七鳃鳗g型溶菌酶的分子特征和抗菌活性

Characterization and Antibacterial Activity of g-Type Lysozyme from Lampetra japonica

溶菌酶是先天免疫系统中对抗细菌病原体感染的一种关键蛋白.本研究从七鳃鳗中克隆g型溶菌酶基因.其酶基因c DNA为701 bp(Gen Bank序列号KP204854),开放阅读框为555 bp,编码由184个氨基酸组成的多肽,理论分子质量为20.24 k D,等电点为5.48,含有1个半胱氨酸残基,无信号肽.实时荧光定量PCR分析表明,七鳃鳗g型溶菌酶基因在各组织中广泛表达,其中在肠中表达量最高.脂多糖(LPS)体内刺激七鳃鳗后发现,溶菌酶在口腔腺和头肾表达量显著升高.以溶壁微球菌和哈维弧菌为底物检测重组g型溶菌酶的活性时,均表现出抗菌活性,最适p H为7.5,最适温度为35℃.扫描电镜分析表明,重组酶能够使溶壁微球菌破裂.以上结果均表明,g型溶菌酶在七鳃鳗的先天免疫系统防御病菌感染中起到重要作用.

Lysozyme is a key protein of the innate immune system to fight bacterial pathogen infections.In this study,we cloned a g-type lysozyme gene（ Gen Bank accession number KP204854） from lamprey（ Lampetra japonica）. The c DNA is 701 bp in length with an open reading frame（ ORF） of 555 bp encoding a polypeptide of 184 amino acids. The theoretical molecular weight of the lysozyme is 20. 24 k D with a theoretical p I of 5. 48. One cysteine residue is contained in the protein and no putative signal peptide was found. Real-time PCR results showed that the lamprey g-type lysozyme transcript was detected in multiple tissues with the highest expression in intestine. Furthermore,upon in vivo stimulation of lampreys with lipopolysaccharide（ LPS）,the lysozyme gene was significantly up regulated in buccal gland and head kidney. The bacteriolytic activity of the recombinant lysozyme was detected by Micrococcus lysodeikticus and Vibrio harveyi as substrates,the optimal p H and temperature were 7. 5 and35℃,respectively. In addition,M. lysodeikticus cells were found to be damaged when treated with the lysozyme by scanning electron microscopy（ SEM） analysis. These data suggest that the identified g-type lysozyme should be involved in the innate immune responses of lamprey against pathogen infection.