摘要
目的:构建稳定表达pLVshRNA-EGFP-EBVsponge的SNU719细胞系。方法:合成能与EB病毒(EBV)编码的EBV-miR-BART16、EBV-miR-BART4-5p、EBV-miR-BART5-5p及EBV-miR-BART22 miRNAs特异结合的miRNA海绵(miRNA sponge)序列,用PITA软件预测构建的sponge是否结合非特异性miRNAs;构建pLVshRNA-EGFP-EBVsponge载体,与慢病毒包装质粒pH1、pH2共转染293T细胞,包装产生慢病毒颗粒,感染SNU719细胞,经嘌呤霉素筛选获得稳定表达细胞株;Real-time PCR检测细胞中EBV-miR-BART16、EBV-miRBART4-5p、EBV-miR-BART5-5p及EBV-miR-BART22表达水平。结果:成功构建pLVshRNA-EGFP-EBVspong(命名为pEGFP-EBV-sp);与对照组相比,pEGFP-EBV-sp组中4种microRNA表达水平显著降低,差异有统计学意义(P<0.05)。结论:成功构建稳定表达pEGFP-EBV-sp的SNU719细胞系。
Objective： To construct SNU719 cell line stably expressing pLVshRNA-EGFP-EBV- sponge. Methods： The miRNA sponge sequence of EBV-miR-BART16, EBV-miR-BART4-5p and EBV-miR-BARTS-5p which could encode with EB virus （EBV） and miRNA sponge sequence which specific binding with EBV-miR-BART22 miRNAs were synthesized. PITA software was adopted to predict the constructed sponge was binding with non-specific miRNAs. Constructing pLVshRNA-EGFP- EBVsponge vector, which were co-transfected with the pill and pH2 packing plasmid into 293T cells. The SNU719 cells were transfected with packing lentivirus transduction particles. The SNU719 cell strain with stable expression of cell strains was screened with puromycin. Then, the expression level of the four miRNAs were detected by real-time PCR. Results： The pLVshRNA-EGFP-EBVsponge （named pEGFP-EBV-sp） was successfully constructed. Comparing with control group, the expression level of 4 microRNA were significantly decreased, differences were statistically significant （ P 〈 0.05 ）. Conclusion： The stably expressing pLVshRNA-EGFP-EBVsponge of SNU719 cell line was successfully constructed.
出处
《贵州医科大学学报》
CAS
2017年第4期435-439,共5页
Journal of Guizhou Medical University
基金
中国医学科学院病原生物学研究所基本科研业务费项目(2012IPB107)
高等学校博士学科点专项科研基金(20111106120056)
