A recombinant DNA polymerase, methods of making, kits, and use in probe-based qPCR amplification

Abstract

The application belongs to the technical field of molecular biology, and discloses a recombinant DNA polymerase, a preparation method, a kit and application in probe qPCR amplification. The recombinant DNA polymerase provided by the application comprises an amino acid fragment as shown in SEQ ID NO: 1 and / or a variant sequence with more than 90 % homology with the amino acid fragment shown in SEQ ID NO: 1. The recombinant DNA polymerase has excellent anti-inhibition ability and thermal stability, can be well applied to probe qPCR to realize qualitative and quantitative detection of a target sequence in a sample to be detected, especially has good detection effect on complex samples, and has important significance in the application in the fields of clinical diagnosis, food safety detection and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology, and in particular relates to a recombinant DNA polymerase, a preparation method, a kit and an application in probe-based qPCR amplification. Background Art

[0002] qPCR technology refers to a technology that introduces fluorescent chemicals on the basis of traditional polymerase chain reaction. It achieves quantitative detection of amplification products by detecting changes in the intensity of fluorescent signals in the reaction system. It is widely used in various fields of life sciences such as clinical disease diagnosis, environmental sample testing, food safety testing, and biological research.

[0003] Based on how the fluorescent signal is generated in qPCR technology, it can be divided into dye-based qPCR and probe-based qPCR. Dye-based qPCR is low-cost, but the nonspecific amplification present in the reaction has a significant impact on test results, resulting in a high false-positive rate. Compared to dye-based qPCR, probe-based qPCR utilizes the specific binding ability of probes containing fluorescent chemicals to the target sequence, effectively reducing the impact of nonspecific amplification present in the reaction on test results. It has the advantages of high specificity, high accuracy, and short detection time, and is therefore more widely used in fields such as biomedical diagnosis, pathogen detection, agricultural science, and food safety.

[0004] Currently, the DNA polymerase used in probe-based qPCR is Taq DNA polymerase, which possesses 5'-3' exonuclease activity and can degrade the probe bound to the target sequence during DNA chain extension, separating the fluorescent quencher and reporter groups, thereby generating a fluorescent signal. However, Taq DNA polymerase has poor inhibition resistance and less than ideal thermal stability, making it difficult to apply to probe-based qPCR detection of complex samples, resulting in significant limitations. Summary of the Invention

[0005] The first purpose of the present invention is to provide a recombinant DNA polymerase in order to solve the problems of poor inhibition resistance and low thermal stability of DNA polymerase in the existing probe-based qPCR.

[0006] The second object of the present invention is to provide a method for preparing the above-mentioned recombinant DNA polymerase.

[0007] The third object of the present invention is to provide the use of the above-mentioned recombinant DNA polymerase in probe-based qPCR amplification.

[0008] Specifically, the recombinant DNA polymerase provided by the present invention includes an amino acid fragment as shown in SEQ ID NO: 1 and / or a variant sequence having more than 90% homology with the amino acid fragment as shown in SEQ ID NO: 1.

[0009] Furthermore, the amino acid sequence of the recombinant DNA polymerase is shown in SEQ ID NO: 1.

[0010] The preparation method of the above-mentioned recombinant DNA polymerase provided by the present invention comprises: S1, taking the coding gene of the recombinant DNA polymerase and recombining it into a plasmid vector to obtain a recombinant plasmid; S2, taking the recombinant plasmid and introducing it into a host cell to obtain an enzyme expression strain; S3, taking the enzyme expression strain and inducing expression to obtain the recombinant DNA polymerase.

[0011] Furthermore, in step S1, the nucleotide sequence of the gene encoding the recombinant DNA polymerase is shown in SEQ ID NO: 2.

[0012] Furthermore, in step S1, the plasmid vector is selected from one or more of pET-28a, pET-24a, pET-24b and pET-24c.

[0013] Furthermore, in step S2, the host cell is selected from one or more of Escherichia coli DH5α, Escherichia coli BL21 and Escherichia coli BL21 (DE3).

[0014] Furthermore, in step S3, the induced expression includes: taking the enzyme expression strain for expansion culture to obtain a seed solution; taking an inducing expression agent and adding it to the seed solution for induced expression culture to obtain the recombinant DNA polymerase.

[0015] Furthermore, the inoculation amount of the enzyme expression strain in the expanded culture is 1-3%, the temperature is 35-40° C., and the time is 12-48 hours.

[0016] Furthermore, the inducing agent is IPTG, and the final concentration of the addition is 0.05-0.2 mM.

[0017] Furthermore, the induced expression culture temperature is 25-30°C and the time is 16-18 hours.

[0018] The present invention also provides the use of the recombinant DNA polymerase in probe-based qPCR amplification.

[0019] The probe method qPCR amplification reaction kit provided by the present invention comprises the above-mentioned recombinant DNA polymerase.

[0020] Beneficial effects:

[0021] The recombinant DNA polymerase provided by the present invention includes an amino acid fragment as shown in SEQ ID NO: 1 and / or a variant sequence having more than 90% homology with the amino acid fragment as shown in SEQ ID NO: 1. The recombinant DNA polymerase has excellent anti-inhibition ability and thermal stability, and can be well applied to probe-based qPCR to achieve qualitative and quantitative detection of target sequences in test samples, especially for complex samples. It has good detection effect and is of great significance in applications in clinical diagnosis, food safety testing and other fields. DETAILED DESCRIPTION

[0022] With the goal of "obtaining a DNA polymerase that combines 5'-3' exonuclease activity with excellent inhibition resistance and thermal stability for application in probe-based qPCR, particularly with good detection results for complex samples," the inventors, after extensive and in-depth research, creatively fused a domain with 5'-3' exonuclease catalytic activity to a structurally optimized KOD DNA polymerase and performed structural optimization, thereby obtaining the recombinant DNA polymerase provided by the present invention. This recombinant DNA polymerase not only possesses good 5'-3' exonuclease activity but also excellent inhibition resistance and thermal stability.

[0023] In the present invention, the recombinant DNA polymerase comprises an amino acid fragment as shown in SEQ ID NO: 1 and / or a variant sequence having more than 90% homology with the amino acid fragment as shown in SEQ ID NO: 1. The variant sequence is obtained by substituting, deleting, or adding one or more amino acids to the amino acid sequence as shown in SEQ ID NO: 1, and the substitution, deletion, or addition does not affect the biological activity of the recombinant DNA polymerase.

[0024] In some specific embodiments, the recombinant DNA polymerase preferably includes an amino acid fragment with a sequence as shown in SEQ ID NO: 1 and a His tag connected to the N-terminus or C-terminus of the amino acid fragment shown in SEQ ID NO: 1. In this case, the His tag introduced into the recombinant DNA polymerase facilitates its purification, thereby achieving the effect of improving the purity of the recombinant DNA polymerase.

[0025] In some specific embodiments, the amino acid sequence of the recombinant DNA polymerase is preferably as shown in SEQ ID NO: 1. In this case, the spatial structure of the recombinant DNA polymerase is more ideal, and it has better enzyme activity, anti-inhibition ability and thermal stability.

[0026] Based on the purpose of obtaining the above-mentioned recombinant DNA polymerase, the present invention also provides a method for preparing the recombinant DNA polymerase. The preparation method comprises: S1, recombining the encoding gene of the recombinant DNA polymerase into a plasmid vector to obtain a recombinant plasmid; S2, introducing the recombinant plasmid into a host cell to obtain an enzyme-expressing strain; S3, inducing expression in the enzyme-expressing strain to obtain the recombinant DNA polymerase.

[0027] In the present invention, the nucleotide sequence of the gene encoding the recombinant DNA polymerase can be deduced based on the amino acid sequence of the recombinant DNA polymerase, and due to the degeneracy of the codons, it has diversity. Those skilled in the art can make adaptive designs based on actual conditions. The present invention does not impose any particular limitation on the nucleotide sequence of the gene encoding the recombinant DNA polymerase.

[0028] In some specific embodiments, the nucleotide sequence of the gene encoding the recombinant DNA polymerase is shown in SEQ ID NO: 2. In this case, the nucleotide sequence of the gene encoding the recombinant DNA polymerase is codon-optimized for Escherichia coli, so that the recombinant DNA polymerase can be better expressed in Escherichia coli, a common host cell for genetic engineering, thereby obtaining a large amount of recombinant DNA polymerase.

[0029] In the present invention, the plasmid vector is a type of small circular double-stranded DNA molecule that is independent of the chromosomal DNA of the host cell and can be replicated and transcribed in the host cell. It is a tool commonly used in genetic engineering. Those skilled in the art can make adaptive choices based on actual needs. The present invention does not impose any particular limitation on the plasmid vector.

[0030] In some specific embodiments, specific examples of the plasmid vector include but are not limited to one or more of pET-28a, pET-24a, pET-24b and pET-24c.

[0031] In the present invention, "recombining the encoding gene of the recombinant DNA polymerase into a plasmid vector" means using a restriction enzyme to cut the encoding gene of the recombinant DNA polymerase and the plasmid vector so that the two produce homologous ends, and using the homologous ends to achieve splicing of the recombinant DNA polymerase and the plasmid vector to obtain the recombinant plasmid. This is a technique commonly used in the field of genetic engineering and is not particularly limited in the present invention.

[0032] In the present invention, the host cell is a cell used to carry and express the gene encoding the recombinant DNA polymerase, which has a protein expression system for synthesizing the recombinant DNA polymerase. Specifically, it can be a prokaryotic cell such as Escherichia coli, or a eukaryotic cell such as yeast or mammalian cells. It is a tool commonly used in the field of genetic engineering. Those skilled in the art can make adaptive choices according to actual needs. The present invention does not impose any particular limitation on the host cell.

[0033] In some specific embodiments, specific examples of the host cell include, but are not limited to, one or more of Escherichia coli DH5α, Escherichia coli BL21, and Escherichia coli BL21 (DE3).

[0034] In the present invention, "introducing the recombinant plasmid into the host cell" means temporarily destroying or changing the cell membrane of the host cell by using chemical reagents, gene guns, electroporation, viral vector-mediated infection, liposome-mediated infection and other technical means to allow the recombinant plasmid to enter the cell and allow the host cell to express the recombinant DNA polymerase. This is a technique commonly used in the field of genetic engineering and is not particularly limited in the present invention.

[0035] In the present invention, the induced expression is to activate the regulatory elements on the recombinant plasmid using specific induction conditions, thereby initiating the expression of the gene encoding the recombinant DNA polymerase in the host cell. It is a technology commonly used in the field of genetic engineering and is not particularly limited in the present invention.

[0036] In the present invention, the induced expression preferably includes: taking the enzyme expression strain for expansion culture to obtain seed liquid; taking an inducing expression agent and adding it to the seed liquid for induced expression culture to obtain the recombinant DNA polymerase.

[0037] In the present invention, the expanded culture is for activating the enzyme-expressing strain and also makes the cell density in the culture medium in a relatively ideal state, which is conducive to the subsequent induced expression culture. It is a technology commonly used in the field of genetic engineering and is not particularly limited in the present invention.

[0038] In some specific embodiments, the conditions for the expanded culture include that the inoculation amount of the enzyme expression strain is preferably 1-3%, such as 1%, 1.05%, 1.3%, 1.5%, 2%, 2.5%, 3% or any value therebetween; the temperature is preferably 35-40°C, such as 35°C, 36°C, 37°C, 38°C, 39°C, 40°C or any value therebetween; and the time is 12-48h, such as 12h, 14h, 18h, 20h, 25h, 30h, 38h, 40h, 48h or any value therebetween.

[0039] In the present invention, the inducible expression agent is used to activate the regulatory elements on the recombinant plasmid, thereby initiating the expression of the coding gene of the recombinant DNA polymerase in the host cell. This is a technology commonly used in the field of genetic engineering and is not particularly limited in the present invention.

[0040] In some specific embodiments, the expression inducing agent is preferably IPTG, and its final added concentration is preferably 0.05-0.2 mM, such as 0.05 mM, 0.08 mM, 0.1 mM, 0.13 mM, 0.18 mM, 0.2 mM or any value therebetween.

[0041] In some specific embodiments, the conditions for the induced expression culture include a temperature preferably of 25-30°C, such as 25°C, 26°C, 28°C, 29°C, 30°C or any value therebetween; and a time preferably of 16-18h, such as 16h, 16.3h, 17h, 17.5h, 18h or any value therebetween.

[0042] Based on the 5'-3' exonuclease activity, excellent anti-inhibition ability and thermal stability of the above-mentioned recombinant DNA polymerase, the present invention also provides the use of the recombinant DNA polymerase in probe-based qPCR amplification.

[0043] Based on the application of the above-mentioned recombinant DNA polymerase in probe-based qPCR amplification, the present invention further provides a probe-based qPCR amplification reaction kit, which includes the above-mentioned recombinant DNA polymerase.

[0044] The embodiments of the present invention are described in detail below. The examples of the embodiments are intended to explain the present invention and are not to be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in this area or according to the product specifications. Reagents or instruments used that do not specify the manufacturer are all conventional products that can be obtained commercially.

[0045] The amino acid and nucleotide sequences involved in the present invention are shown in Table 1.

[0046] Table 1.

[0047]

[0048] Example 1

[0049] This example is used to illustrate a recombinant DNA polymerase and its preparation. The amino acid sequence of the recombinant DNA polymerase is shown in SEQ ID NO: 1. Its preparation specifically includes:

[0050] 1. Preparation of recombinant plasmid: (1) Based on the amino acid sequence of the recombinant DNA polymerase, perform codon optimization in E. coli and synthesize the recombinant DNA polymerase encoding gene (nucleotide sequence shown in SEQ ID NO: 2);

[0051] (2) Adding NdeⅠ and XhoⅠ restriction sites to both ends of the recombinant DNA polymerase encoding gene, synthesizing it, and digesting it with NdeⅠ and XhoⅠ enzymes before recombining it into the His-tagged vector pET24a(+) to obtain a recombinant plasmid;

[0052] (3) The recombinant plasmid was transformed into Escherichia coli DH5α using a bacterial transformation kit (Thermo Fisher Scientific, catalog number K2711) and referring to the instructions. The recombinant plasmid was placed in LB liquid medium (Solebo, catalog number L1010) and cultured overnight at 37°C and 200 rpm. The plasmid was extracted from the culture medium using a plasmid extraction kit (Merck, catalog number PLN350) and referring to the instructions to obtain a large amount of recombinant plasmid.

[0053] 2. Preparation of enzyme expression strain: The recombinant plasmid was transformed into Escherichia coli BL21 (DE3) using a bacterial transformation kit, and the transformed cells were inoculated into LB liquid culture medium containing 50 mg / L kanamycin sulfate. The culture was shaken at 37°C and 200 rpm overnight to obtain the enzyme expression strain.

[0054] 3. Preparation of recombinant DNA polymerase: (1) Inoculate 1% of the expression strain into fresh LB liquid medium containing 50 mg / L kanamycin sulfate, and culture at 37°C and 200 rpm for 12 h to obtain seed solution;

[0055] (2) Adjust the OD of the seed solution 600 to 0.8, then add IPTG with a final concentration of 0.1 mmol / mL, and culture at 18°C ​​and 200 rpm for 16 h to obtain the induced expression culture medium;

[0056] (3) Take the induced expression culture medium and centrifuge it at 4°C and 10,000 rpm for 10 minutes to collect the cell mud; resuspend 1 g of cell mud in 5 mL of 50 mM Tris-HCl buffer (pH = 7.5), and ultrasonically disrupt the cells in an ice bath for 10 minutes in the form of working 4 seconds and resting 4 seconds. Centrifuge it at 4°C and 10,000 rpm for 10 minutes, and collect the supernatant to obtain the crude enzyme solution;

[0057] (4) Purify the crude enzyme solution using a Ni-NTA affinity chromatography column (Shanghai Biotech, catalog number C597593) according to the instructions to obtain a pure enzyme solution, which was then freeze-dried at -80°C to obtain recombinant DNA polymerase.

[0058] Example 2

[0059] This example is used to illustrate the thermal stability of the recombinant DNA polymerase provided in Example 1. The commercially available Taq DNA polymerase (Yingxin Laboratory, catalog number TX20089) was used for testing. The test specifically includes:

[0060] (1) Recombinant DNA polymerase and Taq DNA polymerase were mixed with nuclease-free water to prepare a test enzyme solution with a concentration of 0.05 μg / μL. The test enzyme solution was incubated at 90°C for 0, 2, 4, 6, 8 and 10 h. The enzyme-catalyzed reaction was carried out at 74°C for 30 min using salmon sperm DNA as a template / primer. After the reaction was completed, the solution was immediately placed on ice to terminate the reaction.

[0061] The reaction system of the enzyme-catalyzed reaction includes 1 ng of salmon sperm DNA, 200 μmol / L of dNTPs, 1 μCi [α-32P] of dCTP, 20 mmol / L of Tris-HCl, 2 mmol / L of MgCl2, 5 mmol / L of (NH4)2SO4, 10 mmol / L of KCl, 0.1 g / L of BSA and 0.05% of Triton X-100, pH 8.0.

[0062] (2) 5 μL of the reaction product was added to an ion exchange paper disc. After the spot dried, it was rinsed three times with 2×SSC buffer (containing 300 mmol / L NaCl and 30 mmol / L Na3C6H5O7, pH 7.0). Finally, it was washed once with anhydrous ethanol after an ice bath and dried. The infiltrated radioactive material was measured using a liquid scintillation counter. Among them, 1 unit of enzyme activity (1U) is defined as the amount of enzyme that catalyzes the incorporation of 10 nmol of dNTPs into DNA. The results are shown in Table 2.

[0063] Table 2.

[0064]

[0065] The test results shown in Table 2 indicate that, compared to the known Taq DNA polymerase, the recombinant DNA polymerase provided in Example 1 of the present invention has an enzyme activity of up to 16.59 U / mL, indicating excellent enzyme activity. Furthermore, the recombinant DNA polymerase has a half-life of approximately 8 hours at 90°C, indicating excellent thermal stability.

[0066] Example 3

[0067] This example is used to illustrate the anti-inhibition ability of the recombinant DNA polymerase provided in Example 1, and uses commercially available Taq DNA polymerase (Yingxin Laboratory, catalog number TX20089). The test specifically includes:

[0068] (1) Recombinant DNA polymerase and Taq DNA polymerase were mixed with nuclease-free water to prepare a test enzyme solution with a concentration of 0.05 μg / μL;

[0069] (2) Take the vector pUC19 and mix it with nuclease-free water to prepare a concentration of 1×10 6 copies / μL of the DNA sample to be tested I; 1 mL of the collected sputum was mixed with 0.1 mL of sodium hydroxide solution (0.1 M), liquefied for 30 min, and 10 mL of Tris-HCl buffer (1 mM, pH = 7.5) was added and mixed evenly to obtain the interference solution; the final concentration was 1×10 6 The vector pUC19 was added in an amount of 100 copies / μL and mixed with the interfering solution to obtain the DNA sample II to be tested;

[0070] (3) qPCR amplification was performed on the DNA sample I and the DNA sample II using the primer-probe system shown in Table 3, and the CT value of the sample was obtained based on the measured fluorescence signal.

[0071] Table 3.

[0072]

[0073] The qPCR amplification reaction system consisted of 2 μL of DNA sample I (or II), 0.4 μL of 10 mM dNTPs, 10 μL of 2× PCR buffer, 0.4 μL of each upstream and downstream primer (10 μM), 0.4 μL of 10 μM probe, 1 μL of enzyme solution, and ddH₂O to a final volume of 20 μL. The PCR protocol consisted of a 3-min initial denaturation at 95°C followed by 45 cycles of denaturation at 95°C for 10 s and annealing and extension at 55°C for 30 s. The results are shown in Table 4.

[0074] Table 4.

[0075]

[0076] The test results shown in Table 4 indicate that, compared to the existing Taq DNA polymerase, the recombinant DNA polymerase provided in Example 1 of the present invention has a lower CT value for the test DNA sample I with the same copy number, and still has a good detection effect for the test DNA sample II to which the interfering solution is added. In other words, the recombinant DNA polymerase provided in Example 1 of the present invention has excellent DNA polymerase catalytic activity and good anti-inhibition ability.

[0077] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention without departing from the principles and purpose of the present invention.

Claims

1. A recombinant DNA polymerase, characterized in that The amino acid sequence of the recombinant DNA polymerase is shown in SEQ ID NO:

1.

2. The method for preparing the recombinant DNA polymerase according to claim 1, wherein The preparation method comprises: S1, taking the coding gene of the recombinant DNA polymerase and recombining it into a plasmid vector to obtain a recombinant plasmid; S2, taking the recombinant plasmid and introducing it into a host cell to obtain an enzyme expression strain; S3, taking the enzyme expression strain and inducing expression to obtain the recombinant DNA polymerase.

3. The method for preparing the recombinant DNA polymerase according to claim 2, wherein In step S1, the nucleotide sequence of the gene encoding the recombinant DNA polymerase is shown in SEQ ID NO:

2.

4. The method for preparing a recombinant DNA polymerase according to claim 2, wherein In step S1, the plasmid vector is selected from one or more of pET-28a, pET-24a, pET-24b and pET-24c.

5. The method for preparing the recombinant DNA polymerase according to claim 2, wherein In step S2, the host cell is selected from one or more of Escherichia coli DH5α, Escherichia coli BL21 and Escherichia coli BL21 (DE3).

6. The method for preparing a recombinant DNA polymerase according to claim 2, wherein: In step S3, the induced expression includes: taking the enzyme expression strain for expansion culture to obtain a seed solution; taking an inducing expression agent and adding it to the seed solution for induced expression culture to obtain the recombinant DNA polymerase.

7. The method for preparing a recombinant DNA polymerase according to claim 6, wherein: The inoculation amount of the enzyme expression strain in the expanded culture is 1-3%, the temperature is 35-40° C., and the time is 12-48 hours.

8. The method for preparing a recombinant DNA polymerase according to claim 6, wherein: The inducing agent is IPTG, and the final concentration of the addition is 0.05-0.2 mM.

9. The method for preparing a recombinant DNA polymerase according to claim 6, wherein: The induction expression culture temperature is 25-30° C. and the time is 16-18 hours.

10. Use of the recombinant DNA polymerase according to claim 1 in probe-based qPCR amplification for non-therapeutic or non-diagnostic purposes.

11. A probe-based qPCR amplification reaction kit, characterized in that: The kit comprises the recombinant DNA polymerase according to claim 1.

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