CN105463003A - 一种消除卡那霉素耐药基因活性的重组载体及其构建方法 - Google Patents
一种消除卡那霉素耐药基因活性的重组载体及其构建方法 Download PDFInfo
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Abstract
本发明提供了一种消除卡那霉素耐药基因活性的重组载体,以消除生物体内耐药细菌,解决细菌具有卡那霉素耐药性的问题。所述消除卡那霉素耐药基因活性的重组载体的特征在于,包括消除了氯霉素抗性的pCas9载体和针对卡那霉素抗性基因kan的gRNA核酸序列:KR58或KR208,其具体核酸序列分别如下:KR58:GCCGCGAT?TAAATTCCAACA或KR208:CAATGATG?TTACAGATGAGA。其构建方法主要包括:利用基因编辑新工具CRISPR/Cas9系统携带该间区核酸,去除重组载体上的氯霉素抗性基因后转化入减毒沙门菌等疫苗载体菌,重组菌与卡那霉素耐药菌共培养,重组菌细胞内的重组载体通过接合方式进入卡那霉素耐药菌,有效抑制卡那霉素抗性基因kan的活性,使原来耐药的细菌在卡那霉素培养基上不能生长。
Description
技术领域
本发明涉及病原细菌学领域,具体涉及一种消除耐药基因活性的gRNA的重组载体及其构建方法。
背景技术
随着抗生素的广泛应用,细菌耐药性现象越来越严重。卡那霉素属于氨基糖甙类抗生素,对金葡菌、绿脓杆菌、大肠杆菌、变形杆菌均有效。临床上主要用于敏感菌特别是耐药性绿脓杆菌引起的尿路感染、呼吸道及肺部感染等。大肠杆菌等多种细菌含有卡那霉素耐药基因。
耐药细菌的消除是解决细菌耐药性的重要方法。环境中的耐药细菌可通过物理学或化学方法进行消毒灭菌。但生物机体内的耐药细菌只能以特异性抗菌化学或生物药物进行抑制或杀灭[1-3]。然而,噬菌体宿主特异性太强,只能裂解某些特定型别的致病菌,还存在可能引起人体过敏的问题[1];细菌素抑菌菌谱太窄,规模化生产工艺尚未成熟,且也可能存在诱导耐药性问题[2];新型抗生素仍然是抗生素,必然存在诱导耐药性问题[3]。因此,抑制或破坏耐药基因成为控制细菌耐药性的重要方法之一。在现有的研究中,部分中药可以消除耐药质粒基因而消除大肠杆菌耐药性[4],但其消除效率不高,针对104的CFU/ml的大肠杆菌,耐药性消除率最高者仍不足12.5%[4]。
近年来,一种全新的CRISPR(clusteredregularlyinterspacedshortpalindromicrepeats)技术的出现使我们能对任意物种的基因组进行定点编辑。CRISPR原本是细菌和古细菌中的一种适应性免疫防御系统,可保护宿主菌免受噬菌体或质粒等有害外源核酸的再次侵袭。CRISPR系统分为3种类型:Ⅰ型、Ⅱ型和Ⅲ型。目前Ⅱ型系统已被成功改造为人工核酸酶切系统,即CRISPR/Cas9系统,用于基因编辑,且具有制作简单、成本低、作用高效等优点[5]。目前该技术已在科学研究中得到广泛应用,例如,人多能干细胞基因的高效敲除;斑马鱼的高通量定点基因突变和大范围分型;基因敲低小鼠的制备;修饰生殖细胞DNA以预防小鼠肌肉营养不良症;植物基因组基因编辑以及微生物基因组编辑和转录控制等。这些研究大都是对基因组上的基因进行操作,往往用于疾病的预防或治疗,基因改造有利于生物体的生存。
在将上述技术应用于抑制或破坏质粒耐药基因时,至少存在以下两个技术难点:1)耐药基因本身对细菌的生存有利,破坏耐药基因反而是对细菌的生存不利,因而必须“逆向”突破生物基因演化的阻力;2)质粒拷贝数往往较高,相应耐药基因拷贝数也同样较高,抑制破坏质粒耐药基因必须研发更为强力的核酸剪辑工具。事实上,如果控制不好,耐药基因确实可以引发CRISPR系统的突变甚至大片段缺失,使之丧失功能而得以保存对细菌有利的耐药基因[7]。
基于上述背景,本研究选择pET-28a上的kan为靶基因,利用生物信息学技术设计了3条引导RNA(gRNA),研究了CRISPR/Cas系统在抑制质粒耐药基因活性中的应用价值,获得了具有破坏kan基因活性的2条gRNA序列及其重组载体。
参考文献:
1.李菁华,孙延波.噬菌体疗法在耐药性细菌感染中应用的研究进展.吉林大学学报(医学版),2013,39(3):630-633.
2.章昱,周云芳.细菌素的抗细菌耐药研究及应用现状.微生物学免疫学进展,2015,43(6):76-79.
3.李文月.细菌耐药监测与抗菌药物合理应用.中国现代药物应用,2015,9(9):147-150
4.张鼎,李宏全,马海利,等.鸡源大肠杆菌耐药性分析及中药对大肠杆菌耐药性消除作用的研究.畜牧兽医学报,2015,46(6):1018-1025.
5.PetersJM,SilvisMR,ZhaoD,HawkinsJS,GrossCA,QiLS.BacterialCRISPR:accomplishmentsandprospects.CurrOpinMicrobiol.2015;27:121-126.
6.CarattoliA.Plasmidsandthespreadofresistance.IntJMedMicrobiol.2013;303(6-7):298-304.
7.JiangW,ManivI,ArainF,WangY,LevinBR,MarraffiniLA.DealingwiththeevolutionarydownsideofCRISPRimmunity:bacteriaandbeneficialplasmids.PLoSGenet.2013,9(9):e1003844.
发明内容
本发明的目的在于构建一种消除卡那霉素耐药基因活性的gRNA重组载体,以消除生物体内耐药细菌,解决细菌具有卡那霉素耐药性的问题。
本发明还要解决的技术问题是提供该重组载体的构建方法。
为此,本发明的基本思路是:设计一种针对卡那霉素抗性基因kan的间区核酸序列(即编码gRNA的DNA序列),利用基因编辑新工具CRISPR/Cas9系统携带该间区核酸,去除重组载体上的氯霉素抗性基因后转化入减毒沙门菌等疫苗载体菌,重组菌与卡那霉素耐药菌共培养,重组菌细胞内的重组载体通过接合方式进入卡那霉素耐药菌,有效抑制卡那霉素抗性基因kan的活性,使原来耐药的细菌在卡那霉素培养基上不能生长。
为了解决上述问题,本发明的技术方案如下:
本发明提供了一种消除卡那霉素耐药基因活性的重组载体,其特征在于,包括消除了氯霉素抗性的pCas9载体和针对卡那霉素抗性基因kan的gRNA核酸序列,所述gRNA核酸序列命名为KR58或KR208,其具体核酸序列分别如下:
KR58:GCCGCGATTAAATTCCAACA;或者
KR208:CAATGATGTTACAGATGAGA。
本发明还提供了一种构建上述消除卡那霉素耐药基因活性的gRNA重组载体的方法,其特征在于,包括如下步骤:
1)设计具有如上所述的核酸序列的kan基因特异性gRNA序列;
2)构建含有步骤1)所设计的gRNA序列的重组载体,构建步骤主要包括:a)根据步骤1)所设计的任意一个核酸序列(本发明中,KR58或者KR208),人工合成一对单链DNA片段;b)对步骤a)中所得的序列组合磷酸化后退火形成双链DNA片段;c)酶切pCas9质粒后,进行琼脂糖凝胶回收;d)将步骤c)得到的pCas9质粒与步骤2)获得的双链DNA片段连接,得到重组载体;
3)去除重组载体中的氯霉素抗性基因,以避免应用于机体时引入新的耐药基因,即氯霉素耐抗性基因。
进一步地,上述步骤3)中的用于去除重组载体中的氯霉素抗性基因的引物的核酸序列(划线部分为BglII识别位点)为:
△CamF:GAAGATCTTTTAGCTTCCTTAGCTCCTG;
△CamR:GAAGATCTATTTTTTTAAGGCAGTTATTGGT。
该方法虽然在本发明中仅针对卡那霉素,但其原理和方法很容易引申应用于消除其他抗生素抗性基因。
本发明的技术方案达到了如下的有益效果:
1)针对“破坏耐药基因会对细菌的生存不利,必须“逆向”突破生物基因演化的阻力”的技术难点,本发明设计并筛选了特异性强的kan特异性gRNA,预测了脱靶效应以及与靶基因的结合能力,构建了重组载体,并经实验结果的实践验证,获得了2条有效的gRNA,达到了有效抑制卡那霉素抗性基因kan的活性。
2)本发明采用可对DNA双链进行剪辑的核酸酶Cas9的表达载体,插入gRNA获得重组载体。一方面利用了CRISPR/Cas9系统在基因编辑中的高效性,另一方面以质粒形式来产生可切割靶基因的Cas9-gRNA复合体,因质粒可自我复制,拷贝数较高,因此也可抑制高数量的靶基因。
3)本发明可用于开发具有消除耐药细菌的功能益生菌或疫苗载体菌产品,具有重要的市场价值,可望产生良好的社会和经济价值。
附图说明
图1是本发明方法构建的重组载体pCas9△Cam-KR58。
图2是本发明方法构建的重组载体pCas9△Cam-KR208。
具体实施方式
为了阐明本发明的技术方案及技术目的,下面结合附图及具体实施方式对本发明做进一步的介绍。
实施例1:
本实施例1为重组载体pCas9△Cam-KR58和pCas9△Cam-KR208的构建方法。
1.kan基因特异性gRNA的设计:
pET28a为常用商品化分子克隆载体。直接选取pET28a中kan基因序列进行gRNA靶点扫描,获得75个潜在gRNA序列靶位点。遵循尽量减少脱靶几率以及尽量增强与靶基因结合的亲和力的原则,经过对这些潜在位点进行宿主菌基因组脱靶分析,选取若干脱靶几率低而高亲和力结合靶基因的gRNA序列,并经过随后的实验研究验证,确定了两个具有理想的降解kan基因活性的gRNA序列,分别命名为KR58和KR208,其具体核苷酸序列分别如下:
KR58:GCCGCGATTAAATTCCAACA
KR208:CAATGATGTTACAGATGAGA
2.pCas9-KR58和pCas9-KR208的构建:
使用pCas9载体(Addgene)用于克隆和转录针对特定靶基因的gRNA,并编码Cas9蛋白。特异性gRNA可引导Cas9蛋白切割和降解靶基因。构建方法包括如下具体步骤:
1)为将KR58和KR208分别克隆进pCas9,人工合成以下两对单链DNA片段:
KR58F:5’-AAACGCCGCGATTAAATTCCAACAG-3’
KR58R:5’-AAAACTGTTGGAATTTAATCGCGGC-3’;
KR208F:5’-AAACCAATGATGTTACAGATGAGAG-3’
KR208R:5’-AAAACTCTCATCTGTAACATCATTG-3’。
2)gRNA磷酸化:对KR58F与KR58R组合用T4PNK磷酸化后退火形成双链DNA片段KR58;KR208F与KR208R组合用T4PNK磷酸化后退火形成双链DNA片段KR208;
gRNA磷酸化体系包括:10xT4BufferNEB5μL,上游序列KR58(或208)F1μL,下游序列KR58(或208)R1μL,T4PNK1μL,添加ddH2O至总体积为50μL。
混匀,37℃反应2h后,加1mol/LNaCl2.5μL,混匀,95℃水浴5min,使其缓慢冷却,稀释十倍后备用。
3)pCas9质粒用BsaI酶酶切后进行琼脂糖凝胶回收:
其中,BsaI酶切体系包括:NEBuffer20μL,100xBSA2μL,BsaI酶4μL,pCas9质粒120μL,添加ddH2O至总体积200μL。
混匀后,于37℃恒温静置,酶切过夜,次日,进行琼脂糖电泳,并进行胶回收,定量。Takara胶回收试剂盒。
4)gRNA与pCas9连接:将步骤3)得到的pCas9质粒分别与步骤2)获得的KR58及KR208连接,得到重组载体pCas9-KR58及pCas9-KR208;
其中,连接体系包括:pCas9酶切质粒1μL,磷酸化的sgRNA2μL,10xT4DNALigaseBuffer2μL,T4DNALigase1μL,添加H2O至总体积为20μL。于16℃反应2h。
此后,于2.5μL连接产物中加入100μLDH5a感受态,混匀,置于冰上30min。于42℃中热激75s,再次置于冰上3min,向离心管中加入LB800μL,混匀后于37℃摇床中,160rpm振荡1h左右。3800rpm离心6min,吸取上清仅留约100μL,吹打混匀后,40μL涂布于含有氯霉素的LB固定培养基,于37℃培养箱,正置30min,待菌液完全吸收后倒置培养皿,过夜。挑取单菌落转接液体培养基培养后分别取1μL菌液作模板,用位于pCas9上的正向引物DocF(5’-GAAACAAGCGCTCATGAGCCCG-3’)分别和KR58R、KR208R等反向引物组合成2对PCR引物进行PCR扩增筛选得到重组载体pCas9-KR58及pCas9-KR208。
3.pCas9-KR58和pCas9-KR208中氯霉素抗性基因CamR的去除:
pCas9载体中CamR基因位于8886-219,将此部分去除即可得到消除氯霉素抗性的载体。以pCas9中220-8885部分的DNA序列为参照,设计了一对引物(划线部分为BglII识别位点):
△CamF:GAAGATCTTTTAGCTTCCTTAGCTCCTG
△CamR:GAAGATCTATTTTTTTAAGGCAGTTATTGGT
分别以pCas9-KR58及pCas9-KR208作模板,用上述F和R引物进行扩增,扩增产物用BglII酶切并凝胶回收后再进行自连接,电转化大肠杆菌DH5a,挑取多个单菌落扩培,通过提取质粒凝胶分析和直接菌液PCR筛选获得去除CamR基因的重组载体pCas9△Cam-KR58及pCas9△Cam-KR208。
其中,pCas9△Cam的PCR扩增体系包括:上游引物△CamF1μL,下游引物△CamR1μL,缓冲液5μL,dNTP(10M)1μL,LATag2μL,添加ddH2O至总体积为50μL。
其中,pCas9△Cam的PCR程序为:预变性95℃4分钟,再经94℃变性30秒,58℃复性30秒,72℃延伸8分钟,30个循环,最后72℃延伸10分钟。
PCR扩增后,其扩增产物用BglII酶切并凝胶回收后再进行自连接。其酶切体系及条件、连接产物的转化和筛选等实验方法,与步骤2中的相关实验方法步骤3)、4)相似。
实施例2:
本实施例2为重组载体pCas9△Cam-KR58和pCas9△Cam-KR208的应用举例之一:通过转化抑制受体菌卡那霉素耐药性。自然界存在受体菌直接吸收环境中DNA的现象,即转化现象。
在将pCas9△Cam-KR58和pCas9△Cam-KR208分别转化进含pET28a的大肠杆菌中时,其具体步骤如下:首先,制备含pET-28a大肠杆菌的感受态细胞;然后,分别取1μL重组载体加入100μL感受态,混匀,置于冰上30min后,于42℃中热激75s,再次置于冰上3min,向离心管中加入LB800μL,混匀后于37℃摇床中,160rpm振荡1h左右;随后,吸取约50μL菌液涂布于LB琼脂平板,于37℃培养箱,正置30min,待菌液完全吸收后倒置培养皿,过夜;最后,挑取单菌落转接液体培养基培养后,分别取1μL菌液作模板,用位于pCas9上的正向引物DocF(5’-GAAACAAGCGCTCATGAGCCCG-3’)分别和KR58R、KR208R等反向引物组合成2对PCR引物进行PCR扩增,筛选得到分别转入了pCas9△Cam-KR58和pCas9△Cam-KR208的含pET-28a大肠杆菌重组菌。
重组菌培养液分别在不含抗生素和含卡那霉素的LB平板上计数,比较计数结果显示,重组菌在不含抗生素的LB平板上可正常生长,但在含卡那霉素的LB平板上已经不能生长(表1)。说明pCas9△Cam-KR58/pET-28a和pCas9△Cam-KR208/pET-28a中的kan基因特异性gRNA(即KR58和KR208)均发挥了抑制细菌卡那霉素耐药性的作用,可引导Cas9蛋白结合于kan基因的特定位置并降解了kan基因。
表1.pCas9△Cam-KR58和pCas9△Cam-KR208转化后含pET-28a受体菌的生长情况
pCas9△Cam-KR58 | pCas9△Cam-KR208 | pCas9△Cama | -b | |
LB | + | + | + | + |
K+LB | - | - | + | + |
注:a表示转化入不携带特异性gRNA空载体pCas9△Cam的pET28a受体菌;b表示不转化质粒的pET28a大肠杆菌;K+LB表示含卡那霉素的LB平板。
实施例3:
本实施例3为重组载体pCas9△Cam-KR58和pCas9△Cam-KR208的应用举例之二:通过接合抑制受体菌卡那霉素耐药性。自然界存在细菌之间通过性菌毛传递DNA的现象,即接合现象。
在将pCas9△Cam-KR58和pCas9△Cam-KR208分别导入减毒沙门菌作为供体菌时,其制备减毒沙门菌感受态细胞,重组载体转化、筛选方法与实施例2中的转化大肠杆菌感受态相同,并分别以含pET28a的大肠杆菌为受体菌进行接合实验,即:将含重组载体的减毒沙门菌和含pET-28a的大肠杆菌在37℃条件下共孵育8h,在不含抗生素和含卡那霉素的LB平板上分别计数,考察pET28a大肠杆菌接受pCas9△Cam-KR58或pCas9△Cam-KR208的生长情况。结果显示,接合后受体大肠杆菌不能在卡那霉素平板上生长(表2)。这说明pCas9△Cam-KR58或pCas9△Cam-KR208通过接合进入受体大肠杆菌,并抑制了其中kan基因的活性。
表2.pCas9△Cam-KR58和pCas9△Cam-KR208接合后受体菌的生长情况
pCas9△Cam-KR58 | pCas9△Cam-KR208 | pCas9△Camc | -d | |
LB | + | + | + | + |
K+LB | - | - | + | + |
注:c表示与不携带特异性gRNA空载体pCas9△Cam的供体菌接合的pET28a受体菌;d表示不与供体菌接合的pET28a大肠杆菌;K+LB表示含卡那霉素的LB平板。
以上显示和描述了本发明的基本原理、主要特征和本发明的优点。本行业的技术人员应该了解,本发明不受上述实施例的限制,上述实施例和说明书中描述的只是说明本发明的原理,在不脱离本发明精神和范围的前提下,本发明还会有各种变化和改进,本发明要求保护范围由所附的权利要求书、说明书及其等效物界定。
。
Claims (3)
1.一种消除卡那霉素耐药基因活性的重组载体,其特征在于,包括消除了氯霉素抗性的pCas9载体和针对卡那霉素抗性基因kan的gRNA核酸序列,KR58或者KR208,其具体核酸序列分别如下:
KR58:GCCGCGATTAAATTCCAACA;
KR208:CAATGATGTTACAGATGAGA。
2.一种构建如权利要求1所述的消除卡那霉素耐药基因活性的重组载体的方法,其特征在于,包括如下步骤:
1)设计具有如上所述的核酸序列的kan基因特异性gRNA序列;
2)构建含有步骤1)所设计的gRNA序列的重组载体,构建步骤主要包括:a)根据步骤1)所设计的任一个核酸序列,人工合成一对单链DNA片段;b)对步骤a)中所得的序列组合磷酸化后退火形成双链DNA片段;c)酶切pCas9质粒后,进行琼脂糖凝胶回收;d)将步骤c)得到的pCas9质粒与步骤2)获得的双链DNA片段连接,得到重组载体;
3)去除重组载体中的氯霉素抗性基因。
3.如权利要求2所述的一种构建消除卡那霉素耐药基因活性的重组载体的方法,其特征在于,步骤3)中的用于去除重组载体氯霉素抗性基因的引物的核酸序列为:
△CamF:GAAGATCTTTTAGCTTCCTTAGCTCCTG;
△CamR:GAAGATCTATTTTTTTAAGGCAGTTATTGGT。
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