CN115011604B - 铜绿假单胞菌IV型菌毛蛋白PilA的适配体PilA-1及用途 - Google Patents
铜绿假单胞菌IV型菌毛蛋白PilA的适配体PilA-1及用途 Download PDFInfo
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Abstract
本申请属于生物化学和医学微生物技术领域，涉及铜绿假单胞菌IV型菌毛蛋白PilA的适配体PilA‑1及用途。本发明以铜绿假单胞菌Ⅳ菌毛中外段蛋白PilA为靶标，利用SELEX技术通过12轮筛选与其特异性结合的核酸适配体，通过大通量测序分析适配体的碱基序列。获得铜绿假单胞菌Ⅳ菌毛蛋白PilA核酸适配体PilA‑1的序列，其核酸序列为：PilA‑1：5’‑CACGGTCCAACGGTCGTTCGCATGCCTATCCACGG‑3’。并通过软件对获得的碱基序列进行分析，模拟出它的二级结构，分析其亲和力大小，并探究适配体PilA‑1对铜绿假单胞菌菌膜形成的影响。
Description
技术领域
本发明属于医学微生物和分子生物学技术领域，涉及铜绿假单胞菌IV型菌毛蛋白PilA的适配体PilA-1及用途。
背景技术
铜绿假单胞菌(Pseudomonasaeruginosa，PA)，又名绿脓杆菌，可产生绿脓素和荧光素，在培养基上呈蓝绿色荧光，感染伤口时形成绿色脓液。铜绿假单胞菌为重要的条件致病菌，是医院常见病原体之一，引起一些列严重的化脓性感染。它存在于在人的肠道、皮肤、呼吸道等，当人的免疫力低下或者防御机制遭受破坏，以及治疗操作或手术后的患者等易导致铜绿假单胞菌的感染，且居非发酵细菌感染首位。随着抗菌药物的广泛应用，铜绿假单胞菌的耐药性越来越严重，并逐年增加趋势，给临床治疗带来了极大困难，导致高致死率。目前国内外对铜绿假单胞菌的耐药机理进行深入的研究。研究表明，铜绿假单胞菌的耐药机制异常复杂，其中在体内极易形成菌膜的成为其耐药的主要原因之一。铜绿假单胞菌菌膜浮游细菌吸附到固体表面，接着形成小菌落，三级结构的发育和整个群落被菌表多糖包被。铜绿假单胞菌菌膜是浮游细菌附着在物体表面形成的被多聚基质包裹着细菌，成为具有屏障功能的细菌结构群体，从而逃避机体的免疫应答和抗菌药物的杀灭作用，并且在细胞形态和生理上会发生很大的变化，因此抑制生物膜的形成具有重要的意义。
铜绿假单胞菌拥有极性细丝的IV型菌毛，通过它的抽搐运动参与附着于物体和宿主组织表面。菌毛伸展和收缩介导的抽搐运动是铜绿假单胞菌生物膜形成所必需的。PilA是IV型菌毛中上段蛋白，由PilA的数千个亚单位组成，通过复杂的组装机快速聚合和解聚。它和小pilins具有相似的棒棒糖样拓扑结构，具有高度保守的疏水N-端α-螺旋，与可变C-端反平行β-折叠。成熟菌毛蛋白的第1个，24个残基将亚单位固定在内膜上，直到它们被组装机器聚合。在浮游细菌的抽搐运动和粘附占有重要作用，因此PilA是菌膜形成的关键蛋白之一。有研究表明，IV型菌毛中上段的PilA蛋白在细菌耐药性中起到了关键作用。研究PilA蛋白的存在形式以及结构有助于深入研究铜绿假单胞菌的生物膜耐药机制，为细菌耐药机制及干预提供依据。因此通过改变IV型菌毛蛋白PilA的结构是抑制铜绿假单胞菌的菌膜的形成的有效途径。
目前，适配体的研究是一个研究热点。它是能够与许多靶标分子(药物，蛋白，无机或有机分子)发生高特异性和高亲合性结合的一类单链核酸(DNAorRNA)。采用配体指数富集系统(systematicevolutionofligandsbyexponentialenrichment，SELEX)筛选高特异性和高亲和性的适配体。由于适配体结构稳定，且易修饰基团，在医学研究上常被用于靶向治疗和检测等方面。继2004年针对血管内皮生长因子(VEGF)的适配体—Macugen，被批准用于治疗年龄相关的黄斑变性(AMD)以后，另外两种用于眼睛相关疾病治疗的适配体药物—Zimura和Fovista也分别进入二期和三期临床试验(HuangYF,ShangguanDH,LiuHP,PhillipsJA,ZhangXL,ChenY,TanWH.MolecularAssemblyofan Aptamer-DrugConjugateforTargetedDrugDeliverytoTumorCells.Chembiochem2009,10(5):862-868.)。还有一些适配体正处于临床试验的不同阶段，各种医学实验正在进行测试。但迄今尚无针对铜绿假单胞菌IV型菌毛蛋白PilA的适配体报道或公开，因此本发明拟考虑核酸适配体应用于改变PilA蛋白结构来有效抑制菌毛的抽搐运动及粘附性能(原理如图1所示)。开发针对IV型菌毛蛋白PilA的核酸适配体，将为抑制铜绿假单胞菌菌膜的形成，为干预其多重耐药提供有力支持，并为铜绿假单胞菌的靶向治疗和检测奠定基础，具有重要的临床价值。
发明内容
本发明的目的是提供以铜绿假单胞菌Ⅳ菌毛蛋白PilA为靶标的核酸适配体PilA-1序列。
本发明的进一步目的是提供所述核酸适配体序列的用途，可以作为一种抑制铜绿假单胞菌菌膜形成的核酸药物，也根据核酸序列设计铜绿假单胞菌靶向治疗方案。
本发明铜绿假单胞菌Ⅳ菌毛蛋白PilA核酸适配体PilA-1序列制备的方案是：
铜绿假单胞菌IV型菌毛蛋白PilA的适配体，适配体是以铜绿假单胞菌IV型菌毛中上段蛋白PilA为靶标的核酸适配体序列，其核酸序列为：PilA-1：5’-CACGGTCCAACGGTCGTTCGCATGCCTATCCACGG-3’。
作为优选，所述铜绿假单胞菌Ⅳ菌毛蛋白PilA核酸适配体PilA-1在制备靶向治疗铜绿单胞菌感染药物中的用途。
作为优选，所述药物为菌膜抑制药物。
作为优选，所述铜绿假单胞菌Ⅳ菌毛蛋白PilA核酸适配体PilA-1在制备检测铜绿假单胞菌的探针中的用途。
先从上海生工购买基因表达纯化蛋白PilA.随后利用适配体的SELEX体外筛选技术，以收集纯化后的PilA蛋白为靶标，从78个碱基的随机ssDNA文库(5'-GGGAGCTCAGAATAAACGCTCAA-N35-TTCGACATGAGGCCCGGATC-3')中筛选出与PilA蛋白特异结合的核酸适配体，将筛选出来的序列用上游引物P1(5'-GGGAGCTCAGAATAAACGCTCAA-3')和磷酸标记的下游引物P2(5'-PO4-GATCCGGGCCTCATGTCGAA-3')进行扩增。然后再酶切成ssDNA成下来一轮文库，筛选12轮后送至上海生工进行高通量测测序。其中PilA-1序列重复率达到38.59％，表明序列在测序样本中重复出现次数高，是在12轮筛选中保留下来的优势序列。
PilA-1：CACGGTCCAACGGTCGTTCGCATGCCTATCCACGG。
然后合成适配体序列，对其亲和力进行分析，并且考察不同浓度适配体(0、0.25、0.5、1.0、2.5、5.0μM)对菌膜抑制作用。
本发明中，所述核酸序列构成的适配体可与铜绿假单胞菌Ⅳ菌毛蛋白PilA特异性结合，可用于抑制铜绿假单胞菌的菌膜及靶向治疗。所述核酸序列构成的适配体也可以作为检测PilA蛋白的探针或靶点。
本发明具有如下良好的效果：
1、本发明获得以铜绿假单胞菌Ⅳ菌毛蛋白PilA为靶标的核酸适配体PilA-1序列，其核酸序列分别为：5’-CACGGTCCAACGGTCGTTCGCATGCCTATCCACGG-3’。
2、本发明铜绿假单胞菌Ⅳ菌毛蛋白PilA核酸适配体PilA-1序列在制备药物或其它制品中的作用巨大。
3、本发明含有铜绿假单胞菌Ⅳ菌毛蛋白PilA核酸适配体PilA-1序列的药物为菌膜抑制剂和铜绿假单胞菌的靶向治疗提供有力支持，其将具有重要的临床价值。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作一简单地介绍，显而易见地，下面描述中的附图是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1适配体抑制菌膜形成的原理图；
图2适配体亲和力检测结果；
图3荧光倒置显微镜观察适配体抑制菌膜形成；
图4扫描电镜观察适配体抑制菌膜的形成。
具体实施方式
下面将结合本申请实施例中的附图，对本申请实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本申请一部分实施例，而不是全部的实施例。基于本申请中的实施例，本领域技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本申请保护的范围。
为了便于更好的理解本发明应用，下面将结合相关实例附图对本发明作进一步的解释，附图中给出了本发明装置。
实施例1：核酸适配体筛选
筛选初始文库设计为长度为78个碱基的随机ssDNA文库，两端分别为23个碱基和20个碱基的引物结合位点，中间为35个碱基的随机序列，文库容量约为435。文库序列为5'-GGGAGCTCAGAATAAACGCTCAA-N35-TTCGACATGAGGCCCGGATC-3'。同时设计了筛选过程对应的PCR引物，其中，上游引物P1：5'-GGGAGCTCAGAATAAACGCTCAA-3'和磷酸标记的下游引物P2：5'-PO4-GATCCGGGCCTCATGTCGAA-3'。用于ssDNA次级文库的制备。上述文库和引物均为上海生物工程技术服务有限公司合成纯化。
1.筛选过程各种溶液配制
(1)蛋白包被液：0.05mol/L的碳酸盐缓冲液，Na2CO31.59g，NaHCO32.93g，pH9.6，4℃保存(保存一个月)。
(2)电泳缓冲液(5×TBE缓冲液)：Tris 5.4g，硼酸2.75g，2ml0.5M EDTA溶液，加双蒸水溶解定容到100ml，调节pH至8.0后4℃冰箱中保存备用。
(3)2.5％琼脂糖凝胶：1.25g琼脂糖粉加入50ml1×TBE，微波炉煮沸溶解，取出后室温冷却至45℃左右加入2μl溴化乙溴化乙锭，搅拌均匀后倒胶，待胶凝固后使用。
(4)1×SELEX结合缓冲液(1×bindingbuffer)：20mmol/LHEPES(pH7.35)，120mmol/LNaCl，1mmol/LCaCl2，5mmol/LKCl，1mmol/LMgCl2,pH7.35。分别称取0.4766gHepes，0.7013gNaCl，0.0373gKCl，0.0147gCaCl2.2H2O以及0.0203gMgCl2.6H2O溶解于80mL灭菌纯水中，调节pH值调节至7.35，定容至100mL容量瓶后于4℃冰箱保存。
(5)PBS缓冲液(pH7.4)：称取NaCl8g，KCl0.2g，KH2PO40.24g，Na2HPO4˙12H2O3.63g，用超纯水将其定容至1000ml，调节pH至7.4。置于灭菌锅中高压蒸汽灭菌20min，室温条件下贮存备用。
(6)SELEX洗涤缓冲液：结合缓冲液+0.05％Tween20。
(7)SELEX洗脱缓冲液：20mmol/LTris-HCl(pH8.3),1mmol/LDTT,4mol/L异硫氰酸胍。
2.核酸适配体的筛选及高通量测序
(1)准备8个酶标板孔，加200μl的蛋白包被液，在加入PilA蛋白(第一轮10ug),用封孔膜封好，4℃包被过夜或37度包被两小时。再准备8个酶标板孔(反筛或者対照组)加入200μl的3％的BSA溶液，用封孔膜封好，4℃包被过夜或37℃包被两小时。
(2)装有PilA蛋白的8个酶标孔弃去包被液，用PBS洗涤2-5次，拍干后将200μl的3％的BSA溶液加入其中，用封孔膜封好，37℃恒温水浴箱孵育120min。(利用BSA封闭酶标孔未结合上PilA的位点)。
(3)将ssDNA文库(首轮用量为每孔2ug)溶于200μl的SELEX结合缓冲液中，置95℃中变性5min，而后立即置于冰上放置10min,使文库保存单链状态。(8孔即16ug，1600μl的SELEX结合缓冲液)
(4)对照组(反筛)的8个酶标孔用PBS洗涤4次，将维持单链状态的ssDNA加入到用PBS洗涤好的酶标板中，每个孔200μL，37℃孵育30-60min。
(5)将实验组(固定PilA蛋白)的8个孔用PBS洗涤2-5次，将第(4)步孵化后酶标板孔中的液体转移到实验组酶标板孔中，封孔膜封好，防止水分蒸发，每个孔200μl，37℃孵育30-60min。将上轮的液体吸出保存于EP管，以防失败。
(6)孵育后弃去上清液(上清液用EP管收集，以防实验失败，可以从这里开始重新孵育)。SELEX洗涤缓冲液洗涤2-5次。
(7)拍干后，加入200μl的SELEX洗脱缓冲液于95℃加热10min，用移液枪将上清液转入干净的EP管中。(加苯酚、异戊醇和氯仿复合物去除蛋白。取上清液)。
(8)加入上清液3倍体积的无水乙醇(预冷)和1/10体积的醋酸钠(3mol/LNaAC)于-80℃放置40min(或者-20℃静置30分钟以上)，12000r/min离心10min。
(9)去上清，将预冷的无水乙醇加入沉淀中，洗涤，12000r/min离心10min，去上清，重复上述步骤一次，开盖于室温中放置10min。
(10)待乙醇完全挥发后，加入20μL的TE缓冲液(灭菌)溶解DNA沉淀作为下一轮的PCR模板。
(11)以上一轮筛选得到的适配体为PCR模板DNA，进行PCR扩增以获得增强的次级文库。PCR所用的引物为上游引物P1及磷酸标记的下游引物P2。
(12)将PCR产物进行λ酶切。根据酶的说明说来换算酶和双链DNA的量，降解带磷酸根的那条单链，获得ssDNA次级文库。
(13)紫外分光光度计测ssDNA量确定下来一轮文库的浓度。
(14)将第12轮筛选的文库送至上海生工进行高通量测序。经高通量测序，测得序列共6856条，每条序列重复次数从1条至几万条不等，因为进行了多轮的筛选，所以相同序列重复率高。这使得PCR显著扩增了适配体库中的主要序列，降低了复杂性。PilA-1序列重复率达到38.59％，表明序列在测序样本中重复出现次数高，是在12轮筛选中保留下来的优势序列。
PilA-1：CACGGTCCAACGGTCGTTCGCATGCCTATCCACGG。
实施例2：核酸适配体亲和力分析
对适配体PilA-1进行FAM标记，在被PilA蛋白包被的96孔板中分别加入0-120nM浓度的适配体，37℃孵育40min。通过荧光仪测量荧光值，并结合Kd值计算公式进行亲和性分析。
适配体的亲和力检测结果如图2所示，荧光值在一定范围与适配体的浓度成正比关系，这说明适配体与PilA蛋白具有较高的亲和力。结合公式进行亲和性能的分析，得到适配体对PilA蛋白的Kd值，适配体PilA-1的Kd值为26.15±2.21nM，达到了nM级别，亲和力较理想。
实施例3：核酸适配体抑制铜绿假单胞菌菌膜形成
1.荧光倒置显微镜观察菌膜情况
如图3所示，通过荧光倒置显微镜直观评价适配体PilA-1对铜绿假单胞菌菌膜的干预情况。
⑴将干净的直径20mm的玻璃细胞生长盖玻片加至12孔细胞培养板中，取108CFU/mL铜绿假单胞菌以1％接种于1mL无菌LB培养基中，混匀；
⑵加入不同浓度梯度的适配体入细胞培养孔板混合(以结构中适配体成分最终浓度分别为0、0.25、0.5、1.0、2.5、5.0μM)，于37℃、摇床100r/min依次孵育24h、48h、72h；
⑶弃去孔板中液体，1mL固定剂处理10min，在遮光条件下，用SYTO9绿色荧光核酸染色剂对菌膜进行染色荧光倒置显微镜下镜检不同时间段内靶标菌菌膜生长情况。
2.扫描电镜观察适配体抑制菌膜的形成
如图4所示，通过扫描电镜直观评价适配体PilA-1对铜绿假单胞菌菌膜的干预情况。
⑴将干净的直径20mm的玻璃细胞生长盖玻片加至12孔细胞培养板中，取108CFU/mL铜绿假单胞菌以1％接种于2mL无菌LB培养基中，混匀；
⑵加入不同浓度梯度的适配体入细胞培养孔板混合(以结构中适配体成分最终浓度分别为0、0.25、0.5、1.0、2.5、5.0μM)，于37℃、摇床100r/min依次孵育24h、48h、72h；
⑶培养完成后，取出盖玻片用无菌水缓慢冲洗3次，自然风干，对样品进行喷金处理，用扫描电镜下观察不同时间段内靶标菌菌膜生长情况。
以上结合具体实施方式和范例性实例对本申请进行了详细说明，不过这些说明并不能理解为对本申请的限制。本领域技术人员理解，在不偏离本申请精神和范围的情况下，可以对本申请技术方案及其实施方式进行多种等价替换、修饰或改进，这些均落入本申请的范围内。本申请的保护范围以所附权利要求为准。
Claims (5)
1.铜绿假单胞菌IV型菌毛蛋白PilA的适配体PilA-1，其特征在于：其核酸序列为：PilA-1：5’-CACGGTCCAACGGTCGTTCGCATGCCTATCCACGG-3’。
2.如权利要求1的所述铜绿假单胞菌Ⅳ菌毛蛋白PilA核酸适配体PilA-1，其特征在于：适配体是以铜绿假单胞菌IV型菌毛中上段蛋白PilA为靶标的核酸适配体序列。
3.如权利要求1的所述铜绿假单胞菌Ⅳ菌毛蛋白PilA核酸适配体PilA-1在制备靶向治疗铜绿单胞菌感染药物中的用途。
4.如权利要求3所述的应用，其特征在于，所述药物为菌膜抑制药物。
5.如权利要求1的所述铜绿假单胞菌Ⅳ菌毛蛋白PilA核酸适配体PilA-1在制备检测铜绿假单胞菌的探针中的用途。
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