Pfu DNA polymerase mutant having reverse transcriptase activity and use thereof

Abstract

Provided are a Pfu DNA polymerase mutant having a reverse transcriptase activity and the use thereof. The Pfu DNA polymerase mutant comprises K467R / F588L / W769R, R382H / R385H / V390I, I38L / R97M, E665K / E735K and K118I / N713V, wherein the K118I / N713V has the strongest reverse transcription activity. In addition, the Pfu DNA polymerase mutant also retains the polymerase activity of a wild-type enzyme. The mutant has a high fidelity due to the proofreading activity thereof compared with the lower fidelity of a conventional reverse transcriptase, and the mutant has a high heat resistance, so that the mutant can generate cDNA with a high efficiency and a high fidelity by means of using RNA as a substrate, and then the cDNA is amplified under standard PCR reaction conditions. In the whole process, additional addition of the reverse transcriptase is not required, and a single-enzyme "one-step" RT-PCR is realized.

Description

A Pfu DNA polymerase mutant with reverse transcriptase activity and its application Technical Field

[0001] The invention belongs to the technical field of polymerases, and particularly relates to a Pfu DNA polymerase mutant with reverse transcriptase activity and application thereof. Background Art

[0002] With the outbreak of the COVID-19 pandemic, rapid and accurate detection of RNA viruses has become increasingly important. Reverse transcription real-time quantitative PCR (RT-qPCR) is currently the gold standard for detecting RNA viruses.

[0003] Current RT-qPCR requires the combined use of reverse transcriptase and Taq DNA polymerase. Because the reverse transcriptase currently used is not heat-resistant, the entire detection process is divided into two steps: reverse transcription must first be performed at a lower temperature, and then the temperature must be raised for PCR. If the RNA to be detected has a higher-order structure, the sample must also be pretreated, which is cumbersome and time-consuming. Furthermore, the activity of both enzymes will be affected after mixing, such as by extending the reverse transcription time. Additionally, Taq DNA polymerase will compete with the reverse transcriptase for template binding, resulting in a significant decrease in detection sensitivity under conditions with less template. Therefore, developing a DNA polymerase with high reverse transcriptase activity is of great significance for the field of RNA detection.

[0004] As one of the most commonly used B-family polymerases, Pfu DNA polymerase was first isolated and purified from the thermophilic archaeon Pyrococcus furiosus in 1991. Its 3'→5' exonuclease activity and higher activity than Taq DNA polymerase were demonstrated. Pfu DNA polymerase has a molecular weight of approximately 90 kDa and catalyzes deoxyribonucleotide (DNA) polymerization in the 5'→3' direction, as well as 3'→5' exonuclease (proofreading) activity, but lacks reverse transcription activity. Pfu DNA polymerase has five distinct domains, including the 3'→5' exonuclease domain: the N-terminal domain (1-130, 327-368), the 3'→5' exonuclease domain (131-326), the palm domain (369-450, 501-588), the finger domain (451-500), and the thumb domain (589-775).

[0005] Summary of the Invention

[0006] Given that naturally occurring DNA polymerases can no longer meet the functional requirements of various studies, the present invention aims to rationally design a Pfu DNA polymerase based on the Pfu DNA polymerase, in order to obtain a Pfu DNA polymerase mutant with high reverse transcriptase activity while retaining the polymerase activity of the wild-type enzyme, thereby realizing a single-enzyme "one-step" RT-PCR.

[0007] The technical solutions of the present invention are as follows:

[0008] The first aspect of the present invention provides a Pfu DNA polymerase mutant having reverse transcriptase activity, specifically any one of the following:

[0009] K467R / F588L / W769R mutant, wherein the lysine (Lys, K) at position 467 of the Pfu DNA polymerase amino acid sequence as shown in SEQ ID NO. 1 is mutated to arginine (Arg, R), the phenylalanine (Phe, F) at position 588 is mutated to leucine (Leu, L), and the tryptophan (Trp, W) at position 769 is mutated to arginine; or

[0010] R382H / R385H / V390I mutant, wherein the arginine at positions 382 and 385 of the Pfu DNA polymerase amino acid sequence as shown in SEQ ID NO.1 is mutated to histidine (His, H), and the valine at position 390 (Val, V) is mutated to isoleucine (Ile, I); or

[0011] I38L / R97M mutant, wherein the isoleucine at position 38 of the Pfu DNA polymerase shown in the amino acid sequence of SEQ ID NO. 1 is mutated to leucine, and the arginine at position 97 is mutated to methionine (Met, M); or

[0012] The E665K / E735K mutant is a mutant in which the glutamic acid (Glu, E) at positions 665 and 735 of the Pfu DNA polymerase shown in the amino acid sequence of SEQ ID NO.1 are simultaneously mutated to lysine; or

[0013] The K118I / N713V mutant is a mutant in which the lysine at position 118 of the Pfu DNA polymerase shown in the amino acid sequence of SEQ ID NO.1 is mutated to isoleucine, and the asparagine (Asn, N) at position 713 is mutated to valine.

[0014] The present invention uses the amino acid sequence of Pfu DNA polymerase (i.e., wild type) as the basis for rational design. Through homology modeling and structure-based prediction of its reverse transcription activity mutation sites, 16 mutation sites are predicted. The present invention uses polymerase chain reaction engineering to obtain the following mutant proteins: I38L, R97M, K118I, I137L, R382H, Y385H, V390I, K467R, Y495L, T515I, I522L, F588L, E665K, N713V, E735K, W769R. The activity of the mutants is then tested by reverse transcription RNA. If reverse transcriptase activity is detected, additional mutation sites are introduced to improve reverse transcriptase activity. Screening results show that the introduction of any of the four mutation sites, I137L, Y495L, T515I, and I522L, will weaken or even completely lose the reverse transcription activity of the polymerase; among them, the introduction of the I137L and I522L mutation sites will cause the mutant's reverse transcription to weaken progressively, and the amount of reverse transcription product will increase significantly at high enzyme levels; the introduction of the Y495L and T515I mutation sites will cause the mutant to lose reverse transcription activity. Through extensive screening work, the present invention ultimately obtained Pfu DNA polymerase mutants with efficient and stable reverse transcriptase activity, among which K118I / N713V had the strongest reverse transcription activity, followed by E665K / E735K.

[0015] The second aspect of the present invention provides a biological material related to the Pfu DNA polymerase mutant of the present invention, comprising the following:

[0016] (a) Gene encoding a Pfu DNA polymerase mutant;

[0017] (b) a recombinant expression vector containing the gene in (a);

[0018] (c) A recombinant cell containing the gene in (a) or the recombinant expression vector in (b).

[0019] In one embodiment of the present invention, the gene sequence encoding the wild-type enzyme is shown in SEQ ID NO. 2, and the gene encoding the Pfu DNA polymerase mutant can be obtained by Primer-directed mutagenesis based on the sequence shown in SEQ ID NO. 2.

[0020] Among the above-mentioned biological materials, recombinant expression vectors include plasmid vectors and viral vectors.

[0021] The third aspect of the present invention provides the use of the above-mentioned biological material in preparing a Pfu DNA polymerase mutant, specifically: cloning the correctly sequenced gene encoding the Pfu DNA polymerase mutant into an expression vector, then transforming the recombinant expression vector into a host cell, and purifying it after expression.

[0022] The fourth aspect of the present invention provides use of the Pfu DNA polymerase mutant of the present invention in preparing a reverse transcription reaction reagent or system, wherein the Pfu DNA polymerase mutant is preferably an E665K / E735K mutant or a K118I / N713V mutant.

[0023] The Pfu DNA polymerase mutant described in the present invention has reverse transcriptase activity, so it can reverse transcribe and synthesize cDNA using RNA as a template; at the same time, because the mutant also retains the polymerase activity of the wild-type enzyme, compared with the lower fidelity of conventional reverse transcriptases, the calibration activity of the mutant itself gives it high fidelity.

[0024] Preferably, the above reverse transcription reaction system also includes a PCR reaction solution.

[0025] The fifth aspect of the present invention provides an application of the Pfu DNA polymerase mutant of the present invention in RT-PCR reaction or RNA detection, specifically: using RNA as a template, using the Pfu DNA polymerase mutant of the present invention to reverse transcribe and synthesize cDNA and amplify the cDNA.

[0026] In the above application, the Pfu DNA polymerase mutant is preferably a K118I / N713V mutant; experimental data show that the mutant has reverse transcription activity in the temperature range of 56-74°C, with 68°C being the optimal temperature.

[0027] The Pfu DNA polymerase mutant of the present invention not only has catalytic 5'→3' DNA polymerization activity and 3'→5' proofreading activity, but also has reverse transcription activity. In addition, its high heat resistance enables it to efficiently and highly fidelity generate cDNA using RNA as a substrate, and then amplify the cDNA under standard PCR reaction conditions. The entire process does not require the addition of additional reverse transcriptase, thus realizing a single-enzyme "one-step" RT-PCR.

[0028] The sixth aspect of the present invention provides a method for detecting RNA based on RT-qPCR, specifically: using the RNA to be tested as a template, using the Pfu DNA polymerase mutant described in the present invention to perform real-time fluorescence quantitative PCR, and qualitatively or quantitatively analyzing the RNA to be tested based on the fluorescence results.

[0029] In the above method, the amount of RNA to be tested in the real-time fluorescence quantitative PCR mixture can be quantified based on the amount of fluorescent signal generated by the cleavage of the intercalating dye or the labeled target probe; wherein the intercalating dye includes but is not limited to SYBRGreen, EvaGreen, etc.

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

[0031] The present invention obtains a Pfu DNA polymerase mutant through primer-directed mutagenesis. Unlike the wild-type Pfu DNA polymerase, which exhibits limited reverse transcriptase activity under very stringent reaction conditions, the mutant provided by the present invention (especially the mutant K118I / N713V) has efficient and stable reverse transcriptase activity. It 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 can significantly improve the efficiency of real-time fluorescence quantitative PCR detection of target ribonucleic acid (RNA). At the same time, it can also simplify the protocol and facilitate protocol optimization, such as adjusting the buffer composition to the most effective component for a single enzyme and adjusting factors such as the enzyme amount, reaction temperature, and reaction time to conditions most conducive to the enzymatic reaction. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 shows the protein purification results of the Pfu DNA polymerase mutant Pfu-M12 of the present invention. In the figure, M is a protein marker, lane 1 is a cell lysate, lane 2 is a supernatant after lysis of bacteria, lane 3 is a supernatant after treatment at 80°C for 30 min, lane 4 is a 10 mM imidazole elution product, lane 5 is a 20 mM imidazole elution product, lane 6 is a 50 mM imidazole elution product, lane 7 is a 100 mM imidazole elution product, and lane 8 is a 200 mM imidazole elution product.

[0033] Figure 2 is a graph showing the reverse transcriptase activity test results of some Pfu DNA polymerase mutants prepared by the present invention. In the figure, MMLV is a commercial MMLV reverse transcriptase positive control, RTX is an RTX reverse transcription control, -E is a negative control without enzyme addition, -RNA is a negative control without RNA addition, WT is a wild-type Pfu DNA polymerase, and M3, M6, M8, M10, and M12 are different mutants.

[0034] Figure 3 is a comparison of the reverse transcriptase activity of the mutant Pfu-M12 of the present invention at different temperatures. In the figure, MMLV is the positive control, and lanes 1-12 are the reverse transcription products of Pfu-M12 at 56°C, 57°C, 58°C, 60°C, 62°C, 64°C, 66°C, 68°C, 70°C, 72°C, 73°C, and 74°C, respectively.

[0035] FIG4 is a graph showing the detection results of the effect of the mutant Pfu-M12 of the present invention on reverse transcription under different dosage conditions.

[0036] FIG5 is a graph showing the detection results of the effect of the reverse transcription reaction system on the reverse transcription yield in the present invention.

[0037] FIG6 is a comparison of the reverse transcription detection results before and after optimization of the reverse transcription reaction system in the present invention. In the figure, lane 1 is the MMLV control, lane 2 is the negative control, lanes 3 and 4 are before optimization, and lanes 5 and 6 are after optimization.

[0038] Figure 7 is a graph showing the polymerase activity detection results of the mutant Pfu-M12 of the present invention. In the figure, M is a marker, lanes 1-4 are 1kb, 2kb, 4kb, and 6kb fragments amplified by PCR of wild-type Pfu DNA polymerase, and lanes 5-8 are 1kb, 2kb, 4kb, and 6kb fragments amplified by PCR of Pfu-M12, respectively.

[0039] Figure 8 is a graph showing the product detection results of RT-PCR mediated by the mutant Pfu-M12 of the present invention. In the figure, M is a marker, lane 1 is a positive control, lane 2 is WT-Pfu, lane 3 is a control without adding RNA template, lane 4 is a control after RNase treatment, lanes 5 and 6 are PCR without a reverse transcription step, and lanes 7 and 8 are RT-PCR. DETAILED DESCRIPTION

[0040] The technical solution of the present invention is further described below with reference to specific examples. It should be understood that the specific examples described herein are only used to illustrate and explain the present invention and are not used to limit the present invention.

[0041] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0042] In the following examples, if specific techniques or conditions are not specified, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. All reagents or instruments used, if the manufacturer is not specified, are conventional products that can be purchased through regular channels.

[0043] The term "biologically active fragment" refers to any fragment, derivative, homolog, or analog of a Pfu DNA polymerase or mutant sequence thereof that possesses in vivo or in vitro reverse transcriptase activity specific to a biomolecule. In some embodiments, the biologically active fragment, derivative, homolog, or analog of a Pfu DNA polymerase mutant possesses any degree of biological activity of the Pfu DNA polymerase mutant in any in vivo or in vitro assay.

[0044] In some embodiments, the biologically active fragment may optionally include any number of contiguous amino acid residues of the Pfu DNA polymerase mutant sequence. The present invention also includes polynucleotides encoding any such biologically active fragment and / or degenerate nucleic acid sequence.

[0045] Biologically active fragments can be derived from post-transcriptional processing or from translation of alternatively spliced ​​RNA, or can be produced by engineering, bulk synthesis or other suitable manipulations. Biologically active fragments include fragments expressed in natural or endogenous cells, as well as fragments produced in expression systems such as bacteria, yeast, plants, insects or mammalian cells.

[0046] When referring to a gene, "mutant" means that the gene has at least one base (nucleotide) change, deletion, or insertion relative to the native or wild-type gene.

[0047] When the term "Pfu DNA polymerase mutant" is used in the present invention, it refers to a Pfu DNA polymerase mutant polypeptide that has been tested and exhibits enhanced reverse transcriptase activity, either collectively or individually, depending on the context. The term "Pfu DNA polymerase mutant" also includes variant sequences and / or degenerate nucleic acid sequences.

[0048] "Naturally occurring" or "wild-type" refers to a form found in nature. For example, a naturally occurring or wild-type polypeptide or polynucleotide sequence is a sequence found in an organism that has not been intentionally modified by human manipulation.

[0049] In some embodiments, methods (and related kits, systems, apparatus, and compositions) for performing a ligation reaction are provided, comprising or consisting of contacting a Pfu DNA polymerase mutant or a biologically active 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 Pfu DNA polymerase mutant or the biologically active fragment thereof.

[0050] The Pfu DNA polymerase mutants of the present invention can be expressed in any suitable host system, including bacterial, yeast, fungal, baculovirus, plant or mammalian host cells.

[0051] For bacterial host cells, useful promoters for transcription of the Pfu DNA polymerase mutants include those obtained from the Escherichia coli lac operon, the Streptomyces coelicolor agarase gene (dagA), the Bacillus subtilis levansucrase gene (sacB), the Bacillus licheniformis alpha-amylase gene (amyL), the Bacillus stearothermophilus maltogenic amylase gene (amyM), the Bacillus amyloliquefaciens alpha-amylase gene (amyQ), the Bacillus licheniformis penicillinase gene (penP), the Bacillus subtilis xylA and xylB genes, prokaryotic β-lactamase genes (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).

[0052] For filamentous fungal host cells, useful promoters for transcription of the Pfu DNA polymerase mutant include promoters obtained from the genes for Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral 1-amylase, Aspergillus niger acid-stable α-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline proteinase, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acetamidase, and Fusarium oxysporum trypsin-like protease (WO 96 / 00787), the NA2-tpi promoter (a hybrid of the promoters from the Aspergillus niger neutral α-amylase and Aspergillus oryzae triose phosphate isomerase genes), and mutants, truncations, and hybrid promoters thereof.

[0053] In yeast hosts, promoters useful for transcription of the Pfu DNA polymerase mutants can be derived from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP), and Saccharomyces cerevisiae 3-phosphoglycerate kinase. Other useful promoters for yeast host cells are described in Romanos et al., 1992, Yeast 8:423-488.

[0054] For baculovirus expression, promoters for transcription of the Pfu DNA polymerase mutant can be derived from insect cell lines of the order Lepidoptera (moths and butterflies), such as Spodoptera frugiperda, and used as hosts. Gene expression is controlled by a strong promoter, such as pPolh.

[0055] Plant expression vectors are based on the Ti plasmid of Agrobacterium tumefaciens, or on tobacco mosaic virus (TMV), potato virus X, or rainbow bean mosaic virus. A commonly used constitutive promoter in plant expression vectors is the cauliflower mosaic virus (CaMV) 35S promoter.

[0056] For mammalian expression, cultured mammalian cell lines such as Chinese hamster ovary (CHO), COS (including human cell lines such as HEK and HeLa) can be used to produce Pfu DNA polymerase mutants. Mammalian expression vectors include adenovirus vectors, pSV and pCMV series plasmid vectors, vaccinia virus and retroviral vectors, and baculovirus. 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.

[0057] The control sequence for expression can be a suitable transcription terminator sequence, i.e., a sequence that is recognized by the host cell to terminate transcription. The terminator sequence is operably linked to the 3' end of the nucleic acid sequence encoding the polypeptide. Any terminator that works in the selected host cell can be used.

[0058] For example, exemplary transcription terminators for filamentous fungal host cells can be obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease.

[0059] Exemplary terminators for yeast host cells can be obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase.

[0060] The control sequence can also be a suitable leader sequence, i.e., the non-translational region of an mRNA that is very important to host cell translation. The leader sequence is operably linked to the 5' end of the nucleotide sequence encoding the polypeptide. Any leader sequence that functions in the selected host cell can be used. Exemplary leader sequences for filamentous fungal host cells are obtained from the genes of Aspergillus oryzae TAKA amylase and Aspergillus nidulans triosephosphate isomerase. Leader sequences that are suitable for yeast host cells are 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).

[0061] The control sequence can also be a polyadenylation sequence, which is operably linked to the 3' end of the nucleic acid sequence and, when transcribed, is recognized by the host cell as a signal to add polyadenylic acid residues to the transcribed mRNA. Any polyadenylation sequence that works in the selected host cell can be used in the present invention. Exemplary polyadenylation sequences for filamentous fungal host cells can be derived from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Fusarium oxysporum trypsin-like protease, and Aspergillus niger α-glucosidase.

[0062] The control sequence may also be a signal peptide coding region, which encodes an amino acid sequence linked to the amino terminus of a polypeptide and directs the encoded polypeptide into the cell's secretory pathway. The 5' end of the coding sequence of the nucleic acid sequence may inherently contain a signal peptide coding region that is naturally linked in translation reading frame to the coding region segment encoding the secreted polypeptide. Alternatively, the 5' end of the coding sequence may contain a signal peptide coding region that is exogenous to the coding sequence. Where the coding sequence does not naturally contain a signal peptide coding region, an exogenous signal peptide coding region may be required.

[0063] Alternatively, the foreign signal peptide coding region may simply replace the natural signal peptide coding region to enhance secretion of the polypeptide.However, any signal peptide coding region that directs the expressed polypeptide into the secretory pathway of the chosen host cell may be used.

[0064] Effective signal peptide coding regions for bacterial host cells are those obtained from the genes for Bacillus NCIB 11837 maltoamylase, Bacillus stearothermophilus α-amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis β-lactamase, Bacillus stearothermophilus neutral protease (nprT, nprS, nprM), and Bacillus subtilis prsA. Signal peptides are further described by Simonen and Palva, 1993, Microbiol Rev 57: 109-137.

[0065] An effective signal peptide coding region for a filamentous fungal host cell can be that obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase, Humicola insolens cellulase, and Humicola lanuginosa lipase.

[0066] Useful signal peptides for yeast host cells can be derived from the genes for Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae invertase. Signal peptides for other host cell systems are also well known.

[0067] The control sequence can also be a propeptide coding region encoding an amino acid sequence located at the amino terminus of the polypeptide. The resulting polypeptide is referred to as a proenzyme or propolypeptide (or in some cases, a zymogen). A propolypeptide is typically inactive and can be converted into a mature active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. The propeptide coding region can be obtained from the genes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Saccharomyces cerevisiae α-factor, Rhizomucor miehei aspartic proteinase, and Myceliophthora thermophila lactase (WO95 / 33836).

[0068] When both the signal peptide and the propeptide region are present at the amino terminus of a 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.

[0069] It may also be necessary to add regulatory sequences that allow expression of the Pfu DNA polymerase mutant relative to the growth regulation of the host cell. Examples of regulatory systems are those that respond to chemical or physical stimuli (including the presence of regulatory compounds) that cause gene expression to be turned on or off. In prokaryotic host cells, suitable regulatory sequences include lac, tac, and trp operating systems. In yeast host cells, suitable regulatory systems include, for example, the ADH2 system or the GAL1 system. In filamentous fungi, suitable regulatory sequences include the TAKAa-amylase promoter, the Aspergillus niger glucoamylase promoter, and the Aspergillus oryzae glucoamylase promoter. The regulatory systems of other host cells are also well known.

[0070] Other examples of regulatory sequences are sequences that allow gene amplification. In eukaryotic systems, these include the dihydrofolate reductase gene that is amplified in the presence of methotrexate and the metallothionein genes that are amplified with heavy metals. In these cases, the nucleic acid sequence encoding the polypeptide of the present invention will be operably linked to the regulatory sequence.

[0071] In a specific embodiment, comprise recombinant expression vector, it comprises the polynucleotide of the Pfu archaeal dna polymerase mutant of coding engineering, and one or more expression regulatory regions, for example promotor and terminator, and replication origin, and this depends on the type of the host that they are imported.Above-mentioned various nucleic acid and control sequence can link together to produce recombinant expression vector, and described recombinant expression vector can comprise one or more restriction sites easily, to allow to insert or replace the nucleotide sequence of coding Pfu archaeal dna polymerase mutant in these sites.Perhaps, the nucleotide sequence of Pfu archaeal dna polymerase mutant can be expressed by nucleotide sequence or the nucleic acid construct that comprises this sequence being inserted in the suitable expression vector.When making up expression vector, encoding sequence is positioned in the carrier, makes encoding sequence effectively connected with suitable control sequence to express.

[0072] Described recombinant expression vector can be any carrier (for example plasmid or virus), and it can carry out recombinant DNA procedure easily, and can cause the expression of Pfu archaeal dna polymerase mutant polynucleotide sequence.The selection of carrier depends on the compatibility of carrier and the host cell of importing carrier usually.Described carrier can be linear or closed circular plasmid.

[0073] The expression vector can be an autonomously replicating vector, i.e., a vector existing as an extrachromosomal entity, which replicates independently of chromosomal replication, such as a plasmid, an extrachromosomal element, a minichromosome or an artificial chromosome. The vector can include any means for ensuring self-replication. Alternatively, the vector can be a vector that is integrated into the genome and replicates together with the chromosome into which it is integrated when the host cell is imported. In addition, a single vector or plasmid, or two or more vectors or plasmids, can be used that together comprise the total DNA or a transposon to be imported into the host cell genome.

[0074] The expression vectors of the present invention preferably contain one or more selectable markers that allow for easy selection of transformed cells. Selectable markers are genes whose products provide biocide or viral resistance, resistance to heavy metals, prototrophy for auxotrophs, and the like. Examples of bacterial selectable markers are the dal genes from Bacillus subtilis or Bacillus licheniformis, or markers that confer antibiotic resistance such as ampicillin, kanamycin, chloramphenicol (Example 1), or tetracycline resistance. Suitable markers for yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for filamentous fungal host cells include, but are not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase), sC (sulfate adenylate transferase), and trpC (anthranilate synthase), and their equivalents. Embodiments for use in Aspergillus cells include the amdS and pyrG genes of Aspergillus nidulans or Aspergillus oryzae and the bar gene of Streptomyces hygroscopicus.Selectable markers for insect, plant, and mammalian cells are also well known.

[0075] The expression vectors of the present invention preferably contain elements that allow the vector to be integrated into the host cell genome or to autonomously replicate in the cell independent of the genome. To integrate into the host cell genome, the vector may rely on the nucleic acid sequence encoding the polypeptide or any other element of the vector to integrate the vector into the genome by homologous or nonhomologous recombination.

[0076] Alternatively, the expression vector may comprise additional nucleic acid sequences for directing integration into the host cell's genome by homologous recombination. The additional nucleic acid sequences enable the vector to be integrated into the host cell genome at a precise location in the chromosome. The integration element may be any sequence homologous to the target sequence in the host cell genome. Additionally, the integration element may be a non-coding or coding nucleic acid sequence. On the other hand, the vector may be integrated into the host cell's genome by non-homologous recombination.

[0077] In some embodiments, the vector can be used to replicate autonomously in the host cell. For autonomous replication, the vector can further include an origin of replication that enables the vector to replicate autonomously in the host cell in question. The example of bacterial replication origin is P15Aori, or the replication origin of the plasmid pBR322, pUC19, pACYC177 (the plasmid has P15Aori) or pACYC184 allowed to be replicated in Escherichia coli, and the replication origin of pUB110, pE194, pTA1060 or pAM31 allowed to be replicated in bacillus. The example of the replication origin used in the yeast host cell is a combination of 2 micron replication origins ARS1, ARS4, ARS1 and CEN3 and a combination of ARS4 and CEN6. The replication origin can be a sudden change that makes it temperature-sensitive in the host cell (see Ehrlich, 1978, Proc Natl Acad Sci. USA 75:1433).

[0078] The nucleotide sequence of more than one copy of the Pfu DNA polymerase mutant can be inserted into the host cell to increase the production of the gene product. The increase in the nucleotide sequence copy number can be obtained by integrating at least one extra sequence copy into the host cell genome or by including an amplifiable selectable marker gene in the nucleotide sequence, wherein cells containing amplifiable selectable marker gene copies can be selected by cultivating cells in the presence of a suitable selection agent, thereby selecting the extra copy of the nucleotide sequence.

[0079] Expression vectors for the Pfu DNA polymerase mutant polynucleotides are commercially available. Suitable commercial expression vectors include the p3xFLAG™ expression vector from Sigma-Aldrich Chemicals, St. Louis Mo., which includes a CMV promoter and an hGH polyadenylation site for expression in mammalian host cells, and a pBR322 origin of replication and an 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, Gene 57:193-201).

[0080] Suitable host cells for expressing polynucleotides encoding Pfu DNA polymerase mutants are well known in the art and include, but are not limited to, bacterial cells, such as Escherichia coli, Lactobacillus kefir, Lactobacillus brevis, Lactobacillus minor, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris); insect cells, such as Drosophila S2 and Spodoptera exigua Sf9 cells; animal cells, such as CHO, COS, BHK, 293 and Bowes melanoma cells; and plant cells.

[0081] The polynucleotide for expressing the Pfu DNA polymerase mutant can be introduced into cells by various methods known in the art, including electroporation, bioballistic particle bombardment, liposome-mediated transfection, calcium chloride transfection, and protoplast fusion.

[0082] The polynucleotide of encoding the Pfu archaeal dna polymerase mutant can be prepared by standard solid phase method according to known synthetic method.In some embodiments, the fragment of up to about 100 bases can be synthesized separately, then connected (for example, by enzymatic or chemical litigation method or polymerase-mediated method) to form any required continuous sequence.For example, polynucleotide can be prepared by chemical synthesis, and described chemical synthesis uses for example, by Beaucage et al., 1981, the classical phosphoramidite method of Tet Lett22:1859-69 description, or by Matthes et al., 1984, the method for EMBO J.3:801-05 description.According to the phosphoramidite method, synthetic oligonucleotide, for example, in automatic DNA synthesizer, purification, annealing, connection and cloning in suitable carrier. In addition, essentially any nucleic acid can be obtained from a variety of commercial sources, for example, Midland Certified Reagent Company, Midland, Tex.; Great American Gene Company, Ramona, Calif.; ExpressGen Inc. Chicago, 111.; and Operon Technologies Inc., Alameda, Calif.

[0083] The engineered Pfu DNA polymerase mutant expressed in the host cell can be recovered from the cells and / or culture medium using any one or more well-known protein purification techniques, including lysozyme treatment, sonication, filtration, salting-out, ultracentrifugation, and chromatography. Suitable solutions for cracking and efficiently extracting proteins from bacteria (e.g., E. coli) are available from Sigma-Aldrich, St. Louis, Mo., under the trade name CelLyticB.™.

[0084] Chromatographic techniques for isolating the Pfu DNA polymerase mutants include reverse 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, molecular shape, and the like, and will be apparent to those skilled in the art.

[0085] In some embodiments, affinity technology can be used for separating the Pfu DNA polymerase mutant. For affinity chromatography purification, any antibody specifically bound to the Pfu DNA polymerase mutant can be used. In order to produce antibodies, various host animals can be immunized by injection of compound, including but not limited to rabbit, mouse, rat etc. The compound can be connected to a suitable carrier, such as bovine serum albumin, by a side chain functional group or a joint connected to a side chain functional group. According to host species, various adjuvants can be used to increase immune response, including but not limited to Freund's adjuvant (complete and incomplete), mineral gels such as aluminum hydroxide, surfactants such as lysolecithin, Pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (BCG) and Corynebacterium parvum.

[0086] Example 1 Preparation of Pfu DNA polymerase mutants

[0087] Compared to wild-type Pfu DNA polymerase, the Pfu DNA polymerase mutants of the present invention exhibit higher reverse transcriptase activity. By rationally designing the wild-type Pfu DNA polymerase and engineering, characterizing, and screening it through polymerase chain reaction, the present invention obtained several mutants that are both thermostable and possess both reverse transcriptase and DNA polymerase activity. Among them, the K118I / N713V mutant (i.e., Pfu-M12) exhibited the strongest reverse transcriptase activity.

[0088] In this example, the Pfu DNA polymerase mutant was prepared by the following process:

[0089] The Pfu DNA polymerase mutant gene was connected to pET23a to obtain the pET23a-Pfu mutant plasmid, which was then transformed into Escherichia coli BL21 (DE3). A single colony was inoculated into LB liquid medium containing 50 μg / mL ampicillin antibiotics and cultured on a shaker at 37°C and 220 rpm. When the bacterial OD 600 When the pH reached 0.8, the cells were transferred to an 18°C ​​shaker and induced overnight with IPTG. The cells were harvested by centrifugation at 6000 rpm for 10 minutes, washed with Buffer A (20 mM Tris-Cl, pH 8.2, 100 mM KCl, 0.1 mM DTT), resuspended in Buffer A, supplemented with PMSF to a final concentration of 1 mM, disrupted by high pressure, and centrifuged at 18000 rpm for 30 minutes. The supernatant was collected. 10 μL DNase I, 10 μL DNase I Buffer, and 5 μL RNase A were added to the disrupted supernatant. The supernatant was incubated at 37°C for 30 minutes to degrade nucleic acids. The cells were then incubated in an 80°C waterbath for 30 minutes to remove most host-derived proteins. The cells were centrifuged at 18000 rpm for 30 minutes, and the supernatant was collected.

[0090] After the supernatant was filtered, Ni-NTA purification was performed. 20mM and 50mM imidazole were each washed for 10 column volumes (added 3 times), and 100mM, 150mM, and 200mM were each washed for 3 column volumes, and samples were taken for SDS-PAGE detection. The eluted fractions containing the high-purity target protein were collected and ultrafiltered to Buffer A. The purified target protein was concentrated using a Millipore 50-kDa ultrafiltration tube at 4°C and 4000rpm, and the solution was changed to remove the imidazole. The enzyme concentration was quantitatively determined using a BCA kit. The determination steps were carried out according to the operating instructions. BSA was used as a standard to prepare a standard solution and draw a standard curve to calculate the protein concentration of the purified Pfu mutant. The purified protein was collected, and after the purity was identified by SDS-polyacrylamide gel and the activity was verified, the protein was divided into small portions, quickly frozen in liquid nitrogen, and stored at -80°C.

[0091] All Pfu DNA polymerase mutants and wild-type Pfu DNA polymerase in this example were N-terminally tagged to facilitate purification. The amino acid sequence of the wild-type Pfu DNA polymerase with an N-terminal His tag is shown in SEQ ID NO. 3, and its gene sequence is shown in SEQ ID NO. 2.

[0092] The purification results of Pfu-M12 are shown in Figure 1. The molecular weight of Pfu-M12 polymerase is approximately 90 kDa, consistent with the theoretical molecular weight. Treatment at 80°C for 30 minutes removed most of the contaminating proteins (Lane 3). The eluted product was concentrated using an ultrafiltration tube with 100 mM imidazole and the protein concentration of Pfu-M12 was determined to be 2.84 mg / mL using a Bradfold protein assay kit.

[0093] Example 2 Verification of reverse transcriptase activity of Pfu DNA polymerase mutant

[0094] The reverse transcriptase activity of the mutant was detected by reverse transcription RNA. The detection process is as follows:

[0095] (1) Add 5 pmol of synthetic RNA (TEMP.A. RNA) and 5 pmol of the corresponding reverse primer (25FAM) to a PCR tube, add 0.4 μg of Pfu polymerase or mutant, and RNase Inhibitor, and mix well. Heat to 80°C in a thermal cycler for 1 minute, then anneal at a rate of 0.1°C / s until the temperature reaches 25°C, maintaining for 2 minutes. At this point, the RNA forms a ternary complex with the enzyme and primer.

[0096] (2) Add 0.2 μL of 10 mM dNTPs and 1 μL of 10× PCR Buffer to the annealed mixture, and add DEPC water to make up to 10 μL.

[0097] (3) Place the reverse transcription system in a thermal cycler and incubate at 72°C for 30 seconds. Then, quickly cool to 12°C to reduce polymerase activity and terminate the reaction. Add a blocker complementary to the RNA template to prevent the RNA template from affecting the detection results.

[0098] (4) Add an equal volume of 2× RNA loading buffer and incubate at 75°C for 5 min. After the reaction is completed, separate the products using 20% ​​denaturing polyacrylamide gel electrophoresis (nucleic acid PAGE) and visualize them using a gel imaging instrument.

[0099] All extension experiments were repeated three times.

[0100] The activity comparison of Pfu-M12 with Pfu-M3 (K467R / F588L / W769R mutant), Pfu-M6 (R382H / R385H / V390I mutant), Pfu-M8 (I38L / R97M mutant), and Pfu-M10 (E665K / E735K mutant) is shown in Figure 2. It can be clearly seen that Pfu-M12 has the highest reverse transcription activity.

[0101] Example 3 Effect of Temperature on Pfu-M12 Reverse Transcriptase Activity

[0102] Referring to Example 2, to determine the optimal reverse transcription temperature for Pfu-M12, a total of 12 temperature gradients were set for reverse transcription: 56°C, 57°C, 58°C, 60°C, 62°C, 64°C, 66°C, 68°C, 70°C, 72°C, 73°C, and 74°C. When the thermal cycler reached the set temperature, the reaction sample was quickly added and allowed to react for 30 seconds.

[0103] The experimental results are shown in FIG3 . Pfu-M12 has reverse transcription activity in the temperature range of 56-74° C., and the reverse transcription activity is highest at 68° C.

[0104] Example 4 Effect of enzyme amount on Pfu-M12 reverse transcription

[0105] Referring to Example 2 (reverse transcription temperature was changed to 68° C.), in this example, the amount of enzyme in the reverse transcription system was adjusted to 0.1 μg-1 μg, with each 0.1 μg forming a gradient, for a total of 10 enzyme concentration conditions.

[0106] The experimental results are shown in Figure 4. The size of the reverse transcription extension products obtained with the addition of 0.1 μg and 1 μg of Pfu-M12 is consistent, indicating that the amount of Pfu-M12 enzyme is not the cause of the smaller reverse transcription products compared to commercial MMLV. Furthermore, the optimal amount of Pfu-M12 for DNA polymerase activity in a 10 μL PCR system was determined to be between 0.1 μg and 0.2 μg.

[0107] Example 5 Optimization of reverse transcription reaction system

[0108] To improve reverse transcription yield, some components of the reverse transcription system were adjusted in this example. Specifically, the reverse transcription activity of Pfu-M12 was measured in reaction buffers containing 2.0-2.7 mM MgCl2, 10-60 mM Tris-HCl, 0-14 mM KCl, and 0-18 mM (NH4)2SO4.

[0109] The experimental results are shown in Figure 5. From the results, we can see that: Mg 2+ When the concentration is 2.5-2.6mM, as Mg 2+ As the concentration continued to increase, the reverse transcription product decreased. With increasing Tris-HCl concentration, the reverse transcription activity gradually weakened, with 10mM Tris-HCl being the optimal concentration for reverse transcription. With increasing KCl and (NH4)2SO4 levels in the reaction system, the reverse transcription activity also gradually increased. When the KCl concentration reached 12mM and the (NH4)2SO4 concentration reached 8mM, the reverse transcription activity of Pfu-M12 reached its peak and then began to decline. Therefore, the optimal conditions for Pfu-M12 reverse transcription are: 10mM Tris-HCl (pH 8.8), 2.5mM MgCl2, 12mM KCl, and 8mM (NH4)2SO4.

[0110] Under the condition of a reverse transcription time of 20s, the reverse transcription of the buffer before and after optimization was compared, as shown in Figure 6. The results were analyzed using ImageJ software, and the results showed that the reverse transcription yield increased by about 40% after optimization.

[0111] Example 6 Detection of polymerase activity of Pfu-M12

[0112] EvaEZ polymerase activity fluorescence assay kit TMThe polymerase activity of the Pfu-M12 mutant was detected by Fluorometric Polymerase Activity Assay Kit. Commercial Pfu DNA polymerase purchased from Shanghai Bioengineering was used as the standard. A standard curve of enzyme activity was drawn. Pfu-M12 was diluted to 0.1 μg / μL, and a gradient addition was performed. The initial slope was measured and substituted into the polymerase activity standard curve. The specific polymerase activity of Pfu-M12 was calculated to be 3145.54 U / mg.

[0113] After measuring polymerase activity, this example further examined the elongation properties of Pfu-M12 during PCR. Using the pRGEB32 plasmid as a template, fragments of 1 kb, 2 kb, 4 kb, and 6 kb were amplified using PCR at a 2-minute extension time. The results, shown in Figure 7, demonstrate that Pfu-M12's polymerase activity and elongation properties were not affected, with fragments up to 4 kb still generated within 2 minutes, consistent with wild-type Pfu DNA polymerase.

[0114] Example 7 Feasibility Verification of Pfu-M12 for RT-PCR Detection

[0115] This example investigates the feasibility of using Pfu-M12 alone for RT-PCR using HIV RNA as a template. PCR protocols were set up both with and without reverse transcription to test whether Pfu-M12 can directly amplify RNA without the reverse transcription step.

[0116] Following the MMLV instruction manual, MMLV reverse-transcribed cDNA served as a template, and Pfu-M12 was used for PCR as a positive control. Negative controls included direct RNA amplification with wild-type Pfu, amplification without RNA template, and amplification with RNase-treated template. The RT-PCR system and protocol settings are shown in Tables 1 and 2.

[0117] Table 1 RT-PCR system:

[0118] Table 2 RT-PCR program:

[0119] The results, as shown in Figure 8, indicate that products of the expected size can be obtained, demonstrating the feasibility of using the Pfu DNA polymerase mutant for RT-PCR. Furthermore, using Pfu-M12 for RT-PCR eliminates the need for an additional reverse transcription step (as shown in lanes 5 and 6) because Pfu-M12 is heat-resistant and can amplify simultaneously with transcription.

[0120] In summary, the present invention obtains a Pfu DNA polymerase mutant through site-directed mutagenesis. Compared with the wild-type Pfu DNA polymerase, the mutant has efficient and stable reverse transcriptase activity, can efficiently convert cDNA using RNA substrate as a substrate, and amplify the cDNA under standard reaction conditions without the need for additional reverse transcriptase, which can significantly improve the efficiency of detecting target RNA by real-time fluorescence quantitative PCR.

[0121] It should be noted that the above trial implementation examples are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described with reference to the preferred embodiments of the present invention, it should be understood by those skilled in the art that various changes can be made in form and details without departing from the spirit and scope of the present invention.

Claims

1. A Pfu DNA polymerase mutant having reverse transcriptase activity, characterized in that The Pfu DNA polymerase mutant is any of the following: The K467R / F588L / W769R mutant is a mutant in which the lysine at position 467 of the Pfu DNA polymerase shown in the amino acid sequence of SEQ ID NO.1 is mutated to arginine, the phenylalanine at position 588 is mutated to leucine, and the tryptophan at position 769 is mutated to arginine; or R382H / R385H / V390I mutants are obtained by mutating the arginine at positions 382 and 385 of the Pfu DNA polymerase as shown in SEQ ID NO.1 to histidine, and mutating the valine at position 390 to isoleucine; or I38L / R97M mutant is a mutant in which the isoleucine at position 38 of the Pfu DNA polymerase shown in the amino acid sequence of SEQ ID NO.1 is mutated to leucine, and the arginine at position 97 is mutated to methionine; or The E665K / E735K mutant is a mutant in which the glutamic acid at positions 665 and 735 of the Pfu DNA polymerase shown in the amino acid sequence of SEQ ID NO.1 are simultaneously mutated to lysine; or The K118I / N713V mutant is a mutant in which the lysine at position 118 of the Pfu DNA polymerase shown in the amino acid sequence of SEQ ID NO.1 is mutated to isoleucine, and the asparagine at position 713 is mutated to valine.

2. A gene encoding the Pfu DNA polymerase mutant according to claim 1.

3. A recombinant expression vector containing the gene according to claim 2.

4. A recombinant cell containing the gene according to claim 2 or the recombinant expression vector according to claim 3.

5. Use of the gene according to claim 2, the recombinant expression vector according to claim 3, or the recombinant cell according to claim 4 in preparing a Pfu DNA polymerase mutant.

6. Use of the Pfu DNA polymerase mutant according to claim 1 in preparing a reverse transcription reaction reagent or system.

7. The use according to claim 5, characterized in that: The Pfu DNA polymerase mutant is an E665K / E735K mutant or a K118I / N713V mutant.

8. Use of the Pfu DNA polymerase mutant of claim 1 in RT-PCR reaction or RNA detection, specifically: using RNA as a template, using the Pfu DNA polymerase mutant of claim 1 to reverse transcribe and synthesize cDNA and amplify the cDNA.

9. The use according to claim 8, characterized in that: The Pfu DNA polymerase mutant is a K118I / N713V mutant, and the reverse transcription temperature is 56-74°C.

10. A method for detecting RNA based on RT-qPCR, characterized in that: The method is not aimed at disease diagnosis and treatment. The method specifically comprises: using the RNA to be tested as a template, performing real-time fluorescence quantitative PCR using the Pfu DNA polymerase mutant described in claim 1, and performing qualitative or quantitative analysis on the RNA to be tested based on the fluorescence results.

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