TSG101基因敲低对猪伪狂犬病病毒体外增殖的影响

doi:10.11843/j.issn.0366-6964.2024.09.035

畜牧兽医学报 ›› 2024, Vol. 55 ›› Issue (9): 4110-4120.doi: 10.11843/j.issn.0366-6964.2024.09.035

TSG101基因敲低对猪伪狂犬病病毒体外增殖的影响

王梦迪²(), 王昱旻¹(), 张震², 鲁秀香¹, 王恒¹, 樊文杰¹, 姚晨¹, 刘鹏翔¹, 马延杰¹, 褚贝贝¹, 王江¹^,*(), 杨国宇¹^,²^,*()

1. 河南农业大学农业农村部动物生化与营养重点开放实验室，郑州 450046
2. 河南牧业经济学院食品与生物工程学院，郑州 450046

收稿日期:2023-12-12 出版日期:2024-09-23 发布日期:2024-09-27
通讯作者: 王江,杨国宇 E-mail:mengdi.ok@163.com;2622364663@qq.com;wangjiang@henau.edu.cn;haubiochem@163.com
作者简介:王梦迪(1989-)，女，河南南阳人，博士，主要从事动物生理生化研究，E-mail：mengdi.ok@163.com
王昱旻(1999-)，女，硕士生，主要从事动物生理生化研究，E-mail：2622364663@qq.com
第一联系人：
王梦迪和王昱旻为同等贡献作者
基金资助:
河南省科技攻关(232102310381);河南牧业经济学院博士科研启动资金资助(2022HNUAHEDF033);国家重点研发计划(2021YFD1301201)

Effect of TSG101 Gene Knockdown on Proliferation of Pseudorabies Virus in vitro

Mengdi WANG²(), Yumin WANG¹(), Zhen ZHANG², Xiuxiang LU¹, Heng WANG¹, Wenjie FAN¹, Chen YAO¹, Pengxiang LIU¹, Yanjie MA¹, Beibei CHU¹, Jiang WANG¹^,*(), Guoyu YANG¹^,²^,*()

1. Key Laboratory of Animal Biochemistry and Nutrition, Ministry of Agriculture and Rural Affiars, Henan Agricultural University, Zhengzhou 450046, China
2. College of Food and Bioengineering, Henan University of Animal Husbandry and Economy, Zhengzhou 450046, China

Received:2023-12-12 Online:2024-09-23 Published:2024-09-27
Contact: Jiang WANG, Guoyu YANG E-mail:mengdi.ok@163.com;2622364663@qq.com;wangjiang@henau.edu.cn;haubiochem@163.com

摘要/Abstract

摘要：

本研究旨在使用慢病毒包装敲低技术构建敲低肿瘤易感基因(tumor susceptibility gene 101，TSG101)的慢病毒质粒及稳转PK15细胞系，并验证该敲低细胞系对伪狂犬病病毒(pseudorabies virus，PRV)感染的影响。针对TSG101基因构建并筛选, 成功获得重组慢病毒，用获得的重组慢病毒感染PK15细胞，并使用嘌呤霉素进行筛选，获得稳转敲低TSG101基因的PK15细胞系。通过Real-time PCR以及Western blot方法对所构建的细胞系进行编辑效率检测；使用CCK-8法对筛选出的细胞系进行细胞活力及生长状况检测；通过流式细胞术、Real-time PCR、Western blot以及子代病毒滴度方法验证该细胞系对PRV复制的影响；最后对PRV的吸附、进入以及复制阶段的试验探究敲低TSG101基因对于PRV感染有影响的阶段。结果表明，1)本研究所构建的细胞系中TSG101的表达明显降低，并将该细胞系命名为sh-TSG101细胞系；2)细胞活性检测结果显示，敲低TSG101基因对细胞活性及生长情况无显著影响；3)使用PRV-GFP、PRV-QXX感染sh-TSG101，体外试验证明敲低TSG101对PRV的增殖有显著的抑制作用。4)病毒的吸附、进入以及复制试验证明，敲低TSG101在PRV的复制阶段有明显的抑制作用。综上，本研究成功构建TSG101基因敲低的PK15细胞系，体外敲低TSG101能够显著抑制PRV增殖，为PRV等DNA病毒性疾病的防控提供了新思路，并为进一步探究TSG101调控PRV感染的分子机制奠定了基础。

关键词: 肿瘤易感基因101, 伪狂犬病病毒, PK15细胞

Abstract:

The aim of this study is to use lentiviral packaging knockdown technology to construct lentiviral plasmids for knocking down tumor susceptibility gene 101 (TSG101) and stably transfect PK15 cell lines, and to verify the impact of this knockdown cell line on PRV infection. A recombinant lentivirus was successfully constructed and screened for the TSG101 gene, and PK15 cells were infected with the obtained recombinant lentivirus. Puromycin was used for screening to obtain PK15 cell lines that knocked down the TSG101 gene. Perform editing efficiency testing on the constructed cell line using Real time PCR and Western blot methods; Use CCK-8 method to detect cell viability and growth status of the selected cell lines; Verify the effect of this cell line on PRV replication through flow cytometry, Real time PCR, Western blot, and progeny virus titer methods; Finally, experiments were conducted on the adsorption, entry, and replication stages of PRV to explore the impact of knocking down the TSG101 gene on PRV infection. The results showed that: 1) the expression of TSG101 was significantly reduced in the cell line constructed in this study, and the cell line was named sh-TSG101 cell line; 2) The results of cell viability testing showed that knocking down the TSG101 gene had no significant effect on cell viability and growth; 3) Using PRV-GFP and PRV-QXX to infect sh-TSG101, in vitro experiments have shown that knocking down TSG101 has a significant inhibitory effect on PRV proliferation. 4) The adsorption, entry, and replication experiments of the virus have shown that knocking down TSG101 has a significant inhibitory effect on the replication stage of PRV. In summary, this study successfully constructed PK15 cell lines with TSG101 gene knockdown. Knocking down TSG101 in vitro can significantly inhibit PRV proliferation, providing new ideas for the prevention and control of DNA viral diseases such as PRV, and laying a foundation for further exploring the molecular mechanism of TSG101 regulating PRV infection.

Key words: TSG101, pseudorabies virus, PK15 cells

中图分类号:

S852.659.1

王梦迪, 王昱旻, 张震, 鲁秀香, 王恒, 樊文杰, 姚晨, 刘鹏翔, 马延杰, 褚贝贝, 王江, 杨国宇. TSG101基因敲低对猪伪狂犬病病毒体外增殖的影响[J]. 畜牧兽医学报, 2024, 55(9): 4110-4120.

Mengdi WANG, Yumin WANG, Zhen ZHANG, Xiuxiang LU, Heng WANG, Wenjie FAN, Chen YAO, Pengxiang LIU, Yanjie MA, Beibei CHU, Jiang WANG, Guoyu YANG. Effect of TSG101 Gene Knockdown on Proliferation of Pseudorabies Virus in vitro[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(9): 4110-4120.

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基因 Genes	引物序列(5′→3′) Primer sequence	序列大小/bp Sequence size	用途 Purpose
shRNA1	F: CCGGGGAACAATTCCTGTGCCTTATCTCGAGATAAGGCACAGGAATTGTTCCTTTTTG R: AATTCAAAAAGGAACAATTCCTGTGCCTTATCTCGAGATAAGGCACAGGAATTGTTCC		基因敲低
shRNA2	F: CCGGGCTGGACACATACCCATATAACTCGAGTTATATGGGTATGTGTCCAGCTTTTTG R: AATTCAAAAAGCTGGACACATACCCATATAACTCGAGTTATATGGGTATGTGTCCAGC		基因敲低
shRNA3	F: CCGGGCAACAGGGCCACCAAATACTCTCGAGAGTATTTGGTGGCCCTGTTGCTTTTTG R: AATTCAAAAAGGAACAATTCCTGTGCCTTATCTCGAGATAAGGCACAGGAATTGTTCC		基因敲低
TSG101	F: CAGGGCCACCAAATACTTCCT R: GGAGGGTATCCGGATGGGTA		实时荧光定量 PCR
β-actin	F: CTGAACCCCAAAGCCAACCGT R: TTCTCCTTGATGTCCCGCACG	317	实时荧光定量 PCR
PRV gB	F: GGCATCGCCAACTTCTTCC R: CCTCGTCCACGTCGTCCTC	220	实时荧光定量 PCR

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TSG101基因敲低对猪伪狂犬病病毒体外增殖的影响

Effect of TSG101 Gene Knockdown on Proliferation of Pseudorabies Virus in vitro

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