CN110964684A - 表达荧光素酶的产碳青霉烯酶肺炎克雷伯菌及其用途 - Google Patents
表达荧光素酶的产碳青霉烯酶肺炎克雷伯菌及其用途 Download PDFInfo
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Abstract
本发明利用质粒构建和电转化技术,构建了含有荧光素酶簇(luciferase gene cassette,lux)的产碳青霉烯酶肺炎克雷伯菌,最终获得了可稳定表达荧光素酶的产碳青霉烯酶肺炎克雷伯菌(KPC‑KP‑LUX)。该菌稳定性好,操作简便,同时为筛选合适的杀菌方法、抗生素、制备动物模型等提供重要的可视化研究工具。
Description
技术领域
本发明涉及微生物医药领域,具体涉及一种含有荧光素酶基因,并能稳定表达荧光素酶的产碳青霉烯酶肺炎克雷伯菌及其构建方法,以及该菌株在筛选杀菌方法或者筛选药物以及制备动物模型等方面的用途。
背景技术
细菌对抗生素的耐药性及其快速和广泛传播已成为全世界面临的严重健康威胁。数据表明,目前全球每年约有70万人死于耐药性感染,如果没有恰当措施,到2050年将达到1000万。
碳青霉素烯类抗生素对常见耐药菌具有广泛的特异性和有效性,是针对难以治疗的革兰氏阴性肠杆菌科如肺炎克雷伯菌感染的最有效药物,已成为治疗耐药菌感染的关键药物。近年来研究表明:耐碳青霉烯酶肠杆菌(CRE)是目前最重要的感染发生率在世界各地迅速增加。细菌可通过多种机制对碳青霉烯类产生耐药性,其中碳青霉烯酶可使细菌耐受所有β-内酰胺类抗生素、赋予细菌高耐药性,且能够通过横向基因转移在菌间快速传递,对临床治疗造成了极大冲击。该酶有五种常见亚型(KPC、IMP、OXA、NDM和VIM)。产碳青霉烯酶肺炎克雷伯菌株(Klebsiella pneumoniae carbapenemase-producing K.pneumoniae,KPC-KP)属于革兰氏阴性肠杆菌,是院内感染和外科手术感染最常见的病原菌之一,KPC-KP感染与青霉素及其他抗生素使用相关,研究发现,KPC-KP能够产生碳青霉烯耐药,给临床治疗带来了很大的挑战。
由于新型抗菌药物研发时间远远长于细菌耐药变异的时间。因此,除了继续开发新型抗菌药物外,对已有抗菌措施的有效应用以及制定合理有效的抗生素联合应用方案已成为临床抗菌治疗,控制耐药菌的流行和感染的重要措施。因此,需要一种有力的工具评估现行抗生素使用的疗效、新的药物,例如抗生素或其他杀菌方法效果。
生物发光是生物体内在酶的作用下发光的现象。生物发光现象广泛存在细菌、昆虫等生物体中。利用萤光素酶基因(luciferas gene cassette,LUX)基因标记细胞的生物发光技术具有操作简便、结果直观、不依赖底物、对机体损伤小等优点。该技术已广泛应用于肿瘤生长监测和转移示踪、基因治疗、目标基因表达的检测等多个领域。
但尚未有人将生物发光技术应用到产碳青霉烯酶肺炎克雷伯菌,以获得方便的可视化研究工具。
发明内容
本发明将萤光素酶基因LUX A/B/C/D/E转化至泛宿主质粒pBBR1,构建pBBR1-LUXA/B/C/D/E质粒,并将通过电转化至产碳青霉烯酶肺炎克雷伯菌,经过传代,筛选可稳定表达荧光素酶的产碳青霉烯酶肺炎克雷伯菌,并将其用于筛选合适的杀菌方法或者筛选药物以及制备动物模型等方面。因此,
本发明提供一种质粒,所述质粒包括荧光素酶基因簇LUX A/B/C/D/E。
优选的,所述质粒为泛宿主质粒,更优选的,所述质粒为泛宿主质粒pBBR1。
本发明还提供一种产碳青霉烯酶肺炎克雷伯菌(KPC-KP-Lux),所述产碳青霉烯酶肺炎克雷伯菌能稳定表达荧光素酶。
本发明还提供一种上述产碳青霉烯酶肺炎克雷伯菌的构建方法,所述构建方法包括:
(1)制备包含LUX A/B/C/D/E基因簇的质粒;
(2)制备电转化感受态产碳青霉烯酶肺炎克雷伯菌;
(3)将步骤(1)获得的包含LUX A/B/C/D/E基因簇的质粒电转化感受态产碳青霉烯酶肺炎克雷伯菌,挑选阳性菌落。
优选的,所述质粒为泛宿主质粒。
优选的,所述步骤(1)包括:(a)将LUX A/B/C/D/E基因簇克隆至泛宿主质粒;(b)将环状质粒电转化至大肠杆菌感受态细胞;(c)筛选阳性大肠杆菌;(d)从阳性大肠杆菌中提取包含LUX A/B/C/D/E基因簇的泛宿主质粒。
进一步,所述步骤(a)包括①、LUXA/B/C/D/E基因簇扩增;②、pBBR1MCS-1双酶切;③、将步骤②的酶切片段胶回收后与步骤①扩增的LUXA/B/C/D/E基因簇使用Gibson连接方法连接获得环状质粒。
更进一步,步骤①LUX A/B/C/D/E基因簇扩增时采用高保真酶,其退火温度低于推荐温度2-4℃。
本发明还提供一种上述产碳青霉烯酶肺炎克雷伯菌在评估杀菌方法中的应用。
本发明还提供一种上述产碳青霉烯酶肺炎克雷伯菌在筛选药物中的应用。
本发明还提供一种上述产碳青霉烯酶肺炎克雷伯菌在制备动物模型中的应用。
优选的,所述动物模型可以是造烧伤模型、手术模型、感染模型等。
本发明还提供一种杀菌方法的评估方法,所述评估方法包括利用上述产碳青霉烯酶肺炎克雷伯菌对杀菌方法进行评估。
本发明还提供一种药物筛选方法,所述药物筛选方法包括利用上述产碳青霉烯酶肺炎克雷伯菌对药物进行筛选。
所述评估方法不是治疗方法。该评估方法可以是体内或者体外的评估方法,该评估方法用来评估候选杀菌方法,对候选杀菌方法的杀菌效果进行检测和比较,以确定哪些杀菌方法可以作为杀灭产碳青霉烯酶肺炎克雷伯菌,哪些不能,或者,比较不同杀菌方法的杀菌程度,即杀菌效果不是必然的,只是一种可能性。
所述药物筛选方法不是治疗方法。该药物筛选方法可以是体内或者体外的筛选方法,该方法用来筛选药物,对候选药物的药效进行检测和比较,以确定哪些候选药物可以作为药物,哪些不能作为药物,或者,比较不同药物的药效敏感程度,即治疗效果不是必然的,只是一种可能性。
本发明还提供一种动物模型的制备方法,所述制备方法包括将上述产碳青霉烯酶肺炎克雷伯菌注入动物体内。
优选的,所述动物模型可以是造烧伤模型、手术模型、感染模型等。所述体内注入方式可以是鼻饲、灌胃、腹腔注射、尾静脉注射等。
优选的,所述菌株在动物体内富集浓度不低于1*106Cfu/mL。
细菌注射量可根据不同注入方式进行调整,例如,腹腔注射时,所述菌株接种量不低于1*108Cfu,最为优选的,所述菌株接种量不低于5*108Cfu。
本发明还提供一种产碳青霉烯酶肺炎克雷伯菌的杀菌方法,所述杀菌方法包括利用紫外照射、84消毒液或者NaOH对携带产碳青霉烯酶肺炎克雷伯菌的物品进行杀菌。
优选的,所述携带产碳青霉烯酶肺炎克雷伯菌的物品包括医疗器械,医疗材料等医疗物品,例如手术床,手术器械,医用床,医用床单等等,或者携带产碳青霉烯酶肺炎克雷伯菌的食品或土壤等等。
优选的,所述杀菌方法包括紫外照射30min以上,以10%的84消毒液,或者1M的NaOH对物品进行消毒。
本发明的良好技术效果包括:
1、首次利用泛宿主质粒将荧光素酶转化至产碳青霉烯酶肺炎克雷伯菌,从而获得生物发光的产碳青霉烯酶肺炎克雷伯菌,方便的观察产碳青霉烯酶肺炎克雷伯菌的各种性能,包括生长,体内分布等各种性能;
2、本发明使用电转化方法制备感受态细胞,操作简便易得,而且转化效率高,转化后细菌稳定表达荧光素酶概率为70%。优选的,LUX A/B/C/D/E基因簇扩增时采用高保真酶,其退火温度低于推荐温度2-3℃时,扩增效率最高。
3、利用本发明表达萤光素酶的产碳青霉烯酶肺炎克雷伯菌可以便捷,快速、直观的筛选合适的药物,例如各种抗生素,这一点对于耐药产碳青霉烯酶肺炎克雷伯菌尤为重要,为患者快速找到合适的抗生素可以使患者早日康复。
4、利用本发明表达萤光素酶的产碳青霉烯酶肺炎克雷伯菌筛选到适合的杀菌方法,如实施例所示的紫外线、84消毒液和NaOH都可以有效的杀灭携带产碳青霉烯酶肺炎克雷伯菌的物品,可以有效的对医疗物品进行杀菌,防止院内感染;
5、可视化观察不同的理化措施对于KPC-KP的杀菌效果,对临床合理进行环境杀菌,为采取接触预防、患者隔离、环境清洁等更多干预措施提供依据,对于预防控制院内感染具有重要的意义;
6、可视化追踪KPC-KP在实验动物体内的分布情况,对于优化抗菌药物使用方案,具有重要的指导意义。
以上只是概括了本发明的一些方面,不是也不应该认为是在任何方面限制本发明。本说明书提到的所有专利和出版物都是通过参考文献作为整体而引入本发明的。本领域的技术人员应认识到,对本发明可作某些改变并不偏离本发明的构思或范围。下面的实施例进一步详细说明本发明,不能认为是限制本发明或本发明所说明的具体方法的范围。
附图说明
图1:pBBR1-LUX A/B/C/D/E质粒构建策略图,其中A为pBBR1-LUX A/B/C/D/E质粒构建示意图,上:PCR线性扩增luxA/B/C/D/E基因基团,下:HindIII/KpnI对PBBR1质粒进行双酶切。B为pBBR1-LUX A/B/C/D/E质粒转化到大肠杆菌示意图。
图2:pBBR1-LUX A/B/C/D/E质粒在大肠杆菌荧光素酶功能验证,其中(A)图中,DH5a-pBBR1为仅转化质粒pBBR1的对照组大肠杆菌,DH5a-pBBR1-LUX A/B/C/D/E为转化了pBBR1-LUX A/B/C/D/E的实验组大肠杆菌,(B)图为DH5a-pBBR1-LUX A/B/C/D/E在小动物活体成像仪上的结果。
图3:pBBR1-LUX A/B/C/D/E质粒在产碳青霉烯酶肺炎克雷伯菌荧光素酶功能验证,其中(A)图中,KPC-KP-pBBR1为仅转化质粒pBBR1的对照组产碳青霉烯酶肺炎克雷伯菌,KPC-KP-pBBR1-LUX A/B/C/D/E为转化了pBBR1-LUX A/B/C/D/E的实验组产碳青霉烯酶肺炎克雷伯菌,(B)图为KPC-KP-pBBR1-LUX A/B/C/D/E在小动物活体成像仪上的结果。
图4:不同传代菌株的荧光强度,P3-P10依次表示第三代——第十代。
图5:不同浓度KPC-KP-pBBR1-LUX A/B/C/D/E的Luminescence荧光强度。
图6:不同理化杀菌方法对KPC-KP-pBBR1-LUX A/B/C/D/E的杀菌效果。
图7:小鼠腹腔注射产碳青霉烯酶肺炎克雷伯菌后在体内的播散和扩增结果。
图8:小鼠腹腔注射产酸克雷伯菌死亡后,细菌分布情况。
具体实施方式
下面通过制备例和实验例对本发明进行详细说明,以使本发明的特征和优点更清楚。但应该指出,制备和检测实验例用于理解本发明的构思,本发明的范围并不仅仅局限于本文中所列出的制备例和实验例。
下述制备例和实验例中所使用的实验方法如无特殊说明,均为常规方法。所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
1、试验材料:
产碳青霉烯酶肺炎克雷伯菌株(KPC-KP)购自ATCC,经全自动微生物分析仪(美国BD)鉴定,确定为氯霉素敏感(药敏结果的判断参照美国临床实验室标准化研究所的推荐标准)。大肠杆菌(Escherichia coli,E.coli)DH5a感受态细胞、质粒小提试剂盒购于天根生化科技有限公司。胶回收试剂盒购于德国QIAGEN公司。高保真DNA聚合酶Q5、高保真DNA连接酶、KpnI、HindIII限制性内切酶均购于美国NEB公司。pBAV1k-T5-LUX和PBBR1MCS-1购于淼灵质粒平台,氯霉素购自美国Thermo。
2、检测仪器:
Veritas微孔板光度计(Turner BioSystems)、NanoDropTM One超微量紫外分光光度计(Thermo)、IVIS Lumina LT小动物活体成像仪(珀金埃尔默)。
3、细菌培养
KPC-KP固体培养基为哥伦比亚血平板基础培养基(OXOID)添加8%脱脂羊血(北京冬歌博业生物科技有限公司),液体培养基为脑心浸液培养基(Brain Heart Infusion,BHI)(OXOID)。大肠杆菌DH5a的培养基为LB(Luria-Bertani,OXOID)培养基,如需固体培养,则在培养基中加入15%琼脂(OXOID)。
4、统计学分析:
利用GraphPad Prism 7.0软件进行统计分析,采用Kolmogorov-Simrnov对荧光值数据进行正态性检验。E.coli-pBBR1-lux和KPC-KP-pBBR1-lux荧光信号检测数据为偏态分布,荧光值用中位数表示,两组间采用Wilcoxon秩和检验。不同浓度KPC-KP-pBBR1-lux荧光信号检测数据为偏态分布,荧光值用均值表示,采用OneWay anova检验。以P<0.05为差异有统计学意义。
实施例1含有荧光素酶基因的泛宿主质粒的构建
1、LUXA/B/C/D/E基因簇扩增:
(1)设计LUX基因簇的引物对
LUX F:agggaacaaaagctgggtacGATTCAATTGTGAGCGGATAAC(SEQ ID NO.1);
LUX R:gctgcaggaattcgatatcaATTTGTCCTACTCAGGAGAG(SEQ ID NO.2)。
引物中的小写加粗序列20bp为该序列与pBBR1的互补序列。
pBAV1k-T5-LUX质粒长度10546bp,其luxA/B/C/D/E基因簇受T5启动子操纵,共5663bp,我们采用使用Q5高保真DNA聚合酶和含有LUX基因簇的引物对,引物设计过程中加入与pBBR1相同的序列(如图1A所示),扩增pBAV1k-T5-LUX质粒的LUX A/B/C/D/E基团。
(2)LUXA/B/C/D/E基因簇PCR扩增
表1:LUXA/B/C/D/E基因簇扩增PCR反应体系
配置好上述反应体系后进行PCR反应,PCR程序设置如表2:
表2:PCR程序设置
程序进行至4℃时,取出反应产物,关闭PCR仪后进行后续PCR产物鉴定。
同时,申请人发现,依据高保真DNA聚合酶网站推荐的退火温度(61℃),获得DNA片段的效果并不好。经梯度PCR实验,其退火温度低于推荐温度2-4℃时(即57-59℃),扩增效率最高,确定使用58℃退火。
(3)琼脂糖凝胶电泳:1)配制适当浓度(1.5%)琼脂糖凝胶:称取1.5g琼脂糖(GeneStar)称重后溶至100ml TAE中,微波炉加热至琼脂糖完全溶解,溶液澄清透明。冷却40-50℃,加入10μl核酸染料(Gene star)。混匀后倾倒至组装好的胶槽内,室温静置30分钟。2)组装电泳装置后上样:胶孔中加入DNA Ladder(Gene Star)及PCR产物(如有需要,加入0.66μl10×loading buffer)各6μl,恒压120V电泳10分钟。紫外凝胶成像仪下观查PCR条带是否阳性,并将阳性条带位置与DNA Ladder比对,条带分子量与目标DNA(5000bp左右)一致,鉴定为阳性。
(4)PCR产物测序并分析:PCR产物凝胶回收:收集正确大小的DNA:用洁净刀片在紫外灯下切取目的DNA片段后称重,将切取的凝胶收集至1.5ml离心管。利用DNA凝胶回收与纯化试剂盒。按100μg凝胶加入300μl溶胶缓冲液,50℃震荡孵育,观察胶完全融化后加入异丙醇(每100μg凝胶加入100μl异丙醇)。将600μl上述液体转移至离心柱内,12000rpm离心1分钟,弃套管内废液,再将离心柱插入套管内,若液体有剩余,则将剩余液体加入同一离心柱内重复此步骤。离心柱内加500μl清洗液,12000rpm离心1分钟,弃套管内废液。小心取出离心柱,插入新的离心管,12000rpm离心1分钟后打开盖子,室温静置两分钟去除残留的异丙醇H2O。将离心柱置入一个新的1.5ml离心管内,并加入50μl无核酸酶水,室温放置2分钟后12000rpm离心1分钟,收集回收的DNA。将回收的DNA标记后送往测序公司(华大基因)进行一代测序。测序结果与pBAV1k-T5-LUX质粒进行对比分析,测序结果表明为正确质粒,进行后续实验。
2.pBBR1MCS-1双酶切:
pBBR1是大小为4707bp的革兰氏阴性菌泛宿主质粒,该质粒带有氯霉素耐药基因(Chloramphenicol resistance gene,cmR)、pBBR1复制起始位点(pBBR1ori)、pBBR1(pBBR1rep)调节因子。该质粒的3321/3281bp处具有HindIII和KpnI限制性内切酶的单一酶切位点,采用HindIII/KpnI酶切的方法获得线性化的PBBR1质粒。其示意图如图1.A所示。pBBR1MCS-1质粒HindIII/KpnI酶切体系如表3所示。
表3:pBBR1MCS-1质粒HindIII/KpnI酶切体系
3.将上述酶切片段胶回收后与扩增的LUXA/B/C/D/E基因簇使用Gibson连接方法连接:
将上述两个线性化片段经鉴定和凝胶回收后,使用2×NEBuilder HiFi DNAAssembly连接酶连接,连接成功的环状质粒产物命名为pBBR1-lux,示意图如图1.B。pBBR1MCS-1 HindIII/KpnI酶切位点连接LUXA/B/C/D/E基因簇体系如表4。
表4:pBBR1MCS-1 HindIII/KpnI酶切位点连接LUXA/B/C/D/E基因簇体系(20μl)
4.产物转化至大肠杆菌感受态细胞:
连接后产物转化至大肠杆菌感受态细胞,阳性细菌筛选后,扩增细菌,提取质粒并进行一代测序鉴定,鉴定测序正确后的质粒命名为pBBR1-LUX A/B/C/D/E。含有pBBR1-LUXA/B/C/D/E的大肠杆菌收集并保存。
5.表达荧光素酶功能验证
挑取转化pBBR1-LUX A/B/C/D/E(pBBR1-lux)质粒的大肠杆菌阳性单菌落于含有氯霉素的LB液体培养基,37℃培养4小时后鉴定荧光值。阳性菌株传至第三代后,使用Veritas微孔板光度计检测其生物发光值(Luminesence),选取第三代仍具有Luminesence的菌落命名为DH5a-pBBR1-lux(DH5a-pBBR1-LUX A/B/C/D/E),使用IVIS Lumina LT小动物活体成像仪检测结果显示,DH5a-pBBR1-lux具有成像仪上具有可见荧光,如图2B,并且如图2A所示,DH5a-pBBR1-lux荧光值高达15000,和对照组DH5a-pBBR1仅为68相比,具有显著性的差异。
实施例2含有荧光素酶的产碳青霉烯酶肺炎克雷伯菌荧光素基因表达的检测
1.产碳青霉烯酶肺炎克雷伯菌电转化感受态制备
挑取产碳青霉烯酶肺炎克雷伯菌(KPC-KP)单个菌落接种于液体BHI培养基,37℃静置培养过夜。取过夜培养的产碳青霉烯酶肺炎克雷伯菌菌液以1:10的比例接种于新鲜的BHI培养基中,37℃静置培养至OD600=0.3~0.4。取出对数期菌液,冰浴30min后,4℃,4000rcf离心收集菌体,菌体分别用预冷的无菌双蒸水和预冷的1%蔗糖溶液洗涤两后,即为KPC-KP感受态细胞。
2.PBBR1-LUX A/B/C/D/E质粒电转化:
取5μl pBBR1-LUX A/B/C/D/E质粒加入到100μlKPC-KP感受态细胞中,混匀后冰浴10分钟。将上述混合物转移至预冷的0.1cm电击杯(美国伯乐)中,在电场强度:22.5kV/mm;电阻:200Ω;电容:25μF进行电转化。电击结束后,立即向电击杯中加入950μl,37℃预热的BH I培养基,于37℃静置培养1h后涂布于含25μg/ml氯霉素的哥伦比亚血平板培养基。37℃培养24h,挑取阳性菌落进行鉴定。
阳性菌落经过三代培养后,使用Veritas微孔板光度计检测其Luminesence值,选取具有Luminesence信号的菌落命名为KPC-KP-pBBR1-lux(KPC-KP-pBBR1-LUX A/B/C/D/E),使用IVIS Lumina LT小动物活体成像仪检测结果显示,KPC-KPpBBR1-lux具有成像仪上具有可见荧光,如图3B所示。
如图3A所示,KPC-KP-pBBR1-lux荧光值高达12207/108Cfu和对照组KPC-KP-pBBR1仅为88.67/108Cfu相比,具有显著性的差异。同时,也显著性的高于DH5a-pBBR1-lux的荧光值。
阳性菌落冻存于-70℃。一个月后划平板复苏得到的单菌落为第三代菌株(P3),将P1经1:100稀释后培养至对数期(OD600=0.5-0.8)所得菌株为第四代菌株(P4)。依此类推得到P5、P6、P7、P8、P9、P10菌株。将上述菌株稀释至108cfu/ml,检测其荧光强度,其结果如图4所示,本发明的菌落可以稳定传代,到第十代,其荧光值依然可以保持在8000/108Cfu左右。
实施例3:不同浓度KPC-KP-pBBR1-lux荧光素酶强度检测
挑取KPC-KP-pBBR1-lux单菌落菌株,37℃培养4小时后,使用NanoDropTM One超微量紫外分光光度计检测细菌OD值。依据1OD=109菌落形成单位(colony-forming unit,Cfu)计算细菌浓度。将所得菌株按照1:10稀释6个梯度,在酶标仪上检测Luminesence值,结果如图4所示,Luminesence值随菌落浓度降低而降低,当菌株浓度为107Cfu和106Cfu时,其荧光强度分别为2617和338,菌株浓度为105Cfu时,其荧光强度为131(114,140),与阴性对照[78.67(72,82)]差异不明显,因此,建议该细菌用于动物实验时,所述菌株在动物体内富集浓度不能低于106Cfu/mL。
实施例4:不同处理方式对KPC-KP-pBBR1-lux杀菌效果的验证
我们采用了六种不同物理化学方法处理。物理方法:用接种环(1μl)对KPC-KP-pBBR1-lux进行三区划线,37℃敷箱中培养2h,紫外照射30min。化学方法:使用酒精(75%)、84消毒液(10%)、H2O2(30%)、NaOH(0.1M、1M)分别浸泡细菌接种环(1ul)30min,分别挑取1μl KPC-KP-pBBR1-lux进行三区划线。上述处理后的KPC-KP-pBBR1-lux平板37℃培养8h后,使用小动物成像仪观察KPC-KP-pBBR1-lux荧光值。
结果显示,紫外灯、84消毒液以及1M的NaOH杀菌效果最强,能够完全杀菌:平板上未见KPC-KP-pBBR1-lux。酒精、H2O2和0.1M的NaOH有一定杀菌能力,但不能够完全杀灭KPC-KP-pBBR1-lux(不完全杀菌:平板上可见KPC-KP-pBBR1-lux,如图6所示)。
实施例5:KPC-KP-pBBR1-lux用于筛选抗生素
我们设计了不同抗生素杀菌方案。首先模拟感染环境的酸碱度、氧气含量,是否含有细胞因子等,可以模拟痰液、血液、尿液、粪便等环境。检测不同的抗生素浓度在特定的对于该菌的杀菌作用。优选不同抗生素使用浓度。
由于临床上碳青霉烯耐药的肺炎克雷伯菌多为多重耐药菌,我们依据临床上的耐药菌治疗方案,优选特定条件下联合使用抗生素时不同的抗生素比例。
实施例6:KPC-KP-pBBR1-lux用于制备感染动物模型
将KOX-D1/pBBR1-LUX稀释成2*109/ml,5*108/ml,1*108/ml。通过灌胃和腹腔注射方式感染C57小鼠(雄性,6-8周龄)。在小动物成像仪上观察细菌播散路径或定植位置。
如图7所示,在注射后即时以及注射后2h均可以方便的观察到细菌在体内的定植位置,可以根据它们的实时定制位置分析细菌的播散路径。
又如,如图8所示,我们小鼠腹腔注射2*109产碳青霉烯酶肺炎克雷伯菌,小鼠死亡后,通过生物荧光,我们发现细菌局限在肺部分布,从而进一步了解细菌的播散路径。
以上详细描述了本发明的优选实施方式,但是,本发明并不限于上述实施方式中的具体细节,在本发明的技术构思范围内,可以对本发明的技术方案进行多种简单变型,这些简单变型均属于本发明的保护范围。
另外需要说明的是,在上述具体实施方式中所描述的各个具体技术特征,在不矛盾的情况下,可以通过任何合适的方式进行组合,为了避免不必要的重复,本发明对各种可能的组合方式不再另行说明。
此外,本发明的各种不同的实施方式之间也可以进行任意组合,只要其不违背本发明的思想,其同样应当视为本发明所公开的内容。
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<110> 首都医科大学附属北京地坛医院
<120> 表达荧光素酶的产碳青霉烯酶肺炎克雷伯菌及其用途
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Claims (10)
1.一种产碳青霉烯酶肺炎克雷伯菌,其特征在于,所述产碳青霉烯酶肺炎克雷伯菌能稳定表达荧光素酶。
2.一种产碳青霉烯酶肺炎克雷伯菌的构建方法,其特征在于,所述构建方法包括:
(1) 制备包含LUX A/B/C/D/E基因簇的质粒;
(2) 制备电转化感受态产碳青霉烯酶肺炎克雷伯菌;
(3)将步骤(1)获得的包含LUX A/B/C/D/E基因簇的质粒电转化感受态产碳青霉烯酶肺炎克雷伯菌,挑选阳性菌落。
3.如权利要求3所述构建方法,其特征在于,所述质粒为泛宿主质粒。
4.一种权利要求1所述产碳青霉烯酶肺炎克雷伯菌的应用,其特征在于,所述应用包括在筛选杀菌方法中的应用,在筛选药物中的应用,或者,在制备动物模型中的应用。
5.一种杀菌方法的评估方法,其特征在于,所述评估方法包括利用权利要求1所述产碳青霉烯酶肺炎克雷伯菌对杀菌方法进行评估。
6.一种药物筛选方法,其特征在于,所述药物筛选方法包括利用权利要求1所述产碳青霉烯酶肺炎克雷伯菌对药物进行筛选。
7.一种动物模型的制备方法,其特征在于,所述制备方法包括将权利要求1所述产碳青霉烯酶肺炎克雷伯菌注入动物体内。
8.如权利要求7所述的制备方法,其特征在于,所述注入产碳青霉烯酶肺炎克雷伯菌体内方式包括鼻饲、灌胃、腹腔注射或者尾静脉注射。
9.一种产碳青霉烯酶肺炎克雷伯菌的杀菌方法,其特征在于,所述杀菌方法包括利用紫外照射、84消毒液或者NaOH对携带产碳青霉烯酶肺炎克雷伯菌的物品进行杀菌。
10.如权利要求9所述的杀菌方法,其特征在于,所述杀菌方法包括紫外照射30min以上,以10%的84消毒液或者1M的NaOH对物品进行消毒。
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