CN110563996B - 一种革兰氏阴性细菌群体感应淬灭聚合物 - Google Patents
一种革兰氏阴性细菌群体感应淬灭聚合物 Download PDFInfo
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Abstract
本发明涉及一种革兰氏阴性细菌群体感应淬灭聚合物，属于生物医药领域。主要技术方案如下：将模板分子与功能单体在乙腈中超声溶解，静置过夜；向模板分子与功能单体的混合物中加入交联剂和引发剂，聚合反应得到预聚合物；将预聚合物取出后，用去离子水清洗真空干燥，收集预聚物颗粒；将预聚物颗粒洗脱，洗脱后的预聚物颗粒冲洗，真空干燥即得到群体感应淬灭聚合物。本发明设计的革兰氏阴性细菌群体感应淬灭聚合物，可以实现群体淬灭，且制备简便，成本低。
Description
技术领域
本发明属于生物医药领域，具体的涉及一种革兰氏阴性细菌群体感应淬灭聚合物。
背景技术
近年来，抗生素的过度使用已成为日益严重的社会问题。患者无论所患疾病轻重与否，都会使用抗生素，这使得病原微生物的耐药性越来越强，因此急需寻找新的治疗策略。
细菌的群体感应(Quorum sensing,QS)是细菌细胞之间信息传递的特殊方法。细菌利用信号分子在细胞间的扩散来感知自身和周围环境中其它细菌的数量变化，当信号分子的浓度达到一定阈值时即可启动菌体中相关基因的表达，调控相关的生物学功能，如，毒力因子的产生、胞外多糖的合成、生物被膜的形成等。
以酰基高丝氨酸内酯(Acyl-homoserineLactones,AHLs)为信号分子的LuxR/I型群体感应系统广泛存在于革兰氏阴性菌中，包括多种临床致病菌，如铜绿假单胞菌(Pseudomonas aeruginosa)中的LasR/I系统和hlR/I系统、假结核耶尔森氏菌(Yersiniapseudotuberculosis)中的YtbR/I系统等。该系统由AHL分子、LuxI(自诱导物合成酶)和LuxR(自诱导物受体蛋白)组成。随着细胞密度的增大，LuxI诱导合成AHL信号分子并扩散至胞外，当其浓度达到一定的阈值，AHL即与LuxR的N-端区域结合，激活LuxR的C-端区域发生寡聚化，进而与下游相应的启动子结合，调控生物被膜的形成、毒力基因的表达等。至今已发现许多革兰氏阴性菌均能产生AHLs，它们的差别仅在于酰基侧链的长度或结构，但其作用方式基本相似。酰基高丝氨酸内酯的结构通式为
如能在菌体外，干扰AHLs信号分子与受体蛋白的结合，阻断LuxR/I型群体感应系统信号通路，则有望抑制革兰氏阴性病原菌毒力因子的表达，降低其致病性。这种调控方式即为群体淬灭(Quorum quenching)，群体淬灭并非杀菌或抑菌，而是通过干扰微生物的QS系统，阻止QS依赖型基因的表达进而防御病原菌的感染，因此可有效解决致病菌的耐药问题。
分子印迹的设想来源于天然“抗原-抗体”的特异性结合特征。当模板分子与功能单体接触时会形成多重作用点，通过聚合过程这种作用被记忆下来，当模板分子除去后，聚合物中就形成了与模板分子空间构型相匹配且具有多重作用位点的“空穴”，该聚合物称为分子印迹聚合物(MIPs)。
本发明以革兰氏阴性菌群体感应系统信号分子AHLs的结构类似物为模板分子，制备对革兰氏阴性细菌群体感应系统具有群体感应淬灭功能的分子印迹聚合物(Molecularimprinting polymers，MIPs)，即群体感应淬灭聚合物。MIPs中形成的“空穴”可对AHLs信号分子进行特异性的吸附，阻断其与受体蛋白的结合，从而实现群体淬灭。
发明内容
本发明以2-乙酰基-γ-丁内酯(A)为模板分子，丙烯酸(AA)或甲基丙烯酸甲酯(MMA)为功能单体，利用本体聚合法制备一系列革兰氏阴性细菌的群体感应淬灭聚合物。2-乙酰基-γ-丁内酯的结构式为：
本发明的技术方案如下：一种革兰氏阴性细菌群体感应淬灭聚合物，以2-乙酰基-γ-丁内酯为模板分子，丙烯酸或甲基丙烯酸甲酯为功能单体，制备而得。
进一步的制备方法如下：(1)将模板分子与功能单体在乙腈中超声溶解，4℃静置过夜；向模板分子与功能单体的混合物中加入交联剂和引发剂，60℃聚合反应24小时，得到预聚合物；
(2)将预聚合物取出后，用去离子水清洗6次，50℃条件下真空干燥12小时后，进行研磨过筛80目，收集预聚物颗粒；
(3)以甲醇和乙酸为洗脱剂，将预聚物颗粒在95℃下洗脱24小时，洗脱后的预聚物颗粒用甲醇冲洗，直至pH为7，再经真空干燥24小时至恒重即得到群体感应淬灭聚合物。
本发明的有益效果为：
(1)本发明利用分子印迹技术制备革兰氏阴性细菌群体感应系统淬灭聚合物具有较高的可行性，但很难直接以信号分子AHLs为模板分子。这是因为，革兰氏阴性细菌群体感应信号分子AHLs的价格极为昂贵，仅以市售的N-3-氧-己酰高丝氨酸内酯为例，50mg价格即达到近2000元。合成群体感应淬灭聚合物的成本过高。本发明选择了2-乙酰基-γ-丁内酯作为模板分子，主要原因有三点：第一、2-乙酰基-γ-丁内酯与AHLs具有极为相似的五元环内酯结构，这种结构在群体感应淬灭聚合物中留下的印迹，完全能够识别AHLs分子的五元环内酯结构，实现对其的特异性吸附。第二，2-乙酰基-γ-丁内酯分子的价格较低，能够满足大量合成的需要，生产成本较低。第三，2-乙酰基-γ-丁内酯分子与AHLs信号分子相比，分子中无长链脂肪烃尾部。以其为模板分子制备群体感应系统淬灭聚合物时，在洗脱模板分子，即在聚合物中留下印迹“空穴”这一步更为容易。避免因AHLs分子尾部过长，与聚合物发生缠绕，而难以洗脱的缺点。
(2)本发明设计的革兰氏阴性细菌群体感应淬灭聚合物，可以实现革兰氏阴性细菌的群体淬灭，且制备简便，成本低。
附图说明
图1 A-AA-MIPs扫描电镜图；
图2 AA-NIPs扫描电镜图；
图3 AA-NIPs与A-AA-MIPs红外光谱图；
图4 A-MMA-MIPs扫描电镜图；
图5 MMA-NIPs扫描电镜图；
图6 MMA-NIPs与A-MMA-MIPs红外光谱图；
图7粘质沙雷氏菌与群体感应淬灭聚合物共培养12小时颜色变化图(深色为红色)；
图8为本发明的制备过程示意图。
具体实施方式
下面结合具体实施例对本发明做进一步的说明，若无特殊说明，本发明所用原料及设备均为本领域的常规技术。
实施例1 A-AA-MIPs的制备
取108μL模板分子2-乙酰基-γ-丁内酯加入装有15mL乙腈的圆底烧瓶中溶解，再加入0.2882g功能单体丙烯酸。用封口膜将瓶口封住，超声8min，4℃冷却过夜。隔天，将圆底烧瓶放入50℃油浴锅中，预聚合反应4小时，随后向250mL的锥形瓶中加入交联剂3.950g乙二醇二甲基丙烯酸酯和20mL水，再将预聚物倒入锥形瓶中，加入0.050g偶氮二异丁腈和6mL乙醚，超声脱气20min。随后将锥形瓶密封后置于气浴恒温振荡器60℃聚合反应24小时。聚合结束后，用去离子水清洗聚合物6次，50℃真空干燥12小时。聚合物经研磨过筛(80目)，产物颗粒置于4℃保存。
将0.5g聚合物用滤纸包好放入索氏提取装置中，95℃下用甲醇和乙酸的混合溶液(9：1)洗脱24小时。洗脱后的聚合物用甲醇冲洗，直至pH＝7，再真空干燥24小时(50℃)至恒重即得到群体感应淬灭聚合物A-AA-MIPs。
图1A-AA-MIPs扫描电镜图：A-AA-MIPs颗粒形貌规整，粒径大小均匀，表面具有较多的脱除模板分子后形成的“空穴”，其直径约为100nm左右。
实施例2 AA-NIPs的制备
以实施例1相同方法制备AA-NIPs，制备过程中不加入模板分子A。
图2AA-NIPs扫描电镜图：AA-NIPs颗粒未形成“空穴”。
图3AA-NIPs与A-AA-MIPs红外光谱图：2953.14cm-1、2955.07cm-1、2954.11cm-1为-CH2-和-CH3的伸缩振动峰，1655.96cm-1附近的吸收峰应为未完全反应的C＝C振动吸收峰。其中，AA-NIPs C＝O的伸缩振动峰出现在1728.29cm-1处，而A-AA-MIPs C＝O的伸缩振动峰分别在1731.18cm-1和1732.15cm-1处，两者均向长波数方向移动，说明在形成聚合物的过程中，功能单体中的羰基与模板分子A发生了相互作用，致使羰基的特征伸缩频率发生了改变。此外，振动峰发生了红移，也说明在此处发生了氢键作用，这为A-AA-MIPs的特异识别性创造了条件。
实施例3 A-MMA-MIPs的制备
以实施例1相同方法制备A-MMA-MIPs，制备过程中加入功能单体为0.400g甲基丙烯酸甲酯。
图4A-MMA-MIPs扫描电镜图：A-MMA-MIPs颗粒形貌规整，粒径大小均匀，模板分子脱除后，在其中形成了众多直径约为200nm左右“空穴”。
实施例4 MMA-NIPs的制备
以实施例1相同方法制备MMA-NIPs，制备过程中不加入模板分子，功能单体为0.400g甲基丙烯酸甲酯。
图5MMA-NIPs扫描电镜图：MMA-NIPs颗粒中未形成“空穴”。
图6MMA-NIPs与A-MMA-MIPs红外光谱图：2954.11cm-1、2957.97cm-1、2955.07cm-1为-CH2-或-CH3的伸缩振动峰，1639cm-1附近的吸收峰应为未完全反应的C＝C振动吸收峰。其中，无模板分子的MMA-NIPs的C＝O伸缩振动峰出现在1728.29cm-1处。而在A-MMA-MIPs的红外光谱中，C＝O的伸缩振动峰分别在1731.18cm-1和1732.15cm-1处，两者均向长波数方向移动，说明在形成聚合物的过程中，功能单体中的羰基与模板分子A发生了相互作用，致使羰基的特征伸缩频率发生了改变。此外，振动峰发生了红移，也说明在此处发生了氢键作用，这为A-MMA-MIPs的特异识别性创造了条件。
实施例7 聚合物对粘质沙雷氏菌群体感应影响
LB培养基：胰蛋白胨10g，酵母粉5g，氯化钠10g加入去离子水定容至1升，120℃灭菌30min。
在超净工作台中，取一个灭菌后的24孔板，每孔加入100μL含有粘质沙雷氏菌的LB培养基。孔板第1～4列加入群体感应淬灭聚合物和空白聚合物，从左到右分别为：AA-NIPs、A-AA-MIPs、A-MMA-MIPs、MMA-NIPs。孔板A～D行加入2mg、4mg、6mg、0mg的聚合物，分别是。随后在30℃下振荡培养，12小时后测其OD600nm值及培养基的颜色变化。
粘质沙雷氏菌与两种群体感应淬灭聚合物及空白聚合物共培养12小时后菌体的OD600nm值列于表1。实验组与对照组的OD600nm基本保持一致，说明群体感应淬灭聚合物及空白聚合物均不影响菌体的正常生长。
表1粘质沙雷氏菌与群体感应淬灭聚合物共培养12小时OD600nm值。
粘质沙雷氏菌属于革兰氏阴性细菌，可产生红色灵杆素，该色素的生成受菌体中SpnI/SpnR群体感应系统的调控。将两种群体感应淬灭聚合物及空白聚合物添加到菌体中共培养12小时后(图7)，空白组菌体及添加空白聚合物组的菌体中都已有红色灵杆素的产生，而实验组红色灵杆素的产生受到明显抑制，且随着群体感应淬灭聚合物添加量的增加，菌液的颜色明显变浅，当添加量为6mg时，中有孔板边缘有少许红色出现。群体感应淬灭聚合物A-MMA-MIPs的群体淬灭能力要优于A-AA-MIPs。
以上结果表明群体感应淬灭聚合物能够在不影响菌体的正常生长的情况下，有效阻断革兰氏阴性细菌群体感应系统，实现群体淬灭。
Claims (1)
1.一种革兰氏阴性细菌群体感应淬灭聚合物，其特征在于，以2-乙酰基-γ-丁内酯为模板分子，丙烯酸或甲基丙烯酸甲酯为功能单体,制备而得；
所述聚合物的制备方法如下：
(1)将模板分子与功能单体在乙腈中超声溶解8min，4℃静置过夜；再将模板分子与功能单体的混合物在50℃温度下预聚合反应4小时，随后在所述混合物中加入交联剂和引发剂，超声脱气20min,60℃聚合反应24小时，得到预聚合物；
(2)将预聚合物取出后，用去离子水清洗6次，50℃条件下真空干燥12小时后，进行研磨过筛80目，收集预聚物颗粒；
(3)以甲醇和乙酸为洗脱剂，将预聚物颗粒在95℃下洗脱24小时，洗脱后的预聚物颗粒用甲醇冲洗，直至pH为7，再经真空干燥24小时至恒重即得到群体感应淬灭聚合物；
步骤(3)中所述甲醇与所述乙酸的体积比为9:1；所述真空干燥温度为50℃。
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