中国林蛙皮肤抗菌肽基因的cDNA克隆及抗菌、抗癌和溶血活性的测定

Cloning of cDNAs encoding skin antimicrobial peptide precursors from Chinese brown frogs, Rana chensinensis and determination of antimicrobial, anticancer and haemolysis activity

摘要:

采用RT-PCR和3￠RACE方法, 从中国林蛙皮肤总RNA中克隆出了6条编码不同抗菌肽前体的cDNA序列, 分别命名为: preprotemporin-1CEa、preprotemporin-1CEb、preprotemporin-1CEc、preprobrevinin-1CEa、preprobrevinin-1CEb和preprochensinin-1。cDNA碱基序列全长为289~315 bp, 编码59~65个氨基酸。6个抗菌肽前体由3部分结构域组成: 22个氨基酸残基组成的信号肽、多个酸性氨基酸残基组成的中间序列、高度变异的成熟肽。preprotemporin-1CEa、preprotemporin-1CEb和preprotemporin-1CEc属于temporin-1家族抗菌肽前体, 具有肽链短, C-端酰胺化的特点; preprobrevinin-1CEb和preprobrevinin-1CEa属于brevinin-1家族抗菌肽前体, 在肽链的C-端含有RANA盒结构, 可在2个半胱氨酸残基间形成二硫键, 组成7肽环; preprochensinin-1在已知多种数据库中没有发现序列同源的多肽, 被鉴定为新抗菌肽。人工合成temporin-1CEa、temporin-1CEb、brevinin-1CEa和chensinin-1四种中国林蛙皮肤抗菌肽, 活性检测结果表明: 它们对金黄葡萄球菌等3种革兰氏阳性细菌的生长具有明显抑制作用, 同时抑制乳腺癌MCF-7细胞和宫颈癌HeLa细胞生长。

Abstract:

Amphibian skin antimicrobial peptides exhibit a broad spectrum of antimicrobial activity against Gram-positive and Gram-negative bacterium and cytotoxic activity responsible for inhibiting the growth of cancer cells. In this present study, six cDNAs encoding antimicrobial peptide precursors were cloned from the skin of Chinese brown frog, Rana chensinensis by RT-PCR and 3￠-RACE procedure and identified as preprotemporin-1CEa, preprotemporin-1CEb, preprotemporin-1CEc, preprobrevinin-1CEa, preprobrevinin-1CEb, and preprochensinin-1, respectively. The nucleotide sequences of cDNA encoding 59-65 amino acid composed of 289-315 bp. Preprotemporin-1CEa, preprotemporin-1CEb and preprotemporin-1CEc are members of temporin family, which usually are short, hydrophobic, and C-terminally a-amidated antimicrobial peptides. Preprobrevinin-1CEa and preprobrevinin-1CEb were identified as the members of the brevinin-1 family of antimicrobial peptides since both peptides contain “RANA box” that it’s responsible for forming Cys-bridged cyclic heptapeptides at the C-terminal region of peptide. The nucleotide acid sequence and the deduced amino acid Sequence of preprochensinin-1 were not found to be identity with any known amphibian skin defensive peptides, so, preprochensinin-1 was identified as a novel peptide precursor. Four of bioactive peptides: temporin-1Cea, temporin-1Ceb, brevinin-1CEa and chensinin-1 were synthesized to investigate their antimicrobial, anticancer and haemolysis activities. The results showed that all of the synthesized antimicrobial peptides in this study inhibited the growth of the Gram-positive bacterium, and exhibited the anticancer activity against the growth of MCF-7 cells and HeLa cells. Analysis of the R. chensinensis bioactive peptides and their gene expression will be beneficial for preservation of this species.