WO2020124481A1 - 酶活性提高的dna聚合酶及其应用 - Google Patents

酶活性提高的dna聚合酶及其应用 Download PDF

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WO2020124481A1
WO2020124481A1 PCT/CN2018/122334 CN2018122334W WO2020124481A1 WO 2020124481 A1 WO2020124481 A1 WO 2020124481A1 CN 2018122334 W CN2018122334 W CN 2018122334W WO 2020124481 A1 WO2020124481 A1 WO 2020124481A1
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dna polymerase
nucleic acid
amplification
host cell
activity
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French (fr)
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张晓红
兰茜
张周刚
郑越
董宇亮
章文蔚
徐崇钧
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BGI Shenzhen Co Ltd
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BGI Shenzhen Co Ltd
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C—CHEMISTRY; METALLURGY
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)

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  • the invention relates to the field of enzyme engineering, in particular to a DNA polymerase with improved enzyme activity and its application.
  • PCR Polymerase chain reaction
  • the PCR reaction requires the participation of DNA polymerases, templates, primers, and dNTPs. Among them, DNA polymerases play a vital role in PCR reactions. Therefore, there have been many reports on the modification of DNA polymerases to suit different applications, such as Increase the rate of amplification, tolerance to inhibitors, etc.
  • multiple PCR over conventional PCR is that it adds more than two pairs of primers to the same PCR reaction system and simultaneously amplifies multiple nucleic acid fragments. It also has the characteristics of high efficiency, systemicness, and economic simplicity. Due to the amplification of multiple fragments, multiplex PCR not only requires a high activity of DNA polymerase, but also requires a certain degree of amplification uniformity, that is, the amplification of different fragments in the same system is better.
  • an object of the present invention is to propose a DNA polymerase with increased enzyme activity, which has a significantly improved enzyme activity compared to the wild-type DNA polymerase activity.
  • the DNA polymerase can be used in multiple PCR reaction systems. Due to the high activity of the DNA polymerase provided by the present invention, it can be applied to PCR reactions with low template content, mixed with inhibitory substances or long fragment templates.
  • the PCR process has higher requirements for DNA polymerase, and the properties such as the activity of DNA polymerase directly affect the amplification efficiency of the PCR process.
  • the polymerase used for the polymerization reaction is usually provided in the form of a kit, the information of the DNA polymerase used is rarely disclosed, and it is usually expensive. It restricts the application in the reaction system that requires high performance of DNA polymerase.
  • Multiple PCR is to add more than two pairs of primers to the same PCR reaction system, and amplify multiple nucleic acid fragments at the same time, which has the characteristics of high efficiency, systematicness and economic simplicity. Due to the amplification of multiple fragments, multiplex PCR not only requires a high activity of DNA polymerase, but also requires a certain degree of amplification uniformity, that is, the amplification of different fragments in the same system is better. It has been found through research that the homogeneity or yield of wild-type DNA polymerase in multiplex PCR is poor and cannot meet most application requirements. Therefore, it is urgent to find a DNA polymerase that can be applied to multiplex PCR.
  • the present invention can effectively overcome this problem through different multiplex PCR amplification systems and DNA polymerases screened by activity detection, and independent research and development and production can greatly reduce costs.
  • the selected mutants may be used not only for multiplex PCR, but also for PCR or long fragment amplification with low template input or containing inhibitors (such as salt and blood).
  • the present invention provides a DNA polymerase, which has at least one of the following mutation sites compared with the amino acid sequence shown in SEQ ID NO:2: E315K, E507R, E507H, E524K, L552R, D578N, E742R, E742K, A743R, A743K, K53S/E507K, K56N/E507K, K56S/E507K, K56T/E507K, K56Q/E524K, L245M/E524K, L245M/L552R S357C, K56Q/E507K, E57D/E507K, K56Q/E507R, K56T/E524K, E57D/E524K, E57D/E742R, L245M/E315K.
  • amino acid sequence shown in SEQ ID NO:2 E315K, E507R, E507H, E524K, L
  • the above DNA polymerase may further include the following technical features:
  • the DNA polymerase is a mutant of Taq DNA polymerase.
  • the activity of the DNA polymerase is increased by at least 0.5 times compared with the activity of the unmutated Taq DNA polymerase.
  • the activity of the DNA polymerase is at least doubled compared to the activity of unmutated Taq DNA polymerase.
  • the invention provides an isolated nucleic acid molecule that encodes the DNA polymerase according to the first aspect of the invention.
  • the invention provides a construct comprising the isolated nucleic acid molecule according to the second aspect of the invention.
  • the construct is a plasmid.
  • the isolated nucleic acid molecule is operably linked to a promoter.
  • the promoter is selected from one of the following: lambda-PL promoter, tac promoter, trp promoter, araBAD promoter, and trc promoter.
  • the invention provides a host cell containing the construct according to the third aspect of the invention.
  • the host cell used to express the protein of interest may be a prokaryotic cell or a eukaryotic cell.
  • prokaryotic cells are used to express DNA polymerases, such as Escherichia coli.
  • the eukaryotic cells suitable for the present invention may be plant cells, animal cells (eg, Drosophila cells, CHO cells, C. elegans cells, etc.), fungal cells (eg, Saccharomyces cells, Pichia pastoris cells, etc.).
  • the invention provides a method for producing a DNA polymerase, the DNA polymerase being the DNA polymerase according to the first aspect of the invention, the production method comprising: cultivating a host cell, The host cell is the host cell according to the fourth aspect of the present invention; the host cell is subjected to an induction treatment so that the host cell expresses the DNA polymerase; and the DNA polymerase is isolated.
  • the host cell is E. coli.
  • the invention provides a kit comprising the DNA polymerase according to the first aspect of the invention.
  • the kit containing DNA polymerase is used in PCR reactions to increase the efficiency of amplification.
  • the kit described above may be further added with the following technical features:
  • the kit further includes at least one of the following: one or more nucleotides, one or more buffers, one or more primers, one or more Species terminator.
  • the terminator is dideoxynucleotide.
  • the invention provides a method of amplifying a nucleic acid molecule, the method comprising: mixing at least one nucleic acid template with at least one DNA polymerase to obtain a mixture, the DNA polymerase being The DNA polymerase according to the first aspect of the present invention; the mixture is subjected to an amplification process so as to obtain a nucleic acid molecule that is wholly or partially complementary to the at least one nucleic acid template.
  • the above method for amplifying nucleic acid molecules may further include the following technical features:
  • the minimum content of the at least one nucleic acid template is 0.001 pg/ ⁇ l.
  • the content of the template used is 0.2-2ng/ ⁇ l.
  • the DNA polymerase provided by the present application can significantly improve the polymerization activity, and thus can be used for the amplification of low-level template substances. For example, when performing single-plex PCR amplification, when the nucleic acid content of the nucleic acid template is 1 pg/ ⁇ l, perform 20 cycles to obtain obvious amplification bands; even if the nucleic acid content of the nucleic acid template is 0.001 pg/ ⁇ l, perform After 20 cycles, there are still weak bands. When the nucleic acid content of the nucleic acid template is 0.001 pg/ ⁇ l, 30-40 cycles are performed, and there is a clear amplification band.
  • the length of the nucleic acid molecule that is completely or partially complementary to the at least one nucleic acid template can be up to 10 kb. Due to the high polymerization activity of the DNA polymerase provided by the present application, it can be applied to the amplification of long-length nucleic acid templates, and the length of the obtained amplified fragments can reach 10 kb. For example, it can reach more than 2kb, more than 3kb, more than 5kb, more than 8kb and so on.
  • DNA polymerase provided by the present application can also be used for the amplification of short fragment nucleic acid templates, for example, for the amplification of nucleic acid templates of 100 bp or more, 200 bp or more, and 500 bp or more.
  • the present invention provides a method for amplifying multiple nucleic acid molecules, comprising: mixing at least two nucleic acid templates with at least one DNA polymerase to obtain a mixture, wherein the DNA polymerase is based The DNA polymerase according to the first aspect of the invention; the mixture is subjected to an amplification process so as to obtain a nucleic acid molecule that is wholly or partially complementary to the at least two nucleic acid templates.
  • the mutant provided by the present invention has improved polymerization activity and DNA affinity compared with the corresponding naturally occurring DNA polymerase, and the amplification effect in multiplex PCR is significantly improved. Moreover, it can be used for the amplification of low-template substances and the amplification of long fragments.
  • FIG. 1 is an electrophoresis diagram of 8-fold human housekeeping gene amplification of wild-type Taq DNA polymerase and its mutants according to an embodiment of the present invention.
  • FIG. 2 is an electrophoresis diagram of 8-fold human housekeeping gene amplification of wild-type Taq DNA polymerase and its mutants according to an embodiment of the present invention.
  • FIG. 3 is an electrophoresis diagram of 8-fold human-derived housekeeping gene amplification of wild-type Taq DNA polymerase and its mutants according to an embodiment of the present invention.
  • FIG. 4 is an electrophoresis diagram of a five-fold mouse-derived housekeeping gene of wild-type Taq DNA polymerase and its mutants according to an embodiment of the present invention.
  • FIG. 5 is an electrophoresis diagram of the amplification of a house gene of a five-fold mouse derived from wild-type Taq DNA polymerase and a mutant thereof according to an embodiment of the present invention.
  • FIG. 6 is an electrophoresis diagram of the amplification of a house gene of a five-fold mouse derived from wild-type Taq DNA polymerase and its mutants according to an embodiment of the present invention.
  • DNA polymerase refers to a protein, polypeptide or polypeptide fragment that exhibits DNA polymerase activity.
  • DNA polymerase activity refers to the ability to synthesize complementary DNA strands using DNA as a template.
  • mutant refers to a DNA sequence or a wild-type amino acid sequence that has one or more mutations compared to the wild type. Of course, this mutation can occur at the nucleic acid level or the amino acid level.
  • the present invention provides a DNA polymerase having at least one of the following mutation sites compared to the amino acid sequence shown in SEQ ID NO:2: E315K, E507R, E507H, E524K, L552R, D578N, E742R, E742K, A743R, A743K, K53S/E507K, K56N/E507K, K56S/E507K, K56T/E507K, K56Q/E524K, L245M/E524K, L245M/L552R, E315K/S357C E507K, E57D/E507K, K56Q/E507R, K56T/E524K, E57D/E524K, E57D/E742R, L245M/E315K.
  • the amino acid sequence shown in SEQ ID NO: 2 is the amino acid sequence of wild-type Taq DNA polymerase.
  • the DNA polymerase provided in this application exhibits a mutation at a single site or a combination of two sites, making the activity of the DNA polymerase comparable to that of the wild-type
  • the activity of Taq DNA polymerase has increased by at least 0.5 times.
  • the activity of the DNA polymerase is at least doubled compared to the activity of the wild-type DNA polymerase.
  • the activity of the DNA polymerase is at least 2-fold higher than the activity of the wild-type DNA polymerase.
  • the DNA polymerase has improved amplification uniformity compared to wild-type DNA polymerase.
  • “Amplification uniformity” herein refers to the consistency of the amplification ability of the DNA polymerase for each nucleic acid template and corresponding primer when there are multiple nucleic acid templates and multiple primers in the same reaction system.
  • the DNA polymerase has an E315K mutation compared to the amino acid sequence shown in SEQ ID NO:2.
  • the DNA polymerase has an E507R mutation compared to the amino acid sequence shown in SEQ ID NO:2.
  • the DNA polymerase has an E507H mutation compared to the amino acid sequence shown in SEQ ID NO:2.
  • the DNA polymerase has the E524K mutation compared to the amino acid sequence shown in SEQ ID NO:2.
  • the DNA polymerase has an L552R mutation compared to the amino acid sequence shown in SEQ ID NO:2.
  • the DNA polymerase has a D578N mutation compared to the amino acid sequence shown in SEQ ID NO:2.
  • the DNA polymerase has an E742R mutation compared to the amino acid sequence shown in SEQ ID NO:2.
  • the DNA polymerase has an E742K mutation compared to the amino acid sequence shown in SEQ ID NO:2.
  • the DNA polymerase has an A743R mutation compared to the amino acid sequence shown in SEQ ID NO:2.
  • the DNA polymerase has an A743K mutation compared to the amino acid sequence shown in SEQ ID NO:2.
  • the DNA polymerase has a K53S/E507K mutation compared to the amino acid sequence shown in SEQ ID NO:2.
  • the DNA polymerase has a K56N/E507K mutation compared to the amino acid sequence shown in SEQ ID NO:2.
  • the DNA polymerase has a K56S/E507K mutation compared to the amino acid sequence shown in SEQ ID NO:2.
  • the DNA polymerase has a K56T/E507K mutation compared to the amino acid sequence shown in SEQ ID NO:2.
  • the DNA polymerase has a K56Q/E524K mutation compared to the amino acid sequence shown in SEQ ID NO:2.
  • the DNA polymerase has the L245M/E524K mutation compared to the amino acid sequence shown in SEQ ID NO:2.
  • the DNA polymerase has the L245M/L552R mutation compared to the amino acid sequence shown in SEQ ID NO:2.
  • the DNA polymerase has the E315K/S357C mutation compared to the amino acid sequence shown in SEQ ID NO:2.
  • the DNA polymerase has a K56Q/E507K mutation compared to the amino acid sequence shown in SEQ ID NO:2.
  • the DNA polymerase compared with the amino acid sequence shown in SEQ ID NO: 2, the DNA polymerase has an E57D/E507K mutation.
  • the DNA polymerase has a K56Q/E507R mutation compared to the amino acid sequence shown in SEQ ID NO:2.
  • the DNA polymerase has a K56T/E524K mutation compared to the amino acid sequence shown in SEQ ID NO:2.
  • the DNA polymerase has the E57D/E524K mutation compared to the amino acid sequence shown in SEQ ID NO:2.
  • the DNA polymerase has an E57D/E742R mutation compared to the amino acid sequence shown in SEQ ID NO:2.
  • the DNA polymerase has the L245M/E315K mutation compared to the amino acid sequence shown in SEQ ID NO:2.
  • the wild-type Taq DNA polymerase is used for mutation design to improve the affinity of the enzyme for DNA, thereby increasing the activity of DNA polymerase. At the same time, it is considered that if the polymerization activity of the DNA polymerase is too strong and the affinity with the DNA is too strong, it may bring some other undesirable effects, for example, it may result in poor uniformity of amplification and so on. Therefore, combining multiple factors, multiple mutation sites were designed and verified by DNA polymerase activity and multiplex PCR amplification experiments.
  • the invention constructs a sequence containing Taq DNA polymerase mutant by genetic engineering means, and transforms E. coli to express and purify Taq DNA polymerase. Among them, plasmids containing sequences encoding different Taq DNA polymerase mutants were constructed by means of site-directed mutation PCR. Then, the constructed plasmids containing sequences encoding different Taq DNA polymerase mutants were transferred into E. coli for culture and expression induction. Finally, the DNA polymerase was purified and extracted by affinity chromatography and ion exchange chromatography. To obtain Taq DNA polymerase with different mutations.
  • the enzyme activity of the mutant was measured by the following method: using primer-bound M13ssDNA as a template-primer complex, DNA strand extension occurred under the action of DNA polymerase to obtain an extended double-stranded DNA product. Incorporate fluorescent molecules into the reaction product, and calculate the DNA polymerase activity by measuring the amount of double-stranded DNA. Through this method, Taq DNA polymerase mutants with increased polymerase activity were selected.
  • mutants provided in this application a multiplex PCR reaction system was used to verify whether the mutants could meet the requirements of multiplex PCR reactions. Using the mutant provided by the present application, 8-fold PCR amplification of the housekeeping gene of human origin and 5-fold PCR amplification of the housekeeping gene of mouse origin were performed respectively, and the polymerization activity and PCR amplification effect of the mutant and wild-type DNA polymerase were compared. After experimental verification, the mutants provided in this application can meet the requirements for DNA polymerase in multiplex PCR.
  • the gene sequence of wild-type Taq DNA polymerase is shown in SEQ ID NO: 1, and the encoded amino acid sequence is shown in SEQ ID NO: 2.
  • the expression plasmid pET29a-Taq containing wild-type Taq DNA polymerase coding sequence was purchased from Kingsray Technology Co., Ltd. Among them, 6 Hiss were fused at the N-terminus of the amino acid sequence during design to facilitate subsequent protein purification.
  • mutation design of wild-type Taq DNA polymerase was carried out to increase the affinity of the enzyme for DNA and the activity of DNA polymerase. At the same time, it is considered that if the polymerization activity of the DNA polymerase is too strong and the affinity with the DNA is too strong, it may bring some other undesirable effects, for example, it may cause poor uniformity of amplification and so on. Therefore, combining various factors, the inventors designed the following mutations (the specific mutation sites are shown in Table 4 below):
  • the Taq DNA polymerase mutant is to mutate the amino acid sequence of wild-type Taq DNA polymerase such as E507R, E742R, E524K, etc.: specifically by designing forward and reverse mutation primer pairs, using pfu DNA polymerase for mutation extension, the primers used As shown in Table 1:
  • the numbers Taq-21 to Taq-50 are all combined mutations, which means that the corresponding mutants have mutations at two positions relative to the wild-type TaqDNA polymerase.
  • Such combined mutations are given in Table 1.
  • the primers used for site-directed mutagenesis first perform site-directed mutagenesis at a mutation site to obtain a mutant product, and then perform site-directed mutagenesis on the mutant product to obtain two site-mutated mutations compared to wild-type Taq DNA polymerase product.
  • Wild-type Taq DNA polymerase and its mutants are all expressed by the promoter of pET28a, and all have 6 His tags fused at the N-terminus.
  • the His tags can be used for affinity purification of Ni columns during purification.
  • Wild type and mutant plasmids were transformed into BL21 competent cells (purchased from Quanshijin Biotechnology Co., Ltd.), and then single colonies were picked in 5ml LB medium containing kana-resistant (50 ⁇ g/ml), 37°C, 200rpm/ min, incubate overnight.
  • Taq DNA polymerase Ni column affinity A solution After loading the sample, continue to rinse the column with 10CV Taq DNA polymerase Ni column affinity A solution, and then on Taq DNA polymerase Ni column affinity B solution (50mM Tris, 500mM NaCl, 0.5% Triton X-100, 5% Glycerol, 500mM Imidazole, pH 7.8) eluted in a linear gradient in the range of 0-50% (10CV) and collected the protein of interest.
  • Affinity-purified samples were diluted 10-fold with Taq dilution (50 mM Tris, 0.5% Triton X-100, 5% Glycerol, pH 7.8). Then perform anion exchange chromatography, the specific steps are as follows:
  • Taq DNA polymerase ion A solution ((20mM Tris, 1M NaCl, 5% Glycerol, pH7.8) accounted for 0-100 % (15CV) linear gradient elution, and collect the protein of interest.
  • Taq DNA polymerase ion B solution ((20mM Tris, 1M NaCl, 5% Glycerol, pH7.8) accounted for 0-100 % (15CV) linear gradient elution, and collect the protein of interest.
  • the target protein obtained after purification is dialyzed and stored for subsequent determination and analysis.
  • the purified wild-type Taq DNA polymerase and its mutants were tested for polymerase activity.
  • M13 ssDNA combined with primers is used as a template-primer complex, and DNA strand extension occurs under the action of DNA polymerase to obtain an extended double-stranded DNA product.
  • the sequence of the bound primer is (SEQ ID NO: 75): 5'-AGCGAACCTCCCGACTTGCGGGAGG-3'.
  • the formulations of the 10x PCR buffer used in the present invention are: 100 mM Tris, 500 mM KCl, 15 mM MgCl 2 , 25% Glycerol, 0.5 mg/mL BSA.
  • the reaction system for Taq DNA polymerase activity detection is shown in Table 5 below:
  • the enzyme activities of various DNA polymerase mutants all show an increase.
  • the polymerization activity of the DNA polymerase mutant (E315K) is the wild-type Taq DNA 2.46 times of polymerase and so on.
  • the purified wild-type Taq DNA polymerase and its mutants were subjected to 8-fold human housekeeping gene and 5-fold murine housekeeping gene amplification, respectively. test.
  • the template for the 8-fold amplification of the housekeeping gene of human origin is human genomic DNA.
  • the amplification primers and the size of the target fragment are shown in Table 7 below:
  • the amplified bands on the agarose gel electrophoresis are brighter or increased, indicating that the activity of the mutant DNA polymerase is increased compared to the wild-type DNA polymerase. .
  • some mutant DNA polymerases have enhanced uniformity between the amplified bands, indicating that the mutant DNA polymerase can improve the uniformity of amplification.
  • Some of the bands in the figure have a tailing phenomenon due to the presence of certain non-specific amplification phenomena, and have no effect on the polymerization activity of DNA polymerase and the improvement of the uniformity of amplification.
  • the template of the five-fold amplification of the housekeeping gene of mouse origin is derived from the cDNA of Chinese hamster ovary cells (CHO).
  • the specific preparation process is: total RNA extracted from CHO cells is used as a template, and the sequence containing polyA is used as a primer.
  • Reverse transcriptase SuperScript II is used to reverse Transcriptase performs reverse transcription to obtain CHO cDNA.
  • first, second, etc. are for descriptive purposes only, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of indicated technical features.
  • the features defined as “first” and “second” may include at least one of the features explicitly or implicitly.
  • the meaning of “plurality” is at least two, such as two, three, etc., unless specifically defined otherwise.

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Abstract

提供的是一种酶活性提高的Taq DNA聚合酶及其应用。所述DNA聚合酶与SEQ ID NO:2所示氨基酸序列相比，具有如下突变位点中的至少一种：E315K、E507R、E507H、E524K、L552R、D578N、E742R、E742K、A743R、A743K、K53S/E507K、K56N/E507K、K56S/E507K、K56T/E507K、K56Q/E524K、L245M/E524K、L245M/L552R、E315K/S357C、K56Q/E507K、E57D/E507K、K56Q/E507R、K56T/E524K、E57D/E524K、E57D/E742R、L245M/E315K。

Description

酶活性提高的DNA聚合酶及其应用
优先权信息
无。
技术领域
本发明涉及酶工程领域，具体涉及一种酶活性提高的DNA聚合酶及其应用。
背景技术
聚合酶链式反应(PCR)是一种用于放大扩增特定的DNA片段的分子生物学技术，它不仅可用于基因分离、克隆和核酸序列分析等基础研究,还可用于疾病的诊断、测序或任何有DNA，RNA的地方。
PCR反应中需要DNA聚合酶、模板、引物、dNTP的参与，其中DNA聚合酶对PCR反应起着至关重要的作用，因此，关于DNA聚合酶改造以适用于不同应用的报道已有很多，如提高扩增速率、对抑制剂的耐受程度等。
多重PCR较常规PCR的特点是其在同一PCR反应体系里加上二对以上引物，同时扩增出多个核酸片段，其还具有高效性、系统性以及经济简便性的特点。由于扩增多条片段，多重PCR不仅要求DNA聚合酶具有较高的活性，还需要有一定的扩增均一性，即对同一体系中不同片段的扩增均较好。
虽然目前有很多关于DNA聚合酶改造的研究，但是能否用于多重的却不明确。基于多重PCR应用的优越性的可预见的广泛的应用范围，急需找到一种能应用于多重PCR的DNA聚合酶。
发明内容
本发明旨在至少在一定程度上解决相关技术中的技术问题之一。为此，本发明的一个目的在于提出一种酶活性提高的DNA聚合酶，该DNA聚合酶的酶活性相较于野生型的DNA聚合酶活性，有了显著提升。该DNA聚合酶能够应用于多重PCR反应体系中。由于本发明提供的DNA聚合酶的高活性，可以适用于模板含量低、混有抑制物质或者长片段模板的PCR反应。
PCR过程对于DNA聚合酶的要求较高，DNA聚合酶的活性等性质直接影响到PCR过程的扩增效率。但是由于用于聚合反应的聚合酶通常均是以试剂盒的形式提供，所用到的DNA聚合酶的信息很少公开，而且通常价格较贵。限制了对于DNA聚合酶性能要求较高的反应 体系中的应用。
多重PCR是在同一PCR反应体系里加上二对以上引物，同时扩增出多个核酸片段，具有高效性、系统性以及经济简便性的特点。由于扩增多条片段，多重PCR不仅要求DNA聚合酶具有较高的活性，还需要有一定的扩增均一性，即对同一体系中不同片段的扩增均较好。经研究发现，野生型的DNA聚合酶在多重PCR中的扩增均一性或产量较差，无法满足大部分应用需求，因此急需寻找开发出一种能够应用于多重PCR的DNA聚合酶。
本发明通过不同的多重PCR扩增体系及活性检测筛选出的DNA聚合酶能够有效的克服该问题，且自主研发生产能大大地降低成本。所筛选出的突变体可能不仅能应用于多重PCR，也能用于低模板投入量或含有抑制剂(如盐、血液)的PCR或长片段扩增。
为此，根据本发明的第一方面，本发明提供了一种DNA聚合酶，所述DNA聚合酶与SEQ ID NO:2所示氨基酸序列相比，具有如下突变位点中的至少一种：E315K、E507R、E507H、E524K、L552R、D578N、E742R、E742K、A743R、A743K、K53S/E507K、K56N/E507K、K56S/E507K、K56T/E507K、K56Q/E524K、L245M/E524K、L245M/L552R、E315K/S357C、K56Q/E507K、E57D/E507K、K56Q/E507R、K56T/E524K、E57D/E524K、E57D/E742R、L245M/E315K。本发明所提供的DNA聚合酶与野生型的DNA聚合酶(SEQ ID NO:2所示氨基酸序列)相比，具有提高的聚合活性和DNA亲和力，在多重PCR反应体系中扩增效果明显改善。
根据本发明的实施例，以上所述DNA聚合酶可以进一步包括如下技术特征：
在本发明的一些实施例中，所述DNA聚合酶为Taq DNA聚合酶的突变体。
在本发明的一些实施例中，所述DNA聚合酶的活性与未突变的Taq DNA聚合酶活性相比，至少提高了0.5倍。
在本发明的一些实施例中，所述DNA聚合酶的活性与未突变的Taq DNA聚合酶活性相比，至少提高了1倍。
根据本发明的第二方面，本发明提供了一种分离的核酸分子，所述分离的核酸分子编码本发明第一方面所述的DNA聚合酶。
根据本发明的第三方面，本发明提供了一种构建体，包含本发明第二方面所述的分离的核酸分子。
在本发明的一些实施例中，所述构建体为质粒。
在本发明的一些实施例中，所述分离的核酸分子可操作地连接启动子。
在本发明的一些实施例中，所述启动子选自下列中的一种：λ-PL启动子、tac启动子、trp启动子、araBAD启动子和trc启动子。
根据本发明的第四方面，本发明提供了一种宿主细胞，所述宿主细胞含有本发明第三 方面所述的构建体。用来表达目的蛋白(DNA聚合酶)的宿主细胞可以是原核细胞或者真核细胞。在至少一些实施例中，利用原核细胞表达DNA聚合酶，例如大肠杆菌(Escherichia coli)。适用于本发明的真核细胞可以是植物细胞、动物细胞(例如果蝇细胞、CHO细胞、C.elegans细胞等)、真菌细胞(例如酿酒细胞、巴斯德毕赤酵母细胞等)。
根据本发明的第五方面，本发明提供了一种DNA聚合酶的生产方法，所述DNA聚合酶为本发明第一方面所述的DNA聚合酶，所述生产方法包括：培养宿主细胞，所述宿主细胞为本发明第四方面所述的宿主细胞；将所述宿主细胞进行诱导处理，使得所述宿主细胞表达所述DNA聚合酶；分离获得所述DNA聚合酶。
在本发明的一些实施例中，所述宿主细胞为大肠杆菌。
根据本发明的第六方面，本发明提供了一种试剂盒，包括本发明第一方面所述的DNA聚合酶。含有DNA聚合酶的试剂盒用于PCR反应中，可以提高扩增的效率。
根据本发明的实施例，以上所述的试剂盒可以进一步附加如下技术特征：
在本发明的一些实施例中，所述试剂盒还包括下列中的至少一种：一种或多种核苷酸，一种或多种缓冲液，一种或多种引物，一种或多种终止剂。
在本发明的一些实施例中，所述终止剂为双脱氧核苷酸。
根据本发明的第七方面，本发明提供了一种扩增核酸分子的方法，所述方法包括：将至少一种核酸模板与至少一种DNA聚合酶混合，得到混合物，所述DNA聚合酶为本发明第一方面所述的DNA聚合酶；对所述混合物进行扩增处理，以便获得与所述至少一种核酸模板全部或部分互补的核酸分子。
根据本发明的实施例，以上扩增核酸分子的方法可以进一步包括如下技术特征：
在本发明的一些实施例中，所述至少一种核酸模板的最低含量为0.001pg/μl。通常进行DNA扩增时，所用到的模板的含量为0.2-2ng/μl。利用本申请提供的DNA聚合酶，可以显著提高聚合活性，从而能够用于低含量模板物质的扩增。例如在进行单重PCR扩增时，当核酸模板的核酸含量为1pg/μl时，进行20个循环，即可有明显的扩增条带；即便核酸模板的核酸含量为0.001pg/μl，进行20个循环，仍有较弱的条带。而当核酸模板的核酸含量为0.001pg/μl，进行30-40个循环，有明显的扩增条带。
在本发明的一些实施例中，与所述至少一种核酸模板全部或部分互补的核酸分子的长度可达10kb。由于本申请所提供的DNA聚合酶的高的聚合活性，因此可以应用于长片段核酸模板的扩增，所获得的扩增片段的长度可以达到10kb。例如可以达到2kb以上，3kb以上，5kb以上，8kb以上等等。当然应用本申请提供的DNA聚合酶也可以用于短片段核酸模板的扩增，例如用于100bp以上，200bp以上，500bp以上的核酸模板的扩增。
根据本发明的第八方面，本发明提供了一种扩增多个核酸分子的方法，包括：将至少 两种核酸模板与至少一种DNA聚合酶混合，得到混合物，所述DNA聚合酶为本发明第一方面所述的DNA聚合酶；对所述混合物进行扩增处理，以便获得与所述至少两种核酸模板全部或部分互补的核酸分子。
本发明所取得的有益效果为：本发明所提供的突变体与相应的天然存在的DNA聚合酶相比具有提高的聚合活性和DNA亲和力，在多重PCR中的扩增效果明显改善。而且可以用于低模板物质的扩增以及长片段分子的扩增。
附图说明
本发明的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解，其中：
图1是根据本发明的一个实施例提供的野生型Taq DNA聚合酶及其突变体的8重人源持家基因扩增电泳图。
图2是根据本发明的一个实施例提供的野生型Taq DNA聚合酶及其突变体的8重人源持家基因扩增电泳图。
图3是根据本发明的一个实施例提供的野生型Taq DNA聚合酶及其突变体的8重人源持家基因扩增电泳图。
图4是根据本发明的一个实施例提供的野生型Taq DNA聚合酶及其突变体的5重鼠源持家基因扩增电泳图。
图5是根据本发明的一个实施例提供的野生型Taq DNA聚合酶及其突变体的5重鼠源持家基因扩增电泳图。
图6是根据本发明的一个实施例提供的野生型Taq DNA聚合酶及其突变体的5重鼠源持家基因扩增电泳图。
具体实施方式
下面详细描述本发明的实施例，所述实施例的示例在附图中示出，其中自始至终相同或类似的标号表示相同或类似的元件或具有相同或类似功能的元件。下面通过参考附图描述的实施例是示例性的，旨在用于解释本发明，而不能理解为对本发明的限制。
为了对于本申请有更为直观的理解，下面对本申请中存在的术语进行解释和说明。本领域技术人员需要理解的是，这些解释和说明仅为了理解更为方便，不应看做是对本申请保护范围的限制。
术语“DNA聚合酶”是指表现出DNA聚合酶活性的蛋白质、多肽或者多肽片段。
术语“DNA聚合酶活性”、“聚合活性”、“DNA聚合活性”是指以DNA为模板，合 成互补DNA链的能力。
术语“突变体”、“突变”或者“突变型”等，是指相比较于野生型的DNA序列或者野生型的氨基酸序列，具有一个或者多个突变。当然这种突变可以发生在核酸水平上或者氨基酸水平上。
在本文中，当表示突变位点时，依照本领域通常的表述方式，即为“突变前氨基酸缩写+位点+突变后氨基酸缩写”，例如“E315K”，其中“E”代表突变前的氨基酸，“315”为相应的突变位点，“K”代表突变后的氨基酸。其中“E”和“K”均是采用本领域通用的当个字母缩写代表氨基酸。当表述组合突变时，两个突变之间用“/”连接，例如突变位点“K53S/E507K”代表相较于野生型，在第53个氨基酸和第507个氨基酸同时发生了突变。
根据本发明的实施例，本发明提供了一种DNA聚合酶，所述DNA聚合酶与SEQ ID NO:2所示氨基酸序列相比，具有如下突变位点中的至少一种：E315K、E507R、E507H、E524K、L552R、D578N、E742R、E742K、A743R、A743K、K53S/E507K、K56N/E507K、K56S/E507K、K56T/E507K、K56Q/E524K、L245M/E524K、L245M/L552R、E315K/S357C、K56Q/E507K、E57D/E507K、K56Q/E507R、K56T/E524K、E57D/E524K、E57D/E742R、L245M/E315K。其中SEQ ID NO:2所示氨基酸序列即为野生型的Taq DNA聚合酶的氨基酸序列。本申请所提供的DNA聚合酶与野生型的DNA聚合酶的氨基酸序列相比，表现为单个位点的突变，或者两个位点的组合突变，使得DNA聚合酶的活性相较于野生型的Taq DNA聚合酶的活性，至少提高了0.5倍。在至少一种具体实施方式中，所述DNA聚合酶的活性相较于野生型的DNA聚合酶的活性至少提高了1倍。在另一种具体实施方式中，所述DNA聚合酶的活性相较于野生型的DNA聚合酶的活性至少提高了2倍。在至少一些实施中，所述DNA聚合酶相较于野生型的DNA聚合酶具有提高的扩增均一性。本文中“扩增均一性”是指同一反应体系中有多种核酸模板和多种引物时，DNA聚合酶对于每种核酸模板和相应引物的扩增能力的一致性。
在本发明的至少一些实施方式中，与SEQ ID NO:2所示氨基酸序列相比，所述DNA聚合酶具有E315K突变。
在本发明的至少一些实施方式中，与SEQ ID NO:2所示氨基酸序列相比，所述DNA聚合酶具有E507R突变。
在本发明的至少一些实施方式中，与SEQ ID NO:2所示氨基酸序列相比，所述DNA聚合酶具有E507H突变。
在本发明的至少一些实施方式中，与SEQ ID NO:2所示氨基酸序列相比，所述DNA聚合酶具有E524K突变。
在本发明的至少一些实施方式中，与SEQ ID NO:2所示氨基酸序列相比，所述DNA聚 合酶具有L552R突变。
在本发明的至少一些实施方式中，与SEQ ID NO:2所示氨基酸序列相比，所述DNA聚合酶具有D578N突变。
在本发明的至少一些实施方式中，与SEQ ID NO:2所示氨基酸序列相比，所述DNA聚合酶具有E742R突变。
在本发明的至少一些实施方式中，与SEQ ID NO:2所示氨基酸序列相比，所述DNA聚合酶具有E742K突变。
在本发明的至少一些实施方式中，与SEQ ID NO:2所示氨基酸序列相比，所述DNA聚合酶具有A743R突变。
在本发明的至少一些实施方式中，与SEQ ID NO:2所示氨基酸序列相比，所述DNA聚合酶具有A743K突变。
在本发明的至少一些实施方式中，与SEQ ID NO:2所示氨基酸序列相比，所述DNA聚合酶具有K53S/E507K突变。
在本发明的至少一些实施方式中，与SEQ ID NO:2所示氨基酸序列相比，所述DNA聚合酶具有K56N/E507K突变。
在本发明的至少一些实施方式中，与SEQ ID NO:2所示氨基酸序列相比，所述DNA聚合酶具有K56S/E507K突变。
在本发明的至少一些实施方式中，与SEQ ID NO:2所示氨基酸序列相比，所述DNA聚合酶具有K56T/E507K突变。
在本发明的至少一些实施方式中，与SEQ ID NO:2所示氨基酸序列相比，所述DNA聚合酶具有K56Q/E524K突变。
在本发明的至少一些实施方式中，与SEQ ID NO:2所示氨基酸序列相比，所述DNA聚合酶具有L245M/E524K突变。
在本发明的至少一些实施方式中，与SEQ ID NO:2所示氨基酸序列相比，所述DNA聚合酶具有L245M/L552R突变。
在本发明的至少一些实施方式中，与SEQ ID NO:2所示氨基酸序列相比，所述DNA聚合酶具有E315K/S357C突变。
在本发明的至少一些实施方式中，与SEQ ID NO:2所示氨基酸序列相比，所述DNA聚合酶具有K56Q/E507K突变。
在本发明的至少一些实施方式中，与SEQ ID NO:2所示氨基酸序列相比，所述DNA聚合酶具有E57D/E507K突变。
在本发明的至少一些实施方式中，与SEQ ID NO:2所示氨基酸序列相比，所述DNA聚 合酶具有K56Q/E507R突变。
在本发明的至少一些实施方式中，与SEQ ID NO:2所示氨基酸序列相比，所述DNA聚合酶具有K56T/E524K突变。
在本发明的至少一些实施方式中，与SEQ ID NO:2所示氨基酸序列相比，所述DNA聚合酶具有E57D/E524K突变。
在本发明的至少一些实施方式中，与SEQ ID NO:2所示氨基酸序列相比，所述DNA聚合酶具有E57D/E742R突变。
在本发明的至少一些实施方式中，与SEQ ID NO:2所示氨基酸序列相比，所述DNA聚合酶具有L245M/E315K突变。
利用野生型Taq DNA聚合酶进行突变设计，来提高酶对DNA的亲和力，从而提高DNA聚合酶的活性。同时考虑到若DNA聚合酶的聚合活性过强，与DNA的亲和力过强，可能会带来一些其他的不佳影响，例如可能会导致扩增的均一性较差等等。因此，综合多种因素，设计了多个突变位点，并通过DNA聚合酶酶活性以及多重PCR扩增试验进行了验证。
本发明通过基因工程手段，构建了含有编码Taq DNA聚合酶突变体的序列，并且转化E.Coli进行Taq DNA聚合酶的表达和纯化。其中通过定点突变PCR的手段构建了含有编码不同Taq DNA聚合酶突变体序列的质粒。然后将构建好的的含有编码不同Taq DNA聚合酶突变体序列的质粒转入E.Coli中进行培养并诱导表达，最后通过亲和层析及离子交换层析的方法对DNA聚合酶进行纯化提取，从而得到含有不同突变的Taq DNA聚合酶。
为了验证所获得的DNA聚合酶的酶活性。通过如下方法对突变体的酶活性进行了测定：使用结合有引物的M13ssDNA为模板-引物复合体，在DNA聚合酶的作用下发生DNA链的延伸，得到延长的双链DNA产物。将荧光分子参入反应产物，通过检测双链DNA量来计算DNA聚合酶的活性。通过该方法筛选出了聚合酶活性提高的Taq DNA聚合酶突变体。
对于本申请提供的突变体，利用多重PCR的反应体系，对突变体是否能够满足多重PCR反应的要求进行了验证。利用本申请提供的突变体，分别进行人源持家基因的8重PCR扩增和鼠源持家基因的5重PCR扩增，比较突变体和野生型DNA聚合酶的聚合活性以及PCR扩增效果。经过实验验证，本申请所提供的突变体能够满足多重PCR中对于DNA聚合酶的要求。
下面将结合实施例对本发明的方案进行解释。本领域技术人员将会理解，下面的实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例1 Taq DNA聚合酶及其突变体表达质粒的构建
具体实施步骤如下：
(1)野生型Taq DNA聚合酶表达质粒的构建
野生型的Taq DNA聚合酶的基因序列为SEQ ID NO:1所示，其编码的氨基酸序列为SEQ ID NO:2所示。
野生型Taq DNA聚合酶的核酸序列(SEQ ID NO:1)
Figure PCTCN2018122334-appb-000001
Figure PCTCN2018122334-appb-000002
野生型Taq DNA聚合酶的氨基酸序列(SEQ ID NO:2)
Figure PCTCN2018122334-appb-000003
含有野生型Taq DNA聚合酶编码序列的表达质粒pET29a-Taq购自金斯瑞科技有限公司。其中，设计时在氨基酸序列的N端融合有6个His以利于后续蛋白的纯化。
(2)突变体Taq DNA聚合酶表达质粒的构建
考虑到野生型Taq DNA聚合酶的活性，对野生型Taq DNA聚合酶进行突变设计，来提高酶对DNA的亲和力，来提高DNA聚合酶的活性。同时考虑到若DNA聚合酶的聚合活性过强，与DNA的亲和力过强，可能会带来一些其他的不佳影响，例如可能会导致扩增的均一性较差等等。因此，综合多种因素，发明人设计了如下突变(具体的突变位点如下表4所示)：
Taq DNA聚合酶突变体为将野生型Taq DNA聚合酶的氨基酸序列如E507R、E742R、E524K等进行突变：具体通过设计正反向突变引物对，使用pfu DNA聚合酶进行突变延伸，所使用的引物如下表1所示：
表1构建Taq DNA聚合酶突变体表达质粒过程的PCR引物
Figure PCTCN2018122334-appb-000004
Figure PCTCN2018122334-appb-000005
Figure PCTCN2018122334-appb-000006
具体的反应体系如下表2所示：
表2构建Taq DNA聚合酶突变体表达质粒过程的PCR体系
反应组分 体积(μl)
10×pfu缓冲液(含有MgSO 4) 2.5
2.5mM dNTPs 2
10μM正向引物 0.7
10μM反向引物 0.7
pfu DNA聚合酶 0.5
50ng/μl模板(pET29a-Taq或突变体) 1
H 2O 17.6
PCR反应条件如下表3所示：
表3构建Taq DNA聚合酶突变体表达质粒过程的PCR条件
Figure PCTCN2018122334-appb-000007
反应结束后，加入1μl DpnI于37℃消化2h，然后取5μl消化后的产物转化E.Coli感受态细胞DH5α，观察转化结果。然后从平板上挑取单克隆进行培养后提取质粒，再通过测序比对分析得到的突变体是否正确。
所构建的突变体具体如下表4所示：
表4 Taq DNA聚合酶突变体突变位置及突变类型
Figure PCTCN2018122334-appb-000008
Figure PCTCN2018122334-appb-000009
表4中，编号Taq-21～Taq-50均为组合突变，即代表相应的突变体相对于野生型的TaqDNA聚合酶，有两个位点发生突变，这种组合突变利用表1中给出的应用于定点突变的引物，先进行一个突变位点的定点突变，获得突变产物，然后再对该突变产物进行定点突变，得到相较于野生型Taq DNA聚合酶，发生两个位点突变的产物。
实施例2 Taq DNA聚合酶及其突变体的诱导表达和纯化
野生型Taq DNA聚合酶及其突变体均通过pET28a的启动子启动表达，并且均在N-端融合有6个His标签，纯化时可利用His标签进行Ni柱亲和纯化。
野生型及突变体质粒转化BL21感受态细胞(购自全式金生物科技有限公司)，然后挑取单菌落于5ml含卡那抗性(50μg/ml)LB培养基中，37℃，200rpm/min，过夜培养。次日按1:100的比例进行稀释，分别转接于新鲜的1500ml含卡那抗性(50μg/ml)的LB培养基中， 37℃，200rpm振荡培养至OD600≈0.6，然后按照终浓度为0.5mM的量加入诱导剂IPTG，37℃，200rpm/min培养4h诱导表达，最后8000rpm/min的条件离心10分钟，收集诱导后的菌液沉淀。
收集菌体沉淀后，使用Taq DNA聚合酶Ni柱亲和A液(50mM Tris，500mM NaCl，0.5％Triton X-100，5％Glycerol，10mM Imidazole，pH7.8)重悬，在冰水浴条件下进行超声破菌，超声条件为：变幅杆直径10mm，超声强度为40％，超声2s，间歇3s，超声30min。将破碎后的菌液置于75℃水浴30min，然后在转速13000rpm、4℃下离心30min，收集上清。
将上步准备好的样品进行亲和纯化，具体如下：
按照AKTA操作流程，用过滤脱气MillQ水冲洗工作泵和系统，0.5ml/min流速下接上预装柱HisTrap FF 5ml，用H 2O冲洗5CV，再用5CV的Taq DNA聚合酶Ni柱亲和A液平衡柱子，然后将之前处理好的样品以5ml/min上样至层析柱。上样完毕后，继续用10CV的Taq DNA聚合酶Ni柱亲和A液冲洗柱子，然后在Taq DNA聚合酶Ni柱亲和B液(50mM Tris,500mM NaCl，0.5％Triton X-100,5％Glycerol,500mM Imidazole,pH7.8)在占比0-50％(10CV)范围内线性梯度洗脱，并收集目的蛋白。
将经亲和纯化得到的样品用Taq稀释液(50mM Tris,0.5％Triton X-100,5％Glycerol,pH7.8)进行10倍稀释。然后进行阴离子交换层析，具体步骤如下：
按照AKTA操作流程，用过滤脱气MillQ水冲洗工作泵和系统，0.5ml/min流速下接上预装柱HisTrap FF 5ml，用H 2O冲洗5CV，再用5CV的Taq DNA聚合酶离子A液(20mM Tris,50mM NaCl,5％Glycerol,pH7.8)平衡柱子，然后将稀释处理好的样品以5ml/min上样至层析柱。上样完毕后，继续用10CV的Taq DNA聚合酶离子A液冲洗柱子，然后在Taq DNA聚合酶离子B液((20mM Tris,1M NaCl,5％Glycerol,pH7.8)在占比0-100％(15CV)范围内线性梯度洗脱，并收集目的蛋白。
纯化后得到的目的蛋白经过透析、储存，用于后续的测定及分析。
实施例3 Taq DNA聚合酶及其突变体的聚合活性测定及分析
对经纯化得到的野生型Taq DNA聚合酶及其突变体进行聚合酶活性的测定。活性测定中使用结合有引物的M13 ssDNA为模板-引物复合体，在DNA聚合酶的作用下发生DNA链的延伸，得到延长的双链DNA产物。将荧光分子参入反应产物，通过检测双链DNA量来计算DNA聚合酶的活性。其中结合的引物序列为(SEQ ID NO:75)：5’-AGCGAACCTCCCGACTTGCGGGAGG-3’。本发明中所用的10x PCR缓冲液的配方均为：100mM Tris，500mM KCl，15mM MgCl 2，25％Glycerol，0.5mg/mL BSA。
Taq DNA聚合酶活性检测的反应体系如下表5所示：
表5 Taq DNA聚合酶聚合活性检测体系
反应组分 体积(μl)
10x PCR缓冲液 2.5
50nM M13ssDNA模板-引物复合体 2
10mM dNTP 1
4ng/μl Taq DNA聚合酶及其突变体 1
H 2O 补足至25μl
将上述配好的反应体系置于PCR仪中，72℃反应5min后加入0.5μl的0.5M EDTA终止反应，然后用Qubit dsDNA HS Assay Kit检测双链DNA的量，通过计算得到突变体相对于野生型的聚合活性。
Taq DNA聚合酶及其突变体的相对聚合活性如下表6所示：
表6 Taq DNA聚合酶及其突变体的相对聚合活性
Figure PCTCN2018122334-appb-000010
Figure PCTCN2018122334-appb-000011
从上表6可以看出，相较于野生型Taq DNA聚合酶，多种DNA聚合酶突变体的酶活性均表现出提高，例如DNA聚合酶突变体(E315K)的聚合活性是野生型Taq DNA聚合酶的2.46倍等等。
实施例4 Taq DNA聚合酶及其突变体的多重PCR测试及分析
为了获得能够应用于多重PCR扩增的DNA聚合酶突变体，分别对经纯化得到的野生型Taq DNA聚合酶及其突变体进行8重的人源持家基因和5重的鼠源持家基因扩增测试。
(1)人源持家基因8重扩增
人源持家基因8重扩增的模板为人的基因组DNA，扩增引物及目的片段大小如下表7所示：
表7人源持家基因8重扩增引物及目的片段大小
Figure PCTCN2018122334-appb-000012
Figure PCTCN2018122334-appb-000013
人源持家基因8重扩增的PCR反应体系如下表8所示：
表8人源持家基因8重扩增反应体系
反应组分 体积(μl)
10×PCR Buffer 5
10mM dNTPs 1
5μM Primer Mix 2
10ng/μl YH gDNA 2
0.1mg/ml Taq 0.5
H 2O 39.5
人源持家基因8重扩增的PCR条件如下表9所示：
表9人源持家基因8重扩增条件
Figure PCTCN2018122334-appb-000014
人源持家基因8重扩增完成后，加入10μl 6x DNA Loading Buffer(60mM Tris,60mM EDTA,60％Glycerol,Orange G)后充分混匀后用1.3％的琼脂糖胶进行电泳分析，挑选出能扩增出所有条带或条带较野生型多，或产量提高，或均一性提高的突变体。具体结果见附图1～图3。
图1到图3所示的结果来看，琼脂糖凝胶电泳图的扩增条带较亮或者扩增条带增多，表明相较于野生型的DNA聚合酶，突变型DNA聚合酶活性提高。同时相较于野生型的DNA聚合酶，有些突变型DNA聚合酶的扩增条带各条带之间均一性增强，表明突变型DNA聚合酶能够提高扩增的均一性。而图中有些条带出现了拖尾现象，是由于存在某些非特异性扩增的现象，和DNA聚合酶的聚合活性以及对提高扩增均一性没有影响，可以不必考虑。
(2)鼠源持家基因5重扩增
鼠源持家基因5重扩增的模板来源中国仓鼠卵巢细胞(CHO)的cDNA，具体制备过程为：从CHO细胞提取的total RNA为模板，以含有polyA的序列为引物用逆转录酶SuperScript II Reverse Transcriptase进行逆转录，从而得到CHO的cDNA。
鼠源持家基因5重扩增引物及目的片段大小如下表10所示：
表10鼠源持家基因5重扩增引物及目的片段大小
Figure PCTCN2018122334-appb-000015
鼠源持家基因5重扩增的PCR反应体系如下表11所示：
表11鼠源持家基因5重扩增反应体系
反应组分 体积(μl)
10×PCR Buffer 5
10mM dNTPs 1
5μM Primer Mix 2
10ng/μl CHO cDNA 2
0.1mg/ml Taq 0.5
H2O 39.5
鼠源持家基因5重扩增的PCR条件如下表12所示：
表12鼠源持家基因5重扩增条件
Figure PCTCN2018122334-appb-000016
Figure PCTCN2018122334-appb-000017
鼠源持家基因5重扩增完成后，加入10μl 6x DNA Loading Buffer(60mM Tris,60mM EDTA,60％Glycerol,Orange G)后充分混匀后用1.3％的琼脂糖胶进行电泳分析，挑选出能扩增出所所有条带或条带较野生型多，或产量提高，或均一性提高的突变体。具体结果见附图4～图6。
图4到图6所示的结果来看，琼脂糖凝胶电泳图的扩增条带较亮或者扩增条带增多，表明相较于野生型的DNA聚合酶，突变型DNA聚合酶活性提高。同时相较于野生型的DNA聚合酶，有些突变型DNA聚合酶的扩增条带各条带之间均一性增强，表明突变型DNA聚合酶能够提高扩增的均一性。而图中有些条带出现了拖尾现象，是由于存在某些非特异性扩增的现象，和DNA聚合酶的聚合活性以及对提高扩增均一性没有影响，可以不必考虑。
综合不同突变型DNA聚合酶对于人源持家基因8重扩增的结果，对于鼠源持家基因5重扩增的结果，选择聚合酶活性以及扩增均一性提高的DNA聚合酶，同时结合上述实施例对于不同突变型DNA聚合酶相对于野生型DNA聚合酶的聚合活性测定结果，确定了突变：E315K、E507R、E507H、E524K、L552R、D578N、E742R、E742K、A743R、A743K、K53S/E507K、K56N/E507K、K56S/E507K、K56T/E507K、K56Q/E524K、L245M/E524K、L245M/L552R、E315K/S357C、K56Q/E507K、E57D/E507K、K56Q/E507R、K56T/E524K、E57D/E524K、E57D/E742R、L245M/E315K中的至少一种，可以显著提高DNA聚合酶活性，应用于多重PCR中，扩增效果明显改善。而且可以用于低模板物质的扩增以及长片段分子的扩增。
在本发明的描述中，术语“第一”、“第二”等仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括至少一个该特征。在本发明的描述中，“多个”的含义是至少两个，例如两个，三个等，除非另有明确具体的限定。
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。

Claims (19)

  1. 一种DNA聚合酶，其特征在于，所述DNA聚合酶与SEQ ID NO:2所示氨基酸序列相比，具有如下突变位点中的至少一种：
    E315K、E507R、E507H、E524K、L552R、D578N、E742R、E742K、A743R、A743K、K53S/E507K、K56N/E507K、K56S/E507K、K56T/E507K、K56Q/E524K、L245M/E524K、L245M/L552R、E315K/S357C、K56Q/E507K、E57D/E507K、K56Q/E507R、K56T/E524K、E57D/E524K、E57D/E742R、L245M/E315K。
  2. 根据权利要求1所述的DNA聚合酶，其特征在于，所述DNA聚合酶为Taq DNA聚合酶的突变体。
  3. 根据权利要求1所述的DNA聚合酶，其特征在于，所述DNA聚合酶的活性与未突变的Taq DNA聚合酶活性相比，至少提高了0.5倍。
  4. 根据权利要求1所述的DNA聚合酶，其特征在于，所述DNA聚合酶的活性与未突变的Taq DNA聚合酶相比，至少提高了1倍。
  5. 一种分离的核酸分子，其特征在于，所述分离的核酸分子编码权利要求1～4中任一项所述的DNA聚合酶。
  6. 一种构建体，其特征在于，包含权利要求5所述的分离的核酸分子。
  7. 根据权利要求6所述的构建体，其特征在于，所述构建体为质粒。
  8. 根据权利要求6所述的构建体，其特征在于，所述分离的核酸分子可操作地连接启动子。
  9. 根据权利要求8所述的构建体，其特征在于，所述启动子选自下列中的一种：λ-PL启动子、tac启动子、trp启动子、araBAD启动子和trc启动子。
  10. 一种宿主细胞，其特征在于，所述宿主细胞含有权利要求6～9中任一项所述的构建体。
  11. 权利要求1～4中任一项所述的DNA聚合酶的生产方法，其特征在于，包括：
    培养宿主细胞，所述宿主细胞为权利要求13所述的宿主细胞；
    将所述宿主细胞进行诱导处理，使得所述宿主细胞表达所述DNA聚合酶；
    分离获得所述DNA聚合酶。
  12. 根据权利要求11所述的生产方法，其特征在于，所述宿主细胞为大肠杆菌。
  13. 一种试剂盒，其特征在于，包括权利要求1～4中任一项所述的DNA聚合酶。
  14. 根据权利要求13所述的试剂盒，其特征在于，还包括下列中的至少一种：
    一种或多种核苷酸，一种或多种缓冲液，一种或多种引物，一种或多种终止剂。
  15. 根据权利要求14所述的试剂盒，其特征在于，所述终止剂为双脱氧核苷酸。
  16. 一种扩增核酸分子的方法，其特征在于，所述方法包括：
    将至少一种核酸模板与至少一种DNA聚合酶混合，得到混合物，所述DNA聚合酶为权利要求1所述的DNA聚合酶；
    对所述混合物进行扩增处理，以便获得与所述至少一种核酸模板全部或部分互补的核酸分子。
  17. 根据权利要求16所述的方法，其特征在于，所述至少一种核酸模板的最低含量为0.001pg/μl。
  18. 根据权利要求16所述的方法，其特征在于，与所述至少一种核酸模板全部或部分互补的核酸分子的长度可达10kb。
  19. 一种扩增多个核酸分子的方法，其特征在于，包括：
    将至少两种核酸模板与至少一种DNA聚合酶混合，得到混合物，所述DNA聚合酶为权利要求1所述的DNA聚合酶；
    对所述混合物进行扩增处理，以便获得与所述至少两种核酸模板全部或部分互补的核酸分子。
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EP4455279A4 (en) * 2021-12-24 2025-10-22 Bgi Shenzhen DNA POLYMERASE MUTANT AND ITS USE
CN116064462A (zh) * 2022-08-26 2023-05-05 江苏伟禾生物科技有限公司 一种Taq DNA聚合酶突变体及其制备方法
WO2024159661A1 (zh) * 2023-02-01 2024-08-08 南京普济生物有限公司 一种具有dna聚合酶活性的多肽及其应用

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