摘要
【目的】明确分化群抗原209(CD209)变体CD209v-1与罗非鱼机体免疫功能的相关性,为深入探析CD209及其家族成员在鱼类免疫系统中的作用机制提供理论依据,同时为罗非鱼细菌性疾病的防治及新型疫苗研发打下基础。【方法】采用PCR扩增尼罗罗非鱼CD209v-1基因(OnCD209v-1)开放阅读框(ORF),利用Berry-Psite、InterProScan、TMHMM 2.0、ExPASy、SignalP 4.1及SWISS-MODEL等在线软件进行生物信息学分析,并以实时荧光定量PCR检测OnCD209v-1基因在尼罗罗非鱼各组织中的表达情况。随后克隆OnCD209v-1基因胞外段构建pET-OnCD209v-1重组原核表达质粒,经原核诱导表达后,通过细菌结合试验和凝集试验体外探究rOnCD209v-1蛋白对多种水生动物致病菌的识别结合及凝集功能。【结果】OnCD209v-1基因ORF长780 bp,共编码259个氨基酸残基;OnCD209v-1基因编码蛋白相对分子量为29.424 kD,理论等电点(pI)为6.54,在第113~246位氨基酸间存在1个保守的C型凝集素(CTL)结构域。OnCD209v-1氨基酸序列与横线扁鼻丽鱼CD209氨基酸序列的相似性最高(86.49%),基于CD209氨基酸序列相似性构建的系统发育进化树也显示尼罗罗非鱼与横线扁鼻丽鱼先聚在同一分支上,二者的亲缘关系较近。OnCD209v-1基因在尼罗罗非鱼各组织中均有表达,尤其以肝脏和肠道中的相对表达量较高,其次是血液。原核诱导表达获得的rOnCD209v-1蛋白能识别并结合多种致病菌,且在Ca2+存在的情况下对嗜水气单胞菌、大肠杆菌、无乳链球菌、副溶血弧菌和金黄色葡萄球菌具有凝集活性。【结论】OnCD209v-1基因是罗非鱼CD209基因的变体,具有1个保守的CTL结构域,广泛表达于罗非鱼各组织中;原核诱导表达获得的rOnCD209v-1蛋白能识别并结合嗜水气单胞菌、大肠杆菌、哈维氏弧菌、无乳链球菌、副溶血弧菌及金黄色葡萄球菌等常见致病菌,在Ca2+存在的情况下对多种病原菌具有凝集活性。即OnCD209v-1基因作为重要的免疫分子参与了罗非鱼响应细菌感染的免疫过程。
【Objective】This paper clarified the correlation between the differentiated group antigen 209(CD209)variant CD209v-1 and the immune function of tilapia,exploring the mechanism of CD209 and its family members in the fish immune system,and laying a foundation for the prevention and cure of tilapia bacterial disease and the research and deve-lopment of new vaccines.【Method】The open reading frame(ORF)of the Oreochromis niloticus CD209v-1 gene(OnCD209v-1)was amplified by PCR.Bioinformatics analysis was performed using online softwares including BerryPsite,InterProScan,TMHMM 2.0,ExPASy,SignalP 4.1 and SWISS-MODEL.OnCD209v-1 gene expression in various tissues of O.niloticus was detected by real-time fluorescence quantitative PCR.Subsequently,the extracellular segment of OnCD209v-1 gene was cloned to construct pET-OnCD209v-1 recombinant prokaryotic expression plasmid.After prokaryotic-induced expression,the recognition,binding,and agglutination function of rOnCD209v-1 protein on a variety of aquatic animal pathogenic bacteria were explored by bacterial binding assay and pooled test in vitro.【Result】The ORF of OnCD209v-1 gene was 780 bp and encoded 259 amino acid residues;the relative molecular weight of the encoding protein of OnCD209v-1 gene was 29.424 kD.The theoretical isoelectric point(pI)was 6.54,and there was a conserved C-type lectin(CTL)domain between amino acids 113 and 246.The OnCD209v-1 amino acid sequence had the highest similarity to Simochromis diagramma CD209 amino acid sequence(86.49%).The phylogenetic evolutionary tree based on the CD209 amino acid sequence similarity also showed that the O.niloticus was clustered with S.diagramma,and was most closely related to S.diagramma.OnCD209v-1 gene existed in all analyzed tissues,with higher relative expression in the liver and intestine especially,followed by blood.The rOnCD209v-1 protein obtained by prokaryotic-induced expression recognized and bound a variety of pathogenic bacteria and had agglutination activity in Aeromonas hydrophila,Escherichia coli,Streptococcus agalactiae,Vibrio parahemolyticus and Staphylococcus aureus in the presence of Ca2+.【Conclusion】The OnCD209v-1 gene is a variant of tilapia CD209 gene,with a conserved CTL domain,and is widely expressed in tilapia tissues;rOnCD209v-1 obtained by prokaryotic-induced expression can recognize and bind common pathogenic bacteria,such as A.hydrophila,E.coli,Vibrio harveyi,S.agalactiae V.parahaemolyticus and S.aureus,with agglutination activity in a variety of pathogens in the presence of Ca2+.The OnCD209v-1 gene participates as an important immune molecule in the immune process of tilapia’s response to bacterial infection.
作者
宋长江
喻大鹏
赵天珍
唐怀庆
丘金珠
梁富
郑汉生
黄梅燕
黄瑜
宋海霞
SONG Chang-jiang;YU Da-peng;ZHAO Tian-zhen;TANG Huai-qing;QIU Jin-zhu;LIANG Fu;ZHENG Han-sheng;HUANG Mei-yan;HUANG Yu;SONG Hai-xia(Zhongshan Institute for Quality and Safety Inspection of Agricultural Products,Zhongshan,Guangdong 528400,China;College of Fisheries,Guangdong Ocean University,Zhanjiang,Guangdong 524088,China;Shenzhen Institute of Guangdong Ocean University,Shenzhen,Guangdong 510000,China)
出处
《南方农业学报》
CAS
CSCD
北大核心
2023年第4期1244-1252,共9页
Journal of Southern Agriculture
基金
国家自然科学基金项目(32073006)
广东省自然科学基金项目(2022A1515010553)
中山市社会公益与基础研究项目(2020B2066)。
