CN116370652B - 一种高效递送外源核酸进行细菌基因沉默的纳米载体系统构建及应用 - Google Patents
一种高效递送外源核酸进行细菌基因沉默的纳米载体系统构建及应用 Download PDFInfo
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Abstract
本发明涉及生物技术领域，尤其涉及一种高效递送外源核酸进行细菌基因沉默的纳米载体系统构建及应用。本发明首次构建了针对于细菌的金纳米簇递送系统，为外源核酸高效靶向递送进入细菌提供了优良的平台，该递送系统能够实现对siRNA进入细菌胞内的高效靶向递送，通过干扰细菌内关键基因，能够在短时间内实现细菌的有效阻控，将对于实现耐药基因的靶向沉默或耐药菌靶向清除，开发新型绿色阻控技术解决细菌耐药性难题意义重大。
Description
技术领域
本发明涉及生物技术领域，尤其涉及一种高效递送外源核酸进行细菌基因沉默的纳米载体系统构建及应用。
背景技术
细菌耐药性问题已经成为目前全球性的公共卫生问题，肠杆菌科细菌中各种耐药基因的出现和广泛流行严重威胁着人类和动物的健康。在缺乏新的抗菌药物的情况下，替加环素和多粘菌素成为治疗多重耐药革兰氏阴性菌，特别是耐碳青霉烯肠杆菌感染的“最后一道防线”。但质粒介导可水平转移的耐药基因在全球范围内的流行，严重制约着多粘菌素和替加环素在治疗耐碳青霉烯肠杆菌感染中应用。耐药细菌的出现降低了传统药物小分子的临床治疗效果，而加大治疗剂量又可能导致机体损伤与药物残留。因此，设计和开发新方法以对抗细菌耐药性显得尤为迫切。
RNA干扰(RNA interference，缩写为RNAi)技术有望实现耐药致病菌的靶向消除，以克服传统抗菌药物的弊端。RNA干扰过程主要是利用短的反义RNA通过阻碍与其有互补序列基因的转录或翻译来抑制基因的表达，诱发基因沉默现象。近年来，基于RNAi机制的有害生物防治策略发展迅速，用于防治有害生物的RNA分子制剂被称为核酸农药。核酸农药相较于化学杀菌剂，其靶向性强、无残留且开发成本低，目前在医疗领域已得到发展应用。尽管RNAi技术发展迅速且前景广阔，然而其应用仍受到诸多限制。一方面，由于siRNA分子本身带有负电，不能够通过被动扩散的形式穿过磷脂双分子层，且裸siRNA很容易受到酶促降解，这使得siRNA分子的递送效率较低；虽然目前已有诸多基因递送系统被广泛研究，如病毒载体、脂质体、聚合物和多肽等，但是它们都存在各自的缺点。另一方面，RNAi递送至真核生物细胞的成功案例较多，但是否也能高效递送至微生物细胞中以发挥功能却鲜有报道。因此，更为高效且安全地将siRNA递送至细菌靶部位以实现其沉默作用的载体亟待被开发，以扩充实现RNAi技术在多领域的高效应用。
随着纳米技术的发展，基于生物分子的纳米材料逐渐引起科研人员的广泛关注。金纳米簇(AuNCs)作为一种新型的纳米材料，因其具备优良的生物相容性、荧光性、超小尺寸等优点，在生物传感、化学催化和体内成像等诸多领域取得了显著的研究成果。此外，金纳米簇可作为一种优良的递送材料，应用案例如下：通过表面功能化AuNCs与抗癌药物直接偶联，形成药物递送体系，以用于癌症的治疗；AuNCs还可以形成自组装纳米颗粒，同时完成药物包埋装载；AuNCs可以与还原氧化石墨烯材料联用(AuNCs/RGO)，搭载抗癌药物DOX，用于肿瘤治疗等。基于金纳米簇是一种优良的药物递送系统，结合RNAi是一种高效的基因干预手段。然而，由于RNAi的递送成功受多因素的干扰，易被降解，不易被成功传递至目标细胞，从而对其递送系统及其构建提出了更高的要求。若建立一种能够负载siRNA的金纳米材料将其高效递送至目标菌，将对于实现特定基因如耐药基因的靶向沉默或耐药菌靶向清除，开发新型绿色阻控技术解决细菌耐药性难题意义重大。
发明内容
本发明提供了一种高效递送外源核酸进行细菌基因沉默的纳米载体系统构建及应用，该递送系统能够实现对siRNA进入细菌胞内的高效靶向递送，通过干扰细菌内关键基因，能够在短时间内实现细菌的有效阻控，为解决致病细菌感染及细菌耐药性问题提供一条新的途径，解决了现有技术中存在的问题。
本发明所采用的技术方案之一是：
提供了采用纳米金簇递送系统将外源核酸高效递送至细菌胞内实现基因沉默在抑制细菌生长中的应用。
进一步地，基因沉默所涉及的沉默基因源于细菌基因组或细菌胞内质粒；沉默基因为必需基因或耐药基因。
进一步地，所述纳米金簇递送系统包括金纳米簇内核及修饰在金纳米颗粒上的聚乙烯亚胺(PEI)分子，形成AuNCs@PEI；所述外源核酸为DNA或RNA分子；AuNCs@PEI以静电吸附作用与外源核酸分子结合。
进一步地，所述金纳米簇AuNCs@PEI的直径小于12nm，表面带正电位；所述金纳米簇AuNC@PEI与外源核酸的负载质量比例≥1.5，优选1.5～5。
进一步地，所述外源核酸为siRNA，所述siRNA为DNA连接酶编码基因siRNA。
进一步地，所述siRNA包括一对ligAsiRNA及其末端修饰的两个脱氧核苷酸T；ligAsiRNA sense序列如SEQ ID No1所示，antisense序列如SEQ ID No2所示。在序列表中，将上述序列中的U均改成了T。
进一步地，ligAsiRNA sense序列为：GGAAAUUCCCGACGCUGAA；
antisense序列为：UUCAGCGUCGGGAAUUU CC。该序列通过在末端修饰两个脱氧核苷酸T，使ligAsiRNA更稳定。修饰后上述序列为：ligAsiRNA sense：GGAAAUUCCCGACGCUGAAdTdT；antisense序列为：dTdTUUCAGCGUCGGGAAUUU CC。
进一步地，负载ligAsiRNA的金纳米簇递送系统的制备包括如下步骤：
(1)向HAuCl4溶液中加入GSH，加热并温和搅拌，制备AuNCs；
(2)向AuNCs溶液中加入PEI，室温反应，制备AuNCs@PEI；
(3)将AuNC@PEI与siRNA按照一定比例共孵育，得到负载ligAsiRNA的金纳米簇。
进一步地，步骤(1)中：HAuCl4与GSH的摩尔比为1：1.5～1：3；HAuCl4与GSH加热至40～70℃，优选温和搅拌转速为500rpm，恒温搅拌时间优选为24h～36h；
步骤(2)中：AuNCs与PEI反应质量比例区间优选为1：2～1：10；AuNCs与PEI反应温度优选为25℃，反应时间优选为2h～5h；
步骤(3)中：AuNC@PEI与siRNA按照一定比例反应，优选质量比为1：1～5：1，反应时间优选为10min。
进一步地，所述细菌为包含/不包含耐药基因的细菌、耐药菌或耐药致病菌。
进一步地，所述细菌为革兰氏阴性细菌，所述革兰氏阴性细菌为肠杆菌。
进一步地，所述肠杆菌为大肠杆菌，所述大肠杆菌为大肠杆菌MG1655。
进一步地，大肠杆菌MG1655中DNA连接酶I编码基因ligA为关键基因。进一步地，前述应用采用将外源核酸分子负载至金纳米簇AuNC@PEI，并将其与细菌共孵育1～4h的方法向细菌胞内递送，以此实现短时间内靶向抑制细菌生长。
本发明所采用的技术方案之二是：
负载ligAsiRNA的金纳米簇递送系统在制备抑制含耐药基因的细菌、耐药菌或耐药致病菌生长的药物中的应用。
本发明所采用的技术方案之三是：
负载ligAsiRNA的金纳米簇递送系统在制备治疗肠杆菌感染的药物中的应用。
本发明所采用的技术方案之四是：
一种药物，包括负载ligAsiRNA的金纳米簇及药学上可接受的载体，该药物靶向致病菌、耐药菌并实现抑菌。
进一步地，前述核酸递送系统将siRNA递送至细菌胞内的应用可以作为工具，在特异性基因沉默，基因功能研究等领域具有重要的应用价值。
本发明的有益效果：
本发明首次构建了针对于细菌的金纳米簇递送系统，为外源核酸高效靶向递送进入细菌提供了优良的平台，目前尚未见这样的报道。通过采用本发明的技术方案，金纳米簇可以为细菌中关键基因ligA siRNA提供有效的保护，提高siRNA的稳定性以及细菌靶向性，将ligA siRNA高效递送至大肠杆菌细胞胞内，短时间内实现有效ligA的沉默，阻断DNA复制冈崎片段的连接过程，以此抑制大肠杆菌正常的生长，为解决肠杆菌感染及耐药性问题提供一条新的途径。
本发明构建的负载siRNA的金纳米材料可实现高效递送至耐药病原菌，将对于实现耐药基因的靶向沉默或耐药菌靶向清除，开发新型绿色阻控技术解决细菌耐药性难题意义重大。
附图说明
图1为负载ligAsiRNA纳米递送系统的制备流程图；
图2为siRNA纳米金簇递送载体AuNCS@PEI的表征；
图3为琼脂糖凝胶电泳检测纳米金簇递送系统AuNCS@PEI对核酸的负载能力；
图4为AuNCS@PEI对siRNA的递送效率图；
图5为荧光定量PCR技术分析ligA基因的转录水平变化；
图6为AuNCS@PEI递送siRNA对大肠杆菌MG1655的抑制效果图。
具体实施方式
为能清楚说明本方案的技术特点，下面通过具体实施方式，结合附图，对本发明进行详细阐述。
在本发明中，若非特指，所有的份、百分比均为重量单位，所采用的设备和原料等均可从市场购得或是本领域常用的。下述实施例中的方法，如无特别说明，均为本领域的常规方法。
实施例1纳米金簇递送载体AuNCs@PEI的制备
在室温下将新鲜制备的HAuCl4(2mM)和GSH(3mM)溶液于室温下混合并加热至40℃～80℃，温和搅拌24h～36h，此时形成强烈橙色发射的AuNCs的水溶液；向上述溶液中加入乙腈，离心收集得到沉淀物，用超纯水和乙腈的混合物洗涤3次进一步纯化所得沉淀物GSH-AuNCs。将纯化的GSH-AuNCs真空冷冻干燥后取溶于超纯水中，加入PEI于室温搅拌2h～5h，加入乙腈，12000rpm离心10min将产物与溶液分离，进一步使用乙腈洗涤3次进行纯化得到AuNCs@PEI；待真空冷冻干燥后，取100mg AuNCs@PEI溶于100mL超纯水中获得AuNCs@PEI以备用,完整的金纳米簇核酸递送系统制备流程图如图1所示。
利用透射电镜(TEM)，傅里叶红外(FT-IR)，Zetasizer(NanoZS，马尔文)技术等表征AuNCs@PEI的形貌、电位等相关性质。如图2所示，AuNC@PEI大小在6～13nm左右。
实施例2AuNC@PEI负载SiRNA量的优化及凝胶电泳分析
为确定AuNC@PEI的负载能力，将AuNC@PEI与双链siRNA以不同质量比(0.5:1、1:1、1.5:1和2:1)孵育10min；制备1％的琼脂糖凝胶电泳，通过电泳条带的亮度以分析AuNC@PEI负载SiRNA的含量。
结果如图3所示：游离siRNA条带清晰明亮，随着AuNC@PEI的加入，条带逐渐减弱，这说明AuNC@PEI对siRNA有较好的负载效率，当AuNC@PEI：siRNA质量比为1.5：1，可以完全负载siRNA。AuNC@PEI负载siRNA比例优选区间为1.5：1～5：1。
实施例3AuNC@PEI对siRNA的递送效率分析
在siRNA上修饰Cy3荧光探针，将其负载至AuNC@PEI材料，保持Cy3-siRNA终浓度为500nM，将构建的材料与对数生长期的大肠杆菌于37℃条件下共孵育1h；孵育完成后，5000rpm离心洗涤，在沉淀中加入hoechst33342荧光染料染色20min，离心收集菌体，分别在荧光显微镜的橙色荧光和蓝色荧光的条件下观察。若蓝色荧光和橙色荧光有重叠部分，表明siRNA能够被递送至细胞胞内。
结果如图4所示：本发明构建的纳米金簇AuNCs@PEI能够将siRNA有效递送至细菌胞内。而siRNA本身不能有效内化至细菌胞内，且siRNA的递送效率与纳米金簇和外源核酸质量比密切相关，随着AuNC@PEI与siRNA质量比例的增高，蓝色荧光和橙色荧光重叠比率增多，这表明siRNA的递送效率也随之升高。
实施例4荧光定量PCR技术分析ligA基因的转录水平变化
取-80℃保存的大肠杆菌MG1655活化并接种于新鲜的LB培养基，经37℃过夜培养后，按1：100比例转接至20mL LB培养基中，37℃培养2～3h，此时OD600nm值为0.3左右。
将菌液平分等份，分别依次加入H2O，siRNA(500nM)，AuNCs@PEI(13μg)，siRNA(500nm)+AuNCs@PEI(13μg)，siRNA序列如SEQ ID No1和SEQ ID No2所示，将其充分混匀后置于37°，培养2h。将菌体5000rpm离心5min收集沉淀，用PBS洗涤3次后收集菌体待用。
使用TRIzolTM RNA提取试剂盒(12183555,Invitrogen)进行RNA提取，所有RNA提取过程中均使用无酶耗材，使用诺维赞公司的II反转录试剂盒对上述提取的RNA进行DNA去除及反转录，最终获得cDNA。使用BestarSybrGreen qPCR Mastermix(DBI)荧光定量PCR酶和LightCycler 480II(Roche)荧光定量PCR仪进行qPCR实验，选取16S rRNA为参照基因。qPCR反应体系是：2×Bestar SybrGreen qPCR Mastermix 10μL，引物F 0.5μL，引物R 0.5μL，DEPC水8μL，模板cDNA1μL；反应程序是95℃3min，然后95℃10s,60℃35s重复45个循环，最后95℃15s，60℃1min结束程序。测定并记录Ct值，所得数据以16S rRNA作为内参基因，根据2^(-ΔΔCт)的计算方法对目的基因相对表达量进行计算。
实验共设计了目标基因扩增引物2对，内参基因扩增引物3对，引物序列如下表1所示。根据扩增的产物的溶解曲线，ligA-RT-2F，ligA-RT-2R，16S-RT-3F，16S-RT-3R荧光扩增曲线呈标准的S型；扩增产物溶解曲线单个尖锐峰，扩增产物特异性好，由此我们方案中保留该对引物。
表1引物序列
荧光定量PCR结果如图5所示，通过计算大肠杆菌ligA基因的相对表达量，发现负载有ligAsiRNA的AuNCs@PEI处理组明显低于其他组，这表明构建的纳米金簇载体AuNCs@PEI能够成功递送核酸siRNA，实现细菌胞内基因的有效沉默，从而靶向调控细菌生理功能。
实施例5AuNCS@PEI递送siRNA对大肠杆菌MG1655的抑菌效果
取-80℃保存的大肠杆菌MG1655活化活化并接种于新鲜的LB培养基，经37℃过夜培养后按1：100比例转接至20mL LB培养基中，37℃培养2～3h，此时OD600nm值为0.3左右。
分别取培养后的菌液，依次加入H2O，siRNA(500nM)，AuNCs@PEI，siRNA(500nm)+AuNCs@PEI，将其充分混匀后置于37°，培养2h。5000rpm离心5min收集菌体细胞，用PBS洗涤3次后按照以下操作处理细胞：加入100μL 1×Binding buffer，轻轻吹匀至单细胞悬液；加入5μL Annexin V-FITC和5μL PI Staining solution，轻轻吹匀,避光、室温(20～25℃)孵育10min进行细胞染色；加入400μL 1×Binding buffer，轻轻混匀，样品在1h内用流式细胞仪检测细胞凋亡率。
结果如图6所示：由于编码DNA连接酶I的ligA基因对于大肠杆菌是必需基因，ligA基因的沉默会影响DNA的复制和修复过程，最终会导致大肠杆菌生长抑制或死亡；结果显示，负载有ligAsiRNA的AuNCs@PEI处理组细胞凋亡数量明显高于未负载核酸的AuNCs@PEI组和siRNA组。
由于ligA基因为耐药菌或非耐药菌均含有的必须基因，通过以上试验也可以推断，负载有ligAsiRNA的AuNCs@PEI的递送系统对耐药菌、耐药性致病菌、含耐药基因的细菌也具有生长抑制或死亡作用，这对于开发新型绿色阻控技术解决细菌耐药性难题意义重大。
以上对本发明所提供的外源核酸递送至细菌的纳米金簇递送系统的构建及其在抑菌方面的应用进行了详细介绍。上述具体实施方式不能作为对本发明保护范围的限制，对于本技术领域的技术人员来说，对本发明实施方式所做出的任何替代改进或变换均落在本发明的保护范围内。
本发明未详述之处，均为本技术领域技术人员的公知技术。
Claims (5)
1.采用纳米金簇递送系统将外源核酸高效递送至细菌胞内实现基因沉默在非诊疗目的抑制细菌生长中的应用；其特征在于，所述基因沉默所涉及的沉默基因源于细菌基因组或细菌胞内质粒；所述沉默基因为必需基因或耐药基因；
所述外源核酸为siRNA，所述siRNA为DNA连接酶编码基因siRNA；
所述siRNA为一对ligAsiRNA及其末端修饰的两个脱氧核苷酸T；ligAsiRNA sense序列为：GGAAAUUCCCGACGCUGAA；antisense序列为：UUCAGCGUCGGGAAUUUCC；该序列通过在末端修饰两个脱氧核苷酸T，使ligAsiRNA更稳定；修饰后上述序列为：ligAsiRNA sense：GGAAAUUCCCGACGCUGAA dTdT；antisense序列为：dTdTUUCAGCGUCGGGAAUUUCC；
所述纳米金簇递送系统包括金纳米簇内核及修饰在金纳米颗粒上的聚乙烯亚胺分子，形成AuNCs@PEI；
所述细菌为革兰氏阴性细菌，所述革兰氏阴性细菌为大肠杆菌MG1655；
该应用是采用将外源核酸分子以1:5比例负载至金纳米簇AuNCs@PEI，并将其与细菌共孵育1～4h的方法向细菌胞内递送，以此实现短时间内靶向抑制细菌生长。
2.根据权利要求1所述的应用，其特征在于，所述AuNCs@PEI以静电吸附作用与外源核酸分子结合。
3.根据权利要求2所述的应用，其特征在于，所述金纳米簇AuNCs@PEI的直径小于12nm，表面带正电位。
4.根据权利要求1所述的应用，其特征在于，负载ligAsiRNA的金纳米簇递送系统的制备包括如下步骤：
向HAuCl4溶液中加入GSH，加热并温和搅拌，制备AuNCs；
向AuNCs溶液中加入PEI，室温反应，制备AuNCs@PEI；
将AuNCs@PEI与siRNA按照一定比例共孵育，得到负载ligAsiRNA的金纳米簇。
5. 根据权利要求4所述的应用，其特征在于，步骤（1）中：HAuCl4与GSH的摩尔比为1：1.5～1：3；HAuCl4与GSH加热至40～70°C，温和搅拌转速为500 rpm，恒温搅拌时间为24h～36h；
步骤（2）中：AuNCs与PEI反应质量比例区间为1：2～1：10；AuNCs与PEI反应温度为25°C，反应时间为2 h～5h；
步骤（3）中：AuNCs@PEI与siRNA按照一定比例反应，质量比为1：1～5：1，反应时间为10min。
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