摘要
目的构建Cofilin-1过表达的Huh-7细胞株。方法应用RT-PCR克隆出Cofilin-1的完整编码序列,将其亚克隆到慢病毒载体pWPT中,构建慢病毒载体表达质粒pWPT-Cofilin-1;将慢病毒表达质粒pWPT-Cofi-lin-1或pWPT-GFP与慢病毒包装质粒pMD2.G、psPAX2共转染293T细胞,获得携带Cofilin-1完整编码序列的慢病毒和携带GFP基因的慢病毒,将两种慢病毒分别感染Huh-7细胞;通过Real-TimePCR和Western blot检测Huh-7细胞中的Cofilin-1的过表达情况。结果测序结果显示成功构建重组慢病毒表达质粒pWPT-Cofilin-1;慢病毒有效感染Huh-7细胞,依据GFP绿色荧光可测定感染有效率超过95%;Cofilin-1在Huh-7细胞中的mRNA量和蛋白水平都成功的过表达。结论本实验的完成,为进一步研究Cofilin-1基因在肝癌中的生物功能和作用机制奠定了基础。
Objective Construct the Huh-7 cells strains which is overexpression of Cofilin-1 gene. Methods The complete CDS of Cofilin-1 gene was acquired by RT-PCR,and then subcloned into the lentiviral vector pWPT to construct the expression plasmid of lentiviral vector pWPT-Cofilin-1.The plasmid was identified by sequencing.Recombinant lentivirus was produced by 293 T cells following co-transfection of pWPT-Cofilin-1 or pWPT-GFP with the packaging plasmids pMD2.G and psPAX2.The recombinant lentivirus Cofilin-1 or GFP were respectively used to infect Huh-7 cells.The overexpression of Cofilin-1 in Huh-7 cells was detected by Real-Time PCR and Western blotting analysis. Results The recombinant lentivirus expression plasmid pWPT-Cofilin-1 has been constructed successfully.The efficiency of recombinant lentivirus infection was greater than 95% according to the green fluorescent.The mRNA and protein of Cofilin-1 was overexpressed in Huh-7 cells. Conclusion This study is the foundation for the further study of relationship between Cofilin-1 and hepatocellular carcinoma.
出处
《中南医学科学杂志》
CAS
2012年第1期65-69,共5页
Medical Science Journal of Central South China
