Anti-Taq DNA polymerase antibody and hot-start Taq DNA polymerase and their application

Abstract

The present invention belongs to the technical field of antibody preparation, and more particularly to anti-Taq DNA polymerase antibodies and hot-start Taq DNA polymerase and applications thereof. The amino acid sequences of CDR1-3 on the light chain variable region of the antibody are shown in SEQ ID NO.3-5, respectively, and the amino acid sequences of CDR1-3 on the heavy chain variable region are shown in SEQ ID NO.8-10, respectively. The antibody provided by the present invention has high affinity and recognition specificity with Taq DNA polymerase, can effectively block the 5'-3' polymerase activity of Taq DNA polymerase at room temperature, and has good blocking applicability to 450-550aa regional Taq DNA polymerase and its mutants, is expected to be applied to modify Taq DNA polymerase to prepare hot-start Taq DNA polymerase and effectively reduce nonspecific amplification in PCR, etc., and has good application prospects in the fields of nucleic acid amplification and gene diagnosis.

Description

Technical Field

[0001] The invention relates to the technical field of antibody preparation, in particular to an anti-Taq DNA polymerase antibody and a hot-start Taq DNA polymerase and applications thereof. Background Art

[0002] Polymerase Chain Reaction (PCR) is a molecular biology technique for amplifying specific DNA fragments in vitro. It is widely used in gene detection, biochemical tests and other fields. Its reaction principle is similar to the natural replication process of double-stranded DNA molecules, that is, DNA is denatured and melted into single strands under high temperature conditions in vitro, and primers and single strands are combined according to the principle of base complementary pairing under low temperature conditions. When the temperature is the optimal reaction temperature of DNA polymerase, DNA polymerase synthesizes daughter DNA complementary to the parent DNA along the direction from phosphate to pentose (5'-3'). After multiple cycles of denaturation, annealing, extension, etc., a large number of specific DNA fragments can be obtained. DNA polymerase is the key to achieving automatic and repeated amplification of specific DNA sequences in PCR.

[0003] Taq DNA polymerase is a thermostable (heat-resistant) DNA polymerase extracted from Thermus aquaticus. It can withstand temperatures of ≥90°C and maintain its DNA polymerase activity, so it can meet the requirements of PCR reactions that require high temperature conditions. In practical applications, the PCR reaction system using Taq DNA polymerase does not require the addition of polymerase in each cycle, making the PCR reaction simpler and faster, thereby reducing experimental costs. However, under low temperature conditions, Taq DNA polymerase still has a certain polymerase activity, so it will cause annealing between the primer and non-specific DNA at the start of PCR, causing the amplification of non-specific fragments.

[0004] The hot-start Taq DNA polymerase technology is used to control the activity of Taq DNA polymerase under high temperature conditions and inactivate it under low temperature, which is an effective way to enhance the specificity of PCR amplification and improve the sensitivity of amplification. The commonly used method is to use antibodies to modify the hot-start enzyme, that is, under low temperature conditions, the monoclonal antibody against Taq DNA polymerase and the polymerase are incubated to form a complex, thereby neutralizing the polymerization activity of the polymerase, so as to inhibit the non-specific amplification of the polymerase caused by the non-specific annealing of the primers or primer dimers under low temperature conditions or in the heating stage before pre-denaturation; after high temperature denaturation, the antibody and the polymerase are depolymerized, releasing the active Taq DNA polymerase, thereby resuming the PCR amplification reaction.

[0005] Most of the existing Taq DNA polymerase antibodies are mouse monoclonal antibodies, which have low affinity with Taq DNA polymerase, weak blocking effect of polymerase polymerization activity, and often require a higher antibody dose. In addition, most of the existing Taq DNA polymerase antibodies are developed for wild-type, and the blocking efficiency for different mutants of Taq DNA polymerase varies greatly, and the versatility of polymerase blocking activity needs to be improved. Therefore, the development of a new broad-spectrum adaptable Taq DNA polymerase with high blocking activity has a wide range of application value. Summary of the invention

[0006] In view of the problems in the prior art that the anti-Taq DNA polymerase antibody has a weak blocking effect on the polymerase polymerization activity and / or poor versatility, the present invention provides a novel rabbit-derived anti-Taq DNA polymerase antibody and a hot-start Taq DNA polymerase containing the antibody, and provides the use of the antibody and the hot-start Taq DNA polymerase in the preparation of hot-start PCR reaction reagents. To achieve the above-mentioned purpose, the present invention is specifically implemented by the following technical solutions:

[0007] The first aspect of the present invention provides an anti-Taq DNA polymerase antibody, comprising a light chain variable region and a heavy chain variable region, wherein the amino acid sequences of the complementary determining regions CDR1, CDR2 and CDR3 on the light chain variable region are shown as SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5, respectively; and the amino acid sequences of the complementary determining regions CDR1, CDR2 and CDR3 on the heavy chain variable region are shown as SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10, respectively.

[0008] Furthermore, the amino acid sequence of the light chain variable region is shown in SEQ ID NO.2, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.7.

[0009] Furthermore, the amino acid sequence of the antibody light chain is shown in SEQ ID NO.1, and the amino acid sequence of the heavy chain is shown in SEQ ID NO.6.

[0010] Furthermore, the antibody is a full-length antibody or an antigen-binding region of the full-length antibody; the antigen-binding region is selected from at least one of Fab, F(ab)2, Fab', F(ab')2, Fv, (Fv)2, scFv and sc(Fv)2.

[0011] The second aspect of the present invention provides a nucleic acid molecule, a recombinant vector or a host cell comprising the nucleic acid molecule, wherein the nucleic acid molecule encodes the anti-Taq DNA polymerase antibody as described above.

[0012] Furthermore, the recombinant vector is a mammalian expression vector pBR322, and the host cell is a human kidney epithelial cell.

[0013] The third aspect of the present invention provides a hot-start Taq DNA polymerase, wherein the hot-start Taq DNA polymerase comprises Taq DNA polymerase and the anti-Taq DNA polymerase antibody as described above.

[0014] Furthermore, the weight ratio of the anti-TaqDNA polymerase antibody to the TaqDNA polymerase is 0.5-500:1.

[0015] Furthermore, the weight ratio of the anti-Taq DNA polymerase antibody to the Taq DNA polymerase is 0.8-500:1.

[0016] Furthermore, the weight ratio of the anti-Taq DNA polymerase antibody to the Taq DNA polymerase is 0.8-1.5:1.

[0017] The fourth aspect of the present invention provides the use of the anti-Taq DNA polymerase antibody or the hot-start Taq DNA polymerase described above in preparing a hot-start PCR reaction reagent.

[0018] The advantages and positive effects of the present invention are:

[0019] The rabbit-derived monoclonal antibody against Taq DNA polymerase provided by the present invention has high affinity and recognition specificity with Taq DNA polymerase, can effectively block the 5'-3' polymerase activity of Taq DNA polymerase at room temperature, and has good blocking applicability to various mutant types of Taq DNA polymerase in the 450-550aa region, is expected to be used for modifying Taq DNA polymerase to prepare hot-start Taq DNA polymerase and effectively reduce nonspecific amplification in PCR, etc., and has good application prospects in the fields of nucleic acid amplification and gene diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0021] Figure 1 This is the SDS-PAGE gel electrophoresis diagram of the recombinant TaqDNA polymerase purified in Example 1 of the present invention;

[0022] Figure 2This is a graph showing the antibody titer of rabbit serum immunized with Taq DNA polymerase using enzyme-linked immunosorbent assay in Example 2 of the present invention;

[0023] Figure 3 The expression map of the mammalian pBR322 vector used for constructing the anti-Taq DNA polymerase rabbit monoclonal antibody expression vector in Example 2 of the present invention, from left to right are the vectors pre-carrying the antibody light chain constant region and heavy chain constant region. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments. The embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0025] Based on the information contained in the present invention, it is easy for those skilled in the art to make various changes to the precise description of the present invention without departing from the spirit and scope of the appended claims. It should be understood that the scope of the present invention is not limited to the defined processes, properties or components, because these embodiments and other descriptions are only for the purpose of illustrating specific aspects of the present invention. In fact, various changes that a person skilled in the art or related fields can obviously make to the embodiments of the present invention are covered within the scope of the appended claims.

[0026] In order to better understand the present invention rather than limit the scope of the present invention, all the numerals and other numerical values ​​used in the present invention indicating dosage, percentage, etc., should be understood as modified by the word "about" in all cases. Therefore, unless otherwise specified, the numerical parameters listed in the specification and the appended claims are approximate values, which may be changed according to the different ideal properties attempted to be obtained. Each numerical parameter should at least be regarded as being obtained based on the reported significant figures and by conventional rounding methods. The meaning of the terms "include", "comprise", "contain", "have" and the like is non-restrictive, that is, other steps and other ingredients that do not affect the results can be added.

[0027] In addition, it should be noted that, unless otherwise defined, in the context of the present invention, the scientific and technical terms used should have the meanings commonly understood by those of ordinary skill in the art.

[0028] The terms "monoclonal antibody", "monoclonal antibody", "antibody" and other similar terms have the same meaning and are used interchangeably to refer to rabbit monoclonal antibodies that specifically bind to Taq DNA polymerase. The modifier "rabbit" indicates that the complementary determining regions (CDRs) of the antibody are derived from rabbit immunoglobulin sequences. The term "Taq DNA polymerase" is also called Taq enzyme, and the two can be used interchangeably.

[0029] Antibody is an immunoglobulin molecule that can specifically bind to a target antigen or epitope through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. In the present invention, the term "antibody" should be interpreted in the broadest sense and includes different antibody structures, including but not limited to so-called full-length antibodies, antibody fragments (containing antigen binding regions) and their genetic or chemical modifications, as long as they show the desired antigen binding activity.

[0030] A typical antibody molecule (full-length antibody) consists of two identical light chains (L) and two identical heavy chains (H). Light chains can be divided into two types, namely κ chain and λ chain; heavy chains can be classified into five types, namely μ, δ, γ, α and ε chains, and antibodies are defined as IgM, IgD, IgG, IgA and IgE, respectively. The amino acid sequences near the N-terminus of the heavy and light chains vary greatly, while the amino acid sequences of other parts are relatively constant. The regions near the N-terminus of the light and heavy chains where the amino acid sequences vary greatly are called variable regions (V), and the regions near the C-terminus where the amino acid sequences are relatively stable are called constant regions (C). The heavy chain variable region (VH) and the light chain variable region (VL) are usually the most variable parts of the antibody and contain antigen recognition sites. The VH and VL regions can be further subdivided into hypervariable regions (HVR) and framework regions (FR). The hypervariable regions are also called complementarity determining regions (CDRs), which are ring-shaped structures. The heavy chain CDR and the light chain CDR are closely together and cooperate with each other through the FR region, forming a surface that can complement the three-dimensional structure of the target antigen or epitope, which determines the specificity of the antibody and is the site where the antibody recognizes and binds to the antigen. The FR region is the more conservative part of VH and VL. They are generally in a β-folded configuration and are connected by three CDRs that form a connecting loop. Each VH and VL is usually composed of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0031] CDRs and FRs can be identified according to the Kabat definition, the Chothia definition, the accumulation of both the Kabat definition and the Chothia definition, the AbM definition, the contact definition, the IMGT unique numbering definition and / or the conformational definition, or any CDR determination method known in the art. As used herein, the Kabat numbering system is used to define.

[0032] The light chain constant region (CL) and the heavy chain constant region (CH) are not directly involved in the binding of antibodies to antigens, but they exhibit different effector functions, such as participating in antibody-dependent cytotoxicity of antibodies. The length of CL of different types of Ig (κ or λ) is basically the same, but the length of CH of different classes of Ig is different, such as IgG, IgA and IgD include CH1, CH2 and CH3, while IgM and IgE include CH1, CH2, CH3 and CH4. The amino acid sequences of the heavy chain and light chain constant regions of antibodies are well known in the art.

[0033] A full-length antibody is the most complete antibody molecular structure and has a typical Y-shaped molecular structure. Therefore, in the context of the present invention, "full-length antibody", "complete antibody" and "Y-shaped antibody" have the same meaning and can be used interchangeably.

[0034] Antibody fragments are one or more parts or fragments of a full-length antibody, which substantially retain the same biological function or activity as the full-length form. Specifically, antibody fragments at least include the same CDR region as the full-length antibody, and more preferably have the same variable region, thereby retaining complete antigen recognition and binding sites, and can bind to the same antigen as the full-length antibody, especially to the same epitope. In typical examples, antibody fragments include: Fab, F(ab)2, Fab', F(ab')2, Fv, (Fv)2, scFv, sc(Fv)2, and these antibody fragments can be obtained by conventional techniques in the art.

[0035] (i) Fab: Antigen-binding fragment (Fab) is a monovalent fragment consisting of a complete light chain (variable region and constant region) and a part of the heavy chain (variable region and the first constant region). By proteolytic cleavage of the full-length antibody, fragments such as Fab, F(ab')2, and Fab' can be obtained. For example, under the action of papain, IgG can be degraded into two Fab fragments and one Fc fragment; under the action of pepsin, IgG can be degraded into one F(ab')2 fragment and one pFc' fragment. The F(ab')2 fragment is further reduced to form two Fab' fragments. Since Fab has an antigen-binding region and a part of the constant region, it not only has the same antibody-antigen affinity and excellent tissue penetration as scFv, but also has a more stable structure.

[0036] (ii) F(ab)2: A bivalent fragment consisting of two Fabs linked by a disulfide bridge at the hinge region.

[0037] (iii) Fv: The variable fragment (Fv) is located at the N-terminus of the antibody Fab fragment, contains only the variable region, and is composed of the variable regions of a light chain and a heavy chain. It is a dimer of VH and VL non-covalently bound (VH-VL dimer). The three CDRs of each variable region interact with each other to form an antigen binding site on the surface of the VH-VL dimer, which has the ability to recognize and bind to antigens, although the affinity is lower than that of the intact antibody.

[0038] (iv) (Fv)2: consists of two Fv fragments covalently linked together.

[0039] (v) scFv: Single-chain variable fragment (scFv) is an Fv fragment composed of a single polypeptide chain, which is composed of a heavy chain variable region (VH) and a light chain variable region (VL) connected by a flexible linker (generally composed of 10-25 amino acids), which retains the binding specificity of the original antibody to the antigen. The linker in the present invention is not particularly limited as long as it does not hinder the expression of the antibody variable regions connected to its two ends. Compared with full-length antibodies, scFv has the characteristics of small molecular weight, so it has higher penetration and lower immune side effects.

[0040] (vi) The sc(Fv)2 fragment is composed of two heavy chain variable regions and two light chain variable regions connected via a linker or the like.

[0041] The terms "monoclonal antibody" or "single antibody" and the like are used interchangeably and refer to a homogeneous antibody population, i.e., the individual antibodies constituting the population are identical except for a small amount of mutations and / or post-translational modifications (e.g., isomerization, amidation) that may occur naturally. "Monoclonal antibodies" are highly specific and exhibit a single binding specificity and affinity for the same or substantially identical epitopes on an antigen. The modifier "monoclonal" indicates that the antibody is obtained from a substantially homogeneous antibody population and should not be construed as limiting the source or preparation method of the antibody. The antibody can be prepared by a variety of methods, including but not limited to hybridoma methods, phage display methods, yeast display methods, recombinant DNA methods, single cell screening, or single cell sequencing methods.

[0042] The term "specific binding" is a well-known term in the art, and a molecule exhibits "specific binding" if it reacts with a specific target antigen or epitope more frequently, more rapidly, longer lastingly, and / or with greater affinity than with other target antigens or epitopes. "Specific binding" or "preferential binding" does not necessarily require (although it can include) exclusive binding.

[0043] In order to make the above-mentioned objects and advantages of the present invention more obvious and easy to understand, specific embodiments of the present invention are described in detail below.

[0044] An embodiment of the present invention provides an anti-Taq DNA polymerase antibody, comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region each comprise three complementary determining regions (CDRs), respectively named CDR1, CDR2 and CDR3; wherein the amino acid sequences of CDR1, CDR2 and CDR3 on the light chain variable region are respectively shown in SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5, and the amino acid sequences of CDR1, CDR2 and CDR3 on the heavy chain variable region are respectively shown in SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10.

[0045] The invention uses the polymerase domain of Taq DNA polymerase as an antigen to immunize rabbits, and prepares rabbit-derived monoclonal antibodies against Taq DNA polymerase. The antibody has high affinity and recognition specificity with Taq DNA polymerase, can specifically bind to Taq DNA polymerase, effectively block the 5'-3' polymerase activity of Taq DNA polymerase at room temperature, has good blocking activity, has good applicability to various mutant types of Taq DNA polymerase in the 450-550aa region, and can effectively block the polymerase activity of different mutant types of Taq DNA polymerase. The antibody is expected to be applied to modifying Taq DNA polymerase to prepare hot-start Taq DNA polymerase, can effectively reduce nonspecific amplification and generation of primer dimers in PCR during PCR amplification, improve the amplification efficiency and amplification specificity of target fragments, and has good application prospects in the fields of nucleic acid amplification and gene diagnosis.

[0046] Optionally, the light chain variable region and the heavy chain variable region each include four framework regions (FRs), and the four FRs and three CDRs are arranged alternately in sequence to form a variable region. The amino acid sequence of VL of the antibody of the present invention is shown in SEQ ID NO.2, and the amino acid sequence of VH is shown in SEQ ID NO.7.

[0047] Optionally, the antibody of the present invention further comprises a light chain constant region and a heavy chain constant region, wherein CL and VL in each antibody constitute a light chain, and CH and VH constitute a heavy chain. The constant region of an antibody can usually be obtained by public query, such as: searching for rabbit IgG gamma C reign to obtain CH through the IMGT online database (www.imgt.org), and searching for rabbit IgG Kappa C reign to obtain CL. Specifically, the amino acid sequence of the light chain comprising the light chain constant region is shown in SEQ ID NO.1, and the amino acid sequence of the heavy chain comprising the heavy chain constant region is shown in SEQ ID NO.6.

[0048] Alternatively, the antibody of the present invention may be a full-length antibody or an antigen-binding region of the full-length antibody; the antigen-binding region refers to a region that substantially maintains the same biological function or activity as the full-length antibody, specifically, the antigen-binding region includes the CDR region as described above, and more preferably has the variable region as described above, thereby retaining a complete antigen recognition and binding site, and being able to bind to the same antigen as the full-length antibody, especially to the same epitope. The antigen-binding region is selected from at least one of Fab, F(ab)2, Fab', F(ab')2, Fv, (Fv)2, scFv or sc(Fv)2.

[0049] Yet another embodiment of the present invention provides a nucleic acid molecule, a recombinant vector or a host cell comprising the nucleic acid molecule, wherein the nucleic acid molecule encodes the anti-Taq DNA polymerase antibody as described above.

[0050] Nucleic acid molecules can be in the form of DNA (such as cDNA or genomic DNA or synthetic DNA) or RNA (such as mRNA or synthetic RNA). DNA can be single-stranded or double-stranded, and can also be a coding strand or a non-coding strand.

[0051] The sequence of the nucleic acid molecule can be obtained by conventional means such as deduction of codon coding rules based on the amino acid sequence of the antibody. The full-length sequence of the nucleic acid molecule or its fragment can usually be obtained by PCR amplification, recombination or artificial synthesis. The obtained nucleic acid molecule is inserted into an expression vector, then transfected into a host cell, and the transfected host cell is cultured under specific conditions to express the antibody of the present invention.

[0052] The original vector for constructing the recombinant vector is various vectors conventional in the art, as long as it can hold the nucleic acid molecule. Typical vectors include plasmids, viral vectors, bacteriophages, cosmids and minichromosomes. Plasmids are the most common vector forms, and therefore, in the context of the present invention, vectors and plasmids are used interchangeably. The vector can be a cloning vector (i.e., used to transfer the nucleic acid molecule to a host and multiply in a host cell) or an expression vector (i.e., containing necessary genetic elements to allow the nucleic acid molecule inserted into the vector to be expressed in a host cell). The nucleic acid molecule of the present invention can be inserted into a suitable vector to form a cloning vector or an expression vector carrying the nucleic acid molecule. This is a known technology and will not be described in detail herein.

[0053] The nucleic acid molecules encoding the heavy and light chains of the antibodies of the present invention can be constructed into two vectors, respectively, which can be introduced into the same or different host cells. When the heavy and light chains are expressed in different host cells, each chain can be separated from the host cells expressing them, and the separated heavy and light chains are mixed and incubated under suitable conditions to form antibodies. In other embodiments, the nucleic acid molecules encoding the heavy and light chains of the rabbit antibodies of the present invention can also be cloned into a vector, and each nucleic acid sequence is connected to a suitable promoter downstream; for example, each nucleic acid sequence encoding the heavy and light chains can be operably connected to different promoters, or the nucleic acid sequences encoding the heavy and light chains can be operably connected to a single promoter, so that both the heavy and light chains can be expressed by the same promoter. The choice of expression vector / promoter depends on the type of host cell used to produce the antibody.

[0054] In the present invention, the recombinant vector is transfected or transformed into the host cell using conventional techniques. When the host is a prokaryotic organism such as Escherichia coli, competent cells that can absorb DNA are harvested after the exponential growth phase and treated with CaCl2 or MgCl2; or by microinjection, electroporation or liposome packaging. When the host is a eukaryotic organism, the following DNA transfection methods can be selected: calcium phosphate coprecipitation, microinjection, electroporation, liposome packaging, etc.

[0055] The host cell can be a prokaryotic or eukaryotic cell. Representative examples include: Escherichia coli, Streptomyces, Salmonella typhimurium, yeast, Drosophila S2 or Sf9 cells, mammalian CHO, COS7, 293 series cells, etc. After obtaining the host cell transfected or transformed with the recombinant vector as described above, the antibody can be expressed by culturing under suitable conditions, and then separated to obtain purified antibody.

[0056] In a preferred embodiment, the recombinant vector is a mammalian expression vector pBR322, and the host cell is a human kidney epithelial cell (293F cell).

[0057] Another embodiment of the present invention provides a hot-start Taq DNA polymerase, wherein the hot-start Taq DNA polymerase comprises Taq DNA polymerase and the anti-Taq DNA polymerase antibody as described above.

[0058] The anti-Taq DNA polymerase antibody or its antigen binding region is incubated with Taq DNA polymerase at room temperature, and a complex is formed by non-covalent binding to obtain a hot-start Taq DNA polymerase. The Taq DNA polymerase modified by the antibody has reduced polymerase activity at low temperatures.

[0059] Optionally, the weight ratio of the anti-Taq DNA polymerase antibody to the Taq DNA polymerase is 0.5-500:1, preferably 0.8-500:1, and more preferably 0.8-1.5:1.

[0060] Optionally, the Taq DNA polymerase includes but is not limited to the Taq DNA polymerase 450-550aa fragment and mutants within the fragment region.

[0061] Another embodiment of the present invention provides the use of the anti-Taq DNA polymerase antibody or the hot-start Taq DNA polymerase described above in preparing a hot-start PCR reaction reagent.

[0062] The use of the anti-Taq DNA polymerase antibody or hot-start Taq DNA polymerase in preparing hot-start PCR reaction reagents has the same advantages as the anti-Taq DNA polymerase antibody described above over the prior art, which will not be described in detail here.

[0063] The present invention is further described below in conjunction with specific examples. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as the conditions described in the Molecular Cloning Laboratory Guide (Fourth Edition) published by Cold Spring Harbor Laboratory, or usually under the conditions recommended by the manufacturer.

[0064] Example 1 Preparation of Taq DNA polymerase recombinant protein antigen

[0065] The present invention selects the polymerase domain (450-550AA) of Thermus Aquaticus polA (Taq DNA polymerase, hereinafter referred to as Taq enzyme) as an antigen, constructs the gene sequence corresponding to the region into a pET-28a-sumo vector, and transfects it into Escherichia coli Rosetta for expression to obtain a Taq enzyme recombinant protein with high purity, which is used as an antigen for immunizing animals. The Taq enzyme 450-550AA gene fragment is synthesized by Jiangsu Saisuofei Biotechnology Co., Ltd.

[0066] The complete amino acid sequence of Taq enzyme can be found in Swiss-Prot No. P19821, or in NCBI Accession No. NP: AAA27507.1, and its nucleotide sequence can be found in NCBI Accession No. NM: J04639.1. Among them, the polymerase domain is a fragment of amino acids from position 450 to position 550 (450-550AA), and the sequence of the amino acid fragment is as follows:

[0067] RLDVDYLRALQLEVAAEIARLEAEVGRLAGHPFNLNSRDPLERVLFDELGIPAIGPTEKEGKRSTTAAVLAA LREAHGIVEKILDYREITKLKSTKIDPLP (see SEQ ID NO. 11).

[0068] 1.1. Construction of protein expression plasmid

[0069] The synthetic Taq enzyme 450-550AA gene fragment was used as a template and a pair of specific primers for PCR amplification. The PCR reaction system was as follows: 1μL template, 1μL forward primer (10mM), 1μL reverse primer (10mM), 25μL 2×Gloria Hi-FiPCR Master Mix with GC Buffer (from Wuhan Abotek Biotechnology Co., Ltd., catalog number RK20717) and 22μL N.F H2O; the PCR amplification program included: 98℃ pre-denaturation for 30s, followed by 30 cycles of 98℃ for 10s, 64℃ for 30s, and 72℃ for 30s, and finally kept at 72℃ for 5min. The resulting reaction solution was stored at 4℃. The nucleotide sequences of the forward primer and reverse primer are as follows:

[0070] Taq-F: CTGGTTCCGCGTGGATCCCCGCGCCTGGACGTGGCCTATCTCA (see SEQ ID NO. 12);

[0071] Taq-R: CGATTTAGTGGTGGTGATGGTGATGATGATGCGGCAAGGGGTCAATGTA (see SEQ ID NO. 13).

[0072] After the amplified PCR product was purified, it was loaded onto the commercial expression vector pET-28a-SUMO by homologous recombination. After sequencing verification, the plasmid pET-28a-Taq in which the target gene was successfully inserted was preserved. The sequencing work was completed by Jinkairui Biotechnology Co., Ltd.

[0073] 1.2. Taq protein expression

[0074] The pET-28a-Taq plasmid was transformed into the E. coli Rosetta strain and incubated overnight at 37°C on an LB plate containing 100 μg / mL ampicillin to obtain a single colony transformant. The single colony transformant was inoculated into 2 mL LB culture medium (containing 100 μg / mL ampicillin) and cultured at 37°C and 220 rpm for 3-4 hours until the OD600nm was about 0.4-0.6. After that, 2 mL of the culture of each strain was transferred to 400 mL LB expression medium and cultured at 37°C and 220 rpm for another 3-4 hours. When the OD600nm reached about 0.45-0.55, 0.8 mM IPTG was added and induced at 37°C for 3-4 hours. The induced bacterial solution was centrifuged at 4000 rpm for 10 minutes, the supernatant was discarded, and the bacteria were stored in a -20°C freezer.

[0075] First disruption: Take 30mL of lysis solution (50mM Tris+300mM NaCl) to suspend the bacteria in a centrifuge bottle. Transfer the suspended bacterial solution to a 50mL round-bottom centrifuge tube, put it in an ice box, and fix it with ice. Select the amplitude changer, put it in the lysis chamber, power 350W, lysis time 3s, interval time 3s, countdown 5min, then place it in an ice-water mixture to cool for 5min, and repeat the above steps to lysis for 5min. After lysis, centrifuge at 9000rpm for 10min to obtain the supernatant and precipitate.

[0076] Second disruption: Measure 30 mL of lysis solution (2 M urea + PBS buffer) and pour it into the precipitate. Blow the precipitate evenly and transfer it to a 50 mL round-bottom centrifuge tube. Set the power to 350 W, lysis time to 3 s, interval time to 3 s, and disruption for 0 to 5 min (the disruption time is determined by the amount and texture of the precipitate, and the bacterial solution is placed in ice). Centrifuge at 9000 rpm for 10 min to obtain the supernatant.

[0077] 1.3. Taq protein purification

[0078] The Taq enzyme was extracted and purified from the supernatant by affinity chromatography using Ni column affinity chromatography resin (purchased from Huiyan, catalog number HQ060212001L). The SDS-PAGE electrophoresis of the purified protein is shown in Figure 1 As shown, lane "MK" represents marker, lanes "0.4" and "0.2" represent 0.4 mg / mL and 0.2 mg / mL BSA, lane "Upper 1" represents supernatant, lane "FT" represents flow-through during the purification process, lane "50" represents the first supernatant column purification, and the protein concentration is 0.5 mg / mL; lane "250-1" represents the second column purification, and the supernatant protein concentration is 0.5 mg / mL, and lane "250-2" represents the third column purification, and the supernatant protein concentration is 0.5 mg / mL.

[0079] Recombinant Taq enzyme contains Taq enzyme protein fragment (450-550AA) and SUMO tag. Figure 1 It can be seen that the Taq enzyme is mainly expressed in the supernatant, and the protein purity of the recombinant Taq enzyme obtained by affinity chromatography purification is greater than 90%.

[0080] Example 2 Preparation of anti-Taq DNA polymerase rabbit antibody

[0081] New Zealand white rabbits were immunized with recombinant Taq enzyme as antigen. Based on the single B lymphocyte screening and culture technology, B lymphocytes were first sorted and cultured from the spleen cells of rabbits immunized with the antigen. Then, RNA was extracted from the antigen-specific B lymphocytes and reversely transcribed into cDNA. The naturally paired heavy chain variable region (VH) and light chain variable region (VL) genes encoding rabbit antibodies were amplified by PCR. Then, the IMGT online database (www.imgt.org) was queried to obtain the constant region amino acid sequence. Finally, the complete light chain and heavy chain genes were loaded into expression vectors, respectively, and the host cells were co-transformed or transfected and cultured. The rabbit monoclonal antibodies against Taq enzyme were isolated, purified and screened. The amino acid sequence of the antibody is shown in Table 1. For ease of description, the full length of the light chain is represented by FL, the light chain variable region is represented by VL, the light chain complementary determining regions CDR1, CDR2 and CDR3 are represented by LCDR1, LCDR2 and LCDR3, respectively, the full length of the heavy chain is represented by FH, the heavy chain variable region is represented by VH, and the heavy chain complementary determining regions CDR1, CDR2 and CDR3 are represented by HCDR1, HCDR2 and HCDR3, respectively.

[0082] Table 1 Sequence information of rabbit monoclonal antibodies in Example 2

[0083]

[0084] 2.1 Animal Immunization

[0085] Recombinant Taq enzyme (purified in Example 1) was used as an antigen and 2 New Zealand white rabbits were immunized at a dose of 200 μg / rabbit. Before the first immunization, the immunogen was mixed with an equal amount of complete Freund's adjuvant (purchased from Sigma) to prepare an emulsion, which was injected subcutaneously at multiple points in the abdomen and back of the rabbits. After the first immunization, 100 μg of the immunogen was mixed with an equal amount of incomplete Freund's adjuvant (purchased from Sigma) to prepare an emulsion every 3 weeks, which was injected subcutaneously at multiple points in the abdomen and back of the rabbits for two booster immunizations.

[0086] 2.2. Detection of rabbit polyclonal antibody titer against Taq enzyme

[0087] After three immunizations, rabbit serum samples were collected and diluted 1:243000. The titer of the serum against Taq DNA polymerase protein was determined by enzyme-linked immunosorbent assay (ELISA).450nm Rabbits with titers exceeding 0.2 were boosted with 200 μg of antigen once, and the animals were sacrificed three days later to remove the spleen. The ELISA method for detecting the immune serum titer includes the following steps:

[0088] 1) Coating with NeutrAvidin protein: dilute NeutrAvidin protein with 1× PBS buffer (final concentration is 2 μg / mL), add the diluted NeutrAvidin protein to a 384-well plate at 25 μL / well, level the 384-well plate, centrifuge briefly (stop after rpm reaches 1000), and incubate the plate at 4°C overnight.

[0089] 2) Coating with biotinylated antigen: dilute the recombinant Taq enzyme with 1× PBS buffer (final concentration is 1 μg / mL), add the diluted recombinant Taq enzyme to a 384-well plate at 25 μL / well, and incubate at room temperature for 1 hour.

[0090] 3) Blocking: Take out the well plate and wash 5 times by adding 75 μL of washing buffer (PBS containing 0.05% (v / v) Tween-20) to each well using a plate washer; use a spray gun to add 50 μL / well of blocking buffer (PBS containing 1% BSA, 0.5% gelatin and 5% sucrose) into the plate and incubate at room temperature for 1 hour to block irrelevant binding sites.

[0091] 4) Serial dilution and sample loading of the test serum: Repeat the washing process in 3) to clean the well plate, and serially dilute the test serum in a 96-well plate. Use dilution buffer (PBS containing 1% BSA) to dilute the serum three times starting from 1:1000, for a total of 8 serial dilutions. The dilution ratio can be adjusted according to the actual situation.

[0092] 5) Secondary antibody incubation: Repeat the washing process in 3) to clean the well plate, dilute the horseradish peroxidase (HRP)-coupled goat anti-rabbit IgG with dilution buffer at a dilution ratio of 1:5000, use a dispenser to add the diluted goat anti-rabbit IgG-HRP into the well plate at 25 μL / well for binding to the target protein, and incubate at room temperature in the dark for 1 hour.

[0093] The results of immune serum titer test are shown in Figure 2 The results showed that rabbit A had a high serum titer, so this rabbit was selected for subsequent preparation of rabbit monoclonal antibodies using Taq enzyme.

[0094] 2.3. Isolation and sorting of B lymphocytes from spleen

[0095] B lymphocytes in the spleen are separated by conventional methods, and then antigen-specific B lymphocytes are sorted. For related methods, please refer to the patent "Method for efficiently separating single antigen-specific B lymphocytes from spleen cells (publication number: CN110016462A, publication date: 2019-07-16)" and the patent "A B lymphocyte in vitro culture system and application (publication number: CN111518765A, publication date: 2020-08-11)".

[0096] 2.4 Cloning of the gene encoding rabbit monoclonal antibody

[0097] After culture, the supernatant of B cells was used to identify positive clones by antigen-coated ELISA. The cells of positive clones were collected and lysed and then analyzed by Quick-RNA TM RNA was extracted using a MicroPrep kit (purchased from ZYMO, catalog number R1100-250) and reverse transcribed into cDNA. Using cDNA as a template, the naturally paired rabbit monoclonal antibody VL and VH genes were amplified from the cDNA of the corresponding positive clone using the PCR method. The PCR reaction system included: 4 μL cDNA, 1 μL forward primer (10 mM), 1 μL reverse primer (10 mM), 12.5 μL 2×Gloria Hi-Fi (from Wuhan Abotek Biotechnology Co., Ltd., catalog number RK20717) and 6.5 μL H2O. The PCR amplification program included 98°C pre-denaturation for 30 s, followed by 40 cycles of 98°C for 10 s, 64°C for 30 s, and 72°C for 30 s, and finally kept at 72°C for 5 min. The resulting reaction solution was stored at 4°C. The primer pair sequences (5'-3') for amplifying the VL and VH genes are as follows, where F and R represent the forward and reverse primers, respectively:

[0098] VL-F: tgaattcgagctcggtacccATGGACACGAGGGCCCCCAC (see SEQ ID NO. 14);

[0099] VL-R: cacacacgatggtgactgTTCCAGTTGCCACCTGATCAG (see SEQ ID NO. 15);

[0100] VH-F: tgaattcgagctcggtacccATGGAGACTGGGCTGCGCTG (see SEQ ID NO. 16);

[0101] VH-R: gtagcctttgaccaggcagcCCAGGGTCACCGTGGAGCTG (see SEQ ID NO. 17).

[0102] 2.5. Expression and large-scale production of rabbit monoclonal antibodies

[0103] The heavy chain genes and light chain genes of several selected rabbit monoclonal antibodies were loaded on expression vectors respectively. The mammalian expression vector pBR322 used carried the rabbit monoclonal antibody light chain constant region (CL) and heavy chain constant region (CH) genes. The expression profile is shown in Figure 3 Among them, pBR322origin and f1 origin are replication promoters, Ampcillin is a resistance gene, CMV promoter is a transcription promoter, SV40 PAterminator is a tailing signal, Light chain constant is the nucleic acid sequence of the light chain constant region (left picture), and Heavy chain constant is the nucleic acid sequence of the heavy chain constant region (right picture).

[0104] The mammalian expression vector pBR322 carrying the CL and CH genes was linearized with XbaI and NheI restriction endonucleases respectively. After the VL and VH genes containing the signal peptides amplified by the above PCR were purified, they were constructed into the above expression vector by homologous recombination to obtain an expression vector containing complete light chain and heavy chain genes. The expression vector was successfully constructed by sequencing. The expression vector containing the light chain gene and the heavy chain gene were transfected into 293F cells together, and cultured for 72-96 hours after transfection to obtain the culture supernatant containing the recombinant antibody that recognizes the Taq enzyme. The recombinant rabbit monoclonal antibody that recognizes the Taq enzyme was purified from the culture supernatant after transfection using proteinA affinity gel resin (purchased from Tiandi Renhe, item number SA023100). The antibody purity was verified to be ≥95% using 12% SDS-PAGE gel electrophoresis, and the concentration was 0.4 mg / mL. The purified antibody was divided and stored at -20°C for later use.

[0105] The signal peptide of this embodiment adopts the antibody expression signal peptide sequence commonly used in the art, such as the light chain variable region upstream signal peptide "MDTRAPTQLLGLLLLWLPGARC" of the patent "Anti-human interferon α2 rabbit monoclonal antibody and its application (publication number: CN116063487A, publication date: 2023-05-05)" and the patent "High-affinity Human IL-5 rabbit monoclonal antibody and its application (publication number: CN115819578A, publication date: 2023-03-21)", and the heavy chain variable region upstream has a signal peptide "METGLRWLLLVAVLKGVQC".

[0106] Example 3 5'-3' polymerase activity blocking test of anti-Taq DNA polymerase rabbit antibody

[0107] 3.1 Substrate synthesis

[0108] Two partially complementary single-stranded DNA substrates pol-sub1 (S1+S2, sequence see SEQ ID NO.18-19) are used, annealed under specific conditions, and the single-stranded DNA substrates are complementary and combined, and double-stranded DNA products are synthesized under the extension of Taq DNA polymerase (sequence see SEQ ID NO.20). After the polymerase reaction is completed, the free single-stranded DNA in the system is first digested with exonuclease I (Exonuclease I (E.coli)), and then heat denaturation is performed to inactivate exonuclease I, and then specific primers (S1f+S2f, sequence see SEQ ID NO.21-22) are used to quantify the amount of double-stranded DNA products using qPCR. The aforementioned sequence (5'-3') is as follows:

[0109] S1: CTGAGGAGAAGTCTGCCGTTACTGCCCTGTGGGGC TAGGTGAA (see SEQ ID NO.18);

[0110] S2:GTCATGCAACTGACTCTGTAG TTCACCTA (see SEQ ID NO.19);

[0111] dsDNA Substrate:

[0112] CTGAGGAGAAGTCTGCCGTTACTGCCCTGTGGGGC TAGGTGAA CTACAGAGTCAGTTGCATGAC (see SEQ ID NO. 20), the underlined regions are S1 and S2 complementary regions;

[0113] S1f: CTGAGGAGAAGTCTGCCGT (see SEQ ID NO. 21);

[0114] S2f: GTCATGCAACTGACTCTGT (see SEQ ID NO. 22).

[0115] The gene sequences were synthesized by Jiangsu Saisuofei Biotechnology Co., Ltd., and the four sequences (S1, S2, S1f and S2f) were diluted with 1×TE buffer to a concentration of 10 μM.

[0116] Take a clean 1.5mL Eppendorf tube and prepare the following system: 100μL of S1 (10μM), 200μL of S2 (10μM) and 10×Annealing Buffer (from ABclonal, catalog number RM20821) 100μL, and add H2O to 1mL. Place the above reaction system in a water bath that has been heated to 95℃, heat at 95℃ for 5min, then cool to room temperature and place on ice. Use this mixture as a substrate, named pol-sub1, and divide it into aliquots. If not used immediately after dividing, freeze it in a -20℃ refrigerator in time.

[0117] 3.2. Taq enzyme binds to rabbit antibody to form a complex

[0118] Taq DNA polymerase was diluted with PBS buffer (from ABclonal, catalog number RM00012) to a concentration of 1 ng / μL. The following system was configured: 5×Thermo Pol buffer 2μL (purchased from NEB, catalog number B9004S), dNTPs (10mM) 0.1μL, Taq enzyme 1μL, rabbit immune serum 1μL (or antibody 1μL) and H2O 3.9μL, total volume 8μL. The mixed system was incubated at room temperature for 10 minutes to allow the antibody and enzyme to fully bind.

[0119] The control group includes a control group without enzyme and without antibody (including polyclonal antibody and monoclonal antibody), the polyclonal antibody is immune serum, and the monoclonal antibody is the rabbit antibody separated in Example 2. The 5'-3' polymerase activity of the Taq enzyme in the enzyme-free control group is 100% blocked, and the configuration system is as follows: 5×Thermo Pol buffer 2μL, dNTPs (10mM) 0.1μL, PBS buffer 1μL, rabbit immune serum 1μL (or antibody 1μL) and H2O 3.9μL, with a total volume of 8μL. In the non-antibody control group, the 5'-3' polymerase activity of the Taq enzyme is not blocked, and the configuration system is as follows: 5×Thermo Pol buffer 2μL, dNTPs (10mM) 0.1μL, Taq enzyme (1ng / μL) 1μL and H2O 4.9μL, with a total volume of 8μL. In addition, a negative control group IgG group was set up, and the anti-Taq enzyme antibody was replaced with an equal amount of rabbit IgG, and the other systems were the same as the non-antibody control group.

[0120] 3.3. Taq enzyme-antibody complex polymerizes substrate to form double-stranded DNA

[0121] Add 2 μL of pol-sub1 substrate (concentration of 1 μM) in 3.1 to the reaction system of Taq enzyme-antibody complex in 3.2, and immediately incubate at 37°C for 1 hour to start the polymerase reaction and form double-stranded DNA. After the reaction is completed, use exonuclease I to digest and remove excess single-stranded DNA; take a new PCR plate, mix each well according to the following system: 1 μL of 10×Exonuclease IReaction Buffer (from ABclonal, catalog number RM20130), 7.9 μL of ddH2O, 0.1 μL of Exonuclease I (from ABclonal, catalog number RM20519, 20U / μL), add 1 μL of the product after the above polymerization reaction after mixing, and set the following program in the PCR instrument to start the reaction: 37°C 30min, 95°C 2min, 4°C 5min.

[0122] 3.4. qPCR quantification of polymerization products

[0123] The qPCR reaction system includes: 2×Universal SYBR Green Fast qPCR Mix (from ABclonal, catalog number RK21203) 5μL, ddH2O 3.6μL, template (product of step 3.3) 1μL, upstream primer N1f (10μM) 0.2μL, downstream primer SN2f (10μM) 0.2μL, total volume 10μL; reaction program: 95℃3min, Stage 1; 95℃5S, 60℃30-34S, Stage 2, 40cycles; 95℃15S, 65℃1min, 95℃15S, Melt Curve. The Ct value of each well reaction was counted. When the Ct value was higher, it meant that the double-stranded DNA content in the template was lower, and the polymerization activity of the Taq DNA polymerase used to synthesize double-stranded DNA was lower, and the two were negatively correlated.

[0124] 3.5. Rabbit antibody blocking polymerase activity test

[0125] A variety of Taq enzyme mutants were used, including Taq01, Taq02, Taq03, Taq04 and Taq05, wherein the preparation methods of Taq01, Taq02 and Taq04 refer to patent CN116515791A (publication date: 2023-03-23), which are E507K, E520K, and E537K mutations, respectively, Taq03 is a fragment of the 450-550aa region of the sequence shown in UniProtP19821, prepared by Example 1, and Taq05 is a commercially available product from ABclonal (item number RK26000).

[0126] Rabbit monoclonal antibody B and immune serum polyclonal antibody A were used to test the blocking activity of Taq enzyme mutants. The Taq enzyme working amount was 1-3ng, the antibody working amount was 0.5μg, and the negative control group R-IgG antibody amount was 0.5μg. At the same time, a no-enzyme control group and a no-antibody control group (equivalent water replacement) were set up, with 3 parallels in each group. The results are shown in Table 2. ΔCt (Ab-H2O) in Table 2 indicates the blocking activity of Taq enzyme antibody to Taq enzyme after removing the interference of the no-enzyme system. The higher the ΔCt (Ab-H2O), the lower the activity of Taq enzyme, that is, the higher the blocking activity of Taq enzyme antibody.

[0127] Table 2 Blocking activity of rabbit monoclonal and polyclonal antibodies against different Taq enzymes

[0128]

[0129] As can be seen from Table 2, Taq antibody has a significant inhibitory effect on the polymerase activity of Taq01, Taq02 and Taq03.

[0130] In addition, the mass of Taq enzyme was controlled to be 1 ng, and the mass ratio of rabbit monoclonal antibody B to Taq enzyme was adjusted by diluting the antibody dosage. The blocking activity test results of Taq antibody and different Taq enzymes at different mass ratios are shown in Table 3, where ΔCt represents ΔCt(Ab-H2O). Table 3 Blocking activity test results of rabbit monoclonal antibody B / Taq polymerase at different mass ratios

[0131] Closure activity

[0132]

[0133] As can be seen from Table 3, when the mass ratio of rabbit monoclonal antibody B to Taq enzyme is 0.8:1 or above, it has good blocking activity for various mutants such as Taq01, 02, 03, 04, and 05.

[0134] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. An anti-Taq DNA polymerase antibody, characterized in that It comprises a light chain variable region and a heavy chain variable region, wherein the amino acid sequences of the complementary determining regions CDR1, CDR2 and CDR3 on the light chain variable region are shown as SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5, respectively; the amino acid sequences of the complementary determining regions CDR1, CDR2 and CDR3 on the heavy chain variable region are shown as SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10, respectively.

2. The anti-Taq DNA polymerase antibody according to claim 1, characterized in that The amino acid sequence of the light chain variable region is shown in SEQ ID NO.2, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.

7.

3. The anti-Taq DNA polymerase antibody according to claim 2, characterized in that The amino acid sequence of the antibody light chain is shown in SEQ ID NO.1, and the amino acid sequence of the heavy chain is shown in SEQ ID NO.

6.

4. The anti-Taq DNA polymerase antibody according to claim 1, characterized in that The antibody is a full-length antibody or an antigen-binding region of the full-length antibody; the antigen-binding region is selected from at least one of Fab, F(ab)2, Fab', F(ab')2, Fv, (Fv)2, scFv and sc(Fv)2.

5. A nucleic acid molecule, a recombinant vector or a host cell comprising the nucleic acid molecule, characterized in that: The nucleic acid molecule encodes the anti-Taq DNA polymerase antibody according to any one of claims 1 to 4.

6. A hot-start Taq DNA polymerase, characterized in that The method comprises Taq DNA polymerase and the anti-Taq DNA polymerase antibody according to any one of claims 1 to 4.

7. The hot-start Taq DNA polymerase according to claim 6, characterized in that The weight ratio of the anti-Taq DNA polymerase antibody to the Taq DNA polymerase is 0.5-500:

1.

8. The hot-start Taq DNA polymerase according to claim 7, characterized in that The weight ratio of the anti-Taq DNA polymerase antibody to the Taq DNA polymerase is 0.8-500:

1.

9. The hot-start Taq DNA polymerase according to claim 8, characterized in that The weight ratio of the anti-Taq DNA polymerase antibody to the Taq DNA polymerase is 0.8-1.5:

1.

10. Use of the anti-Taq DNA polymerase antibody according to any one of claims 1 to 4 or the hot-start Taq DNA polymerase according to any one of claims 6 to 9 in preparing a hot-start PCR reaction reagent.

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