CD163基因敲除iPAMs的构建及其感染PRRSV的特征分析

doi:10.16431/j.cnki.1671-7236.2024.08.025

Abstract

Abstract: 【Objective】 The aim of this study was to obtain immortalized porcine alveolar macrophages (iPAMs) with cluster of differentiation 163 (CD163) gene knockout,and investigate the role of CD163 protein in the invasion process of Porcine respiratory and reproductive syndrome virus (PRRSV) at the cellular level.【Method】 The sgRNA plasmid (pX330-sgCD163) targeting the exon 7 of CD163 gene was transfected into iPAMs,followed by monoclonal cell selection and genotype identification using a limited dilution method after 48 h of transfection.CD163 gene knockout iPAMs were screened by Western blotting and off-target analysis.The PRRSV infection characteristics of CD163 gene knockout iPAMs were analyzed by Real-time quantitative PCR,Western blotting,indirect immunofluorescence assay (IFA) and 50% tissue culture infective dose (TCID₅₀).【Result】 Sequencing results showed that one of the 100 monoclonal cells was CD163 gene knockout iPAMs,with an efficiency of 1%.Western blotting results showed that there was no expression of CD163 protein in CD163 gene knockout iPAMs,and no off-target effects were detected at the predicted off-target sites.Real-time quantitative PCR results showed that compared with wild-type iPAMs group,the viral copy number of CD163 gene knockout iPAMs group was extremely significantly decreased (P＜0.01).The results of Western blotting and IFA showed that there was no expression of PRRSV-N protein in CD163 gene knockout iPAMs group after 24 h of post-infection.TCID₅₀ assays also revealed that there was no cytopathic lesions in CD163 gene knockout iPAMs group.【Conclusion】 This study successfully constructed CD163 gene knockout iPAMs using CRISPR/Cas9 editing system,which completely resisted PRRSV infection.The establishment of this cell line provided novel research materials for investigating the interaction mechanism between PRRSV and CD163 protein in future studies.

Key words: CRISPR/Cas9; CD163 gene; immortalized porcine alveolar macrophages (iPAMs); Porcine reproductive and respiratory syndrome virus (PRRSV)

CLC Number:

S852.65⁺9.6

DONG Zexia, LIN Xin, ZHOU Qilyu, WANG Nan, HUANG Lei, LIU Zhiguo, FENG Zheng, MU Yulian. Construction and PRRSV Infection Characteristic Analysis of CD163 Gene Knockout iPAMs[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(8): 3471-3483.

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Construction and PRRSV Infection Characteristic Analysis of CD163 Gene Knockout iPAMs

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