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史雨红, 陈炯, 高姗姗, 沈广强, 陆新江, 李明云. 2012: 花鲈Wap65-2基因的克隆、理化性质及其表达与哈维氏弧菌感染的相关性. 动物学研究, 33(5): 481-486. DOI: 10.3724/SP.J.1141.2012.05481
引用本文: 史雨红, 陈炯, 高姗姗, 沈广强, 陆新江, 李明云. 2012: 花鲈Wap65-2基因的克隆、理化性质及其表达与哈维氏弧菌感染的相关性. 动物学研究, 33(5): 481-486. DOI: 10.3724/SP.J.1141.2012.05481
SHI Yu-Hong, CHEN Jiong, GAO Shan-Shan, SHEN Guang-Qiang, LU Xin-Jiang, LI Ming-Yun. 2012: Cloning, physical and chemical property analysis of the Japanese sea bass Wap65-2 gene and its expression following Vibrio harveyi infection. Zoological Research, 33(5): 481-486. DOI: 10.3724/SP.J.1141.2012.05481
Citation: SHI Yu-Hong, CHEN Jiong, GAO Shan-Shan, SHEN Guang-Qiang, LU Xin-Jiang, LI Ming-Yun. 2012: Cloning, physical and chemical property analysis of the Japanese sea bass Wap65-2 gene and its expression following Vibrio harveyi infection. Zoological Research, 33(5): 481-486. DOI: 10.3724/SP.J.1141.2012.05481

花鲈Wap65-2基因的克隆、理化性质及其表达与哈维氏弧菌感染的相关性

Cloning, physical and chemical property analysis of the Japanese sea bass Wap65-2 gene and its expression following Vibrio harveyi infection

  • 摘要: Wap65-2 (warm temperature acclimation related 65 kDa protein-2)是鱼类中发现的一种血浆糖蛋白, 与细菌感染性免疫应激紧密相关。该研究首次克隆了花鲈Wap65-2基因全长cDNA序列。它由1 601个核苷酸组成, 包含一个大的开放阅读框, 预期编码一个由436个氨基酸组成、相对分子质量为4.87×104的前体蛋白, N端19个残基为信号肽序列。序列和系统进化树分析表明, 花鲈Wap65-2与欧洲海鲈进化关系最近, 两者氨基酸同源性高达80.7%。健康花鲈Wap65-2基因mRNA主要在肝中表达, 心和肌肉中少量表达。qRT-PCR分析揭示, 哈维氏弧菌(Vibrio harveyi)感染感染12 h后, 花鲈肝中Wap65-2基因mRNA表达显著上调, 24 h时达到峰值, 为健康对照的6.89倍。原核表达花鲈Wap65-2并制备抗血清。Western blot分析表明, 哈维氏弧菌感染12 h后, 花鲈血清中Wap65-2含量显著增加, 36 h时达到最大值, 为健康对照的5.33倍。综上所述, 花鲈Wap65-2基因的表达与其细菌感染性免疫应激紧密相关。

     

    Abstract: The warm temperature acclimation related 65 kDa protein-2 (Wap65-2), a teleost plasma glycoprotein, plays an important role in immune regulation against bacterial infection. Here, for the first time we determined the full length cDNA sequence of the Japanese sea bass Wap65-2 gene (1 601 bp in length excluding the 3'-polyA tail). The sequence contains an open reading frame that encodes a protein of 436 amino acids with a molecular weight of 4.87?104. The predicted protein had a signal peptide in the N-terminal domain containing 19 residues. Sequence comparison and phylogenetic tree analysis showed that the Japanese sea bass Wap65-2 has a relatively high similarity to the Dicentrarchus labrax Wap65-2. In the healthy Japanese sea bass, Wap65-2 mRNA was expressed mainly in the liver and weakly in the heart and muscle. qRT-PCR results revealed that liver Wap65-2 transcripts were significantly increased after a Vibrio harveyi infection, and peaked 24 hour post injection (6.89 fold increase). The Japanese sea bass Wap65-2 protein was expressed in Escherichia coli and subsequently used for antiserum preparation. Western blot analysis showed that Wap65-2 was significantly increased in V. harveyi infected Japanese sea bass and reached a maximum of 5.33-fold increase at 36 h. In conclusion, the alteration of Japanese sea bass Wap65-2 expression was tightly associated with the progression of the V. harveyi bacterial infection.

     

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