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目的分析旋毛虫(Trichinella spiralis)肌幼虫排泄分泌蛋白(excretory-secretory protein,ESP)的组分,寻找其抗肿瘤活性组分。方法收集纯净的旋毛虫脱囊肌幼虫,制备旋毛虫肌幼虫ESP。采用15%十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分离ESP,将分离获得的所有蛋白条带进行胰蛋白酶酶解,采用液质联用质谱法(LC-MS/MS)进行鉴定。利用Gene Ontology(GO)对已鉴定蛋白质进行细胞组分、分子功能和生物过程的富集分析。结果经SDS-PAGE电泳分离获得清晰的蛋白质条带,相对分子质量(Mr)为10 000~142 000。质谱分析共鉴定出162种蛋白质,其中63种为已明确鉴定的蛋白质,34种为假定蛋白质,65种为未鉴定的蛋白质。鉴定出6种与抗肿瘤相关的蛋白质,即原肌球蛋白、组蛋白H2A、剪切聚腺苷酸化特异因子亚基2、Kazal 4型丝氨酸蛋白酶抑制剂、犰狳极性蛋白和真核起始因子4A。GO富集分析显示,已鉴定的蛋白质具有54种不同的分子功能,参与细胞构成并参与382种生物过程。结论旋毛虫肌幼虫ESP组分复杂,有多种未鉴定的蛋白质,从已知的63种蛋白质中筛选出6种与抗肿瘤相关的蛋白质。
Objective To analyze the components of excretory-secretory protein (ESP) of Trichinella spiralis muscle larvae and to search for their antitumor activity components. Methods Purified Trichinella spiralis larvae were collected to prepare Trichinella spiralis larvae ESP. ESP was separated by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and all protein bands isolated were subjected to tryptic digestion and analyzed by liquid chromatography-mass spectrometry (LC-MS / MS). Gene Ontology (GO) was used to enrich the identified proteins for cellular components, molecular functions and biological processes. Results A clear protein band was obtained by SDS-PAGE electrophoresis. The relative molecular mass (Mr) was 10 000 ~ 142 000. A total of 162 proteins were identified by mass spectrometry, of which 63 were specifically identified, 34 were putative proteins and 65 were unidentified proteins. Six anti-tumor related proteins were identified, namely tropomyosin, histone H2A, cut polyadenylation specific factor subunit 2, Kazal type 4 serine protease inhibitor, armadillo polar protein and eukaryotic Start factor 4A. GO enrichment analysis showed that the identified proteins have 54 different molecular functions involved in cellular constitution and are involved in 382 biological processes. Conclusion The ESP component of Trichinella spiralis larvae is complex and has many unidentified proteins. Six anti-tumor related proteins were screened from 63 known proteins.