Double-antibody blocked Taq DNA polymerase as well as construction method and application thereof

Abstract

The invention discloses a double-antibody blocked Taq DNA polymerase as well as a construction method and application thereof. The Taq DNA polymerase is blocked by a Taq DNA polymerase monoclonal antibody and a thermosensitive fusion nano antibody; the thermosensitive fusion nano antibody is composed of a heat-labile protein tag and a nano antibody capable of being specifically combined with a Taq DNA polymerase 5 '-3' polymerization active structural domain. The preparation method of the double-antibody closed Taq DNA polymerase comprises the following steps: constructing a thermosensitive fusion nano antibody and purifying the thermosensitive fusion nano antibody; and co-incubating the purified thermosensitive fusion nano antibody and a commercial monoclonal antibody with Taq DNA polymerase according to a ratio to realize double-antibody blocking. The double-antibody closed Taq DNA polymerase prepared by the invention has the advantages of low cost, higher fluorescence signal value and amplification efficiency in real-time fluorescent quantitative PCR, high sensitivity, high specificity and the like, and is suitable for conventional PCR and real-time fluorescent quantitative PCR amplification.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a double-antibody blocked Taq DNA polymerase, its construction method, and its application. Background Technology

[0002] The optimal temperature for common Taq DNA polymerase is 72℃, at which point its activity is at its best. Below this temperature, the enzyme exhibits weaker activity. During the PCR amplification process, as the temperature rises from low to high, Taq DNA polymerase exhibits weaker activity, making the system prone to mismatches or primer dimer formation. Therefore, to improve the specificity of PCR amplification and reduce the reaction mismatch rate, enzyme activity can be artificially controlled. The enzyme can be inactive before reaching 72℃, avoiding chain mismatches during the heating process (or reaction system preparation), thus ensuring amplification specificity. Alternatively, monoclonal or polyclonal antibodies that bind to Taq DNA polymerase can be used to block the active site of Taq DNA polymerase at room temperature, releasing the enzyme upon antibody denaturation at high temperatures. Due to its good stability and high sensitivity, antibody blocking is currently the most commonly used method for preparing Taq DNA polymerase for nucleic acid detection.

[0003] Nanobodies possess the smallest structure with complete antigen recognition capabilities, with a molecular weight only 1 / 10 that of traditional antibodies. They exhibit characteristics such as small molecular weight, ease of modification, strong stability, water solubility, and deeper chimerism with antigens. Traditional antibodies are expressed in mammalian cells and yeast, resulting in long production cycles and high costs. Nanobodies, unlike traditional antibodies, can be efficiently expressed in *E. coli*, are easy to mass-produce, and are low-cost. In existing technologies, Chinese patent CN107119029A proposes using a single nanobody to block Taq enzyme for hot-start, verifying the feasibility of nanobodies in this field. However, the single-blocking mode still has room for improvement in inhibition efficiency and specificity. To further enhance the blocking effect, Chinese patent CN107502600A discloses a dual-blocking strategy of "nanobody + chemical modification." While this scheme reduces costs to some extent, chemical modification may introduce uncertainties, such as affecting enzyme activity, reducing reaction consistency, or increasing process complexity.

[0004] Therefore, designing a new Taq DNA polymerase nanobody and performing multiple blocking techniques can effectively reduce costs, while improving the sensitivity and specificity of Taq DNA polymerase is an urgent problem to be solved in this field. Summary of the Invention

[0005] To address the problems of high cost, poor specificity, and low sensitivity in existing technologies, this invention provides a double-antibody blocked Taq DNA polymerase, its construction method, and its application.

[0006] To achieve the above objectives, the present invention provides the following technical solution: One objective of this invention is to provide a dual-antibody blocking Taq DNA polymerase system, comprising: Taq DNA polymerase, a monoclonal antibody against Taq DNA polymerase, and a thermosensitive fusion nanobody; wherein the thermosensitive fusion nanobody comprises a heat-sensitive protein tag and a nanobody capable of binding to the 5'-3' polymerization active domain of Taq DNA polymerase, and the amino acid sequence of the nanobody is shown in SEQ ID NO:1.

[0007] Furthermore, the heat-sensitive protein tag is a GST tag or an MBP tag.

[0008] The second objective of this invention is to provide a method for constructing a double-antibody blocked Taq DNA polymerase, comprising the following steps: S1. Construct a recombinant plasmid capable of expressing thermosensitive fusion nanobodies, transform it into expression cells and induce expression, and purify to obtain thermosensitive fusion nanobodies; S2, thermosensitive fusion nanobody, Taq DNA polymerase monoclonal antibody and Taq DNA polymerase are mixed in proportion and incubated at 20-25℃ to achieve double antibody blocking of Taq DNA polymerase.

[0009] Furthermore, the expression cells mentioned in step S1 are Escherichia coli or yeast cells.

[0010] Further, the expression vector of the recombinant plasmid in step S1 is one of the pET series, pBAD-His or pGEX series plasmid vectors.

[0011] Furthermore, the molar ratio of the Taq DNA polymerase, monoclonal antibody, and thermosensitive fusion nanobody is 1:0.5:(0.5~1).

[0012] The third objective of this invention is to provide an application of double-antibody blocked Taq DNA polymerase in the preparation of nucleic acid detection kits, wherein the nucleic acid detection includes conventional PCR, real-time quantitative PCR, rapid nucleic acid detection, or multiplex nucleic acid detection.

[0013] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows: The dual-blocking Taq DNA polymerase provided in this invention utilizes thermosensitive nanobodies and commercially available monoclonal antibodies, significantly reducing the amount of monoclonal antibody used and lowering costs. The synergistic dual-blocking strategy provides better inhibition of Taq enzyme at room temperature, effectively reducing non-specific amplification and primer dimer formation, resulting in higher amplification efficiency. The thermosensitive fusion nanobodies rapidly and thoroughly denature at high temperatures, ensuring full release of Taq enzyme activity during PCR cycles, thereby improving PCR amplification efficiency and fluorescence signal values, and providing higher detection sensitivity. Attached Figure Description

[0014] Figure 1 This is an SDS-PAGE electrophoresis image of the purified nanobody (His-T2-9) in Example 1 of this invention; Figure 2 This is an SDS-PAGE electrophoresis image of the purified thermosensitive nanobody GST-T2-9 expressed in Example 1 of this invention. Figure 3 This is a comparison of the real-time fluorescence quantitative PCR amplification curves of the monoclonal antibody-blocked Taq DNA polymerase prepared in Comparative Example 1 of the present invention; Figure 4 This is a comparison of the Taq DNA polymerase real-time fluorescence quantitative PCR amplification curves of double antibody blocking (molar ratio 1:0.5:1) and monoclonal antibody blocking in Example 2 of the present invention. Figure 5 This is a comparison of the Taq DNA polymerase real-time fluorescence quantitative PCR amplification curves of double antibody blocking (molar ratio 1:0.5:0.5) and monoclonal antibody blocking in Example 3 of the present invention. Detailed Implementation 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.

[0015] Unless otherwise specified, the experimental methods in the following examples are conventional methods, performed in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions.

[0016] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0017] In the following examples, “codon optimization” refers to the redesign of genes by using preferred codons and avoiding codons with low utilization or rarity. Every organism exhibits some degree of codon utilization difference or preference, and those most frequently used are preferred codons.

[0018] 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.

[0019] Example 1 This embodiment provides the construction, expression, and purification of thermosensitive fusion nanobodies, with the specific steps as follows: 1. Referring to the amino acid sequence shown in SEQ ID NO:1, General Biotechnology (Anhui) Co., Ltd. was commissioned to optimize the codons of the gene sequence using Escherichia coli as the expression host. The optimized full-length Taq DNA polymerase nanobody gene was then inserted into the NcoI and EcoRI restriction sites of pET25b plasmid and the Spe I and Hind III restriction sites of pET42b plasmid, respectively. The synthesized recombinant plasmids were named pET 25b-T2-9 and pET 42b-T2-9, respectively. 2. The pET42b-T2-9 plasmid was transformed into Escherichia coli BL21(DE3) competent cells; single colonies were picked and inoculated into ZYM self-induction medium containing kanamycin sulfate, and cultured at 30℃ and 230 rpm for 20 hours to induce expression, resulting in pET25b-T2-9 / BL21(DE3) bacterial suspension; subsequent steps will be used to prepare thermosensitive fusion nanobodies with different tags. 3. Preparation of His-tagged nanobodies: Collect bacterial cells, sonicate to disrupt, centrifuge to collect the supernatant, filter the pET 42b-T2-9 / BL21(DE3) supernatant through a 0.22 μm filter membrane, and then pass it through AKTA-FPLC with 1 mL HisTrap. TM HP column purification: Samples were loaded at a flow rate of 1 mL / min, and impurities were eluted with Tris-HCl (pH 8.0) containing 40 mM imidazole. The target protein was then eluted with Tris-HCl (pH 8.0) containing 250 mM imidazole to obtain His-T2-9. The His-tagged nanobody (His-T2-9) was then dialyzed overnight at 4°C in 50 mM Tris-HCl (pH 8.0) buffer to obtain the target protein. The purity of the target protein was detected by SDS-PAGE electrophoresis, and the results are as follows: Figure 1As shown, lanes 1-5 represent the precipitate, supernatant, flow-through buffer after nickel column chromatography, His-T2-9 purified by affinity chromatography, and His-T2-9 purified by dialysis, respectively, after pET 25b-T2-9 / BL21(DE3) induction and fragmentation. Lanes 4 and 5 show that high-purity thermosensitive fusion nanobody His-T2-9 was successfully obtained. 4. Preparation of GST-tagged nanobodies: Bacterial cells were collected, sonicated, and the supernatant was collected by centrifugation. The pET 42b-T2-9 / BL21(DE3) supernatant was filtered through a 0.22 μm filter and purified by AKTA-FPLC using a 1 mL GSTrap™ FF column. The sample was loaded at a flow rate of 1 mL / min and equilibrated with 25 mM Tris, 100 mM NaCl, pH 8.0 buffer. The target protein was eluted with 25 mM Tris, 100 mM NaCl, pH 8.0, 10 mM GSH buffer. The GST-tagged nanobody (GST-T2-9) was dialyzed overnight at 4°C in 50 mM Tris-HCl (pH 8.0) buffer to obtain the target protein. The purity of the target protein was detected by SDS-PAGE electrophoresis. The results are shown below. Figure 2 As shown, lanes 1-4 are respectively the supernatant, precipitate, flow-through after GST chromatography, and GST-T2-9 purified by GST chromatography after pET 42b-T2-9 / BL21(DE3) induction and fragmentation. The results of lane 4 show that high-purity thermosensitive fusion nanobody GST-T2-9 was obtained. 5. Determination of the thermal stability of thermosensitive fusion nanobodies The two types of purified and dialyzed thermosensitive fusion nanobodies (protein concentration 1 mg / ml) were incubated at 37℃, 60℃ and 95℃ for 15 min, respectively. After incubation, they were centrifuged at 13000 rpm and 4℃, and the supernatant was collected. The protein concentration was determined using NANODROP ONEC (Thermo Scientific). The results are shown in Table 1. The ST-tagged nanobody GST-T2-9 is sensitive to high temperature. As the temperature increases, the protein denaturation rate gradually increases.

[0020] Table 1. Denaturation rate of thermosensitive fused nanobodies at different temperatures

[0021] Example 2 This embodiment provides the preparation of dual-antibody blocked Taq DNA polymerase, the steps of which are as follows: Take Taq DNA polymerase (TaKaRa Ex Taq DNA Polymerasr, RR001A), commercial Taq monoclonal antibody (Z087-03), and purified thermosensitive fusion nanobody GST-T2-9 prepared in Example 1, mix them at a molar ratio of 1:0.5:1, and place them at 20-25℃ for 30 min to prepare dual-antibody blocked Taq DNA polymerase.

[0022] Example 3 Taq DNA polymerase (TaKaRa Ex Taq DNA Polymerasr, RR001A), commercial Taq monoclonal antibody (Z087-03), and purified thermosensitive fusion nanobody GST-T2-9 prepared in Example 1 were mixed at a molar ratio of 1:0.5:0.5 and placed at 20-25℃ for 30 min to prepare double antibody-blocked Taq DNA polymerase.

[0023] Comparative Example 1 1. Preparation: Taq DNA polymerase (TaKaRa Ex Taq DNA Polymerasr, RR001A) was mixed with Taq DNA polymerase monoclonal antibody (nearshore protein, Z087-03) and thermosensitive fusion nanobody GST-T2-9 at a molar ratio of 1:1. The mixtures were then placed at 20-25℃ for 30 min to obtain Taq DNA polymerase blocked with monoclonal antibody and Taq DNA polymerase blocked with nanobody, respectively.

[0024] Example 4 This embodiment is a performance test of the blocked Taq DNA polymerases prepared in Examples 2 and 3 and Comparative Example 1.

[0025] The testing platform uses the Flash 10 fully automated nucleic acid detection and analysis system from Kaiyu Biotechnology (Beijing) Co., Ltd., and the detection system and procedures adopt the nucleic acid detection system for novel coronavirus 2019-nCoV and influenza A and B (Kayu Biotechnology (Beijing) Co., Ltd.).

[0026] The amplification curve of the Taq DNA polymerase blocked with the monoclonal antibody prepared in Comparative Example 1 after being tested on the platform is shown below. Figure 3As shown in Table 2, the fluorescence signal values ​​indicate that the fluorescence signal of the Taq DNA polymerase blocked by GST-T2-9 is stronger than that of the Taq DNA polymerase blocked by monoclonal antibody in the FAM channel (influenza A), but weaker in the HEX channel (influenza B), ROX channel (COVID-19), and CY5 channel (internal reference of the human genome). The CT value differences are shown in Table 2, and the CT values ​​of the Taq DNA polymerase blocked by GST-T2-9 are all lower than those of the Taq DNA polymerase blocked by monoclonal antibody.

[0027] Table 2 Detection results of fluorescence signals of different antibodies

[0028] The amplification curve of the double-blocked Taq DNA polymerase prepared in Example 2 after being tested on the platform is shown below. Figure 4 As shown, using the thermosensitive nanobody prepared in Comparative Example 1 as a control, the fluorescence signal results showed that the fluorescence signals of the Taq DNA polymerase blocked by GST-T2-9 in the FAM channel (influenza A), HEX channel (influenza B), ROX channel (COVID-19), and CY5 channel (internal reference of the human genome) were all stronger than those of the Taq DNA polymerase blocked by monoclonal antibody; the differences in CT values ​​are shown in Table 3, and the CT values ​​of the double antibody blocking were 0.1~0.2 higher than those of the monoclonal antibody.

[0029] Table 3 Detection results of fluorescence signals of different antibodies

[0030] The amplification curve of the double-blocked Taq DNA polymerase prepared in Example 3 after being tested on the platform is shown in the figure below. Figure 5 As shown in the results, the fluorescence signal values ​​indicate that the fluorescence signals of Taq DNA polymerase blocked by GST-T2-9 are stronger than those blocked by monoclonal antibody in the FAM channel (influenza A), HEX channel (influenza B), ROX channel (COVID-19), and CY5 channel (internal reference of the human genome). The differences in CT values ​​are shown in Table 4. The CT value of the double antibody blocking method is 0.1 to 0.2 higher than that of the monoclonal antibody blocking method.

[0031] Table 4 Detection results of fluorescence signals of different antibodies

[0032] 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 diabody-encapsulated Taq DNA polymerase, characterized in that, The Taq DNA polymerase is blocked by a Taq DNA polymerase monoclonal antibody and a heat-sensitive fusion nanobody; the heat-sensitive fusion nanobody comprises a heat-labile protein tag and a nanobody capable of binding to the 5'-3' polymerization activity domain of the Taq DNA polymerase, and the amino acid sequence of the nanobody is shown as SEQ ID NO:

1.

2. The dual antibody-encapsulated Taq DNA polymerase according to claim 1, wherein, The heat-labile protein tag is a GST tag or an MBP tag.

3. The method of constructing a diabody to inactivate Taq DNA polymerase according to claim 1, wherein, The method comprises the following steps: S1, constructing a recombinant plasmid capable of expressing a heat-sensitive fusion nanobody, transforming the recombinant plasmid into an expression cell and inducing expression, and purifying to obtain the heat-sensitive fusion nanobody; S2, mixing the heat-sensitive fusion nanobody, the Taq DNA polymerase monoclonal antibody and the Taq DNA polymerase in a certain proportion, incubating at 20-25 DEG C, and achieving double-antibody blocking of the Taq DNA polymerase.

4. The method for constructing a diabody to inactivate Taq DNA polymerase according to claim 3, wherein, The expression cell in step S1 is an E. coli or a yeast cell.

5. The method for constructing a diabody to inactivate Taq DNA polymerase according to claim 4, wherein, The expression vector of the recombinant plasmid in step S1 is one of a pET series, a pBAD-His or a pGEX series plasmid vector.

6. The method for constructing a diabody to inactivate Taq DNA polymerase according to claim 4, wherein, The molar ratio of the Taq DNA polymerase, the monoclonal antibody and the heat-sensitive fusion nanobody is 1:0.5:(0.5-1).

7. Use of the bispecific antibody blocking Taq DNA polymerase according to claim 1 for the preparation of a nucleic acid detection kit, characterized in that, The nucleic acid detection comprises conventional PCR, real-time fluorescent quantitative PCR, rapid nucleic acid detection or multiplex nucleic acid detection.

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