尼罗罗非鱼淋巴毒素α基因克隆与表达分析  

Cloning and Expression Analysis of Gene Lymphotoxin α in Nile Tilapia(Oreochromis Niloticus)

作　　者：邹子鸿 郑琦 黄瑜[1] 汤菊芬[1] 王蓓[1] 鲁义善[1] 简纪常[1] 蔡佳[1] ZOU Zi-hong;ZHENG Qi;HUANG Yu;TANG Ju-fen;WANG Bei;LU Yi-shan;JIAN Ji-chang;CAI Jia(Fisheries College of Guangdong Ocean University,Guangdong Provincial Key Laboratory of Pathogenic Biology and Epidemiology for Aquatic Economic Animals&Key Laboratory of Control for Diseases of Aquatic Economic Animals of Guangdong Higher Education Institutes,Zhanjiang 524088,China)

机构地区：[1]广东海洋大学水产学院广东省水产经济动物病原生物学及流行病学重点实验室暨水产经济动物病害控制广东省普通高等学校重点实验室

出　　处：《广东海洋大学学报》2019年第6期1-8,共8页Journal of Guangdong Ocean University

基　　金：国家自然科学基金(31302226,31572651);广东海洋大学优秀硕士学位论文培育项目(201833)

摘　　要：【目的】克隆尼罗罗非鱼(Oreochromis niloticus)淋巴毒素α基因(lymphotoxin alpha, LTα),分析该基因的组织分布,探讨该基因在抗菌抗病毒免疫过程中的重要作用。【方法】通过RACE技术获得尼罗罗非鱼淋巴毒素α基因(On-LTα)的cDNA全长,通过生物信息学分析其序列结构特征;利用实时荧光定量PCR检测基因的组织分布特征,以及健康尼罗罗非鱼感染无乳链球菌后该基因的表达变化,并进一步分离出尼罗罗非鱼非特异性毒性细胞(NCC),检测脂多糖(LPS)和聚肌胞苷酸(Poly I:C)刺激后,On-LTα在NCC中的表达变化。【结果】On-LTα基因序列全长为2769 bp,包含705 bp开放阅读框(ORF),编码234个氨基酸(GenBank登录号:MK770358)。该蛋白属于跨膜蛋白,具有肿瘤坏死因子(TNF)家族保守结构域。实时荧光定量结果表明,On-LTα在健康尼罗罗非鱼11种组织中均有表达,在脾脏中的表达量最高;无乳链球菌刺激后,该基因在脾脏中的表达量显著升高;LPS与PolyI:C刺激后,On-LTα在NCC中表达量也显著上调。【结论】On-LTα参与了尼罗罗非鱼抗菌抗病毒的免疫应答过程。【Objective】To clone and analyze the lymphotoxin alpha gene(LTα),analyze the transcript distribution in different tissues,and to explore the important role of lymphotoxin alpha(LTα)in the antibacterial and antiviral immunity of Oreochromis niloticus.【Method】The full-length cDNA of lymphotoxinαfrom Nile Tilapia(On-LTα)was obtained by RACE.The sequence of On-LTαwas analyzed by bioinformatics method.The tissue distribution and expression profiles of On-LTαfollowing Streptococcus lactobacillus immunization were investigated by Real-time quantitative PCR.Furthermore,On-LTαtranscript levels in the isolated non-specific toxic cells(NCC)after lipopolysaccharide(LPS)and Poly I:C stimulation were also examined【.Results】On-LTαgene sequence is 2 769 bp including a 705 bp open reading frame(ORF)which encodes a polypeptide of 234 amino acids.(GenBank accession number:MK770358).On-LTαprotein belonged to the transmembrane protein and possessed a conserved domain of the tumor necrosis factor(TNF)family.Quantification PCR found that On-LTαwas constitutively expressed in all 11 tissues tested.In the healthy Nile tilapia,the highest expression being detected in the spleen.The relative expression levels of On-LTαin the spleen were significantly increased during S.agalactia infection.The isolated NCCs cultured in vitro could also induce the significant upregulation of On-LTαunder LPS and Poly I:C stimulation.【Conclusion】On-LTαmight be involved in the antimicrobial and antiviral immune responses of tilapia.

关 键 词：尼罗罗非鱼 淋巴毒素α基因 基因克隆 基因表达 非特异性细胞毒性细胞(NCC) 

分 类 号：Q954.4[生物学—动物学]