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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 multifunctional chemokines that are an important part of the fish innate immune system. In this study, cDNA sequence of CCL4-like (aCCL-like) gene from sweetfish (Plecoglossus altivelis, ayu) was obtained by library sequencing. It encodes a propeptide consisting of 123 amino acids and the N-terminal 22 amino acids are signal peptide sequences. The mature peptide contains four cysteine residues and can form two disulfide bonds. Phylogenetic tree analysis revealed that aCCL4-like was the closest to CCL4 in Salmo salar (atalantic salmon). Real-time quantitative PCR (RT-qPCR) analysis revealed that mRNA of aCCL4-like gene was expressed in all 7 tissues of healthy smelt, and the highest expression of liver was observed. In the eelston After infection with Listonella anguillarum, the expression of aCCL4-like mRNA in each tissue changed significantly, but the changes of intestinal tissue were the most obvious among different time points. Subsequently, aCCL4-like was expressed in prokaryotic cells and the antiserum was prepared. Western blot analysis revealed that there was no significant change in serum CCL4-like content at 4 h after injection of Leishmania elutingis and a significant increase at 8 h . In conclusion, the change of aCCL4-like gene expression is closely related to the infection process of L. elersei. It is speculated that aCCL4-like may play an important role in the immune response against the infection of pathogenic bacteria.