CN106818746B - N15多肽在制备蓝藻抑制剂中的用途 - Google Patents
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Abstract
本发明公开了N15多肽在制备蓝藻抑制剂中的用途,N15多肽是一个具有15个氨基酸的短肽,它具有多种生物学功能,包括调节NF‑κB转录因子结合目的基因的能力,影响NF‑κB目的基因产物蛋白质的转录和表达;阻断PCNA结合DNA聚合酶,从而阻止细胞DNA合成抑制细胞增殖;抑制上皮细胞增生,调节表皮增生及分化,使皮肤角化过程正常化等,本发明阐述了它新的生物学功能,具有较好的实用性和产业化前景。
Description
技术领域
本发明涉及生物技术领域,具体指N15多肽在制备蓝藻抑制剂中的用途。
背景技术
蓝藻之所以又称蓝细菌,是因为蓝藻是最原始、最古老的藻类. 其结构简单,无典型的细胞核,所以又称蓝细菌,属于原核生物。已知蓝藻约2000种,中国已有记录的约900种。蓝藻有极大的适应性,分布很广。在一些营养丰富的水体中,有些蓝藻常于夏季大量繁殖,并在水面形成一层蓝绿色而有腥臭味的浮沫,称为“水华”,大规模的蓝藻爆发,被称为“绿潮”(和海洋发生的赤潮对应)。绿潮引起水质恶化,严重时耗尽水中氧气而造成鱼类的死亡。
发明内容
本发明的目的在于提供一种多肽,该多肽能够抑制蓝藻生长,具有在制备细菌抑制剂中的用途,它已被现有技术公开,名为N15 多肽。
N15多肽是一个具有15个氨基酸的短肽,其序列为甲硫氨酸- 脯氨酸-酪氨酸-丝氨酸-苏氨酸-谷氨酸-亮氨酸-异亮氨酸-苯丙氨酸 -酪氨酸-异亮氨酸-谷氨酸-甲硫氨酸-天门冬氨酸-脯氨酸,它具有多种生物学功能,包括调节NF-κB转录因子结合目的基因的能力,影响NF-κB目的基因产物蛋白质的转录和表达;阻断PCNA结合 DNA聚合酶,从而阻止细胞DNA合成抑制细胞增殖;抑制上皮细胞增生,调节表皮增生及分化,使皮肤角化过程正常化等。
发明人发现在研究中发现N15可以抑制革兰氏阴性菌(大肠杆菌)、蓝细菌的生长,N15多肽能够对各种大肠杆菌和蓝藻的生长产生抑制作用。N15多肽抑制蓝藻增殖可能是通过抑制DNA复制和异形胞分化基因的表达,起到抑制蓝藻增殖的。
本发明还提供了一种N15多肽制备的抗蓝藻药物,为N15多肽的水溶液,浓度大于等于100μg/mL。
本发明的有益效果:N15多肽是一种结构清晰,易于工业化生产的多肽,它可以抑制蓝藻的增殖,效果好且无毒副作用。
附图说明
图1为N15对鱼腥蓝细菌PCC 7120的抑菌曲线。
图2为缺氮诱导48小时对照组和N15实验组鱼腥蓝细菌PCC 7120异形胞的分化情况。
图3为荧光定量RT-PCR检测N15对蓝藻hetR基因mRNA表达量的影响。
图4为荧光定量RT-PCR检测N15对蓝藻ntcA基因mRNA表达量的影响。
具体实施方式
下面结合附图和具体实验例对本发明作进一步的论证说明,以下实验例仅是对本发明的解释而本发明并不局限于以下实验例。
实验例1:N15抑制了鱼腥蓝细菌的生长
实验方法:
将鱼腥蓝细菌PCC 7120在液体BG11培养基中培养至OD750 (波长750nm处的吸光度)在0.3~0.4左右,添加N15至终浓度为 100μg/mL,同时设置不添加N15的菌株作为对照。每隔24小时用分光光度计测定菌液的OD750,连续测定8天后根据数据绘制N15 的抑菌曲线,结果如图1所示。
实验结果表明,加入N15多肽至终浓度为100ug/mL的鱼腥蓝细菌PCC 7120的生长和不加入N15的对照菌株比较,受N15多肽抑制鱼腥蓝细菌的生长较为缓慢,加入N15对鱼腥蓝细菌PCC 7120 的生长具有抑制作用。
实验例2:N15推迟了鱼腥蓝细菌的异形胞分化时间
实验方法:
鱼腥蓝细菌PCC 7120是一种丝状细菌,其菌丝体能进行光合作用。在环境中缺乏氮源的条件下,会分化出具有固氮功能的异形胞。为了观察N15处理对异形胞发育的影响,本研究中在对鱼腥蓝细菌进行缺氮处理的同时,加入100ug/mL的N15,同时设置不加入N15的对照组,诱导后进行显微镜观察。
实验结果:
鱼腥蓝细菌PCC 7120的营养细胞在形成异形胞的过程中,产氧的光系统II复合物在异形胞中失活,因此在荧光显微镜下,只有营养细胞能够发红色荧光,而异形胞没有荧光。在进行缺氮诱导的同时加入100μg/mL的N15,分别在24小时、48小时、72小时、 96小时进行显微镜观察,结果显示对照组的鱼腥蓝细菌在缺氮诱导 24小时就有异形胞的产生,而加入N15的鱼腥蓝细菌直到96小时以后才有异形胞生成,缺氮诱导48小时的照片如图2所示,加入 N15的鱼腥蓝细菌PCC 7120没有异形胞产生,菌丝体的所有细胞在荧光显微镜下都发红色荧光,而对照组菌丝有异形胞产生,这些异形胞在荧光显微镜下也没有红色荧光。由此我们得知,N15推迟了鱼腥蓝细菌PCC7120的异形胞发育生成的时间。
实验例3:N15抑制了异形胞分化基因的表达
实验方法:
为了观察N15处理对异形胞发育的影响,本研究中在对鱼腥蓝细菌进行缺氮处理的同时,加入100μg/mL的N15,同时设置不加入N15的对照组,分别提取0hour、6hour、12hour、24hour的对照组和实验组,每个样品收集50mL菌体,提取RNA,并且用DNasel 去除残余的基因组DNA。利用紫外分光光度计对总RNA定量后, 使用TakaRa公司的反转录试剂盒反转录RNA合成cDNA。具体操作步骤参见产品说明书。用TakaRa公司的试剂盒PrimeScriptTM RT Reagent Kit(Perfect Real Time)进行实时荧光定量反转录PCR。具体操作参见产品说明书。分析软件使用ROCHE LightCycler480 进行分析,定量方法为相对定量法。
实验结果:
异形胞在发育过程中需要在许多基因的参与,其中hetR和ntcA 基因是异形胞发育的关键基因,与异形胞发育的多个后期基因的转录激活依赖于hetR和ntcA。我们通过实时荧光定量反转录PCR实验检测了N15对这几个异形胞分化相关基因表达的影响,实验结果如图3~4所示。
和对照组相比,N15加入6hour、12hour、24hour后,较明显的抑制了鱼腥蓝细菌中hetR和ntcA基因的表达,由此我们得知 N15对蓝藻生长的抑制是通过抑制蓝细菌中异形胞分化相关基因的表达来实现的。
Claims (2)
1.N15多肽在制备蓝藻抑制剂中的用途;所述N15多肽是一个具有15个氨基酸的短肽,其序列为甲硫氨酸-脯氨酸-酪氨酸-丝氨酸-苏氨酸-谷氨酸-亮氨酸-异亮氨酸-苯丙氨酸-酪氨酸-异亮氨酸-谷氨酸-甲硫氨酸-天门冬氨酸-脯氨酸。
2.一种N15多肽制备的抗蓝藻药物,其特征在于:所述抗蓝藻药物为N15多肽的水溶液,浓度大于等于100μg/mL;所述N15多肽是一个具有15个氨基酸的短肽,其序列为甲硫氨酸-脯氨酸-酪氨酸-丝氨酸-苏氨酸-谷氨酸-亮氨酸-异亮氨酸-苯丙氨酸-酪氨酸-异亮氨酸-谷氨酸-甲硫氨酸-天门冬氨酸-脯氨酸。
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