CN113621585B - 一种Taq DNA聚合酶与核酸内切酶嵌合体及其制备方法与应用 - Google Patents
一种Taq DNA聚合酶与核酸内切酶嵌合体及其制备方法与应用 Download PDFInfo
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Classifications
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- C12N9/1241—Nucleotidyltransferases (2.7.7)
- C12N9/1252—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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Abstract
本发明公开了一种Taq DNA聚合酶与核酸内切酶嵌合体及其制备方法与应用,属于生物技术领域。本发明的嵌合体是野生型Taq DNA聚合酶经缺失突变和定点突变,以及在N末端融合一段高效亲和双链DNA结构域和瓣状核酸内切酶1等方式改造而成。本发明的嵌合体在野生型Taq DNA聚合酶的基础上,大幅提高了在PCR反应中的扩增效率和延伸速率,经化学修饰热启动后,适用于各类常规PCR、快速PCR、长片段PCR和热启动PCR。同时,该嵌合体具有5’‑3’核酸外切酶活性,可应用于探针法qPCR中。该嵌合体对PCR抑制剂具有较强的耐受能力,适合在抗凝血或常规滤纸收集的血液中直接扩增DNA。
Description
技术领域
本发明属于生物技术领域,具体涉及一种Taq DNA聚合酶与核酸内切酶嵌合体及其制备方法与应用。
背景技术
实时荧光定量PCR(Real-time quantitative PCR,qPCR)技术是在传统聚合酶链式反应(PCR)技术的基础上引入荧光化学物质,以达到实时监测PCR反应过程的目的,弥补了普通PCR只能采用终点法观察及无法定量分析模板的缺陷,成为了当今基因研究的重要工具。TaqMan探针法qPCR因其污染小、实时监测、定量线性范围广、特异性强、灵敏度高等特点,已经广泛应用于生物医学、农业科学、环境科学、食品安全等众多领域。
快速和精准一直是当今检测领域发展的需求及目标。传统的TaqMan探针法qPCR技术需对样本进行核酸提取,此过程不仅耗时长,且易造成样本间核酸交叉污染和损失。而直扩型TaqMan探针法qPCR技术省去了核酸提取步骤,缩短检测时间的同时不会因提取而造成核酸损失,大大提高检测结果的准确性。使用具有抑制剂耐受性的耐热DNA聚合酶是实现PCR直扩的关键。Taq DNA聚合酶作为所有耐热DNA聚合酶种类中发现并应用最早,同时也是抑制剂耐受性研究较为成熟的一类DNA聚合酶,对其采用化学修饰实现热启动是一种可逆封闭DNA聚合酶活性的方法。热启动DNA聚合酶的使用能有效优化扩增的目标产物,同时抑制非特异性产物的生成,是目前TaqMan探针法qPCR试剂中的核心组分。尽管近年来涌现了多种具有耐受抑制剂能力的DNA聚合酶,然而这些DNA聚合酶大多数因缺少5’-3’核酸外切酶活性而不能水解TaqMan探针,因此无法应用在直扩型TaqMan探针法qPCR中。
综上所述,亟需研究开发一种兼具抑制剂耐受能力强和高5’-3’核酸外切酶活性的热启动DNA聚合酶,以在直扩型TaqMan探针法qPCR检测中进行应用。
发明内容
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Taq DNA聚合酶与核酸内切酶嵌合体。
本发明的另一目的在于提供上述Taq DNA聚合酶与核酸内切酶嵌合体的制备方法。
本发明的再一目的在于提供上述Taq DNA聚合酶与核酸内切酶嵌合体的应用。
本发明的目的通过如下技术方案实现:
一种Taq DNA聚合酶与核酸内切酶嵌合体,命名为pFEN1-STaq,其氨基酸序列如SEQ ID NO.1所示。
所述的嵌合体与氨基酸序列如SEQ ID NO.2所示的野生型Taq DNA聚合酶(NCBI登录号为P19821.1)相比,具有以下特点:1~289位氨基酸缺失;626位谷氨酸(Glu)突变为精氨酸(Arg);707位异亮氨酸(Ile)突变为苏氨酸(Thr);708位谷氨酸(Glu)突变为谷氨酰胺(Gln);742位谷氨酸(Glu)突变为赖氨酸(Lys);且N末端融合了双链DNA结合蛋白,在双链DNA结合蛋白的N末端再融合了氨基酸序列如SEQ ID No.5所示的瓣状核酸内切酶1片段。
所述的双链结合蛋白优选为氨基酸序列如SEQ ID NO.3所示的Sso7d(NCBI登录号为WP_009990119.1)。双链结合蛋白可以显著提高DNA聚合酶对模板DNA的亲和力。
所述的瓣状核酸内切酶1片段与氨基酸序列如SEQ ID NO.6所示的野生型瓣状核酸内切酶1(NCBI登录号为WP_011012561.1)相比,具有以下特点:328~340位氨基酸缺失。与野生型Taq DNA聚合酶相比,瓣状核酸内切酶1有着更强的5’-3’核酸外切酶活性。瓣状核酸内切酶1可以使融合后的蛋白获得5’-3’核酸外切酶活性,能够水解TaqMan探针。
所述的双链DNA结合蛋白的C末端与经改造后Taq DNA聚合酶的N末端通过氨基酸序列如SEQ ID NO.4所示的连接子连接;所述的瓣状核酸内切酶1结构域的C末端与所述的双链DNA结合蛋白的N末端通过氨基酸序列如SEQ ID NO.7所示的连接子连接。
编码上述Taq DNA聚合酶与核酸内切酶嵌合体的DNA分子。
所述的DNA分子的核苷酸序列如SEQ ID NO.8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。
一种重组表达载体,是将编码上述Taq DNA聚合酶与核酸内切酶嵌合体的DNA分子克隆入表达载体得到。
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。
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。
所述的工程细胞优选为大肠杆菌细胞;更优选为大肠杆菌T7 Express-lysY/Iq。
上述Taq DNA聚合酶与核酸内切酶嵌合体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:
1)取上述重组工程细胞株,接种于SB培养基中,培养,得到种子液;
2)取所得种子液接种于SB培养基中,培养,得到菌液;
3)向所得菌液中加入IPTG至终浓度为0.1~0.5mmol/L,诱导细胞表达蛋白,离心收集菌体沉淀;
4)向所得菌体沉淀中加入裂解缓冲液重悬菌体,超声破碎菌体,离心取上清液;
5)所得上清液75℃孵育20~30min,冰浴10~20min,离心,0.22μm微孔滤膜过滤,取上清液;
6)通过镍离子亲和层析收集含有目的蛋白的样品洗脱液,即获得所述的嵌合体。
优选地,步骤1)和步骤2)中所述的SB培养基均含50μg/mL卡那霉素。
优选地,步骤1)中所述的培养的条件为37℃,150~200r/min振荡培养。
优选地,步骤2)中所述的培养的条件为37℃,150~200r/min振荡培养至OD600=0.6~0.8。
优选地,步骤2)中所述的种子液按1:100的体积比接种于SB培养基。
优选地,步骤3)中所述的诱导的条件为18℃诱导12~16h。
优选地,步骤3)所述的离心的条件为4℃以12000rpm的转速离心20~30min。
优选地,步骤4)中所述的裂解缓冲液的用量按每克菌体沉淀中加入5mL计。
优选地,步骤4)中所述的超声的条件为功率250W,超声5.5s,间隔5.5s,持续30min。
优选地,步骤4)和步骤5)中所述的离心的条件为4℃以12000rpm的转速离心10~20min。
优选地,步骤4)中所述的裂解缓冲液的组分为:50mmol/L Tris-HCl、50mmol/LNaCl、5%(v/v)甘油,pH9.0。
优选地,步骤6)中所述的镍离子亲和层析所用的结合缓冲液(Buffer A)的组分为:50mmol/L Tris-HCl、50mmol/L NaCl、5%(v/v)甘油,pH9.0;所用的洗脱缓冲液(BufferB)的组分为:50mmol/L Tris-HCl、50mmol/L NaCl、500mmol/L咪唑、5%(v/v)甘油,pH9.0。
一种热启动DNA聚合酶,是上述嵌合体的聚合酶活性位点赖氨酸侧链氨基上经特异性结合酸酐类化合物得到。该热启动DNA聚合酶通过可逆的封闭其聚合酶活性,以实现所述嵌合体的热启动修饰。
所述的酸酐类化合物优选为马来酸酐或柠康酸酐;更优选为柠康酸酐。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。
上述热启动DNA聚合酶的制备方法,包括如下步骤:
(1)将所述的嵌合体透析到Tris-HCl缓冲液中;
(2)加入酸酐类化合物,混合均匀,反应;
(3)将反应后的混合液透析到储存缓冲液中,即获得稳定的热启动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80℃,孵育10min仍无聚合酶活性释放;在95℃热激,8~10min才可释放活性。
所述的Tris-HCl缓冲液优选浓度为10~50mmol/L、pH=9~10的Tris-HCl缓冲液。
所述的嵌合体与酸酐类化合物的配比优选为摩尔比1:2500~1:3500。
所述的反应的条件优选为温度37℃~42℃、时间3~4h。
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,1mmol/LDTT,0.5%(v/v)Tween-20,pH7.4。
一种直扩PCR试剂盒,包含PCR用水、PCR反应缓冲液、引物、dNTPs中的至少一种,和上述嵌合体或热启动DNA聚合酶。
优选地,所述的PCR反应缓冲液的组成如下:20~50mmol/L Tris-HCl、10~30mmol/L KCl、5~20mmol/L(NH4)2SO4、3~6mmol/L MgSO4、0.05~0.10%(v/v)Triton X-100、0.1~0.3mol/L海藻糖、0.1~0.2mol/L L-肉毒碱、0.1~0.4%(v/v)NP-40,pH值为8.0~9.0。
优选地,所述的嵌合体或热启动DNA聚合酶在体系中的酶活力单位为1.25~2.5U/μL。
优选地,所述的dNTPs在体系中的浓度为100~300μmol/L。
优选地,所述的引物在体系中的浓度为0.2~0.4μmol/L。
一种直扩qPCR试剂盒,包含qPCR用水、qPCR反应缓冲液、引物、探针和dNTPs中的至少一种,和上述热启动DNA聚合酶。
优选地,所述的qPCR反应缓冲液的组成如下:10~30mmol/L Tris-HCl、10~30mmol/LKCl、5~20mmol/L(NH4)2SO4、3~6mmol/L MgSO4、0.05~0.10%(v/v)Triton X-100、pH值为8.0~9.0。
优选地,所述的热启动DNA聚合酶在体系中的酶活力单位为1.25~2.5U/μL。
优选地,所述的dNTPs在体系中的浓度为100~300μmol/L。
优选地,所述的引物在体系中的浓度为0.2~0.4μmol/L,所述的探针在体系中的浓度为0.3~0.4μmol/L。
上述直扩qPCR试剂盒在直扩型探针法qPCR中的应用。
所述的应用的具体操作为:采用所述直扩qPCR试剂盒对生物样本直接进行扩增(无需核酸提取),获得靶基因产物。
上述嵌合体,或上述热启动DNA聚合酶,或上述直扩PCR试剂盒,或上述直扩qPCR试剂盒在生物样本扩增和/或检测的应用。
所述的生物样本来源不受限制,包括但不限于全血。
本发明相较于现有技术具有如下的优点和有益效果:
1、本发明所述Taq DNA聚合酶与核酸内切酶嵌合体(pFEN1-STaq),与野生型TaqDNA聚合酶相比,有更高的耐受抑制剂的能力;同时在其N端融合的瓣状核酸内切酶1相较于野生型Taq DNA聚合酶,有更高的5’-3’核酸外切酶活性。
2、本发明所述热启动DNA聚合酶(hot-startpFEN1-STaq),在80℃及以下温度完全没有活性释放,降低了qPCR反应过程中非特异性产物扩增的可能性,提高了qPCR反应的特异性和灵敏度。
3、本发明所述嵌合体或热启动DNA聚合酶,直扩PCR至少可耐受40%(v/v)的全血,直扩TaqMan探针法qPCR至少可耐受8%(v/v)的全血,对血液等样品中存在的PCR抑制剂的抗性明显提高,无需经过复杂的基因组DNA提取和纯化步骤,不仅节约时间成本和物料设备成本,而且能够避免操作时发生样本间的交叉污染,使得检测更加方便、快捷、准确。同时,还为不能直接进行基因组DNA提取的珍贵微量血样中的DNA检测提供了一种可选择的方法。
附图说明
图1为Taq DNA聚合酶与核酸内切酶嵌合体的可溶性分析结果图;其中,泳道M为(10-180kDa)宽范围蛋白上样Marker,泳道1为诱导表达前菌体细胞破碎液,泳道2为诱导表达后菌体细胞破碎液,泳道3为细胞破碎后离心处理所得的上清液,泳道4为破碎液上清75℃加热30min离心处理后上清液。
图2为Taq DNA聚合酶与核酸内切酶嵌合体的纯化结果图;其中,泳道M为(10-180kDa)宽范围蛋白上样Marker,泳道1为诱导表达后菌体细胞破碎液,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离心处理后上清液,泳道4为层析流出组分,泳道5-14为层析洗脱组分。
图3为本发明嵌合体与市售野生型Taq DNA聚合酶的全血PCR直扩对比结果图;其中,泳道1以50ng从血液样本提纯的基因组DNA为模板,泳道2-6分别以5%(v/v)、10%(v/v)、20%(v/v)、30%(v/v)、40%(v/v)全血样本为模板。
图4为本发明热启动DNA聚合酶在不同信号通道下的qPCR扩增曲线结果图;其中,图A为FAM信号,图B为ROX信号、图C为HEX信号。
图5为本发明热启动DNA聚合酶在不同全血含量中的qPCR扩增对比结果图;其中,图A为全血含量0%(v/v)、图B为全血含量2%(v/v)、图C为全血含量4%(v/v)、图D为全血含量6%(v/v)、图E为全血含量8%(v/v)。
图6为本发明热启动DNA聚合酶在8%全血含量中qPCR扩增的重复结果图。
具体实施方式
下面结合具体实施例,进一步阐述本发明,但引用实施例仅用于说明本发明而不用于限制本发明的范围。本领域专业人员在没有进行创造性劳动的前提下做出的基于本发明的其他实施例,都属于本发明的权利保护范围。
除有特别说明,本发明中用到的各种试剂、原料均为可以从市场上购买的商品或者可以通过公知的方法制得的产品。
实施例1构建含编码嵌合体的核苷酸序列的重组载体
(1)根据所述Taq DNA聚合酶与核酸内切酶嵌合体的氨基酸序列(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,人工合成编码所述嵌合体的DNA分子,具体如SEQ ID NO.8所示。
(2)将编码Taq DNA聚合酶与核酸内切酶嵌合体的DNA分子与表达载体pET-28a进行同源重组。嵌合体扩增引物序列如下:
Taq-FP:5'-CCGCGCGGCAGCCATATGGGCGTGCCGATCGGTGA-3'(SEQ ID NO.9);
Taq-RP:5'-GACGGAGCTCGAATTTTATTCCTTCGCAGATAACC-3'(SEQ ID NO.10)。
pET-28a线性化引物序列如下:
pET-28a-FP:5'-AATTCGAGCTCCGTCGACAA-3'(SEQ ID NO.11);
pET-28a-RP:5'-ATATGGCTGCCGCGCGGCAC-3'(SEQ ID NO.12)。
分别以人工合成编码所述嵌合体的DNA分子及pET-28a空载为模板,加入25μL 2×Pfu Max HiFi PCR ProMix(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。嵌合体DNA分子的扩增条件为:98℃30s;98℃10s、58℃30s、68℃1.5min,共25个循环;68℃5min。质粒线性化PCR扩增程序为:98℃30s;98℃10s、58℃30s、68℃2.5min,共25个循环;68℃5min。琼脂糖凝胶电泳回收全长约3kb的嵌合体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5μL2×Hipro DNAAssembly Mix(广州英赞生物科技有限公司,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。
(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所述嵌合体的DNA分子的重组载体。
实施例2表达Taq DNA聚合酶与核酸内切酶嵌合体的转化体制备
将实施例1获得的重组载体转化到宿主细胞E.coliT7 Express-lysY/Iq中,挑取单菌落,接种至液体SB(含50μg/mL的硫酸卡那霉素)培养基培养至OD600为0.8,加入IPTG至终浓度为0.1mmol/L,18℃诱导16h,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嵌合体。
实施例3Taq DNA聚合酶与核酸内切酶嵌合体在重组大肠杆菌中的表达
将实施例2获得的能够表达嵌合体的阳性转化体菌种,接种至含50μg/mL硫酸卡那霉素的60mL SB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硫酸卡那霉素的SB培养基中,37℃摇床中震荡培养至OD600为0.8;向摇瓶中加入IPTG至终浓度为0.1mmol/L,18℃继续震荡诱导16h;离心收集诱导后菌体并称重,记录菌体湿重,储存于-20℃。
实施例4Taq DNA聚合酶与核酸内切酶嵌合体的纯化
1、诱导表达菌体超声破碎
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、50mmol/L NaCl、5%(v/v)甘油,pH9.0)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20min,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。各取样20μL,以诱导表达前菌体细胞破碎液作为对照,SDS-PAGE蛋白电泳检测,结果如图1所示。
2、镍离子亲和层析纯化
选用的层析柱是HisTrapTM HP 5mL(购自GE Healthcare),结合缓冲液为缓冲液A:50mmol/L Tris-HCl、50mmol/L NaCl、5%(v/v)甘油,pH9.0;洗脱缓冲液为缓冲液B:50mmol/LTris-HCl、50mmol/L NaCl、500mmol/L咪唑、5%(v/v)甘油,pH9.0,0.22μm滤膜过滤备用。
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,再用缓冲液A平衡柱子,然后用样品泵将步骤1所述上清液进行上样,上样结束后,先用缓冲液A清洗柱子,再用缓冲液B进行10%的梯度洗脱和10~60%的线性洗脱,并收集洗脱组分。洗脱峰取样20μL,SDS-PAGE蛋白电泳检测,结果如图2(泳道5-14)所示。
实施例5嵌合体的DNA聚合酶活性和热稳定性测试
取实施例4制备的Taq DNA聚合酶与核酸内切酶嵌合体,按照以下方法测定DNA聚合酶活性。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/LBSA、0.1%Tween-20、4%DMSO组成的pH 8.0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。结果显示嵌合体的聚合酶活性浓度为80U/μL。
将嵌合体稀释至聚合酶活性浓度为1U/μL,按下述方法测试嵌合体的热稳定性。将稀释后的酶于95℃分别孵育0、1、2、3、4、5h后,分别取样进行DNA聚合酶活性测定,结果显示嵌合体具有较高的热稳定性,其在95℃下的半衰期为3h。
实施例6嵌合体在PCR检测中全血耐受性研究
以健康人自愿捐赠的血液样本为模板,在反应体系中的添加量分别为5%(v/v)、10%(v/v)、20%(v/v)、30%(v/v)、40%(v/v),同时设立不加入全血的对照组别,以50ng从血液样本提纯的基因组DNA为模板,用本发明中的Taq DNA聚合酶与核酸内切酶嵌合体和市售野生型Taq DNA聚合酶(NEB,货号M0273S)同时扩增基因,扩增引物和具体操作如下:
β-Actin-FP:5’-CAGCGGAACCGCTCATTGCCAATGG-3’(SEQ ID NO.13);
β-Actin-RP:5’-TCACCCACACTGTGCCCATCTACGA-3’(SEQ ID NO.14)。
其中嵌合体25μL PCR反应体系为:20mmol/L Tris-HCl、10mmol/LKCl、10mmol/L(NH4)2SO4,5mmol/L MgSO4、0.05%Triton X-100、0.3mol/L海藻糖、0.2mol/L L-肉毒碱、0.4%NP-40、200μmol/L dNTPs、0.2μmol/L上下游引物、1.25U嵌合体,pH 8.8;所述市售野生型Taq DNA聚合酶反应体系参考商品说明书进行配制。PCR反应程序如下:95℃3min;95℃15s、60℃30s、68℃1min,35个循环;68℃5min。将嵌合体和市售野生型Taq DNA聚合酶所得PCR产物进行2%琼脂糖凝胶电泳。结果显示所述市售野生型Taq DNA聚合酶几乎没有耐受全血的能力,Taq DNA聚合酶与核酸内切酶嵌合体在PCR反应中至少可耐受40%(v/v)全血,其全血耐受能力与野生型Taq DNA聚合酶相比提高了约40倍(图3)。
实施例7热启动DNA聚合酶的制备及测活
将高纯度的Taq DNA聚合酶与核酸内切酶嵌合体过夜透析到10~50mmol/LTris-HCl(pH为9.0~10.0)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1:2500混匀,并于37℃反应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。
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%(v/v)甘油,1mmol/L DTT,0.5%(v/v)Tween-20,pH7.4)中,95℃,热激10min后按实施例5的测活方法测定热启动DNA聚合酶的DNA聚合酶活性。结果表明,热启动DNA聚合酶的聚合酶活性浓度为5U/μL。
实施例8热启动DNA聚合酶在qPCR检测中的标准曲线建立
分别以拷贝数浓度为1×108-1×103copies/μL的新型冠状病毒序列上的特异性基因片段nCoV-ORF1ab(基因的NCBI登录号为43740578)重组质粒(pMD-18T-nCoV-ORF1ab,nCoV-ORF1ab插入的位置为限制性酶切位点EcoRⅤ)、新型冠状病毒核壳蛋白基因nCoV-E(基因的NCBI登录号为43740570)重组质粒(pMD-18T-nCoV-E,nCoV-E插入的位置为限制性酶切位点EcoRⅤ)和新型冠状病毒核壳蛋白基因片段nCoV-N(基因的NCBI登录号为43740575)重组质粒(pMD-18T-nCoV-N,nCoV-N插入的位置为限制性酶切位点EcoRⅤ)为模板,用本发明中的热启动DNA聚合酶扩增基因,扩增引物探针和具体操作如下:
ORF1ab-FP:5’-CCCTGTGGGTTTTACACTTAA-3’(SEQ ID NO.15);
ORF1ab-RP:5’-ACGATTGTGCATCAGCTGA-3’(SEQ ID NO.16)
ORF1ab-Probe:5’-FAM-CCGTCTGCGGTATGTGGAAAGGTTATGG-BHQ1-3’(SEQ IDNO.17)
E-FP:5’-ACAGGTACGTTAATAGTTAATAGCGT-3’(SEQ ID NO.18);
E-RP:5’-ATATTGCAGCAGTACGCACACA -3’(SEQ ID NO.19);
E-probe:5’-ROX-ACACTAGCCATCCTTACTGCGCTTCG-BHQ1-3’(SEQ ID NO.20)
N-FP:5’-GGGGAACTTCTCCTGCTAGAAT-3’(SEQ ID NO.21);
N-RP:5’-CAGACATTTTGCTCTCAAGCTG-3’(SEQ ID NO.22);
N-probe:5’-HEX-TTGCTGCTGCTTGACAGATT-BHQ1-3’(SEQ ID NO.23)。
其中热启动DNA聚合酶25μLqPCR反应体系为:10mmol/L Tris-HCl、20mmol/LKCl、5mmol/L(NH4)2SO4,6mmol/L MgSO4、0.05%Triton X-100、200μmol/L dNTPs、0.2μmol/L上下游引物、0.3μmol/L探针、2.5U热启动DNA聚合酶,pH 8.3。qPCR反应程序如下:95℃10min;95℃10s、60℃30s,45个循环。结果显示所述热启动DNA聚合酶的扩增效率近乎100%,可实现不同信号通道下普通样本的定性和定量检测(图4)。
实施例9热启动DNA聚合酶在qPCR检测中全血耐受性研究
以拷贝数浓度为1×108-1×103copies/μL的新型冠状病毒核壳蛋白基因nCoV-E重组质粒(pMD-18T-nCoV-E,nCoV-E插入的位置为限制性酶切位点EcoRⅤ)为模板,全血样本在反应体系中的添加量分别为0%(v/v)、2%(v/v)、4%(v/v)、6%(v/v)、8%(v/v),用本发明中的热启动DNA聚合酶扩增基因,扩增引物探针和具体操作如下:
E-FP:5’-ACAGGTACGTTAATAGTTAATAGCGT-3’;
E-RP:5’-ATATTGCAGCAGTACGCACACA-3’;
E-probe:5’-ROX-ACACTAGCCATCCTTACTGCGCTTCG-BHQ1-3’。
其中热启动DNA聚合酶25μLqPCR反应体系为:10mmol/L Tris-HCl、20mmol/LKCl、5mmol/L(NH4)2SO4,6mmol/L MgSO4、0.05%Triton X-100、200μmol/L dNTPs、0.2μmol/L上下游引物、0.3μmol/L探针、2.5U热启动DNA聚合酶,pH 8.3。qPCR反应程序如下:95℃10min;95℃10s、60℃30s,45个循环。结果显示所述热启动DNA聚合酶在qPCR反应中至少可耐受8%(v/v)全血,同时扩增效率近乎100%,可实现全血直扩qPCR中对模板的定量分析(图5)。
实施例10全血直扩qPCR的重复性检测及最低检测限
以拷贝数浓度为1×107copies/μL、1×105copies/μL、1×103copies/μL、1×102copies/μL、1×101copies/μL、1×100copies/μL的新型冠状病毒胞膜蛋白基因nCoV-E重组质粒(pMD-18T-nCoV-E,nCoV-E插入的位置为限制性酶切位点EcoRⅤ)为模板,每个浓度设置3个平行实验作为组内重复性检测,再将该实验重复3次作为组间重复性检测。全血样本在反应体系中的添加量为8%(v/v),用本发明中的热启动DNA聚合酶扩增基因,扩增引物探针和具体操作如下:
E-FP:5’-ACAGGTACGTTAATAGTTAATAGCGT-3’;
E-RP:5’-ATATTGCAGCAGTACGCACACA -3’;
E-probe:5’-ROX-ACACTAGCCATCCTTACTGCGCTTCG-BHQ1-3’。
其中热启动DNA聚合酶25μLqPCR反应体系为:10mmol/L Tris-HCl、20mmol/LKCl、5mmol/L(NH4)2SO4,6mmol/L MgSO4、0.05%Triton X-100、200μmol/L dNTPs、0.2μmol/L上下游引物、0.3μmol/L探针、2.5U热启动DNA聚合酶,pH 8.3。qPCR反应程序如下:95℃10min;95℃10s、60℃30s,45个循环。结果显示1×107-1×101copies/μL浓度条件下的组内重复和组间重复的变异系数均小于1.0%,所述热启动DNA聚合酶在8%(v/v)全血qPCR反应中有良好的重复性(图6),最低检测限为每uL样本中10-100copies的模板。
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。
<110> 华南理工大学
<120> 一种Taq DNA聚合酶与核酸内切酶嵌合体及其制备方法与应用
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<223> Taq DNA聚合酶与核酸内切酶嵌合体的氨基酸序列
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Met Gly Val Pro Ile Gly Glu Ile Ile Pro Arg Lys Glu Ile Glu Leu
1 5 10 15
Glu Asn Leu Tyr Gly Lys Lys Ile Ala Ile Asp Ala Leu Asn Ala Ile
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Tyr Gln Phe Leu Ser Thr Ile Arg Gln Lys Asp Gly Thr Pro Leu Met
35 40 45
Asp Ser Lys Gly Arg Ile Thr Ser His Leu Ser Gly Leu Phe Tyr Arg
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Thr Ile Asn Leu Met Glu Ala Gly Ile Lys Pro Val Tyr Val Phe Asp
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Gly Glu Pro Pro Glu Phe Lys Lys Lys Glu Leu Glu Lys Arg Arg Glu
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Ala Arg Glu Glu Ala Glu Glu Lys Trp Arg Glu Ala Leu Glu Lys Gly
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Glu Ile Glu Glu Ala Arg Lys Tyr Ala Gln Arg Ala Thr Arg Val Asn
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Glu Met Leu Ile Glu Asp Ala Lys Lys Leu Leu Glu Leu Met Gly Ile
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Pro Ile Val Gln Ala Pro Ser Glu Gly Glu Ala Gln Ala Ala Tyr Met
145 150 155 160
Ala Ala Lys Gly Ser Val Tyr Ala Ser Ala Ser Gln Asp Tyr Asp Ser
165 170 175
Leu Leu Phe Gly Ala Pro Arg Leu Val Arg Asn Leu Thr Ile Thr Gly
180 185 190
Lys Arg Lys Leu Pro Gly Lys Asn Val Tyr Val Glu Ile Lys Pro Glu
195 200 205
Leu Ile Ile Leu Glu Glu Val Leu Lys Glu Leu Lys Leu Thr Arg Glu
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Lys Leu Ile Glu Leu Ala Ile Leu Val Gly Thr Asp Tyr Asn Pro Gly
225 230 235 240
Gly Ile Lys Gly Ile Gly Leu Lys Lys Ala Leu Glu Ile Val Arg His
245 250 255
Ser Lys Asp Pro Leu Ala Lys Phe Gln Lys Gln Ser Asp Val Asp Leu
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Tyr Ala Ile Lys Glu Phe Phe Leu Asn Pro Pro Val Thr Asp Asn Tyr
275 280 285
Asn Leu Val Trp Arg Asp Pro Asp Glu Glu Gly Ile Leu Lys Phe Leu
290 295 300
Cys Asp Glu His Asp Phe Ser Glu Glu Arg Val Lys Asn Gly Leu Glu
305 310 315 320
Arg Leu Lys Lys Ala Ile Lys Ser Gly Gly Gly Ser Gly Gly Gly Gly
325 330 335
Ser Gly Gly Gly Gly Ser Met Ala Thr Val Lys Phe Lys Tyr Lys Gly
340 345 350
Glu Glu Lys Glu Val Asp Ile Ser Lys Ile Lys Lys Val Trp Arg Val
355 360 365
Gly Lys Met Ile Ser Phe Thr Tyr Asp Glu Gly Gly Gly Lys Thr Gly
370 375 380
Arg Gly Ala Val Ser Glu Lys Asp Ala Pro Lys Glu Leu Leu Gln Met
385 390 395 400
Leu Glu Lys Gln Lys Lys Gly Gly Val Thr Ser Pro Lys Ala Leu Glu
405 410 415
Glu Ala Pro Trp Pro Pro Pro Glu Gly Ala Phe Val Gly Phe Val Leu
420 425 430
Ser Arg Lys Glu Pro Met Trp Ala Asp Leu Leu Ala Leu Ala Ala Ala
435 440 445
Arg Gly Gly Arg Val His Arg Ala Pro Glu Pro Tyr Lys Ala Leu Arg
450 455 460
Asp Leu Lys Glu Ala Arg Gly Leu Leu Ala Lys Asp Leu Ser Val Leu
465 470 475 480
Ala Leu Arg Glu Gly Leu Gly Leu Pro Pro Gly Asp Asp Pro Met Leu
485 490 495
Leu Ala Tyr Leu Leu Asp Pro Ser Asn Thr Thr Pro Glu Gly Val Ala
500 505 510
Arg Arg Tyr Gly Gly Glu Trp Thr Glu Glu Ala Gly Glu Arg Ala Ala
515 520 525
Leu Ser Glu Arg Leu Phe Ala Asn Leu Trp Gly Arg Leu Glu Gly Glu
530 535 540
Glu Arg Leu Leu Trp Leu Tyr Arg Glu Val Glu Arg Pro Leu Ser Ala
545 550 555 560
Val Leu Ala His Met Glu Ala Thr Gly Val Arg Leu Asp Val Ala Tyr
565 570 575
Leu Arg Ala Leu Ser Leu Glu Val Ala Glu Glu Ile Ala Arg Leu Glu
580 585 590
Ala Glu Val Phe Arg Leu Ala Gly His Pro Phe Asn Leu Asn Ser Arg
595 600 605
Asp Gln Leu Glu Arg Val Leu Phe Asp Glu Leu Gly Leu Pro Ala Ile
610 615 620
Gly Lys Thr Glu Lys Thr Gly Lys Arg Ser Thr Ser Ala Ala Val Leu
625 630 635 640
Glu Ala Leu Arg Glu Ala His Pro Ile Val Glu Lys Ile Leu Gln Tyr
645 650 655
Arg Glu Leu Thr Lys Leu Lys Ser Thr Tyr Ile Asp Pro Leu Pro Asp
660 665 670
Leu Ile His Pro Arg Thr Gly Arg Leu His Thr Arg Phe Asn Gln Thr
675 680 685
Ala Thr Ala Thr Gly Arg Leu Ser Ser Ser Asp Pro Asn Leu Gln Asn
690 695 700
Ile Pro Val Arg Thr Pro Leu Gly Gln Arg Ile Arg Arg Ala Phe Ile
705 710 715 720
Ala Glu Glu Gly Trp Leu Leu Val Ala Leu Asp Tyr Ser Gln Ile Glu
725 730 735
Leu Arg Val Leu Ala His Leu Ser Gly Asp Arg Asn Leu Ile Arg Val
740 745 750
Phe Gln Glu Gly Arg Asp Ile His Thr Glu Thr Ala Ser Trp Met Phe
755 760 765
Gly Val Pro Arg Glu Ala Val Asp Pro Leu Met Arg Arg Ala Ala Lys
770 775 780
Thr Ile Asn Phe Gly Val Leu Tyr Gly Met Ser Ala His Arg Leu Ser
785 790 795 800
Gln Glu Leu Ala Ile Pro Tyr Glu Glu Ala Gln Ala Phe Ile Glu Arg
805 810 815
Tyr Phe Gln Ser Phe Pro Lys Val Arg Ala Trp Thr Gln Lys Thr Leu
820 825 830
Glu Glu Gly Arg Arg Arg Gly Tyr Val Glu Thr Leu Phe Gly Arg Arg
835 840 845
Arg Tyr Val Pro Asp Leu Glu Ala Arg Val Lys Ser Val Arg Lys Ala
850 855 860
Ala Glu Arg Met Ala Phe Asn Met Pro Val Gln Gly Thr Ala Ala Asp
865 870 875 880
Leu Met Lys Leu Ala Met Val Lys Leu Phe Pro Arg Leu Glu Glu Met
885 890 895
Gly Ala Arg Met Leu Leu Gln Val His Asp Glu Leu Val Leu Glu Ala
900 905 910
Pro Lys Glu Arg Ala Glu Ala Val Ala Arg Leu Ala Lys Glu Val Met
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Glu Gly Val Tyr Pro Leu Ala Val Pro Leu Glu Val Glu Val Gly Ile
930 935 940
Gly Glu Asp Trp Leu Ser Ala Lys Glu
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<223> 野生型Taq DNA聚合酶的氨基酸序列
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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> 64
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 双链DNA结合蛋白Sso7d的氨基酸序列
<400> 3
Met Ala Thr Val Lys Phe Lys Tyr Lys Gly Glu Glu Lys Glu Val Asp
1 5 10 15
Ile Ser Lys Ile Lys Lys Val Trp Arg Val Gly Lys Met Ile Ser Phe
20 25 30
Thr Tyr Asp Glu Gly Gly Gly Lys Thr Gly Arg Gly Ala Val Ser Glu
35 40 45
Lys Asp Ala Pro Lys Glu Leu Leu Gln Met Leu Glu Lys Gln Lys Lys
50 55 60
<210> 4
<211> 4
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 双链DNA结合蛋白与经改造后Taq DNA聚合酶N末端相连的连接肽序列
<400> 4
Gly Gly Val Thr
1
<210> 5
<211> 327
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 瓣状核酸内切酶1片段(pFEN1)的氨基酸序列
<400> 5
Met Gly Val Pro Ile Gly Glu Ile Ile Pro Arg Lys Glu Ile Glu Leu
1 5 10 15
Glu Asn Leu Tyr Gly Lys Lys Ile Ala Ile Asp Ala Leu Asn Ala Ile
20 25 30
Tyr Gln Phe Leu Ser Thr Ile Arg Gln Lys Asp Gly Thr Pro Leu Met
35 40 45
Asp Ser Lys Gly Arg Ile Thr Ser His Leu Ser Gly Leu Phe Tyr Arg
50 55 60
Thr Ile Asn Leu Met Glu Ala Gly Ile Lys Pro Val Tyr Val Phe Asp
65 70 75 80
Gly Glu Pro Pro Glu Phe Lys Lys Lys Glu Leu Glu Lys Arg Arg Glu
85 90 95
Ala Arg Glu Glu Ala Glu Glu Lys Trp Arg Glu Ala Leu Glu Lys Gly
100 105 110
Glu Ile Glu Glu Ala Arg Lys Tyr Ala Gln Arg Ala Thr Arg Val Asn
115 120 125
Glu Met Leu Ile Glu Asp Ala Lys Lys Leu Leu Glu Leu Met Gly Ile
130 135 140
Pro Ile Val Gln Ala Pro Ser Glu Gly Glu Ala Gln Ala Ala Tyr Met
145 150 155 160
Ala Ala Lys Gly Ser Val Tyr Ala Ser Ala Ser Gln Asp Tyr Asp Ser
165 170 175
Leu Leu Phe Gly Ala Pro Arg Leu Val Arg Asn Leu Thr Ile Thr Gly
180 185 190
Lys Arg Lys Leu Pro Gly Lys Asn Val Tyr Val Glu Ile Lys Pro Glu
195 200 205
Leu Ile Ile Leu Glu Glu Val Leu Lys Glu Leu Lys Leu Thr Arg Glu
210 215 220
Lys Leu Ile Glu Leu Ala Ile Leu Val Gly Thr Asp Tyr Asn Pro Gly
225 230 235 240
Gly Ile Lys Gly Ile Gly Leu Lys Lys Ala Leu Glu Ile Val Arg His
245 250 255
Ser Lys Asp Pro Leu Ala Lys Phe Gln Lys Gln Ser Asp Val Asp Leu
260 265 270
Tyr Ala Ile Lys Glu Phe Phe Leu Asn Pro Pro Val Thr Asp Asn Tyr
275 280 285
Asn Leu Val Trp Arg Asp Pro Asp Glu Glu Gly Ile Leu Lys Phe Leu
290 295 300
Cys Asp Glu His Asp Phe Ser Glu Glu Arg Val Lys Asn Gly Leu Glu
305 310 315 320
Arg Leu Lys Lys Ala Ile Lys
325
<210> 6
<211> 340
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 野生型瓣状核酸内切酶1(wt-pFEN1)的氨基酸序列
<400> 6
Met Gly Val Pro Ile Gly Glu Ile Ile Pro Arg Lys Glu Ile Glu Leu
1 5 10 15
Glu Asn Leu Tyr Gly Lys Lys Ile Ala Ile Asp Ala Leu Asn Ala Ile
20 25 30
Tyr Gln Phe Leu Ser Thr Ile Arg Gln Lys Asp Gly Thr Pro Leu Met
35 40 45
Asp Ser Lys Gly Arg Ile Thr Ser His Leu Ser Gly Leu Phe Tyr Arg
50 55 60
Thr Ile Asn Leu Met Glu Ala Gly Ile Lys Pro Val Tyr Val Phe Asp
65 70 75 80
Gly Glu Pro Pro Glu Phe Lys Lys Lys Glu Leu Glu Lys Arg Arg Glu
85 90 95
Ala Arg Glu Glu Ala Glu Glu Lys Trp Arg Glu Ala Leu Glu Lys Gly
100 105 110
Glu Ile Glu Glu Ala Arg Lys Tyr Ala Gln Arg Ala Thr Arg Val Asn
115 120 125
Glu Met Leu Ile Glu Asp Ala Lys Lys Leu Leu Glu Leu Met Gly Ile
130 135 140
Pro Ile Val Gln Ala Pro Ser Glu Gly Glu Ala Gln Ala Ala Tyr Met
145 150 155 160
Ala Ala Lys Gly Ser Val Tyr Ala Ser Ala Ser Gln Asp Tyr Asp Ser
165 170 175
Leu Leu Phe Gly Ala Pro Arg Leu Val Arg Asn Leu Thr Ile Thr Gly
180 185 190
Lys Arg Lys Leu Pro Gly Lys Asn Val Tyr Val Glu Ile Lys Pro Glu
195 200 205
Leu Ile Ile Leu Glu Glu Val Leu Lys Glu Leu Lys Leu Thr Arg Glu
210 215 220
Lys Leu Ile Glu Leu Ala Ile Leu Val Gly Thr Asp Tyr Asn Pro Gly
225 230 235 240
Gly Ile Lys Gly Ile Gly Leu Lys Lys Ala Leu Glu Ile Val Arg His
245 250 255
Ser Lys Asp Pro Leu Ala Lys Phe Gln Lys Gln Ser Asp Val Asp Leu
260 265 270
Tyr Ala Ile Lys Glu Phe Phe Leu Asn Pro Pro Val Thr Asp Asn Tyr
275 280 285
Asn Leu Val Trp Arg Asp Pro Asp Glu Glu Gly Ile Leu Lys Phe Leu
290 295 300
Cys Asp Glu His Asp Phe Ser Glu Glu Arg Val Lys Asn Gly Leu Glu
305 310 315 320
Arg Leu Lys Lys Ala Ile Lys Ser Gly Lys Gln Ser Thr Leu Glu Ser
325 330 335
Trp Phe Lys Arg
340
<210> 7
<211> 15
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 瓣状核酸内切酶1片段与双链DNA结合蛋白N末端相连的连接肽序列
<400> 7
Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
1 5 10 15
<210> 8
<211> 2862
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 编码Taq DNA聚合酶与核酸内切酶嵌合体的核苷酸序列
<400> 8
atgggcgtgc cgatcggtga aatcatccca cgtaaagaaa tcgagctgga gaacctgtac 60
ggtaaaaaaa ttgctatcga cgctctcaac gccatttacc agttcctgtc aactatccgt 120
cagaaagacg gcactccgct catggatagc aagggtcgta ttacctctca cctgtccggc 180
ctgttctacc gtacgatcaa tctgatggaa gcagggatta aaccggtcta tgtgttcgat 240
ggcgaaccgc cagagttcaa aaagaaagag ttggaaaaac gccgtgaagc acgtgaagaa 300
gcggaagaaa aatggcgtga agctctggaa aaaggcgaaa tcgaagaagc gcgtaaatac 360
gcccagcgtg cgacccgtgt caatgaaatg ctgatcgaag acgccaaaaa actgctggaa 420
ttgatgggta tccctatcgt gcaggctcca tctgaaggcg aagctcaagc ggcgtatatg 480
gccgcaaaag gctctgttta tgcgtctgct tcccaagatt acgactccct gctgtttggt 540
gcaccgcgcc tggtgcgtaa cctgaccatc acgggtaagc gtaagttgcc gggtaagaac 600
gtttatgtgg aaattaaacc tgaactgatt attctggaag aggtcctgaa agagctgaaa 660
ctgacacgcg aaaaactgat tgaactggct atcctggttg gcacagacta caacccaggc 720
ggtatcaaag gcatcggtct gaaaaaagcg cttgaaatcg tgcgtcacag taaagatccg 780
ctggctaagt ttcagaaaca gagcgacgtg gacctgtatg caattaaaga gttcttcctg 840
aaccctccgg ttactgataa ctacaacctg gtttggcgcg atccagacga ggagggtatc 900
ctgaaatttc tgtgtgatga acacgatttc tccgaggaac gtgttaaaaa cggtctggag 960
cgtctgaaga aggcgatcaa atctggcggt ggtagcggtg gcggcggttc tggcggtggt 1020
ggcagcatgg cgaccgttaa gtttaagtac aaaggtgaag aaaaggaagt ggacatatcc 1080
aagatcaaga aagtttggcg cgtgggcaag atgatttcat ttacctacga tgaaggtggc 1140
gggaagacgg gtcgtggggc cgtgagcgag aaggacgctc ccaaagaact gttgcaaatg 1200
cttgagaagc agaaaaaagg cggcgtcact agcccgaagg cacttgagga agctccttgg 1260
cctccgcctg agggcgcttt tgtcggattt gtcttgagcc gtaaagaacc gatgtgggcg 1320
gacttactgg cccttgctgc tgctcgtggg ggtcgcgtgc atcgcgcacc ggagccatac 1380
aaagcacttc gtgaccttaa agaagcccgt ggcttgttgg caaaagattt aagtgtcctg 1440
gctttacgcg agggcttggg cttaccaccg ggagatgatc cgatgctttt ggcctatctg 1500
ctggacccga gcaacacgac tccagagggc gttgcccgtc gttatggcgg agaatggacg 1560
gaggaggcgg gagagcgcgc agcgttaagc gagcgtctgt ttgctaatct gtggggacgc 1620
ttagagggag aggagcgcct gttgtggttg taccgtgaag tggaacggcc gctgagtgca 1680
gtgttagctc acatggaagc aaccggggtg cggctggacg ttgcgtattt gcgtgcgctg 1740
tcgttagagg tcgcggagga aatagcccgt ctggaggccg aagtattccg tttggctggc 1800
catcctttca acctgaacag tcgggatcag ctggaacgtg tactttttga tgaactgggg 1860
ctgcccgcca tcggcaaaac cgaaaaaacc ggcaaacgta gcacctctgc ggcagtgctg 1920
gaagcgttac gtgaagctca tccgattgtg gagaaaattc tgcaatatcg cgaattgacg 1980
aaactgaaga gcacctatat tgatccgctg ccagacttaa ttcacccccg taccggacgg 2040
ttgcataccc gcttcaacca gaccgcgacg gcgacagggc ggctgagtag cagcgatccg 2100
aacctgcaaa acattcccgt gcgtaccccg ctgggtcagc gtattcgccg tgctttcatt 2160
gccgaggaag gctggctgct ggtcgcgctg gactactcgc aaatcgaatt gcgtgtgttg 2220
gcccacctgt cgggcgaccg taacttgata cgcgtgtttc aagaaggtcg tgacatacat 2280
actgaaaccg cgtcctggat gtttggagtc ccacgggagg ctgtcgatcc tcttatgcgt 2340
cgtgccgcca aaacaattaa cttcggagtt ctgtacggca tgtcggcaca tcgtttatca 2400
caggaactgg cgattccgta tgaagaagcg caggccttca tagaacgtta tttccaatca 2460
ttccccaagg tgcgggcctg gacacaaaag accctggaag agggccgtcg tcgtggctat 2520
gtagagactc tgttcggacg tcggcggtat gtacccgatc ttgaggcccg tgtgaagtcc 2580
gttcgtaagg cagcagaacg tatggcgttt aacatgccag tccagggcac agcggcggac 2640
ctgatgaaat tagctatggt taagctgttt ccgcgtttgg aagaaatggg cgctcgtatg 2700
ctgttacagg ttcatgacga gttagtatta gaagcaccga aggagcgtgc cgaagccgtg 2760
gcccggttag ccaaagaggt aatggaaggc gtctaccccc ttgcagtccc gcttgaagtc 2820
gaagttggca taggggaaga ctggttatct gcgaaggaat aa 2862
<210> 9
<211> 35
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> Taq-FP
<400> 9
ccgcgcggca gccatatggg cgtgccgatc ggtga 35
<210> 10
<211> 35
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> Taq-RP
<400> 10
gacggagctc gaattttatt ccttcgcaga taacc 35
<210> 11
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> pET-28a-FP
<400> 11
aattcgagct ccgtcgacaa 20
<210> 12
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> pET-28a-RP
<400> 12
atatggctgc cgcgcggcac 20
<210> 13
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> β-Actin-FP
<400> 13
cagcggaacc gctcattgcc aatgg 25
<210> 14
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> β-Actin- RP
<400> 14
tcacccacac tgtgcccatc tacga 25
<210> 15
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> ORF1ab-FP
<400> 15
ccctgtgggt tttacactta a 21
<210> 16
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> ORF1ab-RP
<400> 16
acgattgtgc atcagctga 19
<210> 17
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> ORF1ab-Probe
<400> 17
ccgtctgcgg tatgtggaaa ggttatgg 28
<210> 18
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> E-FP
<400> 18
acaggtacgt taatagttaa tagcgt 26
<210> 19
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> E-RP
<400> 19
atattgcagc agtacgcaca ca 22
<210> 20
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> E-probe
<400> 20
acactagcca tccttactgc gcttcg 26
<210> 21
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> N-FP
<400> 21
ggggaacttc tcctgctaga at 22
<210> 22
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> N-RP
<400> 22
cagacatttt gctctcaagc tg 22
<210> 23
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> N-probe
<400> 23
ttgctgctgc ttgacagatt 20
Claims (10)
1.一种Taq DNA聚合酶与核酸内切酶嵌合体,其特征在于:命名为pFEN1-STaq,其氨基酸序列如SEQ ID NO.1所示。
2.编码权利要求1所述的嵌合体的DNA分子,其特征在于:所述的DNA分子的核苷酸序列如SEQ ID NO.8所示。
3.含有权利要求2所述的DNA分子的重组表达载体或重组工程细胞株。
4.权利要求1所述的嵌合体的制备方法,其特征在于:包括如下步骤:
1)取权利要求3所述的重组工程细胞株,接种于SB培养基中,培养,得到种子液;
2)取所得种子液接种于SB培养基中,培养,得到菌液;
3)向所得菌液中加入IPTG至终浓度为0.1~0.5mmol/L,诱导细胞表达蛋白,离心收集菌体沉淀;
4)向所得菌体沉淀中加入裂解缓冲液重悬菌体,超声破碎菌体,离心取上清液;
5)所得上清液75℃孵育20~30min,冰浴10~20min,离心,0.22μm微孔滤膜过滤,取上清液;
6)进行镍离子亲和层析,蛋白变性电泳检测各个洗脱峰,收集含有目的蛋白的样品,即获得所述的嵌合体;
其中,步骤1)和步骤2)中所述的SB培养基均含50μg/mL卡那霉素;
步骤1)中所述的培养的条件为37℃,150~200r/min振荡培养;
步骤2)中所述的培养的条件为37℃,150~200r/min振荡培养至OD600=0.6~0.8;
步骤2)中所述的种子液按1:100的体积比接种于SB培养基;
步骤3)中所述的诱导的条件为18℃诱导12~16h;
步骤3)所述的离心的条件为4℃以12000rpm的转速离心20~30min;
步骤4)中所述的裂解缓冲液的用量按每克菌体沉淀中加入5mL计;
步骤4)中所述的超声的条件为功率250W,超声5.5s,间隔5.5s,持续30min;
步骤4)中所述的离心的条件为4℃以12000rpm的转速离心10~20min;
步骤4)中所述的裂解缓冲液的组分为:50mmol/L Tris-HCl、50mmol/L NaCl、5%v/v甘油,pH9.0;
步骤6)中所述的镍离子亲和层析所用的结合缓冲液的组分为:50mmol/L Tris-HCl、50mmol/L NaCl、5%v/v甘油,pH9.0;所用的洗脱缓冲液的组分为:50mmol/L Tris-HCl、50mmol/L NaCl、500mmol/L咪唑、5%v/v甘油,pH9.0。
5.一种热启动DNA聚合酶,其特征在于:是权利要求1所述的嵌合体的聚合酶活性位点赖氨酸侧链氨基上经特异性结合酸酐类化合物得到;所述的酸酐类化合物为马来酸酐或柠康酸酐。
6.权利要求5所述的热启动DNA聚合酶的制备方法,其特征在于:包括如下步骤:
(1)将权利要求1所述的嵌合体透析到Tris-HCl缓冲液中;
(2)加入酸酐类化合物,混合均匀,反应;
(3)将反应后的混合液透析到储存缓冲液中,即获得稳定的热启动DNA聚合酶;
所述的Tris-HCl缓冲液是浓度为10~50mmol/L、pH=9~10的Tris-HCl缓冲液;
所述的嵌合体与酸酐类化合物的配比为摩尔比1:2500~1:3500;
所述的反应的条件为温度37℃~42℃、时间3~4h;
所述的储存缓冲液的配方为:20mmol/L Tris-HCl,100mmol/L KCl,0.1mmol/L EDTA,50%甘油,1mmol/LDTT,0.5%v/vTween-20,pH7.4。
7.一种直扩PCR试剂盒,其特征在于:包含PCR用水、PCR反应缓冲液、引物和dNTPs中的至少一种,和权利要求1所述的嵌合体或权利要求5所述的热启动DNA聚合酶;
其中,所述的PCR反应缓冲液的组成如下:20~50mmol/L Tris-HCl、10~30mmol/LKCl、5~20mmol/L(NH4)2SO4、3~6mmol/L MgSO4、0.05~0.10%v/vTriton X-100、0.1~0.3mol/L海藻糖、0.1~0.2mol/L L-肉毒碱、0.1~0.4%v/vNP-40,pH值为8.0~9.0;
所述的嵌合体或热启动DNA聚合酶在体系中的酶活力单位为1.25~2.5U/μL;
所述的dNTPs在体系中的浓度为100~300μmol/L;
所述的引物在体系中的浓度为0.2~0.4μmol/L。
8.一种直扩qPCR试剂盒,其特征在于:包含qPCR用水、qPCR反应缓冲液、引物、探针和dNTPs中的至少一种,和权利要求5所述的热启动DNA聚合酶;
其中,所述的qPCR反应缓冲液的组成如下:10~30mmol/L Tris-HCl、10~30mmol/LKCl、5~20mmol/L(NH4)2SO4、3~6mmol/L MgSO4、0.05~0.10%v/vTriton X-100、pH值为8.0~9.0;
所述的热启动DNA聚合酶在体系中的酶活力单位为1.25~2.5U/μL;
所述的dNTPs在体系中的浓度为100~300μmol/L;
所述的引物在体系中的浓度为0.2~0.4μmol/L;
所述的探针在体系中的浓度为0.3~0.4μmol/L。
9.权利要求8所述的直扩qPCR试剂盒在直扩型探针法qPCR中的应用,其特征在于:
所述的应用的操作为:采用所述的直扩qPCR试剂盒对生物样本直接进行扩增,获得靶基因产物。
10.权利要求1所述的嵌合体,或权利要求5所述的热启动DNA聚合酶,或权利要求7所述的直扩PCR试剂盒,或权利要求8所述的直扩qPCR试剂盒在生物样本扩增和/或检测的应用。
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