人eIF4E基因shRNA慢病毒载体的构建及鉴定

CONSTRUCTION AND IDENTIFICATION OF RECOMBINANT LENTIVIRUS VECTOR EXPRESSING shRNA TARGETING HUMAN eIF4E GENE

目的设计并构建人真核细胞翻译起始因子4E(eIF4E)基因shRNA慢病毒质粒表达载体。方法设计合成人eIF4E基因RNA干扰双链DNA片段,重组到慢病毒质粒PLVTHM上,然后进行PCR鉴定和测序分析。重组质粒与3种包装质粒混合,以LipoD293TMDNA In Vitro包裹后转染293T细胞,收获病毒上清液感染宫颈癌HeLa细胞。结果经过PCR鉴定出的重组载体测序结果与目的序列一致,成功构建人eIF4E基因shRNA慢病毒质粒表达载体及获得相应慢病毒,并成功感染HeLa细胞,可见绿色荧光蛋白表达。结论成功构建人eIF4E基因shRNA慢病毒载体,为进一步研究宫颈癌基因治疗奠定基础。

Objective To design and construct shRNA lentivirus vectors targeting human eIF4E gene.Methods The shRNA oligonucleotides of eIF4E were designed and synthesized,which was then inserted into lentivirus vector PLVTHM.The recombinant sequences were identified by PCR and sequenced.The recombinant vector and three package plasmids were cotransfected to 293T cells with LipoD293TM DNA In Vitro.The recombinant lentivirus was harvested and then infected to HeLa cells.Results The recombinant eIF4E shRNA lentivirus vector was constructed successfully and the recombinant lentivirus was harvested and efficiently infected to HeLa cells.The expression of green fluorescent protein（GFP） was detected.Conclusion The shRNA lentivirus vectors targeting eIF4E gene are successfully constructed,which has laid the foundation for further study on gene therapy for cervical cancer.