旋毛虫成虫排泄分泌蛋白组分分析

Component analysis of excretory/secretory protein from Trichinella spiralis adult worm

目的通过"鸟枪法"(shotgun技术)分析旋毛虫(Trichinella spiralis)成虫排泄分泌蛋白(adult worm excretory/secretory protein,AWESP)的组分,寻找其调节人炎症性肠炎的活性成分。方法制备旋毛虫AWESP,经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分离酶解后,采用shotgun液相色谱-串联质谱系统鉴定,将分析所得的数据通过Masco软件进行数据库检索,完成蛋白的鉴定,并对鉴定出的蛋白从细胞组分、分子功能和生物学途径3个方面进行Gene Ontology(GO)分类。结果 SDS-PAGE分离的旋毛虫AWESP相对分子质量(Mr)约15 000~116 000,质谱分析共获得280个蛋白,通过Masco软件检索已鉴定的蛋白为96种,假定的蛋白为98种,未鉴定的蛋白为86种。已初步发现至少有4种蛋白可能具有潜在的调节人炎症性肠炎的作用,包括半胱氨酸蛋白酶抑制剂、丝氨酸蛋白酶、53 000排泄/分泌抗原和谷胱氨肽转移酶。GO工具分析显示,已鉴定的蛋白具有104种分子功能,参与了363种生物过程。结论经Masco软件检索,旋毛虫AWESP成分复杂多样,已鉴定的蛋白有96种,从已明确的蛋白中初步筛选出4种与调节人炎症性肠炎潜在相关的蛋白。

Objective To analyze the component of adult worm excretory/secretory protein（AWESP） from Trichinella spiralis using the shotgun method, and find out the active component underlying its regulatory effect on colitis in humans. Methods The T. spiralis AWESP was prepared, separated by SDS-PAGE, lysed with trypsin, and analyzed by shotgun LC-MS/MS. The protein components were determined with the Masco software and classified using the Gene Ontology（GO） method in cellular components, molecular functions, and biological processes. Results The AWESPs isolated by SDS-PAGE had a Mr of 15 000-116 000. A total of 280 proteins were revealed by LC-MS/MS, of which 96 were identified by Masco software, 98 were putative, and the remaining 86 were unclear. Preliminary results showed that 4 proteins had regulatory potential for colitis, including cysteine protease inhibitor, serine protease, 53 000 excretory/secretory antigen, and glutathione-S-transferase. GO enrichment analysis showed that the identified proteins had 104 different molecular functions, involved in 363 biological processes. Conclusion As revealed by the Masco software, T. spiralis AWESP has complex components and 96 have been identified in this study. Four of them are preliminarily shown to be associated with the anti-colitis effect of T. spiralis.