摘要
目的 建立稳定表达人ADP核糖基化因子-4(ARF4)的卵巢癌SKVO3细胞系.方法 构建pCDH-CMV-MCS-EF1-Puro/ARF4真核表达载体;经测序验证后转染SKOV3细胞,运用实时定量PCR验证ARF4 mRNA的表达量;再将验证后的重组质粒与慢病毒包装质粒共转染HEK-293T细胞进行包装,收集重组慢病毒颗粒LV-ARF4,感染SKOV3细胞后用嘌呤霉素筛选稳定表达的细胞系;最后用实时定量PCR与Western Blot测定稳定表达的细胞系内ARF4的表达情况.结果 成功构建出pCDH-CMV-MCS-EF1-Puro/ARF4真核表达载体;该载体在SKOV3细胞中可明显上调ARF4 mRNA的表达,并在HEK-293T细胞中成功包装成重组慢病毒颗粒.与对照组相比,实验组SKOV3细胞感染ARF4慢病毒载体后ARF4 mRNA及蛋白的相对表达水平均明显升高,差异均具有统计学意义(均P<0.001).结论 成功构建了重组质粒pCDH-CMV-MCS-EF1-Puro/ARF4及慢病毒载体LV-ARF4,并建立了ARF4稳定转染的SKOV3细胞系,为进一步研究ARF4在卵巢癌中的生物学功能奠定了基础.
Objective To establish ovarian cancer cell line SKVO3 that can stably express human ADP ribosylation factor-4 (ARF4). Methods A eukaryotic expression vector pCDH-CMV-MCS-EF1-Puro/ARF4 was constructed and transfected into SKOV3 cells after verifying by DNA sequencing. The expression of ARF4 mRNA was verified by real-time quantitative PCR (qRT-PCR). Then, the recombinant plasmid with lentiviral packaging plasmids were co-transfected into SKOV3 cells for packaging. The recombinant lentiviral particles LV-ARF4 were collected and transfected into SKOV3 cells, and the stable transfected SKOV3 cell line was screening by culture with puromycin. The expression of ARF4 gene was detected by qRT-PCR and Western Blot. Results A eukaryotic expression vector pCDH-CMV-MCS-EF1-Puro/ARF4 was successfully constructed. The vector could significantly up-regulate the expression of ARF4 mRNA in SKOV3 cells and be successfully packaged into recombinant lentiviral particles in HEK-293T cells. Compared with the control group, the relative expression level of ARF4 mRNA and protein in SKOV3 cells was significantly increased after the infection with LV-ARF4 (all P<0.001). Conclusion The recombinant plasmid pCDH-CMV-MCS-EF1-Puro/ARF4 and lentiviral vector LV-ARF4 were successfully constructed. The establishment of stably infected SKOV3 cell line with LV-ARF4 provides an experimental foundation for further studies on the biological function of ARF4 in ovarian cancer.
出处
《国际生物医学工程杂志》
CAS
2017年第6期410-415,420,共7页
International Journal of Biomedical Engineering
基金
National Natural Sciences Foundation of China(81572900)%Key Project of Research and Development in Hunan Province(2016JC2036)%Natural Science Foundation of Hunan Province(2016JJ2161)%Shenghua Yuying Project of Central South University(502034003)%Free Exploration Program of Central South University(201710533365)%Innovation Project of Central South University (201710533222)
国家自然科学基金(81572900)%湖南省重点研发计划项目(2016JC2036)%湖南省自然科学基金(2016JJ2161)%中南大学升华育英计划(502034003)%中南大学自由探索项目(201710533365)%中南大学创新训练项目(201710533222)
