ALG5对猪流感病毒复制的影响

doi:10.11843/j.issn.0366-6964.2020.07.024

摘要/Abstract

摘要： 在实验室前期利用猪肾细胞（PK-15）CRISPR/Cas9全基因组敲除文库（PK-15-GeCKO）筛选到猪流感病毒（swine influenza virus，SIV）复制相关的宿主因子磷酸十二烷基磷酸酯-葡萄糖基转移酶（ALG5）的前提下，深入研究ALG5与SIV复制的关系。本研究利用CRISPR/Cas9技术构建了包含ALG5基因向导RNA（gRNA）的质粒LentiGuide-puro-ALG5，并通过慢病毒包装感染稳定表达Cas9蛋白的新生猪气管上皮（NPTr）细胞系，采用有限稀释法筛选ALG5基因敲除的单克隆细胞株，通过靶基因组测序及Western blot验证了ALG5基因在NPTr细胞上的敲除水平，通过CCK-8试验验证基因敲除细胞和野生型细胞的细胞活性。结合TCID₅₀和Western blot试验检测了ALG5敲除和过表达对SIV复制的影响。同时，检测了SIV感染NPTr细胞后内源性ALG5蛋白表达量的变化。最后检测了ALG5敲除对猪流感毒株F26复制的影响。结果显示：获得了ALG5敲除的NPTr单克隆细胞系（ΔALG5），ALG5基因敲除细胞与野生型细胞的细胞活性无显著差异。ALG5基因敲除显著抑制SIV的增殖，过表达促进SIV的增殖。在病毒感染条件下，随着病毒的增殖，ALG5的蛋白表达量上调。ALG5基因敲除后，也可以抑制猪流感毒株F26的复制，无毒株特异性。本研究表明，ALG5是影响猪流感病毒复制过程的一个重要宿主因子，为研究猪流感病毒的复制和致病机制奠定了基础。

关键词: ALG5, CRISPR/Cas9, 基因敲除, 猪流感病毒

Abstract: Earlier in the laboratory, we screened the swine influenza virus (SIV) replication-related host factor ALG5(dolichyl-phosphate beta-glucosyltransferase) through CRISPR/Cas9 genome-wide knockout library in pig kidney cells (PK-15-GeCKO). Under this premise, the relationship between ALG5 and SIV replication was studied in depth. In this study, a plasmid LentiGuide-puro-ALG5 containing ALG5 gene guide RNA (gRNA) was constructed by CRISPR/Cas9, and the newborn pig tracheal epithelial (NPTr) cell line stably expressing Cas9 protein was infected by lentiviral packaging. ALG5 knockout monoclonal cell line was screened by limiting dilution method. The knockout level of ALG5 gene on NPTr cells was confirmed by target genome sequencing and Western blot. The cell viability of knockout cells and wild-type cells was verified by CCK-8 experiment. The effects of ALG5 knockout and overexpression on SIV replication were examined in combination with TCID₅₀ and Western blot experiments. At the same time, the expression of endogenous ALG5 protein was detected after SIV infected NPTr cells. Finally, the effect of ALG5 knockout on replication of swine flu strain F26 was tested. NPTr monoclonal cell line that knocked out ALG5 (ΔALG5) was obtained, and there was no significant difference in cell activity between ALG5 knockout cells and wild-type cells. ALG5 gene knockout significantly inhibited SIV proliferation, and overexpression promoted SIV proliferation. Under viral infection conditions, the protein expression of ALG5 was up-regulated as the virus proliferated. ALG5 gene knockout can also inhibit the replication of swine flu strain F26, without strain specificity. This study indicates that ALG5 is an important host factor affecting the replication process of swine influenza virus. This study lays the foundation for studying the replication and pathogenic mechanism of swine influenza virus.

Key words: ALG5, CRISPR/Cas9, gene knockout, swine influenza virus

中图分类号:

S852.659.5

杨帅科, 邹佳辉, 蒋美君, 赵亚馨, 操继跃, 周红波. ALG5对猪流感病毒复制的影响[J]. 畜牧兽医学报, 2020, 51(7): 1719-1727.

YANG Shuaike, ZOU Jiahui, JIANG Meijun, ZHAO Yaxin, CAO Jiyue, ZHOU Hongbo. Effect of ALG5 on Swine Influenza Virus Replication[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(7): 1719-1727.

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