CN114560914B - 一种抑制blaNDM基因表达的肽核酸及其应用 - Google Patents

一种抑制blaNDM基因表达的肽核酸及其应用 Download PDF

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CN114560914B
CN114560914B CN202210222781.3A CN202210222781A CN114560914B CN 114560914 B CN114560914 B CN 114560914B CN 202210222781 A CN202210222781 A CN 202210222781A CN 114560914 B CN114560914 B CN 114560914B
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黄建胜
赵赟安
陈娇丽
赵芯米
徐建芬
武蓉珍
赵志钢
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Abstract

本发明提供一种抑制blaNDM基因表达的肽核酸及其应用，即一种针对blaNDM基因的反义肽核酸，该肽核酸的序列为5′‑GCAATTCCAT‑3′。本发明提供的PNA能特异性抑制blaNDM基因的表达，Western Blot实验检测共培养后菌株的NDM蛋白表达水平降低，肉汤稀释法检测菌株对碳青霉烯类药物亚胺培南及美罗培南耐药水平降低，该PNA能够高效特异地抑制blaNDM基因的表达并逆转碳青霉烯类抗生素耐药性。此外，PNA几乎不与蛋白质作用，因而毒性作用很小。PNA与DNA或RNA的杂交几乎不受杂交体系盐浓度影响。基于上述特性，本发明所述的PNA在NDM相关耐药病原菌抗感染治疗方面显示出广阔的应用前景。

Description

一种抑制blaNDM基因表达的肽核酸及其应用
技术领域
本发明属于生物医药技术领域，具体涉及一种抑制blaNDM基因表达的肽核酸，及利用该肽核酸抑制blaNDM基因表达来逆转携带blaNDM基因菌株的碳青霉烯类抗生素耐药性。
背景技术
新德里金属-β-内酰胺酶(New Delhi metallo-β-lactamase,NDM)是一种与质粒相关的Ambler B类β-内酰胺酶。2008年首次从一名在印度感染的瑞典患者身上分离的肺炎克雷伯菌中发现该酶。随后，NDM带来的耐药性以克隆方式迅速传播到世界各地，并扩散到许多革兰氏阴性病原体中，包括大肠杆菌、铜绿假单胞菌和鲍曼不动杆菌等。目前已发现blaNDM-1至blaNDM-31等数十个亚型，且呈散在分布，但以blaNDM-1和blaNDM-5检出率最高、流行最广泛。
NDM危险性高，因为它会赋予菌株碳青霉烯类抗生素耐药性，而碳青霉烯类抗生素(Carbapenems)是目前抗菌谱最广、抗菌活性最强的抗生素之一,一度被视为治疗严重细菌感染的最后采纳的手段。同时，NDM会伴随有其他耐药基因，其种类覆盖了大部分可用抗生素类别。
尽管β-内酰胺酶抑制剂已被批准用于人类联合用药，但它们主要对丝氨酸(A类)和ampC(C类)β-内酰胺酶有效，对包括NDM在内的金属β-内酰胺酶均无效。研究表明，NDM菌株已占我国碳青霉烯耐药肠杆菌科菌(Carbapenem Resistant Enterobacteriaceae，CRE)构成比的33.5％，且感染致死率最高可达43.1％，严重威胁国民健康。因此，寻找低毒高效的NDM抑制剂意义重大。
发明内容
本发明的目的在于提供一种抑制blaNDM基因表达的肽核酸及其应用，即一种针对blaNDM基因的反义肽核酸，该肽核酸可高效抑制blaNDM基因表达从而逆转相关菌株的碳青霉烯类抗生素耐药性，可用于制备靶向沉默NDM表达及与抗生素联合抗感染的小分子药物。
本发明所提供的肽核酸PNA，其序列为5′-GCAATTCCAT-3′(SEQ ID NO:1)；
本发明还提供所述的肽核酸在抑制blaNDM基因表达中的应用；
本发明还提供一种用于抑制blaNDM基因表达的制品，所述的制品中包含有药理有效浓度的上述的肽核酸；
更进一步的，所述的肽核酸与透膜肽偶联；
作为实施例的具体记载，所述的透膜肽为透膜肽(RXR)4XB。
本发明还提供一种预防治疗NDM相关耐药病原菌的制品，所述的制品中包含有抗生素和上述的肽核酸PNA。
本发明提供的PNA能特异性抑制blaNDM基因的表达，Western Blot实验检测共培养后菌株的NDM蛋白表达水平降低，肉汤稀释法检测菌株对碳青霉烯类药物亚胺培南及美罗培南耐药水平降低，该PNA能够高效特异地抑制blaNDM基因的表达并逆转碳青霉烯类抗生素耐药性。此外，PNA几乎不与蛋白质作用，因而毒性作用很小。PNA与DNA或RNA的杂交几乎不受杂交体系盐浓度影响。基于上述特性，本发明所述的PNA在NDM相关耐药病原菌抗感染治疗方面显示出广阔的应用前景。
附图说明
图1为受试菌株blaNDM基因PCR产物电泳结果。第一泳道至第三泳道分别为Marker、菌株1、菌株2。结果显示两样品存在500bp-750bp间大小一致条带，与预期相符。
图2为Western Blot实验结果。A，灰度值分析，以对照组NDM表达为参照。B，代表性Western Blot条带，DnaK蛋白为内参。*，P<0.05；**，P<0.01；t-test。
具体实施方式
肽核酸(Peptide nucleic acid,PNA)是一种人工合成的核酸类似物，由N-(2-氨基乙基)甘氨酸单元替代天然核酸中的戊糖-磷酸骨架，碱基通过亚甲羰基和骨架连接。PNA的空间结构和距离都与核酸相似，其碱基可以通过Watson-Crick配对与互补的DNA/RNA链特异性结合，其骨架结构呈电中性，与带负电的DNA/RNA结合不存在静电排斥力，且不依赖于盐离子浓度，单体通过多聚酰胺键连接，对核酸酶和蛋白酶具有抗性且热稳定性较好。
本发明提供了一种肽核酸，可以有效阻止blaNDM基因的翻译。所述肽核酸中，N-(2-氨基乙基)甘氨酸单元替代了天然核酸中的戊糖-磷酸骨架，碱基通过亚甲羰基和骨架连接，其碱基可以通过Watson-Crick配对与互补的mRNA链特异性结合。所述肽核酸与透膜肽(RXR)4XB偶联，以提高对细菌的渗透性。本发明所述的抑制blaNDM基因表达的肽核酸，通过人工合成形成，其生产和制备属于现有技术。
本发明选取临床分离的携带blaNDM基因的CRE菌株作为受试菌株，通过质谱鉴定及基因测序确定菌种及NDM亚型，其中携带blaNDM-5基因的大肠杆菌记为菌株1，携带blaNDM-1基因的弗劳地枸橼酸杆菌记为菌株2。PNA通过共培养作用于受试菌株。以肉汤稀释法检测耐药水平，Western Blot实验检测蛋白质表达水平。进一步的，受试菌株为携带blaNDM-1或blaNDM-5基因的CRE菌株。
下面结合附图和实施例，对本发明的具体实施方式作进一步描述。以下实施例仅用于更加清楚地说明本发明的技术方案，而不能以此来限制本发明的保护范围。
实施例1肽核酸PNA的设计合成
从Genebank调取blaNDM基因各亚型的基因信息，比对发现，起始密码子所在区域序列高度保守。针对blaNDM基因起始密码子位点序列设计反义PNA，具体序列如下：5’-GCAATTCCAT-3’，合成时加入(RXR)4XB配基偶联，以提高对细菌的渗透性。
实施例2菌株及blaNDM基因鉴定
第一步，将临床分离菌株1及菌株2转种于血平板，置于37℃培养箱中过夜培养。
第二步，挑取单菌落通过VITEK MS全自动微生物质谱检测仪进行质谱鉴定。单菌落分散点于质谱仪靶板上，再点入0.5μL有机基质溶液，待靶板上的点样液干燥结晶后，将靶板放入质谱仪进行质谱鉴定。
第三步，PCR扩增blaNDM基因。挑取适量菌落溶于无菌水，100℃加热裂解10min获得扩增模板，12000rpm离心5min，取上清备用。配制PCR反应体系如下：
设置反应程序如下：
该反应体系于4℃保存。扩增产物取2μL进行10％琼脂糖凝胶电泳，在多功能紫外仪上观察，结果见图1。
第四步，PCR产物测序，结果进行blast分析。
结合质谱鉴定及测序比对结果，得到，菌株1为大肠杆菌，携带blaNDM-5基因；菌株2为弗劳地枸橼酸杆菌，携带blaNDM-1基因。
实施例3 NDM蛋白表达变化
使用菌株1进行实验，Western Blot测定蛋白质表达差异。
第一步，配制0.5麦氏浊度的菌悬液，使用LB液体培养基1:200稀释。
第二步，取上述稀释后菌液加入PNA，使PNA终浓度分别为5、10、20μM作为实验组；不含PNA的稀释后菌液作为对照组。
第三步，实验组及对照组菌液置于37℃摇床分别培养1h，2h，3h时，取250μL培养液，使用活性蛋白提取试剂盒提取活性蛋白；按照蛋白质提取液：loading buffer为4:1混合，100℃加热3-5min制得蛋白电泳液。
第四步，进行SDS-PAGE电泳。配制分离胶浓度为12％，浓缩胶浓度为5％的SDS-PAGE凝胶，15μL/孔上样，1×SDS电泳缓冲液中进行电泳；80V电泳约30min，至溴酚蓝染料从浓缩胶进入分离胶，再将电压调至120V继续电泳约50min至溴酚蓝到达凝胶底部。
第五步，转膜。从负极到正极按照海绵-滤纸-胶-膜-滤纸-海绵顺序组装固定进行转膜；冰浴条件下，110V转移0.5h。
第六步，免疫反应。剪除多余的NC膜，封闭后将NC膜剪为携带DNAK蛋白(70-90kD)与NDM蛋白(40kD)两部分；分别使用抗DNAK抗体与抗NDM抗体进行一抗孵育，4℃下过夜，抗体均1:2000稀释；回收一抗，加入洗涤液置于摇床上，洗涤4次，每次15min；两部分膜均用1：4000的抗小鼠HPR二抗室温孵育2h；回收二抗，加入洗涤液置于摇床上，洗涤4次，每次15min。
第七步，曝光。将显影液滴加于膜上，均匀充分覆盖膜表面，放入成像仪中观察及拍照。
实验结果如图2所示，发现本发明设计的PNA可显著抑制NDM蛋白表达，并呈高度的浓度依赖性和时间依赖性；PNA与菌株共培养3h后，3个浓度下NDM表达均下调90％以上(P<0.05)，几乎沉默blaNDM基因的表达。
实施例4 MIC测定实验
第一步，配制0.5麦氏浊度的菌悬液，使用液体药敏试验培养基(CAMHB浊度型)1:200稀释。
第二步，取上述稀释后菌液加入PNA，使终浓度分别为1、5、10μM作为实验组；不含PNA的稀释后菌液作为对照组。
第三步，取亚胺培南及美罗培南药敏板条平衡至室温，分别标记为对照组、PNA-1μM、PNA-5μM、PNA-10μM，按50μL/孔加入对应菌液，37℃培养12-16h，肉眼观察各孔菌液清亮程度，肉眼可见清亮的最低药物浓度对应孔浓度即为此药物的MIC。结果见表1。
表1:PNA对携带blaNDM基因CRE的MIC值影响表
实验结果表明，本发明设计的PNA可显著逆转NDM相关耐药菌株对碳青霉烯类抗生素的耐药性。故该PNA可与抗生素联合使用治疗NDM相关耐药病原菌感染。
以上所述仅为本申请的优选实施例而已，并不用于限制本申请，对于本领域的技术人员来说，本申请可以有各种更改和变化。凡在本申请的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本申请的保护范围之内。
序列表
<110> 丽水市中心医院
<120> 一种抑制blaNDM基因表达的肽核酸及其应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 10
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
gcaattccat 10

Claims (6)

1.一种肽核酸，其特征在于，所述的肽核酸序列为5′-GCAATTCCAT-3′；所述肽核酸中，N-(2-氨基乙基)甘氨酸单元替代了天然核酸中的戊糖-磷酸骨架，碱基通过亚甲羰基和骨架连接。
2.一种用于抑制blaNDM基因表达的制品，其特征在于，所述的制品中包含有药理有效浓度的权利要求1所述的肽核酸。
3.如权利要求2所述的制品，其特征在于，所述的肽核酸与透膜肽偶联。
4.如权利要求3所述的制品，其特征在于，所述的透膜肽为透膜肽(RXR)4XB。
5.一种预防治疗NDM相关耐药病原菌的制品，其特征在于，所述的制品中包含有抗生素和权利要求1所述的肽核酸。
6.如权利要求5所述的预防治疗NDM相关耐药病原菌的制品，其特征在于，所述的肽核酸与透膜肽偶联。
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