody light and heavy chain gene fragments and the vector were purified and recovered. Then, the gene fragments encoding the antibody light and heavy chains were respectively ligated into the 3.4A expression vector and transformed into DH5α Escherichia coli competent cells. After colonies grew, single colonies were picked respectively for PCR identification of positive clones, and the positive clones were picked for sequencing to determine the correctness of the sequence. The clones with correct sequencing were selected for plasmid extraction for standby.

[0211] 2. Preparation of recombinant antibody samples

[0212] Resuscitate HEK293 cells in advance, passage and culture them in a 200 ml system until the cell density reaches (3 - 5)×10 6 cells / ml and the cell viability > 95%; centrifuge and wash the cells, resuspend them with the medium, and at the same time adjust the cell density to 2.9×10 6 cells / ml as the cell dilution. Prepare plasmid DNA and transfection reagent dilutions with the medium respectively. Add the transfection reagent dilution to the plasmid DNA dilution, mix well and let it stand at room temperature for 15 min; slowly add this mixture to the cell dilution within 1 min, mix well, sample and count, record and observe the viability of the cells after transfection, and place them in a 35°C constant temperature incubator for culture at a rotation speed of 120 rmp and a CO2 content of 8%. After 13 days, collect the samples by centrifugation. Affinity purification was carried out with a protein A affinity chromatography column to obtain the antibody.

[0213] Example 3: Affinity analysis

[0214] The 26 mutant antibodies obtained above were subjected to affinity detection and analysis. The specific steps were as follows: The binding and dissociation curves of the antigen and antibody were tested on a Biacore8K+ device, and the instrument automatically fitted to obtain the affinity constant, binding rate, and dissociation rate. In the affinity detection results, KD represents the equilibrium dissociation constant, that is, the affinity constant. The smaller the KD value, the higher the affinity; ka represents the binding rate; kd represents the dissociation rate. The results showed that the affinity of the mutant antibody for Taq DNA polymerase was better than that of the wild-type antibody for Taq DNA polymerase.

[0215] Table 7: Affinity detection data

[0216]

[0217]

[0218] In the description of this specification, the description referring to terms such as "one embodiment", "some embodiments", "example", "specific example", or "some examples", etc. means that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0219] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. An anti-Taq DNA polymerase antibody comprising HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, characterized in that: The HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3 are the amino acid sequences shown below: HCDR 1:TYYIY; HCDR 2:GINPTX1GGPVFNEKFKS, where X1 is S or G; HCDR 3:SLLRRGYYFDY; LCDR 1:RASQX2INNYLN, where X2 is D or Y; LCDR 2: YTX3RLHX4, X3 is S, F, I or L, X4 is S, F or Y; LCDR 3:QQDDTLPLT; Optionally, X1 is S; Optionally, X1 is G; Optionally, X2 is D; Optionally, X2 is Y; Optionally, X3 is S; Optionally, X3 is F; Optionally, X3 is 1; Optionally, X3 is L; Optionally, X4 is S; Optionally, X4 is F; Optionally, X4 is Y; Optionally, X1 is S, X2 is D, X3 is S, and X4 is S do not simultaneously hold; Optionally, X1, X2, X3 and X4 are selected from any one of the following combinations:

2. An anti-Taq DNA polymerase antibody, characterized in that: include: a heavy chain variable region and a light chain variable region; The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 17 or a variant thereof, and compared with the amino acid sequence shown in SEQ ID NO: 17, the variant of SEQ ID NO: 17 comprises a mutation at the following site: S55G; The light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 18 or a variant thereof, wherein compared with the amino acid sequence shown in SEQ ID NO: 18, the variant of SEQ ID NO: 18 comprises a mutation in at least one of the following sites: D28Y, S52I / F / L, and S56F / Y; Optionally, the mutation at position 55 is S55G; Optionally, the mutation at position 28 is D28Y; Optionally, the mutation at position 52 is S52I; Optionally, the mutation at position 52 is S52F; Optionally, the mutation at position 52 is S52FL; Optionally, the mutation at position 56 is S56F; Optionally, the mutation at position 56 is S56Y; Optionally, the heavy chain variable region and the light chain variable region are selected from any one of the following combinations:

3. An anti-Taq DNA polymerase antibody comprising HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, characterized in that: The HCDR1, HCDR2, and HCDR3 are the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region defined by the antibody according to claim 2; the LCDR1, LCDR2, and LCDR3 are the LCDR1, LCDR2, and LCDR3 of the light chain variable region defined by the antibody according to claim 2; Optionally, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 is defined by any one of the Kabat, Chothia, IMGT, AbM or Contact systems or a combination of multiple systems; Optionally, the antibody comprises at least one of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4; Optionally, at least a portion of at least one of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4 is derived from at least one of mouse antibody, human antibody, primate antibody, bovine antibody, horse antibody, dairy cow antibody, porcine antibody, sheep antibody, goat antibody, dog antibody, cat antibody, rabbit antibody, camel antibody, donkey antibody, deer antibody, mink antibody, chicken antibody, duck antibody, goose antibody, turkey antibody, fighting cock antibody or mutants thereof; Optionally, the HFR1 comprises an amino acid sequence as shown in SEQ ID NO: 7 or an amino acid sequence having at least 80% homology thereto; The HFR2 comprises an amino acid sequence as shown in SEQ ID NO: 8 or an amino acid sequence having at least 80% homology thereto; The HFR3 comprises an amino acid sequence as shown in SEQ ID NO: 9 or an amino acid sequence having at least 80% homology thereto; The HFR4 comprises the amino acid sequence shown in SEQ ID NO: 10 or an amino acid sequence having at least 80% homology thereto; The LFR1 comprises an amino acid sequence as shown in SEQ ID NO: 11 or an amino acid sequence having at least 80% homology thereto; The LFR2 comprises an amino acid sequence as shown in SEQ ID NO: 12 or an amino acid sequence having at least 80% homology thereto; The LFR3 comprises an amino acid sequence as shown in SEQ ID NO: 13 or an amino acid sequence having at least 80% homology thereto; The LFR4 comprises the amino acid sequence shown in SEQ ID NO: 14 or an amino acid sequence having at least 80% homology thereto.

4. The antibody according to any one of claims 1 to 3, characterized in that The antibody further comprises a constant region; Wherein, the constant region includes at least one of a heavy chain constant region and a light chain constant region; Optionally, at least a portion of at least one of the heavy chain constant region and the light chain constant region is from at least one of a mouse antibody, a human antibody, a primate antibody, a bovine antibody, a horse antibody, a dairy cow antibody, a porcine antibody, a sheep antibody, a goat antibody, a dog antibody, a cat antibody, a rabbit antibody, a camel antibody, a donkey antibody, a deer antibody, a mink antibody, a chicken antibody, a duck antibody, a goose antibody, a turkey antibody, a fighting cock antibody, or a mutant thereof; Optionally, the heavy chain constant region is selected from any one of the heavy chain constant regions of IgG, IgA, IgM, IgE, and IgD, or a combination of multiple constant region segments; Optionally, the heavy chain constant region includes CH1 of IgG, hinge region of IgG, CH2 of IgM, CH3 of IgM and / or CH4 of IgM; Optionally, the light chain constant region comprises a light chain constant region selected from a κ type or a λ type; Optionally, the heavy chain constant region comprises or is the heavy chain constant region shown in SEQ ID NO: 15 or an amino acid sequence having at least 80% identity thereto; or The light chain constant region comprises or is the light chain constant region shown in SEQ ID NO: 16 or an amino acid sequence having at least 80% identity thereto.

5. A nucleic acid, a vector, a cell or a method for preparing the antibody according to any one of claims 1 to 4, characterized in that: The nucleic acid encodes the antibody according to any one of claims 1 to 4; the vector contains the above-mentioned nucleic acid; the cell contains the above-mentioned nucleic acid or vector; and the method comprises culturing the above-mentioned cell.

6. Use of the antibody according to any one of claims 1 to 4 in PCR; Optionally, Taq DNA polymerase is used in the PCR, and the Taq DNA polymerase is modified by the antibody.

7. An antibody-modified Taq DNA polymerase, wherein the Taq DNA polymerase is modified with the antibody according to any one of claims 1 to 4.

8. A composition for PCR reaction, comprising the antibody according to any one of claims 1 to 4 and Taq DNA polymerase.

9. A reagent or a kit, characterized in that: include: The antibody according to any one of claims 1 to 4, the Taq DNA polymerase according to claim 7, or the composition according to claim 8.

10. A method for screening Taq DNA polymerase antibodies, characterized in that: include: a) designing primers for performing amino acid substitution at the sites of X1, X2, X3 and X4 defined in the antibody of claim 1, or at the mutation sites defined in the antibody of claim 2; b) using the nucleic acid, vector or cell of claim 5 as a template and the primers described in a) to construct a mutation library; c) screening Taq DNA polymerase antibodies from the mutant library; Optionally, the mutation library is a single-site saturation mutation library; Optionally, the Taq DNA polymerase antibody includes or is the antibody according to any one of claims 1 to 4.

11. A mutation library, characterized in that The mutant library comprises the antibody according to any one of claims 1 to 4; Optionally, the mutant library is obtained by the method of claim 10.

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