CN114854758A - 基于CRISPR-Cas13a系统靶向杀死沙门氏菌的方法及其应用 - Google Patents

基于CRISPR-Cas13a系统靶向杀死沙门氏菌的方法及其应用 Download PDF

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CN114854758A
CN114854758A CN202210674863.1A CN202210674863A CN114854758A CN 114854758 A CN114854758 A CN 114854758A CN 202210674863 A CN202210674863 A CN 202210674863A CN 114854758 A CN114854758 A CN 114854758A
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唐艺芝
宋子昊
王红宁
雷昌伟
张安云
杨鑫
赵蒙宇
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Abstract

本发明公开了基于CRISPR‑Cas13a系统靶向杀死沙门氏菌的方法及其应用，属于生物技术领域。本发明利用高序列灵敏度的CRISPR‑Cas13a系统特异性靶向沙门氏菌的内源转录本，从而实现特异性的靶向杀菌，该系统可以通过特异性crRNA靶向特定的RNA，在识别到特定RNA后会产生靶向切割活性和混乱切割活性，导致沙门氏菌的特异性死亡。该杀菌方法可以在混合群落和小鼠疾病模型中起作用，显著降低沙门氏菌的种群密度。

Description

基于CRISPR-Cas13a系统靶向杀死沙门氏菌的方法及其应用
技术领域
本发明涉及生物技术领域，具体涉及基于CRISPR-Cas13a系统靶向杀死沙门氏菌的方法及其应用，更具体涉及特异性靶向沙门氏菌的crRNA、CRISPR-Cas13a系统、基于CRISPR-Cas13a系统靶向杀死沙门氏菌的方法及其应用。
背景技术
自从发现青霉素以来，抗生素在对抗细菌感染方面发挥了重要作用。由于在发现新抗生素方面的过度使用和创新停滞，耐药细菌迅速出现，并被认为是世界各地的一个重大公共卫生威胁。为了应对这一趋势，需要并正在开发新的抗菌疗法，如抗生素佐剂和抗菌肽。值得注意的是，通常用于对抗病原体的抗生素也针对共生细菌，扰乱肠道菌群的组成，并导致意想不到的疾病。因此，细菌物种特异性和精确的抗菌技术是可取的，并正在开发。
CRISPR-Cas系统广泛存在于细菌和古菌中，负责对外源DNA(质粒和噬菌体)的适应性细胞免疫。CRISPR/Cas9系统在特定crRNAs(CRISPR RNA)和tracrRNA(反式激活CRISPRRNA)引导下，在目标DNA位点引入了双链断裂(DSBs)，使其成为一种强大的基因编辑工具。同时，DSBs导致复制分叉崩溃和细胞死亡，为CRISPR/Cas9系统提供了一种精确抗菌工具的潜力。然而，非同源末端连接修复DSBs产生的不可预测突变保真度较低，导致目标菌基因组进化未知，耐药性快速发展。相反，CRISPR-cas13a是一种VI型CRISPR系统，靶向并切割单链RNA(ssRNA)，在识别到目标ssRNA后，具有精确且混杂的切割活性，导致特异性基因失活和细菌生长抑制，从而在不损伤DNA的情况下防御噬菌体感染。这种特性显示了巨大的抗菌潜力，但很少有研究使用该系统专门消除病原体种类。
作为一种基于核酸的抗菌药物，一种高效、连续的载体是CRISPR系统所必需的。噬菌体对细菌具有很强的感染活性，是CRISPR系统最常用的载体，可实现基因特异性杀灭细菌。然而，噬菌体狭窄的光谱限制了它们的应用。
发明内容
本发明的目的是提供基于CRISPR-Cas13a系统靶向杀死沙门氏菌的方法及其应用，以解决抗生素等杀菌方法特异性不足的缺点以及抗生素等杀菌方法持续时间不长的问题。
本发明解决上述技术问题的技术方案如下：
本发明提供一种特异性靶向沙门氏菌的crRNA，所述crRNA包括：靶向dnaA、靶向katG或靶向hilA；
所述靶向dnaA的核苷酸序列如SEQ ID NO.1所示或如SEQ ID NO.2所示；
所述靶向katG的核苷酸序列如SEQ ID NO.3所示或如SEQ ID NO.4所示；
所述靶向hilA的核苷酸序列如SEQ ID NO.5所示。
本发明还提供一种CRISPR-Cas13a系统，包括上述的特异性靶向沙门氏菌的crRNA。
进一步地，在所述的CRISPR-Cas13a系统中，还包括lwaCas13a蛋白和氯霉素抗性基因；
所述lwaCas13a蛋白的氨基酸序列如SEQ ID NO.6所示；
所述氯霉素抗性基因的核苷酸序列如SEQ ID NO.7所示。
进一步地，在所述的特异性靶向沙门氏菌的CRISPR-Cas13a系统中，还包括表达所述lwaCas13a蛋白的质粒。
本发明还提供上述的CRISPR-Cas13a系统在靶向杀死沙门氏菌中的应用。
本发明还提供一种基于CRISPR-Cas13a系统靶向杀死沙门氏菌的方法，包括以下步骤：
将lwaCas13a蛋白、表达所述lwaCas13a蛋白的质粒、氯霉素抗性基因和crRNA进行扩增培养，提取出杀菌质粒；
将所述杀菌质粒通过结合作用转移到供体菌中，得到杀菌供体；
将所述杀菌供体投入到含沙门氏菌的环境中进行杀菌。
进一步地，在所述的基于CRISPR-Cas13a系统靶向杀死沙门氏菌的方法中，所述供体为大肠杆菌。
本发明还提供上述的crRNA在沙门氏菌靶向杀菌制剂中的应用。
本发明具有以下有益效果：
1、本发明利用高灵敏度的CRISPR-Cas13a系统特异性靶向沙门氏菌的内源转录本，从而实现特异性的靶向杀菌，该系统可以通过特异性crRNA靶向特定的RNA，在识别到特定RNA后会产生靶向切割活性和混乱切割活性，导致沙门氏菌的特异性死亡。该杀菌方法可以在混合群落和小鼠疾病模型中起作用，显著降低沙门氏菌的种群密度。
2、本发明通过大肠杆菌与沙门氏菌的接合作用，靶向性较好，可以特异性杀死沙门氏菌而不影响其他细菌，杀菌作用时间较长。
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4中的体外移除率结果；
图2为本发明试验例5中的生长抑制率结果；
图3为本发明试验例6中的小鼠体内试验结果。
具体实施方式
以下结合实施例及附图对本发明的原理和特征进行描述，所举实例只用于解释本发明，并非用于限定本发明的范围。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。
基于质粒的偶联是一种可行的CRISPR系统交付方法，具有寄发连续性、可访问性、易于设计、不需要特定受体和适合不同病原体的优点。因此，结合质粒是输送核酸类抗菌药物的合适方法。最近的研究使用偶联质粒以反式或顺式的方式传递CRISPR-Cas9系统，以去除耐药质粒或直接杀死特定细菌，在体外和体内均显示出很高的有效性和可用性。然而，与CRISPR-Cas13a结合的质粒尚未被用于特异性杀灭细菌病原体。
本发明的技术方法为：
本发明的特异性靶向沙门氏菌的crRNA，所述crRNA包括：靶向dnaA、靶向katG或靶向hilA；
所述靶向dnaA的核苷酸序列如SEQ ID NO.1所示或如SEQ ID NO.2所示；
所述靶向katG的核苷酸序列如SEQ ID NO.3所示或如SEQ ID NO.4所示；
所述靶向hilA的核苷酸序列如SEQ ID NO.5所示。
所述靶向dnaA包括：靶向dnaA 1或靶向dnaA 2。所述靶向katG包括：靶向katG 1或靶向katG 2。
Figure BDA0003694370410000041
Figure BDA0003694370410000051
本发明还提供一种CRISPR-Cas13a系统，包括特异性靶向沙门氏菌的crRNA，还包括lwaCas13a蛋白和氯霉素抗性基因；以及还包括表达所述lwaCas13a蛋白的质粒。
所述lwaCas13a蛋白包含启动子和RBS，所述lwaCas13a蛋白的氨基酸序列如SEQID NO.6所示，具体为：
TTGACAGCTAGCTCAGTCCTAGGTATTGTGCTAGCTACTAGAGAAAGAGGAGAAAATCTAGATATGAAAGTGACCAAGGTCGACGGCATCAGCCACAAGAAGTACATCGAAGAGGGCAAGCTCGTGAAGTCCACCAGCGAGGAAAACCGGACCAGCGAGAGACTGAGCGAGCTGCTGAGCATCCGGCTGGACATCTACATCAAGAACCCCGACAACGCCTCCGAGGAAGAGAACCGGATCAGAAGAGAGAACCTGAAGAAGTTCTTTAGCAACAAGGTGCTGCACCTGAAGGACAGCGTGCTGTATCTGAAGAACCGGAAAGAAAAGAACGCCGTGCAGGACAAGAACTATAGCGAAGAGGACATCAGCGAGTACGACCTGAAAAACAAGAACAGCTTCTCCGTGCTGAAGAAGATCCTGCTGAACGAGGACGTGAACTCTGAGGAACTGGAAATCTTTCGGAAGGACGTGGAAGCCAAGCTGAACAAGATCAACAGCCTGAAGTACAGCTTCGAAGAGAACAAGGCCAACTACCAGAAGATCAACGAGAACAACGTGGAAAAAGTGGGCGGCAAGAGCAAGCGGAACATCATCTACGACTACTACAGAGAGAGCGCCAAGCGCAACGACTACATCAACAACGTGCAGGAAGCCTTCGACAAGCTGTATAAGAAAGAGGATATCGAGAAACTGTTTTTCCTGATCGAGAACAGCAAGAAGCACGAGAAGTACAAGATCCGCGAGTACTATCACAAGATCATCGGCCGGAAGAACGACAAAGAGAACTTCGCCAAGATTATCTACGAAGAGATCCAGAACGTGAACAACATCAAAGAGCTGATTGAGAAGATCCCCGACATGTCTGAGCTGAAGAAAAGCCAGGTGTTCTACAAGTACTACCTGGACAAAGAGGAACTGAACGACAAGAATATTAAGTACGCCTTCTGCCACTTCGTGGAAATCGAGATGTCCCAGCTGCTGAAAAACTACGTGTACAAGCGGCTGAGCAACATCAGCAACGATAAGATCAAGCGGATCTTCGAGTACCAGAATCTGAAAAAGCTGATCGAAAACAAACTGCTGAACAAGCTGGACACCTACGTGCGGAACTGCGGCAAGTACAACTACTATCTGCAAGTGGGCGAGATCGCCACCTCCGACTTTATCGCCCGGAACCGGCAGAACGAGGCCTTCCTGAGAAACATCATCGGCGTGTCCAGCGTGGCCTACTTCAGCCTGAGGAACATCCTGGAAACCGAGAACGAGAACGATATCACCGGCCGGATGCGGGGCAAGACCGTGAAGAACAACAAGGGCGAAGAGAAATACGTGTCCGGCGAGGTGGACAAGATCTACAATGAGAACAAGCAGAACGAAGTGAAAGAAAATCTGAAGATGTTCTACAGCTACGACTTCAACATGGACAACAAGAACGAGATCGAGGACTTCTTCGCCAACATCGACGAGGCCATCAGCAGCATCAGACACGGCATCGTGCACTTCAACCTGGAACTGGAAGGCAAGGACATCTTCGCCTTCAAGAATATCGCCCCCAGCGAGATCTCCAAGAAGATGTTTCAGAACGAAATCAACGAAAAGAAGCTGAAGCTGAAAATCTTCAAGCAGCTGAACAGCGCCAACGTGTTCAACTACTACGAGAAGGATGTGATCATCAAGTACCTGAAGAATACCAAGTTCAACTTCGTGAACAAAAACATCCCCTTCGTGCCCAGCTTCACCAAGCTGTACAACAAGATTGAGGACCTGCGGAATACCCTGAAGTTTTTTTGGAGCGTGCCCAAGGACAAAGAAGAGAAGGACGCCCAGATCTACCTGCTGAAGAATATCTACTACGGCGAGTTCCTGAACAAGTTCGTGAAAAACTCCAAGGTGTTCTTTAAGATCACCAATGAAGTGATCAAGATTAACAAGCAGCGGAACCAGAAAACCGGCCACTACAAGTATCAGAAGTTCGAGAACATCGAGAAAACCGTGCCCGTGGAATACCTGGCCATCATCCAGAGCAGAGAGATGATCAACAACCAGGACAAAGAGGAAAAGAATACCTACATCGACTTTATTCAGCAGATTTTCCTGAAGGGCTTCATCGACTACCTGAACAAGAACAATCTGAAGTATATCGAGAGCAACAACAACAATGACAACAACGACATCTTCTCCAAGATCAAGATCAAAAAGGATAACAAAGAGAAGTACGACAAGATCCTGAAGAACTATGAGAAGCACAATCGGAACAAAGAAATCCCTCACGAGATCAATGAGTTCGTGCGCGAGATCAAGCTGGGGAAGATTCTGAAGTACACCGAGAATCTGAACATGTTTTACCTGATCCTGAAGCTGCTGAACCACAAAGAGCTGACCAACCTGAAGGGCAGCCTGGAAAAGTACCAGTCCGCCAACAAAGAAGAAACCTTCAGCGACGAGCTGGAACTGATCAACCTGCTGAACCTGGACAACAACAGAGTGACCGAGGACTTCGAGCTGGAAGCCAACGAGATCGGCAAGTTCCTGGACTTCAACGAAAACAAAATCAAGGACCGGAAAGAGCTGAAAAAGTTCGACACCAACAAGATCTATTTCGACGGCGAGAACATCATCAAGCACCGGGCCTTCTACAATATCAAGAAATACGGCATGCTGAATCTGCTGGAAAAGATCGCCGATAAGGCCAAGTATAAGATCAGCCTGAAAGAACTGAAAGAGTACAGCAACAAGAAGAATGAGATTGAAAAGAACTACACCATGCAGCAGAACCTGCACCGGAAGTACGCCAGACCCAAGAAGGACGAAAAGTTCAACGACGAGGACTACAAAGAGTATGAGAAGGCCATCGGCAACATCCAGAAGTACACCCACCTGAAGAACAAGGTGGAATTCAATGAGCTGAACCTGCTGCAGGGCCTGCTGCTGAAGATCCTGCACCGGCTCGTGGGCTACACCAGCATCTGGGAGCGGGACCTGAGATTCCGGCTGAAGGGCGAGTTTCCCGAGAACCACTACATCGAGGAAATTTTCAATTTCGACAACTCCAAGAATGTGAAGTACAAAAGCGGCCAGATCGTGGAAAAGTATATCAACTTCTACAAAGAACTGTACAAGGACAATGTGGAAAAGCGGAGCATCTACTCCGACAAGAAAGTGAAGAAACTGAAGCAGGAAAAAAAGGACCTGTACATCCGGAACTACATTGCCCACTTCAACTACATCCCCCACGCCGAGATTAGCCTGCTGGAAGTGCTGGAAAACCTGCGGAAGCTGCTGTCCTACGACCGGAAGCTGAAGAACGCCATCATGAAGTCCATCGTGGACATTCTGAAAGAATACGGCTTCGTGGCCACCTTCAAGATCGGCGCTGACAAGAAGATCGAAATCCAGACCCTGGAATCAGAGAAGATCGTGCACCTGAAGAATCTGAAGAAAAAGAAACTGATGACCGACCGGAACAGCGAGGAACTGTGCGAACTCGTGAAAGTGATGTTCGAGTACAAGGCCCTGGAATGA。
所述氯霉素抗性基因的核苷酸序列如SEQ ID NO.7所示，具体为：
TGATCGGCACGTAAGAGGTTCCAACTTTCACCATAATGAAATAAGATCACTACCGGGCGTATTTTTTGAGTTATCGAGATTTTCAGGAGCTAAGGAAGCTAAAATGGAGAAAAAAATCACTGGATATACCACCGTTGATATATCCCAATGGCATCGTAAAGAACATTTTGAGGCATTTCAGTCAGTTGCTCAATGTACCTATAACCAGACCGTTCAGCTGGATATTACGGCCTTTTTAAAGACCGTAAAGAAAAATAAGCACAAGTTTTATCCGGCCTTTATTCACATTCTTGCCCGCCTGATGAATGCTCATCCGGAATTCCGTATGGCAATGAAAGACGGTGAGCTGGTGATATGGGATAGTGTTCACCCTTGTTACACCGTTTTCCATGAGCAAACTGAAACGTTTTCATCGCTCTGGAGTGAATACCACGACGATTTCCGGCAGTTTCTACACATATATTCGCAAGATGTGGCGTGTTACGGTGAAAACCTGGCCTATTTCCCTAAAGGGTTTATTGAGAATATGTTTTTCGTGTCAGCCAATCCCTGGGTGAGTTTCACCAGTTTTGATTTAAACGTGGCCAATATGGACAACTTCTTCGCCCCCGTTTTCACCATGGGCAAATATTATACGCAAGGCGACAAGGTGCTGATGCCGCTGGCGATTCAGGTTCATCATGCCGTTTGTGATGGCTTCCATGTCGGCAGAATGCTTAATGAATTACAACAGTACTGCGATGAGTGGCAGGGCGGGGCGTAA。
本发明的CRISPR-Cas13a系统在靶向杀死沙门氏菌中的应用。
在本发明的基于CRISPR-Cas13a系统靶向杀死沙门氏菌的方法，包括以下步骤：
将lwaCas13a蛋白、表达所述lwaCas13a蛋白的质粒、氯霉素抗性基因和crRNA进行扩增培养，提取出杀菌质粒；
将所述杀菌质粒通过结合作用转移到大肠杆菌供体中，得到杀菌供体；
将所述杀菌供体投入到含沙门氏菌的环境中进行杀菌。
本发明的crRNA在沙门氏菌靶向杀菌制剂中的应用，可以通过冷冻干燥法将杀菌供体菌液进行干燥制成粉剂，包装进入胶囊中作为药品。
试验例1：构建杀菌质粒(CKP)
使用2×Phanta Flash Master Mix(南京诺唯赞生物科技股份有限公司)进行PCR，将pBBR1质粒线性骨架，蛋白lwaCas13a的表达序列，crRNA的骨架克隆成线性骨架DNA，氯霉素抗性基因线性骨架，使用ClonExpress II One Step Cloning Kit(南京诺唯赞生物科技股份有限公司)50℃，反应5分钟，将3个线性骨架连接在一起，加入到大肠杆菌DH5α感受态细胞中，冰上孵育30分钟，42℃热激60秒，冰上孵育5分钟，加入700μL无抗生素液体培养基中，220rpm，37℃复苏40分钟，之后涂布在含有34μg/ml氯霉素和50μg/ml卡那霉素的LB固体培养基上，37℃培养16小时，挑取单菌落扩增，使用Sanger测序检测是否连接成功。之后将成功转入的菌落扩增，使用质粒小提试剂盒(南京诺唯赞生物科技股份有限公司)将质粒提取，用于下一步工作。
试验例2：将杀菌质粒转入大肠杆菌供体
将试验例1中提取的杀菌质粒转化进入质粒供体大肠杆菌S17-1或带有F质粒的大肠杆菌Nissle 1917中，使用带有氯霉素和卡那霉素的LB固体培养基筛选，挑单菌落测序检测是否成功转入。将成功转入杀菌质粒的菌株扩培。
试验例3：将大肠杆菌供体投放到需要杀死沙门氏菌的环境中
将携带杀菌质粒的大肠杆菌投放到需要杀死沙门氏菌的环境中。
试验例4：体外移除实验
将转入了CKP的大肠杆菌S17-1与携带氨苄青霉素抗性基因的鼠伤寒沙门氏菌菌株等量混合，6000rpm，5min离心，取200μL培养基溶液重悬，涂布在固体无抗生素添加的LB固体培养基上，37℃倒置培养24h，将菌液使用1mL的PBS全部从LB固体培养基上洗脱，稀释涂布在添加0.2％阿拉伯糖或0.2％葡萄糖的LB抗性固体培养基上(培养基中含有34μg/mL的氯霉素，50μg/mL卡那霉素，100μg/mL氨苄青霉素)，选择菌落数量在30-200个的平板进行人工计数。通过以下公式检测体外代谢移除率：
体外代谢移除率＝1-阿拉伯糖转接合物活菌数/葡萄糖转接合物活菌数
结果见图1所示，从图1可以看出：具有靶向鼠伤寒沙门氏菌内源转录本的5个crRNA可以具有40-80％之间的清除率。
试验例5：生长抑制实验
将转入了CKP的大肠杆菌S17-1与携带氨苄青霉素抗性基因的鼠伤寒沙门氏菌菌株和大肠杆菌菌株等量混合，6000rpm，5min离心，取200μL培养基溶液重悬，涂布在固体无抗生素添加的LB固体培养基上，37℃倒置培养24h，将菌液使用1mL的PBS全部从LB固体培养基上洗脱，取10uL分别接种到在添加0.2％阿拉伯糖或0.2％葡萄糖的LB抗性液体培养基上(培养基中含有34μg/mL的氯霉素，50μg/mL卡那霉素，100μg/mL氨苄青霉素)，培养16h，使用实时微生物生长曲线检测仪(杰灵仪器制造(天津)有限公司)，稀释涂布在添加0.2％葡萄糖，含有氨苄青霉素、卡那霉素和氯霉素的固体LB培养基上，选择菌落数量在30-200个的平板进行人工计数。
结果见图2所示，从图2可以看出：通过表达CRISPR-Cas13a系统的鼠伤寒沙门氏菌在经过16h的培养下，比未经诱导的组种群密度低2-3个数量级。说明其具有明显的抑制菌落生长活性。
试验例6：小鼠体内实验
使用6～8周龄ICR/KM小鼠(成都达硕实验动物有限公司)进行实验。小鼠随机分为6组，每组5或6只。小鼠被提供足够的水和饲料。购买3天后，在小鼠饮水中添加氨苄西林2mg/ml，处理3天，然后用1x109CFU对鼠伤寒沙门氏菌进行攻毒。1天后，小鼠口服1x109 CFU的大肠杆菌S17-1或携带CKP的大肠杆菌S17-1，接种鼠伤寒杆菌后第1、2、3、4、8天用1.5mL离心管收集小鼠粪便。称量粪便，加入500mL PBS溶液和1-3个玻璃珠(生工生物，上海，中国)，在旋涡混合器(生工生物，上海，中国)下粉碎粪便。将粪便溶液从10-3连续稀释至10-7，置于加有氨苄西林的LB琼脂平板上，37℃孵育16h，人工计算菌落数量，比较8天内鼠伤寒沙门氏菌的减少量。
结果见图3所示，从图3可以看出：接种了携带CRISPR-Cas13a杀菌质粒(CKP)的大肠杆菌S17-1的小鼠，在第8天时鼠伤寒沙门氏菌的群落密度会出现显著性下降，而其他对照组或者接种不含有质粒的S17-1，无靶向crRNA的CKP，鼠伤寒沙门氏菌种群密度不会出现显著下降。说明本系统可以在动物体内起作用。
以上所述仅为本发明的较佳实施例，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
序列表
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Thr Cys Cys Thr Ala Gly Gly Thr Ala Thr Thr Gly Thr Gly Cys Thr
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Ala Gly Cys Thr Ala Cys Thr Ala Gly Ala Gly Ala Ala Ala Gly Ala
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Ala Ala Cys Thr Ala Cys Cys Ala Gly Ala Ala Gly Ala Thr Cys Ala
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Ala Ala Ala Ala Gly Thr Gly Gly Gly Cys Gly Gly Cys Ala Ala Gly
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Ala Gly Cys Ala Ala Gly Cys Gly Gly Ala Ala Cys Ala Thr Cys Ala
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Thr Cys Thr Ala Cys Gly Ala Cys Thr Ala Cys Thr Ala Cys Ala Gly
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Ala Gly Ala Gly Ala Gly Cys Gly Cys Cys Ala Ala Gly Cys Gly Cys
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Ala Ala Cys Gly Ala Cys Thr Ala Cys Ala Thr Cys Ala Ala Cys Ala
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Ala Cys Gly Thr Gly Cys Ala Gly Gly Ala Ala Gly Cys Cys Thr Thr
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Cys Gly Ala Cys Ala Ala Gly Cys Thr Gly Thr Ala Thr Ala Ala Gly
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Ala Ala Ala Gly Ala Gly Gly Ala Thr Ala Thr Cys Gly Ala Gly Ala
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Ala Ala Cys Thr Gly Thr Thr Thr Thr Thr Cys Cys Thr Gly Ala Thr
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Cys Gly Ala Gly Ala Ala Cys Ala Gly Cys Ala Ala Gly Ala Ala Gly
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Cys Ala Cys Gly Ala Gly Ala Ala Gly Thr Ala Cys Ala Ala Gly Ala
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Thr Cys Cys Gly Cys Gly Ala Gly Thr Ala Cys Thr Ala Thr Cys Ala
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Cys Ala Ala Gly Ala Thr Cys Ala Thr Cys Gly Gly Cys Cys Gly Gly
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Ala Ala Gly Ala Ala Cys Gly Ala Cys Ala Ala Ala Gly Ala Gly Ala
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Ala Cys Thr Thr Cys Gly Cys Cys Ala Ala Gly Ala Thr Thr Ala Thr
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Cys Thr Ala Cys Gly Ala Ala Gly Ala Gly Ala Thr Cys Cys Ala Gly
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Ala Ala Cys Gly Thr Gly Ala Ala Cys Ala Ala Cys Ala Thr Cys Ala
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Ala Ala Gly Ala Gly Cys Thr Gly Ala Thr Thr Gly Ala Gly Ala Ala
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Gly Ala Thr Cys Cys Cys Cys Gly Ala Cys Ala Thr Gly Thr Cys Thr
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Gly Ala Gly Cys Thr Gly Ala Ala Gly Ala Ala Ala Ala Gly Cys Cys
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Ala Gly Gly Thr Gly Thr Thr Cys Thr Ala Cys Ala Ala Gly Thr Ala
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Cys Thr Ala Cys Cys Thr Gly Gly Ala Cys Ala Ala Ala Gly Ala Gly
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Gly Ala Ala Cys Thr Gly Ala Ala Cys Gly Ala Cys Ala Ala Gly Ala
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Ala Thr Ala Thr Thr Ala Ala Gly Thr Ala Cys Gly Cys Cys Thr Thr
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Cys Thr Gly Cys Cys Ala Cys Thr Thr Cys Gly Thr Gly Gly Ala Ala
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Ala Thr Cys Gly Ala Gly Ala Thr Gly Thr Cys Cys Cys Ala Gly Cys
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Thr Gly Cys Thr Gly Ala Ala Ala Ala Ala Cys Thr Ala Cys Gly Thr
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Gly Thr Ala Cys Ala Ala Gly Cys Gly Gly Cys Thr Gly Ala Gly Cys
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Ala Ala Cys Ala Thr Cys Ala Gly Cys Ala Ala Cys Gly Ala Thr Ala
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Ala Gly Ala Thr Cys Ala Ala Gly Cys Gly Gly Ala Thr Cys Thr Thr
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Cys Gly Ala Gly Thr Ala Cys Cys Ala Gly Ala Ala Thr Cys Thr Gly
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Ala Ala Ala Ala Ala Gly Cys Thr Gly Ala Thr Cys Gly Ala Ala Ala
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Ala Cys Ala Ala Ala Cys Thr Gly Cys Thr Gly Ala Ala Cys Ala Ala
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Gly Cys Thr Gly Gly Ala Cys Ala Cys Cys Thr Ala Cys Gly Thr Gly
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Cys Gly Gly Ala Ala Cys Thr Gly Cys Gly Gly Cys Ala Ala Gly Thr
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Ala Cys Ala Ala Cys Thr Ala Cys Thr Ala Thr Cys Thr Gly Cys Ala
1125 1130 1135
Ala Gly Thr Gly Gly Gly Cys Gly Ala Gly Ala Thr Cys Gly Cys Cys
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Ala Cys Cys Thr Cys Cys Gly Ala Cys Thr Thr Thr Ala Thr Cys Gly
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Cys Cys Cys Gly Gly Ala Ala Cys Cys Gly Gly Cys Ala Gly Ala Ala
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Cys Gly Ala Gly Gly Cys Cys Thr Thr Cys Cys Thr Gly Ala Gly Ala
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Ala Ala Cys Ala Thr Cys Ala Thr Cys Gly Gly Cys Gly Thr Gly Thr
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Cys Cys Ala Gly Cys Gly Thr Gly Gly Cys Cys Thr Ala Cys Thr Thr
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Cys Ala Gly Cys Cys Thr Gly Ala Gly Gly Ala Ala Cys Ala Thr Cys
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Cys Thr Gly Gly Ala Ala Ala Cys Cys Gly Ala Gly Ala Ala Cys Gly
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Ala Gly Ala Ala Cys Gly Ala Thr Ala Thr Cys Ala Cys Cys Gly Gly
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Cys Cys Gly Gly Ala Thr Gly Cys Gly Gly Gly Gly Cys Ala Ala Gly
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Ala Cys Cys Gly Thr Gly Ala Ala Gly Ala Ala Cys Ala Ala Cys Ala
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Ala Gly Gly Gly Cys Gly Ala Ala Gly Ala Gly Ala Ala Ala Thr Ala
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Cys Gly Thr Gly Thr Cys Cys Gly Gly Cys Gly Ala Gly Gly Thr Gly
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Gly Ala Cys Ala Ala Gly Ala Thr Cys Thr Ala Cys Ala Ala Thr Gly
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Ala Gly Ala Ala Cys Ala Ala Gly Cys Ala Gly Ala Ala Cys Gly Ala
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Ala Gly Thr Gly Ala Ala Ala Gly Ala Ala Ala Ala Thr Cys Thr Gly
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Ala Ala Gly Ala Thr Gly Thr Thr Cys Thr Ala Cys Ala Gly Cys Thr
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Ala Cys Gly Ala Cys Thr Thr Cys Ala Ala Cys Ala Thr Gly Gly Ala
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Cys Ala Ala Cys Ala Ala Gly Ala Ala Cys Gly Ala Gly Ala Thr Cys
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Gly Ala Gly Gly Ala Cys Thr Thr Cys Thr Thr Cys Gly Cys Cys Ala
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Ala Cys Ala Thr Cys Gly Ala Cys Gly Ala Gly Gly Cys Cys Ala Thr
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Cys Ala Gly Cys Ala Gly Cys Ala Thr Cys Ala Gly Ala Cys Ala Cys
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Gly Gly Cys Ala Thr Cys Gly Thr Gly Cys Ala Cys Thr Thr Cys Ala
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Ala Cys Cys Thr Gly Gly Ala Ala Cys Thr Gly Gly Ala Ala Gly Gly
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Cys Ala Ala Gly Gly Ala Cys Ala Thr Cys Thr Thr Cys Gly Cys Cys
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Thr Thr Cys Ala Ala Gly Ala Ala Thr Ala Thr Cys Gly Cys Cys Cys
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Cys Cys Ala Gly Cys Gly Ala Gly Ala Thr Cys Thr Cys Cys Ala Ala
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Gly Ala Ala Gly Ala Thr Gly Thr Thr Thr Cys Ala Gly Ala Ala Cys
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Gly Ala Ala Ala Thr Cys Ala Ala Cys Gly Ala Ala Ala Ala Gly Ala
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Ala Gly Cys Thr Gly Ala Ala Gly Cys Thr Gly Ala Ala Ala Ala Thr
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Cys Thr Thr Cys Ala Ala Gly Cys Ala Gly Cys Thr Gly Ala Ala Cys
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Ala Gly Cys Gly Cys Cys Ala Ala Cys Gly Thr Gly Thr Thr Cys Ala
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Ala Cys Thr Ala Cys Thr Ala Cys Gly Ala Gly Ala Ala Gly Gly Ala
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Thr Gly Thr Gly Ala Thr Cys Ala Thr Cys Ala Ala Gly Thr Ala Cys
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Cys Thr Gly Ala Ala Gly Ala Ala Thr Ala Cys Cys Ala Ala Gly Thr
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Thr Cys Ala Ala Cys Thr Thr Cys Gly Thr Gly Ala Ala Cys Ala Ala
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Ala Ala Ala Cys Ala Thr Cys Cys Cys Cys Thr Thr Cys Gly Thr Gly
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Cys Cys Cys Ala Gly Cys Thr Thr Cys Ala Cys Cys Ala Ala Gly Cys
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Thr Gly Thr Ala Cys Ala Ala Cys Ala Ala Gly Ala Thr Thr Gly Ala
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Gly Gly Ala Cys Cys Thr Gly Cys Gly Gly Ala Ala Thr Ala Cys Cys
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Cys Thr Gly Ala Ala Gly Thr Thr Thr Thr Thr Thr Thr Gly Gly Ala
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Gly Cys Gly Thr Gly Cys Cys Cys Ala Ala Gly Gly Ala Cys Ala Ala
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Ala Gly Ala Ala Gly Ala Gly Ala Ala Gly Gly Ala Cys Gly Cys Cys
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Cys Ala Gly Ala Thr Cys Thr Ala Cys Cys Thr Gly Cys Thr Gly Ala
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Ala Gly Ala Ala Thr Ala Thr Cys Thr Ala Cys Thr Ala Cys Gly Gly
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Cys Gly Ala Gly Thr Thr Cys Cys Thr Gly Ala Ala Cys Ala Ala Gly
1860 1865 1870
Thr Thr Cys Gly Thr Gly Ala Ala Ala Ala Ala Cys Thr Cys Cys Ala
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Ala Gly Gly Thr Gly Thr Thr Cys Thr Thr Thr Ala Ala Gly Ala Thr
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Cys Ala Cys Cys Ala Ala Thr Gly Ala Ala Gly Thr Gly Ala Thr Cys
1905 1910 1915 1920
Ala Ala Gly Ala Thr Thr Ala Ala Cys Ala Ala Gly Cys Ala Gly Cys
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Gly Gly Ala Ala Cys Cys Ala Gly Ala Ala Ala Ala Cys Cys Gly Gly
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Cys Cys Ala Cys Thr Ala Cys Ala Ala Gly Thr Ala Thr Cys Ala Gly
1955 1960 1965
Ala Ala Gly Thr Thr Cys Gly Ala Gly Ala Ala Cys Ala Thr Cys Gly
1970 1975 1980
Ala Gly Ala Ala Ala Ala Cys Cys Gly Thr Gly Cys Cys Cys Gly Thr
1985 1990 1995 2000
Gly Gly Ala Ala Thr Ala Cys Cys Thr Gly Gly Cys Cys Ala Thr Cys
2005 2010 2015
Ala Thr Cys Cys Ala Gly Ala Gly Cys Ala Gly Ala Gly Ala Gly Ala
2020 2025 2030
Thr Gly Ala Thr Cys Ala Ala Cys Ala Ala Cys Cys Ala Gly Gly Ala
2035 2040 2045
Cys Ala Ala Ala Gly Ala Gly Gly Ala Ala Ala Ala Gly Ala Ala Thr
2050 2055 2060
Ala Cys Cys Thr Ala Cys Ala Thr Cys Gly Ala Cys Thr Thr Thr Ala
2065 2070 2075 2080
Thr Thr Cys Ala Gly Cys Ala Gly Ala Thr Thr Thr Thr Cys Cys Thr
2085 2090 2095
Gly Ala Ala Gly Gly Gly Cys Thr Thr Cys Ala Thr Cys Gly Ala Cys
2100 2105 2110
Thr Ala Cys Cys Thr Gly Ala Ala Cys Ala Ala Gly Ala Ala Cys Ala
2115 2120 2125
Ala Thr Cys Thr Gly Ala Ala Gly Thr Ala Thr Ala Thr Cys Gly Ala
2130 2135 2140
Gly Ala Gly Cys Ala Ala Cys Ala Ala Cys Ala Ala Cys Ala Ala Thr
2145 2150 2155 2160
Gly Ala Cys Ala Ala Cys Ala Ala Cys Gly Ala Cys Ala Thr Cys Thr
2165 2170 2175
Thr Cys Thr Cys Cys Ala Ala Gly Ala Thr Cys Ala Ala Gly Ala Thr
2180 2185 2190
Cys Ala Ala Ala Ala Ala Gly Gly Ala Thr Ala Ala Cys Ala Ala Ala
2195 2200 2205
Gly Ala Gly Ala Ala Gly Thr Ala Cys Gly Ala Cys Ala Ala Gly Ala
2210 2215 2220
Thr Cys Cys Thr Gly Ala Ala Gly Ala Ala Cys Thr Ala Thr Gly Ala
2225 2230 2235 2240
Gly Ala Ala Gly Cys Ala Cys Ala Ala Thr Cys Gly Gly Ala Ala Cys
2245 2250 2255
Ala Ala Ala Gly Ala Ala Ala Thr Cys Cys Cys Thr Cys Ala Cys Gly
2260 2265 2270
Ala Gly Ala Thr Cys Ala Ala Thr Gly Ala Gly Thr Thr Cys Gly Thr
2275 2280 2285
Gly Cys Gly Cys Gly Ala Gly Ala Thr Cys Ala Ala Gly Cys Thr Gly
2290 2295 2300
Gly Gly Gly Ala Ala Gly Ala Thr Thr Cys Thr Gly Ala Ala Gly Thr
2305 2310 2315 2320
Ala Cys Ala Cys Cys Gly Ala Gly Ala Ala Thr Cys Thr Gly Ala Ala
2325 2330 2335
Cys Ala Thr Gly Thr Thr Thr Thr Ala Cys Cys Thr Gly Ala Thr Cys
2340 2345 2350
Cys Thr Gly Ala Ala Gly Cys Thr Gly Cys Thr Gly Ala Ala Cys Cys
2355 2360 2365
Ala Cys Ala Ala Ala Gly Ala Gly Cys Thr Gly Ala Cys Cys Ala Ala
2370 2375 2380
Cys Cys Thr Gly Ala Ala Gly Gly Gly Cys Ala Gly Cys Cys Thr Gly
2385 2390 2395 2400
Gly Ala Ala Ala Ala Gly Thr Ala Cys Cys Ala Gly Thr Cys Cys Gly
2405 2410 2415
Cys Cys Ala Ala Cys Ala Ala Ala Gly Ala Ala Gly Ala Ala Ala Cys
2420 2425 2430
Cys Thr Thr Cys Ala Gly Cys Gly Ala Cys Gly Ala Gly Cys Thr Gly
2435 2440 2445
Gly Ala Ala Cys Thr Gly Ala Thr Cys Ala Ala Cys Cys Thr Gly Cys
2450 2455 2460
Thr Gly Ala Ala Cys Cys Thr Gly Gly Ala Cys Ala Ala Cys Ala Ala
2465 2470 2475 2480
Cys Ala Gly Ala Gly Thr Gly Ala Cys Cys Gly Ala Gly Gly Ala Cys
2485 2490 2495
Thr Thr Cys Gly Ala Gly Cys Thr Gly Gly Ala Ala Gly Cys Cys Ala
2500 2505 2510
Ala Cys Gly Ala Gly Ala Thr Cys Gly Gly Cys Ala Ala Gly Thr Thr
2515 2520 2525
Cys Cys Thr Gly Gly Ala Cys Thr Thr Cys Ala Ala Cys Gly Ala Ala
2530 2535 2540
Ala Ala Cys Ala Ala Ala Ala Thr Cys Ala Ala Gly Gly Ala Cys Cys
2545 2550 2555 2560
Gly Gly Ala Ala Ala Gly Ala Gly Cys Thr Gly Ala Ala Ala Ala Ala
2565 2570 2575
Gly Thr Thr Cys Gly Ala Cys Ala Cys Cys Ala Ala Cys Ala Ala Gly
2580 2585 2590
Ala Thr Cys Thr Ala Thr Thr Thr Cys Gly Ala Cys Gly Gly Cys Gly
2595 2600 2605
Ala Gly Ala Ala Cys Ala Thr Cys Ala Thr Cys Ala Ala Gly Cys Ala
2610 2615 2620
Cys Cys Gly Gly Gly Cys Cys Thr Thr Cys Thr Ala Cys Ala Ala Thr
2625 2630 2635 2640
Ala Thr Cys Ala Ala Gly Ala Ala Ala Thr Ala Cys Gly Gly Cys Ala
2645 2650 2655
Thr Gly Cys Thr Gly Ala Ala Thr Cys Thr Gly Cys Thr Gly Gly Ala
2660 2665 2670
Ala Ala Ala Gly Ala Thr Cys Gly Cys Cys Gly Ala Thr Ala Ala Gly
2675 2680 2685
Gly Cys Cys Ala Ala Gly Thr Ala Thr Ala Ala Gly Ala Thr Cys Ala
2690 2695 2700
Gly Cys Cys Thr Gly Ala Ala Ala Gly Ala Ala Cys Thr Gly Ala Ala
2705 2710 2715 2720
Ala Gly Ala Gly Thr Ala Cys Ala Gly Cys Ala Ala Cys Ala Ala Gly
2725 2730 2735
Ala Ala Gly Ala Ala Thr Gly Ala Gly Ala Thr Thr Gly Ala Ala Ala
2740 2745 2750
Ala Gly Ala Ala Cys Thr Ala Cys Ala Cys Cys Ala Thr Gly Cys Ala
2755 2760 2765
Gly Cys Ala Gly Ala Ala Cys Cys Thr Gly Cys Ala Cys Cys Gly Gly
2770 2775 2780
Ala Ala Gly Thr Ala Cys Gly Cys Cys Ala Gly Ala Cys Cys Cys Ala
2785 2790 2795 2800
Ala Gly Ala Ala Gly Gly Ala Cys Gly Ala Ala Ala Ala Gly Thr Thr
2805 2810 2815
Cys Ala Ala Cys Gly Ala Cys Gly Ala Gly Gly Ala Cys Thr Ala Cys
2820 2825 2830
Ala Ala Ala Gly Ala Gly Thr Ala Thr Gly Ala Gly Ala Ala Gly Gly
2835 2840 2845
Cys Cys Ala Thr Cys Gly Gly Cys Ala Ala Cys Ala Thr Cys Cys Ala
2850 2855 2860
Gly Ala Ala Gly Thr Ala Cys Ala Cys Cys Cys Ala Cys Cys Thr Gly
2865 2870 2875 2880
Ala Ala Gly Ala Ala Cys Ala Ala Gly Gly Thr Gly Gly Ala Ala Thr
2885 2890 2895
Thr Cys Ala Ala Thr Gly Ala Gly Cys Thr Gly Ala Ala Cys Cys Thr
2900 2905 2910
Gly Cys Thr Gly Cys Ala Gly Gly Gly Cys Cys Thr Gly Cys Thr Gly
2915 2920 2925
Cys Thr Gly Ala Ala Gly Ala Thr Cys Cys Thr Gly Cys Ala Cys Cys
2930 2935 2940
Gly Gly Cys Thr Cys Gly Thr Gly Gly Gly Cys Thr Ala Cys Ala Cys
2945 2950 2955 2960
Cys Ala Gly Cys Ala Thr Cys Thr Gly Gly Gly Ala Gly Cys Gly Gly
2965 2970 2975
Gly Ala Cys Cys Thr Gly Ala Gly Ala Thr Thr Cys Cys Gly Gly Cys
2980 2985 2990
Thr Gly Ala Ala Gly Gly Gly Cys Gly Ala Gly Thr Thr Thr Cys Cys
2995 3000 3005
Cys Gly Ala Gly Ala Ala Cys Cys Ala Cys Thr Ala Cys Ala Thr Cys
3010 3015 3020
Gly Ala Gly Gly Ala Ala Ala Thr Thr Thr Thr Cys Ala Ala Thr Thr
3025 3030 3035 3040
Thr Cys Gly Ala Cys Ala Ala Cys Thr Cys Cys Ala Ala Gly Ala Ala
3045 3050 3055
Thr Gly Thr Gly Ala Ala Gly Thr Ala Cys Ala Ala Ala Ala Gly Cys
3060 3065 3070
Gly Gly Cys Cys Ala Gly Ala Thr Cys Gly Thr Gly Gly Ala Ala Ala
3075 3080 3085
Ala Gly Thr Ala Thr Ala Thr Cys Ala Ala Cys Thr Thr Cys Thr Ala
3090 3095 3100
Cys Ala Ala Ala Gly Ala Ala Cys Thr Gly Thr Ala Cys Ala Ala Gly
3105 3110 3115 3120
Gly Ala Cys Ala Ala Thr Gly Thr Gly Gly Ala Ala Ala Ala Gly Cys
3125 3130 3135
Gly Gly Ala Gly Cys Ala Thr Cys Thr Ala Cys Thr Cys Cys Gly Ala
3140 3145 3150
Cys Ala Ala Gly Ala Ala Ala Gly Thr Gly Ala Ala Gly Ala Ala Ala
3155 3160 3165
Cys Thr Gly Ala Ala Gly Cys Ala Gly Gly Ala Ala Ala Ala Ala Ala
3170 3175 3180
Ala Gly Gly Ala Cys Cys Thr Gly Thr Ala Cys Ala Thr Cys Cys Gly
3185 3190 3195 3200
Gly Ala Ala Cys Thr Ala Cys Ala Thr Thr Gly Cys Cys Cys Ala Cys
3205 3210 3215
Thr Thr Cys Ala Ala Cys Thr Ala Cys Ala Thr Cys Cys Cys Cys Cys
3220 3225 3230
Ala Cys Gly Cys Cys Gly Ala Gly Ala Thr Thr Ala Gly Cys Cys Thr
3235 3240 3245
Gly Cys Thr Gly Gly Ala Ala Gly Thr Gly Cys Thr Gly Gly Ala Ala
3250 3255 3260
Ala Ala Cys Cys Thr Gly Cys Gly Gly Ala Ala Gly Cys Thr Gly Cys
3265 3270 3275 3280
Thr Gly Thr Cys Cys Thr Ala Cys Gly Ala Cys Cys Gly Gly Ala Ala
3285 3290 3295
Gly Cys Thr Gly Ala Ala Gly Ala Ala Cys Gly Cys Cys Ala Thr Cys
3300 3305 3310
Ala Thr Gly Ala Ala Gly Thr Cys Cys Ala Thr Cys Gly Thr Gly Gly
3315 3320 3325
Ala Cys Ala Thr Thr Cys Thr Gly Ala Ala Ala Gly Ala Ala Thr Ala
3330 3335 3340
Cys Gly Gly Cys Thr Thr Cys Gly Thr Gly Gly Cys Cys Ala Cys Cys
3345 3350 3355 3360
Thr Thr Cys Ala Ala Gly Ala Thr Cys Gly Gly Cys Gly Cys Thr Gly
3365 3370 3375
Ala Cys Ala Ala Gly Ala Ala Gly Ala Thr Cys Gly Ala Ala Ala Thr
3380 3385 3390
Cys Cys Ala Gly Ala Cys Cys Cys Thr Gly Gly Ala Ala Thr Cys Ala
3395 3400 3405
Gly Ala Gly Ala Ala Gly Ala Thr Cys Gly Thr Gly Cys Ala Cys Cys
3410 3415 3420
Thr Gly Ala Ala Gly Ala Ala Thr Cys Thr Gly Ala Ala Gly Ala Ala
3425 3430 3435 3440
Ala Ala Ala Gly Ala Ala Ala Cys Thr Gly Ala Thr Gly Ala Cys Cys
3445 3450 3455
Gly Ala Cys Cys Gly Gly Ala Ala Cys Ala Gly Cys Gly Ala Gly Gly
3460 3465 3470
Ala Ala Cys Thr Gly Thr Gly Cys Gly Ala Ala Cys Thr Cys Gly Thr
3475 3480 3485
Gly Ala Ala Ala Gly Thr Gly Ala Thr Gly Thr Thr Cys Gly Ala Gly
3490 3495 3500
Thr Ala Cys Ala Ala Gly Gly Cys Cys Cys Thr Gly Gly Ala Ala Thr
3505 3510 3515 3520
Gly Ala
<210> 7
<211> 763
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
tgatcggcac gtaagaggtt ccaactttca ccataatgaa ataagatcac taccgggcgt 60
attttttgag ttatcgagat tttcaggagc taaggaagct aaaatggaga aaaaaatcac 120
tggatatacc accgttgata tatcccaatg gcatcgtaaa gaacattttg aggcatttca 180
gtcagttgct caatgtacct ataaccagac cgttcagctg gatattacgg cctttttaaa 240
gaccgtaaag aaaaataagc acaagtttta tccggccttt attcacattc ttgcccgcct 300
gatgaatgct catccggaat tccgtatggc aatgaaagac ggtgagctgg tgatatggga 360
tagtgttcac ccttgttaca ccgttttcca tgagcaaact gaaacgtttt catcgctctg 420
gagtgaatac cacgacgatt tccggcagtt tctacacata tattcgcaag atgtggcgtg 480
ttacggtgaa aacctggcct atttccctaa agggtttatt gagaatatgt ttttcgtgtc 540
agccaatccc tgggtgagtt tcaccagttt tgatttaaac gtggccaata tggacaactt 600
cttcgccccc gttttcacca tgggcaaata ttatacgcaa ggcgacaagg tgctgatgcc 660
gctggcgatt caggttcatc atgccgtttg tgatggcttc catgtcggca gaatgcttaa 720
tgaattacaa cagtactgcg atgagtggca gggcggggcg taa 763

Claims (8)

1.一种特异性靶向沙门氏菌的crRNA，其特征在于，所述crRNA包括：靶向dnaA、靶向katG或靶向hilA；
所述靶向dnaA的crRNA序列如SEQ ID NO.1所示或如SEQ ID NO.2所示；
所述靶向katG的crRNA序列如SEQ ID NO.3所示或如SEQ ID NO.4所示；
所述靶向hilA的crRNA序列如SEQ ID NO.5所示。
2.一种CRISPR-Cas13a系统，其特征在于，包括如权利要求1所述的特异性靶向沙门氏菌的crRNA。
3.根据权利要求2所述的CRISPR-Cas13a系统，其特征在于，还包括lwaCas13a蛋白和氯霉素抗性基因；
所述lwaCas13a蛋白的氨基酸序列如SEQ ID NO.6所示；
所述氯霉素抗性基因的核苷酸序列如SEQ ID NO.7所示。
4.根据权利要求2所述的CRISPR-Cas13a系统，其特征在于，还包括表达所述lwaCas13a蛋白的质粒。
5.一种如权利要求2-4任一项所述的CRISPR-Cas13a系统在靶向杀死沙门氏菌中的应用。
6.一种基于CRISPR-Cas13a系统靶向杀死沙门氏菌的方法，其特征在于，包括以下步骤：
将lwaCas13a蛋白、表达所述lwaCas13a蛋白的质粒、氯霉素抗性基因和crRNA进行扩增培养，提取出杀菌质粒；
将所述杀菌质粒通过结合作用转移到供体菌中，得到杀菌供体；
将所述杀菌供体投入到含沙门氏菌的环境中进行杀菌。
7.根据权利要求6所述的基于CRISPR-Cas13a系统杀死沙门氏菌的方法，其特征在于，所述供体菌为大肠杆菌。
8.一种如权利要求1所述的crRNA在沙门氏菌靶向杀菌制剂中的应用。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114126631A (zh) * 2019-05-12 2022-03-01 富利安食物科学有限公司 抗菌剂和方法

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110358767A (zh) * 2019-07-30 2019-10-22 湖北大学 一种基于CRISPR-Cas12a系统的运动发酵单胞菌基因组编辑方法及其应用
WO2020229372A1 (en) * 2019-05-12 2020-11-19 Folium Food Science Limited Antibacterial agents & methods
JP2021525105A (ja) * 2018-05-25 2021-09-24 ローカス バイオサイエンシーズ，インク． 標的細菌を死滅させるための方法および組成物
CN113462796A (zh) * 2021-06-15 2021-10-01 浙江大学 核酸等温扩增和CRISPR/Cas13a联合检测微生物的方法和应用

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2021525105A (ja) * 2018-05-25 2021-09-24 ローカス バイオサイエンシーズ，インク． 標的細菌を死滅させるための方法および組成物
WO2020229372A1 (en) * 2019-05-12 2020-11-19 Folium Food Science Limited Antibacterial agents & methods
CN114126631A (zh) * 2019-05-12 2022-03-01 富利安食物科学有限公司 抗菌剂和方法
CN110358767A (zh) * 2019-07-30 2019-10-22 湖北大学 一种基于CRISPR-Cas12a系统的运动发酵单胞菌基因组编辑方法及其应用
CN113462796A (zh) * 2021-06-15 2021-10-01 浙江大学 核酸等温扩增和CRISPR/Cas13a联合检测微生物的方法和应用

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
KOTARO KIGA 等: "Development of CRISPR-Cas13a-based antimicrobials capable of sequence-specific killing of tearget bacteria", vol. 11, no. 1, pages 2 - 3, XP055888259, DOI: 10.1038/s41467-020-16731-6 *
LOTTE BOHEZ 等: "Long-term colonisation-inhibition studies to protect broilers against colonisation with Salmonella Enteritidis, using Salmonella Pathogenicity Island 1 and 2 mutants", VACCINE, vol. 25, no. 21, pages 4265 - 4243 *
SAMANTHA MCLEAN 等: "Peroxynitrite stress is exacerbates by flavohaemoglobin-derived oxidative stress in Salmonella Typhimurium and is relieved by nitric oxide", MICROBIOLOGY, vol. 156, no. 12, pages 3556 - 3565 *
ZHIHAO SONG 等: "Pathogen-Specific Bactericidal Method Mediated by Conjugative Delivery of CRISPR-Cas13a Targeting Bacterial Endogenous Transcripts", vol. 10, no. 4, pages 1 - 14 *
唐正露 等: "肠炎沙门氏菌ssRAB、hilA、hilD基因缺失菌株的构建及其生物学特性", 微生物学通报, vol. 48, no. 4, pages 1195 - 1205 *
孙志坚 等: "沙门氏菌感染的研究进展", vol. 13, pages 6 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114126631A (zh) * 2019-05-12 2022-03-01 富利安食物科学有限公司 抗菌剂和方法

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