摘要
目的通过构建ZNF24基因过表达慢病毒载体,建立ZNF24基因过表达的人结直肠癌HCT116细胞株,为开展后续研究提供物质基础。方法通过PCR扩增ZNF24基因序列片段和3FLAG标签序列片段,并将两产物通过同源重组克隆至慢病毒载体pMT406中,构建成重组表达ZNF24慢病毒质粒pMT-ZNF24。将重组质粒pMT-ZNF24与辅助包装载体质粒pCMV-dR8.9和pCMV-VSV-G一起共转染293T细胞并收集慢病毒。应用孔稀释法检测病毒滴度,用qRT-PCR和蛋白印迹法检测慢病毒转染HCT116细胞后ZNF24的表达水平。结果成功构建了重组载体pMT-ZNF24,并获得相应的病毒,病毒滴度为3.25×10^(9) TU/ml。转染重组ZNF24慢病毒的HCT116细胞中ZNF24表达水平显著高于空白组和阴性对照组细胞。结论构建了ZNF24基因过表达慢病毒载体,并获得相应的病毒和稳定表达ZNF24的HCT116细胞株。
Objective To establish the human colorectal cancer cell line HCT116 with ZNF24 gene overexpression by constructing lentiviral vector of ZNF24 gene overexpression,and provide material basis for subsequent research.Methods The recombinant expression ZNF24 lentiviral plasmid pMT-ZNF24 was constructed by the homologous recombination of ZNF24 gene and 3FLAG tag sequence fragments which was amplified by PCR into lentiviral vector pMT-406.The recombinant plasmid pMT-ZNF24 and the auxiliary packaging vector plasmids pCMV-dR8.9 and pCMV-VSV-G were co-transfected into 293T cells,after which the lentivirus were collected.The virus titer was determined with well dilution method.The lentivirus was transfected into HCT116 cells,and the expression levels of ZNF24 were detected by qRT-PCR and Western blot.Results The recombinant vector pMT-ZNF24 was successfully constructed,and the corresponding virus was obtained.The virus titer was 3.25×10^(9) TU/ml.The expression levels of ZNF24 in cells transfected with recombinant ZNF24 lentivirus were significantly higher than those in blank and negative control.Conclusion The ZNF24 gene overexpression lentiviral vector had been constructed,and the corresponding virus and the HCT116 cell line expressing ZNF24 had been obtained.
作者
田硕
厉建中
TIAN Shuo;LI Jianzhong(Department of Biochemistry,School of Pharmacy,Naval Medical University,Shanghai 200433 China)
出处
《药学实践与服务》
CAS
2023年第4期222-226,共5页
Journal of Pharmaceutical Practice and Service
基金
国家自然科学基金(81570557,30871353)。
