A Taq DNA polymerase mutant and its application

Abstract

This invention discloses a Taq DNA polymerase mutant and its applications. The Taq DNA polymerase mutant is derived from the amino acid sequence SEQ ID NO.1 and contains the following mutations: E9K, L15S, D18R, H20E, H21E, K31E, and R37D. This invention obtains a Taq DNA polymerase mutant through point mutagenesis. Unlike the wild-type Taq DNA polymerase, it possesses highly efficient and stable reverse transcriptase activity, enabling efficient conversion of cDNA using RNA substrates and amplification of the cDNA under standard reaction conditions without the need for additional reverse transcriptase. This significantly improves the efficiency of detecting target ribonucleic acid (RNA) by real-time quantitative PCR.

Description

[0001] This application is a divisional application of patent application number 202210764015.X (the original application was filed on June 29, 2022, and the invention was entitled "A Taq DNA Polymerase Mutant and Its Application"). Technical Field

[0002] This invention belongs to the field of genetic engineering technology and relates to a Taq DNA polymerase mutant and its applications. Background Technology

[0003] Real-time RT-PCR is a method for detecting RNA in samples by detecting the increasing fluorescence signal over time as amplicons are generated in a qPCR reaction. Currently, the most well-known application of RT-PCR is in diagnostic laboratory testing to detect the presence of viral genetic material in patient samples, such as SARS-CoV-2 (COVID-19). RT-PCR is the standard for detecting RNA targets in molecular biology, medicine, and forensic research.

[0004] Taq DNA polymerase is commonly used in molecular biology to amplify nucleic acid amplicones in polymerase chain reaction (PCR). In PCR, a specified DNA fragment (amplicon) is amplified through repeated cycles of three steps: denaturation, annealing, and amplicon extension / expansion. Using qualitative real-time PCR (qPCR), data can be collected during PCR cycles via fluorescence signals generated by dyes or probes, allowing for the measurement and recording of target amplification. Probe-based chemical methods utilize fluorescently labeled target-specific probes that release reporter dyes only upon binding to the target sequence, enabling real-time detection of target amplification as the fluorescence signal intensity increases.

[0005] RT-PCR allows for the detection and amplification of RNA substrates. When qPCR reactions include reverse transcriptase, RNA can be detected through an additional initial cycling step, where reverse transcriptase generates DNA (cDNA) complementary to the RNA substrate; this cDNA can then be amplified by DNA polymerase for quantification. Current RT-PCR protocols rely on a combination of reverse transcriptase and DNA polymerase to generate data. In most cases, Taq polymerase is limited to the amplification of DNA substrates; the few instances where cDNA can be generated from RNA substrates typically depend on very specific buffers and protocols, or on a mutation in the aspartic acid at position 732. Overall, in these cases, Taq activity is generally less potent than that of reverse transcriptase.

[0006] In conclusion, developing a Taq DNA polymerase with high reverse transcriptase activity is of great significance for the field of RNA detection. Summary of the Invention

[0007] To address the shortcomings of existing technologies and practical needs, this invention provides a Taq DNA polymerase mutant and its applications. This invention uses site-directed mutagenesis to obtain the Taq DNA polymerase mutant, which effectively improves the reverse transcription activity of Taq DNA polymerase. The Taq DNA polymerase mutant can efficiently convert RNA substrates into DNA products.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] In a first aspect, the present invention provides a Taq DNA polymerase mutant, wherein the Taq DNA polymerase mutant undergoes the following mutation based on the amino acid sequence SEQ ID NO.1:

[0010] E9K, L15S, D18R, H20E, H21E, K31E, R37D, F66A, K82E, A83F, R85D, P87G, P8 9G, E90K, F92A, I99S, E101K, L102S, L108S, A109F, R110D, G115P, S124I, I1 38S, V155S, L156S, H157E, T164I, L168S, R183D, G187P, E189A, E189I, E18 9K, E189S, K202E, E230A, E230C, E230M, E230Q, E230V, L233S, H235E, M236S D237R, D244R, K247E, L281S, P291G, L294S, V310S, K314E, R349D, L379S, S383I, Y394A, G395P, A442F, E507G, E507I, E507L, E537W, T544I, P550G, D578F, D578R, D578T, D578Y, D732A, D732F, D732G, D732I, D732P, D732Q, D732S, E742A, E742G, E742H, E742I, E742M, E742P, E742S, combinations of E39K and E189K, E3 Combinations of E39K and E230K, E39K and E520K, E39K and E537K, E39K and D578R, E39K and D732R, E39K and E742K, G46D and E189K, G46D and E230K, G46D and N384R, G46D and D578R, E189K and E230K, E189K and E520K, E189K and E537K, E189K and D578R, E189K and D732R, E189K and E742K, E230K and E520K, E23 Combinations of E537K and E230K, E230K and D578R, E230K and D732R, E230K and E742K, E520K and E537K, E520K and D578R, E520K and D732R, E520K and E742K, E537K and D578R, E537K and D732R, E537K and E742K, D578R and D732R, D578R and E742K, D732R and E742K, E39K, E230K and E742K, G46D, E189K and E230K.Combinations of G46D, E189K, and D578R; G46D, E189K, and F667Y; G46D, E189K, and D732R; G46D, E230K, and F667Y; G46D, E230K, and D732R; G46D, N384R, and F667Y; G46D, D578R, and F667Y; E189K, E230K, and E520K; E189K, E230K, and E537K; E189K, E230K, and D578R; E189K, E230K, and D732R Combinations, E189K and E230K and E742K combinations, E189K and E520K and E537K combinations, E189K and E520K and D578R combinations, E189K and E520K / D732R combinations, E189K / E520K / E742K combinations, E189K / E537K / D578R combinations, E189K and E537K and D732R combinations, E189K and E537K and E742K combinations, E189K and D578R and D732R combinations, E189K and D578R and E742K combinations, E189K and D7 Combinations of E230K, E520K, and E537K; combinations of E230K, E520K, and D578R; combinations of E230K, E520K, and D732R; combinations of E230K, E520K, and E742K; combinations of E230K, E537K, and D578R; combinations of E230K, E537K, and D732R; combinations of E230K, D732R, and E742K; combinations of E230K, D578R, and E742K; combinations of E230K, D732R, and E742K. Combinations of E520K, E537K, and D578R; combinations of E520K, E537K, and D732R; combinations of E520K, D578R, and D732R; combinations of E520K, D732R, and E742K; combinations of E537K, D578R, and D732R; combinations of E537K, D578R, and E742K; combinations of D578R, D732R, and E742K; combinations of G46D, E189K, E230K, and F667Y; combinations of G46D, E189K, D578R, and F667Y.

[0011] In this invention, a Taq DNA polymerase mutant is obtained through point mutagenesis. Unlike the wild-type Taq DNA polymerase, which exhibits limited reverse transcriptase activity only under very stringent reaction conditions, the Taq DNA polymerase mutant of this invention can efficiently convert cDNA using RNA as a substrate and amplify the cDNA under standard reaction conditions without the need for additional reverse transcriptase. This significantly improves the efficiency of real-time quantitative PCR detection of target ribonucleic acid (RNA) and simplifies the protocol, facilitating protocol optimization, such as adjusting the buffer composition to the most effective components for a single enzyme.

[0012] SEQ ID NO.1:

[0013] .

[0014] In a second aspect, the present invention provides a nucleic acid molecule containing a nucleic acid sequence encoding the Taq DNA polymerase mutant described in the first aspect.

[0015] Thirdly, the present invention provides an expression vector containing the nucleic acid molecule described in the second aspect.

[0016] Preferably, the expression vector includes a plasmid vector or a viral vector.

[0017] Fourthly, the present invention provides a recombinant cell containing the nucleic acid molecule described in the second aspect or the expression vector described in the third aspect.

[0018] Fifthly, the present invention provides the application of the Taq DNA polymerase mutant described in the first aspect in the preparation of reverse transcription reaction reagents.

[0019] The Taq DNA polymerase mutant obtained by this invention has efficient and stable reverse transcription activity and can be effectively used in the preparation of reverse transcription reaction reagents.

[0020] In a sixth aspect, the present invention provides a reverse transcription kit containing the Taq DNA polymerase mutant described in the first aspect.

[0021] Preferably, the kit further includes PCR reaction solution.

[0022] In a seventh aspect, the present invention provides the application of the Taq DNA polymerase mutant described in the first aspect in reverse transcription reactions.

[0023] Eighthly, the present invention provides a reverse transcription PCR method, the reverse transcription PCR method comprising:

[0024] Using RNA as a template, reverse transcription PCR was performed using the Taq DNA polymerase mutant described in the first aspect.

[0025] In a ninth aspect, the present invention provides the application of the Taq DNA polymerase mutant described in the first aspect in RNA detection.

[0026] Tenthly, the present invention provides an RNA detection method, the RNA detection method comprising:

[0027] Using the RNA to be tested as a template, real-time quantitative PCR was performed using the Taq DNA polymerase mutant described in the first aspect, and the fluorescence results were analyzed.

[0028] In this invention, the amount of RNA to be tested in a real-time quantitative PCR mixture is quantified based on the amount of fluorescence signal generated by the cutting of the target probe with embedded dye or label.

[0029] Preferably, the embedded dye includes SYBRGreen or EvaGreen.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] This invention obtains a Taq DNA polymerase mutant through point mutagenesis. Unlike the wild-type Taq DNA polymerase, it possesses highly efficient and stable reverse transcriptase activity, enabling efficient conversion of cDNA using RNA as a substrate and amplification of the cDNA under standard reaction conditions without the need for additional reverse transcriptase. This significantly improves the efficiency of real-time quantitative PCR detection of target ribonucleic acid (RNA) and simplifies the protocol, facilitating protocol optimization. Attached Figure Description

[0032] Figure 1 The image shows an agarose gel electrophoresis pattern, which illustrates the comparative activities of wild-type (WT) Taq DNA polymerase and Taq DNA polymerase mutants with the following mutation sites: E9K, L15S, D18R, H20E, H21E, K31E, R37D, F66A, K82E, A83F, R85D, P87G, P89G, E90K, F92A, I99S, E101K, L102S, L108S, A109F, R110D, G115P, S124I, I138S, V155S, and L156S.

[0033] Figure 2 The image shows an agarose gel electrophoresis pattern comparing the activities of wild-type (WT) Taq DNA polymerase and Taq DNA polymerase mutants with the following mutation sites: H157E, T164I, L168S, R183D, G187P, E189A, E189I, E189K, E189S, K202E, E230A, E230C, E230M, E230Q, E230V, L233S, H235E, M236S, D237R, D244R, K247E, L281S, P291G, L294S, V310S, K314E, R349D, L379S, S383I, Y394A, G395P, and A442F.

[0034] Figure 3The image shows an agarose gel electrophoresis result comparing the activities of wild-type (WT) Taq DNA polymerase and Taq DNA polymerase mutants with the following mutation sites: E507G, E507I, E507L, E537W, T544I, P550G, D578F, D578R, D578T, D578Y, D732A, D732F, D732G, D732I, D732P, D732Q, D732S, E742A, E742G, E742H, E742I, E742M, and E742P. E742S, E39K / E189K (in this invention, " / " represents the combination of corresponding mutation sites, such as E39K / E189K, which represents the combination of E39K and E189K), E39K / E230K, E39K / E520K, E39K / E537K, E39K / D578R, E39K / D732R, E39K / E742K, G46D / E189K, G46D / E230K;

[0035] Figure 4The image shows an agarose gel electrophoresis pattern comparing the activities of wild-type (WT) Taq DNA polymerase and Taq DNA polymerase mutants with the following mutation sites: G46D / N384R, G46D / D578R, E189K / E230K, E189K / E520K, E189K / E537K, E189K / D578R, E189K / D732R, E189K / E742K, E230K / E520K, E230K / E537K, E230K / D578R, E230K / D732R, and E230K / E742K. K, E520K / E537K, E520K / D578R, E520K / D732R, E520K / E742K, E537K / D578R, E537K / D732R, E537K / E742K , D578R / D732R, D578R / E742K, D732R / E742K, E39K / E230K / E742K, G46D / E189K / E230K, G46D / E189K / D57 8R, G46D / E189K / F667Y, G46D / E189K / D732R, G46D / E230K / F667Y, G46D / E230K / D732R, G46D / N384R / F6 67Y, G46D / D578R / F667Y, E189K / E230K / E520K, E189K / E230K / E537K, E189K / E230K / D578R, E189K / E230 K / D732R, E189K / E230K / E742K, E189K / E520K / E537K, E189K / E520K / D578R, E189K / E520K / D732R, E189K / E520K / E742K, E189K / E537K / D578R, E189K / E537K / D732R, E189K / E537K / E742K, E189K / D578R / D732R;

[0036] Figure 5This is an agarose gel electrophoresis image showing the comparative activities of wild-type (WT) Taq DNA polymerase and Taq DNA polymerase mutants with the following mutation sites: E189K / D578R / E742K, E189K / D732R / E742K, E230K / E520K / E537K, E230K / E520K / D578R, E230K / E520K / D732R, E230K / E520K / E742K, E230K / E537K / D578R, E230K / E537K / D732R, E230K / D578R / D732R, E230K / D732R / E742K, E230K / D578R / E742K. 2K, E230K / D732R / E742K, E520K / E537K / D578R, E520K / E537K / D732R, E520K / D578R / D732R, E520K / D732R / E742K, E537K / D578R / D732R, E537K / D578R / E742K, E537K / D732R / E742K, D578R / D732R / E742K, G46D / E189K / E230K / F667Y or G46D / E189K / D578R / F667Y. Detailed Implementation

[0037] To further illustrate the technical means and effects of this invention, the following description, in conjunction with embodiments and accompanying drawings, provides a further explanation of the invention. It is understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it.

[0038] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0039] The term "bioactive fragment" refers to any fragment, derivative, homolog, or analog of Taq DNA polymerase or a mutant sequence thereof that possesses in vivo or in vitro reverse transcriptase activity specific to a biomolecule. In some embodiments, the bioactive fragment, derivative, homolog, or analog of the Taq DNA polymerase mutant possesses any degree of bioactivity of the Taq DNA polymerase mutant in any in vivo or in vitro assay.

[0040] In some embodiments, the bioactive fragment may optionally comprise any number of consecutive amino acid residues of a Taq DNA polymerase mutant sequence. The invention also includes polynucleotides encoding any such bioactive fragment and / or degenerate nucleic acid sequence.

[0041] Bioactive fragments can originate from post-transcriptional processing or translation of alternatively spliced ​​RNA, or can be generated through engineering, mass synthesis, or other suitable manipulations. Bioactive fragments include those expressed in natural or endogenous cells, as well as those generated in expression systems such as bacterial, yeast, plant, insect, or mammalian cells.

[0042] The phrase “conservative amino acid substitution” or “conservative mutation” refers to the substitution of one amino acid for another amino acid with a shared property. One functional approach to defining the shared property between individual amino acids is to analyze the normalized frequencies of amino acid changes between corresponding proteins from homologous organisms (Schulz (1979) Principles of Protein Structure, Springer-Verlag). Based on such analysis, amino acid groups can be defined where amino acids within a group preferentially exchange with each other and are therefore most similar to each other in their effects on the overall protein structure (Schulz (1979) ibid.). Examples of amino acid groups defined in this way can include: “charged / polar groups,” including Glu, Asp, Asn, Gln, Lys, Arg, and His; “aromatic or cyclic groups,” including Pro, Phe, Tyr, and Trp; and “aliphatic groups,” including Gly, Ala, Val, Leu, Ile, Met, Ser, Thr, and Cys. Within each group, subgroups can also be identified. For example, the charged / polar amino acid group can be further subdivided into subgroups, including: "positively charged subgroup," including Lys, Arg, and His; "negatively charged subgroup," including Glu and Asp; and "polar subgroup," including Asn and Gln. In another example, the aromatic or cyclic group can be subdivided into subgroups, including: "nitrogen-cyclic subgroup," including Pro, His, and Trp; and "phenyl subgroup," including Phe and Tyr. In yet another further example, the aliphatic group can be subdivided into subgroups, including: "large aliphatic nonpolar subgroup," including Val, Leu, and Ile; "aliphatic micropolar subgroup," including Met, Ser, Thr, and Cys; and "small residue subgroup," including Gly and Ala. Examples of conserved mutations include amino acid substitutions within the aforementioned subgroups, such as, but not limited to: Lys replacing Arg, and vice versa, to maintain a positive charge; Glu replacing Asp, and vice versa, to maintain a negative charge; Ser replacing Thr, and vice versa, to maintain a free -OH; and Gln replacing Asn, and vice versa, to maintain a free -NH2. A “conserved variant” is a polypeptide containing one or more amino acids that have been substituted to replace one or more amino acids of a reference polypeptide (e.g., a polypeptide whose sequence is published in a publication or sequence database, or a polypeptide whose sequence has been determined by nucleic acid sequencing) with amino acids having common properties, for example, belonging to the same amino acid group or subgroup as described above.

[0043] When referring to a gene, a "mutant" means a gene that has at least one base (nucleotide) alteration, deletion, or insertion relative to the natural or wild-type gene. The mutation (one or more nucleotide alterations, deletions, and / or insertions) can occur in the coding region of the gene or in introns, the 3'UTR, the 5'UTR, or the promoter region. As a non-limiting example, a mutant gene can be a gene with an insertion in the promoter region that can increase or decrease gene expression; it can be a gene with a deletion that results in the production of a non-functional protein, a truncated protein, a dominant-inactive protein, or no protein; or it can be a gene with one or more point mutations that result in a change in the amino acids encoding the protein or in abnormal splicing of the gene transcript.

[0044] When the terms "Taq DNA polymerase mutant of the present invention" and "Taq DNA polymerase mutant" are used in this Detailed Description section, they refer, together or separately, to a Taq DNA polymerase mutant polypeptide that has been tested and exhibits enhanced reverse transcriptase activity, depending on the context. The terms "Taq DNA polymerase mutant of the present invention" and "Taq DNA polymerase mutant" also include variant sequences and / or degenerate nucleic acid sequences.

[0045] "Naturally occurring" or "wild-type" refers to a form found in nature. For example, naturally occurring or wild-type polypeptide or polynucleotide sequences are sequences that exist in organisms and have not been intentionally modified by human intervention.

[0046] In some embodiments, the present invention relates to methods (and related kits, systems, apparatuses and compositions) for performing ligation reactions, said ligation reactions comprising or consisting of the steps of: contacting a Taq DNA polymerase mutant or a bioactive fragment thereof with a nucleic acid template in the presence of one or more nucleotides, and ligating at least one of the one or more nucleotides using the Taq DNA polymerase mutant or the bioactive fragment thereof.

[0047] In some embodiments, the method of performing the ligation reaction may include linking a double-stranded RNA or DNA polynucleotide chain to a circular molecule. In some embodiments, the method may further include detecting a signal indicating ligation using a sensor. In some embodiments, the sensor is an ISFET. In some embodiments, the sensor may include a detectable tag or detectable reagent in the ligation reaction.

[0048] The Taq DNA polymerase mutant described in this invention can be expressed in any suitable host system, including bacteria, yeast, fungi, baculoviruses, plant or mammalian host cells.

[0049] For bacterial host cells, promoters available for transcription of Taq DNA polymerase mutants include those derived from the following sources: the *Escherichia coli* lac operon, *Streptomyces agarase* gene (dagA), *Bacillus subtilis* gene (sacB), *Bacillus licheniformis* gene (amyL), *Bacillus thermophilus* gene (amyM), *Bacillus amyloliquefaciens* gene (amyQ), *Bacillus licheniformis* gene (penP), *Bacillus subtilis* genes xylA and xylB and prokaryotic β-lactamase (Villa-Kamaroff et al., 1978, Proc. Natl Acad. Sci. USA 75:3727-3731), and the tac promoter (DeBoer et al., 1983, Proc. Natl Acad. Sci. USA 80:21-25).

[0050] For filamentous fungal host cells, promoters available for transcription of Taq DNA polymerase mutants include promoters derived from genes from the following sources: Aspergillus oryzae TAKA amylase, Rhizopus oryzae aspartic protease, Aspergillus niger neutral α-amylase, Aspergillus niger acid-stable α-amylase, Aspergillus niger or Aspergillus avocado glucosylase (glaA), Rhizopus oryzae lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acetamipridase, and Fusarium oxysporum trypsin-like protease (WO96 / 00787), as well as the NA2-tpi promoter (a hybrid of the promoters from the Aspergillus niger neutral α-amylase and Aspergillus oryzae triose phosphate isomerase genes), and their mutant, truncated, and hybrid promoters.

[0051] In yeast hosts, promoters available for transcription of Taq DNA polymerase mutants can be derived from the genes of *Saccharomyces cerevisiae* enolase (ENO-1), *Saccharomyces cerevisiae* galactokinase (GAL1), *Saccharomyces cerevisiae* alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP), and *Saccharomyces cerevisiae* 3-phosphate glycerate kinase. Other promoters useful to yeast host cells are described in Romanose et al., 1992, Yeast 8:423-488.

[0052] For baculovirus expression, promoters available for transcription using Taq DNA polymerase mutants can be derived from lepidopteran insect cell lines, such as the fall armyworm, which serves as the host. Gene expression is controlled by strong promoters, such as pPolh.

[0053] Plant expression vectors are based on the Ti plasmid of Agrobacterium tumefaciens, or on tobacco mosaic virus (TMV), potato virus X, or cowpea mosaic virus. The commonly used constitutive promoter in plant expression vectors is the cauliflower mosaic virus (CaMV) 35S promoter.

[0054] For mammalian expression, cultured mammalian cell lines such as Chinese hamster ovary (CHO) and COS (including human cell lines such as HEK and HeLa) can be used to generate Taq DNA polymerase mutants. Mammalian expression vectors include adenovirus vectors, pSV and pCMV series plasmid vectors, vaccinia virus and retrovirus vectors, and baculoviruses. Cytomegalovirus (CMV) and SV40 promoters are commonly used in mammalian expression vectors to drive gene expression. Non-viral promoters, such as the elongation factor (EF)-1 promoter, are also known.

[0055] The control sequence used for expression can be a suitable transcription terminator sequence, i.e., a sequence recognized by the host cell to terminate transcription. The terminator sequence is operatively linked to the 3' end of the nucleic acid sequence encoding the polypeptide. Any terminator that functions in the selected host cell can be used.

[0056] For example, exemplary transcription terminators for filamentous fungal host cells can be obtained from the genes of Aspergillus oryzae TAKA amylase, Aspergillus niger glucosylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger α-glucosidase, and Fusarium oxysporum trypsin-like protease.

[0057] Exemplary terminators for yeast host cells can be obtained from the genes of Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase.

[0058] The control sequence can also be a suitable leader sequence, i.e., the untranslated region of mRNA that is important for translation in the host cell. The leader sequence is operatively linked to the 5' end of the nucleic acid sequence encoding the polypeptide. Any leader sequence that is functional in the selected host cell can be used. Exemplary leader sequences for filamentous fungal host cells were obtained from the genes of Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase. Leader sequences suitable for yeast host cells were obtained from the genes of Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae α-factor, and Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP).

[0059] The control sequence can also be a polyadenylated sequence operatively linked to the 3' end of a nucleic acid sequence and, during transcription, recognized by the host cell as a signal to add polyadenylated residues to the transcribed mRNA. Any polyadenylated sequence that functions in a selected host cell can be used in this invention. Exemplary polyadenylated sequences for filamentous fungal host cells can be derived from the genes of Aspergillus oryzae TAKA amylase, Aspergillus niger glucosylase, Aspergillus nidulans anthranilate synthase, Fusarium oxysporum trypsin-like protease, and Aspergillus niger α-glucosidase.

[0060] The control sequence can also be a signal peptide coding region, which encodes an amino acid sequence linked to the amino terminus of a polypeptide and introduces the encoded polypeptide into the cellular secretion pathway. The 5' end of the coding sequence of the nucleic acid sequence may inherently contain a signal peptide coding region, which is naturally linked to the coding region of the secretory polypeptide within the translation reading frame. Alternatively, the 5' end of the coding sequence may contain a signal peptide coding region exogenously derived from the coding sequence. An exogenous signal peptide coding region may be required when the coding sequence does not naturally contain one.

[0061] Alternatively, the exogenous signal peptide coding region can simply replace the native signal peptide coding region to enhance peptide secretion. However, any signal peptide coding region in the secretion pathway of the expressed peptide can be used.

[0062] The effective signal peptide coding regions of bacterial host cells are derived from the genes of Bacillus NCIB11837 malt amylase, Bacillus thermophilus α-amylase, Bacillus licheniformis subtilis protease, Bacillus licheniformis β-lactamase, Bacillus thermophilus neutral proteases (nprT, nprS, nprM), and Bacillus subtilis prsA. Simonen and Palva, 1993, Microbiol Rev 57:109-137, further describe the signal peptides.

[0063] The effective signal peptide coding regions of filamentous fungal host cells can be obtained from the signal peptide coding regions of Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase, Aspergillus niger glucosylase, Rhizopus oryzae aspartic protease, Pyropus spp. cellulase, and Pyropus pubescens lipase genes.

[0064] Useful signal peptides for yeast host cells can be derived from the genes of Saccharomyces cerevisiae α-factor and Saccharomyces cerevisiae invertase. Signal peptides from other host cell systems are also well known.

[0065] The control sequence can also be a prepeptide coding region encoding an amino acid sequence located at the amino terminus of the polypeptide. The resulting polypeptide is called a zymogen or polypeptide origin (or in some cases, a zymogen). The polypeptide origin is usually inactive and can be converted into a mature, active polypeptide by catalytic or autocatalytic cleavage of the polypeptide origin. The polypeptide origin coding region can be obtained from the genes of *Bacillus subtilis* alkaline protease (aprE), *Bacillus subtilis* neutral protease (nprT), *Saccharomyces cerevisiae* α-factor, *Rhizopus oryzae* aspartic protease, and *Tricholoma thermophilum* lactase (WO95 / 33836).

[0066] When both the signal peptide and the propeptide region are located at the amino terminus of the polypeptide, the propeptide region is located next to the amino terminus of the polypeptide, and the signal peptide region is located next to the amino terminus of the propeptide region.

[0067] It may also be necessary to add regulatory sequences that allow for the regulation of Taq DNA polymerase mutant expression relative to the growth of the host cell. Examples of regulatory systems are those that respond to chemical or physical stimuli (including the presence of regulatory compounds) to cause gene expression to turn on or off. In prokaryotic host cells, suitable regulatory sequences include the lac, tac, and trp manipulation systems. In yeast host cells, suitable regulatory systems include, for example, the ADH2 or GAL1 systems. In filamentous fungi, suitable regulatory sequences include the TAKA α-amylase promoter, the Aspergillus niger glucosylase promoter, and the Aspergillus oryzae glucosylase promoter. Other host cell regulatory systems are also well known.

[0068] Other examples of regulatory sequences are sequences that allow gene amplification. In eukaryotic systems, these include dihydrofolate reductase genes amplified in the presence of methotrexate and metallothionein genes amplified with heavy metals. In these cases, the nucleic acid sequence encoding the polypeptide of the present invention will be effectively linked to the regulatory sequence.

[0069] One specific implementation includes a recombinant expression vector containing a polynucleotide encoding an engineered Taq DNA polymerase mutant, and one or more expression regulatory regions, such as promoters and terminators, and an origin of replication, depending on the type of host to which they will be introduced. The various nucleic acids and control sequences described above can be linked together to produce a recombinant expression vector, which may include one or more convenient restriction sites to allow insertion or substitution of nucleic acid sequences encoding the Taq DNA polymerase mutant at these sites. Alternatively, the nucleic acid sequence of the Taq DNA polymerase mutant can be expressed by inserting the nucleic acid sequence or a nucleic acid construct containing that sequence into a suitable expression vector. During the construction of the expression vector, the coding sequence is located within the vector such that the coding sequence is effectively linked with suitable control sequences for expression.

[0070] The recombinant expression vector can be any vector (e.g., plasmid or virus) that facilitates recombinant DNA procedures and can induce the expression of Taq DNA polymerase mutant polynucleotide sequences. The choice of vector typically depends on its compatibility with the host cell into which it is introduced. The vector can be a linear or closed circular plasmid.

[0071] The expression vector can be a self-replicating vector, i.e., a vector that exists as an extrachromosomal entity and replicates independently of chromosome replication, such as a plasmid, extrachromosomal element, mini-chromosome, or artificial chromosome. The vector can contain any means to ensure self-replication. Alternatively, the vector can be a vector that integrates into the genome upon introduction into a host cell and replicates along with the chromosome it integrates with. Furthermore, a single vector or plasmid, or two or more vectors or plasmids together containing the total DNA or transposons to be introduced into the host cell genome, can be used.

[0072] The expression vectors described in this invention preferably contain one or more selection markers that allow for easy selection of transformed cells. Selection markers are genes whose products provide resistance to biocides or viruses, resistance to heavy metals, prototrophic auxotrophs, etc. Examples of bacterial selection markers are the dal gene from Bacillus subtilis or Bacillus licheniformis, or markers that confer resistance to antibiotics such as ampicillin, kanamycin, chloramphenicol (Example 1), or tetracycline. Suitable markers for yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selection markers for filamentous fungal host cells include, but are not limited to, amdS (acetamipase), argB (ornithine carbamoyltransferase), bar (phosphinicotinic acid acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotate nucleoside-5'-phosphate decarboxylase), sC (adenosyl sulfate transferase), and trpC (o-aminobenzoic acid synthase) and their equivalents. Implementation schemes for Aspergillus cells include the amdS and pyrG genes of Aspergillus nidulans or Aspergillus oryzae, and the bar gene of Streptomyces hygroscopicus. Selection markers for insect, plant, and mammalian cells are also well known.

[0073] The expression vectors described in this invention preferably contain elements that allow the vector to integrate into the host cell genome or allow the vector to replicate autonomously in the cell independently of the genome. For integration into the host cell genome, the vector may rely on a nucleic acid sequence encoding a polypeptide or any other element of the vector to integrate the vector into the genome via homologous or non-homologous recombination.

[0074] Alternatively, the expression vector may contain additional nucleic acid sequences to guide integration into the host cell's genome via homologous recombination. These additional nucleic acid sequences enable the vector to integrate into the host cell's genome at a precise location within the chromosome. The integrating element can be any sequence homologous to a target sequence in the host cell's genome. Furthermore, the integrating element can be a non-coding or coding nucleic acid sequence. Alternatively, the vector can integrate into the host cell's genome via non-homologous recombination.

[0075] For autonomous replication, the vector may further include an origin of replication that enables the vector to replicate autonomously in the host cell in question. Examples of bacterial origins of replication are P15Aori, or the origins of replication of plasmids pBR322, pUC19, pACYC177 (which contains P15Aori), or pACYC184 that allow replication in *E. coli*, and the origins of replication of pUB110, pE194, pTA1060, or pAM31 that allow replication in *Bacillus*. Examples of origins of replication used in yeast host cells are 2-micron origins of replication ARS1, ARS4, combinations of ARS1 and CEN3, and combinations of ARS4 and CEN6. The origin of replication may be a mutation that makes it sensitive to temperature in the host cell (see, for example, Ehrlich, 1978, ProcNatlAcadSci.USA75:1433).

[0076] One or more copies of the nucleic acid sequence of the Taq DNA polymerase mutant can be inserted into a host cell to increase the production of the gene product. The increase in the nucleic acid sequence copy number can be achieved by integrating at least one additional sequence copy into the host cell genome or by including an amplifiable selection marker gene in the nucleic acid sequence, wherein the additional copy of the nucleic acid sequence can be selected by culturing cells in the presence of a suitable selection reagent to select cells containing copies of the amplifiable selection marker gene.

[0077] The expression vector for the Taq DNA polymerase mutant polynucleotide is commercially available. Suitable commercial expression vectors include the p3xFLAG™ expression vector from Sigma-Aldrich Chemicals, St. Louis Mo., which includes a CMV promoter and hGH polyadenylation site for expression in mammalian host cells, and a pBR322 origin of replication and ampicillin resistance marker for amplification in E. coli. Other suitable expression vectors are pBluescriptII SK(-) and pBK-CMV, which are commercially available from Stratagene, LaJolla CA, and plasmids from pBR322 (GibcoBRL), pUC (GibcoBRL), pREP4, pCEP4 (Invitrogen), or pPoly (Lathe et al., 1987, Gene57:193-201).

[0078] Suitable host cells for expressing polynucleotides encoding Taq DNA polymerase mutants are well known in the art, including but not limited to bacterial cells such as *Escherichia coli*, *Lactobacillus kefiriensis*, *Lactobacillus brevis*, *Lactobacillus microsporum*, *Streptomyces*, and *Salmonella typhimurium* cells; fungal cells such as yeast cells (e.g., *Saccharomyces cerevisiae* or *Pichia pastoris* (ATCC application number 201178)); insect cells such as Drosophila S2 and *Betula spp.* Sf9 cells; animal cells such as CHO, COS, BHK, 293, and Bowes melanoma cells; and plant cells.

[0079] The polynucleotides used to express the Taq DNA polymerase mutant can be introduced into cells using various methods known in the art. Techniques include electroporation, bio-ballistic particle bombardment, liposome-mediated transfection, calcium chloride transfection, and protoplast fusion.

[0080] The polynucleotide encoding the Taq DNA polymerase mutant can be prepared using standard solid-phase methods according to known synthetic techniques. In some embodiments, fragments of up to about 100 bases can be synthesized individually and then ligated (e.g., by enzymatic or chemical reaction methods, or polymerase-mediated methods) to form any desired continuous sequence. For example, the polynucleotide can be prepared by chemical synthesis using, for example, the classic phosphoramidite method described by Beaucage et al., 1981, Tet Lett 22:1859-69, or the method described by Mattes et al., 1984, EMBO J.3:801-05, which is typically practiced in automated synthetic methods. According to the phosphoramidite method, the oligonucleotide is synthesized, purified, annealed, ligated, and cloned into a suitable vector, for example, in an automated DNA synthesizer. Furthermore, virtually any nucleic acid can be obtained from a variety of commercial sources, such as Midland Certified Reagent Company, Midland, Tex.; Great American Gene Company, Ramona, Calif.; ExpressGen Inc., Chicago, Illinois.; and Operaon Technologies Inc., Alameda, Calif.

[0081] Using any one or more well-known protein purification techniques, including lysozyme treatment, sonication, filtration, salting out, ultracentrifugation, and chromatography, engineered Taq DNA polymerase mutants expressed in host cells can be recovered from cells and / or culture media. Suitable solutions for lysing and efficiently extracting proteins from bacteria (e.g., E. coli) are available from Sigma-Aldrich in St. Louis Mo, under the trade name CelLytic B.TM.

[0082] Chromatographic techniques for separating the Taq DNA polymerase mutant include reversed-phase chromatography, high-performance liquid chromatography, ion-exchange chromatography, gel electrophoresis, and affinity chromatography. Purification conditions will depend in part on factors such as net charge, hydrophobicity, hydrophilicity, molecular weight, and molecular shape, and will be apparent to those skilled in the art.

[0083] In some implementations, affinity chromatography can be used to isolate the Taq DNA polymerase mutant. For affinity chromatography purification, any antibody that specifically binds to the Taq DNA polymerase mutant can be used. To generate antibodies, various host animals, including but not limited to rabbits, mice, rats, etc., can be immunized by injecting a compound. The compound can be linked to a suitable vector, such as bovine serum albumin, via a side-chain functional group or a linker connected to the side-chain functional group. Depending on the host species, various adjuvants can be used to enhance the immune response, including but not limited to Freund's adjuvant (complete and incomplete), mineral gels such as aluminum hydroxide, surfactants such as lysophosphatidylcholine, Pluronic acid polyols, polyanionic peptides, peptides, oil emulsions, keyhole hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG and Corynebacterium parvum.

[0084] Example 1

[0085] This embodiment prepares a Taq DNA polymerase mutant.

[0086] Compared to wild-type Taq DNA polymerase, Taq DNA polymerase mutants exhibit higher reverse transcriptase activity. They were engineered, characterized, and screened using polymerase chain reaction, visualized by agarose gel electrophoresis, and initially screened using probe-based real-time quantitative PCR (qPCR). Specific ribonucleic acid (RNA) target sequences were then detected using a typical reverse transcription cycle protocol.

[0087] Taq DNA polymerase mutants were generated by mutagenesis of SEQ NOID.1 using conventional inverse PCR. All mutants were sequenced for verification, expressed and purified in E. coli. C-terminal tags were added to all Taq DNA polymerase mutants and wild-type Taq DNA polymerase (amino acid sequence SEQ ID NO.1, nucleic acid sequence SEQ ID NO.2) to facilitate purification.

[0088] The DNA sequence (wild type) of Taq DNA polymerase with a C-terminal Histag is shown in SEQ ID NO.2.

[0089] SEQ ID NO.2:

[0090]

[0091] qPCR was performed under the following conditions, with the target gene being the 28S gene.

[0092] Forward primer: 5'-CCGCTGCGGTGAGCCTTGAA-3'

[0093] Reverse primer: 5'-TCTCCGGGATCGGTCGCGTT-3'

[0094] Target gene: 28S RNA, derived from total RNA-human tumor cell line: Hela (Biochain Cat#R1255811-50).

[0095] Each 10 μL reaction system contains 1.5 μL 50 ng / μL Taq DNA polymerase, 0.4 μL 10 μM forward primer, 0.4 μL 10 μM reverse primer, 1 μL 10 ng / μL target RNA, 0.4 μL 10 mM equimolar dNTPs, 0.1 μL 1 MdTT, and 1 μL 10× reaction buffer (final composition: 20 mM tris(hydroxymethyl)aminomethane hydrochloride, 80 mM tris(hydroxymethyl)aminomethane acetate, 10 mM ammonium sulfate, 10 mM potassium chloride, 2 mM magnesium sulfate, 3 mM magnesium acetate, 0.1% Triton X-100, pH 8.8 (25 °C)), and water to a final volume of 10 μL.

[0096] The thermal cycler used for qPCR assays was a Bio-Rad T100. The reaction program was as follows: incubation at 60°C for 20 minutes, denaturation at 95°C for 5 minutes, followed by 35 cycles (denaturation at 95°C for 10 seconds, extension at 60°C for 30 seconds), and then incubation at 75°C for 5 minutes. 3 μL of 6× stop dye containing 6× GelRed nucleic acid dye (Biotium Cat#41003) was added to each sample. 10 μL of each sample was loaded into a 2% agarose gel and compared with wild-type (WT) Taq polymerase and a low molecular weight DNA length marker (New England Biolabs, Cat#N3233).

[0097] The results are as follows Figures 1-5 As shown, compared with wild-type Taq polymerase, the amplification products of each Taq DNA polymerase mutant of the present invention all have obvious target product bands, indicating that the Taq DNA polymerase mutants of the present invention can efficiently convert cDNA with RNA as substrate and amplify the cDNA under standard reaction conditions, that is, they have both high-efficiency reverse transcriptase activity and polymerase activity.

[0098] Example 2

[0099] This embodiment provides the application of Taq DNA polymerase mutants in real-time quantitative PCR (qPCR).

[0100] The Taq DNA polymerase mutant obtained by this invention can be used to quantify RNA in samples using conventional qPCR protocols for RNA detection without the need to add additional reverse transcriptase to the reaction mixture.

[0101] In summary, this invention obtains a Taq DNA polymerase mutant through point mutagenesis. Unlike the wild-type Taq DNA polymerase, it possesses highly efficient and stable reverse transcriptase activity, enabling efficient conversion of cDNA using RNA substrates and amplification of the cDNA under standard reaction conditions without the need for additional reverse transcriptase. This significantly improves the efficiency of detecting target ribonucleic acid (RNA) by real-time quantitative PCR, while also simplifying the protocol and facilitating protocol optimization.

[0102] The applicant declares that the detailed method of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A mutant of Taq DNA polymerase, characterized in that, The Taq DNA polymerase mutant is mutated at R85D based on the amino acid sequence of SEQ ID NO.

1.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule contains a nucleic acid sequence encoding the Taq DNA polymerase mutant of claim 1.

3. An expression vector, characterized by, The expression vector contains the nucleic acid molecule of claim 2.

4. The expression vector of claim 3, wherein, The expression vector includes a plasmid vector or a viral vector.

5. A recombinant cell, characterized in that, The recombinant cell contains the nucleic acid molecule of claim 2 or the expression vector of claim 3.

6. Use of the Taq DNA polymerase mutant of claim 1 in the preparation of a reverse transcription reaction reagent.

7. A reverse transcription kit, characterized by, The reverse transcription kit includes the Taq DNA polymerase mutant of claim 1.

8. The reverse transcription kit according to claim 7, characterized in that, The kit further includes a PCR reaction solution.

9. Use of the Taq DNA polymerase mutant of claim 1 in a reverse transcription reaction.

10. A reverse transcription PCR method characterized by, The reverse transcription PCR method includes: Using the Taq DNA polymerase mutant of claim 1 to perform reverse transcription PCR with RNA as a template.

11. Use of the Taq DNA polymerase mutant of claim 1 in RNA detection.

12. A method of detecting RNA, characterized by, The RNA detection method includes: Using the Taq DNA polymerase mutant of claim 1 to perform real-time fluorescent quantitative PCR with the RNA to be detected as a template, and analyzing the fluorescence results.

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