肿瘤坏死因子α(TNFα),主要由活跃的巨噬细胞和单核细胞分泌的细胞因子,导致肿瘤组织出血性坏死,杀死肿瘤细胞,调节炎症反应,在先天免疫中起着至关重要的作用。在这项研究中,获得了日本血吸虫的TNFα(命名为SjTNFα),其全长cDNA为1206bp(GenBank登录号。ON357428),包含185bp的5'UTR,137bp的3'UTR和1002bp的开放阅读框(ORF),以编码333个氨基酸的推定肽,用于构建跨膜结构域和细胞质TNF结构域。其预测的pI为8.69,理论分子量为44.72KDa。多序列比对和系统发育分析表明,SjTNFα与章鱼的同源性最高,它们落入一个统一的分支,并进一步与其他动物聚集在一起。Real-timePCR表明SjTNFα在所有受试者组织中广泛表达,包括脾脏,胰腺,吉尔,心,大脑,视神经瓣,肝脏和肠道,肝脏最高,大脑最低。SjTNFα的相对表达在幼年期的发育期有所不同,鱿鱼产卵前,肝脏在幼年期和产卵期最高,在产卵前的视叶中。感染副溶血性弧菌和嗜水气单胞菌后,SjTNFα在肝脏和ill中的表达均随时间上调,在不同感染的肝脏中,最高表达出现在24h和8h,并在持续4h在吉尔。细胞定位显示SjTNFα分布在HEK293细胞膜上,因为它是一种II型可溶性跨膜蛋白。用不同浓度的LPS刺激HEK293细胞时,NF-κB通路在细胞核中被激活,相应的mRNA通过细胞内信号转导通路转移,导致TNFα的合成和释放,这使得SjTNFα的表达明显上调。这些发现表明,SjTNFα可能在日本血吸虫抵抗细菌攻击的防御中起重要作用,这有助于理解头足类的内在免疫信号通路,并进一步研究宿主与病原体的相互作用。
Tumor necrosis factor α (TNFα), a cytokine mainly secreted by active macrophages and monocytes, causes hemorrhagic necrosis of tumor tissues, kills tumor cells, regulates inflammatory responses, and plays a crucial role in innate immunity. In this study, TNFα of Sepiella japonica (named as SjTNFα) was acquired, whose full-length cDNA was 1206 bp (GenBank accession no. ON357428), containing a 5\' UTR of 185 bp, a 3\' UTR of 137 bp and an open reading frame (ORF) of 1002bp to encode a putative peptide of 333 amino acids for constructing the transmembrane domain and the cytoplasmic TNF domain. Its predicted pI was 8.69 and the theoretical molecular weight was 44.72 KDa. Multiple sequence alignment and phylogenetic analysis showed that SjTNFα had the highest homology to Octopus sinensis, they fell into a unified branch and further clustered with other animals. Real-time PCR indicated that SjTNFα was widely expressed in all subject tissues, including spleen, pancreas, gill, heart, brain, optic lobe, liver and intestine, and exhibited the highest in the liver and the lowest in the brain. The relative expression of SjTNFα varied at the developmental period of juvenile stage, pre-spawning and oviposition in the squid, with the highest in the liver at the juvenile stage and oviposition, and in the optic lobe of pre-spawning. After being infected with Vibrio parahaemolyticus and Aeromonas hydrophila, the expression of SjTNFα in liver and gill were both upregulated with time, and the highest expression appeared at 24 h and 8 h in liver for different infection, and at 4 h in gill consistently. Cell localization showed that SjTNFα distributed on membrane of HEK293 cells because it was a type II soluble transmembrane protein. When HEK293 cells were stimulated with LPS of different concentrations, the NF-κB pathway was activated in the nucleus and the corresponding mRNA was transferred through the intracellular signal transduction pathway, resulting in the synthesis and release of TNFα, which made the expression of SjTNFα was up-regulated obviously. These findings showed that SjTNFα might play an essential role in the defense of S. japonica against bacteria challenge, which contributed to the understanding of the intrinsic immune signaling pathway of Cephalopoda and the further study of host-pathogen interactions.