论文部分内容阅读
白介素-12B(interleukin-12B,IL-12B)是组成IL-12分子的亚基,参与病原菌感染引起的免疫反应。该研究经香鱼(Plecoglossus altivelis)巨噬细胞转录组测序获得其IL-12B基因,包括完整的开放阅读框在内,序列大小为1 152 bp,预测编码的前体蛋白由303个氨基酸组成,N-端22个氨基酸残基为信号肽序列。序列比对及进化树分析表明,香鱼IL-12B与大西洋鲑IL-12B进化关系最近,氨基酸序列同源性达50%。香鱼IL-12B基因mRNA在健康香鱼头肾组织和外周血白细胞中表达量最高,在脾脏、腮和肠等组织也有少量表达。荧光定量PCR分析揭示,鳗利斯顿氏菌(Listonella anguillarum)感染后,香鱼头肾组织和外周血白细胞IL-12B基因mRNA表达显著上调,12 hpi(hours post infection)时达到峰值。随后原核表达了香鱼IL-12B,并制备抗血清。Western blot结果揭示,香鱼血清IL-12B含量在鳗利斯顿氏菌感染后4 hpi时尚无显著变化,8 hpi后显著上调,并表现持续增加。综上,香鱼IL-12B基因的表达变化与鳗利斯顿氏菌感染过程密切相关,揭示香鱼IL-12B基因可能在病原菌感染引起的免疫反应中发挥重要作用。
Interleukin-12B (IL-12B) is a subunit that makes up the IL-12 molecule and is involved in the immune response caused by pathogen infection. In this study, IL-12B gene was obtained from the Plecoglossus altivelis macrophage transcriptome. The complete open reading frame (ORF) of IL-12B gene was 1 152 bp in length. The predicted precursor protein consisted of 303 amino acids, The N-terminal 22 amino acid residues are signal peptide sequences. Sequence alignment and phylogenetic tree analysis showed that the evolutionary relationship between the squid IL-12B and the Atlantic salmon IL-12B was the closest and the amino acid sequence homology was 50%. The expression of IL-12B mRNA in the sweetfish was the highest in the head kidney and peripheral blood leukocytes of healthy fish, but also in the spleen, gills and intestine. Fluorescent quantitative PCR analysis revealed that IL-12B mRNA expression was significantly up-regulated in the head kidney tissues and peripheral leukocytes of the fish after infection with Listonella anguillarum, reaching a peak at 12 hpi (hours post infection). Subsequently, prokaryotic expression of the sweetfish IL-12B, and preparation of antiserum. The results of Western blot revealed that the serum IL-12B level in the fish did not change significantly at 4 hpi after infection with E. anguilla and was significantly up-regulated after 8 hpi, and its performance continued to increase. In summary, the changes of the expression of IL-12B gene in sweetfish are closely related to the infection process of L. elersei, revealing that the IL-12B gene may play an important role in the immune response induced by pathogen infection.