一种新型贻贝抗菌肽的分离纯化及鉴定

摘要

The researches on antibacterial peptides from Mytilus coruscus, an important mytilus on aquiculture, have significant value that helping people to understand the mechanism of innate immune system of this mussel. By using multi-dimensional reverse phase high performance liquid chromatography (RP-HPLC), a novel antimicrobial peptide was isolated from Mytilus coruscus serum. The mass and the N-terminal sequence of this peptide were analyzed by a combination of Mass Spectrometry and Edman degradation. This peptide consisted 55 amino acids and had a 6621.55 D with 6 Cysteines presented in its sequence forming 3 disulfide bonds. A chitin-biding domain was detected by domain analysis and the peptide was named mytichitin-A therefore. The mRNA transcripts of mytichitin-A were mainly detected in gonad, which indicated that mytichitin-A were specifically synthesized and stored in genital system. The expression level of mytichitin-A in gonad was up-regulated and reached to the highest level at 12h after bacterial challenge, which was 9-fold increase compared to that of the control group. These results indicated that mytichitin-A was involved in the host immune response against bacterial infection and might contribute to the clearance of invading bacteria.%厚壳贻贝(Mytilus coruscus)广泛分布于我国东部海域，其体内富含各种抗菌肽分子，是研究软体动物免疫防御机制以及开发抗菌肽来源的新型生物抗生素的重要对象。采用多步反相高效液相色谱对厚壳贻贝血清进行分离纯化，获得一种分子量为6261.55 D的具有抗菌活性的多肽成分；经多肽N端测序和基因克隆，结果表明该抗菌肽由55个氨基酸残基构成，含6个半胱氨酸并形成三对二硫键。结构域分析表明该抗菌肽具有几丁质结合结构域(Chitin-biding domain)，因此将该抗菌肽命名为mytichitin-A。Mytichitin-A对革兰氏阳性菌具有较强的抑制作用，同时对真菌及革兰氏阴性菌也具有抑制作用。荧光定量 PCR 检测表明， mytichitin-A主要在厚壳贻贝的性腺组织中表达且在细菌诱导后12h其表达量达到峰值。研究为深入了解厚壳贻贝抗菌肽的分子多样性及免疫机制奠定了基础。