CN110606871B - 一种小分子多肽rk12及其应用 - Google Patents
一种小分子多肽rk12及其应用 Download PDFInfo
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Abstract
本发明公开了一种小分子多肽RK12，其分子量为1901.30Da、等电点为12.02，其氨基酸序列如SEQ ID:1所示。本发明还公开了一种小分子多肽RK12在制备治疗鲍曼不动杆菌感染药物中的应用。本发明中的多肽RK12分子量小、人工合成方便，对鲍曼不动杆菌的抑菌效果明显。
Description
技术领域
本发明属于生物医学技术领域，具体涉及一种小分子多肽RK12及其在制备抗鲍曼不动杆菌感染药物中的应用。
背景技术
抗菌肽是生物体内经诱导产生的一种具有生物活性的小分子多肽，分子量在1000～7000左右，由10～60个氨基酸残基组成。抗菌肽是当生物受到微生物侵入后机体迅速产生的高效、光谱的抗菌肽类分子，来参与机体的免疫反应。抗菌肽作为机体有效的防御分子是广泛存在的，目前已从微生物、植物、昆虫、节肢动物、两栖动物、哺乳动物甚至人体内鉴定出上千种抗菌肽。抗菌肽抗菌机理复杂，但大多数理论都认为其机制涉及到抗菌肽的阳离子性和疏水性与带负电荷的微生物胞膜的作用，抗菌肽和细菌的细胞膜接触后，引起膜通透性改变，或在细菌细胞膜上形成跨膜的孔洞，最后导致细菌内容物外泄而死亡。因此，抗菌肽在杀灭细菌的速度上要远远高于传统的抗生素，且不像抗生素在低浓度下抑制细菌的生长，抗菌肽对细菌的作用几乎都是致死性的。研究表明：抗菌肽与传统抗生素相比，不易诱导耐药菌株的产生；抗菌谱光，对细菌、真菌、病毒、原虫及癌细胞均有作用。已经在家禽体内发现了3个家族的抗菌肽，包括cathelicidin，liver-expressed antimicrobialpeptide(LEAP)，andβ-defensin。这些抗菌肽对于家禽抵抗细菌性、病毒性疾病具有至关重要的作用，相关这些基因的突变或者缺失对于家禽抗微生物感染的能力将有显著的影响；这些抗菌肽除了具有光谱抗菌活性，同时还有高效的抗真菌、抗病毒、抗原虫和(或)抗肿瘤活性，如Bat5和IMc7能杀灭钩端螺旋体，对白色念珠菌、隐球菌和胞膜病毒和寄生虫都有较好的杀灭作用；一些抗菌肽对疱疹病毒、流感病毒、艾滋病病毒有囊膜病毒有明显的杀伤力；此外，有些抗菌肽同时还具有多种其他的调控功能，如Cathelicidin还具有促进伤口愈合、组织损伤修复、化学趋化作用、促血管生成和抗寄生虫等功能，在调节动物机体免疫上有着重要生物学活性。
发明内容
本发明的目的在于克服现有抗生素长期使用后产生耐药菌技术的缺点，提供一种小分子多肽RK12能治疗临床上耐药的鲍曼不动杆菌；同时RK12具有氨基酸数目少、易合成、生产成本低的特点。
本发明的另一个目的在于提供一种小分子多肽RK12在制备抗鲍曼不动杆菌感染药物中的应用。
本发明的目的通过以下技术方案来实现。
小分子多肽RK12，其特征在于，其分子量为1901.30Da、等电点为12.02，其氨基酸序列如SEQ ID NO.1所示。
作为优选技术方案，所述的小分子多肽RK12采用Fmoc固相合成法制备而成。
上述小分子多肽RK12在制备治疗鲍曼不动杆菌感染药物中的应用。
一种药物组合物，其包含有效成分以及药学上可接受的载体和辅料，其特征在于所述有效成分含有上述RK12多肽。
作为优选技术方案，所述药物组合物的制剂类型为溶液型、胶体溶液型、乳剂型或混悬型。
本发明的有益效果是：
本发明中的多肽RK12为人工合成，具有分子量小、人工合成方便、抑菌效果明显的特点，能够应用于制备抗鲍曼不动杆菌感染药物中，具有较好的市场前景。
附图说明
图1为鲍曼不动杆菌刺激小鼠并采用RK12治疗后主要脏器的载菌量对比图；
图2为RK12治疗后小鼠血液中炎症因子的含量对比图。
具体实施方式
下面结合具体实施方式对本发明做进一步的说明，需要指出的是以下实施方式仅是以例举的形式对本发明所做的解释性说明，但本发明的保护范围并不仅限于此，所有本领域的技术人员以本发明的精神对本发明所做的等效的替换均落入本发明的保护范围。
实施例1
小分子多肽RK12的制备采用Fmoc固相合成法，具体步骤如下。
1.称取树脂0.2g放置于干燥洁净的反应管中，加入适量的N,N-二甲基酰胺(DMF)，活化30分钟，将称取第一氨基酸残基1mmol，DMAP 150mg加入到反应管中，DMF做为溶剂反应3小时；反应完毕后用DMF洗3～6次，加入适当的吡啶和乙酸酐，体积比为1:1，反应30min；反应完毕后DMF洗3～6次；然后用哌啶洗脱氨基酸的保护基团Fmoc，洗脱两次每次15min，再用DMF洗4次，甲醇洗2次。
2.称第二个氨基酸3mmol，HBTU 3mmol于反应管中，加入DIEA 0.5ml，反应40分钟，用DMF洗3～6次，加入哌啶溶液两次洗脱氨基酸的保护基团Fmoc，每次10分钟，再用DMF洗4次，甲醇洗2次。
3.重复第二个步骤直到最后一个氨基酸残基。
4.最后一个氨基酸反应完毕后，用三氟乙酸切割2小时，反应抽滤，得到多肽的三氟乙酸溶液，用***沉淀，离心，再用***洗3～5遍，得到白色固体，经过HPLC脱盐冻干得到多肽样本。
实施例2
小分子多肽RK12的抗菌实验
1.制备鲍曼不动杆菌菌液，37℃恒温培养18h，备用；
2.配制浓度为10mg/ml小分子多肽RK12溶液，将直径为5～7mm的圆形定性滤片消毒烘干后浸入上述不同浓度的小分子多肽RK12溶液中；
3.制备LB培养基，灭菌，备用；
4.溶化LB培养基，冷却至50℃，分别加入鲍曼不动杆菌菌液1ml，轻摇均匀，倒入无菌平皿，轻晃使培养基均匀平铺在平皿上；
5.用消毒过的镊子将滤片整齐、有序的放入冷却的平皿内，盖上陶瓦盖，并做标记，置于37℃恒温培养箱内培养24h；
6.用卡尺测量抑菌圈大小，并比较不同浓度小分子多肽RK12对不同来源的鲍曼不动杆菌(中南大学湘雅医院提供)的作用强度。
表1 小分子多肽RK12对不同来源的鲍曼不动杆菌的抑菌效果
由表1可知，小分子多肽RK12对不同来源的鲍曼不动杆菌的最低抑菌浓度分别为10μg/ml、8.5μg/ml、7.5μg/ml、20μg/ml。
实施例3
小分子多肽RK12在动物模型上的治疗效果
1)模型构建
将鲍曼不动杆菌用LB培养基培养至对数期，之后3000rpm离心5min，用无菌生理盐水洗涤3次，最后用生理盐水重悬，分别配制成1×107CFU/ml的菌悬液，置于4℃冰箱备用。
***性感染鲍曼不动杆菌小鼠动物模型实验选用雄性昆明小鼠，共分为7组，每组6只，每组注射1×107CFU/ml的鲍曼不动杆菌菌悬液，分组如下：1、生理盐水对照组，RK12(1,2,4mg/kg)和多粘菌素E(0.25,0.5,1mg/kg)治组。在接菌之前首先要构建免疫抑制小鼠模型，构建方法如下：
无菌生理盐水配制20mg/ml的环磷酰胺溶液，每天每只小鼠腹腔注射0.2ml环磷酰胺溶液，连续免疫4d，从免疫结束的第二天开始通过尾静脉注射的方式按照分组设计注射不同剂量的鲍曼不动杆菌，给菌刺激后密切观察小鼠的行为和健康情况，如有小鼠死亡，注意记录小鼠的死亡率。鲍曼不动杆菌注射2h后，通过腹腔注射给予生理盐水，RK12和多粘菌素。
2)实验小鼠主要脏器的载菌量检测
48h后处死小鼠并在无菌条件下取出不同分组小鼠的肝脏、脾脏、肺，取一部分组织器官使用电子天平称重，按照1:3(m/v，器官质量/生理盐水体积)的质量体积比例在无菌组织研磨器中进行研磨，后将组织研磨液用无菌生理盐水进行10倍梯度的稀释，最后吸取50μl稀释后的组织研磨液涂在LB固体平板上，倒置于37℃恒温培养箱中培养18h，进行菌落计数，检测结果见图1。
3)实验小鼠血浆中相关炎症因子的检测
小鼠血浆的制备：摘除小鼠眼球，将血液滴到含有抗凝剂的EP管中，1000rpm离心5min，吸取上清液，转移到无菌离心管中，-20℃冻存备用。
相关炎症因子的检测：本实验检测小鼠血浆中IL-6、IL-10、IL-1β和TNF-α的含量变化，采用ELISA检测的方法，检测结果如图2所示。所有试剂盒由达科为生物技术公司提供。
酶联免疫吸附试验(ELISA)是将已知的抗体或者抗原结合在某种固相载体上，并保持其免疫活性。检测时，将待测标本与酶标抗原或者抗体与固相载体上的抗体或者抗原结合，用洗涤的方法将游离的成分洗掉，然后加入酶的作用底物催化显色，进行定性或定量测定。实验步骤主要参考试剂盒说明书，主要实验步骤如下：
标准品的配制：用抗体稀释液将各细胞因子标准品按梯度稀释到一定的浓度，实验中所用标准品的梯度浓度为1000pg/ml、500pg/ml、250pg/ml、125pg/ml、62.5pg/ml、31.3pg/ml、15.6pg/ml。
加样：将稀释后的细胞因子标准品及小鼠血浆样品加入到检测孔中，注意设置空白对照，每孔加入100μl。
加检测抗体：取50μl稀释后的生物素化一抗抗体工作液加入到检测孔中，盖上封板膜，37℃恒温孵育90min。
洗板：扣去板孔内的液体，每孔加入250μl洗涤液，停留3min后弃去板孔内的液体，在滤纸上将残留的液体拍干，重复4次。
加酶：每孔加入100μl辣根过氧化物酶标记的亲和素，盖上封板膜，37℃恒温孵育30min。
显色：洗涤后每孔加入100μl TMB显色液，37℃避光孵育10～30min。
终止：每孔加入100μl 2M的H2SO4终止液，终止反应。
读板：终止反应10min内检测450nm处吸光度值。
4)检测结果分析
由图1可知，经RK12和多粘菌素E治疗后，小鼠体内个器官的鲍曼不动杆菌的数目均明显下降，表示多肽RK12对小鼠模型具有很好的治疗效果。
由图2可知，在小鼠的正常生理状态下，IL-10的浓度为218pg/ml，经鲍曼不动杆菌感染后IL-10的浓度升高到1500pg/ml。经RK12-1(1,2,4mg/kg)和多粘菌素E(0.25,0.5,1mg/kg)治疗后，IL-10的浓度均出现不同程度的下降，与对照组相比均有明显的差异。TNF-α在生理状态下的浓度为48pg/ml，小鼠经鲍曼不动杆菌感染后TNF-α的含量急剧增加，达到116pg/ml。经RK12(1，2，4mg/kg)和多粘菌素E(0.25,0.5,1mg/kg)治疗后，TNF-α的浓度均出现不同程度的下降，与对照组相比均有明显的差异。以上结果说明了多肽RK12在体内可以通过控制上述四种炎症因子的释放来控制***性感染鲍曼不动杆菌的小鼠的炎症反应水平，同时也说明了RK12在体内具有很好的抗炎症作用，为RK12作为抗深部真菌感染药物的开发奠定了基础。
序列表
<110> 湖南生达生物科技有限公司
<120> 一种小分子多肽RK12及其应用
<130> 2019
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 12
<212> PRT
<213> 人工序列()
<400> 1
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Claims (5)
1.小分子多肽RK12，其特征在于，其分子量为1901.30 Da、等电点为12.02，其氨基酸序列如SEQ ID NO.1所示。
2.如权利要求1所述的小分子多肽RK12，其特征在于其采用Fmoc固相合成法制备而成。
3.一种如权利要求1所述小分子多肽RK12在制备治疗鲍曼不动杆菌感染药物中的应用。
4.一种药物组合物，其包含有效成分以及药学上可接受的载体和辅料，其特征在于所述有效成分含有如权利要求1所述多肽。
5.如权利要求4所述的药物组合物，其特征在于所述药物组合物的制剂类型为溶液型、乳剂型或混悬型。
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