CN115261351B - 一种逆转录-聚合双功能酶及其制备方法、应用 - Google Patents
一种逆转录-聚合双功能酶及其制备方法、应用 Download PDFInfo
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Abstract
本发明提供一种逆转录‑聚合双功能酶及其制备方法、应用，涉及基因工程技术领域。该逆转录‑聚合双功能酶通过对野生型Taq DNA聚合酶氨基酸序列进行定向基因突变获得，包括将第583位的天冬酰胺突变为丝氨酸、第587位的精氨酸突变为赖氨酸、第660位的精氨酸突变为天冬氨酸、第667位的苯丙氨酸突变为酪氨酸、第708位的谷氨酸突变为谷氨酰胺、第732位天冬氨酸突变为谷氨酰胺、第742位的谷氨酸突变为精氨酸、第747位的甲硫氨酸突变为精氨酸。使得该逆转录‑聚合双功能酶具有逆转录酶活性、可调控的焦磷酸水解活性和聚合酶活性，可应用于rt‑PCR、rt‑PAP等领域，特异性强、灵敏度高。
Description
技术领域
本发明涉及基因工程领域，且特别涉及一种逆转录-聚合双功能酶及其制备方法、应用。
背景技术
当机体发生疾病时，如恶性肿瘤、外伤、感染重大疾病等，异常坏死细胞会释放大量DNA进入血液循环。循环游离DNA(circulating free DNA， cfDNA)是血液中被降解的DNA片段，大小约150～200bp，在淋巴液、脑脊液、尿液和唾液中也有分布。其中，来源于肿瘤细胞的cfDNA被称为循环肿瘤DNA(circulating tumor DNA，ctDNA)。ctDNA是由肿瘤细胞DNA主动释放或细胞凋零后释放进入体液中，可以通过ctDNA检测肿瘤相关的特异性变异，进而了解肿瘤的特征。ctDNA检测在肿瘤的早期筛查、临床诊断、分子特征预后预测、疗效动态监测、疾病进展动态监测、残留病灶评估、耐药性突变监测、肿瘤复发等方面均具有重要的意义。因此，在大量正常的cfDNA中将ctDNA携带的肿瘤特异性突变检测出来对准确反映疾病状态至关重要。目前常用ctDNA检测技术包括常规实时荧光定量PCR (qPCR)、突变扩增阻滞系统(ARMS)、数字PCR(dPCR)以及新一代测序(NGS)等，各方法的检测原理、灵敏度、特异性各不相同。
但是，血液等体液中的ctDNA会被巨噬细胞实时清除，从而使体液中的ctDNA含量极低，更因为同时存在大量野生型DNA的干扰，低丰度 ctDNA对检测技术的特异性和灵敏度有极高要求。目前大多数肿瘤基因突变监测集中于具有突变DNA丰度较高的肿瘤组织或晚期癌症患者的血浆 ctDNA。常规qPCR法以及ARMS-PCR法一般适用于ctDNA丰度≥1％的组织标本，而组织标本需要通过有创外科手术或穿刺取得，对于非病灶组织 (如血浆或血液、尿液，吐液等体液)中的含量丰度低于1％的ctDNA往往不能准确可靠地检测出来，无法及时进行诊断、指导临床用药、跟踪疗效、发现耐药性位点以及监测复发事件等。数字PCR用于等位基因突变检测的分析灵敏度为0.05％～0.1％，具有灵敏度高、特异性强以及定量检测能力，但需要特殊设备，且通量小、，操作繁琐，对于标本中ctDNA丰度低于 0.1％的体液标本仍然不能取得可靠的结果。
焦磷酸活化聚合反应(Pyrophosphorolysis activated polymerization,PAP)是一种使用3’末端阻断性引物(封闭性引物)，利用Taq DNA聚合酶串联焦磷酸反应和聚合反应的核酸扩增方法。因引物带有3’末端非延伸性核苷酸(3’末端阻断剂)不能被直接延伸。当3’末端阻断性引物在焦磷酸化反应(Pyrophosphorolysis)去除阻断剂后可被聚合酶延伸。因此，PAP技术具有比其它方法更高的特异性和灵敏度。
Taq DNA聚合酶是一种耐热聚合酶，具有极高的热稳定性、5’-3’聚合活性和5’-3’外切核酸酶活性，但无3’-5’外切核酸酶活性。此外，野生型Taq DNA聚合酶具有微弱的焦磷酸水解催化反应，这实际上是聚合反应的逆反应，可以催化ddNMP和PPi反应生成ddNTP。当引物的3’末端标记为2’,3’-双脱氧鸟嘌呤核苷酸(ddGMP)且与模板匹配，Taq DNA 聚合酶会在焦磷酸存在下，对3’末端的ddGMP产生焦磷酸水解作用，继而进行5’-3’的扩增延伸。而当引物的3’末端标记为2’,3’-双脱氧腺嘌呤核苷酸(ddAMP)且与模板配对时，在同样的条件下不会产生焦磷酸水解作用(Qiang Liu and Steve S.Sommer，2000,29:1072-1083)。
目前，商业化的AmpliTaqFS酶和ThermoSequenase酶通过基因改造获得，二者均含有F667Y突变，对于ddNMP的结合没有偏向性，且比野生型的Taq酶的PAP活性高。但是，上述商业化的Taq聚合酶的PAP活性难以调控，且无逆转录酶活性。
发明内容
为了解决上述技术问题，本发明提供一种逆转录-聚合双功能酶及其制备方法、应用。
本发明解决其技术问题是采用以下技术方案来实现的。
本发明的第一方面提供一种逆转录-聚合双功能酶，所述逆转录-聚合双功能酶的核苷酸序列如SEQ ID NO：1所示。
本发明的第二方面提供一种逆转录-聚合双功能酶，所述逆转录-聚合双功能酶的氨基酸序列如SEQ ID NO：2所示。
本发明的第三方面提供一种逆转录-聚合双功能酶，包含野生型Taq DNA聚合酶的氨基酸序列经过如下突变后的氨基酸序列：第583位的天冬酰胺突变为丝氨酸、第587位的精氨酸突变为赖氨酸、第660位的精氨酸突变为天冬氨酸、第667位的苯丙氨酸突变为酪氨酸、第708位的谷氨酸突变为谷氨酰胺、第732位的天冬氨酸突变为谷氨酰胺、第742位的谷氨酸突变为精氨酸、第747位的甲硫氨酸突变为精氨酸。
在本发明的一个示例性实施例中，所述逆转录-聚合双功能酶具有可受二价阳离子调控的逆转录酶活性、焦磷酸水解活性、聚合酶活性以及5’→3’外切酶活性。
本发明的第四方面提供一种逆转录-聚合双功能酶核酸分子，包含编码如上任意一项所述的逆转录-聚合双功能酶的核苷酸序列。
本发明的第五方面提供一种重组表达载体，含有如上所述的逆转录-聚合双功能酶核酸分子。
本发明的第六方面提供一种重组宿主细胞，含有如上所述的重组表达载体。
本发明的第七方面提供一种逆转录-聚合双功能酶的制备方法，包括：
制备如上所述的重组表达载体；
将所述重组表达载体转化到宿主细胞中，并对转化后的所述宿主细胞进行诱导表达；
对诱导表达后的所述宿主细胞进行收集、破碎和纯化后，得到所述逆转录-聚合双功能酶。
在本发明的一个示例性实施例中，所述制备如上所述的重组表达载体步骤包括：
按照预先设计的点突变引物对含有野生型Taq DNA聚合酶基因的载体进行点突变PCR扩增，得到所述重组表达载体。
本发明的第八方面提供如上任意一项所述的逆转录-聚合双功能酶在核酸扩增、肿瘤检测中的应用。
本发明的第九方面提供一种试剂盒，包括如上任意一项所述的逆转录- 聚合双功能酶。
与现有技术相比，本发明的逆转录-聚合双功能酶及其制备方法、应用的有益效果是：
本发明所提供的逆转录-聚合双功能酶通过对野生型Taq DNA聚合酶氨基酸序列进行多个位点的定向基因突变，例如，将第583位的天冬酰胺突变为丝氨酸、将第587位的精氨酸突变为赖氨酸可以提高聚合酶的热稳定性。将第660位的精氨酸突变为天冬氨酸、将第667位的苯丙氨酸突变为酪氨酸可以降低对ddNTP的选择性抵抗作用，并提高渗入的速度。将第 708位的谷氨酸突变为谷氨酰胺、将第732位的天冬氨酸突变为谷氨酰胺、将第742位的谷氨酸突变为精氨酸、将第747位的甲硫氨酸突变为精氨酸可以赋予聚合酶逆转录活性、实现焦磷酸水解活性的可调控。
通过上述位点的突变，得到的逆转录-聚合双功能酶具有逆转录酶活性、 5’→3’外切酶活性、焦磷酸水解活性和聚合酶活性。该逆转录-聚合双功能酶的各种酶活性不同程度地受到二价阳离子的抑制或激活，可以通过二价阳离子(Mn2+、Ca2+、Se2+、Cu2+等)浓度和种类的选择，从而调节逆转录活性、聚合活性和焦磷酸化反应的效率，优化一步法rt-PCR或rt-PAP反应的条件。该逆转录-聚合双功能酶在应用于rt-PCR、rt-PAP等多个领域时，其特异性和灵敏度均得到明显提高。能够将其应用于低丰度ctDNA的检测过程中，实现对肿瘤的早期检测。
附图说明
为了更清楚地说明本发明实施例的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，应当理解，以下附图仅示出了本发明的某些实施例，因此不应被看作是对范围的限定，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他相关的附图。
图1为本发明试验例1提供的突变型Taq DNA酶RT-PCR验证结果；
图2为本发明试验例1提供的野生型Taq DNA酶RT-PCR验证结果；
图3为本发明试验例2提供的突变型Taq DNA酶PAP-PCR验证结果
图4为本发明试验例2提供的野生型Taq DNA酶PAP-PCR验证结果；
图5为本发明试验例3提供的反应体系中添加0.5mM Mn2+痰液样本类型PAP-PCR验证结果；
图6为本发明试验例4提供的反应体系中未添加Mn2+痰液样本类型 PAP-PCR验证结果；
图7为本发明试验例5提供的添加0.3mM Mn2+血浆样本类型PAP-PCR 验证的结果；
图8为本发明试验例6提供的未添加Mn2+血浆样本类型PAP-PCR验证的结果；
图9为本发明试验例7提供的反应体系中添加1mM Mn2+组织样本类型 PAP-PCR验证的结果；和
图10为本发明试验例8提供的反应体系中未添加Mn2+组织样本类型 PAP-PCR验证的结果。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚，下面将对本发明实施例中的技术方案进行清楚、完整地描述。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
下面对本公开实施例的逆转录-聚合双功能酶及其制备方法、应用进行具体说明。
本公开的实施例提供一种逆转录-聚合双功能酶，通过将野生型Taq DNA聚合酶氨基酸序列进行突变获得。具体为将野生型的Taq酶的第583 位的天冬酰胺突变为丝氨酸(N583S)，第587位的精氨酸突变为赖氨酸 (R587K)、第660位的精氨酸突变为天冬氨酸(R660D)、第667位的苯丙氨酸突变为酪氨酸(F667Y)、第708位的谷氨酸突变为谷氨酰胺(E708Q)、第732位的天冬氨酸突变为谷氨酰胺(D732Q)、第742位的谷氨酸突变为精氨酸(E742R)、第747位的甲硫氨酸突变为精氨酸(M747R)。
在具体实施方式中，本公开以野生型Taq DNA聚合酶基因的质粒模板为起始，按照预先设计的点突变引物依次进行N583S、R587K、R660D、F667Y、E708Q、D732Q、E742R、M747R的PCR突变。野生型Taq DNA 聚合酶基因例如可以来自GenBank数据中的D32013.1。
需要说明的是，除非特别定义，本公开所有的科学或技术专业词汇均与本领域大部分一般人员的普通理解一致。以下的文献中本领域中大部分专业名词的一般定义，本专利中所使用的专业名词与上述文献中对该专业名词描述一致。
名词“核苷酸”一般指的是一个核苷通过酯键与一个酸性分子或基团相连而形成的化合物，例如，核苷的磷酸酯，通常有一个、两个或三个磷酸基团共价连接在核苷的糖基团的5号位上。在一些情况下，核苷酸的定义还包括一些典型核苷酸的同系物或类似物。
名词“氨基酸”是指构成蛋白质的基本单位，赋予蛋白质特定的分子结构形态，使它的分子具有生化活性。如本公开所用“氨基酸”包括下列 20种天然氨基酸：丙氨酸(Ala或A)、甘氨酸(Gly或G)、异亮氨酸(Ile或I)、天冬酰胺(Asn或N)、精氨酸(Arg或R)、赖氨酸(Lys或K)、半胱氨酸(Cys 或C)、天冬氨酸(Asp或D)、谷氨酸(Glu或E)、谷胺酰胺(Gln或Q)、组氨酸(His或H)、亮氨酸(Leu或L)、甲硫氨酸(Met或M)、苯丙氨酸(Phe或F)、脯氨酸(Pro或P)、丝氨酸(Ser或S)、苏氨酸(Thr或T)、色氨酸(Trp或W)、缬氨酸(Val或V)和酪氨酸(Tyr或Y)。
名词“核酸”包括脱氧核糖核酸(DNA)，核糖核酸(RNA)，DNA-RNA 杂交体，寡聚核苷酸，适配体(aptamers)，肽核酸(PNAs)，PNA-DNA杂交体，PNA-RNA杂交体等等。包括一切线性形式(单链或双链)或分支状形式的共价相连的核苷酸。一个典型的核酸通常是单链或者双链的，并包含磷酸二脂键。
名词“野生型”指的是从自然界分离到的菌株一般称野生型菌株(wild typestrain)，简称野生型。野生型经突变后形成的带有新性状(基因型)的菌株,称突变株(mutant，或突变体，突变型)。
名词“扩增”指的是目的核酸片段在核酸聚合酶的作用下数目变多的过程，包括但不限于聚合酶链式反应(PCR)，连接酶链式反应(LCR)，核酸序列基础扩增(NASBA)等。
名词“重组宿主细胞”或“宿主细胞”意指包含本发明多核苷酸的细胞，而不管使用何种方法进行插入以产生重组宿主细胞，例如直接摄取、转导、f配对或所属领域中已知的其它方法。外源性多核苷酸可保持为例如质粒的非整合载体或者可整合入宿主基因组中。宿主细胞可为原核细胞或真核细胞。
名词“转化”指将编码基因导入到宿主细胞内部这样的方式将多核苷酸或多肽遗传转化到宿主细胞中。
本公开的实施例中，该逆转录-聚合双功能酶的核苷酸序列如SEQ ID NO：1所示。
SEQ ID NO:1如下：
ATGAGGGGGATGCTGCCCCTCTTTGAGCCCAAGGGCCGGGTCCT CCTGGTGGACGGCCACCACCTGGCCTACCGCACCTTCCACGCCCTGA AGGGCCTCACCACCAGCCGGGGGGAGCCGGTGCAGGCGGTCTACGG CTTCGCCAAGAGCCTCCTCAAGGCCCTCAAGGAGGACGGGGACGCG GTGATCGTGGTCTTTGACGCCAAGGCCCCCTCCTTCCGCCACGAGGCC TACGGGGGGTACAAGGCGGGCCGGGCCCCCACGCCGGAGGACTTTCC CCGGCAACTCGCCCTCATCAAGGAGCTGGTGGACCTCCTGGGGCTGG CGCGCCTCGAGGTCCCGGGCTACGAGGCGGACGACGTCCTGGCCAGC CTGGCCAAGAAGGCGGAAAAGGAGGGCTACGAGGTCCGCATCCTCA CCGCCGACAAAGACCTTTACCAGCTCCTTTCCGACCGCATCCACGCCC TCCACCCCGAGGGGTACCTCATCACCCCGGCCTGGCTTTGGGAAAAGTACGGCCTGAGGCCCGACCAGTGGGCCGACTACCGGGCCCTGACCGG GGACGAGTCCGACAACCTTCCCGGGGTCAAGGGCATCGGGGAGAAG ACGGCGAGGAAGCTTCTGGAGGAGTGGGGGAGCCTGGAAGCCCTCC TCAAGAACCTGGACCGGCTGAAGCCCGCCATCCGGGAGAAGATCCTG GCCCACATGGACGATCTGAAGCTCTCCTGGGACCTGGCCAAGGTGCG CACCGACCTGCCCCTGGAGGTGGACTTCGCCAAAAGGCGGGAGCCC GACCGGGAGAGGCTTAGGGCCTTTCTGGAGAGGCTTGAGTTTGGCAG CCTCCTCCACGAGTTCGGCCTTCTGGAAAGCCCCAAGGCCCTGGAGGAGGCCCCCTGGCCCCCGCCGGAAGGGGCCTTCGTGGGCTTTGTGCTT TCCCGCAAGGAGCCCATGTGGGCCGATCTTCTGGCCCTGGCCGCCGC CAGGGGGGGCCGGGTCCACCGGGCCCCCGAGCCTTATAAAGCCCTCA GGGACCTGAAGGAGGCGCGGGGGCTTCTCGCCAAAGACCTGAGCGT TCTGGCCCTGAGGGAAGGCCTTGGCCTCCCGCCCGGCGACGACCCCA TGCTCCTCGCCTACCTCCTGGACCCTTCCAACACCACCCCCGAGGGG GTGGCCCGGCGCTACGGCGGGGAGTGGACGGAGGAGGCGGGGGAGC GGGCCGCCCTTTCCGAGAGGCTCTTCGCCAACCTGTGGGGGAGGCTTGAGGGGGAGGAGAGGCTCCTTTGGCTTTACCGGGAGGTGGAGAGGC CCCTTTCCGCTGTCCTGGCCCACATGGAGGCCACGGGGGTGCGCCTG GACGTGGCCTATCTCAGGGCCTTGTCCCTGGAGGTGGCCGAGGAGAT CGCCCGCCTCGAGGCCGAGGTCTTCCGCCTGGCCGGCCACCCCTTCA ACCTCAACTCCCGGGACCAGCTGGAAAGGGTCCTCTTTGACGAGCTA GGGCTTCCCGCCATCGGCAAGACGGAGAAGACCGGCAAGCGCTCCA CCAGCGCCGCCGTCCTGGAGGCCCTCCGCGAGGCCCACCCCATCGTG GAGAAGATCCTGCAGTACCGGGAGCTCACCAAGCTGAAGAGCACCTACATTGACCCCTTGCCGGACCTCATCCACCCCAGGACGGGCCGCCTCCA CACCCGCTTCAACCAGACGGCCACGGCCACGGGCAGGCTAAGTAGCT CCGATCCCAACCTCCAGAGCATCCCCGTCAAAACCCCGCTTGGGCAG AGGATCCGCCGGGCCTTCATCGCCGAGGAGGGGTGGCTATTGGTGGC CCTGGACTATAGCCAGATAGAGCTCAGGGTGCTGGCCCACCTCTCCGG CGACGAGAACCTGATCCGGGTCTTCCAGGAGGGGCGGGACATCCACA CGGAGACCGCCAGCTGGATGTTCGGCGTCCCCCGGGAGGCCGTGGAC CCCCTGATGCGCGATGCGGCCAAGACCATCAACTACGGGGTCCTCTAC GGCATGTCGGCCCACCGCCTCTCCCAGGAGCTAGCCATCCCTTACGAG GAGGCCCAGGCCTTCATTGAGCGCTACTTTCAGAGCTTCCCCAAGGT GCGGGCCTGGATTCAGAAGACCCTGGAGGAGGGCAGGAGGCGGGGG TACGTGGAGACCCTCTTCGGCCGCCGCCGCTACGTGCCACAGCTAGA GGCCCGGGTGAAGAGCGTGCGGCGTGCGGCCGAGCGCCGTGCCTTC AACATGCCCGTCCAGGGCACCGCCGCCGACCTCATGAAGCTGGCTAT GGTGAAGCTCTTCCCCAGGCTGGAGGAAATGGGGGCCAGGATGCTCC TTCAGGTCCACGACGAGCTGGTCCTCGAGGCCCCAAAAGAGAGGGC GGAGGCCGTGGCCCGGCTGGCCAAGGAGGTCATGGAGGGGGTGTATC CCCTGGCCGTGCCCCTGGAGGTGGAGGTGGGGATAGGGGAGGACTG GCTCTCCGCCAAGGAG
本公开的实施例中，该逆转录-聚合双功能酶的氨基酸序列如SEQ ID NO：2所示。
SEQ ID NO：2如下：
MRGMLPLFEPKGRVLLVDGHHLAYRTFHALKGLTTSRGEPVQAVY GFAKSLLKALKEDGDAVIVVFDAKAPSFRHEAYGGYKAGRAPTPEDFPR QLALIKELVDLLGLARLEVPGYEADDVLASLAKKAEKEGYEVRILTADKDLYQLLSDRIHALHPEGYLITPAWLWEKYGLRPDQWADYRALTGDESDN LPGVKGIGEKTARKLLEEWGSLEALLKNLDRLKPAIREKILAHMDDLKL SWDLAKVRTDLPLEVDFAKRREPDRERLRAFLERLEFGSLLHEFGLLESP KALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPY KALRDLKEARGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPE GVARRYGGEWTEEAGERAALSERLFANLWGRLEGEERLLWLYREVERP LSAVLAHMEATGVRLDVAYLRALSLEVAEEIARLEAEVFRLAGHPFNLNS RDQLERVLFDELGLPAIGKTEKTGKRSTSAAVLEALREAHPIVEKILQYRE LTKLKSTYIDPLPDLIHPRTGRLHTRFNQTATATGRLSSSDPNLQSIPVKTP LGQRIRRAFIAEEGWLLVALDYSQIELRVLAHLSGDENLIRVFQEGRDIHT ETASWMFGVPREAVDPLMRDAAKTINYGVLYGMSAHRLSQELAIPYEE AQAFIERYFQSFPKVRAWIQKTLEEGRRRGYVETLFGRRRYVPQLEARV KSVRRAAERRAFNMPVQGTAADLMKLAMVKLFPRLEEMGARMLLQV HDELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGIGEDWLSAKE
可以理解，由于编码同一种氨基酸的密码子有多种，多肽的编码序列具有多态性及变异的特点。因此在SEQ ID No.1所示的核苷酸序列中经过取代、缺失或添加一个或几个碱基且能够编码得到具有本公开的逆转录-聚合双功能酶活性的衍生的蛋白，得到的蛋白与逆转录-聚合双功能酶没有明显的功能差异，或者在SEQ ID No.2所示的氨基酸序列中经过取代、缺失或添加一个或几个氨基酸且具有本公开的逆转录-聚合双功能酶活性的蛋白质，也包括在本发明的范围内。
本公开的实施例还提供上述逆转录-聚合双功能酶的制备方法，包括：
S1，按照预先设计的点突变引物对含有野生型Taq DNA聚合酶的表达载体进行点突变PCR扩增，得到所述重组表达载体。
具体地，预先设计的点突变引物如下表1所示。
表1
| 名称 | 引物序列 |
| N583S-F(SEQ ID NO：3) | 5’-CCCAACCTCCAGAGCATCCCCGTCCGCA-3’ |
| N583S-R(SEQ ID NO：4) | 5’-TGCGGACGGGGATGCTCTGGAGGTTGGG-3’ |
| R587K-F(SEQ ID NO：5) | 5’-CAGAGCATCCCCGTCAAAACCCCGCTTGGG-3’ |
| R587K-R(SEQ ID NO：6) | 5’-CCCAAGCGGGGTTTTGACGGGGATGCTCTG-3’ |
| R660D-F(SEQ ID NO：7) | 5’-CCCCTGATGCGCGATGCGGCCAAGACC-3’ |
| R660D-R(SEQ ID NO：8) | 5’-GGTCTTGGCCGCATCGCGCATCAGGGG-3’ |
| F667Y-F(SEQ ID NO：9) | 5’-GCCAAGACCATCAACTACGGGGTCCTCTACGG-3’ |
| F667Y-R(SEQ ID NO：10) | 5’-CCGTAGAGGACCCCGTAGTTGATGGTCTTGGC-3’ |
| E708Q-F(SEQ ID NO：11) | 5’-GTGCGGGCCTGGATTCAGAAGACCCTGGAG-3’ |
| E708Q-R(SEQ ID NO：12) | 5’-CTCCAGGGTCTTCTGAATCCAGGCCCGCAC-3’ |
| D732Q-F(SEQ ID NO：13) | 5’-CCGCTACGTGCCACAGCTAGAGGCCCG-3’ |
| D732Q-R(SEQ ID NO：14) | 5’-CGGGCCTCTAGCTGTGGCACGTAGCGG-3’ |
| E742R-F(SEQ ID NO：15) | 5’-AAGAGCGTGCGGCGTGCGGCCGAGC-3’ |
| E742R-R(SEQ ID NO：16) | 5’-GCTCGGCCGCACGCCGCACGCTCTT-3’ |
| M747R-F(SEQ ID NO：17) | 5’-GCGGCCGAGCGCCGTGCCTTCAACATG-3’ |
| M747R-R(SEQ ID NO：18) | 5’-CATGTTGAAGGCACGGCGCTCGGCCGC-3’ |
可以理解的是，N583S-F表示正向引物、N583S-R表示反向引物。引物的长度控制在25bp～35bp，使得引物可以很好地与野生型Taq DNA聚合酶结合，减少引物二聚体等的形成。
S2，将所述重组表达载体转化到宿主细胞中，并对转化后的所述宿主细胞进行诱导表达。
宿主细胞例如可以为DH5a、TOP10、JM109、大肠杆菌感受态细胞BL21 等，本公开不进行具体的限定。诱导表达过程例如可以通过加入IPTG(异丙基硫代半乳糖苷，Isopropylβ-D-Thiogalactoside)等诱导剂诱导蛋白质表达。
S3，对诱导表达后的所述宿主细胞进行收集、破碎和纯化后，得到所述逆转录-聚合双功能酶。
具体地，收集诱导表达后的宿主细胞(例如菌液等)进行离心、超声破碎、过滤，然后通过层析法进行纯化，例如使用HisTrap纯化柱或Ni-亲和柱对产物进行纯化，得到所述逆转录-聚合双功能酶。
本公开实施例提供一种试剂盒，包含上述的逆转录-聚合双功能酶。该逆转录-聚合双功能酶能够应用于核酸扩增、基因测序、基因位点突变检测以及在疾病检测中。
需要说明的是，本公开的实施例中，试剂盒为至少包括一个设备的任何制品，设备例如为包装或容器。在本公开的一个实施例中，该试剂盒可以仅包含上述的逆转录-聚合双功能酶。
在本公开的其他实施例中，该试剂盒可以进一步包括二价阳离子。该逆转录-聚合双功能酶的焦磷酸化反应的活性受二价阳离子调节。所述二价阳离子选自Mn2+、Ca2+、Se2+和Cu2+中的一种或多种。
在其他实施例中，该试剂盒也可以包含其他组分，用于实现rt-PCR、 rt-PAP等等。例如，该试剂盒可以进一步包括：特异性引物、缓冲液、探针、dNTPs、水中的一种或多种。其中，特异性引物可以根据需要进行设计。
进一步地，在该试剂盒中，该逆转录-聚合双功能酶可以以单一组分的形式存在。该逆转录-聚合双功能酶也可以是，加入到含有其他组分的反应体系中，以混合组分的形式存在，本公开不进行具体限定。
以下结合实施例对本公开的特征和性能作进一步的详细描述。
实施例1
步骤一、Taq DNA聚合酶的定点突变
选取野生型Taq DNA聚合酶基因(GenBank:D32013.1)的质粒作为模板。按照下述步骤(1)～(4)在野生型Taq DNA聚合酶的基因上依次进行N583S、R587K、R660D、F667Y、E708Q、D732Q、E742R、M747R的 PCR突变。突变引物参照上述表1中的内容。
(1)PCR突变扩增
采用QuikChange Site-Directed Mutagenesis Kit(Agilent)进行点突变扩增。PCR反应体系如表2所示。
表2
| 组份 | 体积(μL) |
| 10×reaction buffer | 5 |
| dNTP mix | 1 |
| 10μM正向引物(F) | 1.5 |
| 10μM反向引物(R) | 1.5 |
| Taq DNA聚合酶质粒模板(50ng/μL) | 1 |
| PfuTurbo DNA polymerase | 1 |
| 超纯水 | 39 |
PCR扩增条件为：95℃变性30秒，1个循环；为95℃、30秒，55℃、 1分钟，68℃、6分钟，18个循环。
PCR扩增结束后，将PCR反应管放在冰上。加入1μL的限制性内切片Dpn I，37℃孵育1小时，去除野生型模板。
(2)转化感受态细胞：取分装好的100μL感受态细胞(如DH5a)置于冰浴中解冻，加入5μL步骤(1)的PCR扩增产物，在冰浴中静置30分钟后，置于42℃水浴中放置60～90秒，然后快速将产物转移到冰浴中孵育 2～5分钟。然后加入900μL无菌的不含抗生素的SOC或LB培养液，37℃ 150转/分钟震荡培养45～60分钟。
(3)取100μL已转化的感受态细胞加到含有相应抗生素的LB固体琼脂培养基上，用无菌玻棒轻轻的将细胞均匀涂开。培养箱37℃培养12～16 小时后，挑取单克隆菌接种到5mL含相应抗生素的LB液体培养液中培养。
(4)对细菌中含有突变的质粒进行基因测序之后，得到突变后的Taq DNA聚合酶与设计的突变序列完全一致。
步骤二、Taq DNA聚合酶突变体的表达
将同时含有八个突变位点N583S、R587K、R660D、F667Y、E708Q、 D732Q、E742R、M747R的Taq DNA质粒载体转化到大肠杆菌感受态细胞 BL21中，取100μL感受态细胞涂到含有相应抗生素的LB固体琼脂培养基上，挑取单克隆菌接种于10mL含相应抗生素的LB液体培养液中37℃培养至OD值0.4～0.8，然后进一步扩大培养至1000mL，当OD600(opticaldensity at 600nm)值在0.6～0.8之间时，加入终浓度为0.8～1mM的IPTG诱导蛋白质表达，继续培养4小时。
步骤三、Taq DNA聚合酶的纯化
(1)取步骤二获得的培养好的菌液，5000rpm离心10～30min，离心菌体，加入20mLBinding buffer(50mM Tris，100～500mM NaCl，0～40mM咪唑，pH8.0)重悬菌体，重复超声4s，停6s，总共30min。
(2)对超声破碎的菌液在5000～11000rpm条件下离心30min，取上清液，进行0.45μm的微孔滤膜过滤。
(3)在AKTA纯化系统上，将过滤完的上清液用1mL或5mL HisTrap 纯化柱纯化，用Elution buffer(50mM Tris，100～500mM NaCl，250～500mM 咪唑)洗脱得到突变型TaqDNA聚合酶。再将纯化后的突变型Taq DNA聚合酶进一步用HiLoad 16/60Superdex 200进行凝胶过滤层析，根据蛋白质大小进一步纯化。
(4)使用离心过滤装置对纯化后的蛋白进行浓缩，得到突变型Taq DNA聚合酶(逆转录-聚合双功能酶)。
为了明确实施例1获得的突变型Taq DNA聚合酶的特性，通过下述试验例对该突变型Taq DNA聚合酶的逆转录酶活性和在焦磷酸水解催化的聚合反应(PAP-PCR)中的活性进行验证。
试验例1
将本公开实施例1制备的突变型Taq DNA聚合酶与野生型Taq DNA 聚合酶用RT-PCR方法进行逆转录酶活验证比较。步骤如下：
将突变型Taq DNA聚合酶与野生型Taq DNA聚合酶在同一实验条件下进行检测，选用新型冠状病毒的开放读码框1ab(open reading frame,ORF1ab)为靶序列的PCR反应体系。
反应体系为10×PCR缓冲液5μL、4mM MgCl2、25μM dNTP、0.2μ M上游引物、0.2μM下游引物、0.2μM探针、酶各5U、DEPC水补足至总体积45μL，体系总体积50μL。
上游引物(SEQ ID NO：19)：5’-TGCTAGTTGGGTGATGCGTA-3’；
下游引物(SEQ ID NO：20)：5’-CCACATGGAAATGGCTTGAT-3’；
探针(SEQ ID NO：21)：FAM-5’-TGATGATGGTGCTAGGAGAGTGT GG-3’-BHQ1。
RNA模板：102拷贝/μL、10拷贝/μL(2个重复)、1拷贝/μL(2个重复)，在对应的反应孔各加5μL。
反应程序：第一阶段：62℃10分钟、1循环；第二阶段：95℃2分钟、1循环；第三阶段：95℃10秒、64℃60秒，信号采集、45循环。
如图1所示为突变型Taq DNA聚合酶参与RT-PCR体系验证的结果；如图2所示为野生型Taq DNA聚合酶参与RT-PCR体系验证的结果。
从图1和图2可以看出，在相同体系和相同的反应条件下，野生型Taq DNA聚合酶并无逆转录酶的活性，本公开实施例1获得的突变型Taq DNA 聚合酶表现出了强的逆转录酶的活性。
试验例2
将本公开实施例1制备的突变型Taq DNA聚合酶与野生型Taq DNA 聚合酶用PAP-PCR方法进行活性验证比较。步骤如下：
将突变型Taq DNA聚合酶与野生型Taq DNA聚合酶在同一实验条件下进行检测，选用人类表皮生长因子受体(EGFR)基因中21外显子上的L858R突变(T2573G)为靶标，引物3’端碱基为双脱氧核苷三磷酸 (ddNTP)设计。
反应体系：10×PCR缓冲液5μL、4mM MgCl2、25μM dNTP、0.2μM上游引物、0.2μM下游引物、0.2μM探针、酶各5U、90μM Na4PPi、纯化水补足至总体积45μL，体系总体积50μL。
上游引物(SEQ ID NO：22)：5’-AGATCACAGATTTTGGGC-ddG-3’；
下游引物(SEQ ID NO：23)：5’-AAACAGTCACCCCGTAGCT-ddC-3’；
探针(SEQ ID NO：24)：FAM-5’-CAAACTGCTGGGTGCGGAAGA-3’ -BHQ1。
L858R突变型质粒DNA模板：105拷贝/μL、103拷贝/μL，反应孔中加模板5μL。
反应程序：第一阶段：95℃2分钟、1循环；第二阶段：95℃6秒、 65℃50秒、15循环；第三阶段：95℃6秒、65℃50秒，信号采集、31 循环。
如图3所示为突变型Taq DNA聚合酶参与PAP-PCR体系验证的结果；如图4所示为野生型Taq DNA聚合酶参与PAP-PCR体系验证的结果。
从图3和图4可以看出，在相同体系和相同的反应条件下，野生型Taq DNA聚合酶的焦磷酸水解催化的聚合反应活性较低，而本公开的突变型 Taq DNA聚合酶的活性高，整体表现扩增效率高了10～11个循环(近1000 倍)。
可见，与野生型Taq DNA聚合酶相比，本实施例的突变型Taq DNA聚合酶在焦磷酸水解催化的聚合反应(PAP-PCR)中的活性得到了显著性提高，可应用于PAP-PCR技术中，特异性更强。
试验例3～8
验证不同二价阳性离子(包括Mn2+、Ca2+、Se2+、Cu2+等)的调整对 PAP-PCR的不同影响效果。以Mn2+的调节作用验证二价阳性离子对不同来源核酸标本的扩增效率的影响。步骤如下：
选用人类表皮生长因子受体(EGFR)基因中20外显子上的T790M突变(C2369T)为靶标，引物3’端碱基为双脱氧核苷三磷酸(ddNTP)设计，对二价Mn阳离子可激活突变型TaqDNA聚合酶焦磷酸化反应的活性进行验证。
反应体系：10×PCR缓冲液5μL、4mM MgCl2、25μM dNTP、0.3～1mM Mn2+、0.2μM上游引物、0.2μM下游引物、0.2μM探针、酶5U(本公开实施例的突变型Taq DNA聚合酶)、90μMNa4PPi、纯化水补足至总体积 45μL，体系总体积50μL。
上游引物(SEQ ID NO：25)：5’-CACCGTGCAGCTCATCA-ddT-3’；
下游引物(SEQ ID NO：26)：5’-CTGCGGTGTTTTCACCAGT-ddA-3’；
探针(SEQ ID NO：27)：FAM-5’-TGCAGATCGCAAAGGGCATG-3’ -BHQ1。
不同来源T790M模板制备：将100拷贝/μL、10拷贝/μL、1拷贝/ μL T790M突变型质粒模板(20μL)分别加至已知T790M野生型的痰液、血浆及组织核酸提后的洗脱液中(T790M野生型的痰液、血浆及组织分别预先按照常规的核酸提取步骤进行核酸提取，提取后，80μL洗脱液洗脱)，混匀后分别于对应反应孔中加5μL。
反应程序：第一阶段：95℃2分钟、1循环；第二阶段：95℃6秒、 65℃50秒、15循环；第三阶段：95℃6秒、65℃50秒、信号采集、31 循环。
不同试验例中Mn2+的添加量和样本如下表3所示：
表3
| 项目 | 样本 | Mn2+的添加量，mM |
| 试验例3 | 痰液样本 | 0.5 |
| 试验例4 | 痰液样本 | 0 |
| 试验例5 | 血浆样本 | 0.3 |
| 试验例6 | 血浆样本 | 0 |
| 试验例7 | 组织样本 | 1 |
| 试验例8 | 组织样本 | 0 |
如图5所示为反应体系中添加0.5mM Mn2+痰液样本类型PAP-PCR验证的结果，如图6所示为反应体系中未添加Mn2+痰液样本类型PAP-PCR验证的结果；如图7所示为反应体系中添加0.3mM Mn2+血浆样本类型 PAP-PCR验证的结果；如图8所示为反应体系中未添加Mn2+血浆样本类型 PAP-PCR验证的结果；如图9所示为反应体系中添加1mM Mn2+组织样本类型PAP-PCR验证的结果；如图10所示为反应体系中未添加Mn2+组织样本类型PAP-PCR验证的结果。
从图5～10可以看出，本公开实施例获得的突变型Taq DNA聚合酶对 Mn2+的调节十分敏感，对不同样本类型不同体系进行相应的调整，在野生型基因组DNA未稀释的情况下，发现稀有突变的检测灵敏度均可达到单拷贝。
此外，本公开对其他二价阳性离子(包括Ca2+、Se2+、Cu2+等)也进行了同样的验证试验，验证结果表明，本公开实施例获得的突变型Taq DNA 聚合酶对其他二价阳性离子(包括Ca2+、Se2+、Cu2+等)的调节也非常敏感，对不同样本类型不同体系进行相应的调整，在野生型基因组DNA未稀释的情况下，发现稀有突变的检测灵敏度均可达到单拷贝。
在PAP-PCR检测试剂盒中，将本公开的突变型Taq DNA聚合酶与二价阳性离子联用，可以显著提高检测灵敏度。二价阳性离子为Mn2+、Ca2+、 Se2+、Cu2+等，添加量为0.01～1.05mM，可以适用于痰液、血液、组织等多种样本的PAP-PCR检测。
以上所描述的实施例是本发明一部分实施例，而不是全部的实施例。本发明的实施例的详细描述并非旨在限制要求保护的本发明的范围，而是仅仅表示本发明的选定实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
序列表
<110> 厦门通灵生物医药科技有限公司
<120> 一种逆转录-聚合双功能酶及其制备方法、应用
<130> LZXM22050502CN01
<160> 27
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2496
<212> DNA
<213> DNA聚合酶(DNA polymerase)
<400> 1
atgaggggga tgctgcccct ctttgagccc aagggccggg tcctcctggt ggacggccac 60
cacctggcct accgcacctt ccacgccctg aagggcctca ccaccagccg gggggagccg 120
gtgcaggcgg tctacggctt cgccaagagc ctcctcaagg ccctcaagga ggacggggac 180
gcggtgatcg tggtctttga cgccaaggcc ccctccttcc gccacgaggc ctacgggggg 240
tacaaggcgg gccgggcccc cacgccggag gactttcccc ggcaactcgc cctcatcaag 300
gagctggtgg acctcctggg gctggcgcgc ctcgaggtcc cgggctacga ggcggacgac 360
gtcctggcca gcctggccaa gaaggcggaa aaggagggct acgaggtccg catcctcacc 420
gccgacaaag acctttacca gctcctttcc gaccgcatcc acgccctcca ccccgagggg 480
tacctcatca ccccggcctg gctttgggaa aagtacggcc tgaggcccga ccagtgggcc 540
gactaccggg ccctgaccgg ggacgagtcc gacaaccttc ccggggtcaa gggcatcggg 600
gagaagacgg cgaggaagct tctggaggag tgggggagcc tggaagccct cctcaagaac 660
ctggaccggc tgaagcccgc catccgggag aagatcctgg cccacatgga cgatctgaag 720
ctctcctggg acctggccaa ggtgcgcacc gacctgcccc tggaggtgga cttcgccaaa 780
aggcgggagc ccgaccggga gaggcttagg gcctttctgg agaggcttga gtttggcagc 840
ctcctccacg agttcggcct tctggaaagc cccaaggccc tggaggaggc cccctggccc 900
ccgccggaag gggccttcgt gggctttgtg ctttcccgca aggagcccat gtgggccgat 960
cttctggccc tggccgccgc cagggggggc cgggtccacc gggcccccga gccttataaa 1020
gccctcaggg acctgaagga ggcgcggggg cttctcgcca aagacctgag cgttctggcc 1080
ctgagggaag gccttggcct cccgcccggc gacgacccca tgctcctcgc ctacctcctg 1140
gacccttcca acaccacccc cgagggggtg gcccggcgct acggcgggga gtggacggag 1200
gaggcggggg agcgggccgc cctttccgag aggctcttcg ccaacctgtg ggggaggctt 1260
gagggggagg agaggctcct ttggctttac cgggaggtgg agaggcccct ttccgctgtc 1320
ctggcccaca tggaggccac gggggtgcgc ctggacgtgg cctatctcag ggccttgtcc 1380
ctggaggtgg ccgaggagat cgcccgcctc gaggccgagg tcttccgcct ggccggccac 1440
cccttcaacc tcaactcccg ggaccagctg gaaagggtcc tctttgacga gctagggctt 1500
cccgccatcg gcaagacgga gaagaccggc aagcgctcca ccagcgccgc cgtcctggag 1560
gccctccgcg aggcccaccc catcgtggag aagatcctgc agtaccggga gctcaccaag 1620
ctgaagagca cctacattga ccccttgccg gacctcatcc accccaggac gggccgcctc 1680
cacacccgct tcaaccagac ggccacggcc acgggcaggc taagtagctc cgatcccaac 1740
ctccagagca tccccgtcaa aaccccgctt gggcagagga tccgccgggc cttcatcgcc 1800
gaggaggggt ggctattggt ggccctggac tatagccaga tagagctcag ggtgctggcc 1860
cacctctccg gcgacgagaa cctgatccgg gtcttccagg aggggcggga catccacacg 1920
gagaccgcca gctggatgtt cggcgtcccc cgggaggccg tggaccccct gatgcgcgat 1980
gcggccaaga ccatcaacta cggggtcctc tacggcatgt cggcccaccg cctctcccag 2040
gagctagcca tcccttacga ggaggcccag gccttcattg agcgctactt tcagagcttc 2100
cccaaggtgc gggcctggat tcagaagacc ctggaggagg gcaggaggcg ggggtacgtg 2160
gagaccctct tcggccgccg ccgctacgtg ccacagctag aggcccgggt gaagagcgtg 2220
cggcgtgcgg ccgagcgccg tgccttcaac atgcccgtcc agggcaccgc cgccgacctc 2280
atgaagctgg ctatggtgaa gctcttcccc aggctggagg aaatgggggc caggatgctc 2340
cttcaggtcc acgacgagct ggtcctcgag gccccaaaag agagggcgga ggccgtggcc 2400
cggctggcca aggaggtcat ggagggggtg tatcccctgg ccgtgcccct ggaggtggag 2460
gtggggatag gggaggactg gctctccgcc aaggag 2496
<210> 2
<211> 832
<212> PRT
<213> DNA聚合酶(DNA polymerase)
<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 Ala 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 Ser Ile Pro Val Lys 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 Asp Ala Ala Lys Thr Ile Asn Tyr 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 Gln 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 Gln Leu Glu Ala Arg
725 730 735
Val Lys Ser Val Arg Arg Ala Ala Glu Arg Arg 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> 28
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 3
cccaacctcc agagcatccc cgtccgca 28
<210> 4
<211> 28
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 4
tgcggacggg gatgctctgg aggttggg 28
<210> 5
<211> 30
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 5
cagagcatcc ccgtcaaaac cccgcttggg 30
<210> 6
<211> 30
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 6
cccaagcggg gttttgacgg ggatgctctg 30
<210> 7
<211> 27
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 7
cccctgatgc gcgatgcggc caagacc 27
<210> 8
<211> 27
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 8
ggtcttggcc gcatcgcgca tcagggg 27
<210> 9
<211> 32
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 9
gccaagacca tcaactacgg ggtcctctac gg 32
<210> 10
<211> 32
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 10
ccgtagagga ccccgtagtt gatggtcttg gc 32
<210> 11
<211> 30
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 11
gtgcgggcct ggattcagaa gaccctggag 30
<210> 12
<211> 30
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 12
ctccagggtc ttctgaatcc aggcccgcac 30
<210> 13
<211> 27
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 13
ccgctacgtg ccacagctag aggcccg 27
<210> 14
<211> 27
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 14
cgggcctcta gctgtggcac gtagcgg 27
<210> 15
<211> 25
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 15
aagagcgtgc ggcgtgcggc cgagc 25
<210> 16
<211> 25
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 16
gctcggccgc acgccgcacg ctctt 25
<210> 17
<211> 27
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 17
gcggccgagc gccgtgcctt caacatg 27
<210> 18
<211> 27
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 18
catgttgaag gcacggcgct cggccgc 27
<210> 19
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 19
tgctagttgg gtgatgcgta 20
<210> 20
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 20
ccacatggaa atggcttgat 20
<210> 21
<211> 25
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 21
tgatgatggt gctaggagag tgtgg 25
<210> 22
<211> 18
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 22
agatcacaga ttttgggc 18
<210> 23
<211> 19
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 23
aaacagtcac cccgtagct 19
<210> 24
<211> 21
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 24
caaactgctg ggtgcggaag a 21
<210> 25
<211> 17
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 25
caccgtgcag ctcatca 17
<210> 26
<211> 19
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 26
ctgcggtgtt ttcaccagt 19
<210> 27
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 27
tgcagatcgc aaagggcatg 20
Claims (10)
1.一种逆转录-聚合双功能酶，其特征在于，所述逆转录-聚合双功能酶的核苷酸序列如SEQ ID NO：1所示。
2.一种逆转录-聚合双功能酶，其特征在于，所述逆转录-聚合双功能酶的氨基酸序列如SEQ ID NO：2所示 。
3.根据权利要求1或2所述的逆转录-聚合双功能酶，其特征在于，所述逆转录-聚合双功能酶具有可受二价阳离子调控的逆转录酶活性、焦磷酸水解活性、聚合酶活性以及和5’→3’外切酶活性。
4.一种逆转录-聚合双功能酶核酸分子，其特征在于，包含编码如权利要求1~3任意一项所述的逆转录-聚合双功能酶的核苷酸序列。
5.一种重组表达载体，其特征在于，含有如权利要求4所述的逆转录-聚合双功能酶核酸分子。
6.一种重组宿主细胞，其特征在于，含有如权利要求5所述的重组表达载体。
7.一种逆转录-聚合双功能酶的制备方法，其特征在于，包括：
制备如权利要求5所述的重组表达载体；
将所述重组表达载体转化到宿主细胞中，并对转化后的所述宿主细胞进行诱导表达；
对诱导表达后的所述宿主细胞进行收集、破碎和纯化后，得到所述逆转录-聚合双功能酶。
8.根据权利要求7所述的逆转录-聚合双功能酶的制备方法，其特征在于，所述制备如权利要求5所述的重组表达载体步骤包括：
按照预先设计的点突变引物对含有野生型Taq DNA聚合酶基因的载体进行点突变PCR扩增，得到所述重组表达载体。
9.如权利要求1~3任意一项所述的逆转录-聚合双功能酶在核酸扩增、基因测序以及非疾病诊断与治疗目的的基因位点突变检测中的应用。
10.一种试剂盒，其特征在于，包括如权利要求1~3任意一项所述的逆转录-聚合双功能酶。
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