基于CRISPR/Cas9技术的猪TRIM8基因敲除细胞系构建及其对猪流行性腹泻病毒复制的调控作用

doi:10.16431/j.cnki.1671-7236.2025.08.027

Abstract

Abstract: 【Objective】 The experiment aimed to construct tripartite motif 8 (TRIM8) gene knockout porcine small intestinal epithelial cells (IPEC-J2) using CRISPR/Cas9 technology and investigate the regulatory effect of TRIM8 knockout on Porcine epidemic diarrhea virus (PEDV) replication,provide genetic resources and theoretical basis for an in-depth understanding of the mechanism of TRIM8 gene in antiviral natural immunity and the development of new strategies for PEDV prevention and control. 【Method】 Three sgRNAs (sgRNA1,sgRNA2 and sgRNA3) were designed in the first exon region of the porcine TRIM8 gene transcript,annealed to form double-stranded DNA ligated with linearized pGK1.2 vector,and the products were transformed into Escherichia coli DH5α competent cells for identification.The recombinant vector was transfected into IPEC-J2 cells.Sequences near the knockout site were amplified by PCR,and the efficiency of sgRNA knockout was determined by sequencing.Positive cell clones were identified by T7 endonuclease Ⅰ (M0302) zymography,and the knockout sequences were identified by sequencing of the TA clones.The expression of TRIM8 protein in gene knockout cells was detected by Western blotting.The changes in PEDV replication after TRIM8 gene knockout were detected by 50% tissue culture infective dose (TCID₅₀) and Real-time quantitative PCR. 【Result】 Sequencing of the recombinant vector showed that sgRNAs were successfully ligated with pGK1.2.Analysis of knockout efficiency showed that only sgRNA2 among the three sgRNAs had knockout efficiency.Eight positive monoclonal cells were screened by T7 endonuclease Ⅰ (M0302) digestion of PCR products.Sequencing analysis of TA clone revealed that the sequences of the 2 alleles of the TRIM8 gene missed 5 and 9 bp,respectively.Western blotting results showed that no TRIM8 protein was expressed in TRIM8 gene knockout cells.TCID₅₀ and Real-time quantitative PCR results showed that compared to wild-type IPEC-J2 cells,knockout of TRIM8 gene significantly up-regulated the viral titer of PEDV (P＜0.05),and M gene and N protein of PEDV were also significantly up-regulated (P＜0.05). 【Conclusion】 In this study,TRIM8 knockout IPEC-J2 cells were constructed by CRISPR/Cas9 technology and it improved the ability of PEDV replication in host cells.TRIM8 knockout cells might provide materials for further investigation of TRIM8 gene function and molecular mechanisms regulating PEDV replication.

Key words: pig; TRIM8 gene; Porcine epidemic diarrhea virus (PEDV); CRISPR/Cas9 technique; virus replication

CLC Number:

S852.65^＋1

WANG Wei, BI Zhenbin, GU Shanshen, XIAO Yeyi, ZHOU Yajing, WU Shenglong, BAO Wenbin, WANG Haifei. Construction of Porcine TRIM8 Gene Knockout Cell Line Based on CRISPR/Cas9 Technology and Its Regulatory Effect on Porcine Epidemic Diarrhea Virus Replication[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(8): 3790-3799.

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Construction of Porcine TRIM8 Gene Knockout Cell Line Based on CRISPR/Cas9 Technology and Its Regulatory Effect on Porcine Epidemic Diarrhea Virus Replication

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