齐口裂腹鱼HMGB1基因克隆及其对嗜水气单胞菌胁迫的响应

Cloning of HMGB1 gene from Schizothorax prenanti and its response to Aeromonas hydrophila stress

为了解齐口裂腹鱼高迁移率蛋白B1 (Schizothorax prenanti high mobility group box1,SpHMGB1)的序列特征及其与嗜水气单胞菌胁迫的相关性,实验根据齐口裂腹鱼脾脏TSA库中获得的序列设计引物,采用克隆技术克隆SpHMGB1的核心序列并进行生物信息学分析,采用实时荧光定量PCR (qPCR)技术检测嗜水气单胞菌在体和离体胁迫后组织和巨噬细胞中Sp HMGB1的响应情况。序列分析结果显示,Sp HMGB1开放阅读框长为615 bp,编码204个氨基酸,理论分子量为23.5 ku,等电点为6.79,包含2个基本的HMG盒:A盒和B盒,以及一个酸性尾巴。SpHMGB1二级结构主要由47.06%的α-螺旋和50%的无规则卷曲构成。系统进化树分析显示,SpHMGB1与鱼类HMGB1聚为一支,与斑马鱼和鲫的亲缘关系最近,氨基酸一致性分别为90.7%和90.4%。qPCR检测结果显示,嗜水气单胞菌感染后SpHMGB1具有不同的时空表达趋势,其中血液在72 h表达量最高,肾脏在6 h表达量最高,脾脏在120 h表达量最高;热灭活嗜水气单胞菌刺激巨噬细胞后,SpHMGB1的表达量在0.5～24 h下调,24 h表达量最低,细胞因子IL-1β和TNF-α表达量均有上调趋势。qPCR检测结果提示,SpHMGB1可能参与齐口裂腹鱼细菌感染后的免疫响应。本实验可为进一步研究SpHMGB1在齐口裂腹鱼细菌性疾病中的免疫调节机制提供参考。

In order to understand the immune response of Schizothorax prenanti high mobility group box 1 (SpHMGB1) to Aeromonas hydrophila stress,primers were designed by the sequence obtained from the TSA database of spleen in S.prenanti,then the sequence characterization was analyzed.Finally the response situations were detected by qPCR after A.hydrophila infection in vivo and heat-killed A.hydrophila stimuli in vitro.Sequence analysis results showed that the open reading frame of SpHMGB1 was 615 bp which encoded a peptide of 204 amino acids with a predicted molecular mass of 23.5 Ku and a theoretical isoelectric point of 6.79.The peptide contained two basic HMG boxes:box A and box B,and an acidic tail.The secondary structure was mainly composed of 47.06% of α-helices and 50% of random curls.Phylogenetic tree showed that SpHMGB1 and HMGB1 from Danio rerio and Carassius auratus were clustered together among fish HMGB1 with the high identities of 90.7% and 90.4%,respectively.After A.hydrophila infection,the relative expression of SpHMGB1 was significantly up-regulated at different time in tissues.The highest relative expression of SpHMGB1 appeared in kidney at 6 h,in blood at 72 h and in spleen at 120 h.After heat-killed A.hydrophila stimuli in macrophages,the relative expression of SpHMGB1 was down-regulated within 0.5 -24 h with the lowest at 24 h.Meanwhile the relative expression of IL-1β and TNF-α showed the up-regulated tendency.qPCR results suggested that SpHMGB1 might be involved in the immune response after A.hydrophila infection in S.prenanti.This study provided a theoretical basis for further revealing the immune-modulatory mechanism of SpHMGB1 in bacterial disease of cold-water S.prenanti.