文章摘要
孙盛明,傅洪拓,宣富君,戈贤平,朱健,吴旭干.日本沼虾C型凝集素结构域家族3的cDNA克隆、原核表达和定位分析[J].水产学报,2019,43(11):2317~2326
日本沼虾C型凝集素结构域家族3的cDNA克隆、原核表达和定位分析
Molecular cloning, prokaryotic expression and localization analysis of C-type lectin 3 (MnLec3) cDNA from Macrobrachium nipponense
投稿时间:2018-06-15  修订日期:2018-12-04
DOI:10.11964/jfc.20180611327
中文关键词: 日本沼虾  C型凝集素结构域家族3  嗜水气单胞菌  基因表达
英文关键词: Macrobrachium nipponense  C-type lectins 3  Aeromonas hydrophilia  gene expression
基金项目:国家自然科学基金(31402280,31672633);上海海洋大学水产种质资源发掘与利用教育部重点实验室开放课题(2016KF-02)
作者单位E-mail
孙盛明 中国水产科学研究院淡水渔业研究中心, 农业农村部淡水渔业与种质资源利用重点实验室, 江苏 无锡 214081  
傅洪拓 中国水产科学研究院淡水渔业研究中心, 农业农村部淡水渔业与种质资源利用重点实验室, 江苏 无锡 214081 fuht@ffrc.cn 
宣富君 盐城师范学院, 江苏省滩涂生物资源与环境保护重点建设实验室, 江苏 盐城 224051  
戈贤平 中国水产科学研究院淡水渔业研究中心, 农业农村部淡水渔业与种质资源利用重点实验室, 江苏 无锡 214081  
朱健 中国水产科学研究院淡水渔业研究中心, 农业农村部淡水渔业与种质资源利用重点实验室, 江苏 无锡 214081  
吴旭干 上海海洋大学, 水产种质资源发掘与利用教育部重点实验室, 上海 201306  
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中文摘要:
      C型凝集素(C-type lectin)是一类能与糖类结合的非抗体的蛋白质或糖蛋白家族,为了研究C型凝集素基因在日本沼虾组织分布、细胞定位和细菌感染过程中的表达情况,本研究应用cDNA末端快速克隆(rapid-amplification of cDNA ends,RACE)技术首次克隆了日本沼虾C型凝集素结构域家族3基因(MnLec3)的全长序列,通过实时荧光定量PCR (qRT-PCR)分析MnLec3基因在不同组织、细菌感染后不同时间的表达水平,Western blot和免疫荧光分别分析蛋白的表达水平和细胞定位。结果显示,MnLec3基因cDNA全长1 357 bp,包括125 bp的5'末端非翻译区(UTR)、1 026 bp的开放阅读框(ORF)和206 bp的3'UTR,其中开放阅读框编码341个氨基酸。氨基酸序列比对显示,日本沼虾MnLec3基因含有保守钙结合点(Met 1-Glu17)和糖识别结构域(CRD)。同源性分析结果显示,MnLec3与罗氏沼虾C型凝集素3相似度较高;邻接法(Neighbor-Joining,NJ)进化树分析结果显示,MnLec3与其他甲壳动物C型凝集素聚为一支。通过构建原核表达载体获得体外重组蛋白rMnLec3,并将纯化重组蛋白免疫大鼠获得抗血清,免疫荧光结果显示,绿色荧光信号主要在肝胰腺细胞核中表达。qRT-PCR结果显示,MnLec3在日本沼虾所检测组织中均表达,其中肝胰腺中表达量最高,血细胞次之;与对照组相比,在嗜水气单胞菌刺激12~48 h时MnLec3表达量显著升高,48 h表达量最高,Western blot分析结果显示,MnLec3蛋白表达丰度与基因表达模式基本相似,提示克隆得到的MnLec3参与日本沼虾抵御细菌入侵的免疫过程。
英文摘要:
      C-type lectins are a large family of proteins that exist in all deuterostomia. C-type lectins can bind to carbohydrate moieties normally in a calcium-dependent manner and play important roles in immune defense. This study aims to explore the expression patterns of C-type lectin gene in different tissues, cellular localization and becteria challenge in Macrobrachium nipponense. The cDNA sequence of M. nipponense (MnLec3) was obtained using rapid amplification of cDNA ends method (RACE) and RT-PCR. The expression levels of MnLec3 in different tissues and at different time of artificially challenged with Aeromonas hydrophilia were analyzed by qRT-PCR. The full-length cDNA sequence of MnLec3 was 1 357 bp, which contained a 5' untranslated region of 125 bp, a 3' untranslated region of 206 bp, a 1 026 bp open reading frame (ORF) encoding 341 amino acids. The deduced amino acid sequence of MnLec3 had a signal peptide containing 17 amino acid residues and a carbohydrate recognition domain (CRD). Phylogenetic tree analysis stated that Oriental river prawn has the closest relationship with other crustacean. The expressed recombinant MnLec3 protein and polyclonal antibody were obtained in present study using a conventional method. Furthermore, immunofluorescent staining technique was used to determine cellular localization of MnLec3 in hepatopancreas of prawns. Quantitative real-time RT-PCR analysis showed that the MnLec3 gene was expressed in haemocytes, hepatopancreas, muscles, gill, testis, ovary and intestines with the highest level of expression in the hepatopancreas. Real-time PCR analysis indicated that MnLec3 transcripts level showed significant change in hepatopancreas after the prawn was artificially challenged with A. hydrophilia, followed by return to control levels at 96 h post-injection, which were similar to MnLec3 protein expression abundance using Western Blot. The results suggested that MnLec3 might be involved in the immune response against bacteria.
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