Molecular Cloning of cDNAs Encoding Antimicrobial Peptide Precursors from the Skin of the Chinese Brown Frog, Rana chensinensis
Molecular Cloning of cDNAs Encoding Antimicrobial Peptide Precursors from the Skin of the Chinese Brown Frog, Rana chensinensis
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DOI：
10.2108/zsj.26.220
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发表时间：
2009-04
期刊：
影响因子：
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通讯作者：
D. Shang;F. Yu;Junfeng Li;Junjie Zheng;Lifang Zhang;Yang Li-
中科院分区：
文献类型：
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作者：
D. Shang;F. Yu;Junfeng Li;Junjie Zheng;Lifang Zhang;Yang Li-
皮肤在两栖动物的日常生存中起着关键作用。在本研究中，从中国褐蛙中克隆并鉴定了 6 个编码两栖动物皮肤抗菌肽前体的 cDNA，并鉴定为 preprobrevinin-1CEa、preprobrevinin-1CEb、preprotemporin-1CEa、preprotemporin-1CEb、preprotemporin-1CEc 和 preprochensinin-1。 Preprotemporin-1CEa、CEb 和 CEc 是 temporin 家族的成员，通常是短的、疏水性的、C 末端酰胺化的抗菌肽。 Preprobrevinin-1CEa 和 CEb 被鉴定为 brevinin-1 抗菌肽家族的成员，因为这两种肽均含有负责形成 C 端半胱氨酸桥环七肽的“Rana 盒”。 preprochensinin-1 的核苷酸和推导的氨基酸序列与任何已知的两栖动物皮肤防御肽都不相似。根据推导的6种中国树皮前原肽的氨基酸序列，化学合成了4种生物活性肽，并对其抗菌、细胞毒性和溶血特性进行了评价。所有合成的肽均抑制革兰氏阳性菌的生长。 Brevinin-1CEa 显示出广谱抗菌活性。新型两栖动物皮肤肽 chensinin-1 在 11.6 µM 浓度下具有抗蜡样芽胞杆菌和乳酸链球菌活性，但在 200 µM 浓度下不抑制 MCF-7 和 HeLa 细胞的生长，在 500 µM 浓度下无溶血活性。 Temporin-1CEa表现出最大的抑制MCF-7细胞生长的能力。其抗菌和细胞毒活性可能归因于其高度的β-螺旋确认和两亲性。
Skin plays a key role in the daily survival of amphibians. In the present study, six cDNAs encoding amphibian skin antimicrobial peptide precursors from the Chinese brown frog Rana chensinensis, were cloned and identified as preprobrevinin-1CEa, preprobrevinin-1CEb, preprotemporin-1CEa, preprotemporin-1CEb, preprotemporin-1CEc, and preprochensinin-1. Preprotemporin-1CEa, CEb, and CEc are members of the temporin family, which are usually short, hydrophobic, and C-terminally -amidated antimicrobial peptides. Preprobrevinin-1CEa and CEb were identified as members of the brevinin-1 family of antimicrobial peptides, because both peptides contain a “Rana box” that is responsible for forming C-terminal Cys-bridged cyclic heptapeptides. The nucleotide and deduced amino acid sequences of preprochensinin-1 were not similar to any known amphibian skin defensive peptides. Four bioactive peptides were chemically synthesized according to the deduced amino acid sequences of six prepropeptides from R. chensinensis skin, and their antimicrobial, cytotoxic, and haemolytic properties were evaluated. All of the synthesized peptides inhibited the growth of Gram-positive bacteria. Brevinin-1CEa showed a broad spectrum of antimicrobial activity. The novel amphibian skin peptide chensinin-1 was active against Bacillus cereus and Streptococcus lactis at a concentration of 11.6 µM, but did not inhibit the growth of MCF-7 and HeLa cells at 200 µM, and had no haemolytic activity at a concentration of 500 µM. Temporin-1CEa exhibited the greatest ability to inhibit the growth of MCF-7 cells. Its antimicrobial and cytotoxic activities may be due to its high degree of -helical confirmation and amphipathic nature.