CN111647061A - 一种天蚕素f蛋白抗菌肽及其应用 - Google Patents
一种天蚕素f蛋白抗菌肽及其应用 Download PDFInfo
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Abstract
本发明公开了一种天蚕素F蛋白抗菌肽及其应用，所述天蚕素F蛋白抗菌肽的氨基酸序列如SEQ ID NO.1所示，其对应的基因的核酸序列如SEQ ID NO.2所示。本发明所述的天蚕素F蛋白抗菌肽经过试验验证对大肠杆菌和绿脓杆菌具有很强的抑制作用，可用于制备预防和抵抗细菌感染的伤口的修复的药物使用、以及美容相关行业中的预防和抵抗细菌感染的产品使用，还可以作为食品行业中抑制细菌的保鲜剂使用。
Description
【技术领域】
本发明属于生物医药技术领域，具体涉及一种天蚕素F蛋白抗菌肽及其应用。
【背景技术】
以前的研究表明抗菌肽对细菌、原虫、病毒均有抑制作用。抗菌肽是通过作用于细菌的细胞膜发挥抗菌作用，通过电荷吸附作用于细菌细胞膜上这一特殊机制，使得靶菌株不容易发生突变，因而在医药工业上认为有广阔的应用前景。
目前自然界发现的抗菌肽主要分为防御素(defens in)和天蚕素(cecropin)。天蚕素最早从果蝇基因组发现，后来在很多物种相继被发现。天蚕素一般含有37～39个氨基酸残基，不含半胱氨酸，其N端区域具有强碱性，可形成近乎完美的双亲螺旋结构，而在C端区域可形成疏水螺旋，两者之间有甘氨酸和脯氨酸形成的铰链区，多数多肽的C端被酰胺化，酰胺化对其抗菌活性具有重要作用。不同来源的天蚕素对应的抗菌谱有差异。
近年来，慢性伤口感染给患者和社会带来巨大的压力，细菌感染会进一步延迟创伤愈合，严重的会引起脓毒血症。由于耐药菌的出现，使得抗菌药物对于伤口感染的治疗效果差强人意，因此，寻找新型伤口抗感染药物迫在眉睫。天蚕素抗菌肽因其作用机制独特，不易产生耐药性等优点，将有望成为伤口感染治疗的药物之一。
【发明内容】
为了解决上述问题，本发明提供了一种天蚕素F蛋白抗菌肽，本发明的目的是通过以下方式来实现的：
本发明所述的天蚕素F蛋白(Cecropin F)来源于埃及伊蚊基因组，编号为AAEL000625，简称为CecF，总长为59个氨基酸，其氨基酸序列如下所示：
下划线部分为信号肽部分，发明人将该信号肽部分切除，剩下36个氨基酸，从而得到了本发明的天蚕素F蛋白抗菌肽，其氨基酸序列如下所示：
其基因的核酸序列如下所示：
其中，下划线部分为编码区(ORF),其他部分为非翻译区。
本发明还提供了一种所述的天蚕素F蛋白抗菌肽在制备药物中的应用，所述的药物用于预防和抵抗细菌感染的疾病。
本发明还提供了一种所述的天蚕素F蛋白抗菌肽在制备药物中的应用，所述的药物用于预防和抵抗细菌感染的伤口的修复。
优选地，所述细菌包括大肠杆菌和绿脓假单胞菌。
本发明还提供了一种所述的天蚕素F蛋白抗菌肽在制备预防和抵抗细菌感染的美容产品中的应用。
本发明还提供了一种所述的天蚕素F蛋白抗菌肽在制备抑制细菌的食品保鲜产品中的应用。
本发明还提供了一种所述的天蚕素F蛋白抗菌肽的合成方法，包括如下步骤：
S1、称取n当量树脂放入反应器，加入DCM(二氯甲烷)溶胀半小时，然后抽掉DCM，加入SEQ ID NO.1序列中第一个氨基酸2n当量，加2n当量的DIEA，适量的DMF，DCM(适量是指以可使树脂充分鼓动起来为宜)，DIEA (二异丙基乙胺)、DMF(二甲基甲酰胺)、DCM，氮气鼓泡反应60min。然后加入约5n当量甲醇，反应半小时，抽掉反应液，用DMF、MEOH洗净；
S2、往反应器中加入SEQ ID NO.1序列中第二个氨基酸(也为2n当量)，2n当量HBTU(1-羟基,苯并,三氯唑四甲基六氟磷酸盐)及DIEA，N2 鼓泡反应半小时，洗掉液体，茚三酮检测，然后用吡啶和乙酸酐封端。最后洗净，加入适量的脱帽液去除Fmoc(9-芴甲氧羰基)保护基，洗净，茚三酮检测；
S3、依步骤S2的方式依次加入SEQ ID NO.1序列中不同的氨基酸并进行各种修饰；
S4、将树脂用氮气吹干后从反应柱中取下，倒入烧瓶中，然后往烧瓶中加一定量(切割液和树脂大约以10ml/克的比例)的切割液(组成是95％TFA， 2％乙二硫醇，2％三异丙基硅烷，1％水)，震荡，滤掉树脂，得到滤液；
S5、向S4的滤液中加入大量乙醚，析出粗产物，然后离心，清洗即可得到序列的粗产物；
S6、用高效液相色谱法将S5的粗品提纯至纯度为90.07％。
S7、将S6中纯化好的液体放入冻干机中进行浓缩，冻干成白色粉末，即得到本发明所述的天蚕素F蛋白抗菌肽。
与现有技术相比，本发明具有如下有益效果：本发明的天蚕素F蛋白抗菌肽是一种来源于埃及伊蚊基因组的天蚕素F蛋白，通过试验验证，含有本发明所述的天蚕素F蛋白抗菌肽的药物或制剂对大肠杆菌和绿脓杆菌具有很强的抑制作用。大肠杆菌是临床上的一个机会感染病原菌，在环境中分布广泛，在手术的时候容易感染；绿脓杆菌是一个临床上严重的致病菌，而且这种菌很容易产生耐药性，临床上已经出现多重耐药菌，感染之后会化脓，如果不及时治疗可能会致死。利用本发明的天蚕素F蛋白抗菌肽的药物，一是可以预防和治疗大肠杆菌和绿脓假单胞菌感染，减少抗生素的使用；二是可以防止耐药细菌的产生；三是可以促进伤口愈合；四是可以作为预防和抵抗细菌感染的美容护肤相关的产品使用；五是在食品保鲜行业中可以作为保鲜的抑菌剂使用。
【附图说明】
图1是本发明不同浓度的CecF抗菌肽溶液对大肠杆菌的抑制活性。
图2是本发明CecF抗菌肽抑制大肠杆菌繁殖的最低有效浓度的影响。
图3是本发明CecF抗菌肽抑制绿脓杆菌繁殖的最低有效浓度的影响。
图4是本发明对进行伤口愈合试验的试验组和对照组的小鼠制造伤口。
图5是本发明CecF抗菌肽对小鼠伤口愈合的影响。
图6是本发明对进行伤口细菌感染试验的试验组和对照组的小鼠制造伤口。
图7是本发明伤口感染上细菌的试验组和对照组的小鼠。
图8是本发明CecF抗菌肽对小鼠伤口感染细菌的愈合的影响。
【具体实施方式】
结合以下实施例对本发明的原理和特征进行描述，所举实施例只用于解释本发明，并非用于限定本发明的范围。下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的试验材料，如无特殊说明，均为自常规生化试剂商店购买得到的。以下实施例中的定量试验，均设置三次重复实验，结果取平均值。
实施例1天蚕素F蛋白抗菌肽的合成
S1、称取1倍摩尔当量树脂放入反应器，加入DCM(二氯甲烷)溶胀半小时，然后抽掉DCM，加入SEQ ID NO.1序列中第一个氨基酸2倍摩尔当量，加2倍摩尔当量的DIEA，适量的DMF，DCM(适量是指以可使树脂充分鼓动起来为宜)，DIEA(二异丙基乙胺)、DMF(二甲基甲酰胺)、DCM，氮气鼓泡反应60min。然后加入约5倍摩尔当量甲醇，反应半小时，抽掉反应液，用DMF、MEOH洗净；
S2、往反应器中加入SEQ ID NO.1序列中第二个氨基酸(也为2倍摩尔当量)，2倍摩尔当量HBTU(1-羟基,苯并,三氯唑四甲基六氟磷酸盐) 及DIEA，N2鼓泡反应半小时，洗掉液体，茚三酮检测，然后用吡啶和乙酸酐封端。最后洗净，加入适量的脱帽液去除Fmoc(9-芴甲氧羰基)保护基，洗净，茚三酮检测；
S3、依步骤S2的方式依次加入SEQ ID NO.1序列中不同的氨基酸并进行各种修饰；
S4、将树脂用氮气吹干后从反应柱中取下，倒入烧瓶中，然后往烧瓶中加一定量(切割液和树脂大约以10ml/克的比例)的切割液(组成是95％TFA， 2％乙二硫醇，2％三异丙基硅烷，1％水)，震荡，滤掉树脂，得到滤液；
S5、向S4的滤液中加入大量乙醚，析出粗产物，然后离心，清洗即可得到SEQ IDNO.1序列的粗产物；
S6、用高效液相色谱法将S5的粗品提纯至纯度为90.07％。
S7、将S6中纯化好的液体放入冻干机中进行浓缩，冻干成白色粉末，即得到本发明所述的天蚕素F蛋白抗菌肽。
实施例2 CecF抗菌肽对大肠杆菌和绿脓杆菌繁殖的影响
一、CecF抗菌肽溶液的制备
将实施例1中合成的0.5mg CecF多肽溶于500ul无菌水，配成1.8 mg/ml和0.18mg/ml的溶液，置于-80冰箱备用。
二、细菌活化：取100ul大肠杆菌菌液涂布于无抗的LB(Luria-Bertani) 平板，置于37℃培养箱，培养12小时。
三、CecF抗菌肽抑制细菌生长的应用
(1)取100ul制备好的CecF抗菌肽溶液，在上述活化好的平板上挑取一个单克隆置于抗菌肽溶液混匀，然后涂布于无抗的LB平板。37℃培养12小时，然后取出观察。
(2)结果显示：1.8mg/ml和0.18mg/ml的的CecF抗菌肽溶液对大肠杆菌有明显抑制作用。(见图1)
四、CecF最低抑菌浓度的测定
(1)合成CecF抗菌肽500ug，纯度90.07％，加入125ul无菌水溶解，配成母液浓度为3.6ug/ml。按照10倍梯度稀释，依次为3.6， 0.9,0.225,0.05625,0.0140625，0.003515625，0.00087890625， 0.0002197265325，0.000054931633125，顺次编号为1,2,3,4,5,6,7,8,9，mock为不加蛋白。
(2)将大肠杆菌和绿脓杆菌提前一天活化涂布于LB无抗平板。挑取单克隆于无菌水，稀释10000倍。
(3)取30ul稀释好的多肽溶液和30ul菌液混匀，滴于无抗LB平板。 37℃培养12小时，然后取出观察。
(4)结果显示：CecF抗菌肽抑制大肠杆菌繁殖的最低有效浓度约为0.44 ug/ml,抑制绿脓杆菌繁殖的最低有效浓度约为1.8ug/ml。(见图2，图3)
实施例3 CecF抗菌肽对小鼠伤口的愈合的影响
一、CecF抗菌肽溶液的制备
将实施例1中合成的0.5mgCecF抗菌肽溶于2ml无菌水，配成400ug/ml和10ug/ml的溶液，置于-80冰箱备用。
二、CecF抗菌肽促进伤口愈合的应用
(1)麻醉：选择6-8周龄的昆明鼠，称取体重，根据25g/100ul腹腔注射水合氯醛溶液(10％)。
(2)剪毛和脱毛：待小鼠麻醉后，将其固定在实验台上，用剪刀剪掉小鼠背部及两侧的毛，在剪毛区域均匀涂抹脱毛液，等待约3-5分钟，用蘸有温水的棉棒轻轻擦拭皮肤，彻底清除脱毛液和毛发。
(3)制造伤口：先用碘伏和75％乙醇溶液对背部皮肤进行消毒处理，用消毒的剪刀和镊子在背部剪出大小约5cm的圆形伤口。(见图4)
(4)记录伤口大小和治疗：手机拍照记录伤口大小，之后再次对伤口边缘的皮肤消毒，以免发生感染。将制造好伤口的小鼠分为对照组和实验组，每组各一只；对照组只制造伤口看伤口的愈合情况；实验组制造完伤口后在伤口处加入10ug/ml的CecF蛋白400ul，之后每天加一次CecF蛋白，且每天拍照记录两组的伤口愈合情况。
(5)结果显示：每天在伤口处加入400ul,10ug/mlCecF的蛋白有利于伤口的愈合。(见图5)
三、CecF抗菌肽在伤口感染细菌后对伤口的影响：
(1)麻醉：选择6-8周龄的昆明鼠，称取体重，根据25g/100ul腹腔注射水合氯醛溶液(10％)。
(2)剪毛和脱毛：待小鼠麻醉后，将其固定在实验台上，用剪刀剪掉小鼠背部及两侧的毛，在剪毛区域均匀涂抹脱毛液，等待约3-5分钟，用蘸有温水的棉棒轻轻擦拭皮肤，彻底清除脱毛液和毛发。
(3)制造伤口：先用碘伏和75％乙醇溶液对背部皮肤进行消毒处理，用消毒的剪刀和镊子在背部剪出大小约5cm的圆形伤口。(见图6)
(4)记录伤口大小和治疗：手机拍照记录伤口大小，之后再次对伤口边缘的皮肤消毒，以免发生感染。之后在伤口处涂抹上绿脓杆菌。第二天将伤口感染上细菌的小鼠挑出来并分组，将其分为对照组和实验组，每组各一只(见图7)；对照组小鼠伤口感染后不做任何处理每天观察伤口的愈合情况；实验组小鼠伤口感染后在伤口处加入10ug/ml的CecF抗菌肽400ul，之后每天加一次CecF抗菌肽，且每天拍照记录两组的伤口愈合情况。
(5)结果显示：小鼠伤口感染细菌后,通过每天滴加CecF抗菌肽溶液可加快伤口的愈合。(见图8)
以上所述，仅为本发明较佳的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，可轻易想到的变化或替换，都应涵盖在本发明的保护范围之内。
Claims (6)
1.一种天蚕素F蛋白抗菌肽，其特征在于，所述的天蚕素F蛋白抗菌肽氨基酸序列如SEQID NO.1所示，其基因的核酸序列如SEQ ID NO.2所示。
2.根据权利要求1所述的天蚕素F蛋白抗菌肽在制备药物中的应用，所述的药物用于预防和抵抗细菌感染的疾病。
3.根据权利要求1所述的天蚕素F蛋白抗菌肽在制备药物中的应用，所述的药物用于预防和抵抗细菌感染的伤口的修复。
4.根据权利要求2或3中所述的天蚕素F蛋白抗菌肽在制备药物中的应用，其中所述细菌包括大肠杆菌和绿脓假单胞菌。
5.根据权利要求1所述的天蚕素F蛋白抗菌肽在制备预防和抵抗细菌感染的美容产品中的应用。
6.根据权利要求1所述的天蚕素F蛋白抗菌肽在制备抑制细菌的食品保鲜产品中的应用。
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