WWOX基因慢病毒载体的构建及其在卵巢癌细胞中的表达

摘要

Objective:To construct the lentiviral vector carrying WWOX gene and investigate their expression in ovarian cancer PEOＩ cells. Methods:WWOX gene was acquired from cDNA clone plasmids by RTPCR, and then subcloned into the expression plasmid of lentiviral vector PCDH-CMV-MCS-EFＩ-copGFP to generate the lentiviral vector PCDH-WWOX. The lentiviral vector PCDH-WWOX was identified by restriction enzyme. Recombinant lentiviruse was produced by 293T cells following co-transfection of PCDHWWOX with the packaging plasmids pPACKHＩ-GAG,pPACKHＩ-REV and Pvsv-G. The resulting recombinant lentiviruse Lenti WWOX was then used to infect ovarian cancer PEOＩ cells. WWOX expression in PEOＩ cells was detected by RT-PCR and Western blotting analysis. Results: (1) The recombinant lentiviruse PCDH-WWOX carried the WWOX gene,and it could be expressed in human cell line. (2)The recombinant lentiviruses which carrying WWOX could be product by co-transfection PCDH-WWOX with packaging plasmid to 293T. (3) The recombinant lentiviruse Lenti WWOX could efficiently infect and deliver WWOX and GFP genes to PEOＩ cells. The infection efficiency is about 60％. And WWOX gene can be expressed efficiently in PEO1 by RT-PCR and Western blotting analysis. Conclusion:The recombinant lentiviruse PCDH-WWOX was produced successfully and it can deliver target gene WWOX to PEOＩ cells.%目的:构建及鉴定WWOX基因慢病毒载体,观察WWOX基因在人卵巢癌PEO1细胞株中的表达,为进一步研究WWOX基因的作用机制及其功能奠定基础.方法:采用PCR技术从克隆质粒模板中获得全长WWOX基因,将WWOX基因 亚克隆到慢病毒载体PCDH-CMV-MCS-EFl-copGFP中,构建慢病毒载体表达质粒PCDH-WWOX,通过双酶切验证后,慢病毒表达质粒与慢病毒包装质粒pPACKHl-GAG,pPACKH1-REV,Pvsv-G共转染293T细胞,获得携带WWOX基因及EGFP基因的重组慢病毒Lenti WWOX,并转染靶细胞人卵巢癌PEO1细胞株.通过RT-PCR和Western Blot验证Lenti WWOX在PEO1细胞株中的表达.结果:测序结果显示成功构建重组慢病毒载体表达质粒PCDH-WWOX;表达质粒与辅助包装质粒共转染包装细胞293T后能产生慢病毒颗粒并能有效感染靶细胞PEO1,转导效率约为60%,有WWOX基因mRNA和蛋白水平的高表达.结论:本实验成功构建了携带WWOX基因慢病毒表达载体,包装成病毒后能有效感染卵巢癌PEO1细胞.