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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1601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12h后,花鲈肝中Wap65-2基因mRNA表达显著上调,24h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12h后,花鲈血清中Wap65-2含量显著增加,36h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。
Wap65-2 (warm temperature acclimation related 65 kDa protein-2) is a plasma glycoprotein found in fish that is closely related to bacterial infectious immune stress. In this study, the full-length cDNA sequence of Wap65-2 gene was cloned for the first time. It consists of 1601 nucleotides and contains a large open reading frame. It is expected to encode a precursor protein consisting of 436 amino acids with a relative molecular mass of 4.87 × 104. N-terminal 19 residues are signal peptide sequences. Sequence analysis and phylogenetic tree analysis showed that the evolutionary relationship between Wap65-2 and European sea bass was the closest, with 80.7% amino acid homology. The mRNA expression of Wap65-2 gene in healthy sea bass was mainly expressed in the liver, with little expression in the heart and muscle. qRT-PCR analysis revealed that Wap65-2 gene mRNA expression was significantly up-regulated after Vibrio harveyi infection for 12h and peaked at 24h, 6.89-fold higher than that of healthy controls. Prokaryotic expression of perch Wap65-2 and preparation of antiserum. Western blot analysis showed that the Wap65-2 content of sepal increased significantly after 12 hours of Vibrio harveyi infection and reached the maximum at 36h, 5.33 times of the healthy control. In summary, the expression of Wap65-2 gene is closely related to bacterial infectious immune stress.