猪囊尾蚴囊液蛋白组学分析

doi:10.12140/j.issn.1000-7423.2019.02.005

中国寄生虫学与寄生虫病杂志 ›› 2019, Vol. 37 ›› Issue (2): 144-149.doi: 10.12140/j.issn.1000-7423.2019.02.005

猪囊尾蚴囊液蛋白组学分析

庞建达¹(), 宋伊宁¹, 王昕蕊¹, 刘明远², 孙树民^1,^2,^*()

1 内蒙古民族大学动物科学技术学院,通辽 028000
2 吉林大学人兽共患病研究教育部重点实验室,人兽共患病研究所,长春 130062

收稿日期:2018-11-25 出版日期:2019-04-30 发布日期:2019-05-13
通讯作者: 孙树民
作者简介:
作者简介：庞建达（1994-）,男,硕士研究生,从事人兽共患病方向研究。E-mail：778079399@qq.com
基金资助:
国家重点研发计划课题（No. 2017YFD0501303）;国家自然科学基金（No. 31160504,No. 31460658）;中国博士后科学基金（No. 2012M520674）;内蒙古自治区自然科学基金（No. 2017MS0321,No. 2011MS0404）;内蒙古民族大学博士启动基金（No. BS284）;内蒙古民族大学硕士研究生科研创新项目（No. NMDSS1733,No. NMDSS1861）

Proteomic analysis of cyst fluid of Cysticercus cellulosae

Jian-da PANG¹(), Yi-ning SONG¹, Xin-rui WANG¹, Ming-yuan LIU², Shu-min SUN^1,^2,^*()

1 College of Animal Science and Technology, Inner Mongolia University for Nationalities, Tongliao 028000, China
2 Key Laboratory of Zoonosis Research, Ministry of Education, Institute of Zoonosis, Jilin University, Changchun 130062, China

Received:2018-11-25 Online:2019-04-30 Published:2019-05-13
Contact: Shu-min SUN
Supported by:
Supported by National Key Research and Development Program(No. 2017YFD0501303);National Natural Science Foundation of China(No. 31160504, No. 31460658);China Postdoctoral Science Foundation Project(No. 2012M520674);Inner Mongolia Autonomous Region Natural Science Fund Project(No. 2017MS0321, No. 2011MS0404);Inner Mongolia University for Nationalities Doctoral Startup Fund(No. BS284);Inner Mongolia University for Nationalities Master’s Research and Innovation Project(No. NMDSS1733, No. NMDSS1861)

摘要/Abstract

摘要：

目的利用鸟枪法技术鉴定猪囊尾蚴囊液蛋白组分构成,完善猪囊尾蚴蛋白表达谱,筛选特异性候选诊断标识并分析囊液蛋白组分的功能。方法无菌收集猪囊尾蚴虫体囊液,经处理后采用shotgun液相色谱-串联质谱系统对囊液进行分选与鉴定,通过Mascot 2.2软件进行数据库检索,完成蛋白鉴定。使用Blast2go软件对蛋白序列进行比对和注释,对注释的蛋白组分数据进行Gene Ontology（GO）分析。结果经鸟枪法分析共获得486个蛋白,通过Mascot 2.2软件检索已鉴定蛋白为158种,未知蛋白为328种。初步筛选囊液中含有丰度较高的8 kDa糖蛋白、膜联蛋白、烯醇化酶、胰蛋白酶样蛋白等4个猪囊尾蚴特异性抗原和多种酶类,主要存在于细胞及细胞组分中。其中膜联蛋白主要具有结合功能,主要参与一系列依赖于钙离子的膜型生物活动;烯醇化酶主要具有离子结合功能和水解酶活性,除大量富集在糖酵解途径中,还参与调节DNA结合转录因子过程。GO分析结果显示：蛋白谱分布细胞成分方面,65个蛋白位于细胞中,64个蛋白位于细胞组分中,25个蛋白位于细胞器中,其余位于细胞器、蛋白复合物、细胞外区域和细胞膜中;蛋白谱分子功能方面,72个蛋白具有催化活性,74个蛋白具有结合功能,其余蛋白具有转运蛋白活性、分子功能调节剂、结构分子活性等功能;生物进程分类方面,69个蛋白参与细胞进程,73个蛋白参与代谢进程,11个蛋白参与生物调节、生物过程的调节、信号、对刺激的应答、定位等过程。结论经鸟枪法技术初步筛选出4个丰度较高可作为猪囊尾蚴病诊断标识的候选抗原,鉴定出的酶类可能在猪囊尾蚴与宿主互作过程中起相关作用。

关键词: 猪囊尾蚴, 囊液, 鸟枪法, 诊断标识

Abstract:

Objective The protein components of cyst fluid of Cysticercus cellulosae were analyzed by liquid chromatograph-mass spectrometer (LC-MS/MS) to identify the expression profile of C. cellulosae, The protein profile of Cysticercus fluid can be used to screen for the specific antigens as diagnostic markers and analyze the function of Cysticercus protein components involved in the invasion and survival of Cysticercus in the host. Methods The cystic fluid was aseptically collected from an infected pig and the protein components of the fluid was analyzed by LC-MS/MS after being digested with trypsin. The Blast2Go software was used for the comparison and annotation, and Gene Ontology (GO) was used to analyze classification of the annotated protein components. Results A total of 486 proteins were obtained by LC-MS/MS analysis, 158 of them were known proteins and the rest 328 were unknown proteins after being analyzed by searching with Mascot 2.2 software. The primary searching results identified that cystic fluid contains four highly abundant proteins (8 kDa glycoprotein, annexin, enolase, trypsin-like protein), and other specific antigens and a variety of enzymes, mainly located intracellularly and as cell components, of which annexin has binding functions mainly involved in a series of calcium-dependent membrane-type biological activities. The enolase has ionic binding function and hydrolase activity involved in the regulation of DNA-binding transcription factor and the glycolytic pathway. The results of GO analysis showed that 65 proteins were located intracellularly, 64 proteins located in the cell components, 25 proteins located in the organelles, and the rest located in the organelles, protein complexes, extracellular regions and cell membranes. In terms of molecular functions of identified proteins, 72 proteins have catalytic activities, 74 proteins have binding functions, and the remaining proteins are involved in transporter activities, molecular function regulators, structural molecular activities, etc. In terms of biological process classification, 69 proteins are involved in the cell process, 73 involved in the metabolic process, and 11 involved in the regulation of biological processes, signals, response to stimuli and cell localization. Conclusion The proteomic results of cyst fluid of Cysticercus identified a total of 486 known or unknown proteins, 4 of them are highly abundant proteins that can be used as candidate antigens for immunodiagnosis of cysticercosis. The identified enzymes may play important roles in the interaction between C. cellulosae and the host, providing a basis for further exploration of the invasion and parasitic mechanism of C. cellulosae.

Key words: Cysticercus cellulosae, Cyst fluid, LC-MS/MS, Diagnostic marker

中图分类号:

R383.34

庞建达, 宋伊宁, 王昕蕊, 刘明远, 孙树民. 猪囊尾蚴囊液蛋白组学分析[J]. 中国寄生虫学与寄生虫病杂志, 2019, 37(2): 144-149.

Jian-da PANG, Yi-ning SONG, Xin-rui WANG, Ming-yuan LIU, Shu-min SUN. Proteomic analysis of cyst fluid of Cysticercus cellulosae[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2019, 37(2): 144-149.

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检索项目 Search item	参数Parameter
酶 Enzyme	Trypsin
数据库 Database	Uniprot Taenia
固定修饰 Fixed modifications	Carbamidomethyl
可变修饰 Variable modifications	Oxidation
肽质量耐受性 Peptide mass tolerance	0.002%
片段质量容限 Fragment mass tolerance	相对分子质量(M_r) 0.1
分数过滤 Filter by score	≥ 20

编号 No.	登录号 SwissProt/TrEMBL AC	蛋白名称 Protein name	肽段 Pep count	惟一肽段数 Unique pep count	肽段覆盖率/% Cover percent/%	相对分子质量 M_r	等电点 PI
1	Q9U8G9	囊尾蚴病10 kDa抗原 Cysticercosis 10 kDa antigen	88	8	43.53	9 650	8.89
2	Q8I0G1	排泄分泌抗原m13 Excretion-secretion antigen m13	52	8	50.59	9 820	9.75
3	E5LBB8	胰蛋白酶样蛋白质 Trypsin-like protein	33	17	39.96	5 412	7.09
4	D2U5C3	磷酸烯醇丙酮酸羧激酶Phosphoenolpyruvate carboxykinase	31	23	55.58	5 411	8.49
5	B6E4B3	8 kDa糖蛋白8 kDa glycoprotein	31	5	48.24	9 660	9.78
6	A0A1S5WII1	烯醇化酶Enolase	21	18	45.62	4 661	6.48
7	A0A0R3VSW8	果糖二磷酸醛缩酶 Fructose-bisphosphate aldolase	19	13	47.93	3 971	8.35
8	A0A291I2R3	丝氨酸蛋白酶抑制Serpin	17	15	41.90	4 554	7.63
9	A0A088FV33	胱抑素Cystatin	15	10	52.19	3 065	5.19
10	A0A0R3VUJ9	氨肽酶Aminopeptidase	13	12	11.57	11 410	6.88
11	K7SRY9	肌动蛋白Actin	11	9	30.59	4 174	5.30
12	A0A0R3VWJ0	磷酸甘油酸激酶 Phosphoglycerate kinase	10	7	17.59	4 413	7.56
13	A0A0R3W9G0	腺苷高半胱氨酸酶 Adenosylhomo cysteinase	5	5	12.21	5 182	6.16
14	Q967Z2	钙结合蛋白Calcium binding protein	5	5	17.47	4 557	4.41
15	A0A0R3WCA2	胺氧化酶Amine oxidase	5	4	4.90	8 099	6.55
16	Q5GM20	诊断抗原GP50GP50	5	4	23.51	2 957	8.68
17	Q8MPB9	小热休克蛋白 Small heat-shock protein	5	5	18.15	3 560	5.56
18	G8ZAG5	L-乳酸脱氢酶 L-lactate dehydrogenase	4	4	14.50	3 546	7.09
19	J9PE36	脂肪酸结合蛋白 Fatty acid binding protein	4	4	35.34	1 511	8.61
20	Q9XZL9	膜联蛋白Annexin	1	1	2.59	3 796	5.71

猪囊尾蚴囊液蛋白组学分析

Proteomic analysis of cyst fluid of Cysticercus cellulosae

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