香鱼HMGB1克隆、序列分析及表达特征

Cloning,sequence analysis and mRNA expression pattern of HMGB1 in Sweetfish( Plecoglossus altivelis)

高迁移率族蛋白B1(High mobility group box protein 1,HMGB1)属于晚期细胞因子,与炎症反应相关。为研究香鱼HMGB1在鳗利斯顿氏菌感染引起的炎症反应中的作用,采用随机测序从肝脏组织cDNA文库中获得HMGB1基因,全长1233 bp,编码一个由204个氨基酸组成的23.3 kDa的蛋白。分析表明,该蛋白与胡瓜鱼HMGB1蛋白同源性最高。健康香鱼HMGB1基因在肌肉、脑和肝脏组织中表达量最高,而在鳃和肠中几乎不表达。qRTPCR表明,鳗利斯顿氏菌感染过程中,香鱼肌肉、脑和肝脏组织中HMGB1基因表达显著上调,分别在72、48 h达到峰值。原核表达香鱼HMGB1重组蛋白并制备抗血清,Western blot显示,抗血清能与目的蛋白起特异性反应,能用于研究HMGB1蛋白表达变化,进一步揭示HMGB1基因与鳗利斯顿氏菌感染引起的炎症晚期反应相关性。

HMGB1 （High mobility group box protein1） was a late cytokine involved in inflammatory reaction.This study aimed to analyzethe correlation between sweetfish HMGB1 and the inflammation post Listonella anguli larum infection.The HMGB1 was clonedfrom liver cDNA library by using random sequencing which contains 1233 nucleotides and encoded a protein of 204 amino acids with amolecular weight of 23.3 kDa.Analysis showed that this recombinant protein shared highest homology with rainbow smelt HMGB1.The HMGB1 expression was highest in muscle, brain and liver, while expressed little in gill and intestine.qRT-PCR analysis showed thatsweetfish HMGB1 transcripts were significantly up-regulated in muscle, brain and liver at 72 h or 48 h post Listonella anguillarum infection.Recombinant protein was produced by using prokaryotic expression and the antiserum reacted strongly with recombinant proteinshowed by western blot, suggesting that this antiserum can be used to study the role of HMGB1 in fish late inflammatory reaction post Listonella anguillarum infection.