CN112779238B - 用于丙肝病毒检测的dna聚合酶混合物 - Google Patents
用于丙肝病毒检测的dna聚合酶混合物 Download PDFInfo
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- CN112779238B CN112779238B CN202011577482.9A CN202011577482A CN112779238B CN 112779238 B CN112779238 B CN 112779238B CN 202011577482 A CN202011577482 A CN 202011577482A CN 112779238 B CN112779238 B CN 112779238B
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Abstract
本发明提供了一种用于丙肝病毒检测的DNA聚合酶混合物，具体地本发明通过将野生型DNA聚合酶和突变型DNA聚合酶突变体6混合，获得了更高的PCR扩增效率，表现出了显著的协同效果。
Description
技术领域
本发明属于生物技术领域，具体地说，本发明涉及一种用于丙肝病毒检测的DNA聚合酶混合物。
背景技术
Taq酶是一种来源于耐热性细菌Thermus aquaticus的耐热性DNA聚合酶，分子量94KDa，在镁离子存在的条件下，其最适反应温度为75-80℃，在95℃下的活性半衰期为40分钟，具有5’-3’核酸外切酶活性。由于其具有耐高温的特性，因此广泛用于聚合酶链式反应(PCR)，是核酸扩增、检测等反应的首选用酶。商品化Taq酶使用大肠杆菌原核表达系统进行克隆及表达。
现代分子生物检测技术对PCR反应的灵敏度、精确度、耐用性要求越来越高，野生型Taq酶无法完全满足实际应用的需求。为使其更加适应特定技术的使用，对Taq酶序列的突变改造进行了很多的尝试，如：
添加DNA结合结构域使其具有更强的延伸活性(Wang Y(2004).A novel strategyto engineer DNA polymerases for enhanced processivity and improvedperformance in vitro.Nucleic Acids Res 32,1197–1207))；
通过定点突变、删除结构域使其具有更高的保真性(Suzuki M,Yoshida S,AdmanET,Blank A,Loeb LA(2000)Thermus Aquaticus DNA polymerase I mutants withaltered fidelity.Interacting mutations in the O-Helix.J Biol Chem 275:32728–32735)、更高的DNA聚合活性(Mutant Taq DNA polymerases with improved elongationability as a useful reagent for genetic engineering.Front Microbiol 5:461.doi:10.3389/fmicb.2014.00461)、耐受高浓度抑制剂(Zhang Z,Kermekchiev MB,Barnes WM(2010)Direct DNA amplification from crude clinical samples using aPCR enhancer cocktail and novel mutants of Taq.J Mol Diagn 12:152–161)、降低5’-3’核酸外切酶活性(Vainshtein I,Atrazhev A,Eom SH,Elliott JF,Wishart DS,Malcolm BA(1996)Peptide rescue of an N-Terminal truncation of the Stoffelfragment of Taq DNA polymerase.Protein Sci 5:51785–51792)。
本领域技术人员致力于开发扩增效率更高的DNA聚合酶，以满足现代分子生物检测技术的需要。
发明内容
本发明的目的在于提供一种用于丙肝病毒检测的DNA聚合酶混合物。
在本发明的第一方面，提供了一种DNA聚合酶混合物，所述DNA聚合酶混合物包括野生型DNA聚合酶和突变型DNA聚合酶；其中，所述野生型DNA聚合酶的氨基酸序列如SEQ IDNO.:2所示；
所述突变型DNA聚合酶为突变体6：在SEQ ID NO.:2所示的野生型DNA聚合酶基础上进行突变，并且所述突变体6具有如下突变位点：H785G。
在另一优选例中，所述突变体6的突变位点为：H785G。
在另一优选例中，所述DNA聚合酶混合物中野生型DNA聚合酶和突变型DNA聚合酶的酶活比例约为1～9：9：1；优选地约为2～8：8：2；更优选地约为3～7：7：3。
本发明的第二方面，提供了一种试剂盒，所述试剂盒包含本发明第一方面所述的DNA聚合酶混合物。
在另一优选例中，所述试剂盒还包含PCR缓冲液。
在另一优选例中，所述试剂盒还包含逆转录酶、和/或dNTP。
本发明的第三方面，提供了一种PCR扩增方法，所述方法包括步骤：
(1)提供待检测对象的核酸样本；
(2)制备PCR反应体系并进行PCR反应：
其中，所述步骤(2)中，使用本发明第一方面所述的DNA聚合酶混合物催化底物dNTP分子聚合形成子代DNA。
在另一优选例中，所述核酸样本中包含丙肝病毒cDNA。
在另一优选例中，所述方法为非诊断目的的方法，比如可以对环境样本或者实验室动物或培养细胞样本进行检测。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
具体实施方式
本发明人通过广泛而深入的研究，意外发现野生型DNA聚合酶和DNA聚合酶突变体6的混合物，针对丙肝病毒具有更高的PCR扩增效率，表现出了显著的协同效果。在此基础上，完成了本发明。
在描述本发明之前，应当理解本发明不限于所述的具体方法和实验条件，因为这类方法和条件可以变动。还应当理解本文所用的术语其目的仅在于描述具体实施方案，并且不意图是限制性的，本发明的范围将仅由所附的权利要求书限制。
除非另外定义，否则本文中所用的全部技术与科学术语均具有如本发明所属领域的普通技术人员通常理解的相同含义。如本文所用，在提到具体列举的数值中使用时，术语“约”意指该值可以从列举的值变动不多于1％。例如，如本文所用，表述“约100”包括99和101和之间的全部值(例如，99.1、99.2、99.3、99.4等)。
虽然在本发明的实施或测试中可以使用与本发明中所述相似或等价的任何方法和材料，本文在此处例举优选的方法和材料。
Taq酶
Taq酶广泛用于聚合酶链式反应(PCR)，是核酸扩增、检测等反应的首选用酶。商品化Taq酶使用大肠杆菌原核表达系统进行克隆及表达。
野生型Taq酶DNA序列如下：
ATGCGTGGCATGCTGCCGCTTTTCGAGCCTAAGGGACGCGTTCTTCTTGTGGATGGACATCATCTGGCGTACCGTACCTTTCATGCCCTGAAGGGCCTGACCACTTCGCGTGGGGAACCCGTGCAAGCAGTTTATGGATTCGCCAAATCGTTACTTAAGGCTCTGAAGGAGGATGGTGATGCGGTCATTGTTGTGTTCGACGCAAAAGCTCCCTCGTTCCGTCACGAGGCCTACGGCGGCTATAAAGCTGGGCGTGCACCCACACCTGAGGATTTTCCCCGGCAACTTGCTTTGATAAAGGAATTAGTAGACCTGTTAGGCCTGGCGCGGTTAGAAGTGCCGGGTTACGAAGCAGATGACGTCTTGGCTAGTTTAGCGAAAAAGGCTGAAAAAGAGGGATATGAAGTGCGGATCCTGACCGCGGATAAAGATCTGTATCAACTGTTGTCCGACCGTATTCACGTGCTTCATCCGGAGGGCTACTTGATAACCCCGGCTTGGCTGTGGGAGAAATATGGGCTGCGTCCAGATCAGTGGGCTGATTATCGTGCACTTACAGGCGATGAATCTGATAATCTTCCCGGCGTCAAGGGGATTGGTGAGAAAACCGCCCGTAAACTTTTGGAGGAGTGGGGCAGCTTGGAGGCGCTGTTGAAGAATCTGGATCGTTTGAAACCCGCTATACGGGAAAAAATCTTGGCGCACATGGACGACTTAAAACTGTCTTGGGACCTGGCGAAAGTTCGTACTGATTTGCCGCTGGAGGTCGACTTTGCGAAGCGTCGCGAGCCCGATCGTGAACGTCTTCGCGCATTTCTGGAGCGTTTAGAATTTGGCTCCCTGTTGCATGAGTTTGGTTTGCTTGAAAGCCCGAAGGCACTTGAGGAAGCTCCTTGGCCTCCGCCTGAGGGCGCTTTTGTCGGATTTGTCTTGAGCCGTAAAGAACCGATGTGGGCGGACTTACTGGCCCTTGCTGCTGCTCGTGGGGGTCGCGTGCATCGCGCACCGGAGCCATACAAAGCACTTCGTGACCTTAAAGAAGCCCGTGGCTTGTTGGCAAAAGATTTAAGTGTCCTGGCTTTACGCGAGGGCTTGGGCTTACCACCGGGAGATGATCCGATGCTTTTGGCCTATCTGCTGGACCCGAGCAACACGACTCCAGAGGGCGTTGCCCGTCGTTATGGCGGAGAATGGACGGAGGAGGCGGGAGAGCGCGCAGCGTTAAGCGAGCGTCTGTTTGCTAATCTGTGGGGACGCTTAGAGGGAGAGGAGCGCCTGTTGTGGTTGTACCGTGAAGTGGAACGGCCGCTGAGTGCAGTGTTAGCTCACATGGAAGCAACCGGGGTGCGGCTGGACGTTGCGTATTTGCGTGCGCTGTCGTTAGAGGTCGCGGAGGAAATAGCCCGTCTGGAGGCCGAAGTATTCCGTTTGGCTGGCCATCCTTTCAACCTGAACAGTCGGGATCAGCTGGAACGTGTACTTTTTGATGAACTGGGGCTGCCCGCCATCGGCAAAACCGAAAAAACCGGCAAACGTAGCACCTCTGCGGCAGTGCTGGAAGCGTTACGTGAAGCTCATCCGATTGTGGAGAAAATTCTGCAATATCGCGAATTGACGAAACTGAAGAGCACCTATATTGATCCGCTGCCAGACTTAATTCACCCCCGTACCGGACGGTTGCATACCCGCTTCAACCAGACCGCGACGGCGACAGGGCGGCTGAGTAGCAGCGATCCGAACCTGCAAAACATTCCCGTGCGTACCCCGCTGGGTCAGCGTATTCGCCGTGCTTTCATTGCCGAGGAAGGCTGGCTGCTGGTCGCGCTGGACTACTCGCAAATCGAATTGCGTGTGTTGGCCCACCTGTCGGGCGACGAAAACTTAATACGCGTGTTTCAAGAAGGTCGTGACATACATACTGAAACCGCGTCCTGGATGTTTGGAGTCCCACGGGAGGCTGTCGATCCTCTTATGCGTCGTGCCGCCAAAACAATTAACTTCGGAGTTCTGTACGGCATGTCGGCACATCGTTTATCACAGGAACTGGCGATTCCGTATGAAGAAGCGCAGGCCTTCATAGAACGTTATTTCCAATCATTCCCCAAGGTGCGGGCCTGGATTGAGAAGACCCTGGAAGAGGGCCGTCGTCGTGGCTATGTAGAGACTCTGTTCGGACGTCGGCGGTATGTACCCGATCTTGAGGCCCGTGTGAAGTCCGTTCGTGAGGCAGCAGAACGTATGGCGTTTAACATGCCAGTCCAGGGCACAGCGGCGGACCTGATGAAATTAGCTATGGTTAAGCTGTTTCCGCGTTTGGAAGAAATGGGCGCTCGTATGCTGTTACAGGTTCATGACGAGTTAGTATTAGAAGCACCGAAGGAGCGTGCCGAAGCCGTGGCCCGGTTAGCCAAAGAGGTAATGGAAGGCGTCTACCCCCTTGCAGTCCCGCTTGAAGTCGAAGTTGGCATAGGGGAAGACTGGTTATCTGCGAAGGAA(SEQID NO.:1)
野生型Taq酶氨基酸序列如下：
MRGMLPLFEPKGRVLLVDGHHLAYRTFHALKGLTTSRGEPVQAVYGFAKSLLKALKEDGDAVIVVFDAKAPSFRHEAYGGYKAGRAPTPEDFPRQLALIKELVDLLGLARLEVPGYEADDVLASLAKKAEKEGYEVRILTADKDLYQLLSDRIHVLHPEGYLITPAWLWEKYGLRPDQWADYRALTGDESDNLPGVKGIGEKTARKLLEEWGSLEALLKNLDRLKPAIREKILAHMDDLKLSWDLAKVRTDLPLEVDFAKRREPDRERLRAFLERLEFGSLLHEFGLLESPKALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALRDLKEARGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSERLFANLWGRLEGEERLLWLYREVERPLSAVLAHMEATGVRLDVAYLRALSLEVAEEIARLEAEVFRLAGHPFNLNSRDQLERVLFDELGLPAIGKTEKTGKRSTSAAVLEALREAHPIVEKILQYRELTKLKSTYIDPLPDLIHPRTGRLHTRFNQTATATGRLSSSDPNLQNIPVRTPLGQRIRRAFIAEEGWLLVALDYSQIELRVLAHLSGDENLIRVFQEGRDIHTETASWMFGVPREAVDPLMRRAAKTINFGVLYGMSAHRLSQELAIPYEEAQAFIERYFQSFPKVRAWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKSVREAAERMAFNMPVQGTAADLMKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGIGEDWLSAKE(SEQ ID NO.:2)
本领域的普通技术人员可以使用的常规方法获得本发明的Taq酶基因序列，例如全人工合成或PCR法合成。一种优选的合成法为不对称PCR法。不对称PCR法是用不等量的一对引物，PCR扩增后产生大量的单链DNA(ssDNA)。这对引物分别称为非限制引物与限制性引物，其比例一般为50-100∶1。在PCR反应的最初10-15个循环中，其扩增产物主要是双链DNA，但当限制性引物(低浓度引物)消耗完后，非限制性引物(高浓度引物)引导的PCR就会产生大量的单链DNA。用于PCR的引物可根据本文所公开的本发明的序列信息适当地选择，并可用常规方法合成。可用常规方法如通过凝胶电泳分离和纯化扩增的DNA/RNA片段。
本发明的Taq酶可以通过常规的重组DNA技术进行表达或生产，包括步骤：
(1)用编码本发明蛋白的多核苷酸，或用含有该多核苷酸的重组表达载体转化或转导合适的宿主细胞；
(2)在合适的培养基中培养宿主细胞；
(3)从培养基或细胞中分离、纯化目的蛋白质，从而获得Taq酶。
本领域的技术人员熟知的方法能用于构建含本发明Taq酶的编码DNA序列和合适的转录/翻译控制信号的表达载体，优选市售的载体：pET28。这些方法包括体外重组DNA技术、DNA合成技术、体内重组技术等。所述的DNA序列可有效连接到表达载体中的适当启动子上，以指导mRNA合成。表达载体还包括翻译起始用的核糖体结合位点和转录终止子。此外，表达载体优选包含一个或多个选择性标记基因，以提供用于选择转化的宿主细胞的表型性状。
所述重组载体在5'到3'方向上包括：启动子，目的基因和终止子。如果需要，所述重组载体还可以包括以下元件：蛋白纯化标签；3'多聚核苷酸化信号；非翻译核酸序列；转运和靶向核酸序列；选择标记(抗生素抗性基因、荧光蛋白等)；增强子；或操作子。
用于制备重组载体的方法是本领域普通技术人员所熟知的。表达载体可以是细菌质粒、噬菌体、酵母质粒、植物细胞病毒、哺乳动物细胞病毒或其他载体。总之，只要其能够在宿主体内复制和稳定，任何质粒和载体都可以被采用。
本领域普通技术人员可以采用熟知的方法构建含有本发明启动子和/或目的基因序列的载体。这些方法包括体外重组DNA技术、DNA合成技术、体内重组技术等。
本发明的表达载体，可以用于转化适当的宿主细胞，以使宿主转录目的RNA或表达目的蛋白质。宿主细胞可以是原核细胞，如大肠杆菌、谷氨酸棒杆菌、黄色短杆菌、链霉菌属、农杆菌：或是低等真核细胞，如酵母细胞；或是高等真核细胞，如植物细胞。本领域一般技术人员都清楚如何选择适当的载体和宿主细胞。用重组DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。当宿主为原核生物(如大肠杆菌)时，可以用CaCl2法处理，也可用电穿孔法进行。当宿主是真核生物，可选用如下的DNA转染方法：磷酸钙共沉淀法，常规机械方法(如显微注射、电穿孔、脂质体包装等)。转化植物也可使用农杆菌转化或基因枪转化等方法，例如叶盘法、幼胚转化法、花芽浸泡法等。对于转化的植物细胞、组织或器官可以用常规方法再生成植株，从而获得转基因的植物。
术语“可操作连接”是指将准备转录表达的目的基因以一种本领域的常规方式连接到它的控制序列以被表达。
工程菌的培养和目的蛋白发酵生产
在获得工程细胞后，便可在适合的条件下培养工程细胞，表达本发明的基因序列所编码的蛋白。根据宿主细胞的不同，培养中所用的培养基可选自各种常规培养基，在适于宿主细胞生长的条件下进行培养。当宿主细胞生长到适当的细胞密度后，用合适的方法(如温度转换或化学诱导)诱导选择的启动子，将细胞再培养一段时间。
在本发明中，可采用常规的发酵条件。代表性的条件包括(但并不限于)：
(a)就温度而言，Taq酶的发酵及诱导温度保持在25-37℃；
(b)就诱导期的pH值而言，诱导期pH控制在3-9；
(c)就溶氧(DO)而言，DO控制在10-90％，溶氧的维持可以用氧气/空气混合气体的通入来解决；
(d)就补料而言，补料种类宜包括甘油、甲醇、葡萄糖等碳源，可单独补料或混合补料；
(e)就诱导期IPTG浓度而言，常规诱导浓度都可用于本发明，通常IPTG浓度控制在0.1-1.5mM；
(f)就诱导时间而言，没有特别限制，通常为2-20小时，较佳地为5-15小时。
本发明的目的蛋白Taq酶存在大肠杆菌细胞胞内，通过离心机收集宿主细胞，然后通过高压、机器力、酶解细胞被或其他细胞破碎方法破碎宿主细胞，释放重组蛋白，优选的是高压法。宿主细胞裂解液可通过絮凝、盐析、超滤等方法进行初步纯化后再进行层析、超滤等纯化，也可直接进行层析纯化。
层析技术包括阳离子交换层析、阴离子交换层析、凝胶过滤层析、疏水层析、亲和层析等技术。常用的层析方法包括：
1.阴离子交换层析：
阴离子交换层析介质包括(但不限于)：Q-Sepharose、DEAE-Sepharose。如果发酵样品的盐浓度较高，影响与离子交换介质的结合，则在进行离子交换层析前需降低盐浓度。样品可以用稀释、超滤、透析、凝胶过滤层析等手段进行平衡缓冲液的更换，直至与对应的离子交换柱平衡液系统相似，然后上样，进行盐浓度或pH的梯度洗脱。
2.疏水层析：
疏水层析介质包括(但不限于)：Phenyl-Sepharose、Butyl-Sepharose、Octyle-Sepharose。样品通过添加NaCl、(NH4)2SO4等方式提高盐浓度，然后上样，通过降低盐浓度方法洗脱。通过疏水层析除去疏水性有较大差异的杂蛋白。
3.凝胶过滤层析
疏水层析介质包括(但不限于)：Sephacryl、Superdex、Sephadex类。通过凝胶过滤层析更换缓冲体系，或进一步精纯。
4.亲和层析
亲和层析介质包括(但不限于)：HiTrapTMHeparinHPColumns。
5.膜过滤
超滤介质包括：有机膜如聚砜膜、无机膜如陶瓷膜、金属膜类。通过膜过滤可以达到纯化和浓缩的目的。
本发明的主要优点在于：
本发明的DNA聚合酶混合物，具有显著更高的PCR扩增效率，因此，能够显著提高丙肝病毒的检测效率和检测准确性。
下面结合具体实施例，进一步详陈本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明详细条件的实验方法，通常按照常规条件如美国Sambrook.J等著《分子克隆实验室指南》(黄培堂等译，北京：科学出版社，2002年)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。以下实施例中所用的实验材料和试剂如无特别说明均可从市售渠道获得。
本发明实施例引用专利文献CN201910083410.X中的Taq酶突变体制备及制备方法，引用如下。
实施例1：Taq酶随机突变质粒的构建
用低保真度PCR(Error-PCR)扩增Taq酶的聚合酶活性结构域DNA序列(423－831位氨基酸编码序列)，突变发生率为0.3％，然后与Taq酶的其余编码序列(1－423位氨基酸序列)连接，克隆至pET28a原核表达载体中，得到Taq酶随机突变质粒。具体步骤如下：
1)以Taq-pET28a质粒为模板，设计引物T(1－423)扩增Taq(1－423)片段。
Taq(1-423)DNA Seq
ATGCGTGGCATGCTGCCGCTTTTCGAGCCTAAGGGACGCGTTCTTCTTGTGGATGGACATCATCTGGCGTACCGTACCTTTCATGCCCTGAAGGGCCTGACCACTTCGCGTGGGGAACCCGTGCAAGCAGTTTATGGATTCGCCAAATCGTTACTTAAGGCTCTGAAGGAGGATGGTGATGCGGTCATTGTTGTGTTCGACGCAAAAGCTCCCTCGTTCCGTCACGAGGCCTACGGCGGCTATAAAGCTGGGCGTGCACCCACACCTGAGGATTTTCCCCGGCAACTTGCTTTGATAAAGGAATTAGTAGACCTGTTAGGCCTGGCGCGGTTAGAAGTGCCGGGTTACGAAGCAGATGACGTCTTGGCTAGTTTAGCGAAAAAGGCTGAAAAAGAGGGATATGAAGTGCGGATCCTGACCGCGGATAAAGATCTGTATCAACTGTTGTCCGACCGTATTCACGTGCTTCATCCGGAGGGCTACTTGATAACCCCGGCTTGGCTGTGGGAGAAATATGGGCTGCGTCCAGATCAGTGGGCTGATTATCGTGCACTTACAGGCGATGAATCTGATAATCTTCCCGGCGTCAAGGGGATTGGTGAGAAAACCGCCCGTAAACTTTTGGAGGAGTGGGGCAGCTTGGAGGCGCTGTTGAAGAATCTGGATCGTTTGAAACCCGCTATACGGGAAAAAATCTTGGCGCACATGGACGACTTAAAACTGTCTTGGGACCTGGCGAAAGTTCGTACTGATTTGCCGCTGGAGGTCGACTTTGCGAAGCGTCGCGAGCCCGATCGTGAACGTCTTCGCGCATTTCTGGAGCGTTTAGAATTTGGCTCCCTGTTGCATGAGTTTGGTTTGCTTGAAAGCCCGAAGGCACTTGAGGAAGCTCCTTGGCCTCCGCCTGAGGGCGCTTTTGTCGGATTTGTCTTGAGCCGTAAAGAACCGATGTGGGCGGACTTACTGGCCCTTGCTGCTGCTCGTGGGGGTCGCGTGCATCGCGCACCGGAGCCATACAAAGCACTTCGTGACCTTAAAGAAGCCCGTGGCTTGTTGGCAAAAGATTTAAGTGTCCTGGCTTTACGCGAGGGCTTGGGCTTACCACCGGGAGATGATCCGATGCTTTTGGCCTATCTGCTGGACCCGAGCAACACGACTCCAGAGGGCGTTGCCCGTCGTTATGGCGGAGAATGGACGGAGGAGGCGGGAGAGCGCGCAGCGTTAAGCGAGCGTCTGTTTGCTAATCTGTGGGGACGCTTAGAGGGAGAG(SEQ ID NO.:3)
T1-423_PF：5'ATATCATATGCGTGGCATGCTGCCGCTTTT 3'(SEQ ID NO.:4)
T1-423_PR：5'GCATGAATTCCGTCTCCTCTCCCTCTAAGC 3'(SEQ ID NO.:5)
PCR反应体系及程序：
PCR程序：95℃3分钟，(95℃30秒，60℃30秒，72℃1分钟)×25个循环，72℃3分钟，4℃保存
PCR产物用DNA胶回收试剂盒纯化回收，用NdeI及XhoI酶切，连接到pET28a载体，测序确认序列正确，得到的质粒命名为Taq(1-423)-pET28
2)以Taq-pET28a质粒为模板，使用ClontechPCR RandomMutagenesis Kit(大连宝生物PT3393-2)，设计引物(TMu_F/R)扩增Taq(423-822)片段
Taq(423-832)DNA Seq
GGAGAGGAGCGCCTGTTGTGGTTGTACCGTGAAGTGGAACGGCCGCTGAGTGCAGTGTTAGCTCACATGGAAGCAACCGGGGTGCGGCTGGACGTTGCGTATTTGCGTGCGCTGTCGTTAGAGGTCGCGGAGGAAATAGCCCGTCTGGAGGCCGAAGTATTCCGTTTGGCTGGCCATCCTTTCAACCTGAACAGTCGGGATCAGCTGGAACGTGTACTTTTTGATGAACTGGGGCTGCCCGCCATCGGCAAAACCGAAAAAACCGGCAAACGTAGCACCTCTGCGGCAGTGCTGGAAGCGTTACGTGAAGCTCATCCGATTGTGGAGAAAATTCTGCAATATCGCGAATTGACGAAACTGAAGAGCACCTATATTGATCCGCTGCCAGACTTAATTCACCCCCGTACCGGACGGTTGCATACCCGCTTCAACCAGACCGCGACGGCGACAGGGCGGCTGAGTAGCAGCGATCCGAACCTGCAAAACATTCCCGTGCGTACCCCGCTGGGTCAGCGTATTCGCCGTGCTTTCATTGCCGAGGAAGGCTGGCTGCTGGTCGCGCTGGACTACTCGCAAATCGAATTGCGTGTGTTGGCCCACCTGTCGGGCGACGAAAACTTAATACGCGTGTTTCAAGAAGGTCGTGACATACATACTGAAACCGCGTCCTGGATGTTTGGAGTCCCACGGGAGGCTGTCGATCCTCTTATGCGTCGTGCCGCCAAAACAATTAACTTCGGAGTTCTGTACGGCATGTCGGCACATCGTTTATCACAGGAACTGGCGATTCCGTATGAAGAAGCGCAGGCCTTCATAGAACGTTATTTCCAATCATTCCCCAAGGTGCGGGCCTGGATTGAGAAGACCCTGGAAGAGGGCCGTCGTCGTGGCTATGTAGAGACTCTGTTCGGACGTCGGCGGTATGTACCCGATCTTGAGGCCCGTGTGAAGTCCGTTCGTGAGGCAGCAGAACGTATGGCGTTTAACATGCCAGTCCAGGGCACAGCGGCGGACCTGATGAAATTAGCTATGGTTAAGCTGTTTCCGCGTTTGGAAGAAATGGGCGCTCGTATGCTGTTACAGGTTCATGACGAGTTAGTATTAGAAGCACCGAAGGAGCGTGCCGAAGCCGTGGCCCGGTTAGCCAAAGAGGTAATGGAAGGCGTCTACCCCCTTGCAGTCCCGCTTGAAGTCGAAGTTGGCATAGGGGAAGACTGGTTATCTGCGAAGGAATAA(SEQ ID NO.:6)
TMu_F：5'GGAGAGGAGCGCCTGTTGTGGTTGT 3'(SEQ ID NO.:7)
TMu_R：5'TTATTCCTTCGCAGATAACCAGTCT 3'(SEQ ID NO.:8)
PCR反应体系及程序：
95℃3分钟,(95℃30秒,60℃30秒，68℃2分钟)X25次循环，68℃5分钟，4℃保存
PCR产物用BsmBI及XhoI酶切，然后连接经BsmBI及XhoI酶切的Taq(1-423)-pET28质粒，连接产物转化BL21(DE3)表达宿主菌，统计转化子数量。
实施例2：Taq酶突变体的表达及定向进化筛选
Taq酶突变质粒转化BL21(DE3)表达菌株，诱导表达出Taq酶突变库。将含有Taq酶突变库的BL21(DE3)诱导表达菌用乳液PCR体系分散包裹，进行PCR反应，扩增出含有Taq酶突变片段的DNA。然后把乳液PCR扩增出来的DNA片段用Taq酶特异引物进行高保真PCR二次扩增，将扩增的DNA产物重新克隆至pET28a表达载体中，完成一次筛选过程。之后重复进行乳液PCR-二次高保真PCR-克隆至pET28a表达载体的筛选过程，同时逐步缩短每次筛选中乳液PCR的延伸时间，使具有高延伸活性和高扩增活性的突变体群体得到累积。具体步骤如下：
1)取实施例1转化得到的转化子，接种至LB培养基中，37℃震荡培养6小时，加入终浓度0.1mM的异丙基硫代半乳糖苷(IPTG)，37℃诱导培养3小时。离心收集菌体，用ddH2O洗涤菌体两次，最后用ddH2O重悬菌体，测定菌体溶液在600nm处的光吸收值(OD600值)，用ddH2O将终浓度稀释至OD600＝1.0
2)配制油相溶液
Tween-80 200ul
Triton X-100 25ul
Mineral oil 10ml
以上3种试剂合并，混合均匀
3)水相反应液的配制
将步骤1)配制好的OD600＝1.0的菌体重悬液，用ddH2O稀释100倍，配制以下反应液
10XTaq酶反应液(100mM Tris，500mM KCl，100mM(NH4)2SO4，pH8.0)13ul
pET28_F引物：TACGGTTAACCCTTTGAATCA(SEQ ID NO.:9)
pET28_R引物：GTTACCTGGTTAAACTGTACT(SEQ ID NO.:10)
4)乳液体系的制备
取200ul水相+400ul油相在2ml管中混合，在漩涡振荡器上高速振荡10分钟，取5个PCR管，每个分装100ul混合液，P℃R程序：95℃5分钟，(95℃30秒、55℃30秒、72℃2分钟)X25次循环、72℃5分钟，4℃∞
5)将乳液PCR产物转移至1.5ml管，加入166ul水饱和乙醚，漩涡振荡30秒，12000rpm离心10分钟，转移走下层液相，室温静置10分钟待乙醚挥发，采用酚氯仿法抽提纯化液相产物，然后乙醇沉淀过夜回收产物。
6)高保真PCR二次扩增产物
以步骤4)产物为模板，进行PCR二次扩增
PCR程序如下：95℃5分钟，20次循环X(95℃30秒、62℃30秒、72℃2分钟)72℃5分钟、4℃∞
Taq_F引物：ATGCGTGGCATGCTGCCGCTTTTCGAGCCTAAGGGACG(SEQ ID NO.:11)
Taq_R引物：TTCCTTCGCAGATAACCAGTCTTCCCCTATGCCAACTTCGAC(SEQ ID NO.:12)
7)PCR产物用DNA产物纯化回收试剂盒纯化，然后重新连接pET28a表达载体。至此完成一轮筛选
8)将重新连接pET28a载体的转化子重复步骤(1)-(6)，按下表的程序改变乳液PCR的条件，对突变库逐步添加选择压力
第二轮筛选：95℃5分钟，(95℃30秒、55℃30秒、72℃1.5分钟)X 25次循环、72℃5分钟，4℃∞
第三轮筛选：95℃5分钟，(95℃30秒、55℃30秒、72℃1分钟)X 20次循环、72℃5分钟，4℃∞
第四轮筛选：95℃5分钟，(95℃30秒、55℃30秒、72℃30秒)X 15次循环、72℃5分钟，4℃∞
经4轮筛选后，所得的Taq酶突变体转化子进入实施例3的高通量筛选
实施例3：Taq酶突变体的高通量筛选
从实施例2得到的突变库中随机挑取384个单克隆，经培养及诱导表达后，用高通量PCR反应测试其扩增活性，从中挑选出20个具有高扩增活性的突变体。具体步骤如下：
1)挑取384个单克隆，接种到LB培养基中，37℃培养6小时，加入终浓度0.1mM的异丙基硫代半乳糖苷(IPTG)，37℃诱导培养3小时
2)离心收集诱导培养后菌体，加入含0.1mg/ml溶菌酶的裂解液(50Mm Tris，50MmNaCl，5％甘油pH8.5)，重悬菌体，37℃孵育10分钟，75℃加热30分钟。然后12000rpm离心10分钟，取上清液。
3)取96孔PCR板，每孔加入以下反应组分
PCR程序：95℃5分钟，20次循环X(95℃30秒、62℃30秒、72℃60秒)、4C∞
PCR产物取5ul进行琼脂糖凝胶电泳，对比各单克隆制备的上清液的PCR产物的产量，选出产量最高20个单克隆。各突变体的扩增产量是野生型扩增产量的1.2倍制2倍。
实施例4：优势Taq酶突变体突变位点的确认
对实施例3选出的Taq酶突变体进行的DNA序列测序，确定其氨基酸序列的突变情况，统计高频突变位点及其突变的形式。
表1
| 突变体编号 | 突变氨基酸 |
| 1 | E507A、K508L、E734E、F749K |
| 2 | K508L、V453A、R737K |
| 3 | E734G |
| 4 | F749G、K508L、L764K |
| 5 | E507Q、T757S |
| 6 | H785G |
| 7 | S624T、F749V |
| 8 | E734F、F749V |
| 9 | K508L、R737W、Y672R |
| 10 | E507H、H785L |
| 11 | A518Q、E734M |
| 12 | F495R、F749T |
| 13 | K508L、F749T、E734F |
| 14 | R737P、S624K |
| 15 | T757W、V453G、E507M |
| 16 | F749E、H785G、F495G |
| 17 | E734F、Y672P |
| 18 | T509L、H785K |
| 19 | E734G、T757S、L764Q |
| 20 | K508L、V453A、A518Q |
对扩增活性较好的20个突变体进行测序，统计其氨基酸突变情况如上表，可见：V453、F495、E507、K508、T509、A518、S624、Y672、E734、R737、F749、T757、L764、H785在20个突变体高频重复出现，证明其突变对Taq酶的扩增活性有显著的影响。
实施例5 Taq酶突变体与野生型Taq酶的混合测试
分别将上述制备的Taq酶突变体与野生型Taq酶按设定比例混合，而后进行性能测试。
以下列出了部分代表性的混合酶及测试结果。
表2
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配制的混合酶，每个混合酶的浓度均为10U/ul。按抗体活力：Taq酶活＝1：1的比例加入抗Taq酶抗体(深圳菲鹏生物技术有限公司提供)，37℃孵育30分钟，-20℃保存。
使用丙肝病毒检测试剂盒(PCR-荧光探针法)(中山大学达安基因股份有限公司，国械注准20153402100)，用以上27种混合酶按以下方法配制反应液B替代试剂盒中的反应液B，以野生型及对应的Taq突变酶为阴性对照组。
混合酶或阴性对照 5U/反应
逆转录酶 200U/反应
按照试剂盒的使用方法配制反应体系如下：
反应液A 17ul
反应液B 3ul
阳参样品 40ul
反应条件：55℃15分钟，95℃2分钟，(95℃30秒，65℃1分钟读取荧光)×40个循环，野生型Taq酶Ct值为38.21，扩增Ct值如下表：
表3
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从以上结果可见，当Taq突变酶6与野生型Taq酶混合后，表现出了较为优异的PCR扩增效率的提高；其它Taq突变酶与野生型Taq酶的混合，没有表现出对PCR扩增效率的提高。特别是，当Taq突变酶6与野生型Taq酶以6：4比例混合后，其对HCV阳参扩增的性能比单独的野生型Taq和Taq突变体都显著提高，取得了预料不到的技术效果。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
序列表
<110> 中山大学达安基因股份有限公司
<120> 用于丙肝病毒检测的DNA聚合酶混合物
<130> 020081
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2496
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 1
atgcgtggca tgctgccgct tttcgagcct aagggacgcg ttcttcttgt ggatggacat 60
catctggcgt accgtacctt tcatgccctg aagggcctga ccacttcgcg tggggaaccc 120
gtgcaagcag tttatggatt cgccaaatcg ttacttaagg ctctgaagga ggatggtgat 180
gcggtcattg ttgtgttcga cgcaaaagct ccctcgttcc gtcacgaggc ctacggcggc 240
tataaagctg ggcgtgcacc cacacctgag gattttcccc ggcaacttgc tttgataaag 300
gaattagtag acctgttagg cctggcgcgg ttagaagtgc cgggttacga agcagatgac 360
gtcttggcta gtttagcgaa aaaggctgaa aaagagggat atgaagtgcg gatcctgacc 420
gcggataaag atctgtatca actgttgtcc gaccgtattc acgtgcttca tccggagggc 480
tacttgataa ccccggcttg gctgtgggag aaatatgggc tgcgtccaga tcagtgggct 540
gattatcgtg cacttacagg cgatgaatct gataatcttc ccggcgtcaa ggggattggt 600
gagaaaaccg cccgtaaact tttggaggag tggggcagct tggaggcgct gttgaagaat 660
ctggatcgtt tgaaacccgc tatacgggaa aaaatcttgg cgcacatgga cgacttaaaa 720
ctgtcttggg acctggcgaa agttcgtact gatttgccgc tggaggtcga ctttgcgaag 780
cgtcgcgagc ccgatcgtga acgtcttcgc gcatttctgg agcgtttaga atttggctcc 840
ctgttgcatg agtttggttt gcttgaaagc ccgaaggcac ttgaggaagc tccttggcct 900
ccgcctgagg gcgcttttgt cggatttgtc ttgagccgta aagaaccgat gtgggcggac 960
ttactggccc ttgctgctgc tcgtgggggt cgcgtgcatc gcgcaccgga gccatacaaa 1020
gcacttcgtg accttaaaga agcccgtggc ttgttggcaa aagatttaag tgtcctggct 1080
ttacgcgagg gcttgggctt accaccggga gatgatccga tgcttttggc ctatctgctg 1140
gacccgagca acacgactcc agagggcgtt gcccgtcgtt atggcggaga atggacggag 1200
gaggcgggag agcgcgcagc gttaagcgag cgtctgtttg ctaatctgtg gggacgctta 1260
gagggagagg agcgcctgtt gtggttgtac cgtgaagtgg aacggccgct gagtgcagtg 1320
ttagctcaca tggaagcaac cggggtgcgg ctggacgttg cgtatttgcg tgcgctgtcg 1380
ttagaggtcg cggaggaaat agcccgtctg gaggccgaag tattccgttt ggctggccat 1440
cctttcaacc tgaacagtcg ggatcagctg gaacgtgtac tttttgatga actggggctg 1500
cccgccatcg gcaaaaccga aaaaaccggc aaacgtagca cctctgcggc agtgctggaa 1560
gcgttacgtg aagctcatcc gattgtggag aaaattctgc aatatcgcga attgacgaaa 1620
ctgaagagca cctatattga tccgctgcca gacttaattc acccccgtac cggacggttg 1680
catacccgct tcaaccagac cgcgacggcg acagggcggc tgagtagcag cgatccgaac 1740
ctgcaaaaca ttcccgtgcg taccccgctg ggtcagcgta ttcgccgtgc tttcattgcc 1800
gaggaaggct ggctgctggt cgcgctggac tactcgcaaa tcgaattgcg tgtgttggcc 1860
cacctgtcgg gcgacgaaaa cttaatacgc gtgtttcaag aaggtcgtga catacatact 1920
gaaaccgcgt cctggatgtt tggagtccca cgggaggctg tcgatcctct tatgcgtcgt 1980
gccgccaaaa caattaactt cggagttctg tacggcatgt cggcacatcg tttatcacag 2040
gaactggcga ttccgtatga agaagcgcag gccttcatag aacgttattt ccaatcattc 2100
cccaaggtgc gggcctggat tgagaagacc ctggaagagg gccgtcgtcg tggctatgta 2160
gagactctgt tcggacgtcg gcggtatgta cccgatcttg aggcccgtgt gaagtccgtt 2220
cgtgaggcag cagaacgtat ggcgtttaac atgccagtcc agggcacagc ggcggacctg 2280
atgaaattag ctatggttaa gctgtttccg cgtttggaag aaatgggcgc tcgtatgctg 2340
ttacaggttc atgacgagtt agtattagaa gcaccgaagg agcgtgccga agccgtggcc 2400
cggttagcca aagaggtaat ggaaggcgtc tacccccttg cagtcccgct tgaagtcgaa 2460
gttggcatag gggaagactg gttatctgcg aaggaa 2496
<210> 2
<211> 832
<212> PRT
<213> 人工序列(Artificial sequence)
<400> 2
Met Arg Gly Met Leu Pro Leu Phe Glu Pro Lys Gly Arg Val Leu Leu
1 5 10 15
Val Asp Gly His His Leu Ala Tyr Arg Thr Phe His Ala Leu Lys Gly
20 25 30
Leu Thr Thr Ser Arg Gly Glu Pro Val Gln Ala Val Tyr Gly Phe Ala
35 40 45
Lys Ser Leu Leu Lys Ala Leu Lys Glu Asp Gly Asp Ala Val Ile Val
50 55 60
Val Phe Asp Ala Lys Ala Pro Ser Phe Arg His Glu Ala Tyr Gly Gly
65 70 75 80
Tyr Lys Ala Gly Arg Ala Pro Thr Pro Glu Asp Phe Pro Arg Gln Leu
85 90 95
Ala Leu Ile Lys Glu Leu Val Asp Leu Leu Gly Leu Ala Arg Leu Glu
100 105 110
Val Pro Gly Tyr Glu Ala Asp Asp Val Leu Ala Ser Leu Ala Lys Lys
115 120 125
Ala Glu Lys Glu Gly Tyr Glu Val Arg Ile Leu Thr Ala Asp Lys Asp
130 135 140
Leu Tyr Gln Leu Leu Ser Asp Arg Ile His Val Leu His Pro Glu Gly
145 150 155 160
Tyr Leu Ile Thr Pro Ala Trp Leu Trp Glu Lys Tyr Gly Leu Arg Pro
165 170 175
Asp Gln Trp Ala Asp Tyr Arg Ala Leu Thr Gly Asp Glu Ser Asp Asn
180 185 190
Leu Pro Gly Val Lys Gly Ile Gly Glu Lys Thr Ala Arg Lys Leu Leu
195 200 205
Glu Glu Trp Gly Ser Leu Glu Ala Leu Leu Lys Asn Leu Asp Arg Leu
210 215 220
Lys Pro Ala Ile Arg Glu Lys Ile Leu Ala His Met Asp Asp Leu Lys
225 230 235 240
Leu Ser Trp Asp Leu Ala Lys Val Arg Thr Asp Leu Pro Leu Glu Val
245 250 255
Asp Phe Ala Lys Arg Arg Glu Pro Asp Arg Glu Arg Leu Arg Ala Phe
260 265 270
Leu Glu Arg Leu Glu Phe Gly Ser Leu Leu His Glu Phe Gly Leu Leu
275 280 285
Glu Ser Pro Lys Ala Leu Glu Glu Ala Pro Trp Pro Pro Pro Glu Gly
290 295 300
Ala Phe Val Gly Phe Val Leu Ser Arg Lys Glu Pro Met Trp Ala Asp
305 310 315 320
Leu Leu Ala Leu Ala Ala Ala Arg Gly Gly Arg Val His Arg Ala Pro
325 330 335
Glu Pro Tyr Lys Ala Leu Arg Asp Leu Lys Glu Ala Arg Gly Leu Leu
340 345 350
Ala Lys Asp Leu Ser Val Leu Ala Leu Arg Glu Gly Leu Gly Leu Pro
355 360 365
Pro Gly Asp Asp Pro Met Leu Leu Ala Tyr Leu Leu Asp Pro Ser Asn
370 375 380
Thr Thr Pro Glu Gly Val Ala Arg Arg Tyr Gly Gly Glu Trp Thr Glu
385 390 395 400
Glu Ala Gly Glu Arg Ala Ala Leu Ser Glu Arg Leu Phe Ala Asn Leu
405 410 415
Trp Gly Arg Leu Glu Gly Glu Glu Arg Leu Leu Trp Leu Tyr Arg Glu
420 425 430
Val Glu Arg Pro Leu Ser Ala Val Leu Ala His Met Glu Ala Thr Gly
435 440 445
Val Arg Leu Asp Val Ala Tyr Leu Arg Ala Leu Ser Leu Glu Val Ala
450 455 460
Glu Glu Ile Ala Arg Leu Glu Ala Glu Val Phe Arg Leu Ala Gly His
465 470 475 480
Pro Phe Asn Leu Asn Ser Arg Asp Gln Leu Glu Arg Val Leu Phe Asp
485 490 495
Glu Leu Gly Leu Pro Ala Ile Gly Lys Thr Glu Lys Thr Gly Lys Arg
500 505 510
Ser Thr Ser Ala Ala Val Leu Glu Ala Leu Arg Glu Ala His Pro Ile
515 520 525
Val Glu Lys Ile Leu Gln Tyr Arg Glu Leu Thr Lys Leu Lys Ser Thr
530 535 540
Tyr Ile Asp Pro Leu Pro Asp Leu Ile His Pro Arg Thr Gly Arg Leu
545 550 555 560
His Thr Arg Phe Asn Gln Thr Ala Thr Ala Thr Gly Arg Leu Ser Ser
565 570 575
Ser Asp Pro Asn Leu Gln Asn Ile Pro Val Arg Thr Pro Leu Gly Gln
580 585 590
Arg Ile Arg Arg Ala Phe Ile Ala Glu Glu Gly Trp Leu Leu Val Ala
595 600 605
Leu Asp Tyr Ser Gln Ile Glu Leu Arg Val Leu Ala His Leu Ser Gly
610 615 620
Asp Glu Asn Leu Ile Arg Val Phe Gln Glu Gly Arg Asp Ile His Thr
625 630 635 640
Glu Thr Ala Ser Trp Met Phe Gly Val Pro Arg Glu Ala Val Asp Pro
645 650 655
Leu Met Arg Arg Ala Ala Lys Thr Ile Asn Phe Gly Val Leu Tyr Gly
660 665 670
Met Ser Ala His Arg Leu Ser Gln Glu Leu Ala Ile Pro Tyr Glu Glu
675 680 685
Ala Gln Ala Phe Ile Glu Arg Tyr Phe Gln Ser Phe Pro Lys Val Arg
690 695 700
Ala Trp Ile Glu Lys Thr Leu Glu Glu Gly Arg Arg Arg Gly Tyr Val
705 710 715 720
Glu Thr Leu Phe Gly Arg Arg Arg Tyr Val Pro Asp Leu Glu Ala Arg
725 730 735
Val Lys Ser Val Arg Glu Ala Ala Glu Arg Met Ala Phe Asn Met Pro
740 745 750
Val Gln Gly Thr Ala Ala Asp Leu Met Lys Leu Ala Met Val Lys Leu
755 760 765
Phe Pro Arg Leu Glu Glu Met Gly Ala Arg Met Leu Leu Gln Val His
770 775 780
Asp Glu Leu Val Leu Glu Ala Pro Lys Glu Arg Ala Glu Ala Val Ala
785 790 795 800
Arg Leu Ala Lys Glu Val Met Glu Gly Val Tyr Pro Leu Ala Val Pro
805 810 815
Leu Glu Val Glu Val Gly Ile Gly Glu Asp Trp Leu Ser Ala Lys Glu
820 825 830
<210> 3
<211> 1269
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 3
atgcgtggca tgctgccgct tttcgagcct aagggacgcg ttcttcttgt ggatggacat 60
catctggcgt accgtacctt tcatgccctg aagggcctga ccacttcgcg tggggaaccc 120
gtgcaagcag tttatggatt cgccaaatcg ttacttaagg ctctgaagga ggatggtgat 180
gcggtcattg ttgtgttcga cgcaaaagct ccctcgttcc gtcacgaggc ctacggcggc 240
tataaagctg ggcgtgcacc cacacctgag gattttcccc ggcaacttgc tttgataaag 300
gaattagtag acctgttagg cctggcgcgg ttagaagtgc cgggttacga agcagatgac 360
gtcttggcta gtttagcgaa aaaggctgaa aaagagggat atgaagtgcg gatcctgacc 420
gcggataaag atctgtatca actgttgtcc gaccgtattc acgtgcttca tccggagggc 480
tacttgataa ccccggcttg gctgtgggag aaatatgggc tgcgtccaga tcagtgggct 540
gattatcgtg cacttacagg cgatgaatct gataatcttc ccggcgtcaa ggggattggt 600
gagaaaaccg cccgtaaact tttggaggag tggggcagct tggaggcgct gttgaagaat 660
ctggatcgtt tgaaacccgc tatacgggaa aaaatcttgg cgcacatgga cgacttaaaa 720
ctgtcttggg acctggcgaa agttcgtact gatttgccgc tggaggtcga ctttgcgaag 780
cgtcgcgagc ccgatcgtga acgtcttcgc gcatttctgg agcgtttaga atttggctcc 840
ctgttgcatg agtttggttt gcttgaaagc ccgaaggcac ttgaggaagc tccttggcct 900
ccgcctgagg gcgcttttgt cggatttgtc ttgagccgta aagaaccgat gtgggcggac 960
ttactggccc ttgctgctgc tcgtgggggt cgcgtgcatc gcgcaccgga gccatacaaa 1020
gcacttcgtg accttaaaga agcccgtggc ttgttggcaa aagatttaag tgtcctggct 1080
ttacgcgagg gcttgggctt accaccggga gatgatccga tgcttttggc ctatctgctg 1140
gacccgagca acacgactcc agagggcgtt gcccgtcgtt atggcggaga atggacggag 1200
gaggcgggag agcgcgcagc gttaagcgag cgtctgtttg ctaatctgtg gggacgctta 1260
gagggagag 1269
<210> 4
<211> 30
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 4
atatcatatg cgtggcatgc tgccgctttt 30
<210> 5
<211> 30
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 5
gcatgaattc cgtctcctct ccctctaagc 30
<210> 6
<211> 1236
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 6
ggagaggagc gcctgttgtg gttgtaccgt gaagtggaac ggccgctgag tgcagtgtta 60
gctcacatgg aagcaaccgg ggtgcggctg gacgttgcgt atttgcgtgc gctgtcgtta 120
gaggtcgcgg aggaaatagc ccgtctggag gccgaagtat tccgtttggc tggccatcct 180
ttcaacctga acagtcggga tcagctggaa cgtgtacttt ttgatgaact ggggctgccc 240
gccatcggca aaaccgaaaa aaccggcaaa cgtagcacct ctgcggcagt gctggaagcg 300
ttacgtgaag ctcatccgat tgtggagaaa attctgcaat atcgcgaatt gacgaaactg 360
aagagcacct atattgatcc gctgccagac ttaattcacc cccgtaccgg acggttgcat 420
acccgcttca accagaccgc gacggcgaca gggcggctga gtagcagcga tccgaacctg 480
caaaacattc ccgtgcgtac cccgctgggt cagcgtattc gccgtgcttt cattgccgag 540
gaaggctggc tgctggtcgc gctggactac tcgcaaatcg aattgcgtgt gttggcccac 600
ctgtcgggcg acgaaaactt aatacgcgtg tttcaagaag gtcgtgacat acatactgaa 660
accgcgtcct ggatgtttgg agtcccacgg gaggctgtcg atcctcttat gcgtcgtgcc 720
gccaaaacaa ttaacttcgg agttctgtac ggcatgtcgg cacatcgttt atcacaggaa 780
ctggcgattc cgtatgaaga agcgcaggcc ttcatagaac gttatttcca atcattcccc 840
aaggtgcggg cctggattga gaagaccctg gaagagggcc gtcgtcgtgg ctatgtagag 900
actctgttcg gacgtcggcg gtatgtaccc gatcttgagg cccgtgtgaa gtccgttcgt 960
gaggcagcag aacgtatggc gtttaacatg ccagtccagg gcacagcggc ggacctgatg 1020
aaattagcta tggttaagct gtttccgcgt ttggaagaaa tgggcgctcg tatgctgtta 1080
caggttcatg acgagttagt attagaagca ccgaaggagc gtgccgaagc cgtggcccgg 1140
ttagccaaag aggtaatgga aggcgtctac ccccttgcag tcccgcttga agtcgaagtt 1200
ggcatagggg aagactggtt atctgcgaag gaataa 1236
<210> 7
<211> 25
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 7
ggagaggagc gcctgttgtg gttgt 25
<210> 8
<211> 25
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 8
ttattccttc gcagataacc agtct 25
Claims (9)
1.一种用于丙肝病毒检测的DNA聚合酶混合物，其特征在于，所述DNA聚合酶混合物由野生型Taq DNA聚合酶和突变型Taq DNA聚合酶组成；其中，所述野生型Taq DNA聚合酶的氨基酸序列如SEQ ID NO.2所示；
所述突变型Taq DNA聚合酶为在SEQ ID NO.2所示的野生型Taq DNA聚合酶基础上进行F495R和F749T突变；
并且所述DNA聚合酶混合物中所述突变型Taq DNA聚合酶和所述野生型Taq DNA聚合酶的酶活比例为6～5：4～5。
2.如权利要求1所述的DNA聚合酶混合物，其特征在于，所述DNA聚合酶混合物中所述突变型Taq DNA聚合酶和所述野生型Taq DNA聚合酶的酶活比例为6：4。
3.如权利要求1所述的DNA聚合酶混合物，其特征在于，所述DNA聚合酶混合物中所述突变型Taq DNA聚合酶和所述野生型Taq DNA聚合酶的酶活比例为5：5。
4.一种用于丙肝病毒检测的试剂盒，其特征在于，所述试剂盒包含权利要求1所述的DNA聚合酶混合物。
5.如权利要求4所述的试剂盒，其特征在于，所述试剂盒还包含PCR缓冲液。
6.如权利要求5所述的试剂盒，其特征在于，所述试剂盒还包含逆转录酶。
7.如权利要求5所述的试剂盒，其特征在于，所述试剂盒还包含dNTP。
8.一种非诊断目的用于丙肝病毒检测的PCR扩增方法，其特征在于，所述方法包括步骤：
（1）提供待检测对象的核酸样本；
（2）制备PCR反应体系并进行PCR反应：
其中，所述步骤（2）中，使用权利要求1所述的DNA聚合酶混合物催化底物dNTP分子聚合形成子代DNA。
9.如权利要求8所述的方法，其特征在于，所述核酸样本中包含丙肝病毒cDNA。
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|---|---|---|---|---|
| US9181534B1 (en) * | 2001-12-21 | 2015-11-10 | Agilent Technologies Inc. | High fidelity DNA polymerase compositions and uses thereof |
| CN108546749A (zh) * | 2018-03-27 | 2018-09-18 | 深圳市艾伟迪生物科技有限公司 | 混合型热启动dna聚合酶组合物、pcr扩增试剂盒及其应用 |
| CN111484987A (zh) * | 2019-01-29 | 2020-08-04 | 中山大学达安基因股份有限公司 | 一种具有高扩增活性的耐热dna聚合酶突变体 |
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|---|---|---|---|---|
| US5556772A (en) * | 1993-12-08 | 1996-09-17 | Stratagene | Polymerase compositions and uses thereof |
| WO2001032887A1 (en) * | 1999-10-29 | 2001-05-10 | Stratagene | Compositions and methods utilizing dna polymerases |
| US7960157B2 (en) * | 2002-12-20 | 2011-06-14 | Agilent Technologies, Inc. | DNA polymerase blends and uses thereof |
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| US9181534B1 (en) * | 2001-12-21 | 2015-11-10 | Agilent Technologies Inc. | High fidelity DNA polymerase compositions and uses thereof |
| CN108546749A (zh) * | 2018-03-27 | 2018-09-18 | 深圳市艾伟迪生物科技有限公司 | 混合型热启动dna聚合酶组合物、pcr扩增试剂盒及其应用 |
| CN111484987A (zh) * | 2019-01-29 | 2020-08-04 | 中山大学达安基因股份有限公司 | 一种具有高扩增活性的耐热dna聚合酶突变体 |
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| GR01 | Patent grant |