翘嘴红鲌热休克蛋白60的基因克隆及其表达特征分析(英文)

Molecular Cloning, Characterization and Expression Analysis of Heat Shock Protein 60 in Erythroculter ilishaeformis

利用同源克隆和RACE技术克隆了翘嘴红鲌热休克蛋白HSP60 cDNA全长序列,并对HSP60基因的序列特征进行了分析。EiHSP60 cDNA序列长2 323 bp,开放阅读框为1 728 bp,编码575个氨基酸,其编码蛋白相对分子量为61.26 k D,等电点为5.27。BLAST分析结果表明EiHSP60与哺乳动物、鸟和鱼类等物种的CCT具有较高的同源性。生物信息学分析结果显示,EiHSP60蛋白序列中包含1个典型的分子伴侣蛋白60信号基序。实时荧光定量PCR检测结果显示,EiHSP60在翘嘴红鲌各个组织中均有表达,但在肝脏中的表达量为最高。使用脂多糖对翘嘴红鲌进行攻毒后,在不同时间段取其肝脏总RNA,利用实时荧光定量PCR技术进行EiHSP60 m RNA的表达水平分析。分析结果显示,经过脂多糖攻毒后翘嘴红鲌HSP60呈现时间依赖型的表达模式,且在肝脏中的表达水平呈现上调趋势。上述结果表明,EiHSP60为一种涉及免疫反应的诱导型蛋白,而高表达量的EiHSP60可能在提高翘嘴红鲌免疫反应过程中发挥重要作用。

In the present study, the cDNA of heat shock protein 60 from Erythroculter ilishaeformis（EiHSP60） was cloned by the combination of homology cloning and rapid amplification of cDNA ends（RACE） approaches. The full-length cDNA of EiHSP60 was of 2 323 bp, including an open reading frame（ORF） of 1 728 bp encoding a polypeptide of 575 amino acids with predicted molecular weight of 61.26 k Da and theoretical isoelectric point of 5.27. BLAST analysis revealed that EiHSP60 shared high similarity with other known HSP60 s, and one conserved Chaperonin 60 signature was also identified in EiHSP60, which indicated that EiHSP60 should be a member of the HSP60 family. The m RNA of EiHSP60 was constitutively expressed in all tested tissues of untreated topmouth culter, including brain, muscle, kidney, liver, skin, spleen, heart, gill and intestine with the highest expression level in the liver. Fluorescent real-time quantitative RT-PCR was used to examine the expression of the EiHSP60 gene in topmouth culter after the challenge with LPS. A clear time-dependent expression pattern of EiHSP60 was found after the LPS challenge, and the up-regulated mRNA expression of EiHSP60 in topmouth culter after LPS challenge indicates that the HSP60 gene is inducible and involved in immune responses. These results suggest that EiHSP60 plays an important role in immune responses in topmouth culter.