Anti-Taq enzyme antibody or functional fragment thereof and application thereof

Abstract

The invention relates to the technical field of biology, in particular to an anti-Taq enzyme antibody or a functional fragment thereof and application thereof. The invention not only provides a high-affinity monoclonal antibody (wild type) combined with a Taq enzyme antibody, but also performs site-directed mutagenesis on the wild type, verifies the affinity of each mutation and related application thereof, and provides reference for PCR research in the related field of nucleic acid detection.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and specifically relates to an anti-Taq enzyme antibody or a functional fragment thereof and an application thereof.

[0002] The present invention claims priority to application number 202311056423.0 filed on August 22, 2023, the entire contents of which are incorporated herein by reference. Background Art

[0003] Polymerase chain reaction (PCR) is a method for synthesizing double-stranded DNA in vitro. PCR amplification can amplify a single DNA molecule into millions of copies of DNA molecules through only a few PCR thermal cycles, achieving exponential amplification of DNA. PCR thermal cycles usually include three different steps, namely denaturation, annealing, and extension. In the denaturation step (about 95°C, high temperature zone), the double-stranded DNA is unwound into two single-stranded DNAs. The temperature is then lowered for the annealing step (about 56°C, low temperature zone), which promotes the complementary binding of primers (short DNA sequences) to single-stranded DNA. Finally, under the action of polymerase, free nucleotides in the solution are synthesized into the second complementary strand of DNA. However, the activity of polymerase is low at the annealing temperature, so the temperature needs to be raised to about 72°C. The step at this temperature is called extension. By repeating the three temperature steps, a large amount of DNA can be replicated, and the concentration of the target double-stranded DNA can be exponentially doubled. This DNA replication process is PCR[1].

[0004] Currently, there are many methods to improve the specificity of PCR amplification. The simplest method is to use hot-start enzyme amplification. The optimum temperature of ordinary DNA polymerase is 72°C, at which the enzyme activity is optimal. Below this temperature, the enzyme has weak activity, and both 5'-3' polymerase activity and 3'-5' exonuclease activity can play a role. In this way, during the process of PCR amplification rising from low temperature to high temperature, the enzyme has weak activity, and the system is very easy to mismatch or form primer dimers, especially when the 3' end of the designed primer is G or C. The mismatched chain is difficult to unwind. To improve the specificity of PCR amplification and reduce the reaction mismatch rate, the activity of the enzyme can be artificially controlled to make the enzyme inactive before the hot start temperature. This avoids chain mismatches during the temperature rise process (or configuration of the reaction system), thereby ensuring the specificity of amplification [2].

[0005] In view of the high affinity of monoclonal antibodies for antigen binding, monoclonal antibodies can be used to block Taq polymerase, which can effectively inhibit nonspecific annealing of primers and nonspecific amplification caused by primer dimers under low temperature conditions. Taq enzyme is a basic tool for life science research and is widely used in scientific research institutions such as universities, research institutes and biotechnology companies. The low-cost production of blocking antibodies will greatly reduce the production cost of antibody-based hot-start polymerase. Existing anti-Taq monoclonal antibodies are expensive, used in large quantities, and have relatively poor thermal stability. Therefore, it is urgent to develop anti-Taq monoclonal antibodies with good blocking effect, low cost and good thermal stability.

[0006] [1] Shi Bing. Research on structure and function optimization and application of polymerase chain reaction system[J].[2023-08-09].

[0007] [2] Yisheng Biotechnology Co., Ltd. Taq enzyme 5'-3' polymerase activity blocking monoclonal antibody and its application. 2020, CN 111560073 B Summary of the invention

[0008] The purpose of the present invention is to provide an anti-Taq enzyme antibody combination or a functional fragment thereof, to achieve specific binding with Taq enzyme, to block Taq enzyme activity at low temperature, and finally to apply to Taq enzyme production and PCR experiments.

[0009] The present invention adopts hybridoma technology to screen out specific anti-Taq enzyme antibody combinations (antibody A and antibody B), and mutates their sequences respectively to screen out mutant sequences with high stability, specificity and affinity. The mutated anti-Taq enzyme antibody combinations (antibody A and antibody B) are expressed in a recombinant ExpiCHO manner, which not only avoids the batch difference caused by mouse ascites production, but also has simple production and low cost.

[0010] Therefore, the first object of the present invention is to provide an anti-Taq enzyme antibody combination (antibody A and antibody B, wild type). The second object of the present invention is to provide a mutant of an anti-Taq enzyme antibody combination (antibody A and antibody B) that blocks Taq enzyme and its use. The third object of the present invention is to provide a method for preparing a mutant of an anti-Taq enzyme antibody combination (antibody A and antibody B).

[0011] To achieve the above object, the present invention adopts the following technical solutions:

[0012] An anti-Taq enzyme antibody or a functional fragment thereof, wherein the antibody or the functional fragment thereof comprises the following complementary determining region:

[0013] CDR-VH1: D-X1-YI-X2, where X1 is Y or D, and X2 is N;

[0014] CDR-VH2: W-X1-YPG-X2-GHTQ-X3-NERFKG, wherein X1 is I, X2 is S or T, and X3 is D or Y;

[0015] CDR-VH3: S-X1-FYAM-X2-Y, where X1 is R or A, and X2 is D or N; CDR-VL1: R-X1-SQSIG-X2-SIH, where X1 is A or N, and X2 is T or S;

[0016] CDR-VL2: Y-X1-SES-X2-S, where X1 is A or R, and X2 is I or P;

[0017] and CDR-VL3: Q-X1-TNS-X2-PLT, where X1 is Q and X2 is W or P;

[0018] It was named antibody A;

[0019] or

[0020] The antibody or its functional fragment comprises the following complementarity determining region:

[0021] CDR-VH1: S-X1-W-X2-N, where X1 is F or E, and X2 is I;

[0022] CDR-VH2: D-X1-YPG-X2-GSTHYN-X3-KFKN, wherein X1 is I or N, X2 is S or T, and X3 is E or T;

[0023] CDR-VH3: S-X1-DGYLF-X2-Y, where X1 is G or P, and X2 is A or R;

[0024] CDR-VL1: R-X1-SQSVSTS-X2-YSFIH, where X1 is A and X2 is T or S; CDR-VL2: Y-X1-SN-X2-DS, where X1 is A or R and X2 is L;

[0025] and CDR-VL3: Q-X1-SWEIP-X2-T, where X1 is H and X2 is W or P;

[0026] Named Antibody B.

[0027] Further, in the complementary determining region of antibody A, X2 of CDR-VH1 is N, X1 of CDR-VH2 is I, X2 of CDR-VH3 is D, X1 of CDR-VL1 is A, X2 of CDR-VL2 is I, and X1 of CDR-VL3 is Q;

[0028] or

[0029] In the complementary determining region of antibody B, X2 of CDR-VH1 is I, X1 of CDR-VH2 is I, X2 of CDR-VH3 is A, X1 of CDR-VL1 is A, X2 of CDR-VL2 is L, and X1 of CDR-VL3 is H.

[0030] The CDR segment division described in the present invention adopts the Kabat algorithm.

[0031] "CDR" is used herein to refer to a "complementarity determining region" within an antibody variable sequence. The variable regions of the heavy and light chains each have three CDRs, starting from the N-terminus of the heavy or light chain.

[0032] The antigen binding site may include six CDRs (CDR-VH1, CDR-VH2, CDR-VH3, CDR-VL1, CDR-VL2 and CDR-VL3 in the present invention). A polypeptide comprising a single CDR (e.g., CDR-VH1, CDR-VH2, CDR-VH3, CDR-VL1, CDR-VL2 or CDR-VL3) may be referred to as a "molecular recognition unit". Crystallographic analysis of antigen-antibody complexes has shown that the amino acid residues of CDRs form extensive contacts with the bound antigen, with the most extensive antigen contact being with the heavy chain CDR3. Therefore, the molecular recognition unit may be primarily responsible for the specificity of the antigen binding site. In general, the CDR residues are directly and most substantially involved in influencing antigen binding.

[0033] Further, in the CDR-VH1 of the antibody A, X1 is Y;

[0034] Or in the CDR-VH1 of the antibody A, X1 is D;

[0035] Or in the CDR-VH1 of the antibody A, X2 is N;

[0036] Or in the CDR-VH2 of the antibody A, X1 is I;

[0037] Or in the CDR-VH2 of the antibody A, X2 is S;

[0038] Or in the CDR-VH2 of the antibody A, X2 is T;

[0039] Or in the CDR-VH2 of the antibody A, X3 is D;

[0040] Or in the CDR-VH2 of the antibody A, X3 is Y;

[0041] Or in the CDR-VH3 of the antibody A, X1 is R;

[0042] Or in the CDR-VH3 of the antibody A, X1 is A;

[0043] or X2 in the CDR-VH3 of the antibody A is D; or X2 in the CDR-VH3 of the antibody A is N; or X1 in the CDR-VL1 of the antibody A is A; or X1 in the CDR-VL1 of the antibody A is N; or X2 in the CDR-VL1 of the antibody A is T; or X2 in the CDR-VL1 of the antibody A is S; or X1 in the CDR-VL2 of the antibody A is A; or X1 in the CDR-VL2 of the antibody A is R; or X2 in the CDR-VL2 of the antibody A is X2 is I; or X2 in the CDR-VL2 of the antibody A is P; or X1 in the CDR-VL3 of the antibody A is Q; or X2 in the CDR-VL3 of the antibody A is W; or X2 in the CDR-VL3 of the antibody A is P; or X1 in the CDR-VH1 of the antibody B is F; or X1 in the CDR-VH1 of the antibody B is E; or X2 in the CDR-VH1 of the antibody B is I; or X1 in the CDR-VH2 of the antibody B is I; or X2 in the CDR-VH2 of the antibody B is -VH2 of antibody B, X1 is N; or X2 is S in CDR-VH2 of antibody B; or X2 is T in CDR-VH2 of antibody B; or X3 is E in CDR-VH2 of antibody B; or X3 is T in CDR-VH2 of antibody B; or X1 is G in CDR-VH3 of antibody B; or X1 is P in CDR-VH3 of antibody B; or X2 is A in CDR-VH3 of antibody B; or X2 is R in CDR-VH3 of antibody B; or in the CDR-VL1 of antibody B, X1 is A; or in the CDR-VL1 of antibody B, X2 is T; or in the CDR-VL1 of antibody B, X2 is S; or in the CDR-VL2 of antibody B, X1 is A; or in the CDR-VL2 of antibody B, X1 is R; or in the CDR-VL2 of antibody B, X2 is L; or in the CDR-VL3 of antibody B, X1 is H; or in the CDR-VL3 of antibody B, X2 is W; or in the CDR-VL3 of antibody B, X2 is P.

[0044] Furthermore, each complementary determining region of the antibody A is selected from any one of the following mutation combinations:

[0045]

[0046]

[0047] Or each complementary determining region of the antibody B is selected from any one of the following mutation combinations:

[0048]

[0049]

[0050] Furthermore, the anti-Taq enzyme antibody or its functional fragment also includes light chain framework regions FR1-L, FR2-L, FR3-L and FR4-L and heavy chain framework regions FR1-H, FR2-H, FR3-H and FR4-H; the heavy chain framework regions FR1-H, FR2-H, FR3-H and FR4-H are selected from SEQ ID NO: 1-4 or SEQ ID NO: 5-8 in sequence; the light chain framework regions FR1-L, FR2-L, FR3-L and FR4-L are selected from SEQ ID NO: 9-12 or: SEQ ID NO: 13-16 in sequence.

[0051] Furthermore, the anti-Taq enzyme antibody or its functional fragment further comprises a constant region;

[0052] Preferably, the constant region is selected from the constant region of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD. Preferably, the species of the constant region is cattle, horses, dairy cows, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, geese, turkeys, fighting cocks or humans.

[0053] Preferably, the functional fragment is selected from any one of F(ab')2, Fab', Fab, Fv and scFv of the antibody.

[0054] Further, the antibody is a labeled antibody;

[0055] Preferably, the labeling substances for labeling the antibody include, but are not limited to, latex microspheres, biotin, fluorescent dyes, enzymes that catalyze the color development of substrates, radioactive isotopes, chemiluminescent reagents, and nanoparticle labels.

[0056] On the other hand, the present invention discloses the use of the antibody or its functional fragment in the preparation of a PCR detection kit or in the preparation of a blocking agent.

[0057] In another aspect, the present invention discloses a blocking agent, which includes the antibody or a functional fragment thereof;

[0058] Optionally, the concentration of the antibody in the blocking agent is 1-5 mg / mL.

[0059] The "blocking agent" of the present invention refers to a reagent that can block the activity of taq enzyme.

[0060] On the other hand, the present invention discloses a detection reagent or a kit, wherein the reagent or the kit comprises the antibody or a functional fragment thereof or the labeled antibody.

[0061] On the other hand, the present invention discloses a method for blocking Taq enzyme activity, which comprises adding the antibody or its functional fragment or the antibody conjugate into an immunoassay system.

[0062] In another aspect, the present invention discloses a method for detecting Taq enzyme, comprising:

[0063] A) contacting the antibody or functional fragment thereof, the antibody conjugate, the blocking agent, or the reagent or kit with the sample from the subject under conditions sufficient for a binding reaction to perform a binding reaction; and

[0064] B) Detection of immune complexes produced by the binding reaction.

[0065] Beneficial effects:

[0066] The present invention is designed to combine cell biology and bioinformatics methods to prepare anti-Taq enzyme monoclonal antibodies expressed by hybridoma cell lines, and obtain antibodies with the highest affinity and strongest specificity through sequence mutation. Expression by CHO cells is not only low-cost, but also has good thermal stability, and can effectively avoid effectively inhibiting nonspecific annealing of primers and nonspecific amplification caused by primer dimers under low temperature conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 : PCR profile of anti-Taq antibody combination antibody blocking effect;

[0068] Figure 2 : PCR profile of Chlamydia pneumoniae detection;

[0069] Figure 3 : PCR profile of Mycoplasma pneumoniae detection. DETAILED DESCRIPTION

[0070] Specifically, in order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0071] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the present disclosure belongs. Any methods and materials similar or equivalent to those described herein can be used for the practice or testing of the preparations or unit doses herein. Unless otherwise indicated, the techniques adopted or considered herein are standard methods. Materials, methods and examples are illustrative and non-restrictive only.

[0072] As used herein, the terms "including," "comprising," "having," "may," and variations thereof are intended to be open-ended transitional phrases, terms, or words that do not exclude the possibility of additional actions or structures.

[0073] Unless otherwise indicated, the practice of the present invention will employ conventional techniques of cell biology, molecular biology (comprising recombinant technology), microbiology, biochemistry and immunology, which are within the capabilities of those skilled in the art. This technology is fully explained in the literature, such as "Molecular Cloning: A Laboratory Manual", Second Edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (MJ Gait, 1984); "Animal Cell Culture" (RI Freshney, 1987); "Methods in Enzymology" (Academic Press, Inc.); "Handbook of Experimental Immunology" (DM Weir and CC Black, 1989). ell, ed.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction (Mullis et al., eds., 1994); and Current Protocols in Immunology (JE Coligan et al., eds., 1991), each of which is expressly incorporated herein by reference.

[0074] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0075] Example 1 Preparation of monoclonal antibodies

[0076] 1. Mouse immunization and antibody detection

[0077] Five 6-8 week old SPF female BALB / c mice were selected, and Taq enzyme protein (purchased from Toyobo (Shanghai) Biotechnology Co., Ltd.) was selected as the antigen protein, and mixed with Freund's complete adjuvant in equal volumes and emulsified. The emulsified antigen was used to immunize 6-8 week old SPF female BALB / c mice, and 50 μg of antigen protein was injected into each mouse by foot injection or back subcutaneous injection. Two weeks after the initial immunization, the Taq enzyme was mixed with Freund's incomplete adjuvant and emulsified, and 50 μg of antigen protein was injected into each mouse by foot injection or back subcutaneous injection again. Two weeks later, blood was collected through the tail vein, the supernatant was collected by centrifugation, and the serum titer was tested by ELISA. Immunization was performed every two weeks and the serum titer was tested. After two immunizations, the serum titer was as high as 2.0 or above after a million-fold dilution. The screening serum titer was 10 6 Lymph was taken from the above mice to separate lymphocytes for cell fusion.

[0078] 2. Cell fusion and positive hybridoma cell screening and subcloning

[0079] Screening The obtained hybridoma cell group was screened in three steps: 1) confirming the ability to bind to Taq polymerase; 2) screening for specific antibody pairs.

[0080] 1) In order to screen for high-affinity anti-Taq polymerase antibodies, positive wells were screened by binding to Taq polymerase. Wells with a higher ratio of ELISA positive value to cell number were selected for multiple subcloning to select the monoclonal hybridoma cells with the highest affinity.

[0081] 2) Evaluation of antibody pair screening: The specificity of the 10 antibodies produced by the clones selected in 1) was evaluated in pairs, and the hybridoma cells with the highest antibody reactivity were selected and named anti-Taq-A and anti-Taq-B.

[0082] 3. Production and purification of monoclonal antibodies

[0083] Two groups of 6-8 week old BALB / c mice were selected and injected intraperitoneally with 500 μL paraffin oil to suppress the immune response of mice. One week after the injection, 0.5 ml of anti-Taq-A hybridoma cells were injected intraperitoneally into one group of mice, with a cell number of about 1×106; 0.5 ml of anti-Taq-B hybridoma cells were injected intraperitoneally into the other group of mice. Ascites collection began two weeks later. The collected ascites was precipitated with ammonium sulfate and affinity purified with protein A to obtain two target antibodies, named antibody A (primary antibody) and antibody B (secondary antibody).

[0084] 4. Determination of the potency of antibody A and antibody B against the immobilized Taq enzyme

[0085] Taq enzyme protein was diluted with coating solution (0.05M carbonate buffer with pH=9.5) at 1ug / mL, and 100μL / well was added to the ELISA plate and coated overnight at 4°C. During the detection, each antibody to be tested was diluted to 100ng / mL and incubated with the antigen at 37°C, and HRP-labeled goat anti-mouse polyclonal antibody was used for color development, and the peak absorbance was detected. The results are shown in Table 1 below.

[0086] Table 1 Potency determination

[0087]

[0088] At a lower antibody concentration, that is, when the antibody was 0.1 ng / mL, there was still a signal, indicating that the sensitivity of both antibodies was high and suitable for subsequent tests.

[0089] E. Identification of monoclonal antibody subtypes and gene sequence cloning

[0090] The SBA Clonotyping System-HRP kit from Southern Biothech was used to identify the subtypes of the heavy and light chains of the monoclonal antibody according to the instructions. The specific procedures were as follows:

[0091] 1. Dilute the capture antibody to 1 μg / mL with coating solution (0.05M carbonate and bicarbonate buffer at pH 9.5), add 100 μL / well to the ELISA plate, and coat overnight at 4°C. Wash the plate three times with PBS buffer (washing solution) containing 0.05% Tween-20.

[0092] 2. Dilute the culture supernatant of the hybridoma cells to be tested with diluent (1% BSA, 0.1% PBST) at a ratio of 1:1, add 100 μL / well to the ELISA plate, and incubate at 37°C for 30 minutes. Dilute the corresponding enzyme-labeled antibodies (Ig-HRP, IgG1-HRP, IgG2a-HRP, IgG2b-HRP, IgG3-HRP, IgM-HRP, kappa-HRP, lamda-HRP) at a ratio of 1:3000 with diluent.

[0093] 3. After washing the plate three times with the plate washing solution, add 100 μL of diluted enzyme-labeled antibody to each well and incubate at 37°C for 30 minutes. After washing the plate three more times, add the color developing solution. After about 5 minutes (depending on the strength of the reaction), add 2M sulfuric acid to terminate the reaction and read the OD450 absorbance value. After identification, the heavy chain subtype of the antibody anti-Taq-A is IgG1, and the light chain is Kappa; the heavy chain subtype of the antibody anti-Taq-B is IgG2b, and the light chain is Kappa;

[0094] According to the antibody subtype results, the antibody gene sequence was cloned using a RACE-based method. Hybridoma cells with good growth were collected, and total RNA of hybridoma cells was obtained using a total RNA extraction kit. The mRNA was reverse transcribed into cDNA according to the operating method of Takara's SMARTer RACE manual, and the full-length sequence of the target antibody was amplified.

[0095] Example 2 Verification of antibody performance

[0096] 1. ELISA affinity test

[0097] Dilute the goat anti-mouse to 1ug / mL using coating solution (0.05M carbonate and bicarbonate buffer at pH = 9.5), add 100μL per well to a 96-well ELISA plate, and coat overnight at 4°C. Wash the plate three times with PBS buffer containing 0.05% Tween-20. Dilute the antibody to 1ug / mL using diluent (1% BSA, 0.1% PBST), add 100μL per well to a 96-well ELISA plate, and incubate at 37°C for 30 minutes. Dilute DNA polymerase to 100 ng / mL with diluent, take out the ELISA plate incubated at 37°C, wash the plate three times, add 150 μL of the prepared antibody canine C-reactive protein dilution to the 96-well ELISA plate in row A, add 100 μL of the dilution to each well of the 96-well ELISA plate in rows B to H, take 50 uL of the antibody dilution in the wells of row A, and dilute it 3 times from row B, and incubate at 37°C for 30 minutes. Dilute the avidin labeled with horseradish peroxidase at 1:5000 with diluent, take out the ELISA plate and wash the plate three times, add 100 μL to each well of the 96-well ELISA plate, and incubate at 37°C for 30 minutes. Take out the ELISA plate and wash the plate three times, add the color developing solution, color at room temperature for 3 minutes, add 0.5 M sulfuric acid to terminate the reaction, and read the OD450 value on the plate reader.

[0098] 2. The antibody anti-Taq-A in Example 1 has a heavy chain variable region as shown in SEQ ID NO: 17, wherein the amino acid sequences of the complementary determining regions on the heavy chain variable region are as follows:

[0099] CDR-VH1: DY(X1)-YIN(X2);

[0100] CDR-VH2:WI(X1)-YPGT(X2)-GHTQD(X3)-NERFKG;

[0101] CDR-VH3: SR(X1)-FYAMN(X2)-Y;

[0102] The light chain variable region is shown in SEQ ID NO: 18, wherein the amino acid sequences of the complementary determining regions on the light chain variable region are as follows:

[0103] CDR-VL1: RN(X1)-SQSIGT(X2)-SIH;

[0104] CDR-VL2: YA(X1)-SESP(X2)-S;

[0105] CDR-VL3: QQ(X1)-TNSP(X2)-PLT.

[0106] Based on the antibody anti-Taq-A, mutations were performed on sites related to antibody activity in the complementary determining region. The results are shown in Tables 2 and 3, where X1, X2, and X3 are all mutation sites.

[0107] Table 2 Mutation sites related to antibody activity

[0108]

[0109]

[0110] Table 3 Antibody activity analysis data

[0111]

[0112] As can be seen from the table above, mutation 3 has the best activity effect. Therefore, mutation 3 is used as the backbone sequence to screen other mutation sites with better affinity. Some results are shown in Table 4:

[0113] Table 4 Mutation sites related to antibody affinity

[0114]

[0115]

[0116] Taq enzyme protein 100ng / ml, measure the absorbance value of each mutant OD450, the results are shown in Table 5:

[0117] Table 5 Affinity testing of antibody mutations

[0118] Mutation 3-1 2.5007 Mutation 3-11 2.1232 Mutation 3-21 2.6023 Mutation 3-2 2.3095 Mutation 3-12 2.4408 Mutation 3-22 2.1591 Mutation 3-3 2.2268 Mutation 3-13 2.0563 Mutation 3-23 2.4127 Mutation 3-4 2.1243 Mutation 3-14 2.1253 Mutation 3-24 2.5061 Mutation 3-5 2.5709 Mutation 3-15 2.2476 Mutation 3-25 2.1232 Mutation 3-6 2.1988 Mutation 3-16 2.2083 Mutation 3-26 2.0377 Mutation 3-7 2.3155 Mutation 3-17 2.3500 Mutation 3-27 2.4009 Mutation 3-8 2.3666 Mutation 3-18 2.0038 Mutation 3-28 2.3146 Mutation 3-9 2.2470 Mutation 3-19 2.2504 Mutation 3-29 2.0133 Mutation 3-10 2.3696 Mutation 3-20 2.1352 Mutation 3-30 2.2379

[0119] It can be seen that the affinity of each mutation is higher.

[0120] 3. Anti-Taq-A mutation stability assay

[0121] The concentration of the antibody of the present invention is 1 ug / mL, and the concentration of the Taq enzyme protein is 100 ng / mL.

[0122] The antibodies were diluted in a defined buffer (PBS, 0.05% ProClin TM 300) was heat accelerated at 37°C for 7 days, and the accelerated antibodies were evaluated by SDS-PAGE and ELISA indirect method, and the long-term stability of the antibodies was identified by comparing with 4°C. In addition, the test results were displayed as the deviation between the 4°C value and the accelerated value by repeated freezing and thawing at -20°C for 5 times, and the measurement results are shown in Table 6.

[0123] Table 6 Stability study

[0124]

[0125]

[0126] From the above, it can be seen that mutations 3 to 3-30 can be stably stored at 4°C and remain stable when accelerated at 37°C for 0.5 months.

[0127] 4. The antibody anti-Taq-B in Example 1 has a heavy chain variable region as shown in SEQ ID NO: 19, wherein the amino acid sequences of the complementary determining regions on the heavy chain variable region are as follows:

[0128] CDR-VH1: SE(X1)-WI(X2)-N;

[0129] CDR-VH2: DN(X1)-YPGT(X2)-GSTHYNF(X3)-KFKN;

[0130] CDR-VH3: SG(X1)-DGYLFR(X2)-Y;

[0131] The light chain variable region is shown in SEQ ID NO: 20, wherein the amino acid sequences of the complementary determining regions on the light chain variable region are as follows:

[0132] CDR-VL1:RA(X1)-SQSVSTSS(X2)-YSFIH;

[0133] CDR-VL2: YA(X1)-SNL(X2)-DS;

[0134] and CDR-VL3:QH(X1)-SWEIPP(X2)-T.

[0135] Based on the antibody anti-Taq-B, mutations were performed on sites related to antibody activity in the complementary determining region. The results are shown in Tables 7 and 8, where X1, X2, and X3 are all mutation sites.

[0136] Table 7 Mutation sites related to antibody activity

[0137]

[0138] Table 8 Antibody activity analysis data

[0139]

[0140] As can be seen from the table above, mutation 5 has the best activity effect. Therefore, mutation 5 is used as the backbone sequence to screen other mutation sites with better affinity. Some results are shown in Table 9:

[0141] Table 9 Mutation sites related to antibody affinity

[0142]

[0143]

[0144] Taq enzyme protein 100ng / ml, measure the absorbance value of each mutant OD450, the results are shown in Table 10:

[0145] Table 10 Affinity testing of antibody mutations

[0146] Mutation 5 2.0371 Mutation 5-11 2.2527 Mutation 5-21 2.2212 Mutation 5-1 2.0234 Mutation 5-12 2.3317 Mutation 5-22 2.4107 Mutation 5-2 2.5070 Mutation 5-13 2.4427 Mutation 5-23 2.4417 Mutation 5-3 2.4207 Mutation 5-14 2.2127 Mutation 5-24 2.0567 Mutation 5-4 2.1327 Mutation 5-15 2.5200 Mutation 5-25 2.1327 Mutation 5-5 2.4227 Mutation 5-16 2.6378 Mutation 5-26 2.5252 Mutation 5-6 2.5157 Mutation 5-17 2.007 Mutation 5-27 2.0050 Mutation 5-8 2.5427 Mutation 5-18 2.0257 Mutation 5-28 2.4457 Mutation 5-9 2.3527 Mutation 5-19 2.4207 Mutation 5-29 2.3357 Mutation 5-10 2.5125 Mutation 5-20 2.1237 Mutation 5-30 2.5017

[0147] It can be seen that each mutation has a high affinity.

[0148] 5. Anti-Taq-B mutation stability assay

[0149] The concentration of the antibody of the present invention is 1 ug / mL, and the concentration of the Taq enzyme is 100 ng / mL.

[0150] The antibodies were diluted in a defined buffer (PBS, 0.05% ProClin TM 300) was heat accelerated at 37°C for 7 days, and the accelerated antibody was evaluated by ELISA indirect method, and the long-term stability of the antibody was identified by comparing with 4°C. In addition, the test results were displayed as the deviation between the 4°C value and the accelerated value by repeated freezing and thawing at -20°C for 5 times. The measurement results are shown in Table 11:

[0151] Table 11 Stability study

[0152] 4℃ 7 days 37℃ 7 days 37℃ 14 days Freeze and thaw 5 times WT -9％ 2.5％ 6.4％ 6.5％ Mutation 5 7％ 0.9％ -4.2％ -2.3％ Mutation 5-1 -4％ -7.2％ -8.2％ -3.5％ Mutation 5-2 -4％ 1.7％ -3.0％ -6.3％ Mutation 5-3 6％ 0.0％ -4.0％ -10.2％ Mutation 5-4 11％ 3.4％ -11.4％ -8.7％ Mutation 5-5 8％ 2.8％ 2.1％ 10.5％ Mutation 5-6 1％ 2.9％ 2.1％ -12.3％ Mutation 5-7 -7％ -9.1％ 2.5％ 4％ Mutation 5-8 8％ 4.6％ 0.9％ -2％ Mutation 5-9 -2％ -5.1％ -7.2％ 2％ Mutation 5-10 -14％ 0.9％ 1.7％ 3％ Mutation 5-11 5％ 1.6％ 0.0％ 3％ Mutation 5-12 5％ -0.7％ 3.4％ -3％ Mutation 5-13 4％ 11.4％ 2.8％ -14％ Mutation 5-14 6％ 9.1％ 2.9％ -7％ Mutation 5-15 -5％ -8.0％ 2％ -4％ Mutation 5-16 0.5％ 3.0％ 1％ -4％ Mutation 5-17 3％ -0.5％ -4％ -7％ Mutation 5-18 -7％ 2.3％ 5％ -8％ Mutation 5-19 12％ -2.3％ 14％ -4％ Mutation 5-20 12％ -6.4％ 11％ -5％ Mutation 5-21 1％ -0.5％ 1％ -8％ Mutation 5-22 -3％ -6.3％ 9％ -6％ Mutation 5-23 -4％ -3％ 1％ -3％ Mutation 5-24 9％ 1％ 7％ 4％ Mutation 5-25 -6％ -3％ -4％ -1％ Mutation 5-26 11％ 9％ 1％ -1％ Mutation 5-27 0％ -4％ 10％ 2％ Mutation 5-28 -2％ -1％ 1％ -10％ Mutation 5-29 -5％ 5％ 2％ -7％ Mutation 5-30 5％ -1％ -5％ -2％

[0153] From the above, it can be seen that mutations 5 to 5-30 can be stably stored at 4°C and remain stable when accelerated at 37°C for 0.5 months.

[0154] Example 3 Recombinant Antibody Production and Purification

[0155] In order to study the application performance of antibodies, it is necessary to first produce and purify the recombinant antibodies. From the performance experiments of the above antibodies themselves, it can be seen that antibody A, mutation 3-30, antibody B, and mutation 5-30 have good affinity and stability. It can be inferred that the application performance of antibody A and mutation 3-30, antibody B and mutation 5-30 are equivalent. Due to the limitations of the embodiments themselves, it is impossible to exhaustively demonstrate the effects. Example 3-4 randomly selects mutation 3-5 and mutation 5-16 for subsequent experiments.

[0156] Recombinant antibody production, especially through the Chinese hamster ovary cell (CHO) system, has less host protein, high expression, mature commercialization, and is widely used in the preparation of antibody drugs, with a production capacity of grams or even tens of grams. Compared with mouse ascites antibodies, not only is the production stable, the batch-to-batch difference is small, and the cost is greatly reduced. The specific implementation plan is as follows:

[0157] 1. Plasmid extraction: Extract the plasmid according to the instructions of Thermo Fisher's plasmid extraction kit "PureLink Rapid Low Endotoxin Plasmid Purification Kit".

[0158] 2. Cell transfection: When the cell density of CHO-K1Q cells (cells and their supporting culture medium were purchased from Lonza) was 3*E6 cells / mL, electrotransfection was performed according to the Bio-RAD cell electroporator operating instructions, where the electroporation parameters used were square wave, voltage 300V, and resistance 950Ω. 48 hours after transfection, positive cells were sorted by flow cytometry to form single clones, and the cells with the highest production capacity were selected as the fixed cell line for fermentation, and their expression capacity was 5g / L.

[0159] 3. Cell fermentation: Before fermentation, cells were subcultured every 72 hours. Samples were taken before subculture to test the viable cell density and cell viability. According to the theoretical inoculation density (0.3-0.6)×106 cells / ml and the culture volume, the corresponding volume of cell solution and preheated culture medium were added and cultured in a shaker at 37°C, 8% CO2, 120 rpm, and an amplitude of 25 mm. 4 ~P 14 The reactor (Applikon) was 15L in size (10L in volume) for fermentation. The theoretical inoculation density was (1.0±0.2)×10 6The amount of cell fluid and culture medium added was calculated based on 15L fermenter cells / mL (culture volume of 15L fermenter: 9000mL), and the cell fluid and culture medium were transferred to the injection bottle in the clean bench according to the calculated volume, and then added to the fermenter.

[0160] 4. Purification: Protein G column and cationic conventional purification were used to obtain antibody mutations 3-5 and 5-16, which were named anti-Taq A3-5 antibody and anti-Taq B5-16 antibody, respectively.

[0161] Example 4 Antibody Application Research

[0162] A. Combination application of anti-Taq antibodies and identification of their blocking effect on Taq DNA polymerase

[0163] 1. In order to study the combined application of anti-Taq A3-5 antibody and anti-Taq B5-16 antibody, the present invention studied the combined concentration of the two antibodies. The concentrations of anti-TaqA3-5 antibody and anti-Taq B5-16 antibody were adjusted to 2 mg / mL, and then the volume ratios were 1:0, 0:1, 1:1, 1:2 and 2:1 respectively. 5 μL of antibody combination in different proportions were mixed with 25U Taq DNA polymerase. The PRC program was set according to Table 12.

[0164] Table 12 PCR program setting table

[0165]

[0166] Enter the "Sample Information" interface of the SLAN 96P PCR instrument analysis system to edit the plate; then enter the "Experimental Analysis" interface, click the table option, select Ct, average Ct, CV and unique identifier, click OK, and finally click Analyze to get the final result.

[0167] Note: (1) Definition of anti-Taq antibody blocking power: After the Taq enzyme monoclonal antibody combination is mixed with Taq DNA polymerase and incubated at 25°C for 10 minutes, the amount of Taq enzyme monoclonal antibody that can inhibit 100% of 1U of Taq DNA polymerase activity at 55°C for 10 minutes is defined as 1U (refer to Takara anti-Taq enzyme activity definition). If the slope k of the sample reaction curve is no different from that of anti-Taq (judgment standard: Δ slope k (sample-anti-Taq control) <10), it is determined that the Taq enzyme monoclonal antibody has blocked 100% of the Taq DNA polymerase activity.

[0168] (2) Reaction curve: Export the original reaction curve to Excel and obtain the fluorescence intensity data for each cycle in program segment 2. Use the cycle number as the horizontal axis and the fluorescence value corresponding to the cycle number as the vertical axis to draw a fitting straight line, which is called the reaction curve of each reaction. Record the slope k of each reaction curve. Note: Generally, the data with a cycle number of 5-24 is used as the reaction curve. Because the fluorescence value detected by the instrument is unstable at the beginning of the reaction, the first 3 data are not taken.

[0169] Combinations of different ratios of Taq enzyme monoclonal antibodies were mixed with 25U Taq DNA polymerase for reaction; the slope of the reaction curve is shown in Table 13, wherein the slope K of the antibody group with a 1:1 anti-Taq ratio was mixed with 25UTaq for reaction, and was not significantly different from that of the blank control (anti-Taq, containing only anti-Taq, without Taq enzyme), indicating that the antibody combination with a 1:1 anti-Taq ratio can best block 25U Taq.

[0170] Table 13 Anti-Taq antibody combination to Taq enzyme reaction slope

[0171]

[0172]

[0173] 2. In order to further clarify the effect of Taq enzyme monoclonal antibody combination on Taq enzyme blocking, the sample to be tested was placed at room temperature for 2h and 0h, and the fluorescence value was detected respectively. If the deviation was less than 5%, it indicated that the anti-Taq antibody combination could fully block the active area of ​​Taq DNA polymerase. The specific operation was as follows:

[0174] Add each reagent to each sample in sequence according to Table 14, shake and mix, centrifuge for 5 seconds, and place on ice for later use. (Prepare immediately before use, calculate according to the number of samples + 2 during preparation, 2 is the amount of sample loss); Mix 25μL 5U / μL Taq DNA polymerase and 25μL anti-Taq antibody combination, place at room temperature for 10 minutes; take 5μL of the mixture and add it to the reaction working solution.

[0175] Table 14 Working fluid preparation table

[0176]

[0177] Quickly place the sample centrifuged in the previous step into the fluorescent quantitative PCR instrument and start the test.

[0178] The procedure is as follows:

[0179] The reaction system was 50 μL and the temperature was controlled by a module.

[0180] Instrument program settings:

[0181] Program segment 1 (37°C 20s), number of cycles: 1;

[0182] Program segment 2 (60°C for 25s), number of cycles: 24; fluorescence is detected in this stage.

[0183] like Figure 1 As shown in Table 15, the deviations of the fluorescence values ​​of the samples after 2 h and 0 h were both less than 5%, indicating that the anti-Taq antibody combination can fully block the active area of ​​Taq DNA polymerase and effectively reduce nonspecific amplification.

[0184] Table 15 Anti-Taq antibody combination blocking Taq DNA polymerase activity results

[0185] time 0h 2h deviation Ct mean 2.2315 2.2530 -2.2％

[0186] 3. To explore the blocking ability of different concentrations of anti-Taq antibody combinations, set the PRC program according to Table 12. Enter the "Sample Information" interface of the SLAN 96P PCR instrument analysis system to edit the plate; then enter the "Experimental Analysis" interface, click the table option, select Ct, average Ct, CV and unique identifier, click OK, and finally click Analyze to get the final result.

[0187] The monoclonal antibody to Taq enzyme (25U / μL) was diluted in a gradient manner; mixed with 25U Taq DNA polymerase and reacted; the slope of the reaction curve is shown in Table 16. After 12.5-25U anti-Taq and 25U Taq were mixed and reacted, the slope K had no significant difference from the blank control (anti-Taq buffer), indicating that 12.5-25U anti-Taq could block 25U Taq.

[0188] Table 16 Anti-Taq antibody combination to Taq enzyme reaction slope

[0189] Group Slope K Group Slope K 24U anti-Taq+25U Taq 3.1953 15U anti-Taq+25U Taq 4.0807 23U anti-Taq+25U Taq 3.3791 14.5U anti-Taq+25U Taq 4.5119 22U anti-Taq+25U Taq 3.7643 14U anti-Taq+25U Taq 3.87 21U anti-Taq+25U Taq 3.9411 13.5U anti-Taq+25U Taq 4.3007 20U anti-Taq+25U Taq 4.1484 13U anti-Taq+25U Taq 4.1781 19U anti-Taq+25U Taq 4.1101 12.5U anti-Taq+25U Taq 4.7009 18U anti-Taq+25U Taq 3.6811 12U anti-Taq+25U Taq 69.101 17U anti-Taq+25U Taq 3.9293 anti-Taq buffer 3.6011 TAQ-25U 3152.5

[0190] B. Identification of the effect of anti-Taq antibody combination on the function of Taq DNA polymerase

[0191] 1. Obtain diluted influenza A virus clinical samples: Take influenza A clinical samples, dilute with virus preservation solution (inactivated type) and extract, the Ct value must be within 30±1. (Note: When clinical samples are first obtained, they need to be diluted 20 times for nucleic acid extraction, and the concentration and dilution multiple should be explored.)

[0192] 2. Semi-automatic extraction of influenza A nucleic acid: refer to the instructions of Zhongyuan Huiji's "Nucleic Acid Extraction Kit (Magnetic Bead Method)". Preparation of working solution 1: Prepare the RNA reaction solution, enzyme solution (stored at -20℃ / enzyme solution after acceleration at 37℃), and influenza A primer probe in a ratio of 7:2:1 as needed to make a working solution, vortex mix, and centrifuge in a handheld centrifuge for 5-10s. Preparation of working solution 2: Prepare the new coronavirus reaction solution and enzyme solution (stored at -20℃) in a ratio of 8:2 as needed to make a working solution, vortex mix, and centrifuge in a handheld centrifuge for 5-10s.

[0193] 3. Take 10 μL of each of the above working solutions and add them to the eight-tube PCR strips, with at least four replicates for each working solution. Vortex the extracted influenza A nucleic acid and the new coronavirus internal standard, add 10 μL of influenza A nucleic acid to the eight-tube PCR strips with working solution 1, and add 10 μL of the new coronavirus internal standard to the eight-tube PCR strips with working solution 2. Cover the eight-tube strips tightly (to avoid volatilization and abnormal results), mix well, and centrifuge. Amplification detection: Place the eight-tube strips with the above working solution 1 + influenza A nucleic acid in the PCR instrument and set the following program:

[0194] Table 17 PCR procedure for influenza A nucleic acid detection

[0195]

[0196] Place the above-mentioned working solution 2 + SARS-CoV-2 internal standard eight-tube strip in the PCR instrument and set the following program:

[0197] Table 18 PCR procedure for COVID-19 nucleic acid detection

[0198]

[0199] 4. Influenza A functional test: as shown in Table 19, Figure 2 and Figure 3 As shown in the figure, the enzyme solution to be tested before and after acceleration at 37℃ for 3 days is compared with the control stored at -20℃, and the ΔCt≤1.0, and the amplification curve is a typical S-shaped curve, which means that the functional test and stability of the test product are good. As the temperature rises, the polymerase active site is gradually exposed. Under the optimal annealing and extension conditions of the enzyme, it can be completely separated from the TaqDNA polymerase, so that the enzyme activity reaches the maximum. This gradual release process can effectively protect the activity of the Taq DNA polymerase. On the basis of effectively reducing non-specific amplification, the Taq DNA polymerase can also be gradually released during the denaturation process, so that the effect of high temperature on the enzyme activity is lower, ensuring the thermal stability of the enzyme, and effectively maintaining the persistence of the enzyme activity under the condition of a higher number of amplification cycles (up to 45 cycles).

[0200] Table 19 Precision and stability of anti-Taq antibody combination

[0201]

[0202]

[0203] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes, and they can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and the illustrations shown and described herein.

Claims

1. An anti-Taq enzyme antibody or a functional fragment thereof, characterized in that: The antibody or its functional fragment comprises the following complementarity determining region: CDR-VH1: D-X1-YI-X2, where X1 is Y or D, and X2 is N; CDR-VH2: W-X1-YPG-X2-GHTQ-X3-NERFKG, wherein X1 is I, X2 is S or T, and X3 is D or Y; CDR-VH3: S-X1-FYAM-X2-Y, where X1 is R or A, and X2 is D or N; CDR-VL1: R-X1-SQSIG-X2-SIH, where X1 is A or N, and X2 is T or S; CDR-VL2: Y-X1-SES-X2-S, where X1 is A or R, and X2 is I or P; and CDR-VL3: Q-X1-TNS-X2-PLT, where X1 is Q and X2 is W or P; It was named antibody A; or The antibody or its functional fragment comprises the following complementarity determining region: CDR-VH1: S-X1-W-X2-N, where X1 is F or E, and X2 is I; CDR-VH2: D-X1-YPG-X2-GSTHYN-X3-KFKN, wherein X1 is I or N, X2 is S or T, and X3 is E or T; CDR-VH3: S-X1-DGYLF-X2-Y, where X1 is G or P, and X2 is A or R; CDR-VL1: R-X1-SQSVSTS-X2-YSFIH, where X1 is A and X2 is T or S; CDR-VL2: Y-X1-SN-X2-DS, where X1 is A or R and X2 is L; and CDR-VL3: Q-X1-SWEIP-X2-T, where X1 is H and X2 is W or P; Named Antibody B.

2. The anti-Taq enzyme antibody or its functional fragment as claimed in claim 1, characterized in that: In the complementary determining region of antibody A, X2 of CDR-VH1 is N, X1 of CDR-VH2 is I, X2 of CDR-VH3 is D, X1 of CDR-VL1 is A, X2 of CDR-VL2 is I, and X1 of CDR-VL3 is Q; or In the complementary determining region of antibody B, X2 of CDR-VH1 is I, X1 of CDR-VH2 is I, X2 of CDR-VH3 is A, X1 of CDR-VL1 is A, X2 of CDR-VL2 is L, and X1 of CDR-VL3 is H.

3. The anti-Taq enzyme antibody or its functional fragment as claimed in claim 1, characterized in that: In the CDR-VH1 of the antibody A, X1 is Y; Or in the CDR-VH1 of the antibody A, X1 is D; Or in the CDR-VH1 of the antibody A, X2 is N; Or in the CDR-VH2 of the antibody A, X1 is I; Or in the CDR-VH2 of the antibody A, X2 is S; Or in the CDR-VH2 of the antibody A, X2 is T; Or in the CDR-VH2 of the antibody A, X3 is D; Or in the CDR-VH2 of the antibody A, X3 is Y; Or in the CDR-VH3 of the antibody A, X1 is R; Or in the CDR-VH3 of the antibody A, X1 is A; Or in the CDR-VH3 of the antibody A, X2 is D; Or in the CDR-VH3 of the antibody A, X2 is N; Or in the CDR-VL1 of the antibody A, X1 is A; Or in the CDR-VL1 of the antibody A, X1 is N; Or in the CDR-VL1 of the antibody A, X2 is T; Or in the CDR-VL1 of the antibody A, X2 is S; Or in the CDR-VL2 of the antibody A, X1 is A; Or in the CDR-VL2 of the antibody A, X1 is R; Or in the CDR-VL2 of the antibody A, X2 is 1; Or in the CDR-VL2 of the antibody A, X2 is P; Or in the CDR-VL3 of the antibody A, X1 is Q; Or in the CDR-VL3 of the antibody A, X2 is W; Or in the CDR-VL3 of the antibody A, X2 is P; Or in the CDR-VH1 of the antibody B, X1 is F; Or in the CDR-VH1 of the antibody B, X1 is E; Or in the CDR-VH1 of the antibody B, X2 is 1; Or in the CDR-VH2 of the antibody B, X1 is I; Or in the CDR-VH2 of the antibody B, X1 is N; Or in the CDR-VH2 of the antibody B, X2 is S; Or in the CDR-VH2 of the antibody B, X2 is T; Or in the CDR-VH2 of the antibody B, X3 is E; Or in the CDR-VH2 of the antibody B, X3 is T; Or in the CDR-VH3 of the antibody B, X1 is G; Or in the CDR-VH3 of the antibody B, X1 is P; Or in the CDR-VH3 of the antibody B, X2 is A; Or in the CDR-VH3 of the antibody B, X2 is R; Or in the CDR-VL1 of the antibody B, X1 is A; Or X2 in CDR-VL1 of the antibody B is T; Or in the CDR-VL1 of the antibody B, X2 is S; Or in the CDR-VL2 of the antibody B, X1 is A; Or in the CDR-VL2 of the antibody B, X1 is R; Or in the CDR-VL2 of the antibody B, X2 is L; Or in CDR-VL3 of the antibody B, X1 is H; Or in the CDR-VL3 of the antibody B, X2 is W; Or in the CDR-VL3 of the antibody B, X2 is P.

4. The anti-Taq enzyme antibody or a functional fragment thereof according to any one of claims 1 to 3, characterized in that: Each complementary determining region of the antibody A is selected from any one of the following mutation combinations: Or each complementary determining region of the antibody B is selected from any one of the following mutation combinations:

5. The anti-Taq enzyme antibody or a functional fragment thereof according to any one of claims 1 to 4, characterized in that: The anti-Taq enzyme antibody or its functional fragment also includes light chain framework regions FR1-L, FR2-L, FR3-L and FR4-L and heavy chain framework regions FR1-H, FR2-H, FR3-H and FR4-H; the heavy chain framework regions FR1-H, FR2-H, FR3-H and FR4-H are selected from SEQ ID NOs: 1-4 or SEQ ID NOs: 5-8 in sequence; the light chain framework regions FR1-L, FR2-L, FR3-L and FR4-L are selected from SEQ ID NOs: 9-12 or SEQ ID NOs: 13-16 in sequence.

6. The anti-Taq enzyme antibody or a functional fragment thereof according to any one of claims 1 to 4, characterized in that: The anti-Taq enzyme antibody or its functional fragment further comprises a constant region; Preferably, the constant region is selected from the constant region of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD. Preferably, the species of the constant region is cattle, horses, dairy cows, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, geese, turkeys, fighting cocks or humans. Preferably, the functional fragment is selected from any one of F(ab')2, Fab', Fab, Fv and scFv of the antibody.

7. The anti-Taq enzyme antibody or a functional fragment thereof according to any one of claims 1 to 6, characterized in that: The antibody is a labeled antibody; Preferably, the labeling substances for labeling the antibody include, but are not limited to, latex microspheres, biotin, fluorescent dyes, enzymes that catalyze the color development of substrates, radioactive isotopes, chemiluminescent reagents, and nanoparticle labels.

8. Use of the antibody or functional fragment thereof according to any one of claims 1 to 6 in the preparation of a PCR detection kit or in the preparation of a blocking agent.

9. A detection reagent or kit, characterized in that: The reagent or kit comprises the antibody or functional fragment thereof according to any one of claims 1 to 6 or the labeled antibody according to claim 7.

10. A method for blocking Taq enzyme activity, characterized in that: The antibody or its functional fragment according to any one of claims 1 to 6 or the antibody conjugate according to claim 7 is added to the immunoassay system.

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