本发明的主题是融合单链dna聚合酶bst及其制备方法。本发明的主题还是编码根据bst聚合酶的三种变体：全长、大片段、短片段中的一种的融合聚合酶neqssb-bst的核酸分子，以及融合dna聚合酶用于等温扩增反应的用途。
背景技术：
：：dna聚合酶是在dna复制和修复过程中起重要作用的酶。它们被广泛应用于各个科学领域，并成功用于测序pcr或各种pcr(聚合酶链反应)变体中，在该pcr变体中，它们在体外催化dna合成过程，并且该反应以精确定义的热阶段进行循环。另一种越来越流行的方法是在dna扩增的等温技术中利用dna聚合酶，这些等温技术不基于热循环，并且反应在恒定的延伸温度下进行。迄今为止，已经开发出许多这种用于dna扩增和rna扩增的技术。用于给定技术的合适聚合酶的选择主要取决于其性质。除了基本的聚合能力外，由于存在核酸外切结构域或逆转录酶活性，聚合酶还表现出水解dna分子的能力。这些特征由各个结构域的存在来确定。这些酶中存在的基本结构域是聚合结构域和3'-5'和5'-3'核酸外切结构域。存在如下的聚合酶，在该聚合酶中，核酸外切结构域的缺失使得获得与天然酶相比具有部分改变的特征的功能蛋白。这种类型中最流行的聚合酶是从水生栖热细菌中分离出的taq聚合酶，该taq聚合酶的发现完全改变了分子生物学。不具有5'-3'核酸外切酶活性，聚合酶显示出更高的热稳定性，同时它需要更多的mg2+离子，并且新形成的dna链包含的错误更少。bst聚合酶用于等温扩增技术。它的天然形式包含非活性3'-5'核酸外切结构域和活性5'-3'核酸外切结构域，这些活性可以因在73位(tyr73→phe73和tyr73→ala73)中的点突变而丧失。该聚合酶以及taq聚合酶是a家族的一部分，并且从细菌嗜热脂肪芽孢杆菌中分离出。它的最佳活性约为60℃，并且没有核酸外切酶活性，该聚合酶表现出链置换活性，该链置换活性在环介导的等温扩增(lamp)反应中非常有用。与该家族中的其他聚合酶相比，该聚合酶对临床或环境抑制剂的耐受性有所提高，但考虑到该聚合酶的应用，寻找能够主要能使得其进行性和抗抑制剂性提高的解决方案是重要的。neqssb蛋白是单链dna结合(single-strandedbinding,ssb)蛋白家族的成员。ssb蛋白具有多种氨基酸序列和结构。然而，它们仍然包含一个由约100个氨基酸组成的特征性高度保守的寡核苷酸/寡糖结合(ob)折叠结构域。该结构域广泛存在于表现出ssdna结合能力的蛋白质中，并因此决定了所有ssb蛋白质共有的基本特征-对单链dna的非特异性结合以及后来发现的rna结合能力。ssb蛋白在与ssdna紧密相关的过程中起着关键作用。它们在复制、重组和dna修复中至关重要。这些蛋白质负责与单链dna的相互作用、防止二级结构的产生并防止被核酸酶降解。ssb蛋白的发现可追溯到1960年代上半叶。首先被发现的ssb蛋白是t4噬菌体和大肠杆菌的ssb蛋白。在发现过程中，阐明了它们强的与ssdna相互作用和使用高盐浓度(2mnacl)的ssdna-纤维素珠洗脱蛋白的能力。此外，还发现了该蛋白质对单链dna的非常高的选择性。ssb蛋白在与ssdna相关的过程中的基本作用被以下事实得到证实：这些蛋白存在于所有活生物体以及病毒中。ssb蛋白与ssdna的结合是基于寡核苷酸链残基之间的芳香族氨基酸残基的堆积。而且，带正电荷的氨基酸残基与ssdna分子的磷酸骨架相互作用。尽管neqssb蛋白属于ssb蛋白家族的事实，但它因其特征而与经典ssb蛋白偏离，因此它被称为neqssb样蛋白。该蛋白源自超嗜热古菌nanoarchaeumequitans，其是craenarchaeonignicoccushospitalis的寄生生物。这种微生物的最佳生长条件需要严格的厌氧条件和90℃的温度。有趣的是，nanoarchaeumequitans包含最小的已知基因组，其由490885个碱基对组成。与大多数已知的基因组减少的生物体相反，该微生物包含参与复制、修复和dna重组的全套酶，包括ssb蛋白。neqssb蛋白以及该家族的其他蛋白具有与dna结合的天然能力。它由243个氨基酸残基组成，并且在其结构中包含一个ob结构域，并且与某些病毒ssb蛋白类似，作为单体具有生物活性。报告显示，与其他ssb蛋白相比，neqssb蛋白表现出涉及没有结构偏好地与所有dna形式(ssdna、dsdna)和mrna结合的不同寻常的能力。而且，该蛋白质表现出高的热稳定性。在能保持生物活性的同时，半衰期在100℃下为5分钟，而变性温度(meltingtemperature)为100.2℃。为了满足现代诊断、分子生物学或基因工程提出的要求，需要改进dna聚合酶以在这些科学领域中提供有用的功能。迄今为止引入的修饰主要集中在引入改良的缓冲液、扩增反应增强剂或dna聚合酶的突变。突变使得获得热稳定性和对临床或环境样品中存在的抑制剂的抗性增加的酶。dna聚合酶的作用机制包括几个重要步骤。第一步由酶与dna基质的连接组成。由于3'oh端对核苷酸磷原子的亲核进攻而使所获得的dna-dna复合物将相应的dntps(脱氧核糖核苷酸三磷酸)缔合。最后一步导致磷酸二酯键的产生和焦磷酸的释放。这些酶的聚合作用的负责其最终效率的重要阶段之一是与基质dna结合有关的初始过程。由于这个原因，对已知聚合酶进行修饰是合理的，以便于结合到进行聚合的dna链。这种修饰的示例可以是产生与表现出能与单链和/或双链dna结合的天然能力的蛋白融合的融合dna聚合酶。文献仅呈现了这种融合dna聚合酶的几个示例，其中大多数示例是与主要用于聚合酶链反应的热稳定酶的融合。研究表明，taq、pfu、tpa或koddna聚合酶与超嗜热古菌sulfolobussolfataricus的dna结合蛋白sso7d的融合可使聚合酶的进行性增加5倍至17倍。类似地，在与其结合单链dna的天然ssb蛋白(rb69ssb)融合后，观察到rb69噬菌体的dna聚合酶的进行性(processivity)和忠实性增加。欧洲专利ep1934372b1公开了热球菌zilligi的与古菌sulfolobussolfataricus的ssossb蛋白融合的dna聚合酶，其表明了修饰后的酶的效率和进行性增加。此外，最近报道了taqstoffel聚合酶与能够结合所有类型的dna的neqssb蛋白以及p.furiosus连接酶的dbd结构域的融合。两种融合均使得酶功能特性得到提高，特别是提高了天然酶的进行性和热稳定性，并显著增加了其对临床抑制剂(乳铁蛋白、肝素、全血)的耐受性。还进行了等温反应中使用的聚合酶(诸如bst和)的少量融合。这些聚合酶通过methanopyruskandleri的拓扑异构酶v的hhh(螺旋-发夹-螺旋)结构域连接，这增加了聚合酶与dna的亲和力，而对链置换活性没有负面影响(在融合聚合酶bst和中)。而且，观察到使用质粒和基因组dna的更高的忠实性和扩增效率(在为的情况下)。文献还呈现了从地芽孢杆菌(geobacillussp.)777中分离出的bst样聚合酶的融合。产生了聚合酶与连接酶pyrococcusabyssi的dbd结构域和sto7d蛋白的嵌合体，并且与天然聚合酶相比，该嵌合体表现出在进行性和对抑制剂(尿素、全血、肝素、edta、nacl和乙醇)的抗性方面的增强。技术实现要素：本发明的目的是提供一种与结合所有类型的dna和rna的neqssb蛋白融合的融合dna聚合酶bst。出人意料的是，本发明在很大程度上解决了该问题。本发明的主题是一种与结合所有类型的dna和rna的neqssb蛋白融合的融合dna聚合酶bst。对以下三种bst聚合酶变体进行了修饰：全长-由于点突变而丧失5'-3'活性的dna|bst聚合酶的整个氨基酸序列；大片段-无5'-3'结构域的dna|bst聚合酶；以及短片段-两个核酸外切结构域都缺失的短版本。使用包含六个氨基酸的接头，通过聚合酶n端将bst聚合酶的所有变体与neqssb蛋白融合。本发明的本质是单链dna聚合酶bst或这类dna聚合酶的另一种聚合酶的融合聚合酶，该单链dna聚合酶bst或这类dna聚合酶的另一种聚合酶与neqssb蛋白或与序列在不高于50％的程度上与neqssb相似的蛋白在聚合酶的n端使用示例性氨基酸序列gly-ser-gly-gly-val-asp的接头连接或在不使用接头的情况下直接融合，其中所述聚合酶以三种不同的变体存在。融合dna聚合酶neqssb-bst，其包含以下三种bst聚合酶变体中的一种：-全长-由于点突变而使5'-3'活性丧失的dna聚合酶|bst的整个氨基酸序列；-大片段-无5'-3'结构域的dna聚合酶|bst；-短片段-两个外切核酸结构域都缺失的短版本。融合dna聚合酶neqssb-bst，其与所有类型的dna和rna结合。具有seq1中所示的序列的融合dna聚合酶neqssb-bst。具有seq2中所示的序列的融合dna聚合酶neqssb-bst。具有seq3中所示的序列的融合dna聚合酶neqssb-bst。seq4中所示的编码融合dna聚合酶neqssb-bst全长的核酸分子。seq5中所示的编码融合dna聚合酶neqssb-bst大片段的核酸分子。seq6中所示的编码融合dna聚合酶neqssb-bst短片段的核酸分子。上面限定的编码融合dna聚合酶neqssb-bst的核酸分子。上面限定的融合dna聚合酶neqssb-bst的制备方法，在该制备方法中：-第一步包括在优化的条件下，在微生物振荡器中表达编码该酶的基因：生长温度28℃至37℃，诱导后培养基的温育时间-3h至20h，诱导剂浓度-0.1mm至1mmiptg，-使用超声对所获得的细胞裂解物进行分解，并使用dsdnase消除dna基因组污染。-第二纯化步骤利用使用his-trap珠的金属亲和色谱，-下一步骤包括对制剂的三重透析(10mmtris-hcl，ph7.1，50mmkcl，1mmdtt，0.1mmedta，50％甘油，0.1％tritonx-100)、凝胶过滤和浓缩。-所有过程均在4℃下进行，-使用sds-page电泳测试所获得的蛋白的纯度，并使用evaez荧光聚合酶活性测定试剂盒确定所获得的制剂的单位数量。上面限定的融合单链dna聚合酶bst在体外用于等温扩增反应的用途。序列和附图说明序列1-示出了融合聚合酶neqssb-bst全长的氨基酸序列，序列2-示出了融合聚合酶neqssb-bst大片段的氨基酸序列，序列3-示出了融合聚合酶neqssb-bst短片段的氨基酸序列，序列4-示出了编码融合dna聚合酶neqssb-bst全长的基因的序列，序列5-示出了编码融合dna聚合酶neqssb-bst大片段的基因的序列，序列6-示出了编码融合dna聚合酶neqssb-bst短片段的基因的序列，图1-示出了在融合dna聚合酶纯化的各个阶段中蛋白质的10％聚丙烯酰胺凝胶电泳分离，m-蛋白质量标记物(thermo-fischerscientific)，其具有标准蛋白的质量：116kda；66.2kda；45kda；35kda；25kda；18.4kda；14.4kda；1-重组大肠杆菌top10f′-petneqssb-bst菌株的整个无细胞提取物；2-经过初步热变性的整个无细胞提取物；3-未与his-trap色谱柱结合的部分；4-含有40mm咪唑的his-trap珠的洗涤部分；5-含有100mm咪唑的his-trap珠的洗涤部分；6-在使用500mm咪唑洗脱后收集的含有融合dna聚合酶的部分；图2-示出了关于evagreen染料荧光与从融合dna聚合酶的dna扩增开始的时间的依赖关系的图表，其使得能够计算dna聚合酶的单位数量。图例将反应中使用的dna聚合酶的量以微升分配成曲线。图3-示出了在各种表达条件下的裂解物的10％聚丙烯酰胺凝胶电泳分离，m-蛋白质量标记物(thermo-fischerscientific)，其具有标准蛋白的质量：116kda；66.2kda；45kda；35kda；25kda；18.4kda；14.4kda；1-重组大肠杆菌top10f′-petneqssb-bst菌株在诱导前的整个无细胞提取物；2-用1mmiptg诱导后3小时的整个无细胞提取物，在28℃下进行表达；3-用1mmiptg诱导后4小时的整个无细胞提取物，在28℃下进行表达4-用1mmiptg诱导后5小时的整个无细胞提取物，在28℃下进行表达5-用1mmiptg诱导后6小时的整个无细胞提取物，在28℃下进行表达6-用1mmiptg诱导后20小时的整个无细胞提取物，在28℃下进行表达7-用0.1mmiptg诱导后3小时的整个无细胞提取物，在28℃进行表达；8-用0.1mmiptg诱导后4小时的整个无细胞提取物，在28℃下进行表达9-用0.1mmiptg诱导后5小时的整个无细胞提取物，在28℃下进行表达10-用0.1mmiptg诱导后6小时的整个无细胞提取物，在28℃下进行表达11-用0.1mmiptg诱导后20小时的整个无细胞提取物，在28℃下进行表达12-重组大肠杆菌top10f′-petneqssb-bst菌株在诱导前的整个无细胞提取物；13-用1mmiptg诱导后3小时的整个无细胞提取物，在37℃下进行表达；14-用1mmiptg诱导后4小时的整个无细胞提取物，在37℃下进行表达15-用1mmiptg诱导后5小时的整个无细胞提取物，在37℃下进行表达16-用1mmiptg诱导后6小时的整个无细胞提取物，在37℃下进行表达17-用1mmiptg诱导后20小时的整个无细胞提取物，在37℃下进行表达18-重组大肠杆菌top10f′-petneqssb-bst菌株在诱导前的整个无细胞提取物；19-用0.1mmiptg诱导后3小时的整个无细胞提取物，在37℃下进行表达；20-用0.1mmiptg诱导后4小时的整个无细胞提取物，在37℃下进行表达21-用0.1mmiptg诱导后5小时的整个无细胞提取物，在37℃下进行表达22-用0.1mmiptg诱导后6小时的整个无细胞提取物，在37℃下进行表达23-用0.1mmiptg诱导后20小时的整个无细胞提取物，在37℃下进行表达图4-示出了表示与参考dna聚合酶|bst相比，融合dna聚合酶的活性随温度升高而变化的曲线图。蓝线表示dna聚合酶|bst的结果，红线表示融合dna聚合酶bst全长，紫线表示融合dna聚合酶bst大片段，并且绿线表示融合dna聚合酶bst短片段。使用gelanalyzer程序基于琼脂糖凝胶中获得的pcr产物的强度对活性进行描述。图5-示出了在具有溴化乙锭的1.5％琼脂糖凝胶中的电泳分离，其表示对dna聚合酶的进行性的比较，该进行性被定义为等温pcr期间的扩增速率。反应在各列指示的不同时间段内进行。图6-示出了在1.5％琼脂糖凝胶中的电泳分离，其表示对dna聚合酶对以下抑制剂的抗性的比较：血液乳铁蛋白(a)、土壤多酚(b)。a：a：1-在添加6μg乳铁蛋白的情况下，由dna扩增产生的反应产物2-在添加0.6μg乳铁蛋白的情况下，由dna扩增产生的反应产物3-在添加0.06μg乳铁蛋白的情况下，由dna扩增产生的反应产物4-在添加6ng乳铁蛋白的情况下，由dna扩增产生的反应产物k+在不添加抑制剂的情况下，在dna扩增期间产生的反应产物。b：1-在添加100μg多酚的情况下，由dna扩增产生的反应产物2-在添加10μg多酚的情况下，由dna扩增产生的反应产物3-在添加1μg多酚的情况下，由dna扩增产生的反应产物4-在添加0.1μg多酚的情况下，由dna扩增产生的反应产物，5-在添加0.01μg多酚的情况下，由dna扩增产生的反应产物，k+在不添加抑制剂的情况下，在dna扩增期间产生的反应产物。图7-示出了在具有溴化乙锭的2％琼脂糖凝胶中的电泳分离，其表示在融合dna聚合酶存在下，dna电泳迁移率变动分析的结果。反应混合物包含10pmol(dt76)的荧光素标记(绿色)和2.5pmolpcr产物的100bp(橙色)1-d(t)762-100bp3-d(t)76+100bp+3.3pmol融合dna聚合酶4-d(t)76+100bp+6.6pmol融合dna聚合酶5-d(t)76+100bp+13.2pmol融合dna聚合酶6-d(t)76+100bp+26.4pmol融合dna聚合酶7-d(t)76+100bp+52.8pmol融合dna聚合酶8-d(t)76+100bp+105.6pmol融合dna聚合酶9-d(t)76+100bp+211.2pmol融合dna聚合酶通过实施方式来说明本发明，该实施方式包括但不限于此。具体实施方式实施例：融合dna聚合酶neqssb-bst融合dna聚合酶neqssb-bst通过使用接头使三种不同的bst聚合酶在聚合酶n-端与neqssb蛋白融合而获得，该接头由的六个氨基酸组成，序列为：gly-ser-gly-gly-val-asp。融合dna聚合酶的三种变体的序列在附图seq.1-3(氨基酸序列)和seq.4-6(核苷酸序列)中给出。dna聚合酶在基于大肠杆菌的原核系统中以实验室规模获得。制备-实施例1dna聚合酶制备的第一步包括在以下优化的条件下，在微生物振荡器中表达编码酶的基因：生长温度-30℃，诱导后培养基的温育时间-3h至20h，诱导剂浓度-0.1mm至1mmiptg。在蛋白质纯化过程中，使用超声波将所获得的细胞裂解物进行分解，并使用dsdnase除去dna基因组污染。由于寡聚组氨酸结构域的存在，第二纯化步骤利用了使用his-trap珠的金属亲和色谱法(图1)。接下来的步骤包括制剂的三重透析，直到获得可为dna聚合酶提供稳定性的条件(10mmtris-hclph7.1，50mmkcl，1mmdtt，0.1mmedta，50％甘油，0.1％tritonx-100)、凝胶过滤和增浓。所有过程均在4℃下进行。使用sds-page电泳测试所获得的蛋白的纯度，并使用biotium(usa)的evaez荧光聚合酶活性测定试剂盒根据以下的单位定义来确定所获得的制剂的单位数量：1个活性单位[1u]为在其最佳操作温度65℃下，可在30分钟内结合10nmol核苷酸的dna聚合酶的量(图2)。1升实验室规模的培养物可提供约5mg纯化的制剂，其活性约为10000u，该活性能实现相应数量的扩增反应。制备-实施例2编码融合dna聚合酶的基因的表达在28℃的温度下，在能为液体培养物提供适当的充氧的条件下进行。使用iptg诱导对数期培养物，iptg具有能提供蛋白表达的量-iptg在1mm至0.1mm的范围内，并温育3小时至20小时(图3)。之后，使用金属亲和色谱和离子交换色谱将细胞裂解物机械进行分解并纯化。对所获得的融合dna聚合酶进行透析以获得其储存条件(10mmtris-hclph7.1，50mmkcl，1mmdtt，0.1mmedta，50％甘油，0.1％tritonx-100)，并且基于biotium(usa)的商业evaez荧光聚合酶活性测定试剂盒，根据单位的定义以1u/μl的浓度提供。制备-实施例3在37℃的培养和1mm至0.1mm范围内的iptg诱导3小时至20小时下，获得了编码与neqssb蛋白融合的聚合酶bst的基因的有效表达(图3)。使用色谱技术(金属亲和色谱和离子交换色谱)对离心和机械破碎后的细胞裂解物进行纯化，将其悬浮于配制缓冲液(10mmtris-hclph7.1，50mmkcl，1mmdtt，0.1mmedta，50％甘油，0.1％tritonx-100)中，并以1u/μl的浓度提供。使用biotium(usa)的evaez荧光聚合酶活性测定试剂盒，基于单位的定义了dna单位的数量。本发明主题的酶的性质与参考dna聚合酶bst的比较分析已经表明，另外的结合dna的neqssb蛋白的存在对dna聚合酶的性质具有积极影响。与参考dna聚合酶bst相比，所有获得的dna聚合酶融合变体的热稳定性均增加了约20％。(图4)。而且，与neqssb蛋白融合的dna聚合酶显示出进行性增加了三倍(图5)。与参考聚合酶相比，融合dna聚合酶耐受反应混合物中的临床抑制剂(乳铁蛋白、肝素)和环境抑制剂(腐殖酸、土壤、多酚)的浓度增加了甚至数十倍(图6)。与参考dna聚合酶bst相比，融合dna聚合酶表现出灵敏度增加了几倍，并因此表现出对dna基质的亲和力增加。参考文献[1]patelp.h.,motoshis.,admane.,shinkaia,prokaryoticdnapolymerasei:evolution,structureandbaseflippingmechanismfornucleotideselection,jmolbiol.,2001,308:823-837.[2]steitzt.a.,thejournalofbiologicalchemistry,1999,274:17395-17398[3]riggsm.g.,tudors.,sivaramm.,mcdonoughs.h.constructionofsingleaminoacidsubstitutionmutantsofclonedbacillusstearothermophilusdnapolymeraseiwhichlack5'→3'exonucleaseactivity.biochimbiophysacta,1996；1307(2):178-86.[4]oscorbin,i.p.,belousovae.a.,boyarskikhu.a.,zakabunina.i.,khrapove.a.,filipenkom.l.,derivativesofbst-likegss-polymerasewithimprovedprocessivityandinhibitortolerance,nucleicacidsresearch,2017,45(16):9595-9610[5]phangs.m.,teoc.y.,loe.,wongv.w.cloningandcompletesequenceofthednapolymerase-encodinggene(bstpoli)andcharacterisationofthekienow-likefragmentfrombacillusstearothermophilus.gene.1995；163(1):65-8.[6]nowakm,olszewskim,m,kurj.characterizationofsingle-strandeddna-bindingproteinsfromthepsychrophilicbacteriadesulfotaleapsychrophila,flavobacteriumpsychrophilum,psychrobacterarcticus,psychrobactercryohalolentis,psychromonasingrahamii,psychroflexustorquis,andphotobacteriumprofundum.bmcmicrobiol.2014；14:91.[7]kurj,olszewskim,dtugoleckaa,filipkowskip.single-strandeddna-bindingproteins(ssbs)-sourcesandapplicationsinmolecularbiology.actabiochimpol.2005；52(3):569-74.[8]sigaln,deliush,kornbergt,gefterml,albertsb.adna-unwindingproteinisolatedfromescherichiacoli:itsinteractionwithdnaandwithdnapolymerases.procnatlacadsciusa.1972；69(12):3537-41.[9]olszewskim,balsewiczj,nowakm,maciejewskan,cyranka-czajaa,b,r,kurj.characterizationofasingle-strandeddna-binding-likeproteinfromnanoarchaeumequitans-anucleicacidbindingproteinwithbroadsubstratespecificity.plosone.2015；10(5):e0126563[10]kimk.p.,chos.s.,leek.k.,younm.h.,kwons.t.improvedthermostabilityandpcrefficiencyofthermococcuscelericrescensdnapolymeraseviasite-directedmutagenesis,jbiotechnol.,2011,10；155(2):156-63[11]kermekchievm.b.,kirilovali.,vaile.e.,barnesw.m.,mutantsoftaqdnapolymeraseresistanttopcrinhibitorsallowdnaamplificationfromwholebloodandcrudesoilsamples,nucleicacidsres,2009,37(5):e40[12]patelph,suzukim,admane,shinkaia,loebla.prokaryoticdnapolymerasei:evolution,structure,and"baseflipping"mechanismfornucleotideselection.jmalbiol.2001；308(5):823-37[13]wangy.,prosend.e.,meil.,sullivanj.c.,finneym.,vanderhomp.b.,anovelstrategytoengineerdnapolymerasesforenhancedprocessivityandimprovedperformanceinvitro,2004nucleicacidresearch,32(3)[14]leej.i.,chos.s.,eui-joonkile.j.,kwons.t.,characterizationandpcrapplicationofathermostablednapolymerasefromthermococcuspacificu,enzymeandmicrobialtechnology47(2010)147-152[15]wangf,lis,zhaoh,bianl,chenl,zhangz,zhongx,mal,yux.expressionandcharacterizationoftherkoddnapolymeraseinpichiapastoris.plosone.2015；10(7):e0131757.[16]suns.,gengl.,shamooy.,structureandenzymaticpropertiesofachimericbacteriophagerb69dnapolymeraseandsingle-strandeddnabindingproteinwithincreasedprocessivity,proteins:structure,function,andbioinformatics,2006,65:231-238[17]ssb-polymerasefusionproteins,europeanpatentoffice,ep1934372bl,dateoffiling:08.09.2006,dateofpublication:20.02.2013[18]m,krawczykb,b,r,wysockam,olszewskim.fusionofdna-bindingdomainofpyrococcusfuriosusligasewithtaqstoffeldnapolymeraseasausefultoolinpcrwithdifficulttargets.applmicrobiolbiotechnol.2018；102(2):713-721.[19]olszewskim,m,bilekm,krawczykb.fusionoftaqdnapolymerasewithsingle-strandeddnabinding-likeproteinofnanoarchaeumequitans-expressionandcharacterization.plosone.2017；12(9):e0184162[20]devegam.,lázaroj.m.,menciam.,blancol.,salasm.,improvementofdnapolymeraseamplificationperformancebyfusionofdnabindingmotifs,pans,2010；107(38):16506-16511[21]pavlova.r.,pavlovan.v.,kozyavkins.a.,slesareva.i.,cooperationbetweencatalyticanddna-bindingdomainsenhancesthermostabilityandsupportsdnasynthesisathighertemperaturesbythermostablednapolymerases,biochemistry.2012；51(10):2032-2043.[22]tveith.,kristensent.,fluorescence-baseddnapolymeraseassay.analbiochem.2001；289:96-8.核苷酸和氨基酸序列seq.1.mdeeeliqliiektgksreeiekmveekikafnnlisrrgalllvakklgvlykntpkekkigelesweyvkvkgkilksfglisyskgkfqpiilgdetgtikaiiwntdkelpentvieaigktkinkktgnlelhidsykilesdleikpqkqefvgicivkypkkqtqkgtivskailtsldrelpvvyfndfdweighiykvygklkkniktgkieffadkveeatlkdlkafkgeadgsggvdlknklvlidgnsvayraffalpllhndkgihtnavygftmmlnkilaeeqpthilvafdagkttfrhetfqdakggrqqtppelseqfplvrellkayripayeldhyeaddiigtmaaraeregfavkvisgdrdltqlaspqvtveitkkgitdiesytpetvvekygltpeqivdlkglmgdksdnipgvpgigkktavkllkqfgtvenvlasideikgeklkenlrqyrdlallskqlaaicrdapveltlddivykgedrekvvalfqelgfqsfldkmavqtdegekplagmdfaiadsvtdemladkaalvvevvgdnyhhapivgialanergrfflrpetavadpkflawlgdetkkktmfdskraavalngkgielagvgvvfdlllaaylldpaqaagdvaavakmhqyeavrsdeavygkgakrtvpdeptlaeqlvrkaaaiwaleeplmdelrrneqdrlltelehalagilanmeftgvkvdtkrleqmgaelteqlqaverriyelagqefninspkqlgtvlfdklqlpvlkktktgystsadvleklaphheivehilhyrqlgklqstyiegllkvvhpvtgkvhtmfnqaltqtgrlssvepnlqnipirleegrkirqafvpsepdwlifaadysqielrvlahiaeddnlieafrrwldihtktamdifhvseedvtanmrrqakavnfgivygisdyglaqnlnitrkeaaefieryfasfpgvkqymdnivqeakqkgyvttllhrrrylpditsrnfnvrtfaertamntpiqgsaadiikkamidlsvsvreerlqarlllqghdelileapkeeigrlcrlvpevmeqavtlrvplkvdyhygptwydak长度:1127aa类型：氨基酸序列分子类型：蛋白质seq.2.mdeeeliqliiektgksreeiekmveekikafnnlisrrgalllvakklgvlykntpkekkigelesweyvkvkgkilksfglisyskgkfqpiilgdetgtikaiiwntdkelpentvieaigktkinkktgnlelhidsykilesdleikpqkqefvgicivkypkkqtqkgtivskailtsldrelpvvyfndfdweighiykvygklkkniktgkieffadkveeatlkdlkafkgeadgsggvdladkaalvvevvgdnyhhapivgialanergrfflrpetavadpkflawlgdetkkktmfdskraavalngkgielagvgvvfdlllaaylldpaqaagdvaavakmhqyeavrsdeavygkgakrtvpdeptlaeqlvrkaaaiwaleeplmdelrrneqdrlltelehalagilanmeftgvkvdtkrleqmgaelteqlqaverriyelagqefninspkqlgtvlfdklqlpvlkktktgystsadvleklaphheivehilhyrqlgklqstyiegllkvvhpvtgkvhtmfnqaltqtgrlssvepnlqnipirleegrkirqafvpsepdwlifaadysqielrvlahiaeddnlieafrrwldihtktamdifhvseedvtanmrrqakavnfgivygisdyglaqnlnitrkeaaefieryfasfpgvkqymdnivqeakqkgyvttllhrrrylpditsrnfnvrtfaertamntpiqgsaadiikkamidlsvsvreerlqarlllqghdelileapkeeigrlcrlvpevmeqavtlrvplkvdyhygptwydak长度:816aa类型：氨基酸序列分子类型：蛋白质seq.3.mdeeeliqliiektgksreeiekmveekikafnnlisrrgalllvakklgvlykntpkekkigelesweyvkvkgkilksfglisyskgkfqpiilgdetgtikaiiwntdkelpentvieaigktkinkktgnlelhidsykilesdleikpqkqefvgicivkypkkqtqkgtivskailtsldrelpvvyfndfdweighiykvygklkkniktgkieffadkveeatlkdlkafkgeadgsggvdlelrrneqdrlltelehalagilanmeftgvkvdtkrleqmgaelteqlqaverriyelagqefninspkqlgtvlfdklqlpvlkktktgystsadvleklaphheivehilhyrqlgklqstyiegllkvvhpvtgkvhtmfnqaltqtgrlssvepnlqnipirleegrkirqafvpsepdwlifaadysqielrvlahiaeddnlieafrrwldihtktamdifhvseedvtanmrrqakavnfgivygisdyglaqnlnitrkeaaefieryfasfpgvkqymdnivqeakqkgyvttllhrrrylpditsrnfnvrtfaertamntpiqgsaadiikkamidlsvsvreerlqarlllqgh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