为探究大黄鱼自噬相关基因5 (LcATG5)在病毒感染中免疫应答中的作用，本实验从大黄鱼中克隆得到了自噬相关基因ATG5 (LcATG5)，其开放阅读框 (ORF)全长828个核苷酸，编码275个氨基酸的蛋白质，预测的分子量为32.3 ku，等电点为5.7。氨基酸序列比对和系统进化分析结果显示，LcATG5与其他物种ATG5之间的序列一致性较高，含有1个高度保守的APG5结构域，并且与棘头梅童鱼ATG5的亲缘关系最近。荧光定量PCR结果显示，LcATG5在所检测的11个组织或器官中均有表达，在血液中表达量最高，在脾脏中表达量最低；LcATG5在来源于大黄鱼头肾组织的原代粒细胞、淋巴细胞和巨噬细胞以及细胞系LYCK细胞中也均有表达，在原代粒细胞中表达量相对较高，而在巨噬细胞中相对较低；病毒类似物poly(I:C)刺激后，这4种免疫细胞中LcATG5的表达水平都显著上调，其在LYCK细胞中变化最为显著，刺激后12 h上调了3.93倍。过表达LcATG5的鲤上皮瘤 (epithelioma papulosum cyprini, EPC)细胞受鲤春病毒血症病毒 (spring viremia of carp virus, SVCV)感染48 h后，细胞病变效应 (cytopathic effects, CPE)明显高于对照组，细胞培养上清中SVCV的滴度为10^13.82 TCID₅₀/mL，高于对照组10^9.27 TCID₅₀/mL，同时细胞内SVCV标志基因SVCV-G、SVCV-M和SVCV-P的表达量分别上调了13.77倍、15.72倍和11.39倍，表明LcATG5过表达促进了EPC细胞中SVCV病毒增殖，上述结果为深入研究自噬和自噬相关基因在鱼类病毒感染过程中的作用及机制奠定了基础。

Autophagy is an important process that maintains homeostasis in eukaryotic cells and is involved in cell differentiation, development, and immunity. However, little is known about the function of autophagy-related genes (ATGs) in the immune response of fish. To explore the function of ATG5 in virus infection of the Larimichthys croaker, we cloned a ATG5 gene from large yellow croaker (Larimichthys crocea) and its open reading frame (ORF) was 828 nucleotides long, encoding a protein of 275 amino acids. The predicted molecular weight of LcATG5 was 32.3 ku and its theoretical isoelectric point was 5.7. Homology comparison showed that LcATG5 had a highly conserved APG5 domain as found in other vertebrate ATG5 proteins. Phylogenetic analysis showed that LcATG5 was closely related to Collichthys lucidus ATG5. LcATG5 was detected in all eleven tissues of L. crocea, with highest expression in blood and lowest in spleen. LcATG5 transcripts were also detected in primary head kidney granulocytes, lymphocytes and macrophages, and L. crocea head kidney cell line (LYCK), with highest expression in primary head kidney granulocytes. After induction by poly(I:C), LcATG5 transcripts were significantly increased in these cells, and were more responsive in LYCK cells, with 3.93-fold increase at 12 h. In the LcATG5-overexpressed epithelioma papulosum cyprini (EPC) cells, the cytopathic effects (CPE) caused by spring viremia of carp virus (SVCV) infection were increased at 48 h post-infection. The viral titer of SVCV in the culture supernatant of the LcATG5-overexpressed EPC cells was 10^13.82 TCID₅₀/mL, which was higher than that of control group (10^9.27 TCID₅₀/mL). Besides, the expression levels of three viral genes (SVCV-G, SVCV-M, and SVCV-P) were significantly up-regulated in the LcATG5-overexpressed cells with 13.77-, 15.72-, and 11.39-fold increases, respectively. These results indicate that LcATG5 plays a role in promoting virus replication, which may provide a basis for in-depth study of the function and mechanism of autophagy and ATGs during virus infection in fish.

S 942.3

国家重点研发计划 (2018YFD0900600)；国家自然科学基金 (U1905204，32073007)；财政部和农业农村部：国家现代农业产业技术体系专项 (CARS-47)；福建农林大学“双一流”建设科技创新能力提升培育计划 (KSYLP001)