敲除LTA4H基因的PK-15细胞系构建及其对口蹄疫病毒复制的影响

Construction of PK-15 cell line with LTA4H gene knockout and its impact on the replication of foot-and-mouth disease virus

摘要:

【目的】利用CRISPR/Cas9基因编辑技术构建白三烯A4水解酶(leukotriene A4 hydrolase, LTA4H)基因缺失的猪肾细胞系(porcine kidney-15, PK-15),探究LTA4H对口蹄疫病毒(foot-and-mouth disease virus, FMDV)复制的影响,为开展LTA4H功能研究及调控病毒复制机制研究提供理论依据。【方法】设计2条针对猪LTA4H基因的引导RNA (small guide RNA, sgRNA),分别构建至载体pX459-puro-MCS中;将CRISPR重组质粒转染PK-15细胞,用嘌呤霉素(puromycin)抗生素筛选,并通过有限稀释法筛选单克隆细胞,之后通过Western blotting和测序检测LTA4H基因的敲除,获得LTA4H基因功能缺失细胞系。使用Western blotting、RT-qPCR及病毒滴度测定等方法检测敲除LTA4H基因后对FMDV复制及相关蛋白的表达情况。【结果】获得的单克隆敲除细胞系与野生型细胞相比,能够显著抑制FMDV复制。【结论】本研究成功构建了LTA4H基因敲除的PK-15细胞系,证明LTA4H对FMDV的复制具有促进作用,研究结果为后续LTA4H功能研究提供理论依据。

关键词:CRISPR/Cas9;白三烯A4水解酶;细胞系;口蹄疫病毒

Abstract:

【Objective】 A LTA4H-deleted porcine kidney cell line (PK-15) was constructed by CRISPR/Cas9 to study the effect of LTA4H on the replication of foot-and-mouth disease virus (FMDV), with a view to providing a theoretical basis for revealing the functions of LTA4H and the mechanism of regulating virus replication. 【Methods】 Two small guide RNAs (sgRNAs) targeting porcine LTA4H were designed and integrated into the vector pX459-puro-MCS, respectively. The recombinant plasmid was transfected into PK-15 cells, which were then cultured in the medium supplemented with puromycin, and the monoclonal cells were selected by limiting dilution method. Western blotting and sequencing were performed to detect LTA4H knockout, and LTA4H-deleted cells were obtained. Furthermore, Western blotting, RT-qPCR, and virus titer assay were employed to examine FMDV replication and protein expression after LTA4H knockout. 【Results】 Compared with the wild type cells, the LTA4H-deleted PK-15 cells exhibited inhibited FMDV replication. 【Conclusion】 This study successfully constructed a PK-15 cell line with LTA4H gene knockout, demonstrating that LTA4H can promote the replication of FMDV, and the results provided a theoretical basis for the subsequent research on the function of LTA4H.

Key words:CRISPR/Cas9;leukotriene A4 hydrolase;cell lines;foot-and-mouth disease virus