稳定表达猪瘟病毒NS3-NS4A和NS3pro-NS4A蛋白巨噬细胞系的构建及蛋白质组学分析

doi:10.11843/j.issn.0366-6964.2025.11.040

摘要/Abstract

摘要：

本研究旨在通过构建稳定表达的猪瘟病毒(classical swine fever virus, CSFV)NS3-NS4A和NS3^pro-NS4A蛋白巨噬细胞系，以期更好地探索宿主天然免疫与CSFV的相互作用关系。利用琼脂糖凝胶电泳、连接与转化和提取质粒等方法对重组表达载体进行构建，将构建好的pCMV-CBH-GFP-2A-Puro-CSFV NS3^pro-NS4A、pCMV-CBH-GFP-2A-Puro-CSFV NS3-NS4A和pCMV-CBH-GFP-2A-Puro重组表达载体分别感染HEK-293T细胞包装出慢病毒。然后利用包装成功的慢病毒感染猪肺泡巨噬细胞(3D4/21)，对感染细胞进行单克隆筛选得到稳定转染细胞系，最后用Western blot方法验证稳定细胞系蛋白表达。对其进行蛋白组学分析，筛选出MAPK、PI3K通路的相互作用蛋白，并进行功能分析，利用Western blot检测CDK4、CDC37、CASP3、IKBKG、NFκB1蛋白表达量。结果表明，经MegAlign分析构建的过表达CSFV非结构蛋白NS3-NS4A以及NS3^pro-NS4A慢病毒载体氨基酸序列全部正确，无移码；重组表达载体感染293T细胞24 h后，感染效率可达90%以上，慢病毒包装成功；Western blot分析成功获得稳定表达CSFV NS3-NS4A、NS3^pro-NS4A巨噬细胞系；通过蛋白组学分析，筛选出NS3-NS4A/mock组中MAPK与PI3K通路中上调蛋白11个，下调蛋白24个；经Western blot检测CSFV NS3-NS4A显著降低了MAPK通路中CASP3、NFκB1、IKBKG的表达，PI3K通路中CDK4、CDC37的表达。本研究成功构建了稳定表达NS3-NS4A、NS3^pro-NS4A蛋白的3D4/21细胞系，并对蛋白组学筛选的关键蛋白进行功能分析，为研究CSFV NS3-NS4A调控宿主天然免疫应答的机制提供物质基础。

关键词: 猪瘟病毒, NS3-NS4A, NS3^pro-NS4A, 猪肺泡巨噬细胞, 稳定表达

Abstract:

The purpose of this study is to better explore the interaction between host innate immunity and classical swine fever virus (CSFV) by constructing a stable expressing CSFV NS3/NS4A and pCMV-CBH-GFP-2A-Puro-CSFV NS3^pro-NS4A, pCMV-CBH-GFP-2A-Puro-CSFV NS3-NS4A and pCMV-CBH-GFP-2A-Puro recombinant expression vector were separately infected with HEK-293T cells packaged out of the lentivirus. Then, packaged lentivirus was used to infect porcine alveolar macrophages (3D4/21), monoclonal screening of infected cell lines was performed to obtain stable cell lines, and the protein expression of stable cell lines was verified by Western blot. Proteomic analysis was conducted on it to screen out the interacting proteins of MAPK and PI3K pathways, and functional analysis was carried out. Detection of CDK4, CDC37, CASP3, IKBKG, and NFκB1 Protein Expression Levels via Western blot Analysis. The results showed that the amino acid sequences of NS3-NS4A and NS3^pro-NS4A lentiviral vectors constructed by MegAlign analysis were correct without frameshift. After the recombinant expression vector infected 293T cells for 24 hours, the infection efficiency could reach more than 90%, the lentvirus was successfully packaged, and the stable expression of CSFV NS3-NS4A and NS3^pro-NS4A macrophage cell lines were successfully obtained by Western blot analysis. Through proteomic analysis, 11 up-regulated and 24 down-regulated proteins in the MAPK and PI3K pathways were screened out in the NS3-NS4A/mock group. Western blot analysis revealed that CSFV NS3-NS4A significantly reduced the expression of CASP3, NFκB1, and IKBKG in the MAPK pathway, as well as CDK4 and CDC37 in the PI3K pathway. In this study, 3D4/21 cell lines stably expressing NS3-NS4A and NS3^pro-NS4A proteins were successfully constructed, and conducted functional analysis on the key proteins screened by proteomics, which provides a material basis for studying the mechanism of CSFV NS3-NS4A regulating the host innate immune responses.

Key words: classical swine fever virus, NS3-NS4A, NS3^pro-NS4A, porcine alveolar macrophages, stably expressing

中图分类号:

S852.4

郑晓茹, 王一丹, 张丽红, 杨莹莹, 赵欣如, 李敏, 黄娟, 张乔亚, 曹志. 稳定表达猪瘟病毒NS3-NS4A和NS3^pro-NS4A蛋白巨噬细胞系的构建及蛋白质组学分析[J]. 畜牧兽医学报, 2025, 56(11): 5839-5851.

ZHENG Xiaoru, WANG Yidan, ZHANG Lihong, YANG Yingying, ZHAO Xinru, LI Min, HUANG Juan, ZHANG Qiaoya, CAO Zhi. Construction and Proteomic Analysis of Macrophage Cell Lines Stably Expressing NS3-NS4A and NS3^pro-NS4A of Classical Swine Fever Virus[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(11): 5839-5851.

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Primer名称 Primer name	序列(5′→3′) Sequence
2-3-F1	GCTCTAGAATGGGAAAGATAGATGGCGG
2-3-R1	CAAACGGCAGGCCCTCTAAGCACCCAGC
2-3-F2	GCTGGGTGCTTAGAGGGCCTGCCGTTTG
2-3-R2	ATAAGAATGCGGCCGCTCATAGACCAACTAC
2B-3^pro-F1	GCTCTAGAATGGAAGGGGTATACCTT
2B-3^pro-R2	ATAAGAATGCGGCCGCTCAGTAGCAAGGGTT
3-4A-F1	GCTCTAGAATGGGGCCTGCCGTTTG
3-4A-R1	TTCTCAGCTGTTGATAGACCAACTACTT
3-4A-F2	AAGTAGTTGGTCTATCAACAGCTGAGAA
3-4A-R2	ATAAGAATGCGGCCGCTCATAGCTCCTTCAA
3^pro-4A-R1	TTCTCAGCTGTTGAGTAGCAAGGGTTAT
3^pro-4A-F2	ATAACCCTTGCTACTCAACAGCTGAGAA

通路 Pathway	项目 Item	蛋白序号 Protein No.	蛋白名称 Protein name	基因 Gene	NCBI蛋白登录号 NCBl protein accession No.		置信度/% Max identity
通路 Pathway	项目 Item	蛋白序号 Protein No.	蛋白名称 Protein name	基因 Gene	人 Human	猪 Pig	置信度/% Max identity
MAPK	上调蛋白 Up-regulated protein	1	phospholipase A2 group IVA	PLA2G4A	NP_001298122.2	XP_020919484.1	83
		2	amphiregulin	AREG	NP_001648.1	NP_999541.1	90
		3	tumor protein p53	P53	NP_000537.3	NP_998989.3	99
	下调蛋白 Down-regulated protein	4	tumor protein p53	P53	NP_000537.3	NP_998989.3	99
		5	growth factor receptor bound protein 2	GRB2	NP_002077.1	NP_001131100.1	98
		6	TNFRSF1A associated via death domain	TRADD	NP_001310481.1	XP_020949709.1	99
		7	TAO kinase 3	TAOK3	NP_001333416.1	XP_001924435.2	95
		8	serine/threonine kinase 3	STK3	NP_001243241.1	XP_005655407.3	93
		9	stathmin 1	STMN1	NP_001138926.1	XP_005665169.1	81
		10	protein phosphatase, Mg²⁺/Mn²⁺ dependent 1B	PPM1B	NP_001028729.1	XP_003125227.2	91
		11	mitogen-activated protein kinase kinase 4	MAP2K4	NP_001268364.1	XP_020923549.1	93
		12	inhibitor of nuclear factor kappa B kinase regulatory subunit gamma	IKBKG	NP_001093326.2	NP_001106524.1	96
		13	amphiregulin	AREG	NP_001648.1	NP_999541.1	90
		14	nuclear factor kappa B subunit 1	NFKB1	NP_001158884.1	NP_001041697.1	90
		15	caspase 3	CASP3	NP_001341706.1	NP_999296.1	91

通路 Pathway	项目 Item	蛋白序号 Protein No.	蛋白名称 Protein name	基因 Gene	NCBI蛋白登录号 NCBl protein accession No.		置信度/% Max identity
通路 Pathway	项目 Item	蛋白序号 Protein No.	蛋白名称 Protein name	基因 Gene	人 Human	猪 Pig	置信度/% Max identity
P13K	上调蛋白 Up-regulated protein	1	integrin subunit beta 5	ITGB5	NP_001341693.1	NP_001233598.1	99
		2	BCL2 like 1	BCL2L1	NP_001182.1	NP_999450.1	99
		3	phosphoenolpyruvate carboxykinase 2, mitochondrial	PCK2	NP_001018083.2	NP_001155225.1	96
		4	integrin subunit beta 5	ITGB5	NP_001341693.1	NP_001233598.1	99
		5	thrombospondin 4	THBS4	NP_001293141.1	XP_020940149.1	95
		6	amphiregulin	AREG	NP_001648.1	NP_999541.1	90
		7	tumor protein p53	P53	NP_000537.3	NP_998989.3	99
		8	collagen type Ⅵ alpha 2 chain	COL6A2	NP_001840.3	XP_020938158.1	93
	下调蛋白 Down-regucated protein	9	tumor protein p53	P53	NP_000537.3	NP_998989.3	99
		10	collagen type Ⅵ alpha 2 chain	COL6A2	NP_001840.3	XP_020938158.1	93
		11	cyclin dependent kinase 4	CDK4	NP_000066.1	NP_001116569.1	98
		12	growth factor receptor bound protein 2	GRB2	NP_002077.1	NP_001131100.1	98
		13	protein tyrosine kinase 2	PTK2	NP_001186578.1	XP_020944510.1	96
		14	protein phosphatase 2 regulatory subunit B’alpha	PPP2R5A	NP_001186685.1	NP_001093662.1	99
		15	heat shock protein 90 alpha family class B member 1	HSP90AB1	NP_001258898.1	NP_001231362.1	99
		16	cell division cycle 37, HSP90 cochaperone	CDC37	NP_008996.1	NP_001116568.1	99
		17	eukaryotic translation initiation factor 4B	EIF4B	NP_001287750.1	XR_002343952.1	93
		18	inhibitor of nuclear factor kappa B kinase regulatory subunit gamma	IKBKG	NP_001093326.2	NP_001106524.1	96
		19	amphiregulin	AREG	NP_001648.1	NP_999541.1	90
		20	nuclear factor kappa B subunit 1	NFKB1	NP_001158884.1	NP_001041697.1	90
		21	thrombospondin 1	THBS1	NP_003237.2	NP_001231465.1	90

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