CN111826366A - A kind of direct expansion type hot start DNA polymerase and its preparation method and application - Google Patents
A kind of direct expansion type hot start DNA polymerase and its preparation method and application Download PDFInfo
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- CN111826366A CN111826366A CN202010503288.XA CN202010503288A CN111826366A CN 111826366 A CN111826366 A CN 111826366A CN 202010503288 A CN202010503288 A CN 202010503288A CN 111826366 A CN111826366 A CN 111826366A
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- dna polymerase
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- start dna
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Abstract
Description
技术领域technical field
本发明属于生物技术领域，具体涉及一种直扩型热启动DNA聚合酶及其制备方法与应用。The invention belongs to the field of biotechnology, and in particular relates to a direct expansion type hot-start DNA polymerase and a preparation method and application thereof.
背景技术Background technique
实时荧光定量PCR(Quantitative Real-time PCR，qPCR)技术是在聚合酶链式反应(PCR)的基础上发展起来的，弥补了普通PCR不可以实时监测及定量的缺陷，成为了当今基因研究的重要工具。SYBR GreenΙ染料法qPCR技术以其成本低、重复性好、适用性强等优点广泛应用于医学研究、病原微生物检测和食品安全检测等诸多领域。Real-time fluorescence quantitative PCR (Quantitative Real-time PCR, qPCR) technology is developed on the basis of polymerase chain reaction (PCR), which makes up for the shortcomings of ordinary PCR that cannot be monitored and quantified in real time, and has become the mainstay of genetic research today. important tool. SYBR Green1 dye-based qPCR technology is widely used in many fields such as medical research, pathogenic microorganism detection and food safety detection due to its advantages of low cost, good repeatability and strong applicability.
灵敏、精准一直是检测领域发展的需求及目标，SYBR GreenΙ染料法qPCR技术在应用于核酸检测的过程中，Taq DNA聚合酶的活性会受到样品或者样品提取过程中残留的qPCR抑制剂的干扰。如植物核酸样本中的多糖、血液核酸样本中的免疫球蛋白、土壤核酸样本中的腐殖酸等都会抑制Taq DNA聚合酶活性，从而影响qPCR检测的特异性、荧光信号及灵敏度，造成结果的不可靠和不准确。Sensitivity and accuracy have always been the requirements and goals of the development of the detection field. In the process of applying SYBR Green1 dye-based qPCR technology to nucleic acid detection, the activity of Taq DNA polymerase will be interfered by the residual qPCR inhibitor in the sample or sample extraction process. For example, polysaccharides in plant nucleic acid samples, immunoglobulins in blood nucleic acid samples, and humic acid in soil nucleic acid samples will inhibit the activity of Taq DNA polymerase, thereby affecting the specificity, fluorescence signal and sensitivity of qPCR detection, resulting in inconsistent results. Unreliable and inaccurate.
Taq DNA聚合酶作为qPCR反应的核心组分，具有超强的耐热性和聚合酶活性，其性能直接决定了qPCR检测的速度、特异性及结果的可靠性。热启动DNA聚合酶可在PCR反应前封闭DNA聚合酶的活性，使得PCR反应开始时才释放其活性，可大幅度减少非特异性扩增及引物二聚的形成，是目前qPCR试剂的主要成分。近年来，涌现了多种耐抑制剂的、可以直接对粗样样品进行PCR扩增的Taq DNA聚合酶。但当前的直扩Taq DNA聚合酶可耐受的粗样样品量少，当样本中检测目标含量低时，容易导致假阴性结果(如病毒或细菌感染初期时的样本检测)，且仅适用于普通PCR。用于粗样直接qPCR检测的Taq DNA聚合酶需要更强的耐抑制剂能力，以便在高浓度抑制剂和高浓度染料存在下，保证qPCR检测的特异性及结果的可靠性。因此，亟需研究开发一种用于qPCR的高性能热启动DNA聚合酶。As the core component of qPCR reaction, Taq DNA polymerase has strong heat resistance and polymerase activity, and its performance directly determines the speed, specificity and reliability of qPCR detection. Hot-start DNA polymerase can block the activity of DNA polymerase before the PCR reaction, so that its activity is released at the beginning of the PCR reaction, which can greatly reduce the formation of non-specific amplification and primer dimerization. It is the main component of current qPCR reagents. In recent years, a variety of inhibitor-resistant Taq DNA polymerases have emerged that can directly perform PCR amplification on crude samples. However, the current direct-amplification Taq DNA polymerase can tolerate a small amount of crude samples. When the target content in the sample is low, it is easy to lead to false negative results (such as the detection of samples in the early stage of virus or bacterial infection), and it is only suitable for Ordinary PCR. Taq DNA polymerase for direct qPCR detection of crude samples requires stronger resistance to inhibitors, in order to ensure the specificity of qPCR detection and the reliability of results in the presence of high concentrations of inhibitors and high concentrations of dyes. Therefore, there is an urgent need to develop a high-performance hot-start DNA polymerase for qPCR.
发明内容SUMMARY OF THE INVENTION
本发明的首要目的在于克服现有技术的缺点与不足，提供一种直扩型热启动DNA聚合酶。The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art, and to provide a direct expansion type hot-start DNA polymerase.
本发明的另一目的在于提供上述直扩型热启动DNA聚合酶的制备方法。Another object of the present invention is to provide a method for preparing the above-mentioned direct expansion type hot-start DNA polymerase.
本发明的再一目的在于提供上述直扩型热启动DNA聚合酶的应用。Another object of the present invention is to provide the application of the above-mentioned direct expansion type hot-start DNA polymerase.
本发明的目的通过如下技术方案实现：The object of the present invention is achieved through the following technical solutions:
一种直扩型热启动DNA聚合酶，其氨基酸序列如SEQ ID NO.1所示。A direct-expanding hot-start DNA polymerase, the amino acid sequence of which is shown in SEQ ID NO.1.
所述的直扩型热启动DNA聚合酶与NCBI登录号为P19821.1的野生型TaqDNA聚合酶的氨基酸序列相比，具有以下特点：1～289位氨基酸缺失，626位谷氨酸突变为精氨酸，707位异亮氨酸突变为亮氨酸，708位谷氨酸突变为精氨酸，且N末端融合了双链DNA结合蛋白。Compared with the amino acid sequence of the wild-type Taq DNA polymerase whose NCBI accession number is P19821.1, the direct-expanding hot-start DNA polymerase has the following characteristics:
所述的双链结合蛋白优选为氨基酸序列如SEQ ID NO.2所示的Sso7d。双链结合蛋白可以显著提高DNA聚合酶对模板DNA的亲和力。The double-chain binding protein is preferably Sso7d whose amino acid sequence is shown in SEQ ID NO.2. Double-stranded binding proteins can significantly increase the affinity of DNA polymerases for template DNA.
编码上述直扩型热启动DNA聚合酶的DNA分子。A DNA molecule encoding the above-mentioned direct expansion type hot-start DNA polymerase.
所述的DNA分子的核苷酸序列如SEQ ID NO.3所示。该序列是根据大肠杆菌表达系统特点进行密码子优化而得，能显著提高异源基因在宿主菌中的表达效率。The nucleotide sequence of the DNA molecule is shown in SEQ ID NO.3. The sequence is obtained by codon optimization according to the characteristics of Escherichia coli expression system, and can significantly improve the expression efficiency of heterologous genes in host bacteria.
一种重组表达载体，是将编码上述直扩型热启动DNA聚合酶的DNA分子克隆入表达载体得到。A recombinant expression vector is obtained by cloning the DNA molecule encoding the above-mentioned direct expansion type hot-start DNA polymerase into the expression vector.
所述的表达载体优选为原核表达载体；更优选为pET系列载体；最优选为pET-28a。The expression vector is preferably a prokaryotic expression vector; more preferably a pET series vector; most preferably pET-28a.
一种重组工程细胞株，是将上述重组表达载体转化入工程细胞得到。A recombinant engineering cell strain is obtained by transforming the above recombinant expression vector into engineering cells.
所述的工程细胞优选为大肠杆菌细胞；更优选为BL21(DE3)细胞。该重组工程细胞株可快速、可溶表达上述重组表达载体。The engineered cells are preferably Escherichia coli cells; more preferably BL21(DE3) cells. The recombinant engineered cell line can express the above recombinant expression vector rapidly and soluble.
上述直扩型热启动DNA聚合酶可通过化学合成方法得到，或是通过重组工程细胞株诱导表达、纯化制备得到。从成本考虑，优选为通过重组工程细胞株诱导表达、纯化制备得到；具体包括如下步骤：The above-mentioned direct-expanding hot-start DNA polymerase can be obtained by chemical synthesis, or can be prepared by inducing expression and purification of recombinant engineering cell lines. Considering the cost, it is preferably prepared by inducing expression and purification of recombinant engineering cell lines; it specifically includes the following steps:
取上述重组工程细胞株，接种于含抗生素的LB培养基中，在35～38℃条件下，培养至菌液OD600达到0.7～0.9，再用IPTG诱导细胞表达蛋白，经分离纯化后得到所述的直扩型热启动DNA聚合酶。Take the above-mentioned recombinant engineering cell line, inoculate it in LB medium containing antibiotics, and cultivate it at 35-38 ℃ until the OD 600 of the bacterial liquid reaches 0.7-0.9, and then induce the cells to express proteins with IPTG, and obtain the obtained after separation and purification. The direct-expanding hot-start DNA polymerase described above.
所述的抗生素优选为卡那霉素。The antibiotic is preferably kanamycin.
所述的卡那霉素的用量优选为按50μg/mL LB培养基计算。The dosage of the kanamycin is preferably calculated as 50 μg/mL LB medium.
所述的IPTG的用量优选为按终浓度为0.1～0.5mmol/L计算。The dosage of the IPTG is preferably calculated at a final concentration of 0.1 to 0.5 mmol/L.
所述的诱导的条件优选为温度35～38℃、时间2～4h；更优先为温度37℃、时间3h。The conditions for the induction are preferably a temperature of 35 to 38° C. and a time of 2 to 4 hours; more preferably, a temperature of 37° C. and a time of 3 hours.
所述的分离纯化的方法优选为先经镍离子亲和层析(Ni-NTA层析)，再经阴离子交换柱层析。The method of separation and purification is preferably performed by nickel ion affinity chromatography (Ni-NTA chromatography) first, and then by anion exchange column chromatography.
优选的，所述的镍离子亲和层析所用的结合缓冲液(Buffer A)的配方为：50mmol/L NaCl、50mmol/L Tris-HCl，pH＝8.0；所用的洗脱缓冲液(Buffer B)的配方为：500mmol/L咪唑、50mmol/L Tris-HCl、50mmol/L NaCl，pH＝8.0。Preferably, the formula of the binding buffer (Buffer A) used in the nickel ion affinity chromatography is: 50 mmol/L NaCl, 50 mmol/L Tris-HCl, pH=8.0; the used elution buffer (Buffer B ) formula is: 500mmol/L imidazole, 50mmol/L Tris-HCl, 50mmol/L NaCl, pH=8.0.
优选的，所述的阴离子交换层析所用的结合缓冲液(Buffer C)的配方为：50mmol/L Tris-HCl、100mmol/L NaCl，pH＝8.0；所用的洗脱缓冲液(Buffer D)的配方为：50mmol/LTris-HCl、1mol/L NaCl，pH＝8.0。Preferably, the formula of the binding buffer (Buffer C) used in the anion exchange chromatography is: 50 mmol/L Tris-HCl, 100 mmol/L NaCl, pH=8.0; the used elution buffer (Buffer D) has The formula is: 50mmol/LTris-HCl, 1mol/L NaCl, pH=8.0.
优选的，所述的直扩型热启动DNA聚合酶的聚合酶活性位点赖氨酸侧链氨基上特异性结合有酸酐类化合物，以可逆的封闭其聚合酶活性。Preferably, an acid anhydride compound is specifically bound to the amino group of the lysine side chain of the polymerase active site of the direct expansion type hot-start DNA polymerase to reversibly block its polymerase activity.
所述的酸酐类化合物优选为顺式乌头酸酐或柠槺酸酐；更优选为柠槺酸酐。经酸酐修饰的聚合酶，大大降低了PCR反应过程中非特异性产物扩增和引物二聚体形成的可能性。The acid anhydride compound is preferably cis-aconitic anhydride or citracic anhydride; more preferably citracic anhydride. Anhydride-modified polymerase greatly reduces the possibility of non-specific product amplification and primer-dimer formation during PCR reactions.
上述直扩型热启动DNA聚合酶的制备方法，包括如下步骤：The preparation method of the above-mentioned direct expansion type hot-start DNA polymerase comprises the following steps:
(1)将上述直扩型热启动DNA聚合酶透析到Tris-HCl缓冲液中；(1) dialyzing the above-mentioned direct expansion hot-start DNA polymerase into Tris-HCl buffer;
(2)加入酸酐类化合物，混合均匀，孵育；(2) Add acid anhydride compounds, mix well, and incubate;
(3)将孵育后的酶液透析到储存缓冲液中，即获得稳定的直扩型热启动DNA聚合酶。经该方法制得的热启动DNA聚合酶具有优异的热启动性能，在50℃，孵育10min仍无聚合酶活性释放；在95℃热激，5～10min才可释放活性。(3) Dialyzing the incubated enzyme solution into a storage buffer to obtain a stable direct expansion hot-start DNA polymerase. The hot-start DNA polymerase prepared by this method has excellent hot-start performance, and no polymerase activity is released even after 10 minutes of incubation at 50°C; activity can be released only after 5-10 minutes of heat shock at 95°C.
所述的Tris-HCl缓冲液优选浓度为10～30mmol/L、pH＝9～10的Tris-HCl缓冲液。The Tris-HCl buffer is preferably a Tris-HCl buffer with a concentration of 10-30 mmol/L and pH=9-10.
所述的直扩型热启动DNA聚合酶与所述的酸酐类化合物的配比优选为摩尔比1:50～1:100。The ratio of the direct expansion type hot-start DNA polymerase to the acid anhydride compound is preferably a molar ratio of 1:50 to 1:100.
所述的孵育的条件优选为温度40℃～50℃、时间2～5h。The incubation conditions are preferably a temperature of 40°C to 50°C and a time of 2 to 5 hours.
所述的储存缓冲液的配方优选为：20mmol/L Tris-HCl，100mmol/L KCl，0.1mmol/L EDTA，50％甘油(Glycerol)，1mmol/LDTT，0.5％Tween-20，0.5％
一种PCR反应缓冲液，包含如下组分：20～100mmol/L Tris-HCl、1～3mmol/LMgCl2、5～20mmol/L(NH4)2SO4、20～80mmol/L KCl、0.1～0.4mg/L BSA、0.05％～0.1％Tween-20、1％～6％DMSO，pH值为8～10；所述的PCR反应缓冲液与上述直扩型热启动DNA聚合酶配套使用。A PCR reaction buffer, comprising the following components: 20-100 mmol/L Tris-HCl, 1-3 mmol/LMgCl 2 , 5-20 mmol/L (NH 4 ) 2 SO 4 , 20-80 mmol/L KCl, 0.1-3 mmol/L 0.4mg/L BSA, 0.05%-0.1% Tween-20, 1%-6% DMSO, pH value is 8-10; the PCR reaction buffer is used together with the above-mentioned direct-amplification hot-start DNA polymerase.
所述的PCR反应缓冲液还包含海藻糖、乙基苯基聚乙二醇(NP-40)和L-肉毒碱中的至少一种。The PCR reaction buffer also contains at least one of trehalose, ethylphenyl polyethylene glycol (NP-40) and L-carnitine.
所述的海藻糖在体系中的浓度优选为0.1～0.4mol/L；更优选为0.3mol/L。The concentration of the trehalose in the system is preferably 0.1-0.4 mol/L; more preferably 0.3 mol/L.
所述的乙基苯基聚乙二醇在体系中的浓度优选为0～0.4％；更优选为0.4％。The concentration of the ethylphenyl polyethylene glycol in the system is preferably 0-0.4%; more preferably 0.4%.
所述的L-肉毒碱在体系中的浓度优选为0.1～0.3mol/L；更优选为0.2mol/L。The concentration of the L-carnitine in the system is preferably 0.1-0.3 mol/L; more preferably 0.2 mol/L.
上述PCR反应缓冲液在DNA样本扩增中的应用。The application of the above PCR reaction buffer in the amplification of DNA samples.
一种qPCR反应试剂盒，包含上述PCR反应缓冲液、SYBR Green I、PCR用水和dNTPs中的至少一种，以及上述直扩型热启动DNA聚合酶。A qPCR reaction kit, comprising at least one of the above PCR reaction buffer, SYBR Green I, PCR water and dNTPs, and the above direct expansion type hot-start DNA polymerase.
所述的SYBR Green I在体系中的浓度为0.2～10ng/μL。The concentration of the SYBR Green I in the system is 0.2-10 ng/μL.
所述的直扩型热启动DNA聚合酶在体系中的浓度优选为0.02～0.05U/μL。The concentration of the direct expansion type hot-start DNA polymerase in the system is preferably 0.02-0.05 U/μL.
所述的dNTPs在体系中的浓度优选为100～300μmol/L。The concentration of the dNTPs in the system is preferably 100-300 μmol/L.
上述qPCR反应试剂盒在DNA样本扩增中的应用。Application of the above qPCR reaction kit in DNA sample amplification.
所述的DNA样本来源不受限制，可能含有腐殖酸、多糖、免疫球蛋白、酪蛋白等抑制剂中的至少一种。比如，含有多糖的植物核酸样本、含有免疫球蛋白的血液核酸样本、含有酪蛋白的牛奶核酸样本、含有腐殖酸的土壤核酸样本等。The source of the DNA sample is not limited, and may contain at least one of inhibitors such as humic acid, polysaccharide, immunoglobulin, and casein. For example, plant nucleic acid samples containing polysaccharides, blood nucleic acid samples containing immunoglobulins, milk nucleic acid samples containing casein, soil nucleic acid samples containing humic acid, etc.
本发明相较于现有技术具有如下的优点和有益效果：Compared with the prior art, the present invention has the following advantages and beneficial effects:
(1)本发明的直扩型热启动DNA聚合酶稳定性良好，在qPCR检验中可耐受高浓度的荧光染料和各种高浓度的抑制剂，如10ng/μL SYBR GreenΙ、1.6μg/mL腐殖酸、0.32mg/mL黄芪多糖或600μg/mL IgG。(1) The direct expansion type hot-start DNA polymerase of the present invention has good stability, and can withstand high concentrations of fluorescent dyes and various high concentrations of inhibitors in qPCR testing, such as 10ng/μL SYBR Green1, 1.6μg/mL Humic acid, 0.32mg/mL Astragalus polysaccharide or 600μg/mL IgG.
(2)本发明所述的热启动DNA聚合酶使用便利，配套所述的PCR反应缓冲液后，可以直接用于各种来源、组成复杂的样本中目的基因的多重PCR扩增，至少可以在含50％的全血或20％的牛奶的反应体系中正常扩增目的基因。(2) The hot-start DNA polymerase of the present invention is convenient to use. After matching with the PCR reaction buffer, it can be directly used for multiple PCR amplification of target genes in samples from various sources and complex compositions. The target gene was amplified normally in the reaction system containing 50% whole blood or 20% milk.
(3)本发明所述qPCR反应试剂盒具有特异性强、灵敏度高的特点，可以准确检出浓度低至0.2copies/μL的目的基因。(3) The qPCR reaction kit of the present invention has the characteristics of strong specificity and high sensitivity, and can accurately detect the target gene with a concentration as low as 0.2 copies/μL.
附图说明Description of drawings
图1为Taq DNA聚合酶的纯化结果图；其中，泳道1为(11.4-116.0kDa)宽范围蛋白上样Marker，泳道2为细胞破碎后离心处理所得的上清液，泳道3为破碎液上清75℃加热30min离心处理后上清，泳道4～9分别为Ni-NTA亲和层析纯化时10％～60％Buffer B线性洗脱峰中的Taq DNA聚合酶，泳道10为阴离子交换层析纯化后所得到的Taq DNA聚合酶。Figure 1 is a diagram of the purification results of Taq DNA polymerase; wherein,
图2为本发明HS Sso-Taq与市售热启动DNA聚合酶TaqHS(Takara，R007Q)用于全血直接扩增的对比结果图；其中，A为两种DNA聚合酶用于扩增不同浓度的全血中的人类固醇21-羟化酶基因(CYP21)得到的PCR产物的琼脂糖凝胶电泳图；B为本发明HS Sso-Taq用于在40％(v/v)全血中扩增3种不同长度的DNA片段，得到的PCR产物琼脂糖凝胶电泳图。Fig. 2 is the comparison result diagram of the direct amplification of whole blood with HS Sso-Taq of the present invention and the commercially available hot-start DNA polymerase TaqHS (Takara, R007Q); wherein, A is the use of two DNA polymerases for amplification of different concentrations The agarose gel electrophoresis image of the PCR product obtained from the human sterol 21-hydroxylase gene (CYP21) in the whole blood; B is the HS Sso-Taq of the present invention used for amplification in 40% (v/v) whole blood Agarose gel electrophoresis of PCR products obtained by adding 3 DNA fragments of different lengths.
图3为本发明HS Sso-Taq与市售热启动DNA聚合酶TaqHS(Takara，R007Q)用于直接扩增不同浓度鲜牛奶中的牛线粒体色素细胞b(Cytochrome,cob)基因，得到的PCR产物的琼脂糖凝胶电泳对比结果图。Fig. 3 is the PCR product obtained when HS Sso-Taq of the present invention and commercially available hot-start DNA polymerase TaqHS (Takara, R007Q) are used to directly amplify the bovine mitochondrial pigment cell b (Cytochrome, cob) gene in fresh milk with different concentrations Agarose gel electrophoresis comparison results.
图4为含本发明HS Sso-Taq的SYBR GreenΙ染料法荧光定量PCR试剂与市售同类试剂的扩增灵敏度对比结果图；其中，A为市售品牌Promega(A600A)结果曲线图、B为市售品牌Takara(RR820)结果曲线图、C为本发明HS Sso-Taq结果曲线图、D为不同试剂的扩增灵敏度和扩增效率结果。Fig. 4 is the amplification sensitivity comparison result of the SYBR Green1 dye-based fluorescent quantitative PCR reagent containing HS Sso-Taq of the present invention and commercially available similar reagents; wherein, A is the result curve of the commercially available brand Promega (A600A), and B is the market The curve diagram of the sales brand Takara (RR820), C is the curve diagram of the HS Sso-Taq results of the present invention, and D is the amplification sensitivity and amplification efficiency results of different reagents.
图5为本发明HS Sso-Taq与热启动野生型Taq DNA聚合酶(HS wt-Taq)的耐受SYBRGreenΙ荧光染料性能对比结果图；其中，A为HS Sso-Taq扩增曲线，B为HS Sso-Taq熔解曲线，C为HS wt-Taq扩增曲线，D为HS wt-Taq熔解曲线。Figure 5 is a graph showing the comparison results of the SYBRGreen1 fluorescent dye resistance of HS Sso-Taq of the present invention and hot-start wild-type Taq DNA polymerase (HS wt-Taq); wherein, A is the amplification curve of HS Sso-Taq, and B is HS Sso-Taq melting curve, C is the HS wt-Taq amplification curve, D is the HS wt-Taq melting curve.
图6为本发明HS Sso-Taq与热启动野生型Taq DNA聚合酶(HS wt-Taq)在荧光定量PCR检测中耐受腐殖酸的性能对比结果图；其中，A为HS Sso-Taq扩增曲线，B为HS Sso-Taq熔解曲线，C为HS wt-Taq扩增曲线，D为HS wt-Taq熔解曲线。Figure 6 is a graph showing the comparison results of the humic acid tolerance between HS Sso-Taq of the present invention and hot-start wild-type Taq DNA polymerase (HS wt-Taq) in fluorescence quantitative PCR detection; wherein, A is HS Sso-Taq amplification Amplification curve, B is the melting curve of HS Sso-Taq, C is the amplification curve of HS wt-Taq, D is the melting curve of HS wt-Taq.
图7为本发明HS Sso-Taq与热启动野生型Taq DNA聚合酶(HS wt-Taq)在荧光定量PCR检测中耐受多糖的性能对比结果图；其中，A为HS Sso-Taq扩增曲线，B为HS Sso-Taq熔解曲线，C为HS wt-Taq扩增曲线，D为HS wt-Taq熔解曲线。Figure 7 is a graph showing the comparison results of the performance of HS Sso-Taq of the present invention and the hot-start wild-type Taq DNA polymerase (HS wt-Taq) in the detection of polysaccharide tolerance in fluorescence quantitative PCR; wherein, A is the amplification curve of HS Sso-Taq , B is the melting curve of HS Sso-Taq, C is the amplification curve of HS wt-Taq, and D is the melting curve of HS wt-Taq.
图8为本发明HS Sso-Taq与热启动野生型Taq DNA聚合酶(HS wt-Taq)在荧光定量PCR检测中耐受IgG性能对比结果图。其中，A为HS Sso-Taq扩增曲线，B为HS Sso-Taq熔解曲线，C为HS wt-Taq扩增曲线，D为HS wt-Taq熔解曲线。FIG. 8 is a graph showing the comparison results of the IgG tolerance of the HS Sso-Taq of the present invention and the hot-start wild-type Taq DNA polymerase (HS wt-Taq) in fluorescence quantitative PCR detection. Among them, A is the HS Sso-Taq amplification curve, B is the HS Sso-Taq melting curve, C is the HS wt-Taq amplification curve, and D is the HS wt-Taq melting curve.
具体实施方式Detailed ways
下面结合实施例及附图对本发明做进一步的详细说明，以令本领域技术人员参照说明书文字能够据以实施。The present invention will be further described in detail below with reference to the embodiments and the accompanying drawings, so that those skilled in the art can refer to the description and implement accordingly.
实施例1构建含编码Taq DNA聚合酶的核苷酸序列的重组载体Example 1 Construction of the recombinant vector containing the nucleotide sequence encoding Taq DNA polymerase
(1)根据Taq DNA聚合酶的氨基酸序列(SEQ ID NO.1)，进行大肠杆菌表达系统的密码子优化后，获得能在大肠杆菌中进行高效表达的DNA分子，利用重叠延伸PCR的方法，人工合成编码所述Taq DNA聚合酶的DNA分子，具体如SEQ ID NO.3所示。(1) according to the amino acid sequence (SEQ ID NO.1) of Taq DNA polymerase, after carrying out the codon optimization of Escherichia coli expression system, obtain the DNA molecule that can carry out high-efficiency expression in Escherichia coli, utilize the method of overlapping extension PCR, The DNA molecule encoding the Taq DNA polymerase is artificially synthesized, as shown in SEQ ID NO.3.
(2)将编码Taq DNA聚合酶的DNA分子与表达载体pET-28a进行同源重组。Taq DNA聚合酶扩增引物序列如下：(2) Homologous recombination was performed between the DNA molecule encoding Taq DNA polymerase and the expression vector pET-28a. Taq DNA polymerase amplification primer sequences are as follows:
Taq-FP：5’-GGCATATGGCGACCGTTAAGTTTAAGTAC-3’(SEQ ID NO.4)；Taq-FP: 5'-GGCATATGGCGACCGTTAAGTTTAAGTAC-3' (SEQ ID NO. 4);
Taq-RP：5’-CCATGAATTCTTATTCCTTCGCAGAT-3’(SEQ ID NO.5)。Taq-RP: 5'-CCATGAATTCTTATTCCTTCGCAGAT-3' (SEQ ID NO. 5).
pET-28a线性化引物序列如下：The pET-28a linearization primer sequences are as follows:
pET-28a-FP：5’-GGAATAAGAATTCATGGTTGCGGCCGCA-3’(SEQ ID NO.6)；pET-28a-FP: 5'-GGAATAAGAATTCATGGTTGCGGCCGCA-3' (SEQ ID NO. 6);
pET-28a-RP：5’-ACGGTCGCCATATGCCGCGCGGCACCA-3’(SEQ ID NO.7)。pET-28a-RP: 5'-ACGGTCGCCATATGCCGCGCGGCACCA-3' (SEQ ID NO. 7).
应用PCR对Taq DNA聚合酶DNA分子及pET-28a载体进行扩增，分别以人工合成编码所述Taq DNA聚合酶的DNA分子及pET-28a空载为模板，加入25μL 2×Pfu Max HiFi PCRProMix(广州英赞生物科技有限公司，货号P217A)、各1μL(10μmol/L)上下游引物以及适量的灭菌水，进行PCR扩增。Taq DNA聚合酶DNA分子的扩增条件为：98℃30s；98℃10s、55℃30s、68℃1min，共30个循环；68℃5min。质粒线性化PCR扩增程序为：98℃30s；98℃10s、55℃30s、68℃2.5min，共30个循环；68℃5min。琼脂糖凝胶电泳回收全长约1.8kb的Taq DNA聚合酶DNA基因片段，以及约5kb线性化的pET-28a载体。将回收产物进行同源重组，体系为5μL 2×Hipro DNA Assembly Cloning Mix(广州英赞生物科技，K001A)、回收产物各加50ng，补水至10μL，50℃孵育15min。孵育完后将重组产物转化至DH5a感受态细胞中。The Taq DNA polymerase DNA molecule and pET-28a vector were amplified by PCR, and 25 μL of 2 × Pfu Max HiFi PCRProMix ( Guangzhou Yingzan Biotechnology Co., Ltd., product number P217A), 1 μL (10 μmol/L) upstream and downstream primers and appropriate amount of sterilized water for PCR amplification. The amplification conditions of Taq DNA polymerase DNA molecules are: 98°C for 30s; 98°C for 10s, 55°C for 30s, 68°C for 1min, a total of 30 cycles; 68°C for 5min. The plasmid linearization PCR amplification program was as follows: 98°C for 30s; 98°C for 10s, 55°C for 30s, 68°C for 2.5min, a total of 30 cycles; 68°C for 5min. Agarose gel electrophoresis was used to recover the Taq DNA polymerase DNA gene fragment of about 1.8kb in length, and the linearized pET-28a vector of about 5kb. The recovered products were subjected to homologous recombination, the system was 5 μL of 2×Hipro DNA Assembly Cloning Mix (Guangzhou Yingzan Biotechnology, K001A), 50 ng of each recovered product was added, water was added to 10 μL, and incubated at 50°C for 15 min. After incubation, the recombinant product was transformed into DH5a competent cells.
(3)挑取单菌落进行菌落PCR鉴定，并将阳性单克隆送往测序公司测序验证，培养验证正确的感受态细胞，并提取质粒，所获得的质粒即为含有编码Taq DNA聚合酶的DNA分子的重组载体。(3) Pick a single colony for colony PCR identification, send the positive single clone to a sequencing company for sequencing verification, culture and verify the correct competent cells, and extract the plasmid, the obtained plasmid is the DNA containing the encoding Taq DNA polymerase Molecular recombinant vector.
实施例2表达Taq DNA聚合酶的转化体的制备Example 2 Preparation of transformants expressing Taq DNA polymerase
将实施例1获得的重组载体转化到宿主细胞E.coliBL21(DE3)中，挑取单菌落，接种至液体LB(含50μg/mL的卡那霉素)培养基培养至OD600为0.9，加入IPTG至终浓度为0.1mmol/L，37℃诱导3h，收集菌体超声破碎，SDS-PAGE电泳检测目的蛋白的表达，发现所制备的转化体能够高效表达Taq DNA聚合酶。The recombinant vector obtained in Example 1 was transformed into the host cell E.coliBL21 (DE3), a single colony was picked, inoculated into a liquid LB (containing 50 μg/mL of kanamycin) medium and cultured to an OD 600 of 0.9, adding The final concentration of IPTG was 0.1 mmol/L, induced at 37°C for 3 h, the cells were collected and sonicated, and the expression of the target protein was detected by SDS-PAGE electrophoresis. It was found that the prepared transformants could express Taq DNA polymerase efficiently.
实施例3 Taq DNA聚合酶在重组大肠杆菌中的表达Example 3 Expression of Taq DNA polymerase in recombinant Escherichia coli
将实施例2获得的能够表达Taq DNA聚合酶的阳性转化体菌种，接种至含50μg/mL卡那霉素的60mL LB培养基中，放置于37℃摇床中震荡培养过夜；取过夜培养的种子液，按体积比1:100接种至1L含有50μg/mL卡那霉素的LB培养基中，37℃摇床中震荡培养至OD600为0.7；向摇瓶中加入IPTG至终浓度为0.1mmol/L，37℃继续震荡诱导3h；离心收集诱导后菌体并称重，记录菌体湿重，储存于-20℃。The positive transformant strains that can express Taq DNA polymerase obtained in Example 2 were inoculated into 60 mL of LB medium containing 50 μg/mL kanamycin, and placed in a shaker at 37 °C for overnight culture; The seed solution was inoculated into 1 L of LB medium containing 50 μg/mL kanamycin at a volume ratio of 1:100, and shaken in a shaker at 37 °C until the OD 600 was 0.7; IPTG was added to the shake flask to a final concentration of 0.1 mmol/L, 37°C for further induction by shaking for 3h; the induced cells were collected by centrifugation and weighed, the wet weight of the cells was recorded, and stored at -20°C.
实施例4 Taq DNA聚合酶的纯化Example 4 Purification of Taq DNA polymerase
1、重组菌体超声破碎1. Ultrasonic fragmentation of recombinant bacteria
取-20℃冻存的诱导表达菌体，根据实施例3记录的菌体湿重，按每克菌体加入5mL裂解缓冲液(50mmol/L Tris-HCl、100mmol/L NaCl，pH 8.0)重悬菌体，用超声波细胞破碎仪裂解菌体，超声条件为：功率250W，超声5.5s，停5.5s，持续30min。将裂解后菌体放入高速冷冻离心机，4℃下20000r/min离心15min，取上清液至250mL灭菌玻璃瓶中。上清液于75℃恒温水浴锅孵育30min，4℃下20000r/min离心15min，0.22μm微孔滤膜过滤，取上清液至250mL灭菌玻璃瓶。Get the induced expression thalline stored frozen at -20°C, add 5mL lysis buffer (50mmol/L Tris-HCl, 100mmol/L NaCl, pH 8.0) per gram of thalline according to the wet weight of the thalline recorded in Example 3. The cells were suspended, and the cells were lysed with an ultrasonic cell disruptor. The ultrasonic conditions were: power 250W, ultrasonic for 5.5s, stop for 5.5s, and last for 30min. The lysed cells were placed in a high-speed refrigerated centrifuge, centrifuged at 20,000 r/min for 15 min at 4°C, and the supernatant was taken into a 250 mL sterilized glass bottle. The supernatant was incubated in a constant temperature water bath at 75°C for 30 minutes, centrifuged at 20,000 r/min for 15 minutes at 4°C, filtered with a 0.22 μm microporous membrane, and the supernatant was taken into a 250 mL sterilized glass bottle.
2、镍离子亲和层析纯化2. Nickel ion affinity chromatography purification
选用的层析柱是HisTrapTM HP 5mL(购自GE Healthcare)，结合缓冲液为bufferA：50mmol/L Tris-HCl、50mmol/L NaCl，pH＝8.0；洗脱缓冲液为buffer B：500mmol/L咪唑、50mmol/L Tris-HCl、50mmol/L NaCl，pH＝8，0.22μm滤膜过滤备用。The selected chromatographic column is HisTrap ™ HP 5mL (purchased from GE Healthcare), and the binding buffer is bufferA: 50mmol/L Tris-HCl, 50mmol/L NaCl, pH=8.0; the elution buffer is buffer B: 500mmol/L Imidazole, 50 mmol/L Tris-HCl, 50 mmol/L NaCl, pH=8, and 0.22 μm filter membrane was used for filtration.
将HisTrapTM HP 5mL接入快速蛋白质纯化仪柱位阀中，后用超纯水清洗系统和柱子，再用buffer A平衡柱子，然后用样品泵将步骤1所述上清液进行上样，上样完毕，先用buffer A清洗柱子，再用buffer B进行线性洗脱(10％～60％)，并收集线性洗脱峰样品。对buffer B洗脱峰中收集的1～6管(3mL/管)样品进行取样，用SDS-PAGE蛋白电泳检测，结果如图1(泳道4-9)所示，将纯度较高的5、6管样品合并作为强阴离子交换柱层析样品。Insert HisTrap TM HP 5mL into the column valve of the rapid protein purification instrument, then wash the system and the column with ultrapure water, then equilibrate the column with buffer A, and then use the sample pump to load the supernatant described in
3、强阴离子交换柱层析3. Strong anion exchange column chromatography
所用层析柱为HisTrapTMCaptoTM Q 5mL(购自GE Healthcare)，所用的结合缓冲液为Buffer C：50mmol/L Tris-HCl、100mmol/L NaCl，pH＝8.0；洗脱缓冲液为BufferD：50mmol/L Tris-HCl、1mol/L NaCl，pH＝8.0，0.22μm滤膜过滤备用。The chromatography column used was HisTrap ™ Capto ™ Q 5mL (purchased from GE Healthcare), the used binding buffer was Buffer C: 50 mmol/L Tris-HCl, 100 mmol/L NaCl, pH=8.0; the elution buffer was
将HisTrapTMCaptoTM Q 5mL接入AKTA纯化仪柱位阀中，再用超纯水清洗系统和柱子，用buffer C平衡柱子，然后用样品泵将上述含有目的蛋白的亲和层析样品进行上样，上样完毕，先用buffer C清洗柱子，再用洗脱缓冲液buffer D进行梯度洗脱，并收集洗脱峰。洗脱峰取样20μL，SDS-PAGE蛋白电泳检测，结果如图1所示。Insert the HisTrap TM Capto TM Q 5mL into the column valve of the AKTA purifier, then wash the system and the column with ultrapure water, equilibrate the column with buffer C, and then use the sample pump to carry out the above-mentioned affinity chromatography sample containing the target protein. After sample loading, the column was first washed with buffer C, and then gradient elution was performed with elution buffer buffer D, and the elution peaks were collected. The elution peak was sampled by 20 μL, and SDS-PAGE protein electrophoresis was used for detection. The results are shown in Figure 1.
实施例5 Taq DNA聚合酶活性测试Example 5 Taq DNA polymerase activity test
取实施例4制备的Taq DNA聚合酶，按照以下方法测定酶活。74℃下，以活性化的大马哈鱼精子DNA作为模板/引物，在200μmol/L dNTPs、50mmol/L Tris-HCl、2mmol/L MgCl2、5mmol/L(NH4)2SO4、50mmol/L KCl、0.1～0.4mg/L BSA、0.1％Tween-20、4％DMSO组成的pH8.0的反应体系中进行酶催化反应，30min内，催化10nmoldNTPs掺入到DNA的酶量定义为1U。结果表明，本发明的TaqDNA聚合酶的酶活浓度为62U/μL。Take the Taq DNA polymerase prepared in Example 4, and measure the enzyme activity according to the following method. At 74℃, using activated salmon sperm DNA as template/primer, in 200μmol/L dNTPs, 50mmol/L Tris-HCl, 2mmol/L MgCl 2 , 5mmol/L (NH 4 ) 2 SO 4 , 50mmol /L KCl, 0.1-0.4mg/L BSA, 0.1% Tween-20, 4% DMSO to carry out the enzyme-catalyzed reaction in a pH8.0 reaction system, within 30min, the amount of enzyme that catalyzes the incorporation of 10nm oldNTPs into DNA is defined as 1U . The results showed that the enzyme activity concentration of the TaqDNA polymerase of the present invention was 62 U/μL.
实施例6 HS Sso-Taq的制备及测活Example 6 Preparation and activity measurement of HS Sso-Taq
将高纯度的Taq DNA聚合酶过夜透析到10～30mmol/L Tris-HCl(pH9.0～10.0)缓冲溶液中。并用BCA的方法测定蛋白浓度，后与柠槺酸酐按摩尔比为1:100混匀均匀，并于42℃孵育4h，得到经柠槺酸酐修饰可逆封闭酶活性的热启动DNA聚合酶，命名为HS Sso-Taq。The high-purity Taq DNA polymerase was dialyzed into 10-30 mmol/L Tris-HCl (pH 9.0-10.0) buffer solution overnight. The protein concentration was determined by BCA method, and then mixed with citrate anhydride in a molar ratio of 1:100, and incubated at 42 °C for 4 hours to obtain a hot-start DNA polymerase modified with citrate anhydride to reversibly block the enzymatic activity, named as HS Sso-Taq.
将得到的热启动DNA聚合酶透析到储存缓冲液(20mmol/L Tris-HCl，100mmol/LKCl，0.1mmol/L EDTA，50％Glycerol，1mmol/L DTT，0.5％Tween-20，0.5％
实施例7 HS Sso-Taq的全血直扩能力研究Example 7 Study on the direct expansion ability of HS Sso-Taq in whole blood
(1)对不同浓度的全血进行直扩(1) Direct expansion of whole blood of different concentrations
分别以100ng的从EDTA-2Na抗凝血中提纯的人基因组DNA及10％、20％、40％、50％(v/v)的EDTA-2Na抗凝血(全血由健康人提供，于广东省内取样)为模板，用本发明中的直扩型热启动DNA聚合酶以及购买市售品牌Takara的热启动DNA聚合酶TaqHS(Takara，R007Q)同时扩增人类固醇21-羟化酶基因(CYP21)基因，结果如图2所示。扩增引物和具体操作如下：100ng of human genomic DNA purified from EDTA-2Na anticoagulation and 10%, 20%, 40%, 50% (v/v) EDTA-2Na anticoagulation (whole blood provided by healthy people, in Sampling in Guangdong Province) is a template, with the direct expansion type hot-start DNA polymerase in the present invention and the hot-start DNA polymerase TaqHS (Takara, R007Q) of the commercially available brand Takara to simultaneously amplify the human sterol 21-hydroxylase gene (CYP21) gene, and the results are shown in Figure 2. The amplification primers and specific operations are as follows:
CYP21-FP：5’-GCTCAGCATGGTGGTGGCATAA-3’(SEQ ID NO.8)；CYP21-FP: 5'-GCTCAGCATGGTGGTGGCATAA-3' (SEQ ID NO. 8);
CYP21-RP：5’-CCTCATACCTTCCCCCCCATTT-3’(SEQ ID NO.9)。CYP21-RP: 5'-CCTCATACCTTCCCCCCCATTT-3' (SEQ ID NO. 9).
基于本发明中的PCR反应缓冲液包含50mmol/L Tris-HCl(pH8.0)、3mmol/LMgCl2、5mmol/L(NH4)2SO4、50mmol/L KCl、0.1mg/L BSA、0.05％Tween-20、1％DMSO、250nmol/L CYP21-FP、250nmol/L CYP21-RP，200μmol/L dNTPs，0.05U/μL HS Sso-Taq，适量的灭菌ddH2O。反应程序为95℃10min；95℃15s、60℃30s、72℃30s，共35个循环。The PCR reaction buffer based on the present invention contains 50mmol/L Tris-HCl (pH8.0), 3mmol/LMgCl 2 , 5mmol/L(NH 4 ) 2 SO 4 , 50mmol/L KCl, 0.1mg/L BSA, 0.05 %Tween-20, 1%DMSO, 250nmol/L CYP21-FP, 250nmol/L CYP21-RP, 200μmol/L dNTPs, 0.05U/μL HS Sso-Taq, appropriate amount of sterilized ddH 2 O. The reaction program was 95°C for 10 min; 95°C for 15s, 60°C for 30s, and 72°C for 30s, a total of 35 cycles.
市售品牌Takara热启动DNA聚合酶Taq HS的反应体系包含2.5μL 10×PCRBuffer，200nmol/L CYP21-FP、200nmol/L CYP21-RP，200μmol/L dNTPs，0.05U/μL Taq HS，适量的灭菌ddH2O。反应程序为98℃10s、60℃30s、72℃30s，共35个循环。The reaction system of the commercially available brand Takara hot-start DNA polymerase Taq HS contains 2.5μL 10×PCRBuffer, 200nmol/L CYP21-FP, 200nmol/L CYP21-RP, 200μmol/L dNTPs, 0.05U/μL Taq HS, an appropriate amount of bacteria ddH 2 O. The reaction program was 98 °C for 10 s, 60 °C for 30 s, and 72 °C for 30 s, a total of 35 cycles.
结果如图2A所示。分析结果可得，本发明的直扩型热启动DNA聚合酶至少可耐受50％的全血，并从中扩增得到目的基因。而商品的热启动DNA聚合酶TaqHS不能耐受全血抑制。The results are shown in Figure 2A. According to the analysis results, the direct expansion type hot-start DNA polymerase of the present invention can tolerate at least 50% of whole blood, and the target gene can be amplified from it. The commercial hot-start DNA polymerase TaqHS cannot tolerate whole blood inhibition.
(2)在40％全血中同时扩增3个不同长度的基因片段(2) Simultaneous amplification of 3 gene fragments of different lengths in 40% whole blood
以40％EDTA-2Na抗凝血为模板，用本发明中的直扩型热启动DNA聚合酶同时扩增不同长度的基因片段，扩增基因、引物和具体操作如下：Taking 40% EDTA-2Na anticoagulation as a template, using the direct expansion type hot-start DNA polymerase of the present invention to simultaneously amplify gene fragments of different lengths, the amplified genes, primers and specific operations are as follows:
人类固醇21-羟化酶基因(steroid 21-hydroxylase，CYP21)基因，基因长度为320bp，引物如SEQ ID NO.8、SEQ ID NO.9所示。Human steroid 21-hydroxylase gene (steroid 21-hydroxylase, CYP21) gene, the gene length is 320bp, and the primers are shown in SEQ ID NO.8 and SEQ ID NO.9.
二酰基甘油激酶基因(diacylglycerol kinase，DGK)，基因长度为243bp，The diacylglycerol kinase (DGK) gene is 243bp in length.
DGK-FP：5’-GGAACAAGACACGGCTGGGTT-3’(SEQ ID NO.10)；DGK-FP: 5'-GGAACAAGACACGGCTGGGTT-3' (SEQ ID NO. 10);
DGK-RP：5’-AGCAAGGCAGGGCAGGCAAGT-3’(SEQ ID NO.11)。DGK-RP: 5'-AGCAAGGCAGGGGCAGGCAAGT-3' (SEQ ID NO. 11).
bHLH转录因子基因(bHLH transcription factor，bHLH TF)，基因长度为100bp，bHLH transcription factor gene (bHLH transcription factor, bHLH TF), the gene length is 100bp,
bHLH TF-FP：5’-GTCCTTCCCCCGCTGGAAAC-3’(SEQ ID NO.12)；bHLH TF-FP: 5'-GTCCTTCCCCCGCTGGAAAC-3' (SEQ ID NO. 12);
bHLH TF-RP：5’-GCAGCAGAGATCATCGCGCC-3’(SEQ ID NO.13)。bHLH TF-RP: 5'-GCAGCAGAGATCATCGCGCC-3' (SEQ ID NO. 13).
基于本发明中的PCR反应缓冲液包含50mmol/L Tris-HCl(pH8.0)、3mmol/LMgCl2、5mmol/L(NH4)2SO4、50mmol/L KCl、0.1mg/L BSA、0.05％Tween-20、1％DMSO、200nmol/L CYP21-FP/CYP21-RP、DGK-FP/DGK-RP、bHLH TF-FP/bHLH TF-RP，200μmol/LdNTPs，0.05U/μL HS Sso-Taq，适量模板，适量的灭菌ddH2O。反应程序为95℃10min；95℃15s、60℃30s、72℃30s，共35个循环。The PCR reaction buffer based on the present invention contains 50mmol/L Tris-HCl (pH8.0), 3mmol/LMgCl 2 , 5mmol/L (NH 4 ) 2 SO 4 , 50mmol/L KCl, 0.1mg/L BSA, 0.05 %Tween-20, 1%DMSO, 200nmol/L CYP21-FP/CYP21-RP, DGK-FP/DGK-RP, bHLH TF-FP/bHLH TF-RP, 200μmol/LdNTPs, 0.05U/μL HS Sso-Taq , an appropriate amount of template, an appropriate amount of sterilized ddH 2 O. The reaction program was 95 °C for 10 min; 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 30 s, a total of 35 cycles.
结果如图2B所示。分析结果可得，本发明的直扩型热启动DNA聚合酶可在40％(v/v)的全血含量的PCR体系中，同时扩增得到三个不同长度的基因片段(320bp、243bp、100bp)。这是Taq DNA聚合酶首次于40％(v/v)的全血中，同时完成三个基因的扩增。The results are shown in Figure 2B. The analysis results show that the direct expansion type hot-start DNA polymerase of the present invention can simultaneously amplify three gene fragments of different lengths (320bp, 243bp, 100bp). This is the first time that Taq DNA polymerase has simultaneously amplified three genes in 40% (v/v) whole blood.
实施例8 HS Sso-Taq的牛奶直扩能力测试Example 8 Test of milk direct expansion ability of HS Sso-Taq
分别以2ng的从鲜牛奶中提纯的基因组DNA及5％、10％、15％、20％(v/v)的鲜牛奶(光明优倍)为模板，用本发明中的直扩型热启动DNA聚合酶以及购买市售品牌Takara的热启动DNA聚合酶HS-Taq同时扩增牛线粒体色素细胞b(Cytochrome,cob)基因，扩增引物，扩增长度为424bp和具体操作如下：Take 2ng of genomic DNA purified from fresh milk and 5%, 10%, 15%, 20% (v/v) fresh milk (bright excellent times) as templates respectively, and use the direct expansion type hot start in the present invention. DNA polymerase and the hot-start DNA polymerase HS-Taq purchased from the commercially available brand Takara simultaneously amplify the bovine mitochondrial pigment cell b (Cytochrome, cob) gene, the amplification primer, the amplification length is 424bp, and the specific operations are as follows:
cob-FP：5’-AAGACGAGAAGACCCTATGGAGCTTTA-3’(SEQ ID NO.14)；cob-FP: 5'-AAGACGAGAAGACCCTATGGAGCTTTA-3' (SEQ ID NO. 14);
cob-RP：5’-GATTGCGCTGTTATCCCTAGGGTA-3’(SEQ ID NO.15)。cob-RP: 5'-GATTGCGCTGTTATCCCTAGGGTA-3' (SEQ ID NO. 15).
基于本发明中的PCR反应缓冲液包含50mmol/L Tris-HCl(pH8.0)、3mmol/LMgCl2、5mmol/L(NH4)2SO4、50mmol/L KCl、0.1mg/L BSA、0.05％Tween-20、1％DMSO、200nmol/Lcob-FP、200nmol/Lcob-RP，200μmol/L dNTPs，0.05U/μL HS Sso-Taq，适量的灭菌ddH2O。反应程序为95℃10min；95℃15s、60℃30s、72℃30s，共35个循环。The PCR reaction buffer based on the present invention contains 50mmol/L Tris-HCl (pH8.0), 3mmol/LMgCl 2 , 5mmol/L(NH 4 ) 2 SO 4 , 50mmol/L KCl, 0.1mg/L BSA, 0.05 %Tween-20, 1% DMSO, 200nmol/Lcob-FP, 200nmol/Lcob-RP, 200μmol/L dNTPs, 0.05U/μL HS Sso-Taq, appropriate amount of sterilized ddH 2 O. The reaction program was 95°C for 10 min; 95°C for 15s, 60°C for 30s, and 72°C for 30s, a total of 35 cycles.
市售品牌Takara热启动DNA聚合酶TaqHS的反应体系包含2.5μL10×PCR Buffer，200nmol/Lcob-FP、200nmol/Lcob-RP，200μmol/L dNTPs，0.05U/μL TaqHS，适量的灭菌ddH2O。反应程序为98℃10s、60℃30s、72℃30s，共35个循环。The reaction system of the commercial brand Takara hot-start DNA polymerase TaqHS contains 2.5μL 10× PCR Buffer, 200nmol/Lcob-FP, 200nmol/Lcob-RP, 200μmol/L dNTPs, 0.05U/μL TaqHS, appropriate amount of sterilized ddH 2 O . The reaction program was 98 °C for 10 s, 60 °C for 30 s, and 72 °C for 30 s, a total of 35 cycles.
结果如图3所示。分析结果可得，本发明的直扩型热启动DNA聚合酶至少可耐受20％的鲜牛奶，并从中扩增得到目的基因。而商品的热启动DNA聚合酶TaqHS只能耐受10％的鲜牛奶。与商品的热启动DNA聚合酶相比，本发明的热启动DNA聚合酶具有更强的直扩能力。The results are shown in Figure 3. The analysis results show that the direct expansion type hot-start DNA polymerase of the present invention can tolerate at least 20% of fresh milk, and the target gene can be obtained by amplification therefrom. The commercial hot-start DNA polymerase TaqHS can only tolerate 10% fresh milk. Compared with the commercial hot-start DNA polymerase, the hot-start DNA polymerase of the present invention has stronger direct amplification ability.
实施例9基于HS Sso-Taq的本发明qPCR反应试剂灵敏度测试
以梯度稀释的重组内参基因U6质粒(U6-pMD 18-T，B2M插入的位置为限制性酶切位点EcorⅤ)为模板，最后一个梯度中重组质粒的拷贝数浓度约为0.2copies/μL，用本发明中的qPCR反应液以及购买市售品牌Takara、Promega同类产品同时扩增U6基因，扩增引物和具体操作如下：Using the gradient dilution of the recombinant internal reference gene U6 plasmid (U6-pMD 18-T, the position of B2M insertion is the restriction enzyme site Ecor V) as the template, the copy number concentration of the recombinant plasmid in the last gradient is about 0.2copies/μL, Amplify U6 gene simultaneously with the qPCR reaction solution of the present invention and the similar products purchased from commercially available brands Takara and Promega. The amplification primers and specific operations are as follows:
U6-FP：5’-GCTCGCTTCGGCAGCACATAT-3’(SEQ ID NO.16)；U6-FP: 5'-GCTCGCTTCGGCAGCACATAT-3' (SEQ ID NO. 16);
U6-RP：5’-CGCTTCACGAATTTGCGTGTC-3’(SEQ ID NO.17)。U6-RP: 5'-CGCTTCACGAATTTGCGTGTC-3' (SEQ ID NO. 17).
基于本发明中的qPCR反应液的反应体系包含0.05U/μL HS Sso-Taq、200μmol/LdNTPs、50mmol/L Tris-HCl(pH8.0)、3mmol/L MgCl2、5mmol/L(NH4)2SO4、50mmol/L KCl、0.1mg/L BSA、0.05％Tween-20、4％DMSO、0.4×SYBR Green I、250nmol/L U6-FP、250nmol/L U6-RP，1μL模板，适量的灭菌ddH2O，反应总体积为20μL。反应程序为95℃，10min；95℃5s、60℃20s、read plate，共40个循环；读取熔解曲线。The reaction system based on the qPCR reaction solution in the present invention includes 0.05U/μL HS Sso-Taq, 200 μmol/LdNTPs, 50mmol/L Tris-HCl (pH8.0), 3mmol/L MgCl 2 , 5mmol/L (NH 4 ) 2 SO 4 , 50mmol/L KCl, 0.1mg/L BSA, 0.05%Tween-20, 4%DMSO, 0.4×SYBR Green I, 250nmol/L U6-FP, 250nmol/L U6-RP, 1μL template, appropriate amount Sterilize ddH2O, and the total reaction volume is 20 μL. The reaction program was 95°C, 10 min; 95°C for 5s, 60°C for 20s, read plate, a total of 40 cycles; read the melting curve.
市售品牌Takara同类试剂TB
市售品牌Promega同类试剂
结果如图4所示。分析结果可得，本发明的qPCR反应试剂可准确检测出浓度为0.2copies/μL的靶基因，具有优良的灵敏度，且扩增效率接近100％，梯度间的线性关系为0.999可进行准确的定量及微量核酸检测。和市售的Promega(A600A)、Takara(RR820)相比具有更高的灵敏度及扩增效率。The results are shown in Figure 4. The analysis results show that the qPCR reaction reagent of the present invention can accurately detect the target gene with a concentration of 0.2 copies/μL, has excellent sensitivity, and the amplification efficiency is close to 100%, and the linear relationship between the gradients is 0.999, which can be accurately quantified and trace nucleic acid detection. Compared with commercially available Promega (A600A) and Takara (RR820), it has higher sensitivity and amplification efficiency.
实施例10 HS Sso-Taq在qPCR检测中耐受抑制剂性能研究Example 10 Study on the resistance of HS Sso-Taq to inhibitors in qPCR detection
以1ng重组内参基因B2M质粒(购买得到，B2M-pMD 18-T，B2M插入的位置为限制性酶切位点EcorⅤ)为模板，用等量的HS wt-Taq(热启动野生型Taq DNA聚合酶)及HS Sso-Taq，在不同抑制剂存在的情况下去扩增B2M基因，扩增引物为：Using 1 ng of recombinant internal reference gene B2M plasmid (purchased, B2M-pMD 18-T, the position of B2M insertion is the restriction enzyme site EcorⅤ) as the template, with the same amount of HS wt-Taq (hot-start wild-type Taq DNA polymerization enzyme) and HS Sso-Taq to amplify the B2M gene in the presence of different inhibitors, and the amplification primers are:
B2M-FP：5’-CTATCCAGCGTACTCCAAAG-3’(SEQ ID NO.18)，B2M-FP: 5'-CTATCCAGCGTACTCCAAAG-3' (SEQ ID NO. 18),
B2M-RP：5’-GAAAGACCAGTCCTTGCTGA-3’(SEQ ID NO.19)。B2M-RP: 5'-GAAAGACCAGTCCTTGCTGA-3' (SEQ ID NO. 19).
各种抑制剂测试qPCR反应体系均包含以下通用成分：0.05U/μL HS wt-Taq/HSSso-Taq、200nmol/L B2M-FP、200nmol/L B2M-RP、200μmol/L dNTPs、50mmol/L Tris-HCl、2mmol/L MgCl2、5mmol/L(NH4)2SO4、30～80mmol/L KCl、0.1mg/L BSA、0.05％Tween-20、3％DMSO、0.3mol/L海藻糖、0.2mol/L L-肉毒碱、0.4％NP40，反应体系的pH为8.0。Various inhibitor test qPCR reaction systems contain the following general components: 0.05U/μL HS wt-Taq/HSSso-Taq, 200nmol/L B2M-FP, 200nmol/L B2M-RP, 200μmol/L dNTPs, 50mmol/L Tris -HCl, 2mmol/L MgCl 2 , 5mmol/L (NH 4 ) 2 SO 4 , 30～80mmol/L KCl, 0.1mg/L BSA, 0.05%Tween-20, 3%DMSO, 0.3mol/L trehalose, 0.2mol/L L-carnitine, 0.4% NP40, the pH of the reaction system was 8.0.
(1)耐受SYBR GreenΙ性能测试(1) Resistance to SYBR GreenΙ performance test
在上述的通用组分中再分别加入反应终浓度为0ng/μL、0.5ng/μL、1.0ng/μL、2.0ng/μL、4.0ng/μL、8.0ng/μL、10.0ng/μL的SYBR GreenΙ，反应程序均为95℃10min；95℃5s、60℃20s、read plate，共40个循环；读取熔解曲线。结果如图5所示，从结果可以看出HSwt-Taq可耐受最高SYBR GreenΙ浓度为1.0ng/μL，HS Sso-Taq至少可耐受反应浓度为10.0ng/μL的高浓度染料，相对于HS wt-Taq，HS Sso-Taq具有更强的SYBR GreenΙ耐受能力。SYBR GreenI with final concentrations of 0ng/μL, 0.5ng/μL, 1.0ng/μL, 2.0ng/μL, 4.0ng/μL, 8.0ng/μL and 10.0ng/μL was added to the above general components respectively. , the reaction program is 95 ℃ 10min; 95
(2)耐受腐殖酸性能测试(2) Test of humic acid tolerance
在上述的通用组分中分别加入反应终浓度为0.4×SYBR GreenΙ(HS wt-Taq)、0.8×SYBR GreenΙ(HS Sso-Taq)，再分别添加终浓度为0μg/mL、0.1μg/mL、0.2μg/mL、0.4μg/mL、0.8μg/mL、1.6μg/mL的腐殖酸，反应程序均为95℃10min；95℃5s、60℃20s、read plate，共45个循环；读取熔解曲线。结果如图6所示，从结果可以看出，HS Sso-Taq可耐受反应终浓度为1.6μg/mL的腐殖酸，大大提升了野生型Taq DNA聚合酶抗腐殖酸抑制能力。In the above-mentioned general components, the final concentrations of 0.4×SYBR Green1 (HS wt-Taq) and 0.8×SYBR Green1 (HS Sso-Taq) were added respectively, and the final concentrations were 0 μg/mL, 0.1 μg/mL, 0.2μg/mL, 0.4μg/mL, 0.8μg/mL, 1.6μg/mL of humic acid, the reaction program is 95℃ 10min; 95
(3)耐受多糖性能测试(3) Tolerance polysaccharide performance test
在上述的通用组分中分别加入反应终浓度为0.4×SYBR GreenΙ(HS wt-Taq)、0.8×SYBR GreenΙ(HS Sso-Taq)，再分别添加终浓度为0mg/mL、0.02mg/mL、0.04mg/mL、0.08mg/mL、0.16mg/mL、0.32mg/mL的黄芪多糖，反应程序均为95℃10min；95℃5s、60℃20s、read plate，共45个循环；读取熔解曲线。结果如图7所示，从结果可以看出HS Sso-Taq可耐受高至反应浓度为0.32mg/mL的黄芪多糖，野生型Taq DNA聚合酶只能耐受反应浓度为0.08mg/mL的黄芪多糖。In the above-mentioned general components, the final concentrations of 0.4×SYBR Green1 (HS wt-Taq) and 0.8×SYBR Green1 (HS Sso-Taq) were added respectively, and the final concentrations were 0mg/mL, 0.02mg/mL, Astragalus polysaccharides of 0.04mg/mL, 0.08mg/mL, 0.16mg/mL, 0.32mg/mL, the reaction procedures are all 95°C 10min; 95°
(4)耐受免疫球蛋白IgG性能测试(4) Tolerance immunoglobulin IgG performance test
在上述的通用组分中分别加入反应终浓度为0.4×SYBR GreenΙ(HS wt-Taq)、0.8×SYBR GreenΙ(HS Sso-Taq)，再分别添加终浓度为0μg/mL、75μg/mL、150μg/mL、300μg/mL、600μg/mL的免疫球蛋白IgG，反应程序均为95℃，10min；95℃5s，60℃20s，read plate；共45个循环；读取熔解曲线。结果如图8所示，从结果可以看出HS Sso-Taq可耐受高至反应浓度为600μg/mL的免疫球蛋白IgG，野生型Taq DNA聚合酶几乎不具备抗IgG抑制的性能。In the above-mentioned general components, the final concentrations of 0.4×SYBR Green1 (HS wt-Taq) and 0.8×SYBR Green1 (HS Sso-Taq) were respectively added, and the final concentrations were 0 μg/mL, 75 μg/mL, 150 μg respectively. /mL, 300μg/mL, 600μg/mL of immunoglobulin IgG, the reaction program is 95℃, 10min; 95℃ for 5s, 60℃ for 20s, read plate; a total of 45 cycles; read the melting curve. The results are shown in Figure 8. It can be seen from the results that HS Sso-Taq can tolerate immunoglobulin IgG up to a reaction concentration of 600 μg/mL, and wild-type Taq DNA polymerase has almost no anti-IgG inhibitory performance.
综上所述，本发明的热启动DNA聚合酶具有优异的直扩能力，更强的耐qPCR抑制剂能力，适用于粗样的直接荧光定量PCR。To sum up, the hot-start DNA polymerase of the present invention has excellent direct amplification ability, stronger resistance to qPCR inhibitors, and is suitable for direct fluorescent quantitative PCR of crude samples.
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受所述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。The above-mentioned embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the described embodiments, and any other changes, modifications, substitutions, and combinations made without departing from the spirit and principle of the present invention , simplification, all should be equivalent replacement modes, and are all included in the protection scope of the present invention.
序列表 sequence listing
<110> 华南理工大学<110> South China University of Technology
<120> 一种直扩型热启动DNA聚合酶及其制备方法与应用<120> A kind of direct expansion type hot start DNA polymerase and its preparation method and application
<160> 19<160> 19
<170> SIPOSequenceListing 1.0<170> SIPOSequenceListing 1.0
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<212> PRT<212> PRT
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<220><220>
<223> 直扩型热启动DNA聚合酶氨基酸序列<223> Direct expansion hot-start DNA polymerase amino acid sequence
<400> 1<400> 1
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val ProMet Gly Ser Ser His His His His His His Ser Ser Ser Gly Leu Val Pro
1 5 10 151 5 10 15
Arg Gly Ser His Met Ala Thr Val Lys Phe Lys Tyr Lys Gly Glu GluArg Gly Ser His Met Ala Thr Val Lys Phe Lys Tyr Lys Gly Glu Glu
20 25 30 20 25 30
Lys Glu Val Asp Ile Ser Lys Ile Lys Lys Val Trp Arg Val Gly LysLys Glu Val Asp Ile Ser Lys Ile Lys Lys Val Trp Arg Val Gly Lys
35 40 45 35 40 45
Met Ile Ser Phe Thr Tyr Asp Glu Gly Gly Gly Lys Thr Gly Arg GlyMet Ile Ser Phe Thr Tyr Asp Glu Gly Gly Gly Lys Thr Gly Arg Gly
50 55 60 50 55 60
Ala Val Ser Glu Lys Asp Ala Pro Lys Glu Leu Leu Gln Met Leu GluAla Val Ser Glu Lys Asp Ala Pro Lys Glu Leu Leu Gln Met Leu Glu
65 70 75 8065 70 75 80
Lys Gln Lys Lys Gly Gly Val Thr Ser Pro Lys Ala Leu Glu Glu AlaLys Gln Lys Lys Gly Gly Val Thr Ser Pro Lys Ala Leu Glu Glu Ala
85 90 95 85 90 95
Pro Trp Pro Pro Pro Glu Gly Ala Phe Val Gly Phe Val Leu Ser ArgPro Trp Pro Pro Pro Glu Gly Ala Phe Val Gly Phe Val Leu Ser Arg
100 105 110 100 105 110
Lys Glu Pro Met Trp Ala Asp Leu Leu Ala Leu Ala Ala Ala Arg GlyLys Glu Pro Met Trp Ala Asp Leu Leu Ala Leu Ala Ala Ala Arg Gly
115 120 125 115 120 125
Gly Arg Val His Arg Ala Pro Glu Pro Tyr Lys Ala Leu Arg Asp LeuGly Arg Val His Arg Ala Pro Glu Pro Tyr Lys Ala Leu Arg Asp Leu
130 135 140 130 135 140
Lys Glu Ala Arg Gly Leu Leu Ala Lys Asp Leu Ser Val Leu Ala LeuLys Glu Ala Arg Gly Leu Leu Ala Lys Asp Leu Ser Val Leu Ala Leu
145 150 155 160145 150 155 160
Arg Glu Gly Leu Gly Leu Pro Pro Gly Asp Asp Pro Met Leu Leu AlaArg Glu Gly Leu Gly Leu Pro Pro Gly Asp Asp Pro Met Leu Leu Ala
165 170 175 165 170 175
Tyr Leu Leu Asp Pro Ser Asn Thr Thr Pro Glu Gly Val Ala Arg ArgTyr Leu Leu Asp Pro Ser Asn Thr Thr Pro Glu Gly Val Ala Arg Arg
180 185 190 180 185 190
Tyr Gly Gly Glu Trp Thr Glu Glu Ala Gly Glu Arg Ala Ala Leu SerTyr Gly Gly Glu Trp Thr Glu Glu Ala Gly Glu Arg Ala Ala Leu Ser
195 200 205 195 200 205
Glu Arg Leu Phe Ala Asn Leu Trp Gly Arg Leu Glu Gly Glu Glu ArgGlu Arg Leu Phe Ala Asn Leu Trp Gly Arg Leu Glu Gly Glu Glu Arg
210 215 220 210 215 220
Leu Leu Trp Leu Tyr Arg Glu Val Glu Arg Pro Leu Ser Ala Val LeuLeu Leu Trp Leu Tyr Arg Glu Val Glu Arg Pro Leu Ser Ala Val Leu
225 230 235 240225 230 235 240
Ala His Met Glu Ala Thr Gly Val Arg Leu Asp Val Ala Tyr Leu ArgAla His Met Glu Ala Thr Gly Val Arg Leu Asp Val Ala Tyr Leu Arg
245 250 255 245 250 255
Ala Leu Ser Leu Glu Val Ala Glu Glu Ile Ala Arg Leu Glu Ala GluAla Leu Ser Leu Glu Val Ala Glu Glu Ile Ala Arg Leu Glu Ala Glu
260 265 270 260 265 270
Val Phe Arg Leu Ala Gly His Pro Phe Asn Leu Asn Ser Arg Asp GlnVal Phe Arg Leu Ala Gly His Pro Phe Asn Leu Asn Ser Arg Asp Gln
275 280 285 275 280 285
Leu Glu Arg Val Leu Phe Asp Glu Leu Gly Leu Pro Ala Ile Gly LysLeu Glu Arg Val Leu Phe Asp Glu Leu Gly Leu Pro Ala Ile Gly Lys
290 295 300 290 295 300
Thr Glu Lys Thr Gly Lys Arg Ser Thr Ser Ala Ala Val Leu Glu AlaThr Glu Lys Thr Gly Lys Arg Ser Thr Ser Ala Ala Val Leu Glu Ala
305 310 315 320305 310 315 320
Leu Arg Glu Ala His Pro Ile Val Glu Lys Ile Leu Gln Tyr Arg GluLeu Arg Glu Ala His Pro Ile Val Glu Lys Ile Leu Gln Tyr Arg Glu
325 330 335 325 330 335
Leu Thr Lys Leu Lys Ser Thr Tyr Ile Asp Pro Leu Pro Asp Leu IleLeu Thr Lys Leu Lys Ser Thr Tyr Ile Asp Pro Leu Pro Asp Leu Ile
340 345 350 340 345 350
His Pro Arg Thr Gly Arg Leu His Thr Arg Phe Asn Gln Thr Ala ThrHis Pro Arg Thr Gly Arg Leu His Thr Arg Phe Asn Gln Thr Ala Thr
355 360 365 355 360 365
Ala Thr Gly Arg Leu Ser Ser Ser Asp Pro Asn Leu Gln Asn Ile ProAla Thr Gly Arg Leu Ser Ser Ser Asp Pro Asn Leu Gln Asn Ile Pro
370 375 380 370 375 380
Val Arg Thr Pro Leu Gly Gln Arg Ile Arg Arg Ala Phe Ile Ala GluVal Arg Thr Pro Leu Gly Gln Arg Ile Arg Arg Ala Phe Ile Ala Glu
385 390 395 400385 390 395 400
Glu Gly Trp Leu Leu Val Ala Leu Asp Tyr Ser Gln Ile Glu Leu ArgGlu Gly Trp Leu Leu Val Ala Leu Asp Tyr Ser Gln Ile Glu Leu Arg
405 410 415 405 410 415
Val Leu Ala His Leu Ser Gly Asp Arg Asn Leu Ile Arg Val Phe GlnVal Leu Ala His Leu Ser Gly Asp Arg Asn Leu Ile Arg Val Phe Gln
420 425 430 420 425 430
Glu Gly Arg Asp Ile His Thr Glu Thr Ala Ser Trp Met Phe Gly ValGlu Gly Arg Asp Ile His Thr Glu Thr Ala Ser Trp Met Phe Gly Val
435 440 445 435 440 445
Pro Arg Glu Ala Val Asp Pro Leu Met Arg Arg Ala Ala Lys Thr IlePro Arg Glu Ala Val Asp Pro Leu Met Arg Arg Ala Ala Lys Thr Ile
450 455 460 450 455 460
Asn Phe Gly Val Leu Tyr Gly Met Ser Ala His Arg Leu Ser Gln GluAsn Phe Gly Val Leu Tyr Gly Met Ser Ala His Arg Leu Ser Gln Glu
465 470 475 480465 470 475 480
Leu Ala Ile Pro Tyr Glu Glu Ala Gln Ala Phe Ile Glu Arg Tyr PheLeu Ala Ile Pro Tyr Glu Glu Ala Gln Ala Phe Ile Glu Arg Tyr Phe
485 490 495 485 490 495
Gln Ser Phe Pro Lys Val Arg Ala Trp Leu Arg Lys Thr Leu Glu GluGln Ser Phe Pro Lys Val Arg Ala Trp Leu Arg Lys Thr Leu Glu Glu
500 505 510 500 505 510
Gly Arg Arg Arg Gly Tyr Val Glu Thr Leu Phe Gly Arg Arg Arg TyrGly Arg Arg Arg Gly Tyr Val Glu Thr Leu Phe Gly Arg Arg Arg Tyr
515 520 525 515 520 525
Val Pro Asp Leu Glu Ala Arg Val Lys Ser Val Arg Glu Ala Ala GluVal Pro Asp Leu Glu Ala Arg Val Lys Ser Val Arg Glu Ala Ala Glu
530 535 540 530 535 540
Arg Met Ala Phe Asn Met Pro Val Gln Gly Thr Ala Ala Asp Leu MetArg Met Ala Phe Asn Met Pro Val Gln Gly Thr Ala Ala Asp Leu Met
545 550 555 560545 550 555 560
Lys Leu Ala Met Val Lys Leu Phe Pro Arg Leu Glu Glu Met Gly AlaLys Leu Ala Met Val Lys Leu Phe Pro Arg Leu Glu Glu Met Gly Ala
565 570 575 565 570 575
Arg Met Leu Leu Gln Val His Asp Glu Leu Val Leu Glu Ala Pro LysArg Met Leu Leu Gln Val His Asp Glu Leu Val Leu Glu Ala Pro Lys
580 585 590 580 585 590
Glu Arg Ala Glu Ala Val Ala Arg Leu Ala Lys Glu Val Met Glu GlyGlu Arg Ala Glu Ala Val Ala Arg Leu Ala Lys Glu Val Met Glu Gly
595 600 605 595 600 605
Val Tyr Pro Leu Ala Val Pro Leu Glu Val Glu Val Gly Ile Gly GluVal Tyr Pro Leu Ala Val Pro Leu Glu Val Glu Val Gly Ile Gly Glu
610 615 620 610 615 620
Asp Trp Leu Ser Ala Lys GluAsp Trp Leu Ser Ala Lys Glu
625 630625 630
<210> 2<210> 2
<211> 20<211> 20
<212> PRT<212> PRT
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<220><220>
<223> Sso7d氨基酸序列<223> Sso7d amino acid sequence
<400> 2<400> 2
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val ProMet Gly Ser Ser His His His His His His Ser Ser Ser Gly Leu Val Pro
1 5 10 151 5 10 15
Arg Gly Ser HisArg Gly Ser His
20 20
<210> 3<210> 3
<211> 1893<211> 1893
<212> DNA<212> DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<220><220>
<223> 直扩型热启动DNA聚合酶核苷酸序列<223> Nucleotide sequence of direct expansion hot-start DNA polymerase
<400> 3<400> 3
atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60
atggcgacgg ttaaattcaa gtataagggc gaggagaaag aagtagatat tagcaagatt 120atggcgacgg ttaaattcaa gtataagggc gaggagaaag aagtagatat tagcaagatt 120
aagaaagtgt ggcgcgtagg caaaatgatt tctttcacct atgatgaagg aggtggcaaa 180aagaaagtgt ggcgcgtagg caaaatgatt tctttcacct atgatgaagg aggtggcaaa 180
accggtcgtg gggcggtgtc agaaaaggat gcgccgaagg aacttcttca aatgctggag 240accggtcgtg gggcggtgtc agaaaaggat gcgccgaagg aacttcttca aatgctggag 240
aaacagaaga agggcggagt gaccagcccg aaagcccttg aggaggcacc atggccaccg 300aaacagaaga agggcggagt gaccagcccg aaagcccttg aggaggcacc atggccaccg 300
cctgaaggtg cgtttgtggg gtttgtgctg agccgtaagg aaccgatgtg ggccgatctg 360cctgaaggtg cgtttgtggg gtttgtgctg agccgtaagg aaccgatgtg ggccgatctg 360
ttggcattgg cagccgcacg tggtggccgc gtgcatcgcg ctccagaacc gtacaaagcg 420ttggcattgg cagccgcacg tggtggccgc gtgcatcgcg ctccagaacc gtacaaagcg 420
ctgcgggatc tgaaagaggc tcgtggatta ctggcgaagg atcttagcgt gcttgctctt 480ctgcgggatc tgaaagaggc tcgtggatta ctggcgaagg atcttagcgt gcttgctctt 480
cgcgagggcc tgggtcttcc acctggtgac gatccgatgc ttcttgcata tctgctggat 540cgcgagggcc tgggtcttcc acctggtgac gatccgatgc ttcttgcata tctgctggat 540
ccgagcaaca ccaccccgga gggcgtggct cgtcggtatg gaggtgaatg gaccgaagaa 600ccgagcaaca ccaccccgga gggcgtggct cgtcggtatg gaggtgaatg gaccgaagaa 600
gccggagaac gtgccgcgtt aagcgagcgg ctgtttgcga atctttgggg tcgtcttgag 660gccggagaac gtgccgcgtt aagcgagcgg ctgtttgcga atctttgggg tcgtcttgag 660
ggggaagagc gtctgctgtg gctttatcgt gaagtcgagc gtccgcttag tgcagtgtta 720ggggaagagc gtctgctgtg gctttatcgt gaagtcgagc gtccgcttag tgcagtgtta 720
gcgcacatgg aggccacggg tgttcgttta gacgtggcgt acctgcgtgc gttgagcctg 780gcgcacatgg aggccacggg tgttcgttta gacgtggcgt acctgcgtgc gttgagcctg 780
gaagtggcgg aggaaatagc gcggttggag gcggaagttt ttcgcttggc gggtcaccct 840gaagtggcgg aggaaatagc gcggttggag gcggaagttt ttcgcttggc gggtcaccct 840
tttaatctga atagccgtga tcagctggaa cgtgtcctgt tcgatgaact gggcctgccg 900tttaatctga atagccgtga tcagctggaa cgtgtcctgt tcgatgaact gggcctgccg 900
gccattggga agacagagaa aacaggcaaa cgttcaacca gcgcggcagt gcttgaagct 960gccattggga agacagagaa aacaggcaaa cgttcaacca gcgcggcagt gcttgaagct 960
ttacgtgagg cccacccaat tgtggaaaaa atcctgcagt atcgcgaact gaccaaactg 1020ttacgtgagg cccacccaat tgtggaaaaa atcctgcagt atcgcgaact gaccaaactg 1020
aaaagcacct acattgatcc gctgccggac ctgattcacc cgcgtaccgg acgcctgcat 1080aaaagcacct acattgatcc gctgccggac ctgattcacc cgcgtaccgg acgcctgcat 1080
acccgtttca atcagaccgc gaccgccaca ggccggttga gtagcagcga tcccaactta 1140acccgtttca atcagaccgc gaccgccaca ggccggttga gtagcagcga tcccaactta 1140
cagaacattc cggtgcgtac cccgttaggc cagcgcattc gccgtgcctt tattgcggaa 1200cagaacattc cggtgcgtac cccgttaggc cagcgcattc gccgtgcctt tattgcggaa 1200
gagggctggc tgcttgtggc acttgattat agccaaattg aactgcgtgt gttggctcac 1260gagggctggc tgcttgtggc acttgattat agccaaattg aactgcgtgt gttggctcac 1260
ctgtctggcg atcggaacct tatccgggta tttcaggagg gacgtgatat tcatacagaa 1320ctgtctggcg atcggaacct tatccgggta tttcaggagg gacgtgatat tcatacagaa 1320
accgcgagct ggatgttcgg tgtaccgcgc gaagcagtag atcctctgat gcggcgtgct 1380accgcgagct ggatgttcgg tgtaccgcgc gaagcagtag atcctctgat gcggcgtgct 1380
gctaagacaa tcaattttgg cgtgttatat ggcatgtccg cccatcgtct gagtcaagaa 1440gctaagacaa tcaattttgg cgtgttatat ggcatgtccg cccatcgtct gagtcaagaa 1440
ttggcaatac cgtacgaaga ggcgcaggca tttatcgagc ggtattttca gagttttccg 1500ttggcaatac cgtacgaaga ggcgcaggca tttatcgagc ggtattttca gagttttccg 1500
aaagttcggg catggctgcg taaaaccctg gaggaaggtc gccgtcgggg ctacgttgag 1560aaagttcggg catggctgcg taaaaccctg gaggaaggtc gccgtcgggg ctacgttgag 1560
actctgtttg ggcgtcggcg ttatgttccg gatctggaag cgcgtgtgaa atccgtgcgg 1620actctgtttg ggcgtcggcg ttatgttccg gatctggaag cgcgtgtgaa atccgtgcgg 1620
gaagcagctg aacgcatggc ctttaacatg cctgtgcagg ggaccgccgc tgatctgatg 1680gaagcagctg aacgcatggc ctttaacatg cctgtgcagg ggaccgccgc tgatctgatg 1680
aaactggcta tggtgaaact tttcccacgc ttggaggaga tgggggctcg tatgttgctg 1740aaactggcta tggtgaaact tttcccacgc ttggaggaga tgggggctcg tatgttgctg 1740
caggtccatg acgaactggt tttagaagct ccaaaggaac gcgcagaggc agttgcgcgc 1800caggtccatg acgaactggt tttagaagct ccaaaggaac gcgcagaggc agttgcgcgc 1800
ctggctaagg aagttatgga aggtgtgtat ccgttagcgg tgccgctgga ggttgaagtg 1860ctggctaagg aagttatgga aggtgtgtat ccgttagcgg tgccgctgga ggttgaagtg 1860
ggcataggag aggattggct gagtgcaaaa gaa 1893ggcataggag aggattggct gagtgcaaaa gaa 1893
<210> 4<210> 4
<211> 29<211> 29
<212> DNA<212> DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<220><220>
<223> Taq -FP<223> Taq-FP
<400> 4<400> 4
ggcatatggc gaccgttaag tttaagtac 29ggcatatggc gaccgttaag tttaagtac 29
<210> 5<210> 5
<211> 26<211> 26
<212> DNA<212> DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<220><220>
<223> Taq -RP<223> Taq-RP
<400> 5<400> 5
ccatgaattc ttattccttc gcagat 26ccatgaattc ttattccttc gcagat 26
<210> 6<210> 6
<211> 28<211> 28
<212> DNA<212> DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<220><220>
<223> pET-28a-FP<223> pET-28a-FP
<400> 6<400> 6
ggaataagaa ttcatggttg cggccgca 28ggaataagaa ttcatggttg cggccgca 28
<210> 7<210> 7
<211> 27<211> 27
<212> DNA<212> DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<220><220>
<223> pET-28a-RP<223> pET-28a-RP
<400> 7<400> 7
acggtcgcca tatgccgcgc ggcacca 27acggtcgcca tatgccgcgc ggcacca 27
<210> 8<210> 8
<211> 22<211> 22
<212> DNA<212> DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<220><220>
<223> CYP21-FP<223> CYP21-FP
<400> 8<400> 8
gctcagcatg gtggtggcat aa 22gctcagcatg gtggtggcat aa 22
<210> 9<210> 9
<211> 22<211> 22
<212> DNA<212> DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<220><220>
<223> CYP21-RP<223> CYP21-RP
<400> 9<400> 9
cctcatacct tcccccccat tt 22cctcatacct tcccccccat tt 22
<210> 10<210> 10
<211> 21<211> 21
<212> DNA<212> DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<220><220>
<223> DGK -FP<223> DGK-FP
<400> 10<400> 10
ggaacaagac acggctgggt t 21ggaacaagac acggctgggt t 21
<210> 11<210> 11
<211> 21<211> 21
<212> DNA<212> DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<220><220>
<223> DGK -RP<223> DGK-RP
<400> 11<400> 11
agcaaggcag ggcaggcaag t 21agcaaggcag ggcaggcaag t 21
<210> 12<210> 12
<211> 20<211> 20
<212> DNA<212> DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<220><220>
<223> bHLH TF -FP<223> bHLH TF-FP
<400> 12<400> 12
gtccttcccc cgctggaaac 20
<210> 13<210> 13
<211> 20<211> 20
<212> DNA<212> DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<220> <220>
<223> bHLH TF -RP<223>bHLHTF-RP
<400> 13<400> 13
gcagcagaga tcatcgcgcc 20
<210> 14<210> 14
<211> 27<211> 27
<212> DNA<212> DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<220><220>
<223> cob-FP<223> cob-FP
<400> 14<400> 14
aagacgagaa gaccctatgg agcttta 27aagacgagaa gaccctatgg agcttta 27
<210> 15<210> 15
<211> 24<211> 24
<212> DNA<212> DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<220><220>
<223> cob-RP<223> cob-RP
<400> 15<400> 15
gattgcgctg ttatccctag ggta 24gattgcgctg ttatccctag ggta 24
<210> 16<210> 16
<211> 21<211> 21
<212> DNA<212> DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<220><220>
<223> U6-FP<223> U6-FP
<400> 16<400> 16
gctcgcttcg gcagcacata t 21gctcgcttcg gcagcacata t 21
<210> 17<210> 17
<211> 21<211> 21
<212> DNA<212> DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<220> <220>
<223> U6-RP<223> U6-RP
<400> 17<400> 17
cgcttcacga atttgcgtgt c 21cgcttcacga atttgcgtgt c 21
<210> 18<210> 18
<211> 20<211> 20
<212> DNA<212> DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<220> <220>
<223> B2M-FP<223> B2M-FP
<400> 18<400> 18
ctatccagcg tactccaaag 20
<210> 19<210> 19
<211> 20<211> 20
<212> DNA<212> DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<220><220>
<223> B2M-RP<223> B2M-RP
<400> 19<400> 19
gaaagaccag tccttgctga 20
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| CN113621585A (en) * | 2021-08-18 | 2021-11-09 | 华南理工大学 | Taq DNA polymerase and endonuclease chimera as well as preparation method and application thereof |
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