本发明属于蛋白质工程
技术领域：
：，具体涉及来源于酿脓链球菌的crispr-cas9核酸酶spcas9的高特异性截短变异体tspcas9-v1/v2及其在基因编辑领域中的应用。
背景技术：
：：crispr/cas系统可方便地对基因组特定基因进行的切割编辑，是最新发现的一种革命性的基因编辑技术，近年来，发展尤为迅速。来源于酿脓链球菌(streptococcuspyogenes)的crispr-cas9核酸酶spcas9是目前最为广泛使用的crispr核酸酶[1]，因其具有易用、高效、强特异性和多功能性等优点，已被应用到多个领域，包括医学研究以及生物技术等，比如细胞或动物模型的快速构建、功能基因的便捷筛选以及部分遗传疾病的治疗[2-6]。虽然，crispr-cas9系统的优势促使其能够广泛发展，但是，其仍然存在着许多有待解决的问题。比如spcas9体积过于庞大，难以通过病毒载体将其高效靶向地运载到体内或细胞中，从而限制了其在临床医学治疗上的发展[7]；spcas9由sgrna引导至特定靶序列进行切割编辑时，sgrna可能会与其他类似靶序列的基因位点发生局部匹配，进一步激活cas9切割非靶dna，因而难以精准的靶向特异性疾病基因位点，导致产生脱靶现象，进而限制了该技术在精准医疗方面的应用[8-10]。基于上述问题，寻找一个体积较小，特异性较高的cas9核酸酶，从而有效地实现体内dna片段的精准遗传编辑是十分必要的。技术实现要素：本发明的目的是提供一种来源于酿脓链球菌的crispr-cas9核酸酶spcas9的截短型高特异性变异体及其用途。本发明提供的来源于酿脓链球菌的crispr-cas9核酸酶spcas9的截短型高特异性变异体有两种，一种是将截短型crispr-cas9(tspcas9)核酸酶的第863位的氨基酸h突变成n，记为tspcas9-v1核酸酶；一种是将截短型crispr-cas9(tspcas9)核酸酶的第862位的氨基酸h突变成a，第863位的氨基酸h突变成n，记为tspcas9-v2核酸酶；两种截短型高特异性变异体记为tspcas9-v1/v2；它们具有与野生型crispr-cas9核酸酶相当的基因编辑活性，但比野生型特异性更高，能实现精准编辑。所述野生型spcas9核酸酶的核苷酸序列和氨基酸序列分别为seqidno.1和seqidno.2所示。所述截短型tspcas9核酸酶的核苷酸序列和氨基酸序列分别为seqidno.3和seqidno.4所示。所述截短型高特异性tspcas9-v1核酸酶的核苷酸序列和氨基酸序列分别为seqidno.5和seqidno.6所示，与野生型spcas9的相似度达90％以上。所述截短型高特异性tspcas9-v2核酸酶的核苷酸序列和氨基酸序列分别为seqidno.7和seqidno.8所示，与野生型spcas9的相似度达90％以上。本发明还提供一种多核苷酸序列，可以转录和翻译成所述的crispr-cas9核酸酶(tspcas9-v1/v2)。本发明还提供一种表达载体，其含有上述多核苷酸序列。本发明还提供一种宿主细胞，可以用于转化上述表达载体。本发明还提供所述crispr-cas9核酸酶(tspcas9-v1/v2)的制备方法，具体步骤包括：首先，构建所述crispr-cas9核酸酶的多核苷酸序列表达载体；然后，将所述表达载体转化至宿主细胞，筛选并挑出单克隆；最后，将所述单克隆诱导表达，并通过亲和层析、离子交换等方法从表达产物中分离出所述的crispr-cas9核酸酶。本发明提供的上述crispr-cas9核酸酶、多核苷酸序列以及表达载体均可作为编辑基因组dna的编辑工具，用于基因组dna片段的相关编辑。本发明中，所述的基因编辑可以是单点编辑，也可以是编辑位点大于等于两个的多点编辑。所述编辑的手段包括删除、突变、插入、倒位、移位、重复或易位。本发明中，所述crispr-cas9编辑工具包括与靶标dna片段匹配的引导sgrna。所述的crispr-cas9核酸酶与能够介导它的sgrna组合，从而对目的基因进行编辑。本发明中，所述含有编码crispr-cas9核酸酶多核苷酸序列的载体和与之匹配的引导sgrna一同转入宿主细胞，对基因进行编辑。本发明中，所述单位点或多位点基因编辑，包括利用所述crispr-cas9核酸酶对双链dna进行剪切，并通过宿主细胞的修复系统对断裂的缺口进行修复。本发明中，所述的单位点或多位点的基因编辑，是改变单位点或多位点编辑时的碱基突变特征。与现有技术相比，本发明的(tspcas9-v1/v2)，属于crispr-cas9免疫系统，分别含有seqidno.5、seqidno.6和seqidno.7、seqidno.8的核苷酸序列和氨基酸序列，能够提高基因编辑特异性，实现对基因组dna片段的特定位置的精准编辑，具有潜在的精准生物医学应用价值。附图说明图1为pet21-6his-tev-tspcas9-v1/v2质粒构建图示。图2为pet21-6his-tev-tspcas9-v1/v2-v1/v2质粒筛选与培养。图3为含有tspcas9-v1/v2的质粒测序。图4为tspcas9-v1/v2-v1/v2目标蛋白的纯化方法。图5为tspcas9-v1/v2-v1/v2目标蛋白纯化获取的过程。图6为crispr-cas9目标蛋白纯化的电泳鉴定。图7为sgrna的在靶和脱靶序列。图8为野生型spcas9、tspcas9及tspcas9-v1/v2体外剪切活性的检测。图9为野生型spcas9体外在靶和脱靶效应的检测。图10为截短型tspcas9体外在靶和脱靶效应的检测。图11为截短变异体tspcas9-v1体外在靶和脱靶效应的检测。图12为截短变异体tspcas9-v2体外在靶和脱靶效应的检测。具体实施方式下面通过具体实施例进一步描述本发明。下述实施例中所用的实验方法，如无特定说明，均为常规方法。下述实施例中所用的材料、试剂等，如无特定说明，均为从商业途径获得。一、crispr-cas9核酸酶本发明的tspcas9-v1核酸酶将截短型crispr-cas9(tspcas9)核酸酶的第863位的氨基酸h突变成n，tspcas9-v2核酸酶将截短型crispr-cas9(tspcas9)核酸酶的第862位的氨基酸h突变成a、第863位的氨基酸h突变成n，属于crispr-cas9系统，具有与野生型crispr-cas9核酸酶相当的基因编辑活性，但比野生型更能特异性靶向基因编辑位点，实现精准编辑。二、编码crispr-cas9核酸酶的多核苷酸序列转录和翻译所述crispr-cas9(tspcas9-v1/v2)的多核苷酸序列，包括dna或rna。dna还可以细分为质粒dna、基因组dna或人工合成的dna。编码所述crispr-cas9(tspcas9-v1/v2)的多核苷酸序列，可以利用该领域科研或技术人员所熟悉的相关分子生物学技术来制备，其不局限于重组dna技术和化学合成方法。三、表达载体所述表达载体含有编码所述crispr-cas9核酸酶(tspcas9-v1/v2)的多核苷酸序列。该表达载体可以通过科研或技术人员所熟悉的分子生物学方法来构建，包括dna重组技术和dna合成技术等，主要将crispr-cas9核酸酶(tspcas9-v1/v2)的dna有效连接到载体上的克隆位点中，然后通过转录翻译等过程表达目的蛋白tspcas9-v1/v2。四、宿主细胞所述宿主细胞是用来转化表达crispr-cas9核酸酶的重组质粒。主要包括原核细胞(如细菌)，低等真核细胞(如酵母)，高等真核细胞(如哺乳动物细胞)等。常用的宿主细胞如大肠杆菌dh5α、毕赤酵母、hek293、cho、hela细胞等。五、crispr-cas9核酸酶(tspcas9-v1/v2)及其编码该酶的核苷酸序列和所述表达载体的用途本发明的crispr-cas9核酸酶(tspcas9-v1/v2)及其编码该酶的多核苷酸序列和所述的表达载体能够用于基因组dna片段的编辑或用于制备基因编辑工具。crispr-cas9核酸酶(tspcas9-v1/v2)编辑包括单位点和多位点编辑，其编辑手段包括删除、突变、插入、倒位、移位、重复或易位等。六、基因编辑工具及其方法本发明的基因编辑工具属于crispr-cas9系统，crispr-cas9(tspcas9-v1/v2)在特定的sgrna的引导下可以在目的基因dna片段pam(ngg)位点上游3到4位碱基间剪切底物dna。该编辑过程可以在体内或体内进行。当sgrna是单个的时候可以进行单点编辑，当sgrna是两个或两个以上时可以进行多位点编辑。如本发明的一些实施方式中所列举的，crispr-cas9核酸酶(tspcas9-v1/v2)在sgrna的引导下，可以在体外对底物dna(920bp)进行剪切，其产物长度分别为760bp和260bp。在本发明中，cas9可作为crispr-cas9核酸酶的简称使用，其含义与crispr-cas9核酸酶相同。本发明中的截短型高特异性tspcas9-v1核酸酶是将截短型crispr-cas9(tspcas9)核酸酶的第863位的氨基酸h突变成n后表达得到的蛋白，tspcas9-v2核酸酶是将截短型crispr-cas9(tspcas9)核酸酶的第862位的氨基酸h突变成a、第863位的氨基酸h突变成n后表达得到的蛋白。在进一步描述本发明具体实施方式之前，应理解，本发明的保护范围并不局限为下述特定的具体实施方案，还应理解为，本发明实施例中的术语是为了描述特定的具体实施方案，而不是为了限制本发明的保护范围。下例实施例中未注明具体条件的试验方法，通常按照常规条件操作，或者按照各生产厂商所建议的条件操作。在实施例给出的数值范围中，应理解，除非本发明另有说明，每个数值范围的两个端点以及两个端点之间任何一个数值均可选用。除非另外定义，本发明中使用的所有技术和科学术语与本
技术领域：
：技术人员通常理解的意义相同。除实施例中使用的具体方法、设备、材料外，根据本
技术领域：
：的技术人员对现有技术的掌握及本发明的记载，还可以使用与本发明实施例中所述的方法、设备、材料相似或等同的现有技术的任何方法、设备和材料来实现本发明。除非另外说明，本发明中所公开的实验方法、检测方法、制备方法均采用本
技术领域：
：常规的分子生物学、生物化学、重组dna技术及相关领域的常规技术。实施例1，构建crispr-cas9核酸酶(tspcas9-v1/v2)的质粒1.变异体的设计以pet21-6his-tev-tspcas9质粒，即seqidno.3(3744bp)为模板，将2587位的碱基c和2584-2587位的碱基cacc分别突变为a和gcca，其余位置的碱基保持不变，将其分别命名为tspcas9-v1和tspcas9-v2。其改造设计思路如图1所示，其详细步骤简述如下：利用引物f2587和r2587，f2584和r2584分别对质粒pet21-6his-tev-tspcas9(相当于seqidno.3)进行点突变，然后质粒模板消化，即可获得目的产物tspcas9-v1/v2。(1)采购点突变试剂盒所用点突变试剂盒fastsite-directedmutagenesiskit从天根生化科技(北京)有限公司订购。(2)采购引物所用引物均从上海生工生物工程有限公司订购。它们的序列如下：2587位碱基c突变为a的正反引物：f2587：gatcaacaattaccacaatgcgcatgatgcc(seqidno.9)r2587：tgtggtaattgttgatctctctcaccttata(seqidno.10)2584-2587位的碱基cacc突变为gcca的正反引物：f2584：gatcaacaattacgccaatgcgcatgatgcctac(seqidno.11)r2584：gtaggcatcatgcgcattggcgtaattgttgatc(seqidno.12)。点突变体系如下：pcr反应条件：点突变体系如下：pcr反应条件：该反应体系在37℃孵育1小时后，转化大肠杆菌dh5α(购于tiangen)37℃过夜培养筛选单克隆tspcas9-v1/v2，如图2所示，其核苷酸序列和氨基酸分别为序列为seqidno.5、seqidno.6和seqidno.7、seqidno.8，培养细菌。(3)采购质粒抽提试剂盒所用质粒小提试剂盒tianprepminiplasmidkit从天根生化科技(北京)有限公司订购。抽提质粒，抽提方法见天根质粒小提试剂盒使用说明。(4)样品测序在上海杰李生物技术有限公司通过一代测序对8个样品质粒进行测序，发现样品质粒1和6构建正确，其结果如图3所示。实施例2，制备crispr-cas9(tspcas9-v1/v2)核酸酶2.蛋白表达与纯化2.1蛋白表达(1)打开超净台，用含75％酒精的棉球擦拭桌面以及各种器具耗材，开紫外灯照射20min，启动风机备用；(2)移液枪吸取10μl表达pet21-6his-tev-tspcas9-v1/v2的rosetta(de3)(购于tiangen)菌液转至6ml含有双抗(amp与cm)的lb液体培养基中，37℃，200r/min振荡培养过夜；(3)将过夜培养的菌液按照体积比为1：100转至500ml含双抗的lb(购于生工)液体培养基中，37℃，200r/min振荡培养。在培养过程中，随时检测菌液的od值；(4)当菌液的od值接近0.4～0.8时，加入蛋白诱导剂iptg，使其终浓度为0.1mm，然后16℃，200r/min振荡培养20h；(5)收集菌液，5000r/min离心5min使菌体沉淀，弃上清，并称重pet21-6his-tev-tspcas9-v1/v2菌体。2.2蛋白纯化所述蛋白纯化主要通过镍柱亲和层析技术，如图4所示；其纯化过程包括菌体破碎、蛋白样品离心收集、蛋白样品与镍柱介质共孵育以及目的蛋白的洗脱等，如图5所示。其详细步骤如下：(1)向菌体中加入预先冰浴且pmsf终浓度为0.1mm的裂解液(20mmhepes，500mmkcl，ph7.5；1g菌体加入5ml)，涡旋仪重悬使菌块分散混匀，细胞超声破碎仪破碎细胞，超声3sec停3sec，一次10min，超声两次，超声过程均在冰浴中进行；(2)向破碎的菌液中加入终浓度为10μg/mlrnase(生工)，5μg/mldnasei(生工)，冰浴处理30min后，4℃10000r/min离心45～60min，收集上清；(3)将上清与预先用平衡液(20mmhepes，500mmkcl，1％蔗糖，ph7.5)处理的qiagenni-nta介质孵育，此过程在冰浴上进行，并加以振荡(150r/min)，1.5h后静置，待qiagenni-nta沉淀；(4)将qiagenni-nta装载到重力柱中，biologiclp系统的监测下，分别以流速为2ml/min的平衡液和洗脱液(20mmhepes，500mmkcl，500mm咪唑，1％蔗糖，ph7.5)，20、30、40、50、100、250、500mm洗脱液冲洗qiagenni-nta，并收集蛋白；(5)将不同咪唑浓度下的蛋白溶液跑sds-page(购于epizymescientific)电泳，考马斯亮蓝染色，脱色剂脱色，观察目的蛋白的表达和挂柱效果。所述蛋白tspcas9-v1/v2的纯化结果如图6所示，其显示该目的蛋白tspcas9-v1/v2的纯化情况，杂条带较少，蛋白较为纯净。实施例3，检验crispr-cas9(tspcas9-v1/v2)核酸酶剪切活性3.变异体活性检测所用底物dna(seqidno.13)，主要利用引物qg-f和qg-r通过常规pcr扩增，然后割胶回收获取。(1)采购扩增试剂盒所用扩增试剂盒fasthifidelitypcrkit从天根生化科技(北京)有限公司订购。(2)采购引物所用引物均从上海生工生物工程有限公司订购。它们的序列如下：qg-f：tagtcctgtcgggtttcg(seqidno.14)qg-r：ttccattcgccattcagg(seqidno.15)。其反应体系和扩增条件如下：扩增体系如下：pcr反应条件：(3)采购割胶回收试剂盒所用割胶回收试剂盒axypreptmdnagelextractionkit从axygen公司订购，割胶回收操作均按其说明书进行，可以获得较纯的底物dna(seqidno.13)。cas9与sgrna以等摩尔混合，而根据实验需要，底物dna可调为cas9摩尔质量的0.2～1倍。其反应体系如下将反应体系置37℃孵育，1h后70℃加热10min，最后通过琼脂糖凝胶电泳检测目的蛋白tspcas9-v1/v2的体外切割活性。其结果如图8所示，从图8中可以发现，与野生型spcas9和截短型tspcas9相比，tspcas9-v1和tspcas9-v2在体外均能够剪切底物dna(seqidno.13)(泳道5和6)，生成产物1和产物2。虽然tspcas9-v1/v2在体外的dna剪切活性并不比野生型spcas9的强，但是其体积在小型化上却比野生型spcas9有优势，即我们的tspcas9-v1/v2体积比野生型spcas9小，对方便腺病毒aav运输而言，比野生型spcas9有较大优势。实施例4，crispr-cas9(tspcas9-v1/v2)体外脱靶检测的评价方法4.脱靶效应检测利用不同的sgrna，如图7中1到8号所示检测tspcas9-v1/v2的体外切割活性，从而评价tspcas9-v1/v2的脱靶效应，其反应体系如下：首先，评价野生型spcas9在体外的脱靶效应，如图9所示，与0号sgrna引导的spcas9剪切活性相比(泳道3)，1到8号sgrna引导的spcas9均能够在体外剪切底物dna(seqidno.13)(泳道4到11)，生成产物1和产物2。尽管只有部分sgrna的引导活性强，即1到4号sgrna引导的spcas9体外剪切活性比较强(泳道4到7)，5到8号sgrna引导的spcas9体外剪切活性相对较弱(8号到11号)，但是，该结果依然反映野生型spcas9在体外的脱靶效应比较严重。由此说明，野生型spcas9有较强的脱靶效应，尤其在1到4号sgrna的引导下更为突出。其次，评价截短型tspcas9在体外的脱靶效应，如图10所示，与0号sgrna引导的tspcas9剪切活性相比(泳道3)，1到8号sgrna引导的截短型tspcas9均能够在体外剪切底物dna(seqidno.13)(泳道4到11)，生成产物1和产物2。尤其是1到4号sgrna引导的截短型tspcas9的体外剪切活性相对较强，由此说明截短型tspcas9依然存在较强的脱靶效应。再次，评价截短型tspcas9-v1在体外的脱靶效应，如图11所示，与0号sgrna引导的tspcas9-v1剪切活性相比(泳道3)，1、2和4号sgrna引导的截短型tspcas9-v1体外剪切底物dna(seqidno.13)的能力较为明显(泳道4、5、7)，生成产物1和产物2。其余sgrna引导的tspcas9-v1的在体外的切割活性非常弱，尤其是5到8号sgrna引导的tspcas9-v1几乎没有发挥切割作用，由此说明tspcas9-v1能降低脱靶效应。最后，评价截短型tspcas9-v2在体外的脱靶效应，如图12所示，与0号sgrna引导的tspcas9-v2剪切活性相比(泳道3)，1和4号sgrna引导的截短型tspcas9-v2体外剪切底物dna(seqidno.13)的能力明显减弱(泳道4和7)，生成产物1和产物2。而其余sgrna引导的tspcas9-v2的在体外几乎没有发挥切割作用，由此说明tspcas9-v2能降低脱靶效应。在sgrna与底物dna完全互补的情况下，tspcas9-v1/v2保留了野生型spcas9核酸酶的切割活性；同时，在sgrna与底物dna存在两个碱基错配的情况下，和野生型spcas9和截短型tspcas9相比较，tspcas9-v1/v2对于对底物dna的容错率更低，体外剪切特异性更高。即tspcas9-v1/v2不仅具有野生型crispr-cas9核酸酶基因编辑功能，而且与野生型核酸酶相比，更能特异性靶向基因编辑位点，实现精准编辑，同时体积更小，方便腺病毒aav运输，比野生型spcas9有更大优势，这对将来crispr-cas9系统方便应用到临床医学上提供了潜在的价值。参考文选[1].doudna,j.a.ande.charpentier,genomeediting.thenewfrontierofgenomeengineeringwithcrispr-cas9.science,2014.346(6213):p.1258096.[2].hsu,p.d.,e.s.landerandf.zhang,developmentandapplicationsofcrispr-cas9forgenomeengineering.cell,2014.157(6):p.1262-78.[3].suzuki,k.,etal.,invivogenomeeditingviacrispr/cas9mediatedhomology-independenttargetedintegration.nature,2016.540(7631):p.144-149.[4].jinek,m.,etal.,rna-programmedgenomeeditinginhumancells.elife,2013.2:p.e00471.[5].hille,f.,etal.,thebiologyofcrispr-cas:backwardandforward.cell,2018.172(6):p.1239-1259.[6].karginov,f.v.andg.j.hannon,thecrisprsystem:smallrna-guideddefenseinbacteriaandarchaea.molcell,2010.37(1):p.7-19.[7].niewoehner,j.,etal.,increasedbrainpenetrationandpotencyofatherapeuticantibodyusingamonovalentmolecularshuttle.neuron,2014.81(1):p.49-60.[8].kleinstiver,b.p.,etal.,high-fidelitycrispr-cas9nucleaseswithnodetectablegenome-wideoff-targeteffects.nature,2016.529(7587):p.490-5.[9].pattanayak,v.,etal.,high-throughputprofilingofoff-targetdnacleavagerevealsrna-programmedcas9nucleasespecificity.natbiotechnol,2013.31(9):p.839-43.[10].jamal,m.,etal.,keepingcrispr/cason-target.currissuesmolbiol,2016.20:p.1-12。序列表<110>复旦大学<120>核酸内切酶spcas9高特异性截短变异体tspcas9-v1/v2及其应用<130>2019.06.28<160>16<170>siposequencelisting1.0<210>1<211>4104<212>dna<213>streptococcuspyogenesserotypem1<400>1ggcgacaagaagtactccattgggctcgatatcggcacaaacagcgtcggctgggccgtc60attacggacgagtacaaggtgccgagcaaaaaattcaaagttctgggcaataccgatcgc120cacagcataaagaagaacctcattggcgccctcctgttcgactccggggagacggccgaa180gccacgcggctcaaaagaacagcacggcgcagatatacccgcagaaagaatcggatctgc240tacctgcaggagatctttagtaatgagatggctaaggtggatgactctttcttccatagg300ctggaggagtcctttttggtggaggaggataaaaagcacgagcgccacccaatctttggc360aatatcgtggacgaggtggcgtaccatgaaaagtacccaaccatatatcatctgaggaag420aagcttgtagacagtactgataaggctgacttgcggttgatctatctcgcgctggcgcat480atgatcaaatttcggggacacttcctcatcgagggggacctgaacccagacaacagcgat540gtcgacaaactctttatccaactggttcagacttacaatcagcttttcgaagagaacccg600atcaacgcatccggagttgacgccaaagcaatcctgagcgctaggctgtccaaatcccgg660cggctcgaaaacctcatcgcacagctccctggggagaagaagaacggcctgtttggtaat720cttatcgccctgtcactcgggctgacccccaactttaaatctaacttcgacctggccgaa780gatgccaagcttcaactgagcaaagacacctacgatgatgatctcgacaatctgctggcc840cagatcggcgaccagtacgcagacctttttttggcggcaaagaacctgtcagacgccatt900ctgctgagtgatattctgcgagtgaacacggagatcaccaaagctccgctgagcgctagt960atgatcaagcgctatgatgagcaccaccaagacttgactttgctgaaggcccttgtcaga1020cagcaactgcctgagaagtacaaggaaattttcttcgatcagtctaaaaatggctacgcc1080ggatacattgacggcggagcaagccaggaggaattttacaaatttattaagcccatcttg1140gaaaaaatggacggcaccgaggagctgctggtaaagcttaacagagaagatctgttgcgc1200aaacagcgcactttcgacaatggaagcatcccccaccagattcacctgggcgaactgcac1260gctatcctcaggcggcaagaggatttctacccctttttgaaagataacagggaaaagatt1320gagaaaatcctcacatttcggataccctactatgtaggccccctcgcccggggaaattcc1380agattcgcgtggatgactcgcaaatcagaagagaccatcactccctggaacttcgaggaa1440gtcgtggataagggggcctctgcccagtccttcatcgaaaggatgactaactttgataaa1500aatctgcctaacgaaaaggtgcttcctaaacactctctgctgtacgagtacttcacagtt1560tataacgagctcaccaaggtcaaatacgtcacagaagggatgagaaagccagcattcctg1620tctggagagcagaagaaagctatcgtggacctcctcttcaagacgaaccggaaagttacc1680gtgaaacagctcaaagaagactatttcaaaaagattgaatgtttcgactctgttgaaatc1740agcggagtggaggatcgcttcaacgcatccctgggaacgtatcacgatctcctgaaaatc1800attaaagacaaggacttcctggacaatgaggagaacgaggacattcttgaggacattgtc1860ctcacccttacgttgtttgaagatagggagatgattgaagaacgcttgaaaacttacgct1920catctcttcgacgacaaagtcatgaaacagctcaagaggcgccgatatacaggatggggg1980cggctgtcaagaaaactgatcaatgggatccgagacaagcagagtggaaagacaatcctg2040gattttcttaagtccgatggatttgccaaccggaacttcatgcagttgatccatgatgac2100tctctcacctttaaggaggacatccagaaagcacaagtttctggccagggggacagtctt2160cacgagcacatcgctaatcttgcaggtagcccagctatcaaaaagggaatactgcagacc2220gttaaggtcgtggatgaactcgtcaaagtaatgggaaggcataagcccgagaatatcgtt2280atcgagatggcccgagagaaccaaactacccagaagggacagaagaacagtagggaaagg2340atgaagaggattgaagagggtataaaagaactggggtcccaaatccttaaggaacaccca2400gttgaaaacacccagcttcagaatgagaagctctacctgtactacctgcagaacggcagg2460gacatgtacgtggatcaggaactggacatcaatcggctctccgactacgacgtggatcat2520atcgtgccccagtcttttctcaaagatgattctattgataataaagtgttgacaagatcc2580gataaaaatagagggaagagtgataacgtcccctcagaagaagttgtcaagaaaatgaaa2640aattattggcggcagctgctgaacgccaaactgatcacacaacggaagttcgataatctg2700actaaggctgaacgaggtggcctgtctgagttggataaagcaggcttcatcaaaaggcag2760cttgttgagacacgccagatcaccaagcacgtggcccaaattctcgattcacgcatgaac2820accaagtacgatgaaaatgacaaactgattcgagaggtgaaagttattactctgaagtct2880aagctggtctcagatttcagaaaggactttcagttttataaggtgagagagatcaacaat2940taccaccatgcgcatgatgcctacctgaatgcagtggtaggcactgcacttatcaaaaaa3000tatcccaagcttgaatctgaatttgtttacggagactataaagtgtacgatgttaggaaa3060atgatcgcaaagtctgagcaggaaataggcaaggccaccgctaagtacttcttttacagc3120aatattatgaattttttcaagaccgagattacactggccaatggagagattcggaagcga3180ccacttatcgaaacaaacggagaaacaggagaaatcgtgtgggacaagggtagggatttc3240gcgacagtccggaaggtcctgtccatgccgcaggtgaacatcgttaaaaagaccgaagta3300cagaccggaggcttctccaaggaaagtatcctcccgaaaaggaacagcgacaagctgatc3360gcacgcaaaaaagattgggaccccaagaaatacggcggattcgattctcctacagtcgct3420tacagtgtactggttgtggccaaagtggagaaagggaagtctaaaaaactcaaaagcgtc3480aaggaactgctgggcatcacaatcatggagcgatcaagcttcgaaaaaaaccccatcgac3540tttctcgaggcgaaaggatataaagaggtcaaaaaagacctcatcattaagcttcccaag3600tactctctctttgagcttgaaaacggccggaaacgaatgctcgctagtgcgggcgagctg3660cagaaaggtaacgagctggcactgccctctaaatacgttaatttcttgtatctggccagc3720cactatgaaaagctcaaagggtctcccgaagataatgagcagaagcagctgttcgtggaa3780caacacaaacactaccttgatgagatcatcgagcaaataagcgaattctccaaaagagtg3840atcctcgccgacgctaacctcgataaggtgctttctgcttacaataagcacagggataag3900cccatcagggagcaggcagaaaacattatccacttgtttactctgaccaacttgggcgcg3960cctgcagccttcaagtacttcgacaccaccatagacagaaagcggtacacctctacaaag4020gaggtcctggacgccacactgattcatcagtcaattacggggctctatgaaacaagaatc4080gacctctctcagctcggtggagac4104<210>2<211>1368<212>prt<213>streptococcuspyogenesserotypem1<400>2glya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