CN116410329A - 一种c末端修饰的杂合抗菌蛋白及其药物组合物和应用 - Google Patents
一种c末端修饰的杂合抗菌蛋白及其药物组合物和应用 Download PDFInfo
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- CN116410329A CN116410329A CN202111659134.0A CN202111659134A CN116410329A CN 116410329 A CN116410329 A CN 116410329A CN 202111659134 A CN202111659134 A CN 202111659134A CN 116410329 A CN116410329 A CN 116410329A
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Abstract
本发明提供了一种C末端修饰的抑菌杂合蛋白,具有式(Ⅰ)所示氨基酸序列:X‑Y(Ⅰ);其中,X为被修饰的杂合抗菌蛋白,包括AB469杂合抗菌蛋白或其类似物;Y为X的C末端修饰体,所述C末端修饰体包括含有3‑10个精氨酸残基的多肽。本发明所述C末端修饰的杂合抗菌蛋白,通过在C末端修饰含精氨酸残基的螺旋状两性多肽,显著提升杂合抗菌蛋白对革兰氏阴性菌细胞外膜的透过性,显著提升对革兰氏阴性菌的杀菌效果。
Description
技术领域
本发明涉及药物技术领域,尤其涉及一种C末端修饰的杂合抗菌蛋白及其药物组合物和应用。
背景技术
细菌耐药性问题已经严重威胁到人用抗生素资源与公共卫生安全。动物养殖场的污水、粪便,农场的土壤、水源、粮食、水产品中都能检验出多重耐药性细菌。有报道预测,到2050年将有1000万人死于细菌耐药性疾病。解决细菌耐药性问题最好的办法是研发新的抗生素,然而新的抗生素研发过程较慢且成本昂贵。噬菌体及其裂解酶制剂能高效、快速、特异性地裂解耐药性病原菌,不易使细菌产生耐受性,安全性好,环境友好。
裂解酶主要通过催化细菌细胞壁上的肽聚糖,发挥裂解细胞的作用。革兰氏阳性菌的肽聚糖在细胞壁的最外层,裂解酶可以直接与其发生作用将其裂解。裂解酶制剂在体内、外都能较好地杀灭革兰氏阳性细菌,比如链球菌、金黄色葡萄球菌。但革兰氏阴性菌的细胞壁与阳性菌不同,最外层是由脂多糖、磷脂、蛋白质和脂蛋白等复合物组成的外膜。外膜蛋白是镶嵌在其中的蛋白质的统称,包括脂蛋白、微孔蛋白,细菌通过微孔蛋白与外界物质进行互换,但是只有小分子化合物(分子量小于600)才能通过。细胞壁的最里层是细胞质膜,在外膜与细胞质膜之间是周质空间,其中有2-3nm的肽聚糖。裂解酶的分子量至少在30kD以上,因此裂解酶不能透过阴性菌的外膜,接触周质空间的肽聚糖,致使裂解酶不能直接从菌外裂解革兰氏阴性菌。
为了解决裂解酶不能透过革兰氏阴性菌外膜的问题,一般会使用化学透膜剂来协同裂解酶透过细胞外膜。透膜剂一般来说分为两类,第一类为多价阳离子化合物基团,它能竞争取代连接脂多糖分子的邻近二价阴离子,如多粘菌素及其衍生物,赖氨酸聚合物和氨基糖苷;第二类为螯合剂,螯合剂中EDTA使用最为普遍,但也有质子化形式的弱有机酸做螯合剂。多个研究表明EDTA协同裂解酶穿透细胞壁的作用最强,但是由于它有其他的药理作用,不适合于全身感染的治疗。
中国专利CN201910975220.9公开了一种具有强杀菌效果的杂合抗菌蛋白AB469,对革兰氏阴性菌及其耐药菌具有非常强的杀菌效果,对革兰氏阳性菌也具有较强的杀菌效果,杂合抗菌蛋白AB469相较于野生型ABgp46多了一个结合域,显著提高了在复杂环境下的抗菌活性。虽然杂合抗菌蛋白AB469相较于野生型裂解酶的抑菌活性有了显著提升,是一种可以直接裂解细菌的抗菌物质,但其对革兰氏阴性菌的抑制能力仍有待提升,影响了其进一步的临床应用。
发明内容
为了进一步提高对革兰氏阴性菌的抑菌效果,本发明提供了一种C末端修饰的杂合抗菌蛋白,通过在C末端修饰含精氨酸残基的螺旋状两性多肽,显著提升杂合抗菌蛋白对革兰氏阴性菌细胞外膜的透过性,显著提升对革兰氏阴性菌的杀菌效果。
本发明还提供上述C末端修饰的杂合抗菌蛋白的编码核酸序列、表达载体、工程菌以及含有上述C末端修饰的杂合抗菌蛋白的药物组合物及其应用。
为了实现上述发明目的,本发明提供了一种C末端修饰的抑菌杂合蛋白,具有式(Ⅰ)所示氨基酸序列:
X-Y (Ⅰ)
其中,X为被修饰的杂合抗菌蛋白,包括AB469杂合抗菌蛋白或其类似物;
Y为X的C末端修饰体,所述C末端修饰体包括含有3-10个精氨酸残基的多肽。
优选的,所述AB469杂合抗菌蛋白类似物包括与AB469杂合抗菌蛋白编码序列具有至少70%同源性的蛋白、与AB469杂合抗菌蛋白的催化域编码序列至少具有70%同源性的蛋白、与AB469杂合抗菌蛋白的结合域编码序列至少具有70%同源性的蛋白以及与AB469杂合抗菌蛋白催化域和结合域位置互换组成的蛋白编码序列至少具有70%同源性的蛋白。
优选的,所述C末端修饰体包括含有5-8个精氨酸残基的多肽。
优选的,所述C末端修饰体包括含有3-10个连续的精氨酸残基的多肽。
优选的,所述C末端修饰的抑菌杂合蛋白的氨基酸序列如SEQ ID NO.1或SEQ IDNO.3所示。
优选的,C末端修饰体Y还包括疏水性氨基酸残基。
优选的,所述C末端修饰体为精氨酸残基与疏水性氨基酸残基间隔性排列。
优选的,所述疏水性氨基酸残基为丙氨酸残基。
优选的,所述C末端修饰的抑菌杂合蛋白的氨基酸序列如SEQ ID NO.5所示。
本发明的第二方面提供了一种核苷酸序列,包括上述技术方案所述C末端修饰的抑菌杂合蛋白的编码序列。
优选的,所述核苷酸序列如SEQ ID NO.2、SEQ ID NO.4或SEQ ID NO.6所示。
本发明的第三方面提供了一种表达载体,包括如上述技术方案所述核苷酸序列。
本发明的第四方面提供了一种工程菌,包括前述技术方案所述核苷酸序列,或上述技术方案所述表达载体。
本发明的第五方面提供了一种药物组合物,包括前述技术方案所述C末端修饰的抑菌杂合蛋白,和药学上可接受的辅料。
优选的,所述药物组合物为外用制剂时,所述C末端修饰的抑菌杂合蛋白的质量浓度不超过0.5%。
本发明的第六方面提供了前述技术方案所述C末端修饰的抑菌杂合蛋白、前述技术方案所述核苷酸序列、前述技术方案所述表达载体、前述技术方案所述工程菌或上述技术方案所述药物组合物在制备抑菌食品、药品或保健品中的应用。
优选的,所述抑菌食品、药品或保健品为抑制革兰氏阴性菌的食品、药品或保健品。
优选的,所述革兰氏阴性菌包括鲍曼不动杆菌、铜绿假单胞菌、肺炎克雷伯菌、沙门氏菌和大肠杆菌中的一种或多种。
与现有技术相比,本发明的有益效果:
杂合抗菌蛋白AB469是申请人前期合成的一种对革兰氏阴性菌具有强杀菌效果的杂合抗菌蛋白。在此基础上,申请人意外发现在AB469的C末端修饰一段含有3-10个精氨酸残基的多肽,相较于AB469本发明所述C末端修饰的AB469(以下简称“AB469A”)对革兰氏阴性菌的抑菌效果提升1倍以上(MIC≤50μg/mL),对革兰氏阴性菌的杀菌效果显著提升。
附图说明
图1为AB469A5蛋白的SDS-PAGE图。
图2为AB469A8蛋白的SDS-PAGE图。
具体实施方式
下面将结合附图,对本发明的技术方案进行描述。显然,所描述的实施例仅是本申请一部分实施方式,而不是全部的实施方式;并且附图中所示的结构仅仅是示意性的,并不代表实物。需要说明的是,基于本发明中的这些实施例,本领域普通技术人员所获得的所有其他实施例,都属于本申请保护的范围。
在本发明中,为方便起见,常常使用单数形式例如“一种”、“一个”和“该/所述”;然而,除非上下文明确规定或清楚指示仅为单数,否则单数形式意指包括复数。
术语“编码序列”指能够编码具有相应蛋白或多肽活性的核苷酸序列,如AB469杂合抗菌蛋白的核苷酸序列及其简并序列。简并序列是指该核苷酸序列中,有一个或多个密码子被编码相同氨基酸的简并密码子所取代而产生的序列。该术语还包括能编码具有与相应蛋白或多肽功能相同的蛋白或多肽核苷酸序列中开放阅读框序列的变异形式,这些变异形式包括但不限于:若干个核苷酸的缺失、插入和/或取代,以及在5’和/或3’端添加若干个核苷酸。
术语“类似物”是指与相应蛋白或多肽及其核苷酸序列不同但保留了主要性质的多肽或核苷酸序列,该类似物可以是天然形成的,也可以是通过一种或多种修饰(如置换、添加和/或缺失)引起的多肽或核苷酸序列不同,例如保守修饰的类似物。该属于还包括相应蛋白或多肽的蛋白修饰(如甲基化、乙酰化、磷酸化、泛素化、ADP核糖基化)产物、偶联物(如抗体偶联、多肽偶联等)、缀合物(如药物缀合、聚合物缀合等)以及所有与相应蛋白或多肽总体功能上相同或近似的类似物。
术语“多肽”意指无论其大小、由任意氨基酸组成的聚合物。尽管术语“蛋白质”常用来指相对较大的多肽,“肽”常用来指小的多肽,在此领域中术语“多肽”“蛋白质”和“肽”常部分重叠使用。除非另行注明,否则术语“多肽”一般是指蛋白质、多肽及肽。
编码序列的同源性百分比可由本领域已知的软件进行分析,如GAP分析等。
本发明提供了一种C末端修饰的抑菌杂合蛋白,具有式(Ⅰ)所示氨基酸序列:
X-Y (Ⅰ)
其中,X为被修饰的杂合抗菌蛋白,包括AB469杂合抗菌蛋白或其类似物;Y为X的C末端修饰体,所述C末端修饰体包括含有3-10个精氨酸残基的多肽。本发明研究显示,只有采用特定数量的精氨酸残基对AB469的C末端修饰才能显著提升其对革兰氏阴性菌的杀菌效果;本发明的实施例中对AB469杂合抗菌蛋白采用赖氨酸、组氨酸进行C末端修饰后对革兰氏阴性菌的杀菌效果反而相对于AB469有所降低。
在本发明中,所述AB469杂合抗菌蛋白的核苷酸序列、氨基酸序列以及AB469杂合抗菌蛋白某些类似物(如与AB469催化域序列相似的杂合蛋白、与AB469结合域序列相似的杂合蛋白、AB469的催化域和结合域位置互换的杂合蛋白)的核苷酸序列及氨基酸序列已在中国专利CN201910975220.9中记载,本发明不再赘述。
本发明所述AB469杂合抗菌蛋白类似物包括与AB469杂合抗菌蛋白编码序列具有至少70%、80%、85%、90%、95%、98%、99%、99.9%同源性的蛋白、与AB469杂合抗菌蛋白的催化域编码序列至少具有70%、80%、85%、90%、95%、98%、99%、99.9%同源性的蛋白、与AB469杂合抗菌蛋白的结合域编码序列至少具有70%、80%、85%、90%、95%、98%、99%、99.9%同源性的蛋白以及与AB469杂合抗菌蛋白催化域和结合域位置互换组成的蛋白编码序列至少具有70%、80%、85%、90%、95%、98%、99%、99.9%同源性的蛋白。
在本发明中,所述C末端修饰体包括含有3-10个精氨酸残基的多肽。超过或不足本发明限定的精氨酸残基数量的C末端修饰体无法显著提高AB469对革兰氏阴性菌的杀菌效果。本发明优选的,所述C末端修饰体包括含有5-8个精氨酸残基的多肽,包括该C末端修饰体的AB469A杂合抗菌蛋白对革兰氏阴性菌的杀菌效果可提高8倍以上,MIC≤12.5μg/mL。
在本发明的一些具体实施方式中,所述C末端修饰体包括含有3-10个连续的精氨酸残基的多肽,其序列可以如SEQ ID NO.7-SEQ ID NO.14所示(表1);本发明优选的,所述C末端修饰体包括含有5-8个连续的精氨酸残基的多肽,其序列可以如SEQ ID NO.9、SEQ IDNO.10、SEQ ID NO.11、SEQ ID NO.12所示。
表1
序号 | C末端修饰体序列 |
SEQ ID NO.7 | RRR |
SEQ ID NO.8 | RRRR |
SEQ ID NO.9 | RRRRR |
SEQ ID NO.10 | RRRRRR |
SEQ ID NO.11 | RRRRRRR |
SEQ ID NO.12 | RRRRRRRR |
SEQ ID NO.13 | RRRRRRRRR |
SEQ ID NO.14 | RRRRRRRRRR |
在本发明的一些具体实施例中,C末端修饰由5个连续的精氨酸残基多肽组成(如SEQ ID NO.9所示),修饰后的杂合抑菌蛋白命名为“AB469A5”,其氨基酸序列如SEQ IDNO.1所示,其蛋白编码序列如SEQ ID NO.2所示:
SEQ ID NO.1:
MAILTKDGFGIIRNELFGGKLDQTQVDAINFIVEKATESGLSYPEAAYLLATIYHETGLP 60
SGYRTMQPIKEAGSDNYLRSKKYYPYIGYGYVQLTWKENYGRIGKLIGIDLIKNPEKALE 120
PLIAIQIAIKGMLNGWFTGVGFRRKRPVSKYNKQQYIAARNIINGKDKAELIAKYAIIFE 180
RALRSLGSNSTSNSSTNSGSTGKVSLPNRVIRVTKPIVHGSDVLAIQKALSSLYFYPEKG 240
AKDNGSDSYYGPKTANAVKRFQSVNGLVADGVYGPKTRAAILKKLRRRRR
SEQ ID NO.2:
ccatggctatcctgaccaaagatggctttggcatcattcgcaacgaactgtttggcggca 60
aactggatcagacacaggtggatgccattaattttattgttgaaaaagctaccgaatctg 120
ggttaagttatccggaagcggcctatctgttagcgacgatctatcatgaaacgggtctgc 180
cgagcggttatcgtaccatgcagccaatcaaagaagccggtagtgataattacctccgct 240
ctaaaaaatattatccgtatatcggctatggctatgttcagctgacgtggaaagaaaatt 300
atggtcgtattggtaaactgatcggcatcgacttgatcaaaaatccggaaaaagccttag 360
aaccgctgattgcgattcagattgccatcaaaggtatgctgaatggttggtttacaggtg 420
tgggctttcgtcgcaaacgtccagtgagtaaatataacaagcagcagtatattgctgcac 480
gcaatatcattaatggtaaagataaagcagaactgatcgcgaaatatgccatcatctttg 540
aacgcgccctgcgttctctgggctccaatagtacctctaatagtagcaccaattcaggct 600
ctaccggtaaagtgagtctgccgaatcgtgtgattcgcgtgaccaaaccgattgttcatg 660
gtagcgatgtgctggcaattcagaaagcactgtcaagcctgtatttttatccggaaaaag 720
gtgccaaagataatggttccgatagctattatggtccgaaaaccgccaatgccgttaaac 780
gctttcagtctgttaatggcttagttgcagatggcgtgtatggcccgaaaacccgcgcag 840
ccattctgaaaaaactgcgtcgtcgtcgtcgttaagctt
在本发明的一些具体实施例中,C末端修饰由8个连续的精氨酸残基多肽组成(如SEQ ID NO.12所示),修饰后的杂合抑菌蛋白命名为“AB469A8”,其氨基酸序列如SEQ IDNO.3所示,其蛋白编码序列如SEQ ID NO.4所示:
SEQ ID NO.3:
MAILTKDGFGIIRNELFGGKLDQTQVDAINFIVEKATESGLSYPEAAYLLATIYHETGLP 60
SGYRTMQPIKEAGSDNYLRSKKYYPYIGYGYVQLTWKENYGRIGKLIGIDLIKNPEKALE 120
PLIAIQIAIKGMLNGWFTGVGFRRKRPVSKYNKQQYIAARNIINGKDKAELIAKYAIIFE 180
RALRSLGSNSTSNSSTNSGSTGKVSLPNRVIRVTKPIVHGSDVLAIQKALSSLYFYPEKG 240
AKDNGSDSYYGPKTANAVKRFQSVNGLVADGVYGPKTRAAILKKLRRRRRRRR
SEQ ID NO.4:
ccatggctatcctgaccaaagatggctttggcatcattcgcaacgaactgtttggcggca 60
aactggatcagacacaggtggatgccattaattttattgttgaaaaagctaccgaatctg 120
ggttaagttatccggaagcggcctatctgttagcgacgatctatcatgaaacgggtctgc 180
cgagcggttatcgtaccatgcagccaatcaaagaagccggtagtgataattacctccgct 240
ctaaaaaatattatccgtatatcggctatggctatgttcagctgacgtggaaagaaaatt 300
atggtcgtattggtaaactgatcggcatcgacttgatcaaaaatccggaaaaagccttag 360
aaccgctgattgcgattcagattgccatcaaaggtatgctgaatggttggtttacaggtg 420
tgggctttcgtcgcaaacgtccagtgagtaaatataacaagcagcagtatattgctgcac 480
gcaatatcattaatggtaaagataaagcagaactgatcgcgaaatatgccatcatctttg 540
aacgcgccctgcgttctctgggctccaatagtacctctaatagtagcaccaattcaggct 600
ctaccggtaaagtgagtctgccgaatcgtgtgattcgcgtgaccaaaccgattgttcatg 660
gtagcgatgtgctggcaattcagaaagcactgtcaagcctgtatttttatccggaaaaag 720
gtgccaaagataatggttccgatagctattatggtccgaaaaccgccaatgccgttaaac 780
gctttcagtctgttaatggcttagttgcagatggcgtgtatggcccgaaaacccgcgcag 840
ccattctgaaaaaactgcgtcgtcgtcgtcgtcgtcgtcgttaagctt
在本发明的一些具体实施方式中,所述C末端修饰体除了精氨酸残基外还可以包括疏水性氨基酸残基。在本发明中,所述疏水性氨基酸选自甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、苯丙氨酸和脯氨酸中的一种或多种;在本发明的一些优选实施例中,所述疏水性氨基酸为丙氨酸。
在本发明的一些具体实施方式中,本发明所述C末端修饰体中的精氨酸也可以是不连续排列,如在两个精氨酸残基中插入1-2个的疏水性氨基酸。
在本发明的一些具体实施例中,C末端修饰体为包括6个精氨酸残基并在任意两个精氨酸残基之间插入了2个丙氨酸残基(如SEQ ID NO.15所示)的多肽,其修饰后的杂合抑菌蛋白命名为“AB469A11”,其氨基酸序列如SEQ ID NO.5所示,其蛋白编码序列如SEQ IDNO.6所示:
SEQ ID NO.15:
RAARAARAARAARAAR
SEQ ID NO.5:
MAILTKDGFGIIRNELFGGKLDQTQVDAINFIVEKATESGLSYPEAAYLLATIYHETGLP 60
SGYRTMQPIKEAGSDNYLRSKKYYPYIGYGYVQLTWKENYGRIGKLIGIDLIKNPEKALE 120
PLIAIQIAIKGMLNGWFTGVGFRRKRPVSKYNKQQYIAARNIINGKDKAELIAKYAIIFE 180
RALRSLGSNSTSNSSTNSGSTGKVSLPNRVIRVTKPIVHGSDVLAIQKALSSLYFYPEKG 240
AKDNGSDSYYGPKTANAVKRFQSVNGLVADGVYGPKTRAAILKKLRAARAARAARAARAA 300
R
SEQ ID NO.6:
ccatggctatcctgaccaaagatggctttggcatcattcgcaacgaactgtttggcggca 60
aactggatcagacacaggtggatgccattaattttattgttgaaaaagctaccgaatctg 120
ggttaagttatccggaagcggcctatctgttagcgacgatctatcatgaaacgggtctgc 180
cgagcggttatcgtaccatgcagccaatcaaagaagccggtagtgataattacctccgct 240
ctaaaaaatattatccgtatatcggctatggctatgttcagctgacgtggaaagaaaatt 300
atggtcgtattggtaaactgatcggcatcgacttgatcaaaaatccggaaaaagccttag 360
aaccgctgattgcgattcagattgccatcaaaggtatgctgaatggttggtttacaggtg 420
tgggctttcgtcgcaaacgtccagtgagtaaatataacaagcagcagtatattgctgcac 480
gcaatatcattaatggtaaagataaagcagaactgatcgcgaaatatgccatcatctttg 540
aacgcgccctgcgttctctgggctccaatagtacctctaatagtagcaccaattcaggct 600
ctaccggtaaagtgagtctgccgaatcgtgtgattcgcgtgaccaaaccgattgttcatg 660
gtagcgatgtgctggcaattcagaaagcactgtcaagcctgtatttttatccggaaaaag 720
gtgccaaagataatggttccgatagctattatggtccgaaaaccgccaatgccgttaaac 780
gctttcagtctgttaatggcttagttgcagatggcgtgtatggcccgaaaacccgcgcag 840
ccattctgaaaaaactgcgtgatgatcgtgatgatcgtgatgatcgtgatgatcgtgatg 900
atcgttaagctt
本发明的第二方面提供了一种核苷酸序列,包括上述技术方案所述C末端修饰的抑菌杂合蛋白的编码序列。在本发明的一些具体实施方案中,所述核苷酸序列可以如SEQID NO.2、SEQ ID NO.4或SEQ ID NO.6所示。
本发明的第三方面提供了一种表达载体,包括如上述技术方案所述核苷酸序列。在本发明的一些具体实施例中,所述表达载体可以是包含AB469A编码序列的质粒。
本发明的第四方面提供了一种工程菌,包括前述技术方案所述核苷酸序列,或上述技术方案所述表达载体。在本发明的一些具体实施方式中,所述工程菌可以是包括含有AB469A编码序列质粒载体的酵母菌、大肠杆菌等。
本发明的第五方面提供了一种药物组合物,包括前述技术方案所述C末端修饰的抑菌杂合蛋白,和药学上可接受的辅料。本发明所述药学上可接收的载体包括但不限于稳定剂、赋形剂、填充剂、粘合剂、分散剂、溶剂和矫味剂中的一种或多种。所述药物组合物可被配制成改良释放剂型,包括药物组合物也可以配制成为改良释放剂型,包括延迟(delayed)释放、延缓(extended)释放、延长(prolonged)释放、持续(sustained)释放、脉冲(pulsatile)释放、控释、加速释放和快速释放、靶向释放、程序化释放和胃滞留剂型。这些剂型可以根据本领域技术人员已知的常规方法和技术制备。
在本发明中,所述药物组合物中AB469A杂合抗菌蛋白的质量百分含量可以是0.1-99.99%。在本发明的一些具体实施方式中,当所述药物组合物为外用制剂时,所述C末端修饰的抑菌杂合蛋白的质量浓度不超过0.5%;优选的为0.00001-0.5%。
本发明的第六方面提供了前述技术方案所述C末端修饰的抑菌杂合蛋白、前述技术方案所述核苷酸序列、前述技术方案所述表达载体、前述技术方案所述工程菌或上述技术方案所述药物组合物在制备抑菌食品、药品或保健品中的应用。优选的,所述抑菌食品、药品或保健品为抑制革兰氏阴性菌的食品、药品或保健品;优选的,所述革兰氏阴性菌包括但不限于鲍曼不动杆菌、铜绿假单胞菌、肺炎克雷伯菌、沙门氏菌和大肠杆菌中的一种或多种。
下面结合实施例对本发明提供的技术方案进行详细的说明,但是不能把它们理解为对本发明保护范围的限定。
实施例1 AB469A5杂合抗菌蛋白
1.工程菌的构建
AB469的氨基酸序列:
MAILTKDGFG IIRNELFGGK LDQTQVDAIN FIVEKATESG LSYPEAAYLL 51
ATIYHETGLP SGYRTMQPIK EAGSDNYLRS KKYYPYIGYG YVQLTWKENY 101
GRIGKLIGID LIKNPEKALE PLIAIQIAIK GMLNGWFTGV GFRRKRPVSK 151
YNKQQYIAAR NIINGKDKAE LIAKYAIIFE RALRSLGSNS TSNSSTNSGS 201
TGKVSLPNRV IRVTKPIVHG SDVLAIQKAL SSLYFYPEKG AKDNGSDSYY 251
GPKTANAVKR FQSVNGLVAD GVYGPKTRAA ILKKL
在AB469氨基酸序列的C末端增加如SEQ ID NO.9所示的“RRRRR”多肽,即得AB469A5的氨基酸序列(如SEQ ID NO.1所示)。按照中国专利CN201910975220.9说明书实施例1所示的方法,将AB469A5的编码序列(如SEQ ID NO.2所示)插入pET28a质粒中,得到重组质粒pET28a-AB469A5。
SEQ ID NO.1:
MAILTKDGFGIIRNELFGGKLDQTQVDAINFIVEKATESGLSYPEAAYLLATIYHETGLP 60
SGYRTMQPIKEAGSDNYLRSKKYYPYIGYGYVQLTWKENYGRIGKLIGIDLIKNPEKALE 120
PLIAIQIAIKGMLNGWFTGVGFRRKRPVSKYNKQQYIAARNIINGKDKAELIAKYAIIFE 180
RALRSLGSNSTSNSSTNSGSTGKVSLPNRVIRVTKPIVHGSDVLAIQKALSSLYFYPEKG 240
AKDNGSDSYYGPKTANAVKRFQSVNGLVADGVYGPKTRAAILKKLRRRRR
SEQ ID NO.2:
ccatggctatcctgaccaaagatggctttggcatcattcgcaacgaactgtttggcggca 60
aactggatcagacacaggtggatgccattaattttattgttgaaaaagctaccgaatctg 120
ggttaagttatccggaagcggcctatctgttagcgacgatctatcatgaaacgggtctgc 180
cgagcggttatcgtaccatgcagccaatcaaagaagccggtagtgataattacctccgct 240
ctaaaaaatattatccgtatatcggctatggctatgttcagctgacgtggaaagaaaatt 300
atggtcgtattggtaaactgatcggcatcgacttgatcaaaaatccggaaaaagccttag 360
aaccgctgattgcgattcagattgccatcaaaggtatgctgaatggttggtttacaggtg 420
tgggctttcgtcgcaaacgtccagtgagtaaatataacaagcagcagtatattgctgcac 480
gcaatatcattaatggtaaagataaagcagaactgatcgcgaaatatgccatcatctttg 540
aacgcgccctgcgttctctgggctccaatagtacctctaatagtagcaccaattcaggct 600
ctaccggtaaagtgagtctgccgaatcgtgtgattcgcgtgaccaaaccgattgttcatg 660
gtagcgatgtgctggcaattcagaaagcactgtcaagcctgtatttttatccggaaaaag 720
gtgccaaagataatggttccgatagctattatggtccgaaaaccgccaatgccgttaaac 780
gctttcagtctgttaatggcttagttgcagatggcgtgtatggcccgaaaacccgcgcag 840
ccattctgaaaaaactgcgtcgtcgtcgtcgttaagctt
以重组质粒pET28a-AB469A5作为模板,利用AB469A5的特异性引物(上游引物如SEQ ID NO.16所示,下游引物如SEQ ID NO.17所示)扩增出全部编码区。PCR反应程序设置为:①94℃预变性5min,②94℃变性30s;③55℃复性30s;④72℃延伸1min;⑤72℃终延伸5min。将步骤②③④进行30个循环。将PR产物进行纯化回收,与pET28a载体连接。连接产物转化大肠杆菌BL21(DE3),挑选3个单个菌落接种于LB液体培养基,并送测序公司测序,以验证阅读框的正确性。
AB469A5的特异性引物:
SEQ ID NO.16:5’-tataccatggctatcctgaccaaag-3’
SEQ ID NO.17:5’-ccgcaagcttaacgacgacgacgacgcagttttttcagaatggctgcgcgggtttt-3’
2.重组蛋白的表达和纯化:
工程菌挑单克隆接种于LB培养液(含30mg/L卡那霉素),30℃振荡培养过夜,按1%接种到相同的LB培养液中,30℃振荡培养至光密度值(波长600nm)≈0.6时,加入终浓度为0.05mM的IPTG诱导蛋白表达,继续30℃振荡培养,诱导4h左右,离心收集发酵上清。
发酵上清经阳离子交换和凝胶过滤两步纯化。纯化后的样品置于-20℃冷冻保存备用。用15%SDS-PAGE测定重组蛋白样品的大小和纯度,电泳图如图1所示,杂合抗菌蛋白AB469A5的电泳主带在32kD附近。
实施例2 AB469A8杂合抗菌蛋白
参照实施例1的方法构建AB469A8的重组质粒pET28a-AB469A8,AB469A8的氨基酸序列如SEQ ID NO.3所示、编码序列如SEQ ID NO.4所示。利用AB469A8的特异性引物(上游引物如SEQ ID NO.18所示,下游引物如SEQ ID NO.19所示)对pET28a-AB469A8进行PCR扩增验证后,将重组质粒pET28a-AB469A8导入大肠杆菌中进行重组蛋白表达和纯化。
用15%SDS-PAGE测定重组蛋白样品的大小和纯度,电泳图如图2所示,杂合抗菌蛋白AB469A8的电泳主带在32kD附近。
SEQ ID NO.3:
MAILTKDGFGIIRNELFGGKLDQTQVDAINFIVEKATESGLSYPEAAYLLATIYHETGLP 60
SGYRTMQPIKEAGSDNYLRSKKYYPYIGYGYVQLTWKENYGRIGKLIGIDLIKNPEKALE 120
PLIAIQIAIKGMLNGWFTGVGFRRKRPVSKYNKQQYIAARNIINGKDKAELIAKYAIIFE 180
RALRSLGSNSTSNSSTNSGSTGKVSLPNRVIRVTKPIVHGSDVLAIQKALSSLYFYPEKG 240
AKDNGSDSYYGPKTANAVKRFQSVNGLVADGVYGPKTRAAILKKLRRRRRRRR
SEQ ID NO.4:
ccatggctatcctgaccaaagatggctttggcatcattcgcaacgaactgtttggcggca 60
aactggatcagacacaggtggatgccattaattttattgttgaaaaagctaccgaatctg 120
ggttaagttatccggaagcggcctatctgttagcgacgatctatcatgaaacgggtctgc 180
cgagcggttatcgtaccatgcagccaatcaaagaagccggtagtgataattacctccgct 240
ctaaaaaatattatccgtatatcggctatggctatgttcagctgacgtggaaagaaaatt 300
atggtcgtattggtaaactgatcggcatcgacttgatcaaaaatccggaaaaagccttag 360
aaccgctgattgcgattcagattgccatcaaaggtatgctgaatggttggtttacaggtg 420
tgggctttcgtcgcaaacgtccagtgagtaaatataacaagcagcagtatattgctgcac 480
gcaatatcattaatggtaaagataaagcagaactgatcgcgaaatatgccatcatctttg 540
aacgcgccctgcgttctctgggctccaatagtacctctaatagtagcaccaattcaggct 600
ctaccggtaaagtgagtctgccgaatcgtgtgattcgcgtgaccaaaccgattgttcatg 660
gtagcgatgtgctggcaattcagaaagcactgtcaagcctgtatttttatccggaaaaag 720
gtgccaaagataatggttccgatagctattatggtccgaaaaccgccaatgccgttaaac 780
gctttcagtctgttaatggcttagttgcagatggcgtgtatggcccgaaaacccgcgcag 840
ccattctgaaaaaactgcgtcgtcgtcgtcgtcgtcgtcgttaagctt
SEQ ID NO.18:5’-tataccatggctatcctgaccaaag-3’
SEQ ID NO.19:5’-ccgcaagcttaacgacgacgacgacgacgacgacgcagttttttcagaatggctgcgcgggtttt-3’
实施例3
参照实施例1、2所示的方法,构建下表所示的杂合抗菌蛋白:
表2
名称 | C末端修饰体 | C末端修饰体序列表编号 |
AB469A3 | RRR | SEQ ID NO.7 |
AB469A4 | RRRR | SEQ ID NO.8 |
AB469A6 | RRRRRR | SEQ ID NO.10 |
AB469A7 | RRRRRRR | SEQ ID NO.11 |
AB469A9 | RRRRRRRRR | SEQ ID NO.13 |
AB469A10 | RRRRRRRRRR | SEQ ID NO.14 |
AB469A11 | RAARAARAARAARAAR | SEQ ID NO.15 |
AB469B1 | KKKKK | SEQ ID NO.20 |
AB469B2 | KKKKKKKK | SEQ ID NO.21 |
AB469B3 | HHHH | SEQ ID NO.22 |
AB469B4 | HHHHHHHH | SEQ ID NO.23 |
AB469B5 | RR | SEQ ID NO.24 |
AB469B6 | RRRRRRRRRRR | SEQ ID NO.25 |
AB469B7 | RRRRRRRRRRRR | SEQ ID NO.26 |
实施例4杂合抑菌蛋白对革兰氏阴性菌的抑菌活性比较实验
(1)供试菌株:鲍曼不动杆菌(ATCC 19606)来自美国菌种保存中心。
(2)菌株培养:将菌株甘油管菌株划线到LB琼脂平板上。鲍曼不动杆菌菌保藏在LB液体培养基:将培养鲍曼的LB液体培养基与30%甘油1:1混合均匀后保藏于-80℃。
(3)二倍稀释微孔法:
将AB469杂合抗菌蛋白、实施例1-3制备的杂合抗菌蛋白AB469A1-AB469A11以及AB469B1-AB469B7分别用LB液体培养基稀释到合适起始浓度(200μg/mL、100μg/mL、50μg/mL、25μg/mL、12.5μg/mL、6.25μg/mL、3.125μg/mL、1.56μg/mL)。刮取平板中鲍曼不动杆菌适量至2mL LB液体培养基,混匀,配制0.5麦氏浓度菌液,再用LB液体培养基进行100倍稀释(约106cfu/mL)。
96孔板加样操作:
阴性对照,每孔添加LB液体培养基200μL,作为阴性对照。
实验组:每孔依次添加稀释好的不同蛋白样液100ul,然后每孔依次添加LB液体培养基50μL,最后每孔添加50μL上述稀释好的鲍曼不动杆菌菌液,设两个复孔。
37℃培养24h,观察孔中菌液是否澄清。如果第一孔就是浑浊的,则判定MIC>100μg/mL。实验结果如表3所示。
表3不同C末端修饰的AB469对鲍曼不动杆菌的MIC值
由表3数据可以看出,当C末端修饰体含有3-10个连续或不连续的精氨酸时,与AB469杂合蛋白相比,其对革兰氏阴性菌鲍曼不动杆菌抑菌活性提高了1-23倍,MIC值达到50μg/mL以下,AB469A6对鲍曼不动杆菌的MIC值达到3.125μg/mL。表明本发明通过C末端修饰显著增强了AB469对革兰氏阴性菌的抑菌活性。
当C末端修饰体为2、11、12个精氨酸时,其MIC值均>100μg/mL;当C末端修饰体为赖氨酸(K)和组氨酸(H)时,虽然二者也是碱性氨基酸,但其在C末端修饰后不但无法提升AB469对革兰氏阴性菌抑菌活性,反而抑菌活性更差。以上对比实验表明,只有在修饰本发明限定数量的精氨酸时方可显著提高AB469对革兰氏阴性菌的抑制活性。
实施例5C末端修饰的杂合抑菌蛋白对不同革兰氏阴性菌抑菌活性实验
参照实施例4所示的方法,检测AB469A5和AB469A8对临床分离的耐药及不耐药的鲍曼不动杆菌、铜绿假单胞菌、肺炎克雷伯菌的抗菌活性,具体结果见下表4:
表4 AB469A5和AB469A8对不同革兰氏阴性菌的MIC值(μg/mL)
由表4的数据可以看出,AB469A5和AB469A8对耐药及不耐药的鲍曼不动杆菌、铜绿假单胞菌和肺炎克雷伯菌均具有显著抑菌作用,表明本发明提供的C末端修饰的杂合抗菌蛋白AB469A对于耐药或不耐药的革兰氏阴性菌均有显著的抑菌活性。
实施例5含AB469A5的体外抗菌药物
制造100ml含AB469A5的生物抗菌制剂,其处方为:
制备方法:
a)按照处方和总配制液体积计算辅料需求量,精确称取至洁净容器中。
b)在配料器皿中加入总配制液70%的水,先溶解HPMC、磷酸氢二钠和磷酸二氢钾,充分溶解后再加入甘油,混匀后加入AB469,最后用水定容并且充分混匀。
c)除菌过滤:将配制好的消毒剂通过除菌过滤器,流出的消毒剂接入无菌容器。
d)灌装:将无菌消毒剂灌装于塑料或玻璃瓶。
该抗菌制剂可用于伤口或创面的杀菌消毒,每日1-2次。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以作出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 上海高科生物工程有限公司
<120> 一种C末端修饰的杂合抗菌蛋白及其药物组合物和应用
<141> 2021-12-27
<160> 26
<170> SIPOSequenceListing 1.0
<210> 1
<211> 290
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Met Ala Ile Leu Thr Lys Asp Gly Phe Gly Ile Ile Arg Asn Glu Leu
1 5 10 15
Phe Gly Gly Lys Leu Asp Gln Thr Gln Val Asp Ala Ile Asn Phe Ile
20 25 30
Val Glu Lys Ala Thr Glu Ser Gly Leu Ser Tyr Pro Glu Ala Ala Tyr
35 40 45
Leu Leu Ala Thr Ile Tyr His Glu Thr Gly Leu Pro Ser Gly Tyr Arg
50 55 60
Thr Met Gln Pro Ile Lys Glu Ala Gly Ser Asp Asn Tyr Leu Arg Ser
65 70 75 80
Lys Lys Tyr Tyr Pro Tyr Ile Gly Tyr Gly Tyr Val Gln Leu Thr Trp
85 90 95
Lys Glu Asn Tyr Gly Arg Ile Gly Lys Leu Ile Gly Ile Asp Leu Ile
100 105 110
Lys Asn Pro Glu Lys Ala Leu Glu Pro Leu Ile Ala Ile Gln Ile Ala
115 120 125
Ile Lys Gly Met Leu Asn Gly Trp Phe Thr Gly Val Gly Phe Arg Arg
130 135 140
Lys Arg Pro Val Ser Lys Tyr Asn Lys Gln Gln Tyr Ile Ala Ala Arg
145 150 155 160
Asn Ile Ile Asn Gly Lys Asp Lys Ala Glu Leu Ile Ala Lys Tyr Ala
165 170 175
Ile Ile Phe Glu Arg Ala Leu Arg Ser Leu Gly Ser Asn Ser Thr Ser
180 185 190
Asn Ser Ser Thr Asn Ser Gly Ser Thr Gly Lys Val Ser Leu Pro Asn
195 200 205
Arg Val Ile Arg Val Thr Lys Pro Ile Val His Gly Ser Asp Val Leu
210 215 220
Ala Ile Gln Lys Ala Leu Ser Ser Leu Tyr Phe Tyr Pro Glu Lys Gly
225 230 235 240
Ala Lys Asp Asn Gly Ser Asp Ser Tyr Tyr Gly Pro Lys Thr Ala Asn
245 250 255
Ala Val Lys Arg Phe Gln Ser Val Asn Gly Leu Val Ala Asp Gly Val
260 265 270
Tyr Gly Pro Lys Thr Arg Ala Ala Ile Leu Lys Lys Leu Arg Arg Arg
275 280 285
Arg Arg
290
<210> 7
<211> 879
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
ccatggctat cctgaccaaa gatggctttg gcatcattcg caacgaactg tttggcggca 60
aactggatca gacacaggtg gatgccatta attttattgt tgaaaaagct accgaatctg 120
ggttaagtta tccggaagcg gcctatctgt tagcgacgat ctatcatgaa acgggtctgc 180
cgagcggtta tcgtaccatg cagccaatca aagaagccgg tagtgataat tacctccgct 240
ctaaaaaata ttatccgtat atcggctatg gctatgttca gctgacgtgg aaagaaaatt 300
atggtcgtat tggtaaactg atcggcatcg acttgatcaa aaatccggaa aaagccttag 360
aaccgctgat tgcgattcag attgccatca aaggtatgct gaatggttgg tttacaggtg 420
tgggctttcg tcgcaaacgt ccagtgagta aatataacaa gcagcagtat attgctgcac 480
gcaatatcat taatggtaaa gataaagcag aactgatcgc gaaatatgcc atcatctttg 540
aacgcgccct gcgttctctg ggctccaata gtacctctaa tagtagcacc aattcaggct 600
ctaccggtaa agtgagtctg ccgaatcgtg tgattcgcgt gaccaaaccg attgttcatg 660
gtagcgatgt gctggcaatt cagaaagcac tgtcaagcct gtatttttat ccggaaaaag 720
gtgccaaaga taatggttcc gatagctatt atggtccgaa aaccgccaat gccgttaaac 780
gctttcagtc tgttaatggc ttagttgcag atggcgtgta tggcccgaaa acccgcgcag 840
ccattctgaa aaaactgcgt cgtcgtcgtc gttaagctt 879
<210> 3
<211> 293
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 3
Met Ala Ile Leu Thr Lys Asp Gly Phe Gly Ile Ile Arg Asn Glu Leu
1 5 10 15
Phe Gly Gly Lys Leu Asp Gln Thr Gln Val Asp Ala Ile Asn Phe Ile
20 25 30
Val Glu Lys Ala Thr Glu Ser Gly Leu Ser Tyr Pro Glu Ala Ala Tyr
35 40 45
Leu Leu Ala Thr Ile Tyr His Glu Thr Gly Leu Pro Ser Gly Tyr Arg
50 55 60
Thr Met Gln Pro Ile Lys Glu Ala Gly Ser Asp Asn Tyr Leu Arg Ser
65 70 75 80
Lys Lys Tyr Tyr Pro Tyr Ile Gly Tyr Gly Tyr Val Gln Leu Thr Trp
85 90 95
Lys Glu Asn Tyr Gly Arg Ile Gly Lys Leu Ile Gly Ile Asp Leu Ile
100 105 110
Lys Asn Pro Glu Lys Ala Leu Glu Pro Leu Ile Ala Ile Gln Ile Ala
115 120 125
Ile Lys Gly Met Leu Asn Gly Trp Phe Thr Gly Val Gly Phe Arg Arg
130 135 140
Lys Arg Pro Val Ser Lys Tyr Asn Lys Gln Gln Tyr Ile Ala Ala Arg
145 150 155 160
Asn Ile Ile Asn Gly Lys Asp Lys Ala Glu Leu Ile Ala Lys Tyr Ala
165 170 175
Ile Ile Phe Glu Arg Ala Leu Arg Ser Leu Gly Ser Asn Ser Thr Ser
180 185 190
Asn Ser Ser Thr Asn Ser Gly Ser Thr Gly Lys Val Ser Leu Pro Asn
195 200 205
Arg Val Ile Arg Val Thr Lys Pro Ile Val His Gly Ser Asp Val Leu
210 215 220
Ala Ile Gln Lys Ala Leu Ser Ser Leu Tyr Phe Tyr Pro Glu Lys Gly
225 230 235 240
Ala Lys Asp Asn Gly Ser Asp Ser Tyr Tyr Gly Pro Lys Thr Ala Asn
245 250 255
Ala Val Lys Arg Phe Gln Ser Val Asn Gly Leu Val Ala Asp Gly Val
260 265 270
Tyr Gly Pro Lys Thr Arg Ala Ala Ile Leu Lys Lys Leu Arg Arg Arg
275 280 285
Arg Arg Arg Arg Arg
290
<210> 4
<211> 888
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
ccatggctat cctgaccaaa gatggctttg gcatcattcg caacgaactg tttggcggca 60
aactggatca gacacaggtg gatgccatta attttattgt tgaaaaagct accgaatctg 120
ggttaagtta tccggaagcg gcctatctgt tagcgacgat ctatcatgaa acgggtctgc 180
cgagcggtta tcgtaccatg cagccaatca aagaagccgg tagtgataat tacctccgct 240
ctaaaaaata ttatccgtat atcggctatg gctatgttca gctgacgtgg aaagaaaatt 300
atggtcgtat tggtaaactg atcggcatcg acttgatcaa aaatccggaa aaagccttag 360
aaccgctgat tgcgattcag attgccatca aaggtatgct gaatggttgg tttacaggtg 420
tgggctttcg tcgcaaacgt ccagtgagta aatataacaa gcagcagtat attgctgcac 480
gcaatatcat taatggtaaa gataaagcag aactgatcgc gaaatatgcc atcatctttg 540
aacgcgccct gcgttctctg ggctccaata gtacctctaa tagtagcacc aattcaggct 600
ctaccggtaa agtgagtctg ccgaatcgtg tgattcgcgt gaccaaaccg attgttcatg 660
gtagcgatgt gctggcaatt cagaaagcac tgtcaagcct gtatttttat ccggaaaaag 720
gtgccaaaga taatggttcc gatagctatt atggtccgaa aaccgccaat gccgttaaac 780
gctttcagtc tgttaatggc ttagttgcag atggcgtgta tggcccgaaa acccgcgcag 840
ccattctgaa aaaactgcgt cgtcgtcgtc gtcgtcgtcg ttaagctt 888
<210> 5
<211> 301
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 5
Met Ala Ile Leu Thr Lys Asp Gly Phe Gly Ile Ile Arg Asn Glu Leu
1 5 10 15
Phe Gly Gly Lys Leu Asp Gln Thr Gln Val Asp Ala Ile Asn Phe Ile
20 25 30
Val Glu Lys Ala Thr Glu Ser Gly Leu Ser Tyr Pro Glu Ala Ala Tyr
35 40 45
Leu Leu Ala Thr Ile Tyr His Glu Thr Gly Leu Pro Ser Gly Tyr Arg
50 55 60
Thr Met Gln Pro Ile Lys Glu Ala Gly Ser Asp Asn Tyr Leu Arg Ser
65 70 75 80
Lys Lys Tyr Tyr Pro Tyr Ile Gly Tyr Gly Tyr Val Gln Leu Thr Trp
85 90 95
Lys Glu Asn Tyr Gly Arg Ile Gly Lys Leu Ile Gly Ile Asp Leu Ile
100 105 110
Lys Asn Pro Glu Lys Ala Leu Glu Pro Leu Ile Ala Ile Gln Ile Ala
115 120 125
Ile Lys Gly Met Leu Asn Gly Trp Phe Thr Gly Val Gly Phe Arg Arg
130 135 140
Lys Arg Pro Val Ser Lys Tyr Asn Lys Gln Gln Tyr Ile Ala Ala Arg
145 150 155 160
Asn Ile Ile Asn Gly Lys Asp Lys Ala Glu Leu Ile Ala Lys Tyr Ala
165 170 175
Ile Ile Phe Glu Arg Ala Leu Arg Ser Leu Gly Ser Asn Ser Thr Ser
180 185 190
Asn Ser Ser Thr Asn Ser Gly Ser Thr Gly Lys Val Ser Leu Pro Asn
195 200 205
Arg Val Ile Arg Val Thr Lys Pro Ile Val His Gly Ser Asp Val Leu
210 215 220
Ala Ile Gln Lys Ala Leu Ser Ser Leu Tyr Phe Tyr Pro Glu Lys Gly
225 230 235 240
Ala Lys Asp Asn Gly Ser Asp Ser Tyr Tyr Gly Pro Lys Thr Ala Asn
245 250 255
Ala Val Lys Arg Phe Gln Ser Val Asn Gly Leu Val Ala Asp Gly Val
260 265 270
Tyr Gly Pro Lys Thr Arg Ala Ala Ile Leu Lys Lys Leu Arg Ala Ala
275 280 285
Arg Ala Ala Arg Ala Ala Arg Ala Ala Arg Ala Ala Arg
290 295 300
<210> 6
<211> 912
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
ccatggctat cctgaccaaa gatggctttg gcatcattcg caacgaactg tttggcggca 60
aactggatca gacacaggtg gatgccatta attttattgt tgaaaaagct accgaatctg 120
ggttaagtta tccggaagcg gcctatctgt tagcgacgat ctatcatgaa acgggtctgc 180
cgagcggtta tcgtaccatg cagccaatca aagaagccgg tagtgataat tacctccgct 240
ctaaaaaata ttatccgtat atcggctatg gctatgttca gctgacgtgg aaagaaaatt 300
atggtcgtat tggtaaactg atcggcatcg acttgatcaa aaatccggaa aaagccttag 360
aaccgctgat tgcgattcag attgccatca aaggtatgct gaatggttgg tttacaggtg 420
tgggctttcg tcgcaaacgt ccagtgagta aatataacaa gcagcagtat attgctgcac 480
gcaatatcat taatggtaaa gataaagcag aactgatcgc gaaatatgcc atcatctttg 540
aacgcgccct gcgttctctg ggctccaata gtacctctaa tagtagcacc aattcaggct 600
ctaccggtaa agtgagtctg ccgaatcgtg tgattcgcgt gaccaaaccg attgttcatg 660
gtagcgatgt gctggcaatt cagaaagcac tgtcaagcct gtatttttat ccggaaaaag 720
gtgccaaaga taatggttcc gatagctatt atggtccgaa aaccgccaat gccgttaaac 780
gctttcagtc tgttaatggc ttagttgcag atggcgtgta tggcccgaaa acccgcgcag 840
ccattctgaa aaaactgcgt gatgatcgtg atgatcgtga tgatcgtgat gatcgtgatg 900
atcgttaagc tt 912
<210> 7
<211> 3
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 7
Arg Arg Arg
1
<210> 8
<211> 4
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 8
Arg Arg Arg Arg
1
<210> 9
<211> 5
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 9
Arg Arg Arg Arg Arg
1 5
<210> 10
<211> 6
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 10
Arg Arg Arg Arg Arg Arg
1 5
<210> 11
<211> 7
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 11
Arg Arg Arg Arg Arg Arg Arg
1 5
<210> 12
<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 12
Arg Arg Arg Arg Arg Arg Arg Arg
1 5
<210> 13
<211> 9
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 13
Arg Arg Arg Arg Arg Arg Arg Arg Arg
1 5
<210> 14
<211> 10
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 14
Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg
1 5 10
<210> 15
<211> 16
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 15
Arg Ala Ala Arg Ala Ala Arg Ala Ala Arg Ala Ala Arg Ala Ala Arg
1 5 10 15
<210> 16
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
tataccatgg ctatcctgac caaag 25
<210> 17
<211> 56
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
ccgcaagctt aacgacgacg acgacgcagt tttttcagaa tggctgcgcg ggtttt 56
<210> 18
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
tataccatgg ctatcctgac caaag 25
<210> 19
<211> 65
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
ccgcaagctt aacgacgacg acgacgacga cgacgcagtt ttttcagaat ggctgcgcgg 60
gtttt 65
<210> 20
<211> 5
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 20
Lys Lys Lys Lys Lys
1 5
<210> 21
<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 21
Lys Lys Lys Lys Lys Lys Lys Lys
1 5
<210> 22
<211> 4
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 22
His His His His
1
<210> 23
<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 23
His His His His His His His His
1 5
<210> 24
<211> 2
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 24
Arg Arg
1
<210> 25
<211> 11
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 25
Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg
1 5 10
<210> 26
<211> 12
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 26
Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg
1 5 10
Claims (18)
1.一种C末端修饰的抑菌杂合蛋白,其特征在于,具有式(Ⅰ)所示氨基酸序列:
X-Y(Ⅰ)
其中,X为被修饰的杂合抗菌蛋白,包括AB469杂合抗菌蛋白或其类似物;
Y为X的C末端修饰体,所述C末端修饰体包括含有3-10个精氨酸残基的多肽。
2.根据权利要求1所述的抑菌杂合蛋白,其特征在于,所述AB469杂合抗菌蛋白类似物包括与AB469杂合抗菌蛋白编码序列具有至少70%同源性的蛋白、与AB469杂合抗菌蛋白的催化域编码序列至少具有70%同源性的蛋白、与AB469杂合抗菌蛋白的结合域编码序列至少具有70%同源性的蛋白以及与AB469杂合抗菌蛋白催化域和结合域位置互换组成的蛋白编码序列至少具有70%同源性的蛋白。
3.根据权利要求3所述的抑菌杂合蛋白,其特征在于,所述C末端修饰体包括含有5-8个精氨酸残基的多肽。
4.根据权利要求1所述的抑菌杂合蛋白,其特征在于,所述C末端修饰体包括含有3-10个连续的精氨酸残基的多肽。
5.根据权利要求1-4任意一项所述的抑菌杂合蛋白,其特征在于,所述C末端修饰的抑菌杂合蛋白的氨基酸序列如SEQ ID NO.1或SEQ ID NO.3所示。
6.根据权利要求1所述的抑菌杂合蛋白,其特征在于,C末端修饰体还包括疏水性氨基酸残基。
7.根据权利要求6所述的抑菌杂合蛋白,其特征在于,所述C末端修饰体为精氨酸残基与疏水性氨基酸残基间隔性排列。
8.根据权利要求6或7所述的抑菌杂合蛋白,其特征在于,所述疏水性氨基酸残基为丙氨酸残基。
9.根据权利要求8所述的抑菌杂合蛋白,其特征在于,所述C末端修饰的抑菌杂合蛋白的氨基酸序列如SEQ ID NO.5所示。
10.一种核苷酸序列,其特征在于,包括权利要求1-9任意一项所述C末端修饰的抑菌杂合蛋白的编码序列。
11.根据权利要求10所述的核苷酸序列,其特征在于,所述核苷酸序列如SEQ ID NO.2、SEQ ID NO.4或SEQ ID NO.6所示。
12.一种表达载体,其特征在于,包括如权利要求10或11所述核苷酸序列。
13.一种工程菌,其特征在于,包括权利要求10或11所述核苷酸序列,或权利要求12所述表达载体。
14.一种药物组合物,其特征在于,包括权利要求1-9任意一项所述C末端修饰的抑菌杂合蛋白,和药学上可接受的辅料。
15.根据权利要求14所述的药物组合物,其特征在于,所述药物组合物为外用制剂时,所述C末端修饰的抑菌杂合蛋白的质量浓度不超过0.5%。
16.权利要求1-9任意一项所述C末端修饰的抑菌杂合蛋白、权利要求10-11任意一项所述核苷酸序列、权利要求12所述表达载体、权利要求13所述工程菌或权利要求14-15任意一项所述药物组合物在制备抑菌食品、药品或保健品中的应用。
17.根据权利要求16所述的应用,其特征在于,所述抑菌食品、药品或保健品为抑制革兰氏阴性菌的食品、药品或保健品。
18.根据权利要求17所述的应用,其特征在于,所述革兰氏阴性菌包括鲍曼不动杆菌、铜绿假单胞菌、肺炎克雷伯菌、沙门氏菌和大肠杆菌中的一种或多种。
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