A dCE-KOD DNA polymerase and its preparation method and application

Abstract

The present invention discloses a dCE-KOD DNA polymerase and a preparation method and application thereof. The dCE-KOD DNA polymerase is obtained by fusing a mutant dCE expressing colicin CE at the N-terminus of a KOD DNA polymerase protein sequence, wherein dCE is any one of dCE2, dCE7, dCE8 and dCE9. Compared with natural KOD DNA polymerase, the continuous synthesis ability of dCE-KOD DNA polymerase is effectively improved and it has higher sensitivity to templates. Therefore, the dCE-KOD DNA polymerase provided by the present invention has broad application prospects in gene cloning, high-fidelity amplification of long fragments and PCR diagnosis.

Description

Technical Field

[0001] The invention belongs to the fields of molecular biology and protein engineering, and specifically relates to a dCE-KOD DNA polymerase and a preparation method and application thereof. Background Art

[0002] Polymerase chain reaction (PCR) is the most rapidly popular and widely used molecular biology technology in recent decades. PCR is a gene manipulation technology that simulates the DNA replication process in vivo in vitro to amplify specific DNA fragments in large quantities. In the presence of template DNA, primers, and dNTPs, DNA polymerase can achieve exponential amplification of the target sequence within 2-3 hours by cyclic changes in reaction temperature. PCR technology is widely used in molecular cloning, genome sequencing, forensic identification, and diagnosis of genetic diseases and infectious diseases. It has promoted the development of precision medicine and accelerated the research on the correlation between genes and genetic diseases. However, PCR technology also faces many limitations. For example, blood and tissue samples cannot be used directly for PCR, and DNA needs to be further extracted as an amplification template. At the same time, the length and speed of PCR amplification need to be further improved. These needs drive people to continuously discover and transform DNA polymerases.

[0003] In 1988, Rand K Saiki first applied the thermostable Taq DNA polymerase from Thermus aquaticus YT-1 to PCR, which changed the cumbersome steps of constantly adding polymerase during the PCR cycle and greatly simplified the PCR process. However, since Taq DNA polymerase lacks 3'-5' exonuclease activity, it cannot correct mismatched single nucleotides in time during the in vitro synthesis of DNA, resulting in low fidelity of PCR products. Researchers have successively discovered a series of thermostable DNA polymerases with correction functions, such as Tgo, Vent, Deep, Pfu and KOD1, which significantly improve the fidelity of PCR amplification. However, in general, the role of the DNA polymerases commonly used in PCR in their natural hosts is mainly to repair DNA damage and assist in the completion of gene recombination, so their progressivity and extension rate are low. With the continuous advancement of medical testing and life science technology, there is an urgent need for strategies to improve the extension activity and extension rate of thermostable DNA polymerases while ensuring the sensitivity, specificity and fidelity of PCR amplification. KOD1 DNA polymerase is derived from the thermophilic bacterium Thermococcus kodakarensis KOD1. It has become a target of much attention for modification due to its good heat resistance, high fidelity and high processivity. The inventors hope to further improve its enzymatic properties through genetic engineering modification.

[0004] Colicin produced by Escherichia coli is a protein toxin encoded by a plasmid. The DNA enzymes of the colicin E family include CE2, CE7, CE8 and CE9, which are a class of highly conserved non-specific nucleases. Reports show that the DNA enzyme domains of the four CEs are located at the C-terminus of the protein molecule, about 15 kDa, and can be truncated for expression. At the same time, the dCE protein obtained by mutating the amino acids involved in DNA cleavage in the C-terminal DNA enzyme domain of CE still retains strong non-specific DNA binding activity, but does not cut DNA. At the same time, the preliminary work of the inventor's team found that dCE has good thermal stability. Summary of the invention

[0005] In view of this, the present invention attempts to fuse dCE protein with KOD DNA polymerase for expression, and for the first time finds that dCE can significantly improve the specific amplification efficiency of KOD DNA polymerase, providing a solution to the problem of low continuous synthesis ability of thermostable DNA polymerase.

[0006] The technical solution of the present invention is as follows:

[0007] The first aspect of the present invention provides a dCE-KOD DNA polymerase, specifically: a mutant dCE expressing colicin CE is fused to the N-terminus of the KOD DNA polymerase protein sequence, and the nucleic acid sequence of the dCE is shown in any one of SEQ ID NOs. 1-4.

[0008] The invention introduces a non-specific DNA binding protein dCE into KOD DNA polymerase, thereby significantly improving the specific amplification efficiency of KOD DNA polymerase, wherein dCE is transformed from the C-terminal DNA enzyme domain (CE2, CE7, CE8, CE9) of colicin and named dCE2, dCE7, dCE8, dCE9 respectively, so the inventor names the fusion protein obtained after fusing KOD DNA polymerase as dCE-KOD DNA polymerase.

[0009] Preferably, in the above dCE-KOD DNA polymerase, the KOD DNA polymerase protein sequence is derived from Thermococcus kodakarensis KOD1 strain, and its encoding gene is shown in SEQ ID NO.9.

[0010] More preferably, in the above-mentioned dCE-KOD DNA polymerase, the dCE-KOD DNA polymerase is any one of the following:

[0011] dCE2-KOD, whose amino acid sequence is as shown in SEQ ID NO.5 or an amino acid sequence with equivalent function formed by replacing, deleting or adding one or more amino acid residues in the sequence shown in SEQ ID NO.5; or

[0012] dCE7-KOD, whose amino acid sequence is as shown in SEQ ID NO.6 or an amino acid sequence with equivalent function formed by replacing, deleting or adding one or more amino acid residues in the sequence shown in SEQ ID NO.6; or

[0013] dCE8-KOD, whose amino acid sequence is as shown in SEQ ID NO.7 or an amino acid sequence with equivalent function formed by replacing, deleting or adding one or more amino acid residues in the sequence shown in SEQ ID NO.7; or

[0014] dCE9-KOD, whose amino acid sequence is shown in SEQ ID NO.8 or an amino acid sequence with equivalent function formed by replacing, deleting or adding one or several amino acid residues in the sequence shown in SEQ ID NO.8.

[0015] The amino acid sequences of dCE2-KOD, dCE7-KOD, dCE8-KOD and dCE9-KOD provided in the embodiments of the present invention all carry purification tags. It should be noted that, in the specific implementation process, the fusion protein with the same function as the dCE-KOD DNA polymerase of the present invention obtained by replacing or modifying the tag sequence according to actual needs still belongs to the protection scope of the present invention.

[0016] The second aspect of the present invention provides biological materials related to the dCE-KOD DNA polymerase of the present invention, including but not limited to the following materials:

[0017] a) a gene encoding the dCE-KOD DNA polymerase of the present invention;

[0018] b) a recombinant expression vector containing the gene described in a);

[0019] c) a recombinant cell containing the gene described in a) or the recombinant expression vector described in b) .

[0020] The third aspect of the present invention provides a method for preparing the dCE-KOD DNA polymerase of the present invention, comprising the following steps:

[0021] S1. Design and synthesize primers based on the gene sequence information of KOD DNA polymerase and dCE protein, and amplify the dCE-KOD DNA polymerase gene coding sequence with purification tags and restriction sites by Overlapping PCR technology;

[0022] S2, cloning the dCE-KOD DNA polymerase gene fragment obtained in step S1 into the expression vector pET23a to construct a recombinant expression vector pET23a-dCE-KOD;

[0023] S3. The recombinant expression vector pET23a-dCE-KOD was transformed into Escherichia coli and induced to express the target protein.

[0024] Preferably, the KOD DNA polymerase gene coding sequence in step S1 is as shown in SEQ ID NO.9, and the gene sequence of the dCE protein is as shown in any one of SEQ ID NOs.1-4.

[0025] The fourth aspect of the present invention provides the use of the dCE-KOD DNA polymerase of the present invention in DNA replication. The experimental data of the present invention show that, compared with the natural KOD DNA polymerase, the extension rate of the dCE-KOD DNA polymerase is doubled and the requirement for the template concentration is halved.

[0026] The fifth aspect of the present invention provides use of the dCE-KOD DNA polymerase of the present invention in preparing a PCR amplification reagent or a kit.

[0027] The above-mentioned PCR amplification reagent or kit may also include other reagents and / or consumables for PCR amplification.

[0028] Compared with the prior art, the beneficial effects of the present invention are:

[0029] (1) The present invention has carried out molecular biological transformation on the DNA polymerase in the nucleic acid amplification technology. After the transformation, the continuous synthesis ability of the polymerase is effectively improved, and the sensitivity to the template in the recombinase-mediated nucleic acid amplification reaction is improved, and the amplification rate is also doubled.

[0030] (2) Currently, blood or tissue samples are often difficult to use directly for PCR detection because the sample DNA template concentration is too low. Usually, purified DNA samples need to be extracted. The modified DNA polymerase of the present invention has higher sensitivity to the template and can significantly improve the DNA amplification effect of the recombinase-mediated nucleic acid amplification technology. Therefore, it is expected to achieve direct detection of blood or tissue samples.

[0031] (3) The present invention also provides a specific preparation method and application method of the modified polymerase, which lays a foundation for the practical application of dCE-KOD DNA polymerase. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1This is a schematic diagram of the structure of the recombinant plasmid pET23a-dCE7-KOD in an embodiment of the present invention;

[0033] Figure 2 The SDS-PAGE diagram of the samples during the recombinant expression and purification of dCE7-KOD in the embodiment of the present invention, in which lane M is a marker, lane 1 is a total protein supernatant after bacterial lysis, lane 2 is a precipitate after bacterial lysis, lane 3 is a supernatant after bacterial lysis, lane 4 is a supernatant after heat treatment, and lane 5 is a target protein (about 106 kDa);

[0034] Figure 3 is a schematic diagram of the PCR procedure used in Example 2 of the present invention;

[0035] Figure 4 1 is a comparison result of the amplification rates of KOD DNA polymerase and dCE-KOD DNA polymerase in the embodiment of the present invention, in which lane M is DNA Marker, lane 2 is a 2 kb fragment, lane 4 is a 4 kb fragment, lane 6 is a 6 kb fragment, lane 8 is an 8 kb fragment, and lane 10 is a 10 kb fragment;

[0036] Figure 5 1 is a comparative result diagram of the sensitivity of KOD DNA polymerase and dCE-KOD DNA polymerase to templates in the embodiment of the present invention. In the figure, lane M is DNA Marker, and lanes 1-12 are PCR results of different template concentrations (lane 1-2 is 0.125 ng, lane 3-4 is 0.25 ng, lane 5-6 is 0.5 ng, lane 7-8 is 1 ng, lane 9-10 is 2 ng, and lane 11-12 is 4 ng). DETAILED DESCRIPTION

[0037] The technical solution of the present invention will be described clearly and completely below in conjunction with the embodiments and drawings. It should be understood that the specific implementation methods described herein are only used to illustrate and explain the present invention and are not used to limit the present invention.

[0038] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this application belongs. The term "comprises" and any variation thereof in the description and claims of the present invention are intended to cover non-exclusive inclusions.

[0039] In order to solve the problem of low extension activity and extension speed of KOD DNA polymerase in the prior art, the present invention provides a new solution, specifically: dCE is fused and expressed at the N-terminus of the natural KOD DNA polymerase protein sequence, wherein dCE is specifically: dCE2 (amino acid sequence as shown in SEQ ID NO.1), dCE7 (amino acid sequence as shown in SEQ ID NO.2), dCE8 (amino acid sequence as shown in SEQ ID NO.3) and dCE9 (amino acid sequence as shown in SEQ ID NO.4).

[0040] If no specific techniques or conditions are specified in the following examples, all of them are carried out according to the techniques or conditions described in the literature in the field or according to the product instructions; if the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be obtained commercially.

[0041] The Escherichia coli BL21 (DE3) used in the following examples was purchased from Novagen, the primers used were commissioned to be synthesized by China Sangon Biotechnology Co., Ltd., the plasmid pET23a-dCE containing the dCE gene was commissioned to be synthesized by China Jinkairui Biotechnology Co., Ltd., and the vector pET23a used was purchased from Novagen.

[0042] Example 1 Preparation of dCE-KOD DNA polymerase

[0043] This example takes dCE7-KOD as an example to explain in detail the preparation process of dCE-KOD DNA polymerase.

[0044] (1) Construction of recombinant plasmid pET23a-dCE7-KOD.

[0045] The KOD DNA polymerase gene sequence was synthesized by Jinkairui Company, and a 6×His affinity tag was attached to the N-terminus of the encoded protein. The target gene was amplified by PCR using primer pairs KOD-his-F and KOD-R and cloned into the pET23a vector to construct a recombinant vector. The recombinant vector was named pET23a-KOD after sequencing verification. The corresponding gene fragments were synthesized according to the amino acid sequence of dCE7, and the KOD and dCE7 gene fragments were amplified using primer pairs dCE7-F / dCE7-R and KOD-his-F / KOD-R, respectively. T5 exonuclease-mediated gene cloning was performed through the homologous arms at both ends of the fragments, thereby connecting the dCE7 coding sequence to the 5' end of the KOD coding sequence. The recombinant plasmid was named pET23a-dCE7-KOD after DNA sequencing verification (see Figure 1 ).

[0046] Table 1 Primer information used to construct recombinant plasmid pET23a-dCE7-KO

[0047]

[0048] (2) E. coli recombinant expression and purification of dCE7-KOD, the specific steps are as follows:

[0049] ① The sequencing-verified recombinant expression plasmid pET23a-dCE7-KOD was transformed into Escherichia coli BL21 (DE3), and a single colony was picked and inoculated into LB liquid culture medium containing ampicillin, and cultured overnight at 37°C and 250 rpm.

[0050] ②Inoculate the bacterial solution into 400 mL of LB liquid medium containing ampicillin at a ratio of 1 / 100, and culture at 37°C and 250 rpm until the OD 600 When the value reached 0.6, IPTG (isopropyl-β-D-thiogalactoside) was added to a final concentration of 0.5 mM for induction. At the same time, an expression vector control group was set up, and the bacterial solution was taken after low-temperature induction at 18°C ​​for 16 hours, centrifuged at 6000 rpm for 10 minutes, and the supernatant was removed.

[0051] ③The bacterial precipitate was resuspended in Ni-NTA loading buffer (20 mM Tris-HCl, 0.5 M NaCl, 5 mM imidazole, pH 7.9), and after ultrasonic disruption, centrifuged at 14000 rpm for 30 min, and the supernatant and precipitate were collected for SDS-PAGE.

[0052] ④ Place the supernatant in a 75℃ hot water bath for 30 minutes, centrifuge at 14000rpm for 30 minutes, then add DNase and RNase to the supernatant and incubate at 37℃ for 30 minutes to remove endogenous nucleic acids. Place it in a 75℃ water bath for 15 minutes, centrifuge at 14000rpm for 30 minutes to remove the precipitate.

[0053] ⑤Use AKTA protein purification system, Ni Sepharose 6Fast Flow affinity chromatography column to affinity purify the target protein, loading buffer (20mM Tris-HCl, 0.5M NaCl, 5mM imidazole, pH 7.9), elution buffer (20mMTris-HCl, 0.5M NaCl, 200mM imidazole, pH 7.9). Collect protein elution peak samples and ultrafilter the protein samples at 4℃, and store them in Buffer A (20mM Tris-HCl pH 7.9, 100mM potassium glutamate, 0.1mM EDTA, 1mM DTT, 0.05% (v / v) Nonidet P40, 0.05% (v / v) Tween 20), and store them at -80℃ after quick freezing in liquid nitrogen.

[0054] The electrophoresis results of each sample during the purification of dCE7-KOD protein are shown in Figure 2 As shown in the figure, it can be seen that the present invention successfully prepared dCE7-KOD.

[0055] Example 2 d Detection of amplification rate of CE-KOD DNA polymerase

[0056] In this case, dCE7-KOD was used as an example to compare the amplification rates of KOD DNA polymerase before protein modification and dCE-KOD DNA polymerase after modification.

[0057] Using pRGEB32 plasmid as template, 1U of KOD DNA polymerase and dCE-KOD DNA polymerase were used to amplify 2kb, 4kb, 6kb, 8kb and 10kb fragments, respectively. The primers used are shown in Table 2.

[0058] Table 2 Primer information used to amplify pRGEB32 plasmid

[0059]

[0060] according to Figure 3 Set up the PCR program as shown.

[0061] After the reaction is completed, 4 μL of the sample is electrophoresed in a 0.8% agarose gel. Figure 4 The electrophoresis results showed that the extension rate of dCE7-KOD was faster than that of KOD DNA polymerase. dCE7-KOD could amplify a 10kb fragment in 60 seconds, while KOD DNA polymerase could only amplify a 6kb fragment. This shows that the fusion of dCE protein and KOD DNA polymerase doubled its extension rate.

[0062] Example 3d Detection of amplification sensitivity of CE-KOD DNA polymerase

[0063] In this case, dCE7-KOD was used as an example to compare the template concentrations of KOD DNA polymerase before protein modification and dCE-KOD DNA polymerase after modification.

[0064] Using pRGEB32 plasmid as template, the size of the PCR amplification product was 2 kb (the primers used were the same as those in Example 2). The reaction was carried out under the condition of decreasing the concentration of the template agent (continuous 2-fold dilution gradient). The PCR program was: pre-denaturation at 94°C for 2 min; denaturation at 98°C for 10 s, extension at 68°C for 1 min, 30 cycles; and finally extension at 68°C for 7 min, and storage at 4°C.

[0065] After the reaction is completed, 4 μL of the sample is electrophoresed in a 0.8% agarose gel. Figure 5The electrophoresis results showed that when the concentration of plasmid DNA was 1ng, the KOD DNA polymerase fused with dCE could amplify the target fragment, while the KOD polymerase required 2ng of plasmid DNA, and the amplification yield was far less than that of the dCE-KOD DNA polymerase. This shows that the KOD DNA polymerase fused with dCE has a half lower requirement for the template concentration than the natural KOD DNA polymerase.

[0066] In summary, the present invention effectively improves the extension rate and template sensitivity of the modified DNA polymerase by fusing and expressing dCE at the N-terminus of the KOD DNA polymerase protein sequence, and provides a new approach to solving the problem of low extension activity and extension rate of thermostable DNA polymerase.

[0067] It should be noted that the above embodiments are only part of the embodiments of the present invention rather than all the embodiments, and are only used to illustrate the technical solution of the present invention rather than to limit it. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

Claims

1. Use of dCE-KOD DNA polymerase in DNA replication, characterized in that: The mutant dCE7 of colicin CE was fused to the N-terminus of the KOD DNA polymerase protein sequence, the gene sequence of dCE7 was shown in SEQ ID NO.2, the KOD DNA polymerase was derived from the Thermococcus kodakarensis KOD1 strain, and the amino acid sequence of the dCE-KOD DNA polymerase was shown in SEQ ID NO.

6.

2. The use according to claim 1, characterized in that: The preparation method of the dCE-KOD DNA polymerase comprises the following steps: S1. Design and synthesize primers based on the gene sequence information of KOD DNA polymerase and dCE7, and amplify the dCE-KOD DNA polymerase gene coding fragment with purification tags and restriction sites by Overlapping PCR technology; S2, cloning the dCE-KOD DNA polymerase gene coding fragment obtained in step S1 into the expression vector pET23a to construct a recombinant expression vector pET23a-dCE-KOD; S3. The recombinant expression vector pET23a-dCE-KOD was transformed into Escherichia coli and induced to express the target protein.

3. The use according to claim 2, characterized in that: The gene sequence of the KOD DNA polymerase in step S1 is shown in SEQ ID NO.9.

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