CN108707620A - 一种Gene drive载体及构建方法 - Google Patents
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Abstract
本发明属于生物工程领域，尤其涉及一种Gene drive载体及其构建方法，包括以下步骤：步骤1)cap5A启动子与sgRNA片段的人工合成，步骤2)扩增cas9基因，步骤3)扩增质粒骨架，步骤4)扩增rpsL启动子，步骤5)组装空载体，步骤6)插入靶向的spacer，步骤7)构建载体，本发明尝试结合了新型染色体盒重组酶CcrC2和CRISPR‑Cas9技术，构建了SCCmec killer载体系统，即Gene drive载体，Gene drive载体作用在甲氧西林抗性菌株MRSA，实现了在甲氧西林抗性菌株MRSA中靶向去除葡萄球菌染色体盒SCCmec，从而消除甲氧西林抗性基因。
Description
技术领域
本发明属于生物工程领域，尤其涉及一种能够消除MRSA中甲氧西林抗性基因的Gene drive载体及构建方法。
背景技术
金黄色葡萄球菌能引起肺炎、菌血症、心内膜炎等感染性疾病，而医疗过程中抗生素的滥用导致耐药菌株的发生率居高不下，其中耐甲氧西林金黄色葡萄球菌(methicillin-resistant Staphylococcus aureus，MRSA)的危害尤为严重。MRSA是由敏感菌株从外源获得mecA基因所产生的，该基因编码一种新的青霉素结合蛋白-PBP2a，赋予了金黄色葡萄球菌对β-内酰胺类抗生素的广谱耐药性，导致了β-内酰胺类抗生素在临床治疗中失去效果。其中，mecA基因位于可移动基因岛葡萄球菌染色体盒中，Ccr重组酶能够介导其在葡萄球菌间的水平转移，使甲氧西林抗性菌株的出现变得更加频繁，临床治疗金黄色葡萄球菌引起的疾病越来越困难。
目前针对甲氧西林抗性金黄色葡萄球菌的治疗，仍以抗生素为主导，常用抗生素有万古霉素，妥布霉素等，但近年来，万古霉素和妥布霉素抗性菌株也相继出现。此外广谱抗生素会无差别的杀死绝大部分的细菌，在治疗同时会破坏人原有的微生物区系。为解决甲氧西林抗性菌株频发的问题，亟须发明一种更具针对性的方法来直接消除甲氧西林抗性基因，而尽量少的减少对有益菌以及微生物区系平衡的破坏。
发明内容
本发明的目的在于克服现有技术中存在的不足而提供的一种能够消除MRSA中甲氧西林抗性基因的Gene drive载体及其构建方法。
本发明是这样实现的：一种Gene drive载体，其核酸序列如SEQ ID NO.1所示。
一种Gene drive载体的构建方法，其特征在于，包括以下步骤：
步骤1)cap5A启动子与sgRNA片段的人工合成：分别取引物SK1到SK8，混合于一个PCR管中，加入超纯水和High-Fidelity 2X Master Mix后，进行PCR反应，反应结束后，胶回收扩增产物。
步骤2)扩增cas9基因：在PCR管中加入High-Fidelity 2X Master Mix、引物SK9、引物SK10和酿脓链球菌基因组DNA，然后进行PCR反应，反应结束后，胶回收扩增产物。
步骤3)扩增质粒骨架：在PCR管中加入High-Fidelity 2X Master Mix、引物SK11、引物SK12和质粒pSCCcat(来自于GenBank数据库中KY701738)，进行PCR反应，反应结束后，胶回收扩增产物。
步骤4)扩增rpsL启动子：在PCR管中加入High-Fidelity 2X Master Mix、引物SK13、引物SK14和金黄色葡萄球菌基因组DNA，进行PCR反应，反应结束后，胶回收扩增产物。
步骤5)组装空载体：将步骤1、步骤2、步骤3和步骤4中所有的扩增产物加入到超纯水中，再添加入GibsonMaster Mix(购自New England Biolabs公司)，最后将全部产物转化至大肠杆菌DH5a中。
步骤6)插入靶向的spacer：使用T4多聚核苷酸激酶(T4PNK)对引物SK15和SK16进行了5’末端的磷酸化，将磷酸化的引物稀释后，加入NaCl调节浓度，而后与步骤5获得的空载体进行Golden Gate assembly，将反应液直接转化大肠杆菌DH5a。
步骤7)构建载体：在PCR管中加入High-Fidelity 2X MasterMix、引物SK17、SK18引物和质粒pSCCcat(来自于GenBank数据库中KY701738)，构成PCR反应体系，PCR反应结束后，胶回收扩增产物；同时用Xba1和Xho1双酶切并回收步骤6中的载体，最后与胶回收的扩增产物进行进行Gibson assembly，转化大肠杆菌DH5a，即获得了载体。
一种Gene drive载体的制备方法，所述的引物SK 1的核酸序列如SEQ ID NO.2所示；所述的引物SK 2的核酸序列如SEQ ID NO.3所示；所述的引物SK 3的核酸序列如SEQ IDNO.4所示；所述的引物SK 4的核酸序列如SEQ ID NO.5所示；所述的引物SK 5的核酸序列如SEQ ID NO.6所示；所述的引物SK 6的核酸序列如SEQ ID NO.7所示；所述的引物SK 7的核酸序列如SEQ ID NO.8所示；所述的引物SK 8的核酸序列如SEQ ID NO.9所示；所述的引物SK 9的核酸序列如SEQ ID NO.10所示；所述的引物SK 10的核酸序列如SEQ ID NO.11所示；所述的引物SK 11的核酸序列如SEQ ID NO.12所示；所述的引物SK 12的核酸序列如SEQ IDNO.13所示；所述的引物SK 13的核酸序列如SEQ ID NO.14所示；所述的引物SK 14的核酸序列如SEQ ID NO.15所示；所述的引物SK 15的核酸序列如SEQ ID NO.16所示；所述的引物SK16的核酸序列如SEQ ID NO.17所示；所述的引物SK 17的核酸序列如SEQ ID NO.18所示；所述的引物SK 18的核酸序列如SEQ ID NO.19所示。
本发明具有以下优点：
该发明中ccrC2基因克隆自甲氧西林抗性金黄色葡萄球菌BA01611菌株，通过人为组装，融合了mecA基因的启动子用于ccrC2基因的过表达。Cas9基因克隆自酿脓链球菌，融合了rpsL基因启动子用于其表达。mecA-targeting-sgRNA表达盒融合了截短的去除了核糖体结合位点的cap5A启动子用于其转录，这个表达盒整体由直接基因合成得到。金黄色葡萄球菌温度敏感型复制起始蛋白repF以及氯霉素抗性基因cat均克隆自质粒pKORl。大肠杆菌复制起始ori和氨苄青霉素抗性ampR来源于质粒pUC19，所有元件通过Gibson assembly技术组装成为SCCmec killer载体。
针对目前抗生素滥用导致的耐药菌株频发问题，本发明尝试结合了新型染色体盒重组酶CcrC2和CRISPR-Cas9技术，构建了SCC mec killer载体系统，即Gene drive载体，Gene drive载体作用在甲氧西林抗性菌株MRSA，实现了在甲氧西林抗性菌株MRSA中靶向去除葡萄球菌染色体盒SCC mec，从而消除甲氧西林抗性基因。并且携带了SCC mec killer载体系统的菌株，能够避免再次获得含有mecA基因的可移动元件，进而可以通过现有的抗生素对患者进行治疗。
附图说明
图1为SCC mec killer载体的设计图谱。
图2为SCC mec killer载体去除甲氧西林抗性金黄色葡萄球菌中甲氧西林抗性基因的原理图示。
图3为SCC mec killer载体在家畜源MRSA菌株BA01611中消除甲氧西林抗性基因mecA的效果图。
图4为SCCmec killer载体在医院源MRSA菌株Mu50中消除甲氧西林抗性基因mecA的效果图。
具体实施方式
实施例1
针对目前抗生素滥用导致的耐药菌株频发问题，本发明尝试结合了新型染色体盒重组酶CcrC2和CRISPR-Cas9技术，构建了SCC mec killer载体系统，即Gene drive载体，Gene drive载体作用在甲氧西林抗性菌株MRSA，实现了在甲氧西林抗性菌株MRSA中靶向去除葡萄球菌染色体盒SCC mec，从而消除甲氧西林抗性基因。并且携带了SCC mec killer载体系统的菌株，能够避免再次获得含有mecA基因的可移动元件，进而可以通过现有的抗生素对患者进行治疗。
如图1所示，一种Gene drive载体的构建方法，包括以下步骤：
步骤1)，cap5A启动子与sgRNA片段的人工合成
分别各取1μL的10μM浓度的引物SK1到SK8，混合于一个PCR管中，补加2μL超纯水，再向PCR管中混合10μL的Q5R High-Fidelity 2X Master Mix(购自New England Biolabs公司)，构成PCR反应体系，并于如下条件下进行PCR反应：98℃保持1min；98℃保持10s，55℃保持30s，72℃保持30s，按照上述条件依次做20个循环；最后，72℃保持2min，反应结束后，胶回收扩增产物。
步骤2)，扩增cas9基因
在一个20μL的PCR体系中加入10μL的Q5R High-Fidelity 2X Master Mix，以及0.8μL的引物SK9、0.8μL的引物SK10和1μL酿脓链球菌基因组DNA。于如下条件下进行PCR反应：98℃保持1min；98℃保持10s，55℃保持30s，72℃保持120s，依次做30个循环；最后，72℃保持2min，反应结束后，胶回收扩增产物。
步骤3)，扩增质粒骨架
在一个20μL的PCR体系中含有10μL的Q5R High-Fidelity 2X Master Mix，以及0.8μL的引物SK11、0.8μL的引物SK12引物和1μL质粒pSCCcat(来自于GenBank数据库中KY701738)。于如下条件下进行PCR反应：98度1min；98℃保持10s，55℃保持30s，72℃保持120s，依次做30个循环；72℃保持2min。反应结束后，胶回收扩增产物。
步骤4)，扩增rpsL启动子
在一个20μL的PCR体系中含有10μL的Q5R High-Fidelity 2X Master Mix，以及0.8μL的SK13和SK14引物和1μL金黄色葡萄球菌基因组DNA。于如下条件下进行PCR反应：98度1min；98度10s，55度30s，72度30s，共30循环；72度2min。反应结束后，胶回收扩增产物。
步骤5)，组装空载体
将步骤1)到步骤4)步骤中所得的胶回收扩增产物，以摩尔比1∶1∶1∶1的比例在容器内混合，然后加水补足总体积为10μL，再向容器中添加10μL的GibsonMasterMix(购自New England Biolabs公司)。于50℃下保温30min，然后将全部产物转化至大肠杆菌DH5α中。
步骤6)，插入mecA靶向的spacer
使用T4多聚核苷酸激酶(T4PNK)对引物SK15和引物SK16进行了5’末端的磷酸化，反应体系与条件记载在表1中。
表1
然后将磷酸化的引物稀释十倍后，加入2M NaCl至终溶液浓度为0.1M。然后溶液与步骤5)获得的空载体进行Golden Gate assembly，反应体系与条件记载在表2，然后将反应液直接转化大肠杆菌DH5α。
表2
步骤7)，构建SCCmec killer载体
在一个20μL的PCR体系中加入10μL的Q5R High-Fidelity 2X Master Mix、0.8μL的引物SK17、0.8μL的引物SK18和1μL质粒pSCCcat(来自于GenBank数据库中KY701738)。于如下条件下进行PCR反应：98℃保持1min；98℃保持10s，55℃保持30s，72℃保持60s，共30循环；72℃保持2min，反应结束后，胶回收扩增产物。同时用Xba1和Xho1双酶切并回收第6步中的载体，与PCR扩增产物进行进行Gibson assembly，转化大肠杆菌DH5α。最后获得了SCCmeckiller载体。
实施例2
如图1、图2和图3所示，利用SCCmec killer载体在家畜源MRSA菌株BA01611中消除甲氧西林抗性基因。
(1)制备MRSA菌株BA01611的感受态细胞
接种BA01611菌株的单克隆于10mL的BHI中。37℃摇菌过夜，次日接种约6mL所得液体于100mL新鲜BHI培养基中，调整OD600数值等于0.5。继续振荡培养30min，然后4度、5000g、离心5min，收集菌体弃去培养基，用等体积灭菌水洗涤菌体一次，再次按照第一次的离心条件进行离心，收集菌体弃去培养基，用等体积10％甘油洗涤菌体两次。最后重悬菌体于500uL的10％甘油中，以50uL为一份，分装10支离心管，置于-80度冰箱中保存。
(2)MRSA菌株BA01611的电转化
取一支装有感受态细胞的离心管，加入10uL SCCmec killer质粒(总量5ug)，混匀后室温静置15min，转入1mm间距的电击杯中，以2100V,25uF,100ohm,2.5ms条件进行电击，加入1mL BHI培养基重悬菌液，37℃震荡培养2小时，涂布TSA+Cm(25ug/mL)的平板。
(3)甲氧西林抗性基因去除效率的测定。
任意挑选10个单克隆，用2mL BHI培养基，37℃摇菌过夜。次日，提取基因组DNA，分别用金黄色葡萄球菌特异性引物nuc-F/R，甲氧西林抗性基因特异性引物mecA-F/R，葡萄球菌染色体盒剪切后特异性引物attR1’-F/R进行PCR检测，结果如图3所示，任意挑选的10个单克隆中均丢失了mecA基因，而同时能够检测到葡萄球菌染色体盒SCCmec丢失的痕迹(attR1’位点的出现)。
所以本发明消除了家畜源MRSA菌株BA01611中的甲氧西林抗性基因。
实施例3
如图1、图2和图4所示，利用SCCmec killer载体在医院源MRSA菌株Mu50中消除甲氧西林抗性基因。
(1)制备MRSA菌株Mu50的感受态细胞
接种Mu50菌株的单克隆于10mL的BHI中。37℃摇菌过夜，次日接种约6mL所得液体于100mL新鲜BHI培养基中，调整OD600数值等于0.5。继续振荡培养30min，然后4度、5000g、离心5min，收集菌体弃去培养基，用等体积灭菌水洗涤菌体一次，再次按照第一次的离心条件进行离心，收集菌体弃去培养基，用等体积10％甘油洗涤菌体两次。最后重悬菌体于500uL的10％乙醇中，以50uL为一份，分装10支离心管，置于-80度冰箱中保存。
(2)MRSA菌株Mu50的电转化
取一支装有感受态细胞的离心管，加入10uL SCCmec killer质粒(总量5ug)，混匀后室温静置15min，转入1mm间距的电击杯中，以2100V,25uF,100ohm,2.5ms条件进行电击，加入1mL BHI培养基重悬菌液，37℃震荡培养2小时，涂布TSA+Cm(25ug/mL)的平板。
(3)甲氧西林抗性基因去除效率的测定。
任意挑选10个单克隆，用2mL BHI培养基摇菌过夜。次日，提取基因组DNA，分别用金黄色葡萄球菌特异性引物nuc-F/R，甲氧西林抗性基因特异性引物mecA-F/R，葡萄球菌染色体盒剪切后特异性引物attB-F/R进行PCR检测，结果如图4所示。任意挑选的10个单克隆中均丢失了mecA基因，而同时能够检测到葡萄球菌染色体盒SCCmec丢失的痕迹(attB位点的出现)。
所以本发明消除了医院源MRSA菌株Mu50中的甲氧西林抗性基因。
Claims (3)
1.一种Gene drive载体，其特征在于：其核酸序列如SEQ ID NO.1所示。
2.一种如权利要求1中所述的Gene drive载体的构建方法，其特征在于，包括以下步骤：
步骤1)cap5A启动子与sgRNA片段的人工合成：分别取引物SK1到SK8，混合于一个PCR管中，加入超纯水和High-Fidelity 2X Master Mix后，进行PCR反应，反应结束后，胶回收扩增产物；
步骤2)扩增cas9基因：在PCR管中加入High-Fidelity 2X Master Mix、引物SK9、引物SK10和酿脓链球菌基因组DNA，然后进行PCR反应，反应结束后，胶回收扩增产物；
步骤3)扩增质粒骨架：在PCR管中加入High-Fidelity 2X Master Mix、引物SK11、引物SK12和质粒pSCCcat(来自于GenBank数据库中KY701738)，进行PCR反应，反应结束后，胶回收扩增产物；
步骤4)扩增rpsL启动子：在PCR管中加入High-Fidelity 2X Master Mix、引物SK13、引物SK14和金黄色葡萄球菌基因组DNA，进行PCR反应，反应结束后，胶回收扩增产物；
步骤5)组装空载体：将步骤1、步骤2、步骤3和步骤4中所有的扩增产物加入到超纯水中，再添加入GibsonMaster Mix(购自New England Biolabs公司)，最后将全部产物转化至大肠杆菌DH5a中；
步骤6)插入靶向的spacer：使用T4多聚核苷酸激酶(T4PNK)对引物SK15和SK16进行了5’末端的磷酸化，将磷酸化的引物稀释后，加入NaCl调节浓度，而后与第5步获得的空载体进行Golden Gate assembly，将反应液直接转化大肠杆菌DH5a；
步骤7)构建载体：在PCR管中加入High-Fidelity 2X Master Mix、引物SK17、SK18引物和质粒pSCCcat(来自于GenBank数据库中KY701738)，构成PCR反应体系，PCR反应结束后，胶回收扩增产物；同时用Xba1和Xho1双酶切并回收步骤6中的载体，最后与胶回收的扩增产物进行进行Gibson assembly，转化大肠杆菌DH5a，即获得了载体。
3.根据权利要求2所述的Gene drive载体的制备方法，其特征在于：
所述的引物SK 1的核酸序列如SEQ ID NO.2所示；
所述的引物SK 2的核酸序列如SEQ ID NO.3所示；
所述的引物SK 3的核酸序列如SEQ ID NO.4所示；
所述的引物SK 4的核酸序列如SEQ ID NO.5所示；
所述的引物SK 5的核酸序列如SEQ ID NO.6所示；
所述的引物SK 6的核酸序列如SEQ ID NO.7所示；
所述的引物SK 7的核酸序列如SEQ ID NO.8所示；
所述的引物SK 8的核酸序列如SEQ ID NO.9所示；
所述的引物SK 9的核酸序列如SEQ ID NO.10所示；
所述的引物SK 10的核酸序列如SEQ ID NO.11所示；
所述的引物SK 11的核酸序列如SEQ ID NO.12所示；
所述的引物SK 12的核酸序列如SEQ ID NO.13所示；
所述的引物SK 13的核酸序列如SEQ ID NO.14所示；
所述的引物SK 14的核酸序列如SEQ ID NO.15所示；
所述的引物SK 15的核酸序列如SEQ ID NO.16所示；
所述的引物SK 16的核酸序列如SEQ ID NO.17所示；
所述的引物SK 17的核酸序列如SEQ ID NO.18所示；
所述的引物SK 18的核酸序列如SEQ ID NO.19所示。
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