斯氏并殖吸虫蛋白质组学研究

摘要/Abstract

摘要：

目的对斯氏并殖吸虫（Paragonimus skrjabini）囊蚴、童虫和成虫蛋白质组进行研究。方法在贵州省斯氏并殖吸虫疫源地开阳、毕节采集溪蟹,从中分离囊蚴置于PBS中,灌胃感染SD大鼠3只（10个囊蚴/只）,喂食感染雄性犬3只（20个囊蚴/只）。感染大鼠后1个月解剖取童虫,感染犬后3个月解剖取成虫。将囊蚴、童虫和成虫中加入中性组织裂解液,冰上超声破碎提取总蛋白,Bradford法测定蛋白含量并进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）及双向凝胶电泳,采用PDquest 8.0软件对双向凝胶电泳图像进行分析寻找差异点。对差异点进行酶解、质谱鉴定。最后进行NCBI在线及本地数据库检索。结果 SDS-PAGE显示囊蚴、童虫和成虫总蛋白主要集中分布在相对分子质量（M_r）25 000~116 000。双向凝胶电泳共筛选出51个差异蛋白点,分别为囊蚴20个、童虫25个、成虫6个。因成虫差异点灰度值两两间的比值变化远小于囊蚴与童虫,故未进行质谱鉴定。36个囊蚴和童虫的肽段序列进行分析后发现,在线检索蛋白种类主要为无色杆菌（Achromobacter lyticus）蛋白酶Ⅰ（一种赖氨酸特异性的丝氨酸酶）、脱氢抗坏血酸还原酶蛋白、谷胱甘肽S转移酶DHAR2类似物、热激蛋白Hsp82及Hsp96-β、肌动蛋白和半胱氨酸酶抑制剂等;本地复殖目数据库检索主要蛋白种类是半胱氨酸酶、肌动蛋白和热激蛋白。结论斯氏并殖吸虫囊蚴与童虫蛋白组成有差异,囊蚴含有肌动蛋白,童虫含有解毒及应激蛋白,两者都含有水解酶类及半胱氨酸酶。

关键词: 斯氏并殖吸虫, 蛋白质组, 囊蚴, 童虫, 成虫

Abstract:

Objective To perform a proteomics analysis for metacercariae, juvenile and adult worms of Paragonimus skrjabini. Methods Crabs were collected in P. skrjabini endemic areas of Kaiyang and Bijie in Guizhou Province. Metacercariae were isolated, placed in PBS, and were used to infect three SD rats（10 metacercariae/rat） by intragastric administration and infect three male dogs（20 metacercariae/dog） through feeding. The rats were sacrificed at 1 month after infection to obtain juvenile worms. The dogs were sacrificed at 3 months after infection to obtain adult worms. The metacercariae, juvenile and adult worms were lysed, and total protein was extracted by ultrasonication. The total protein content was determined by Bradford method and separated by SDS-PAGE and two dimensional gel electrophoresis. Images of two dimensional gel electrophoresis were analyzed using the PDquest 8.0 software. The dots with difference were digested and analyzed with mass spectrometry. Finally, online searches in NCBI and local databases were performed. Results Results of SDS-PAGE showed that the total protein of metacercariae, juvenile and adult worms was concentrated within M_r of 25 000-116 000. Fifty-one protein dots with difference were found by two dimensional gel electrophoresis, comprising of 20 dots for metacercariae, 25 for juvenile worms and 6 for adult worms. Thirty-six peptide sequences of metacercariae and juvenile worms were analyzed. They were basically determined to be Achrornobacter lyticus protease Ⅰ（a lysine-specific serine protease）, ascorbate reductase protein, glutathione s-transferase DHAR2 analogue, heat shock proteins Hsp82 and Hsp96-β, actin, cystatin, etc., by online searches, and cysteine, actin and heat shock protein by local searches in the diginea database（downloaded to a local computer from NCBI）. Mass spectrometry was not performed for adult worms, as the variation of grayscale value between their spots was far less than those for metacercariae and juvenile worms. Conclusion One difference is that the metacercariae of P. skrjabini have actin, while the juvenile worms have detoxification proteins and stress proteins. But they both have hydrolases and cysteine enzymes.

Key words: Paragonimus skrjabini, Proteomics, Metacercaria, Juvenile worm, Adult worm

中图分类号:

R383.233

李安梅, 吴玛莉, 黄雨婷, 郭志来. 斯氏并殖吸虫蛋白质组学研究[J]. 中国寄生虫学与寄生虫病杂志, 2017, 35(1): 43-47.

An-mei LI, Ma-li WU, Yu-ting HUANG, Zhi-lai GUO. Proteomics analysis of Paragonimus skrjabini[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2017, 35(1): 43-47.

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