Nanometer antibody for hot start Taq DNA polymerase and construction method thereof

Abstract

The invention belongs to the technical field of antibodies, and discloses a nano antibody for hot start of Taq DNA polymerase and a construction method of the nano antibody. The nano-antibody is a monovalent nano-antibody for sealing a 3 '-5' excision enzyme structural domain of Taq DNA polymerase, a monovalent nano-antibody for sealing a 5 '-3' polymerization active structural domain of Taq DNA polymerase or a bivalent nano-antibody obtained by connecting the monovalent nano-antibody and the monovalent nano-antibody through flexible peptide; the amino acid sequence of the monovalent nano antibody for sealing the 3 '-5' excision enzyme structural domain of the Taq DNA polymerase is shown as SEQ ID NO: 1, the amino acid sequence of the monovalent nano antibody for sealing the 5 '-3' polymerization active structural domain of the Taq DNA polymerase is shown as SEQ ID NO: 2, and the amino acid sequence of the flexible peptide is shown as SEQ ID NO: 3.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of genetic engineering, and particularly relates to a nanobody for hot-start Taq DNA polymerase and a construction method thereof. BACKGROUND

[0002] Polymerase chain reaction (PCR) is a technology for amplifying specific DNA fragments in vitro, and is an important tool in the fields of molecular biology and medicine. The DNA polymerase used in PCR is mainly Taq DNA polymerase, and with the popularization and application of PCR technology, the research on its properties is increasingly important. Taq DNA polymerase has thermal stability, but still has certain polymerase activity at room temperature, which is easy to produce non-specific amplification and primer dimers, reducing the sensitivity and specificity of PCR reaction. To solve the above problems, people invented hot-start PCR, which increases the optimum reaction temperature of Taq DNA polymerase through various ways to achieve the purpose of hot-start PCR, so as to minimize non-specific amplification. Among them, the antigen-antibody combination inhibition method is a common way to increase the optimum reaction temperature of Taq DNA polymerase. The monoclonal antibody or polyclonal antibody against Taq DNA polymerase is obtained by immunizing experimental animals with Taq DNA polymerase. Through this method, in the PCR reaction, the enzyme and the antibody form an antigen-antibody complex at low temperature, so that the polymerase is temporarily inactivated; while at a higher temperature, the antibody denatures, so that the Taq DNA polymerase is released and active, thereby realizing hot-start.

[0003] The conventional antigen-antibody combination inhibition method also has the disadvantage that multiple high-low temperature cycles are easy to cause the peptide bond to break, resulting in inactivation of the antibody; with the development of antibody technology, small antibodies are constantly emerging. Scientists have found that the antibodies of the Camelidae family in the Tylopoda suborder of Artiodactyla naturally lack light chains, and the single-domain antibody composed of only one heavy chain variable region cloned from the heavy chain variable region is called single-domain heavy chain antibody (VHH), which is the smallest functional antigen-binding fragment, also known as nanobody (Nanobody). Due to its unique molecular structure and physicochemical properties, compared with ordinary antibodies, nanobodies have small volume, high affinity, strong resistance to heat and PH; compared with conventional single-domain antibodies, they also have better conformational stability.

[0004] Chinese patent CN117384293A discloses Taq DNA polymerase antibodies and compositions thereof, modified Taq DNA polymerases thereof and applications thereof; the invention uses Taq DNA polymerase as an immunogen to immunize animals, and through cell fusion and screening, hybridoma cell strains and monoclonal antibodies produced thereby are obtained; the above-mentioned antibodies are used to modify Taq DNA polymerase, which can block the active site of Taq DNA polymerase at low temperature; it can effectively reduce the generation of non-specific amplification and primer dimers for PCR; the antibody-modified Taq DNA polymerase provided by the invention has high stability, and still has good hot start and the effect of reducing non-specific amplification and primer dimers after storage.

[0005] At present, most of the commercial antibodies used for hot start are traditional monoclonal or polyclonal antibodies, which have large molecular weight, high production cost, need to be expressed in mammalian cells, and the stability needs to be improved. Nanobodies are derived from heavy chain antibodies variable regions of camelids and other in vivo natural light chain deletion, which are the smallest units known to have complete antigen binding capacity. They have the advantages of small molecular weight, high stability, good water solubility, easy large-scale production in prokaryotic systems such as E. coli, and the like. However, there is no report on nanobodies that can specifically bind and effectively inhibit the activity of Taq DNA polymerase and are suitable for hot start PCR.

[0006] Therefore, it is of great significance to develop a nanobody that can efficiently block Taq DNA polymerase, has low production cost and stable performance, for improving the economy and reliability of hot start PCR technology. SUMMARY

[0007] In order to solve the problems of high cost and complex production in the prior art, the present application provides a nanobody for hot start Taq DNA polymerase and a construction method thereof. The nanobody for hot start Taq DNA polymerase provided by the present application blocks the active site of Taq DNA polymerase at room temperature, and releases the enzyme when the antibody denatures at high temperature to realize its function.

[0008] To achieve the above-mentioned purposes, the present application realizes the following technical solutions: One objective of this invention is to provide a nanobody for hot-starting Taq DNA polymerase, wherein the nanobody is a monovalent nanobody blocking the 3'-5' exonuclease domain of Taq DNA polymerase, a monovalent nanobody blocking the 5'-3' polymerization activity domain of Taq DNA polymerase, or a divalent nanobody obtained by linking the two through a flexible peptide; the amino acid sequence of the monovalent nanobody blocking the 3'-5' exonuclease domain of Taq DNA polymerase is shown in SEQ ID NO:1; the amino acid sequence of the monovalent nanobody blocking the 5'-3' polymerization activity domain of Taq DNA polymerase is shown in SEQ ID NO:2; and the amino acid sequence of the flexible peptide is shown in SEQ ID NO:3.

[0009] The second objective of this invention is to provide a method for preparing nanobodies for hot-start TagDNA polymerase, comprising the following steps: Using Taq DNA polymerase Taq DPI and its truncated variants Taq DPI-1 and Taq DPI-2 as antigens, gene sequences encoding monovalent nanobodies were obtained by immunizing camel-like animals, sharks, or by screening artificially constructed nanobody libraries. Recombinant plasmids capable of expressing monovalent nanobodies were constructed, transformed into expression cells, and induced to express the monovalent nanobodies. The monovalent nanobodies were then purified to obtain bivalent nanobodies by linking them with flexible peptides using genetic engineering techniques.

[0010] Furthermore, the truncated Taq DNA polymerase variant Taq DPI-1 lacks the 5'-3' exonuclease active region of Taq DNA polymerase, specifically amino acids 1-291 from the N-terminus of Taq DNA polymerase; the truncated Taq DNA polymerase variant Taq DPI-2 lacks both the 5'-3' and 3'-5' exonuclease active regions of Taq DNA polymerase, specifically amino acids 1-423 from the N-terminus of Taq DNA polymerase.

[0011] Furthermore, the amino acid sequence of the truncated Taq DNA polymerase derivative Taq DPI-1 is shown in SEQ ID NO:4; the amino acid sequence of the truncated Taq DNA polymerase derivative Taq DPI-2 is shown in SEQ ID NO:5.

[0012] Furthermore, the plasmid vector of the recombinant plasmid is one of pET25b, pET28a, or pBAD-HisB.

[0013] Furthermore, the expression cells are Escherichia coli or yeast cells.

[0014] The present invention also provides a hot-start Taq DNA polymerase comprising nanobodies; the nanobodies are monovalent nanobodies blocking the 3'-5' exonuclease domain of Taq DNA polymerase, monovalent nanobodies blocking the 5'-3' polymerization activity domain of Taq DNA polymerase, or divalent nanobodies obtained by linking the two through a flexible peptide; the amino acid sequence of the monovalent nanobodies blocking the 3'-5' exonuclease domain of Taq DNA polymerase is shown in SEQ ID NO:1; the amino acid sequence of the monovalent nanobodies blocking the 5'-3' polymerization activity domain of Taq DNA polymerase is shown in SEQ ID NO:2; the amino acid sequence of the flexible peptide is shown in SEQ ID NO:3.

[0015] The present invention also provides a hot-start Taq DNA polymerase for use in conventional PCR amplification and real-time quantitative PCR amplification.

[0016] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows: 1. The nanobody for hot-start Taq DNA polymerase provided by this invention can effectively block Taq DNA polymerase at room temperature by efficiently and specifically binding to the key active domains of Taq DNA polymerase, significantly inhibiting non-specific amplification; it can rapidly dissociate in the initial stage of high temperature PCR, so as to fully restore enzyme activity and ensure reaction sensitivity and amplification efficiency.

[0017] 2. The nanobodies for hot-start Taq DNA polymerase provided by this invention can be flexibly constructed in various forms, including monovalent and multivalent nanobodies. By adjusting the combination and valence state, their affinity and inhibitory effect can be further optimized to meet the needs of different application scenarios. Furthermore, the nanobodies provided by this invention can be efficiently expressed in prokaryotic expression systems, significantly reducing production costs compared to traditional mammalian cell expression systems, making them more suitable for large-scale industrial production. Attached Figure Description

[0018] Figure 1 This is an SDS-PAGE electrophoresis image of the Taq DNA polymerase and its truncated form induced to be expressed in Escherichia coli in Example 1 of the present invention; Figure 2 This is an SDS-PAGE electrophoresis image of the purified Taq DNA polymerase and its truncated form from Example 1 of this invention. Figure 3This is an agarose gel electrophoresis image of Taq DNA polymerase activity verification in Example 1 of the present invention; lanes 1-9 in A represent the electrophoresis results of different concentrations of Taq DPI (polymerase I), and lanes 10-18 in A represent different concentrations of commercial Taq DPI (polymerase I); lanes 1-9 in B represent the electrophoresis results of different concentrations of recombinant Taq DPI-1, and lanes 10-18 in B represent the electrophoresis results of different concentrations of recombinant Taq DPI-1. Figure 4 This is an SDS-PAGE electrophoresis image of the purified monovalent and divalent nanobodies in Example 2 of this invention; Figure 5 This is a graph illustrating the hot-start effect of nanobody blocking Taq DNA polymerase in Example 3 of the present invention. Figure 6 This is an amplification curve of different hot-start Taq enzymes in qPCR detection in Example 4 of the present invention; Figure 7 This is a standard curve diagram of hot-start Taq enzyme qPCR detection in Example 4 of the present invention. Detailed Implementation

[0019] The present invention will be further described below with reference to preferred embodiments. The endpoints and any values ​​of the ranges disclosed in the present invention are not limited to the precise ranges or values. These ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges. These numerical ranges should be considered as specifically disclosed herein.

[0020] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0021] In the following examples, "codon optimization" refers to gene redesign that utilizes preferred codons and avoids codons with low utilization or rarity. Every organism exhibits some degree of codon utilization difference or preference, with the most frequently used codons being preferred codons.

[0022] The molecular biology experiments not specifically described in the following examples include vector construction, enzyme digestion, ligation, preparation of competent cells, transformation, and culture medium preparation, which are mainly performed with reference to "Molecular Cloning: A Laboratory Manual" (3rd edition); PCR amplification experiments are performed according to the reaction conditions or kit instructions provided by the vector or DNA template supplier.

[0023] Example 1 This embodiment provides a method for recombinant expression and purification of Taq DNA polymerase and its truncated form, including the following steps: (1) Referring to the protein sequence of Taq DNA polymerase (NCBI Reference Sequence: WP_157628163.1), General Biotechnology (Anhui) Co., Ltd. was commissioned to optimize the gene sequence using Escherichia coli as the expression host. The optimized full-length Taq DNA polymerase gene Taq DPI, the truncated Taq DPI-1 gene (deleted amino acids 1-291 from the N-terminus), and the truncated Taq DPI-2 gene (deleted amino acids 1-423 from the N-terminus) were inserted into the Nde I and EcoRI restriction sites of the pET28a plasmid, and the synthesized plasmids were named pET 28a-Taq DPI, pET28a-Taq DPI-1, and pET 28a-Taq DPI-2, respectively. (2) The synthesized plasmid was transformed into Escherichia coli BL21. The successfully transformed E. coli BL21 was inoculated into 5 mL of LB medium containing kanamycin sulfate and cultured overnight to obtain pET 28a-Taq DPI / BL21(DE3), pET28a-Taq DPI-1 / BL21(DE3), and pET 28a-Taq DPI-2 / BL21(DE3) bacterial suspensions, respectively. The bacterial suspensions obtained above were inoculated into ZYM self-induction medium and induced to express at 30℃ for 20 hours to obtain bacterial suspensions. The expression of the target protein in the bacterial suspensions was detected by SDS-PAGE protein electrophoresis. The results are as follows: Figure 1 As shown, Taq DNA polymerase, as well as the truncated forms Taq DNA polymerase Taq DPI-1 and Taq DNA polymerase Taq DPI-2, were normally expressed in the bacterial cultures of pET 28a-Taq DPI / BL21(DE3), pET 28a-Taq DPI-1 / BL21(DE3), and pET 28a-Taq DPI-2 / BL21(DE3). (3) Collect the bacterial cells by centrifugation at 4500 rpm, resuspend the bacterial cells in 50 mM Tris-HCl (pH 8.0) buffer, sonicate, centrifuge at 10000 r / min for 30 min at 4 °C, and collect the supernatant. Filter the supernatant through a 0.22 μm filter membrane and purify it by AKTA-FPLC using a 1 mL HisTrap™ HP column. Load the sample at a flow rate of 1 mL / min, elute impurities with Tris-HCl (pH 8.0) containing 40 mM imidazole, and elute the target protein with Tris-HCl (pH 8.0) containing 250 mM imidazole. Dialyze the target protein overnight in 50 mM Tris-HCl (pH 8.0) buffer at 4 °C to obtain the target protein. Detect the purity of the target protein using SDS-PAGE electrophoresis. The results are as follows: Figure 2 As shown, the target proteins were all expressed normally, and high-purity Taq DNA polymerase and Taq DNA polymerase truncated forms Taq DPI-1 and Taq DNA polymerase truncated forms Taq DPI-2 were successfully obtained. (4) The relative enzyme activities of the purified Taq DNA polymerase and the truncated Taq DNA polymerase derivatives Taq DPI-1 and Taq DNA polymerase derivative Taq DPI-2 obtained above were determined: Using the pET28a recombinant plasmid (with a 1350 bp DNA fragment inserted at the multiple cloning site) as a template, the DNA fragment was amplified using T7 F / T7 Terminator universal primers, with commercially available Taq DNA polymerase I as a control. The specific PCR system and conditions are as follows:

[0024] The above reagents were thoroughly mixed using a vortex mixer and then subjected to PCR reaction under the following conditions: pre-denaturation at 94°C for 5 min; (94°C for 30 s; 55°C for 30 s; 72°C for 2 min) 25 cycles, 72°C for 5 min; 16°C for ∞.

[0025] PCR products were detected by agarose gel electrophoresis, and the results are shown in the figure. Figure 3 The purified Taq DNA polymerase and its truncated form, Taq DPI-1, exhibit the same polymerization activity as commercial Taq DNA polymerase.

[0026] Example 2 This embodiment provides the screening, expression, and purification of monovalent nanobodies, including the following steps: (1) Using purified full-length Taq DNA polymerase protein as an immune antigen, Nanchang Dajia Technology Co., Ltd. was commissioned to inject the antigen into alpacas for immunization, and a phage display library was constructed. (2) The purified Taq DNA polymerase, truncated Taq DPI-1 and truncated Taq DPI-2 proteins were used as antigens to coat an enzyme-labeled plate, and phages that bind to the above antigens were screened; the phages obtained by screening were sequenced to obtain the nanobody sequences. The four nanobody genes with unique sequences obtained from the screening were entrusted to General Biotech (Anhui) Co., Ltd. for synthesis and directly cloned into the pET25b expression vector, and labeled as pET25b-T1-20, pET25b-T2-13, pET25b-T2-49 and pET25b-T2-9, respectively. (3) Using the Gibson assembly seamless cloning method, two different monovalent nanobody genes (T1-20 and T2-49) were linked by sequences encoding flexible peptides to construct a bivalent nanobody expression vector. The specific construction method and primers used are as follows: This experiment uses the Gibson assembly seamless cloning method to construct a recombinant expression vector for bivalent nanobodies, which requires cloning the linearized vector, flexible fragment, and monovalent nanobodies separately.

[0027] Amplification of the linearized vector fragment: The vector was linearized using PCR, and the primers used are shown in Table 1 below: Table 1 Primers used for vector linearization (from top to bottom, SEQ ID NO. 6~9 in the sequence listing)

[0028] Amplification of the vector fragment: Information on the amplification of the vector fragment is shown in Table 2. Table 2. Vector fragment amplification information

[0029] Table 3 PCR reaction system

[0030] The system shown in Table 3 was used, and the PCR reaction conditions were as follows: Step 1: 94℃, 5 min pre-denaturation; Step 2: 94℃, 30 s; 58℃, 30 s; 72℃, 3 min, for a total of 28 cycles; Step 3: 72℃, 10 min; After amplification, the target fragment was separated by 1% agarose gel electrophoresis and the gel was cut and recovered.

[0031] Cloning of flexible linker fragments and VHH gene: Two monovalent nanobodies use the flexible peptide chain GGGSGGGGSGGGGGSGGGG as a linker, so the DNA sequence encoding this peptide chain was synthesized first.

[0032] Table 4. Primers for flexible peptide chain amplification (from top to bottom, SEQ ID NO. 10~11 in the sequence listing)

[0033] Table 5. Primers for flexible peptide linker amplification (from top to bottom, SEQ ID NO. 12~17 in the sequence listing).

[0034] Using the primers shown in Tables 4 and 5, flexible peptides and adapter fragments were amplified for subsequent ligation experiments with monovalent nanobodies.

[0035] Table 6 Primers for VHH gene fragment amplification (from top to bottom, SEQ ID NO. 18~23 in the sequence listing)

[0036] The VHH gene fragment was amplified using the primers shown in Table 6.

[0037] Table 7 Amplification information of flexible peptide chain fragments

[0038] Amplification of flexible peptide fragments: Information on the amplification of flexible peptide fragments is shown in Table 7. The reaction system, PCR amplification procedure, and gel recovery process are the same as above.

[0039] Amplification of VHH gene fragment: Based on the four unique nanobody genes, primers were designed to amplify the VHH fragment. The VHH fragment amplification information is shown in Table 8. Table 8 Amplification information of VHH gene fragment

[0040] The reaction system, PCR amplification procedure, and gel recovery process are the same as above; Recombination reaction: The carrier fragment, flexible peptide chain fragment, and VHH fragment recovered from the gel were seamlessly cloned and recombined according to the following combination and reaction system. The specific polymerization forms are shown in Table 9, and the bivalent nanobodies were numbered and named. Table 9. Protein sequence composition of bivalent nanobodies

[0041] Table 10 Recombination reaction system (15 µL)

[0042] All reaction solutions shown in Table 10 were mixed thoroughly, briefly centrifuged, and then incubated in a 50°C metal bath for 1 h for recombination ligation. The ligation product was transformed into *E. coli* BL21(DE3). Strains with correct sequencing were selected for nanobody expression and nickel chromatography purification. SDS-PAGE verification results are shown in [Table 10]. Figure 4 ;Depend on Figure 4 All nanobodies were successfully expressed.

[0043] Example 3 This embodiment provides verification of the effectiveness of nanobody blocking Taq DNA polymerase. The purified Taq DNA polymerase was incubated with nanobodies in different molar ratios (enzyme to antibody molar ratio of 1:1, 0.5:1, 1.5:1) at 25°C for 45 minutes to prepare a hot-start Taq enzyme mixture.

[0044] Using partially complementary primer pairs (as shown in Table 11) as templates, and employing the PCR reaction system shown in Table 12, fluorescence signals were monitored at 37°C to assess the inhibition effect at room temperature. Subsequently, the system was heated to 95°C and then cooled to 37°C to monitor enzyme activity recovery.

[0045] The results are as follows Figure 5 As shown, the monovalent nanobody T2-49 and the bivalent nanobodies 9-1 and 20-6, when in appropriate proportions, can effectively inhibit Taq enzyme activity at room temperature and restore enzyme activity after high-temperature dissociation, exhibiting good hot-start characteristics.

[0046] Table 11 Primers used to monitor enzyme activity recovery (from top to bottom, SEQ ID NO. 24~25 in the sequence listing)

[0047] Table 12 PCR reaction system used in Example 3

[0048] Example 4 The wheat-derived γ-gliadin gene template at known concentrations was serially diluted to a concentration of 5 × 10⁻⁶. -1 5×10 -2 5×10 -3 5×10 -4 5×10 -5 The hot-start effect of enzyme antibody, unblocked Taq DNA polymerase I, and commercially available antibody-blocked Taq DNA polymerase I was compared using ng / µL.

[0049] Table 13 Primers used in Example 4 (from top to bottom, SEQ ID NO. 26~28 in the sequence listing)

[0050] Table 14 qPCR reaction system

[0051] Mix the above reagents thoroughly. The qPCR reaction conditions are: 95℃, 2 min pre-denaturation; 95℃, 15 s; 58℃, 30 s (read fluorescence signal) for 45 cycles. The qPCR reaction system is shown in Table 14. After the qPCR reaction is completed, the obtained data are analyzed.

[0052] Test results as follows Figure 6 As shown, the higher the initial template concentration, the lower the number of cycles (Ct) required to reach the instrument detection threshold; for example... Figure 6 The amplification curves show that in the 20-6 blocked Taq DNA polymerase I system, the Ct value does not exhibit a linear relationship within the low template concentration range.

[0053] Based on the above experimental results, a standard curve was established between the template concentration log value and the corresponding Ct value, as follows: Figure 7 As shown, in the unblocked Taq DNA polymerase I system, the template concentration log value and the corresponding Ct value are not linearly related; in the 9-1 blocked Taq DNA polymerase I system, the template concentration from 5 × 10⁻⁶ to 10⁻⁶ Ct values ​​is linearly related. -5 ng / µL to 5×10 -1 Within the range of ng / µL, its log value showed a good linear relationship with the corresponding Ct value. Compared with the commercial antibody-blocked Taq DNA polymerase I system, the standard curve and fluorescence signal value were basically consistent.

[0054] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the inventive concept of the present invention, and these all fall within the protection scope of the present invention.

Claims

1. A Nanobody for hot-starting Taq DNA polymerase, characterized in that, The nanobody is a monovalent nanobody blocking the 3'-5' exonuclease domain of Taq DNA polymerase, a monovalent nanobody blocking the 5'-3' polymerization activity domain of Taq DNA polymerase, or a bivalent nanobody obtained by connecting the two through a flexible peptide; the amino acid sequence of the monovalent nanobody blocking the 3'-5' exonuclease domain of Taq DNA polymerase is shown in SEQ ID NO: 1; the amino acid sequence of the monovalent nanobody blocking the 5'-3' polymerization activity domain of Taq DNA polymerase is shown in SEQ ID NO: 2; and the amino acid sequence of the flexible peptide is shown in SEQ ID NO:

3.

2. A method of preparing a Nanobody for hot-starting Taq DNA polymerase as claimed in claim 1, characterized in that, The method comprises the following steps: The monovalent nanobody is obtained by screening a gene sequence encoding the monovalent nanobody from an animal immunized with Taq DNA polymerase Taq DPI and its Taq DNA polymerase truncation Taq DPI-1, Taq DNA polymerase truncation Taq DPI-2 as antigens, or from a nanobody library constructed artificially, constructing a recombinant plasmid capable of expressing the monovalent nanobody, transforming the recombinant plasmid into expression cells and inducing expression, and purifying the monovalent nanobody; and the bivalent nanobody is obtained by connecting the monovalent nanobody through a flexible peptide by genetic engineering.

3. The method for preparing a Nanobody for hot-starting Taq DNA polymerase according to claim 2, wherein the amino acid sequence of the Nanobody is SEQ ID NO:

1. The Taq DNA polymerase truncation Taq DPI-1 lacks the 1-291 amino acids at the N terminus of Taq DNA polymerase, and the Taq DNA polymerase truncation Taq DPI-2 lacks the 1-423 amino acids at the N terminus of Taq DNA polymerase. 4.The method for preparing the nanobody for hot-starting Taq DNA polymerase according to claim 2, wherein the amino acid sequence of the nanobody is SEQ ID NO:

1. The amino acid sequence of the Taq DNA polymerase truncation Taq DPI-1 is shown in SEQ ID NO: 4, and the amino acid sequence of the Taq DNA polymerase truncation Taq DPI-2 is shown in SEQ ID NO:

5.

5. The method for preparing a nanobody for hot-start Taq DNA polymerase according to claim 2, wherein, The plasmid vector of the recombinant plasmid is pET25b, pET28a, or pBAD-HisB. 6.The method for preparing a nanobody for hot-starting Taq DNA polymerase according to claim 2, wherein, The expression cells are Escherichia coli or yeast cells.

7. A hot start Taq DNA polymerase, characterized in that, The method comprises the following steps:

8. The hot start Taq DNA polymerase of claim 7 is used for conventional PCR amplification and real-time fluorescent quantitative PCR amplification.

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