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

Abstract

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

CLC Number:

S852.659.1

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.

Figures/Tables 9

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

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