应用CRISPR/Cas9基因敲除技术研究B4GalT7基因对丙型肝炎病毒感染的影响

Importance of B4GalT7 gene in hepatitis C virus infection in by CRISPR/Cas9-mediated gene knockout cells

目的应用CRISPR/Cas9系统建立B4GalT7基因敲除的Huh-7.5细胞株,进而验证其对丙型肝炎病毒(HCV)感染的影响。方法针对人源B4GalT7基因设计3个不同的sgRNA,构建同时表达Cas9和sgRNA的慢病毒转移质粒,制备重组慢病毒并转导Huh-7.5细胞,用嘌呤霉素筛选细胞克隆,提取细胞克隆的基因组DNA,对sgRNA靶点附近的DNA片段进行PCR扩增,纯化PCR产物测序分析鉴定,并用Western blot进一步验证B4GalT7蛋白表达情况。成功建立基因敲除细胞株后,比较其与野生型Huh-7.5细胞株对HCV的易感性。结果成功建立了B4GalT7基因敲除的Huh-7.5细胞株,证明HCV对该细胞株的感染能力明显低于野生型Huh-7.5细胞株。结论 B4GalT7基因敲除可显著减弱HCV对细胞的感染。

Objective To study the importance of B4GalT7 in HCV infection by making B4GalT7 knockout Huh-7.5cell lines using the CRISPR/Cas9 technology. Methods Three B4GalT7-specific sgRNAs were designed and expressed using the pLentiCRISPR/Cas9-sgRNA plasmids in which both Cas9 and sgRNA could be simultaneously expressed from a single vector.Upon lentivirus transduction and selection with puromycin,B4GalT7-knockout Huh-7.5cell lines were determined by PCR amplification,DNA sequence analysis,and Western blot.The susceptibility of B4GalT7-knockout cells and parental Huh-7.5cells to HCV infection was compared. Results The B4GalT7-knockout Huh-7.5cells were successfully constructed.B4GalT7 knockout resulted in a significantly reduction of HCV infection in Huh-7.5cells. Conclusions B4GalT7 knockout remarkably reduced the susceptibility of Huh-7.5cells to HCV infection.