基于蛋白质组学技术对竹叶青蛇毒染毒家兔模型血清蛋白质变化的检测及其意义

doi:10.13481/j.1671-587X.20220206

摘要/Abstract

摘要： 目的

探讨竹叶青蛇蛇毒模型中血清蛋白的表达，阐明竹叶青蛇蛇毒在染毒过程中标志蛋白的表达情况。

方法

将12只雌雄不限日本大耳兔随机分为假手术组和模型组，每组6只。模型组家兔给予20 mg·kg^－1竹叶青蛇毒肌肉注射，假手术组家兔给予等量生理盐水注射；4 h后，2组日本大耳兔用戊巴比妥钠麻醉后心脏采血，分离血清，采用绝对定量同位标记（TMT）联合高效液相色谱-串联质谱（HPLC-TMS）定量蛋白组学技术对2组家兔血清进行差异蛋白分析，采用Proteome Discover 2.4等数据库筛选差异蛋白。

结果

假手术组与模型组在主成分分析（PCA分析）坐标图上分成明显2簇；差异蛋白鉴定分析共鉴定分析出199个差异蛋白，其中表达上调的蛋白有139个，上调蛋白前5位分别为骨骼肌快肌肌钙蛋白Ⅰ（TNNI2）、小清蛋白α（PV）、烯醇化酶2（ENO2）、肌红蛋白（MB）和肌酸激酶（CK）；表达下调蛋白有60 个，下调蛋白前5位分别为载脂蛋白C1（APOC1）、扣带蛋白（CGN）、载脂蛋白CI（APOCI）、前蛋白转化酶枯草溶菌素9（PCSK9）和转钴胺素蛋白Ⅰ（TCN1）。基因本体（GO）分析，参与胞外调节功能过程的差异蛋白最多，其次为钙离子调节和蛋白降解调节过程蛋白。

结论

竹叶青蛇蛇毒染毒过程中抗原加工及呈递功能有关的蛋白表达水平变化明显，其中CK和溶质载体家族16成员1（SLC16A1）可以作为染毒后损伤的候选靶标。

关键词: 竹叶青蛇毒, TJMT标记定量蛋白组学, 血清差异表达蛋白, 肌酸激酶, 溶质载体家族16成员1

Abstract: Objective

To investigate the expression of serum proteins of the snake venom model of Trimeresurus stejnegeri， and to reveal the expressions of marker proteins in the process of poisoning.

Methods

Twelve Japanese big ear rabbits， male or female， were randomly divided into sham operation group and model group， with 6 rabbits in each group. The rabbits in model group were given intramuscular injection of 20 mg·kg^－1Trimeresurus stejnegeri snake venom， and the rabbits in sham operation group were given the same volume of normal saline injection； four hours later， the rabbits in two groups were anesthetized with sodium pentobarbital，the blood samples were obtained from the hearts of rabbits， and the blood serum was isolated. The differential proteins in serum of the rabbits in two groups were analyzed by tandem mass tag（TMT） combined with high performance liquid chromatography-tandem mass spectrometry （HPLC-TMS） quantitative proteomics technology. The biological changes of the differential proteins were analyzed by Proteome Discover 2.4 and other databases.

Results

The sham operation group and the experimental group were divided into two distinct clusters of differential proteins on the coordinate diagram of PCA analysis. A total of 199 differential proteins were identified and analyzed， including 139 up-regulated proteins； the top 5 up-regulated proteins were troponin I type 2，fast skeletal muscle（TNNI2）， parvalbum alpha（PV）， enolase2（ENO2），myoglobin（MB） and creatine kinase（CK）；there were 60 down-regulated proteins；the top 5 down-regulated proteins were apolipoprotein C1（APOC1），cinggulin（CGN）， proprotein invertase Bacillus subtilis proteinase 9（PCSK9） and transfer cobalamin protein 1（TCN1）.The differential proteins involved in the extracellular regulatory function were the most， followed by calcium ion regulation protein and degradation regulation proteins.

Conclusion

The expression levels of antigen processing and presentation function-related proteins change significantly during the process of infection of Trimeresurus stejnegeri venom， and CK and solute carrier fannily 16 member1（SLC16A1） could be used as candidate targets of post-infection injury.

Key words: Trimeresurus stejnegeri venom, TJMT labeled quantitative proteomics, Serum differentially expressed proteins, Creatine kinase, Solute carrier family 16 member 1

中图分类号:

R994

杨尾莲,王世军,沈芳华,张勇,陈福伟,苏秋香,史超,余沁瑶,陈涛. 基于蛋白质组学技术对竹叶青蛇毒染毒家兔模型血清蛋白质变化的检测及其意义[J]. 吉林大学学报(医学版), 2022, 48(2): 308-316.

Weilian YANG,Shijun WANG,Fanghua SHEN,Yong ZHANG,Fuwei CHEN,Qiuxiang SU,Chao SHI,Qinyao YU,Tao CHEN. Detection of serum protein changes in rabbit model poisoned by Trimeresurus stejnegeri snake venom based on proteomics and its significance[J]. Journal of Jilin University(Medicine Edition), 2022, 48(2): 308-316.

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Accession	Description of protein	Protein symbol	P value	FC
P02643	Troponin Ⅰ type 2, fast skeletal muscle	TNNI2	0.011 1	33.28
P02624	Parvalbumin alpha	PVALB	0.003 7	20.58
A0A5F9C342	2-phospho-D-glycerate hydro-lyase	ENO2	0.001 0	18.16
P02170	Myoglobin	MB	0.006 6	14.58
P00563	Creatine kinase	CK	0.000 2	13.99
P08507	Glycerol-3-phosphate dehydrogenase cytoplasmic	GPD1	0.010 8	12.88
A0A5F9CRZ7	Keratin 15	KRT15	0.023 2	12.27
Q28641	Myosin-4	MYH4	0.013 1	9.98
G1SZ91	Fatty acid binding protein 5	FABP5	0.011 6	9.60
A0A5F9C2V9	Syndecan 4	SDC4	0.004 3	9.29
A0A5F9D578	LIM domain binding 3	LDB3	0.005 8	8.96
P02586	Troponin C, skeletal muscle	TNNC2	0.006 8	8.75
G1U7S4	Phosphoglycerate mutase	PGAM2	0.000 2	8.06
P25704	Beta-enolase	ENO3	0.000 2	6.80
P00489	Glycogen phosphorylase, muscle form	PYGM	0.000 2	6.74
A0A5F9C4E2	Aldo-keto reductase family 1 member B1	AKR1B1	0.044 4	6.28
A0A5F9C3R6	Asparaginase-like protein 1	ASRGL1	<0.01	5.94
P11974	Pyruvate kinase PKM	PKM	<0.01	4.98
P00883	Fructose-bisphosphate aldolase A	ALDOA	0.007 7	4.74
G8ZF20	Alpha-globin 1	HBA1	0.039 6	4.71
G1U685	Chromogranin B	CHGB	0.002 2	4.35
P25230	Antimicrobial protein CAP18	CAP18	0.000 7	4.25
A0A5F9CU78	Nidogen 2	NID2	0.000 4	4.21
G1SNC4	Adenylate kinase isoenzyme 1	AK1	0.012 9	4.21
P00939	Triosephosphate isomerase	TPI1	0.001 8	4.01
A0A5F9DVL7	Tetraspanin	TSPAN8	0.000 2	3.51
G1T821	Fibrinogen like 1	FGL1	0.000 3	3.47
G1TCC1	Solute Carrier Family 16 Member 1	SLC16A1	0.020 2	3.43
A0A5F9D6D6	Collagen alpha-2(I) chain	COL1A2	0.014 8	3.42
G1TBW9	Dynein light chain	DYNLL2	0.023 8	3.41
FC:Foldchange.

Accession	Description of Protein	Protein symbol	P-value	FC
P33047	Apolipoprotein C-1	APOC1	0.029 2	0.122 5
B7NZD9	Cingulin (Predicted)	CGN	0.034 8	0.260 5
B6S6L6	Apolipoprotein C-Ⅰ	apoCⅠ	0.023 5	0.294 2
G1U461	Proprotein convertase 9	PCSK9	0.000 8	0.294 7
G1TA71	Transcobalamin 1	TCN1	0.000 9	0.376 8
P55057	Apolipoprotein C-4	APOC4	0.028 8	0.403 8
A0A5F9D253	Serpin family A member 7	SERPINA7	0.023 2	0.437 0
G1SJM1	Apolipoprotein H	CEP112	0.020 1	0.444 9
G1U9S1	Complement factor properdin	CFP	0.000 1	0.454 8
G1T5K3	Apolipoprotein A-Ⅱ	APOA2	0.013 8	0.462 7
P14461	Fibrinogen alpha chain	FGA	0.004 2	0.466 6
G1SQC7	Pleckstrin	PLEK	0.027 0	0.487 3
G1TFU9	Galectin-3-binding protein	LGALS3BP	0.000 1	0.498 1
G1SE06	Platelet-activating factor acetylhydrolase	PLA2G7	0.005 5	0.522 0
G1U754	Histidine-rich glycoprotein	HRG	0.000 1	0.587 3
G1U9R4	Apolipoprotein B	APOB	0.012 0	0.597 1
A0A5F9CI66	Ceruloplasmin	CP	0.007 9	0.618 5
G1SH58	Carboxypeptidase N subunit 1	CPN1	0.010 7	0.620 3
G1TRF0	Serglycin	SRGN	0.023 2	0.623 3
G1TCW1	Transferrin receptor protein 1	TFRC	0.026 1	0.645 6
G1SGV9	Proteasome subunit beta	PSMB8	0.019 4	0.649 8
P15196	Sex hormone-binding globulin	SHBG	0.017 6	0.649 9
G1TET0	Carboxypeptidase B2	CPB2	0.001 7	0.661 1
G1SQU1	Proteasome subunit beta	PSMB10	0.027 1	0.683 0
G1SER2	Glycoprotein Ib platelet subunit alpha	GP1BA	0.046 2	0.697 1
B7NZD7	Proteasome subunit beta	PSMB4	0.032 3	0.709 8
A0A5F9C295	Proteasome subunit alpha type	PSMA6	0.032 4	0.710 6
G1SZ14	Proteasome subunit alpha type-3	PSMA3	0.028 7	0.719 9
G1U415	Vitronectin	VTN	0.007 5	0.721 9
B7NZD9	Cingulin (Predicted)	CGN	0.034 8	0.726 0
FC:Foldchange.

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