旋毛虫成虫排泄分泌蛋白组分分析

中国寄生虫学与寄生虫病杂志 ›› 2017, Vol. 35 ›› Issue (1): 24-29.

旋毛虫成虫排泄分泌蛋白组分分析

杨小迪¹, 陶志勇¹, 程洋², 吴琦¹, 王小莉¹, 宋迪¹, 徐岚松¹, 薛仁敏¹, 常雪莲¹, 张慧¹, 汪瑞¹, 陈兴智¹, 方强^1,^*()

1蚌埠医学院病原生物学教研室,安徽省感染与免疫重点实验室,蚌埠233030
2江南大学无锡医学院病原感染与免疫组,无锡214122

收稿日期:2016-09-17 出版日期:2017-02-28 发布日期:2017-03-06
通讯作者: 方强
基金资助:
国家自然科学基金（No. 81441120）;安徽省自然科学基金（No. 1508085QH158）;安徽高校科研创新平台团队项目（2016-40）;安徽省高校优秀青年人才支持计划重点项目（No. gxyqZD2016159）;安徽省高等学校自然科学研究重点项目（No. KJ2016A708,KJ2015A146）

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

Xiao-di YANG¹, Zhi-yong TAO¹, Yang CHENG², Qi WU¹, Xiao-li WANG¹, Di SONG¹, Lan-song XU¹, Ren-min XUE¹, Xue-lian CHANG¹, Hui Zhang¹, Rui WANG¹, Xing-zhi CHEN¹, Qiang FANG^1,^*()

1 Department of Microbiology and Parasitology, Anhui Key Laboratory of Infection and Immunity, Bengbu Medical College, Bengbu 233030, China
2 Department of Pathogenic Infection and Immunity, Wuxi Medical School, Jiangnan University, Wuxi 214122, China

Received:2016-09-17 Online:2017-02-28 Published:2017-03-06
Contact: Qiang FANG
Supported by:
Supported by the National Natural Science Foundation of China（No. 81441120）;The Natural Science Fund of Anhui（No. 1508085QH158）;The Scientific Research Innovation Team Project of Anhui Colleges and Universities（2016-40）;Key Supporting Project of Prominent Youth in University of Anhui（No. gxyqZD2016159）;The Key Project of Anhui Education Department（No. KJ2016A708,KJ2015A146）

摘要/Abstract

摘要：

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（M_r）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。

关键词: 旋毛虫, 成虫, 排泄分泌蛋白, shotgun技术, 炎症性肠炎

Abstract:

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 M_r 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.

Key words: Trichinella spiralis, Excretory/secretory protein, Adult worm, Shotgun, Inflammatory bowel disease

中图分类号:

R383.15

杨小迪, 陶志勇, 程洋, 吴琦, 王小莉, 宋迪, 徐岚松, 薛仁敏, 常雪莲, 张慧, 汪瑞, 陈兴智, 方强. 旋毛虫成虫排泄分泌蛋白组分分析[J]. 中国寄生虫学与寄生虫病杂志, 2017, 35(1): 24-29.

Xiao-di YANG, Zhi-yong TAO, Yang CHENG, Qi WU, Xiao-li WANG, Di SONG, Lan-song XU, Ren-min XUE, Xue-lian CHANG, Hui Zhang, Rui WANG, Xing-zhi CHEN, Qiang FANG. Component analysis of excretory/secretory protein from Trichinella spiralis adult worm[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2017, 35(1): 24-29.

图/表 3

图1

表1

Shotgun技术鉴定的旋毛虫成虫排泄-分泌蛋白

登录号 Accession No.	蛋白质名称 Protein name	肽段得分 Score	相对分子质量 M_r	等电点 PI	覆盖范围/% Coverage/%	肽段匹配 Peptides matches
A1BQX8 Q9BJM1 E5SK38 Q27071 B0FJW6 E5SGG8 Q32R69 E5S0E7 G4XSU5 E5SA29 E5S554 A1DYF1 E5SUQ1 E5SLU1 E5S2E7 E5S913 E5SXK4 E5SCW1 E5SED3 E5SEF8 E5SAH1 E5SR95 E5S2X3 Q32VU9 E5SNV8 E5S188 E5SPV4 E5RZT8 E5S5P0 E5SKM0 E5SFR7 E5SH15 B0F9R6 E5T0G7 E5SFB3 E5SKL9 E5SUB6 E5RYP1 E5S562 E5SJ09 B0F9R8 E5SS61 E5S9H4 E5SDS1 Q0H8U8	多重半胱氨酸蛋白酶抑制剂结构域样蛋白 Multi cystatin-like domain protein 丝氨酸蛋白酶Serine proteinase 大衣壳蛋白 Large tegument protein 53 000排泄/分泌抗原53 000 excretory/secretory antigen 半胱氨酸抑制剂样蛋白 Cystatin-like protein 脱氧核糖核酸酶II家族蛋白Deoxyribonuclease II family protein 成虫特异性DNA酶II-7 Adult-specific DNase II-7 AP-1复合体β-1亚基AP-1 complex subunit beta-1 多聚半胱氨酸组氨酸同型追踪蛋白2 Poly-cysteine and histidine tailed protein isoform 2 α-淀粉酶Alpha-amylase 5′核苷酸酶5′-nucleotidase 保守半胱氨酸-甘氨酸蛋白2类蛋白 Conserved cysteine-glycine protein 2-like protein 钠钾转运ATP酶β1亚基互作蛋白3 Sodium/potassium-transporting ATPase subunit β1-interacting protein 3 肌动蛋白-5C Actin-5C 肽酶, S1A 亚家族Peptidase, S1A subfamily 促胰液素受体Secretin receptor 脱氧核糖核酸酶Ⅱ超家族DeoxyribonucleaseⅡsuperfamily 丝氨酸蛋白酶抑制家族蛋白 Serine protease inhibitor family protein 精子主要蛋白Major sperm protein 铁硫簇结合蛋白Iron-sulfur cluster-binding protein 肠肽酶Enteropeptidase 钙网蛋白Calreticulin 谷氨酰胺肽环转移酶Glutaminyl-peptide cyclotransferase 新生幼虫特异性DNA酶Ⅱ-7 Newborn larvae-specific DNase Ⅱ-7 果糖-二磷酸醛缩酶Fructose-bisphosphate aldolase 星状蛋白 Protein asteroid 跨膜蛋白85 Transmembrane protein 85 超氧化物歧化酶[Cu-Zn] Superoxide dismutase [Cu-Zn] 蛋白质二硫化物异构酶Protein disulfide-isomerase 切补核酸酶ABC, C亚基 Excinuclease ABC, C subunit 热休克蛋白A Heat shock protein A 肽酰脯氨酰顺反异构酶 Peptidyl-prolyl cis-trans isomerase 丝氨酸蛋白激酶抑制剂 Serine proteinase inhibitor 肽酰脯氨酰顺反异构酶 Peptidyl-prolyl cis-trans isomerase 组织蛋白酶F Cathepsin F 切补核酸酶ABC, C亚基 Excinuclease ABC, C subunit 肽酶家族M13 Peptidase family M13 泛素蛋白家族Ubiquitin family protein (fragment) 溶酶体天冬氨酸蛋白酶 Lysosomal aspartic protease 核氧化还原蛋白 Nucleoredoxin 核苷二磷酸激酶 Nucleoside diphosphate kinase 磷酸丙糖异构酶 Triosephosphate isomerase 胎盘蛋白11 Placental protein 11 氨肽酶 A/Ⅰ Aminopeptidase A/Ⅰ 谷胱甘肽-S-转移酶 Glutathione-S-transferase	9 003 4 035 2 483 1 902 1 605 1 517 1 436 1 422 982 819 819 557 503 492 462 417 407 341 325 296 281 234 230 183 168 164 157 145 144 124 117 94 93 92 82 77 73 70 69 69 65 62 42 41 40	45 600 71 600 84 100 46 600 27 500 36 700 38 200 46 600 47 500 108 000 61 900 16 100 75 100 41 800 28 600 14 100 36 900 39 600 22 200 21 500 70 100 47 900 39 900 37 500 41 100 27 600 50 300 18 900 54 700 30 200 112 100 22 500 42 500 18 500 41 900 36 200 8 200 23 600 45 100 23 300 17 200 30 800 12 100 54 700 24 000	5.53 8.54 8.69 8.21 7.83 8.94 7.68 8.43 7.08 7.84 6.70 8.73 5.52 5.48 8.06 6.51 8.46 6.18 4.74 8.50 7.40 4.67 6.86 9.03 7.05 8.51 8.57 6.35 5.12 5.24 6.86 8.90 6.57 8.29 7.53 7.44 8.02 6.10 6.19 6.28 8.44 7.88 6.79 7.08 6.55	80.89 62.67 59.10 41.50 71.73 67.27 70.11 35.96 60.90 36.82 46.36 40.88 33.73 34.57 44.40 27.42 45.59 29.51 50.49 36.76 13.44 33.17 24.71 25.29 27.25 38.65 12.69 50.56 24.59 11.70 10.37 25.12 14.21 34.68 19.67 10.06 11.28 44.29 23.21 18.14 20.92 24.1 46.67 10.71 10.24	1 4 36 2 17 12 2 10 24 27 20 6 24 11 8 3 12 10 5 7 8 11 6 5 8 9 4 8 11 3 8 5 5 3 5 4 6 3 7 3 3 5 3 4 3

表1

图2

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