厚壳贻贝抗菌肽Mytilin的初步鉴定

Purification and identification of Mytilins from Mytilus coruscus

厚壳贻贝是我国具有重大经济价值的水产养殖贝类,对其抗菌肽的研究有助于人们了解厚壳贻贝的免疫机制。为了解厚壳贻贝血清中抗菌肽Mytilin的分子组成和特性,采用多维高效液相色谱对厚壳贻贝血清进行分离纯化,从中获得3种对革兰氏阳性菌以及阴性菌均有抑制作用的抗菌肽分子,序列分析表明3种抗菌肽具有较高的序列相似性,均属于贻贝抗菌肽Mytilin家族,分别命名为Mytilin-1,Mytilin-2和Mytilin-3。其中,Mytilin-1为34个氨基酸残基构成的多肽,其分子量为3885.17u,含8个半胱氨酸,形成4对二硫键。根据所测Mytilin-1的氨基酸序列设计特异性引物,通过菌落PCR方法筛选厚壳贻贝cDNA文库,获得Mytilin-1的cDNA基因并进行了序列分析。以上研究结果表明,作为贻贝的主要抗菌肽家族,Mytilin在厚壳贻贝血清中具有较高丰度,对其蛋白质序列以及基因序列的研究为深入了解厚壳贻贝Mytilin抗菌肽的分子多样性奠定了基础。

The researches on antibacterial peptides from Mytilus coruscus, an important Mytilus in aquaculture, have significant value helping people to understand the mechanism of innate immune system of this mussel. Here, three peptides with antibacterial activity were purified from Mytilus coruscus serum by multi-dimensional high performance liquid chromatography （HPLC）. The three peptides exhibited complementary antimicrobial properties against both gram-positive and gram-negative bacteria. The mass and the N-terminal sequences of these peptides were analyzed by a combination of Edman degradation and Mass Spectrometry. Based on the results of sequential BLAST, these antibacterial peptides from Mytilus eoruscus serum belong to Mytilin family and are named Mytilin-1, Mytilin-2 and Mytilin-3, respectively ; The molecular mass of these antibacterial peptides are 3885.17 u ,3993.26 u and 3991.39 u, respectively. Among them, Mytilin-1 was characterized as a 34-residues peptide including eight cyctines formed four disulfides. The cDNA sequence coding for the Mytilin-1 precursor was obtained by screening PCR from the cDNA library of Mytilus coruscus blood cell. The precursor of Mytilin-1 contains a putative signal peptide of 22 residues, a processing peptide sequence of 34 amino acids, and a C-terminal extension of 46 residues rich in acidic residues. This study lays the foundation for further research about the molecular diversity and the mechanism of these antibacterial peptides from Mytilus coruscus serum.