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

Yu-Hong SHI, Jiong CHEN, Shan-Shan GAO, Guang-Qiang SHEN, Xin-Jiang LU, Ming-Yun LI
2013 Zoological Research  
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 4.87×10 4 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
more » ... 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。 关键词:Wap65-2 基因; 花鲈; 哈维氏弧菌; 实时荧光定量 PCR; Western blot 中图分类号:Q959.483; Q344.13 文献标志码:A 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 4.87×10 4 . 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. Wap65s (warm temperature acclimation related 65 kDa protein)是鱼类中发现的一类血浆糖蛋白,
doi:10.3724/sp.j.1141.2012.05481 pmid:23019029 fatcat:opinniowdvbyxeijxb25hqgt6i