A mutant of Tth DNA polymerase independent of Mn 2+ and a method for preparing the same and use thereof

Abstract

This invention discloses a method that does not depend on Mn 2+ This invention discloses a Tth DNA polymerase mutant, its preparation method, and its applications. First, it discloses a Tth DNA polymerase mutant with the amino acid sequence shown in SEQ ID NO.1. Further, it discloses the preparation method and applications of the aforementioned Tth DNA polymerase mutant. This invention's Tth DNA polymerase, through amino acid substitution, alters the enzyme's conformation, allowing for the production of Tth DNA polymerases in the absence of Mn. 2+ Under certain conditions, it exhibits reverse transcription and polymerase activity, retains its heat tolerance, and can efficiently reverse transcribe RNA templates with complex secondary structures or high GC content; at the same time, it eliminates dependence on Mn. 2+ The dependency on Mn avoids 2+ It eliminates the problems of high amplification mismatch rate and non-specific amplification, and also eliminates its inhibition of DNA polymerase activity, so it can be used for reverse transcription of crude extracts and is suitable for more application scenarios.

Description

Technical Field

[0001] This invention relates to the field of biotechnology. More specifically, it relates to a method that is independent of Mn. 2+ Tth DNA polymerase mutant, its preparation method and application. Background Technology

[0002] Reverse transcription, catalyzed by reverse transcriptase, synthesizes DNA strands using RNA as a template. The synthesized DNA strand is called complementary DNA (cDNA). With the continuous development of molecular biology, it is now possible to synthesize cDNA in vitro using reverse transcriptase. cDNA synthesis is perhaps the second most important technique in molecular biology today, after polymerase chain reaction (PCR) and its modification. It is widely used for cDNA library generation, cloning, quantitative RT-PCR, RACE (rapid-amplification of cDNA ends) technology, microarray analysis, RNA amplification, and other applications.

[0003] Reverse transcription typically involves the following steps: In the presence of annealing primers, reverse transcriptase binds to the RNA template and incorporates dNTPs to synthesize a complementary DNA strand, i.e., a cDNA strand. The reverse transcriptase then degrades the RNA template in the DNA:RNA complex using RNase H activity. DNA-dependent DNA polymerase activity recognizes single-stranded cDNA as a template and synthesizes a second-stranded cDNA, forming double-stranded cDNA. Throughout this process, reverse transcriptase is generally required to possess the following characteristics: RNA-dependent DNA polymerase activity, DNA-dependent DNA polymerase activity, RNase H activity, thermostability, and sustained synthesis capacity. Among these, the thermostability of reverse transcriptase is a crucial factor affecting cDNA synthesis; increasing the reaction temperature helps denature RNA with secondary structures and / or high GC content, enabling reverse transcriptase to read the sequence.

[0004] Currently, commercially available reverse transcriptases are mainly derived from retroviruses, such as mesothermic enzyme-dependent Moloney mouse leukemia virus reverse transcriptases (M-MuLV RTs) and avian myeloblast virus enzymes (AMV RTs). M-MuLV RTs are monomeric proteins containing an n-terminal polymerase domain and a c-terminal RNase H domain. Studies have shown that RNase H activity is not a necessary condition for the efficient synthesis of cDNA and thermostability of RTs. During cDNA synthesis, they simultaneously cleave the RNA template in the RNA:cDNA hybrid strand. Since the RNA template may be degraded before full-length reverse transcription is completed, some enzyme modification studies introduce mutations into the RNase H domain of the reverse transcriptase to reduce or even completely eliminate RNase H activity, thereby increasing the yield of long-chain cDNAs. Wild-type M-MuLV RTs are typically used at 37℃-42℃ and are rapidly inactivated at higher temperatures. Heterodimidal AMV RTs have even higher optimal temperatures (45℃-50℃, up to 60℃). Therefore, reverse transcriptases derived from retroviruses have limited ability to withstand high temperatures, which is not conducive to the reverse transcription of RNA templates with secondary structures and high GC content. Improving the thermal stability of reverse transcriptases derived from retroviruses remains an important part of enzyme evolution.

[0005] The Tth (Thermus thermophilus) DNA polymerase derived from the thermophilic bacterium Thermus thermophilus HB8 shares 88% homology with Taq DNA polymerase (Taq pol), but its characteristics are quite different from Taq pol. In Mg 2+ Under certain conditions, this enzyme possesses 5′-3′ DNA polymerase activity and 5′-3′ exonuclease activity, but lacks 3′-5′ exonuclease activity, and can be widely used in PCR amplification reactions. In Mn 2+ Under suitable conditions, this enzyme exhibits strong reverse transcription activity at 55-70℃, making it suitable for one-step RT-PCR reactions. It offers the following advantages: a) for one-step RT-qPCR, it avoids the inhibitory effect of commonly used reverse transcriptases like M-MLV on DNA polymerase; b) it has better thermostability, allowing reverse transcription at high temperatures, making it easier to open the secondary structure of the RNA template and improving reverse transcription efficiency; c) it is more resistant to PCR inhibitors than Taq polymerase, allowing for direct amplification of crude samples. However, because Tth DNA polymerase only exhibits activity in Mn... 2+ RTase activity is only exhibited in the presence of Mn, which limits many application scenarios. 2+This increases the error rate in the DNA amplification process, leading to false positive results such as nonspecific amplification, which limits its application in the field of molecular biology.

[0006] Therefore, it is necessary to develop a method that does not depend on Mn. 2+ Reverse transcriptases with good activity are better suited for reverse transcription reactions. Summary of the Invention

[0007] One object of the present invention is to provide a method that is independent of Mn 2+ The Tth DNA polymerase mutant, which in the absence of Mn 2+ It exhibits reverse transcription and polymerase activity under certain conditions, can withstand high temperatures, and is suitable for a wider range of complex applications requiring inhibitors.

[0008] Another object of the present invention is to provide a method for preparing the above-mentioned Tth DNA polymerase mutant and its application.

[0009] To achieve the above objectives, the present invention adopts the following technical solution: This invention first provides a method that is independent of Mn 2+ The Tth DNA polymerase mutant, the amino acid sequence of which is shown in SEQ ID NO.1.

[0010] The present invention relates to a mutant of Tth DNA polymerase, based on the wild-type Tth DNA polymerase (amino acid sequence shown in SEQ ID NO.3) having four single-point amino acid mutations, represented by letter-number-letter, where the first letter is the wild-type amino acid, the number is the amino acid sequence, and the second letter is the mutated amino acid. The specific mutations are: I709K (isoleucine mutated to lysine), R538H (arginine mutated to histidine), I616E (isoleucine mutated to glutamic acid), and Q459R (glutamine mutated to arginine).

[0011] The polynucleotide encoding the above-mentioned Tth DNA polymerase mutant is also within the scope of protection of this invention.

[0012] In a specific embodiment of the present invention, the polynucleotide encoding the above-mentioned Tth DNA polymerase mutant is shown in SEQ ID NO.2.

[0013] Recombinant vectors containing the aforementioned polynucleotides are also within the scope of protection of this invention.

[0014] In a specific embodiment of the present invention, the recombinant vector is pET-21a-TthM1; pET-21a-TthM1 is obtained by inserting the polynucleotide encoding the Tth DNA polymerase mutant as shown in SEQ ID NO.2 between the BamHI and XhoI restriction sites of the pET-21a plasmid, while keeping the other sequences of pET-21a unchanged.

[0015] Recombinant cells containing the above-mentioned polynucleotides or recombinant vectors are also within the scope of protection of this invention.

[0016] In a specific embodiment of the present invention, the host cell of the recombinant cell is a modified BL21 Escherichia coli.

[0017] In a preferred embodiment of the present invention, the host cell is a BL21(DE3) competent cell.

[0018] This invention further provides a method for preparing the above-mentioned Tth DNA polymerase mutant, the method comprising the following steps: a1) Construct a recombinant vector containing the polynucleotide shown in SEQ ID NO.2; a2) Transform the recombinant vector into host cells, induce expression, and obtain bacterial cells; a3) The bacterial cells were broken, centrifuged, and the supernatant was obtained. The supernatant was then purified to obtain the Tth DNA polymerase mutant.

[0019] In a specific embodiment of the present invention, the recombinant vector is the above-mentioned pET-21a-TthM1; the host cell is BL21(DE3) competent cells.

[0020] In a specific embodiment of the present invention, the method for constructing the recombinant vector pET-21a-TthM1 includes the following steps: b1) Using the cloning vector pET-21a-Tth containing the wild-type Tth DNA polymerase shown in SEQ ID NO.4 as a template, five PCR products were amplified by PCR reaction using BamH I-Taq-F / Q459R-R, Q459R-F / R538H-R, R538H-F / I616E-R, I616E-F / I709K-R, and I709K-F / Xho I-Taq-R as primers, respectively. b2) After the PCR products of the five segments were recovered from the gel, they were mixed in a molar ratio of 1:1:1:1:1 and amplified by PCR using BamHI-Taq-F / XhoI-Taq-R primers to obtain PCR products containing BamHI, four mutation sites and XhoI. b3) After double digestion with BamHI and XhoI and gel recovery, the pET-21a empty vector and PCR product were ligated using T4 ligase to obtain the ligation product; b3) The ligation product was transformed into E. coli DH5α competent cells, and positive clones were obtained through resistance selection; plasmids were extracted and sequenced to verify that the recombinant vector pET-21a-TthM1 was successfully constructed.

[0021] In a specific embodiment of the present invention, the purification includes nickel ion affinity chromatography and ion exchange chromatography.

[0022] The present invention further provides the use of the above-mentioned Tth DNA polymerase mutant, and / or, the above-mentioned polynucleotide, and / or, the above-mentioned recombinant vector, and / or, recombinant cells in reverse transcription reactions and / or in the preparation of reverse transcription reaction products.

[0023] The present invention further provides a reverse transcription kit comprising the above-mentioned Tth DNA polymerase mutant.

[0024] The beneficial effects of this invention are as follows: The present invention utilizes Tth DNA polymerase, which alters the enzyme conformation through amino acid substitution, in the absence of Mn. 2+ Under certain conditions, it exhibits both reverse transcription and polymerase activity, retaining the high-temperature tolerance of wild-type Tth DNA polymerase. It can efficiently reverse transcribe RNA templates with complex secondary structures or high GC content; simultaneously, it eliminates dependence on Mn... 2+ The dependency on Mn avoids 2+ It eliminates the problems of high amplification mismatch rate and non-specific amplification, and also eliminates its inhibition of DNA polymerase activity, so it can be used for reverse transcription of crude extracts and is suitable for more application scenarios. Attached Figure Description

[0025] The specific embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.

[0026] Figure 1 The results of SDS-PAGE electrophoresis of Tth DNA polymerase mutant and wild-type Tth DNA polymerase are shown.

[0027] Figure 2 Mg 2+ A comparison of the reverse transcription activities of different DNA polymerases when using primer pairs to detect ACTB03 under environmental conditions.

[0028] Figure 3 Mg 2+ A comparison of the reverse transcription activities of different DNA polymerases when using primer pairs to detect ACTB05 under environmental conditions.

[0029] Figure 4 The figure shows the effect of different NaCl concentrations (20 mM and 50 mM) on the reverse transcription activity of Tth DNA polymerase mutant and MLV-RT. Detailed Implementation

[0030] To more clearly illustrate the present invention, the following description, in conjunction with preferred embodiments and accompanying drawings, further explains the invention. Similar components in the drawings are indicated by the same reference numerals. Those skilled in the art should understand that the specific description below is illustrative rather than restrictive and should not be construed as limiting the scope of protection of the present invention.

[0031] Example 1: Preparation of Tth DNA polymerase mutant and reverse transcription activity test I. Sequence Design of Tth DNA Polymerase Mutants Through sequence alignment and structural analysis, the wild-type Tth DNA polymerase (sequence shown in SEQ ID NO.3, and its polynucleotide shown in SEQ ID NO.4) was modified by selecting specific amino acids for substitution. Specifically, isoleucine (Ile, I) at position 709 was mutated to lysine (Lys, K), arginine (Arg, R) at position 538 was mutated to histidine (His, H), isoleucine (Ile, I) at position 616 was mutated to glutamate (Glue, E), and glutamine (Gln, Q) at position 459 was mutated to arginine (Arg, R), thus obtaining the Tth DNA polymerase mutant (amino acid sequence shown in SEQ ID NO.1, and its polynucleotide shown in SEQ ID NO.2).

[0032] II. Preparation of Tth DNA polymerase mutants 1. Primer design Based on the above mutation sites, specific primers containing mutated bases (underlined bases) were designed, and the primers are shown in Table 1.

[0033] Table 1. Specific primer sequences containing mutant bases used.

[0034] 2. PCR amplification Using the cloned plasmid pET-21a-Tth (which contains the wild-type Tth DNA polymerase gene, the polynucleotide of which is shown in SEQ ID NO.4) as a template, PCR amplification was performed using 2×TranStart® FastPfu PCR SuperMix (purchased from TransGen Biotech) enzymes to obtain PCR amplification products containing 4 mutation sites. Segmented overlapping PCR technology was used to gradually introduce the target mutation sites, ultimately obtaining PCR amplification products containing complete mutation sequences. The specific operation is as follows: Five independent PCR reaction systems were set up, each corresponding to a specific primer pair (BamHI-Taq-F / Q459R-R, Q459R-F / R538H-R, R538H-F / I616E-R, I616E-F / I709K-R, I709K-F / XhoHI-Taq-R). Fragments containing consecutive mutation sites were amplified sequentially to achieve the stepwise introduction of mutation sites, ultimately obtaining five segmented PCR products (each containing one mutation site). The reaction system consisted of a total volume of 50 μL, including 25 μL of 2×TranStart® FastPfu PCRSuperMix, 1 μL of template plasmid, 1 μL each of forward and reverse primers (10 μM), and 22 μL of sterile deionized water. The reaction conditions were as follows: 98℃ pre-denaturation for 3 minutes; 98℃ denaturation for 10 seconds, 58℃ annealing for 30 seconds, 72℃ extension for 2 minutes, for 30 cycles; and a final extension at 72℃ for 10 minutes.

[0035] After separation of the five PCR fragments by 1% agarose gel electrophoresis, the target fragment was recovered using a DNA gel recovery kit (purchased from TransGen Biotech). The recovered target fragments were mixed at a molar ratio of 1:1:1:1:1, and the end primers BamH I-Tth-F and Xho I-Tth-R were added for overlap PCR to splice the complete Tth DNA polymerase mutant gene. The reaction conditions were the same as in the previous step. The product was detected by 1% agarose gel electrophoresis, and the PCR product of the target band of approximately 2500 bp was recovered.

[0036] 3. Carrier Construction After double digestion of the pET-21a vector and the recovered PCR product BamHⅠ and XhoⅠ, the linearized pET-21a vector and the target fragment were recovered using a DNA gel extraction kit and ligated using T4 ligase to obtain the ligation product.

[0037] The ligation product was transformed into 50 μl of cloned *E. coli* DH5α competent cells, plated on a plate containing ampicillin, and incubated overnight at 37°C. The next day, colonies were selected for sequencing to confirm the correct mutation site. Clones with correct sequencing were selected for plasmid extraction, yielding the recombinant vector pET-21a-TthM1 (obtained by inserting the polynucleotide encoding the Tth DNA polymerase mutant, as shown in SEQ ID NO. 2, between the BamHI and XhoI restriction sites of the pET-21a plasmid, while maintaining the other sequences of pET-21a unchanged).

[0038] 4. Protein expression The recombinant vector pET-21a-TthM1 was transformed into 50 μl of BL21(DE3) competent cells. After incubation on ice for half an hour, the cells were heat-shocked at 42°C for 30 s. The cells were then added to 50 μl of SOC medium and cultured in suspension at 37°C for 30 min. The cells were then spread onto ampicillin-containing plates and cultured overnight at 37°C. The next day, single colonies were picked and cultured in 20 ml of LB medium containing ampicillin for 6-8 h. 20 ml of the bacterial culture was then inoculated into 1 L of LB medium containing ampicillin and cultured in suspension at 37°C until the OD600 value reached 0.6. The bacterial culture temperature was then lowered to 25°C, and IPTG was added to a final concentration of 0.1 mM. Induction was continued for 16 h, and the cells were collected at 3000 rpm.

[0039] 5. Protein purification The bacterial cells were homogenized, and the supernatant was collected by high-speed centrifugation to obtain the crude enzyme solution. Since Tth DNA polymerase is a thermostable enzyme, resistant to temperatures above 90°C, heat denaturation can remove impurities from the crude enzyme solution. The crude enzyme solution was heated to 75°C and incubated for 30 min, followed by high-speed centrifugation to collect the supernatant. The Ni-NTA affinity chromatography column was equilibrated with buffer, and the supernatant was passed through the column. Elution was performed using a buffer containing 100 mM imidazole, and the protein solution was collected. Further purification of the protein was achieved using an anion exchange column. The protein bound to the anion exchange column under pH 8.0 conditions, and linear gradient elution was performed using 1 M NaCl buffer. The target protein was collected, and its purity was detected by SDS-PAGE electrophoresis. The target protein was dialyzed into storage buffer for subsequent activity testing.

[0040] SDS-PAGE electrophoresis results are as follows Figure 1 As shown, a high-purity Tth DNA polymerase mutant protein (named Tth DNA polymerase mutant M1, or Tth M1 for short) was obtained.

[0041] II. Reverse Transcription Activity Assay 1. In Mg 2+Reverse transcription activity assay under environmental conditions The test buffer consisted of 50 mM Tris-HCl pH 8.3, 75 mM KCl, 3 mM MgCl2, and 0.01 mM DTT. The test template was 1 ng of hela cell mRNA. Reverse transcription was performed using random primers (Anchored Oligo(dT)20 Primer). The reverse transcription system is shown in Table 2. 0.5 μg of the Tth DNA polymerase mutant was added to the system, along with an equal amount of wild-type Tth DNA polymerase as a negative control and an equal amount of MLV-RT (Transgen Biotech, AT101) as a positive control. The mixture was incubated at 55°C for 15 min to obtain cDNA products through reverse transcription.

[0042] Table 2. Reverse transcription activity assay system

[0043] The cDNA product was diluted 2-fold, and 4 μl was used as a template for qPCR amplification. Amplification efficiency was detected using two primer pairs: ACTB03 (composed of ACTB03-F shown in SEQ ID NO.15 and ACTB03-R shown in SEQ ID NO.16) and ACTB05 (composed of ACTB05-F shown in SEQ ID NO.13 and ACTB05-R shown in SEQ ID NO.14). The amplification steps were: 95℃ for 1 min, 95℃ for 5 s, 60℃ for 30 s, fluorescence signal acquisition, for a total of 40 cycles. The efficiency of reverse transcriptase was determined by the Ct value (a smaller Ct value indicates a higher initial reverse transcription yield).

[0044] Test results are as follows Figure 2 and Figure 3 As shown, in Mg 2+ In the environment, wild-type Tth DNA polymerase (represented as "TthWT" in the figure) does not have reverse transcription ability, while Tth DNA polymerase mutant (represented as "Tth M1" in the figure) has polymerization activity slightly lower than MLV-RT. In the samples detected by primer pair ACTB03, the Ct value of Tth DNA polymerase mutant is comparable to that of MLV-RT amplification. In the samples detected by primer pair ACTB05, the Ct value of Tth DNA polymerase mutant is slightly later than that of MLV-RT reverse transcription product.

[0045] 2. Reverse transcription activity assays were performed under different salt concentrations. The test buffers were prepared by adding NaCl to the following solutions: 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, and 0.01 mM DTT, to achieve final concentrations of 0 mM, 10 mM, 20 mM, 30 mM, 40 mM, and 50 mM. The test template was 1 ng of HeLa cell mRNA. Reverse transcription was performed using random primers, as shown in Table 2. 0.5 μg of the Tth DNA polymerase mutant was added to the system, along with an equal amount of wild-type Tth DNA polymerase as a negative control and an equal amount of MLV-RT as a positive control. The mixture was incubated at 55°C for 15 min to obtain cDNA products.

[0046] Using a 2-fold dilution of cDNA product as a template, the internal reference gene ACTB was amplified by qPCR. The initial cDNA yield was measured using the Ct value. The reaction mixture consisted of: 10 μl of 2×SYBR Green qPCR Master Super Mix (Transgen Biotech), 0.8 μl each of ACTB03-F / ACTB03-R primers, 4 μl of diluted cDNA, and 4.4 μl of Nuclease-Free Water. The reaction conditions were: 95℃ pre-denaturation for 1 min; 95℃ denaturation for 5 seconds, 60℃ annealing and extension for 30 seconds with fluorescence signal acquisition, and 72℃ extension for 2 minutes, for 40 cycles.

[0047] Test results are as follows Figure 4 As shown, MLV-RT tolerates an additional salt concentration of 20 mM. Adding NaCl above 20 mM significantly reduces amplification efficiency. Under 20 mM NaCl conditions, the amplification Ct value of the cDNA product after reverse transcription by MLV-RT is slightly lower than that of the Tth DNA polymerase mutant (represented as "Tth M1" in the figure), indicating that the reverse transcription activity of the Tth DNA polymerase mutant is slightly lower than that of MLV-RT. When the introduced NaCl concentration is 50 mM, the reverse transcription activity of the Tth DNA polymerase mutant is not significantly reduced, but the Ct value of the MLV-RT amplification curve is significantly delayed, indicating that the reverse transcription activity of MLV-RT is significantly reduced under high ion concentration conditions, while the reverse transcription activity of the Tth DNA polymerase mutant is not affected in a high-salt environment.

[0048] Therefore, MLV-RT has high requirements for the working environment and is not very tolerant of complex environments. It is also less tolerant of some one-step qPCR systems with complex environments, and its reverse transcription activity is greatly affected. The Tth DNA polymerase mutant is more tolerant of the environment of crude samples and can tolerate a variety of crude samples, thus improving experimental efficiency.

[0049] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is impossible to exhaustively list all the implementation methods here. All obvious variations or modifications derived from the technical solutions of the present invention are still within the protection scope of the present invention.

Claims

1. A method independent of Mn 2+ The Tth DNA polymerase mutant is characterized by, The amino acid sequence of the Tth DNA polymerase mutant is shown in SEQ ID NO.

1.

2. A polynucleotide encoding the Tth DNA polymerase mutant of claim 1.

3. The polynucleotide according to claim 2, characterized in that, The polynucleotide is shown in SEQ ID NO.

2.

4. A recombinant vector comprising the polynucleotide of claim 2 or 3.

5. Recombinant cells comprising the polynucleotide of claim 2 or the recombinant vector of claim 4.

6. The method for preparing the Tth DNA polymerase mutant according to claim 1, characterized in that, The preparation method includes the following steps: a1) Construct a recombinant vector containing the polynucleotide shown in SEQ ID NO.2; a2) Transform the recombinant vector into host cells, induce expression, and obtain bacterial cells; a3) The bacterial cells were broken, centrifuged, and the supernatant was obtained. The supernatant was then purified to obtain the Tth DNA polymerase mutant.

7. The preparation method according to claim 6, characterized in that, The purification process includes nickel ion affinity chromatography and ion exchange chromatography.

8. The use of the Tth DNA polymerase mutant of claim 1, and / or the polynucleotide of claim 2 or 3, and / or the recombinant vector of claim 4, and / or the recombinant cell of claim 5 in reverse transcription.

9. The use of the Tth DNA polymerase mutant of claim 1, and / or the polynucleotide of claim 2 or 3, and / or the recombinant vector of claim 4, and / or the recombinant cell of claim 5 in the preparation of products by reverse transcription reaction.

10. A reverse transcription kit, characterized in that, The kit includes the Tth DNA polymerase mutant as described in claim 1.

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