香鱼CCL4-like基因的克隆、序列分析及免疫相关性表达变化分析  

Molecular Cloning,Sequences Analysis and Immune-related Expression Changes of aCCL4-like Gene from Ayu(Plecoglossus altivelis)

作　　者：杨旦阳[1] 陈炯[1] 陆新江[1] 史雨红[1] 李明云[1] 

机构地区：[1]宁波大学应用海洋生物技术教育部重点实室,宁波315211

出　　处：《中国细胞生物学学报》2013年第5期676-683,共8页Chinese Journal of Cell Biology

基　　金：国家自然科学基金(批准号:31201970);教育部新世纪优秀人才支持计划(批准号:NCET-08-0928)资助的课题~~

摘　　要：CC型趋化因子是一种多功能的趋化因子,是鱼类先天免疫系统的重要组成部分。该研究通过文库测序方法获得香鱼(Plecoglossus altivelis,ayu)CCL4-like(aCCL-like)基因cDNA序列。它编码一个由123个氨基酸组成的前肽,N-端22个氨基酸为信号肽序列。成熟肽包含4个半胱氨酸残基,可形成2个二硫键。系统进化树分析揭示,aCCL4-like与大西洋鲑(Salmo salar,atalantic salmon)CCL4亲缘关系最近。实时荧光定量PCR(Real-time quantitative PCR,RT-qPCR)分析揭示,aCCL4-like基因mRNA在检测的健康香鱼7个组织中均有表达,其中肝的表达量最高;在鳗利斯顿氏菌(Listonella anguillarum)感染后,各组织中aCCL4-like基因mRNA表达均显著变化,但各时间点变化不一,其中肠组织的变化最为明显。随后原核表达了aCCL4-like并制备相关抗血清,Western blot分析揭示,鳗利斯顿氏菌注射后4 h的血清中CCL4-like含量无显著变化,8 h时显著增加,此后表现为持续增加。综上,aCCL4-like基因的表达变化与鳗利斯顿氏菌的感染过程密切相关,推测aCCL4-like可能在香鱼抗病原菌感染的免疫反应过程中有重要作用。CC chemokines are a kind of multi-functional cytokines and play vital roles in innate immune system of teleost fish. In this study, we obtained a cDNA sequence encoding CCL4-1ike from ayu. The predicted ayu CCL4-1ike （aCCL4-1ike） propeptide was 123 amino acids （aa） long with a 22 aa signal peptide in the N-terminus. The mature peptide contained four conserved cysteine residues and they could form two disulfide bonds. Phylogenetic tree analysis showed that Plecoglossus altivelis CCL4-1ike was closely to Salmo salar CCL4. The Real-time quantitative PCR（RT-qPCR） analysis displayed that aCCL4-like mRNAs were widely existed in kinds of tissues of healthy ayu, and that the highest expression was in the liver. After Listonella anguillarum （L.anguillarum） infection, aCCL4-like mRNAs were dramatically up-regulated in all detected ayu tissues, especially in the intestine. Followed by aCCL4-1ike prokaryotic expression and its preparation of antiserum. Western blot analysis revealedthat the ayu serum aCCL4-1ike was no significant change after bacterial infection after 4 h, but increased suddenly at 8 h and since then continued to raise. In summary, our results suggested that aCCL4-like gene expression level was closely related to the progress of L.anguillarum infection, which predicts aCCL4-1ike＇role in the immune response against microbial infections of ayu.

关 键 词：CC型趋化因子 香鱼 鳗利斯顿氏菌 实时荧光定量PCR 

分 类 号：S917.4[农业科学—水产科学]