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

摘要/Abstract

摘要： 【目的】利用CRISPR/Cas9技术构建三方基序蛋白8(tripartite motif 8，TRIM8)基因敲除的猪小肠上皮细胞(IPEC-J2)，并探究TRIM8基因敲除对猪流行性腹泻病毒(PEDV)复制的调控作用，为深入理解TRIM8基因在抗病毒天然免疫中的作用机制及开发新的PEDV防控策略提供基因资源和理论依据。【方法】在猪TRIM8基因转录本第1外显子区域设计3条sgRNAs(sgRNA1、sgRNA2和sgRNA3)，经退火形成双链DNA，与线性化pGK1.2载体连接，产物转化大肠杆菌DH5α感受态细胞进行鉴定。将重组载体转染IPEC-J2细胞。PCR扩增敲除位点附近序列，并通过测序判断sgRNA敲除效率；利用T7核酸内切酶Ⅰ(M0302)酶切鉴定阳性细胞克隆，通过TA克隆测序鉴定敲除序列；利用Western blotting检测基因敲除细胞中TRIM8蛋白表达情况。利用半数组织培养感染剂量(TCID₅₀)和实时荧光定量PCR检测TRIM8基因敲除后PEDV复制的变化情况。【结果】重组载体测序结果显示，sgRNAs与pGK1.2成功连接。敲除效率分析发现，3个sgRNAs中只有sgRNA2有敲除效率。PCR产物经T7核酸内切酶Ⅰ(M0302)酶切筛选出8个阳性单克隆细胞。TA克隆测序分析发现，TRIM8基因2个等位基因序列分别缺失5和9 bp。Western blotting结果表明，TRIM8基因敲除细胞中未见TRIM8蛋白表达。TCID₅₀和实时荧光定量PCR检测结果显示，与野生型IPEC-J2细胞相比，敲除TRIM8基因后显著上调了PEDV的病毒滴度(P＜0.05)，且PEDV M基因和N蛋白也显著上调(P＜0.05)。【结论】本研究利用CRISPR/Cas9技术构建了TRIM8基因敲除的IPEC-J2细胞，并提高了PEDV在宿主细胞中复制的能力。TRIM8基因敲除细胞可为探究TRIM8基因功能及其调控PEDV复制的分子机制提供材料。

关键词: 猪; TRIM8基因; 猪流行性腹泻病毒(PEDV); CRISPR/Cas9技术; 病毒复制

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

中图分类号:

S852.65^＋1

王威, 毕祯彬, 顾善绅, 肖叶懿, 周雅静, 吴圣龙, 包文斌, 王海飞. 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[J]. 中国畜牧兽医, 2025, 52(8): 3790-3799.

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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