小清蛋白(parvalbumin, PV)是鱼类的主要过敏原,为了解鱼类中不同亚型PV、氨基酸序列及其蛋白修饰情况,以鲢鱼肌肉为研究对象,经分级盐析、高效液相结合尺寸排阻色谱及高分辨质谱分离纯化鉴定PV亚型,并对其氨基酸序列和蛋白修饰进行研究。结果表明,从鲢鱼肌肉中自制的PV含有3种亚型(I、II、III),其平均分子质量分别为12.029 0 kDa、11.581 0 kDa和11.622 0 kDa;PVI、PVII和PVIII的氨基酸序列分别与蛋白质数据库中鱼类PV亚型Q804W2、Q6IMW7和Q9I8V0的氨基酸序列匹配率为74%、100%和99%,且PVII和PVIII分别与已报道的鲢鱼PV亚型gi 209902357和gi 209902355有着89%和88%的相似性。在蛋白修饰中,除PVI只存在酰基化修饰外,其余2个亚型主要存在磷酸化、酰基化、甲基化和泛素化修饰,且PVII的修饰程度明显高于PVIII和PVI。该研究可为鱼类不同PV亚型的提取、纯化和鉴定提供一种新思路,同时为淡水鱼类及其制品安全性的研究提供科学依据。
Parvalbumin (PV) is the main allergen of fish. To understand parvalbumin isotypes, amino acid sequence and protein modification in fish, silver carp muscle was used as research object, PV isotypes were isolated and purified by salting out. High-performance liquid chromatography combined with size exclusion chromatography and high-resolution mass spectrometry method were used in this study, and their amino acid sequence and protein modification were explored. The results showed that PV made from silver carp muscle contained three isotypes (I, II and III), and their average molecular weights were 12.029 0 kDa, 11.581 0 kDa and 11.622 0 kDa, respectively. The amino acid sequences of PVI, PVII and PVIII) matched 74%, 100% and 99% respectively with the amino acid sequences of PV isotypes Q804W2, Q6IMW7 and Q9I8V0 in the protein database, and PVII and PVIII had 89% and 88% similarities with the reported PV isotypes of silver carp gi 209902357 and gi 209902355, respectively. In protein modification, except PVI, which only had acylation modification, the other two isotypes mainly had phosphorylation, acylation, methylation and ubiquitination modification, and the modification degree of PVII was significantly higher than that of PVIII and PVI. This study provides a new idea for the extraction, purification and identification of different PV isotypes of fish, and provide a scientific basis for the safety evaluation of freshwater fish and their products.
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