一种TaqDNA聚合酶突变体及其应用的制作方法

本发明涉及生物
技术领域：
，具体涉及一种taqdna聚合酶突变体及其应用。
背景技术：
：pcr技术己广泛应用于遗传病分子诊断、动植物进出口检疫、临床检验、食品安全监测、亲子鉴定和土壤微生物检测等众多领域。检测的基因模板主要来自从组织、唾液、细胞、痰液、血液、便以及土壤等。血液、土壤等样本中因为还有血红蛋白、血红素、乳铁蛋白、igg和腐殖酸等，这些物质都对taqdna聚合酶有很强的抑制作用。传统的方法是从这些样本中将核酸提取出来，再进行pcr扩增。由于该领域的检测通量越来越大，传统的方法操作步骤繁多，导致工作效率低，耗时耗试剂，时间和财务成本高，样本量大很容易产生交叉污染。因此，选择直扩成为一种趋势，而不经过核酸提取，样本中存在很多抑制扩增的物质，导致扩增不能正常进行，常出现假阴性等结果。为此，提高扩增试剂的耐抑制物效力成为分子生物学行业的热门研究方向，主要是对taqdna聚合酶的改造方面。taqdna聚合酶的编码基因全长2496个碱基，编码832个氨基酸，全酶共分为三个结构域，taqdna聚合酶蛋白多肽链n端1-291个氨基酸构成第一个结构域，该结构域表达5’-3’核酸外切酶活性；肽链c端424-832个氨基酸构成第二个结构域，该结构域表达5’-3’聚合酶活性；位于肽链中间的292-423个氨基酸构成第三个结构域，该区域与taqdna聚合酶的三维空间结构相关。酶的改造和修饰主要有以下方法：基因突变、基因融合、化学修饰、抗体修饰和核酸适配体修饰等。其中在提高耐抑制能力上，主要采用基因突变方法，已经被报告的突变位点很多，如：e189k、e230k、e507k、h28r、l30r、g38r、h75r、e76g、e76k、e90k、e734k、e734g、d732g、e708q、e681m、q680r、h676r、d578n、i553v等。针对耐抑制物的突变，如：e189k、e230k和e507k可提高taqdna聚合酶对sybrgreenⅰdye、全血、sds和盐酸胍等的耐受能力；d452n、d551n可增强酶对肝素的耐受能力；e708q可提供酶对sybrgreenⅰdye、全血和土壤的耐受能力。从已被报道的突变可知，绝大多数突变位点设置在第一结构域和第二结构域，通过影响5’-3’核酸外切酶活性和5’-3’聚合酶活性相关的性能来实现taqdna聚合酶的改变。抑制物主要通过干扰细胞的裂解过程、融合或降低核酸或者抑制dna聚合酶的活性等方面来影响扩增反应，其中多数以抑制dna聚合酶的活性为主。血液中的抑制物，如：血红蛋白、乳铁蛋白等，是通过让taqdna聚合酶失活或者抑制其活性，或者捕获/降解模板dna和引物，从而抑制扩增。目前很久研究都是在第一结构域和第二结构域进行基因突变，筛选能够提高全血耐受能力的突变体，几乎没有关于第三结构域的突变，对第三结构域的研究具有重要意义。技术实现要素：本发明的目的提供taqdna聚合酶突变体及其在pcr领域的应用，本发明所述的taqdna聚合酶突变体是通过对taqdna聚合酶第三结构域进行突变的突变体。本发明的目的可以通过以下措施达到：本发明提供一种突变型taqdna聚合酶，其特征在于，所述突变体在seqidno.1所示的序列中，具有下述的一个或多个氨基酸位置上的氨基酸取代，各取代用三联体表示：字母-数字-字母，其中数字表示突变氨基酸的位置，数字前的字母对应突变涉及的氨基酸，数字后的字母表示用于置换数字前氨基酸的氨基酸：t386a，a407l，f413y。本发明通过筛选taqdna聚合酶第三结构域的突变体，在第三结构域发现了三个有意义的突变位点，分别为：t386a(苏氨酸突变成丙氨酸)、a407l(丙氨酸突变成亮氨酸)、f413y(苯丙氨酸突变成络氨酸)。我们发现，三个位点同时突变或者两两突变，得到的突变体可以在血液直扩中有很好的扩增性能，具体表现出：耐受血液抑制的能力很强，产量高等方面。在本发明的一种具体的实施例中，本发明所述的突变型taqdna聚合酶taq-mut，其特征在于，氨基酸序列如seqidno.2、seqidno.3、seqidno.4或seqidno.5所示，或与所示序列具有80％同一性的具有taqdna聚合酶活性的氨基酸序列；优选的具有85％同一性，更优选的具有90％同一性，最优选的，具有95％同一性，进一步优选，具有99％同一性。本发明还提供了编码所述的突变型taqdna聚合酶的核苷酸序列。在本发明的一种具体的实施方式中，编码所述的突变型taqdna聚合酶的核苷酸序列，如seqidno.7、seqidno.8、seqidno.9或seqidno.10所示。需要说明的是，由于同一氨基酸可能有多种不同的密码子来决定，所以编码上述突变型taqdna聚合酶taq-mut的核苷酸序列也可以是由seqidno.6所示野生型taqdna聚合酶核苷酸序列突变一个或多个核苷酸形成同义突变得到同样可编码本发明所述突变型taqdna聚合酶的氨基酸序列的核苷酸序列。也可以是seqidno.7、seqidno.8、seqidno.9或seqidno.10的同义序列。本发明还提供一种包含本发明所述的核苷酸序列的重组载体。本发明还包含一种重组细胞，包含本发明所述的核苷酸序列或重组载体。本发明还提供本发明所述的突变型taqdna聚合酶、本发明所述的核苷酸序列、本发明所述的重组载体或本发明所述的重组细胞在生物
技术领域：
中的应用。本发明还提供本发明所述的突变型taqdna聚合酶、本发明所述的核苷酸序列、本发明所述的重组载体或本发明所述的重组细胞在pcr领域中的应用。本发明还提供本发明所述的突变型taqdna聚合酶、本发明所述的核苷酸序列、本发明所述的重组载体或本发明所述的重组细胞在包含血液的样品的pcr领域中的应用。在本发明的一种实施例中，包含血液的样品中血液体积浓度可以为10％～40％，发明人发现，本发明所述的突变型taqdna聚合酶对血液的耐受性很好，可以优选的在血液体积浓度为30％～40％的样品中进行pcr扩增，更优选的，血液体积浓度为30％的样品中进行pcr扩增。本发明的有益效果：本发明所述的突变体，可以通过改变酶的构象，提高酶的耐受能力，很好的应用于血液样本的扩增。本发明所述的突变型taqdna聚合酶的酶活虽然没有增强，但其在扩增含高浓度血液样本时，却表现出很好的扩增性能。因此我们推测：从第三结构域中筛选出的t386a(苏氨酸突变成丙氨酸)、a407l(丙氨酸突变成亮氨酸)、f413y(苯丙氨酸突变成络氨酸)突变，是从结构上改变了taqdna聚合酶，是能改变taqdna聚合酶构象的突变体，使得原先能被抑制物封闭的区域结构发生变化，变化后，抑制物不再能封闭该位置，减少或消除抑制物对pcr扩增的抑制。定义：基因突变：dna分子中发生碱基对的替换、增添和缺失，而引起的基因结构的改变，基因突变可以是自发的也可以是诱发的。第一个结构域：taqdna聚合酶蛋白多肽链n端1-291个氨基酸构成第一个结构域，该结构域表达5’-3’核酸外切酶活性。第二个结构域：肽链c端424-832个氨基酸构成第二个结构域，该结构域表达5’-3’聚合酶活性。第三个结构域：位于肽链中间的292-423个氨基酸构成第三个结构域，该区域与taqdna聚合酶的三维空间结构相关。血液直扩：不用提取血液中的核酸，直接以血液为模板进行pcr扩增，用来实现核酸检测，血液中的血红蛋白、乳铁蛋白等对pcr具有抑制作用。附图说明图1是四种突变体taq和野生型taq在不同血液浓度时扩增500bp底物的结果；图2是四种突变体taq和野生型taq在不同血液浓度时扩增1013bp底物的结果；图3是四种突变体taq和野生型taq在不同血液浓度时扩增2000bp底物的结果。具体实施方式下面结合实施例对本发明做进一步说明，应当理解，此处所描述的具体实施例仅用以解释本发明，并不用于限定本发明，凡在本发明的构思前提下对本发明制备方法的简单改进都属于本发明的保护范围之内。下面实施例未注明具体条件的实验方法，通常按照本领域的公知手段。下述实施例中所用的试验材料，如无特殊说明，均为自常规生化试剂商店购买得到的。实施例1四种突变体的获得按照常规方法使用南京诺唯赞生物科技有限公司的maxmastermix(p515)和ultraonestepcloningkit(c115)对taqdna聚合酶(序列如seqidno.1所示)进行定点突变，得到突变体，称为：mut1、mut2、mut3和mut4。mut1的突变位点为：t386a、a407l、f413y(序列如seqidno.2所示)；mut2的突变位点为：t386a、a407l(序列如seqidno.3所示)；mut3的突变位点为：a407l、f413y(序列如seqidno.4所示)；mut4的突变位点为：t386a、f413y(序列如seqidno.5所示)。用于mut1点突变的引物如下表1～表2所示：表1mut1引物序列引物名称5’-3’序列1-1fccaacaccgcccccgagggggtg1-1rtcgggggcggtgttggaagggtccag2-1fgccctcctttccgagaggctctacgccaacctgtggggga2-1rggcgtagagcctctcggaaaggagggcccgctccc表2mut1的载体和插入片段的扩增方式扩增引物对(p515)tm(℃)insert-011-1f+2-1r60vector-012-1f+1-1r60然后将insert-01与vector-01用ultraonestepcloningkit(c115)进行重组获得mut1。用于mut2点突变的引物如下表3～表4所示：表3mut2引物序列引物名称5’-3’序列1-1fccaacaccgcccccgagggggtg1-1rtcgggggcggtgttggaagggtccag2-2fgccctcctttccgagaggctcttcgccaacctgtggggga2-2rggcgaagagcctctcggaaaggagggcccgctccc表4mut2的载体和插入片段的扩增方式扩增引物对(p515)tm(℃)insert-021-1f+2-2r60vector-022-2f+1-1r60然后将insert-02与vector-02用ultraonestepcloningkit(c115)进行重组获得mut2。用于mut3点突变的引物如下表5～表6所示：表5mut3引物序列引物名称5’-3’序列2-1fgccctcctttccgagaggctctacgccaacctgtggggga2-1rggcgtagagcctctcggaaaggagggcccgctccc表6mut3的扩增方式扩增引物对(p515)tm(℃)insert-032-1f+1-1r60然后将insert-03用ultraonestepcloningkit(c115)进行重组获得mut3。用于mut4点突变的引物如下表7～表8所示：表7mut4引物序列引物名称5’-3’序列1-1fccaacaccgcccccgagggggtg1-1rtcgggggcggtgttggaagggtccag2-3fgccgccctttccgagaggctctacgccaacctgtggggga2-3rggcgtagagcctctcggaaagggcggcccgctccc表8mut4的载体和插入片段的扩增方式扩增引物对(p515)tm(℃)insert-041-1f+2-3r60vector-042-3f+1-1r60然后将insert-04与vector-04用ultraonestepcloningkit(c115)进行重组获得mut4。野生型的taqdna聚合酶突变前的核酸序列如seqidno.6所示，突变后的核酸序列如seqidno.7(mut1)、seqidno.8(mut2)、seqidno.9(mut3)、seqidno.10(mut4)所示。实施例2四种突变体taq具有较高的血液耐受能力野生型的taq和四个突变体，按照以下配方，配制成2×pcrmix，得到五种2×pcrmix。2×pcrmix1：200mmtris-hcl，100mmkcl，0.8mmdntp，4mmmgcl2，0.2u/μltaq；2×pcrmix2：200mmtris-hcl，100mmkcl，0.8mmdntp，4mmmgcl2，0.2u/μlmut1；2×pcrmix3：200mmtris-hcl，100mmkcl，0.8mmdntp，4mmmgcl2，0.2u/μlmut2；2×pcrmix4：200mmtris-hcl，100mmkcl，0.8mmdntp，4mmmgcl2，0.2u/μlmut3；2×pcrmix5：200mmtris-hcl，100mmkcl，0.8mmdntp，4mmmgcl2，0.2u/μlmut4。五种2×pcrmix按照以下表20方式混样(50μl反应体系)，为了控制每个孔中模板的投入量一致，以λdna为模板，加入的血液作为杂质，用以验证血液耐受程度。表9pcr反应体系混样方式组分体积2×pcrmix1/2/3/4/525μl10m引物各2μlλdna5ng血液0/5/10/15μlh2oto50μl用于测试的三对引物序列如下表10：表10测试体系引物序列反应程序如下表11：表11pcr反应程序1％的琼脂糖凝胶跑核酸电泳，得到如图1(500bp)、图2(1013bp)、图3(2000bp)所示结果，图中taq-wt是野生型taq的扩增结果，mut1、mut2、mut3、mut4是四个突变体酶的扩增结果。0％表示反应体系中不含血液，10％表示反应体系中含10％的血液(50μl反应体系中含5μl血液)，20％表示反应体系中含20％的血液(50μl反应体系中含10μl血液)，30％表示反应体系中含30％的血液(50μl反应体系中含15μl血液)。图1(500bp)、图2(1013bp)、图3(2000bp)中的marker是dl5000。从图1(500bp)、图2(1013bp)、图3(2000bp)可以得知：与野生型taq相比，mut2、mut3、mut4在3个测试体系中均具有更好的扩增性能，mut1的扩增性能最好，在血液含量高达30％依然扩增很好。实施例3检测四种突变体和野生型taq的酶活将实施例2制备的5种2×pcrmix用本领域常规方法进行酶活检测，得到结果如表12所示。表125种2×pcrmix的酶活检测结果(酶活，单位：mu/μl)酶种类重复一重复二重复三平均值2×pcrmix11921901881902×pcrmix21951831901892×pcrmix31841911831862×pcrmix41901871921902×pcrmix5183197190190由表12的数据可知，突变体酶的酶活并没有增强，而其在扩增含血液样本时，表现出很好的扩增性能。因此结合实施例2的结果我们推测：从第三结构域中筛选出的t386a(苏氨酸突变成丙氨酸)、a407l(丙氨酸突变成亮氨酸)、f413y(苯丙氨酸突变成络氨酸)突变，可以通过改变酶的构象，提高酶的耐受能力，因此可以很好的应用于血液样本的扩增。序列表<110>南京农业大学<120>一种taqdna聚合酶突变体及其应用<160>10<170>siposequencelisting1.0<210>1<211>832<212>prt<213>水生栖热菌(thermusaquaticus)<400>1metargglymetleuproleuphegluprolysglyargvalleuleu151015valaspglyhishisleualatyrargthrphehisalaleulysgly202530leuthrthrserargglygluprovalglnalavaltyrglypheala354045lysserleuleulysalaleulysgluaspglyaspalavalileval505560valpheaspalalysalaproserphearghisglualatyrglygly65707580tyrlysalaglyargalaprothrprogluasppheproargglnleu859095alaleuilelysgluleuvalaspleuleuglyleualaargleuglu100105110valproglytyrglualaaspaspvalleualaserleualalyslys115120125alaglulysgluglytyrgluvalargileleuthralaasplysasp130135140leutyrglnleuleuseraspargilehisvalleuhisproglugly145150155160tyrleuilethrproalatrpleutrpglulystyrglyleuargpro165170175aspglntrpalaasptyrargalaleuthrglyaspgluseraspasn180185190leuproglyvallysglyileglyglulysthralaarglysleuleu195200205gluglutrpglyserleuglualaleuleulysasnleuaspargleu2102152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ggtcctcgaggccccaaaagagagggcggaggccgtggcc2400cggctggccaaggaggtcatggagggggtgtatcccctggccgtgcccctggaggtggag2460gtggggataggggaggactggctctccgccaaggaatga2499<210>10<211>2499<212>dna<213>人工序列(artificialsequence)<400>10atgagggggatgctgcccctctttgagcccaagggccgggtcctcctggtggacggccac60cacctggcctaccgcaccttccacgccctgaagggcctcaccaccagccggggggagccg120gtgcaggcggtctacggcttcgccaagagcctcctcaaggccctcaaggaggacggggac180gcggtgatcgtggtctttgacgccaaggccccctccttccgccacgaggcctacgggggg240tacaaggcgggccgggcccccacgccggaggactttccccggcaactcgccctcatcaag300gagctggtggacctcctggggctggcgcgcctcgaggtcccgggctacgaggcggacgac360gtcctggccagcctggccaagaaggcggaaaaggagggctacgaggtccgcatcctcacc420gccgacaaagacctttaccagctcctttccgaccgcatccacgccctccaccccgagggg480tacctcatcaccccggcctggctttgggaaaagtacggcctgaggcccgaccagtgggcc540gactaccgggccctgaccggggacgagtccgacaaccttcccggggtcaagggcatcggg600gagaagacggcgaggaagcttctggaggagtgggggagcctggaagccctcctcaagaac660ctggaccggctgaagcccgccatccgggagaagatcctggcccacatggacgatctgaag720ctctcctgggacctggccaaggtgcgcaccgacctgcccctggaggtggacttcgccaaa780aggcgggagcccgaccgggagaggcttagggcctttctggagaggcttgagtttggcagc840ctcctccacgagttcggccttctggaaagccccaaggccctggaggaggccccctggccc900ccgccggaaggggccttcgtgggctttgtgctttcccgcaaggagcccatgtgggccgat960cttctggccctggccgccgccagggggggccgggtccaccgggcccccgagccttataaa1020gccctcagggacctgaaggaggcgcgggggcttctcgccaaagacctgagcgttctggcc1080ctgagggaaggccttggcctcccgcccggcgacgaccccatgctcctcgcctacctcctg1140gacccttccaacaccgcccccgagggggtggcccggcgctacggcggggagtggacggag1200gaggcgggggagcgggccgccctttccgagaggctctacgccaacctgtgggggaggctt1260gagggggaggagaggctcctttggctttaccgggaggtggagaggcccctttccgctgtc1320ctggcccacatggaggccacgggggtgcgcctggacgtggcctatctcagggccttgtcc1380ctggaggtggccgaggagatcgcccgcctcgaggccgaggtcttccgcctggccggccac1440cccttcaacctcaactcccgggaccagctggaaagggtcctctttgacgagctagggctt1500cccgccatcggcaagacggagaagaccggcaagcgctccaccagcgccgccgtcctggag1560gccctccgcgaggcccaccccatcgtggagaagatcctgcagtaccgggagctcaccaag1620ctgaagagcacctacattgaccccttgccggacctcatccaccccaggacgggccgcctc1680cacacccgcttcaaccagacggccacggccacgggcaggctaagtagctccgatcccaac1740ctccagaacatccccgtccgcaccccgcttgggcagaggatccgccgggccttcatcgcc1800gaggaggggtggctattggtggccctggactatagccagatagagctcagggtgctggcc1860cacctctccggcgacgagaacctgatccgggtcttccaggaggggcgggacatccacacg1920gagaccgccagctggatgttcggcgtcccccgggaggccgtggaccccctgatgcgccgg1980gcggccaagaccatcaacttcggggtcctctacggcatgtcggcccaccgcctctcccag2040gagctagccatcccttacgaggaggcccaggccttcattgagcgctactttcagagcttc2100cccaaggtgcgggcctggattgagaagaccctggaggagggcaggaggcgggggtacgtg2160gagaccctcttcggccgccgccgctacgtgccagacctagaggcccgggtgaagagcgtg2220cgggaggcggccgagcgcatggccttcaacatgcccgtccagggcaccgccgccgacctc2280atgaagctggctatggtgaagctcttccccaggctggaggaaatgggggccaggatgctc2340cttcaggtccacgacgagctggtcctcgaggccccaaaagagagggcggaggccgtggcc2400cggctggccaaggaggtcatggagggggtgtatcccctggccgtgcccctggaggtggag2460gtggggataggggaggactggctctccgccaaggaatga2499当前第1页1 2 3