【作者】 侯志国； 房文红； 李新苍；

【Author】 HOUZhiguo;FANG Wenhong;LI Xincang;East China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences;

【机构】 农业部东海与远洋渔业资源开发利用重点实验室东海水产研究所中国水产科学研究院； 上海海洋大学水产与生命技术学院；

【摘要】 抗脂多糖因子(ALFs)是一类具有结合脂多糖(LPS)功能的抗菌肽。鉴于大多数ALFs属于阳离子抗菌肽,且其生物学功能得到了较好的阐明,而阴离子型ALFs生物学功能研究相对较少。本研究从拟穴青蟹(青蟹)中鉴定了一种阴离子型ALF(SpALF6),并通过一系列实验验证了其免疫学功能。SpALF6的cDNA序列全长681bp编码115 AA,其成熟肽和核心结构域pI值分别为6.05和6.79,因此属于阴离子型抗菌肽。SpALF6主要表达于血细胞,弧菌和金黄色葡萄球菌刺激可诱导该基因表达。重组SpALF6能抑制副溶血弧菌、气单胞菌、金黄色葡萄球菌、巨大芽孢杆菌及白色念珠球菌生长。进一步研究发现SpALF6能结合副溶血弧菌、金黄色葡萄球菌、巨大芽孢杆菌及白色念珠球菌的菌体,通过对细菌主要多糖的特异性结合实验证明,SpALF6与菌体的结合活性主要通过与LPS及脂磷壁酸的结合实现,而SpALF6的核心结构域虽能结合多种细菌多糖,但不能抑制上述细菌生长。本研究表明阴离子型抗菌肽SpALF6仍然在青蟹抗菌免疫反应中发挥作用。更多还原

【Abstract】 Anti-lipopolysaccharide factors(ALFs) are antimicrobial peptides with binding and neutralizing activities to lipopolysaccharide(LPS) in crustaceans. This study identified and characterized a novel ALF homolog(SpALF6) from the mud crab Scylla paramamosain. The complete cDNA of SpALF6 had 681 bp with a 348 bp open reading frame encoding a protein with115 aa. SpALF6 had a low isoelectric point(p I) for the mature peptide and the LPS-binding domain with the values of 6.05 and 6.79, respectively. SpALF6, mainly distributed in hemocytes,could be upregulated by Vibrioparahemolyticus,and Staphylococcus aureus. Recombinant SpALF6 could inhibit the growth of Gram-negative bacteria(Vibrio Parahemolyticus, Aeromonas hydrophila), Gram-postive bacteria(S. aureus and Bacillus megaterium), and a fungus Candida albicans to varying degrees. Further study showed that it could also bind to all the aforementioned microorganisms except Aeromonas hydrophila. Polysaccharide-binding assay conducted by ELISA showed that LPS and LTA are responsible for bacterial cell binding activity of Gram-negative and Gram-postive bacteria, respectively.更多还原