大菱鲆(Scophthalmus maximus)热休克蛋白SmHsp47相互作用蛋白His-pull down和质谱鉴定

IDENTIFICATION OF PROTEINS INTERACTION WITH SCOPHTHALMUS MAXIMUS HEAT SHOCK PROTEIN SmHsp47 BY HIS-PULL DOWN COMBINED WITH MASS SPECTROMETRY

首次克隆了大菱鲆(Scophthalmus maximus)热休克蛋白SmHsp47基因。该基因cDNA序列全长为1927 bp,其中开放阅读框长度为1218 bp,编码一条长度为405个氨基酸的多肽链。结果表明,大菱鲆SmHsp47基因在肝脏、皮肤、鳃和肠等4个组织中都有表达,表达量最高的组织是肝脏,表达量最低组织是鳃;25℃处理6 h后SmHsp47在皮肤中的表达量增加150多倍。利用大肠杆菌原核表达系统表达了SmHsp47的his标签融合蛋白,然后利用His-pull down技术捕获了SmHsp47的相互作用蛋白质,通过质谱分析鉴定出31种候选蛋白,其中大部分蛋白为参与翻译后修饰,蛋白转换及分子伴侣;此外还包括参与脂转运与代谢、能量产生与转换、细胞骨架、防御机制等过程的蛋白质。从31种候选蛋白中进一步筛选出3种分值较高的蛋白:未知蛋白(uncharacterized protein,A0A6A4TFV0)、胎球蛋白B(Fetuin B,A0A2U9C388)和胶原结合蛋白(collagen-binding protein,A0A2U9B608),可为后续的研究提供方向。

We cloned the SmHsp47 gene of turbot Scophthalmus maximus using the RACE technique.The cDNA sequence of SmHsp47 is 1927 bp in length and includes a 1218 bp open reading frame encoding a 405 amino acid.The results show that SmHsp47 gene was expressed in liver,skin,intestines,and gills,with the highest expression in liver and the lowest in gill.The relative expression of SmHsp47 was increased by 150 times in 25℃ treatment group for 6 h.Using prokaryotic expression technology,the His tag fusion protein of SmHsp47 was expressed.The interactive proteins of SmHsp47 were captured in the His-pull down technology.By LC-MS/MS analysis,31 candidate proteins were identified and most of them were involved in the modification after translation,protein conversion,and molecular partner,and also energy production and conversion,lipid transport and metabolism,carbohydrate transport and metabolism,defense mechanism,and cytoskeleton proteins.Three high-scored proteins were screened,i.e.,uncharacterized protein(A0A6A4TFV0),Fetuin B(A0A2U9C388),and collagen-binding protein(A0A2U9B608),providing a reference for future studies in this regard.