团头鲂IL-12表达及功能的初步研究  

Preliminary study on the expression and function of IL-12 in Megalobrama amblycephala

作　　者：武佳琪 王济秀 刘红[1] WU Jiaqi;WANG Jixiu;LIU Hong(Key Lab of Freshwater Animal Breeding,Ministry of Agriculture and Rural Affairs,Engineering Research Center of Green Development for Conventional Aquatic Biological Industry in the Yangtze River Economic Belt,Ministry of Education,College of Fisheries,Huazhong Agricultural University,Wuhan 430070,China)

机构地区：[1]华中农业大学水产学院,农业农村部淡水生物繁育重点实验室,长江经济带大宗水生生物产业绿色发展教育部工程研究中心,湖北武汉430070

出　　处：《水产学报》2022年第11期2028-2037,共10页Journal of Fisheries of China

基　　金：国家自然科学基金(31972781);现代农业产业技术体系建设专项(CARS-46-08)。

摘　　要：为探究白介素-12 (IL-12)在鱼类免疫过程中发挥的功能,实验以团头鲂为研究对象,克隆了团头鲂IL-12的4个亚基IL-12A、IL-12Ba、IL-12Bb和IL-12Bc的开放阅读框(ORF)序列,长度分别为588、993、939和837 bp。实时荧光定量PCR (RT-qPCR)结果显示,团头鲂IL-12各亚基在检测的10个不同的成鱼健康组织中都有表达,但是在不同组织中的表达模式不同;腹腔注射嗜水气单胞菌后,团头鲂IL-12各亚基在免疫相关组织中表达差异显著;脂多糖(LPS)刺激分离的团头鲂头肾淋巴细胞后,4个IL-12亚基均被快速诱导表达。进一步通过大肠杆菌原核表达系统和变性复性方法得到团头鲂IL-12Bb、IL-12Bc重组蛋白,刺激分离的头肾淋巴细胞后,TNF-α、IL-1β、IFN-γ的表达显著上调,表明获得的重组蛋白具有生物学活性。最后对获得的蛋白进行抑菌活性检测,结果表明,重组IL-12Bb、IL-12Bc单体蛋白对嗜水气单胞菌、大肠杆菌和金黄色葡萄球菌都有明显的抑制作用。综上表明,团头鲂IL-12在调节免疫应答的过程中发挥着重要作用。Interleukin-12(IL-12) is an important cytokine that connects the innate and adaptive immune systems and has been shown to play an increasingly important role in immune and inflammatory responses. The study of IL-12 in fish is mainly focused on cloning and expression, but the study of IL-12 in Megalobrama amblycephala, one of the economically important freshwater fish, has not been reported yet. Therefore, in this study, four IL-12 subunits in M. amblycephala were cloned, sequenced and phylogenetically analyzed;the expressions of IL-12 in healthy tissues and after infection by Aeromonas hydrophila in vivo and stimulated by LPS in vitro were detected;and the function of the recombinant proteins of IL-12Bb and IL-12BC was verified. The results showed that the open reading frame(ORF) sequences of IL-12A、IL-12Ba、IL-12Bb and IL-12Bc were 588, 993, 939 and 837 bp,respectively. Quantitative real-time PCR(RT-qPCR) results showed that the four IL-12 subunits were expressed in all the 10 different healthy tissues tested, but their expression patterns were different. After intraperitoneal injection of A. hydrophila, the M. amblycephala IL-12 subunits were significantly induced in the immune-related tissues. After LPS stimulation, the four IL-12 subunits were rapidly induced in the head-kidney lymphocytes(HKL)of M. amblycephala. Furthermore, the recombinant IL-12Bb and IL-12Bc proteins were obtained by Escherichia coli prokaryotic expression system, and then were used to stimulate HKL. The results showed that the expressions of TNF-α, IL-1β and IFN-γ in the stimulated HKL were significantly up-regulated, indicating that the obtained recombinant proteins had biological activity. Finally, the antimicrobial activity of the obtained protein was tested,and the results showed that the recombinant IL-12Bb and IL-12Bc monomers had obvious inhibitory effects on A.hydrophila, E. coli and Staphylococcus aureus. The above results of this study indicate that IL-12 plays an important role in the process of regulating immune response

关 键 词：团头鲂 白介素12 细菌刺激 基因表达 原核表达 抑菌活性 

分 类 号：Q812[生物学—生物工程] S941.42[农业科学—水产养殖]