慢病毒eIF4E-shRNA的构建及其感染Hela细胞株的初步研究

发布时间：2018-04-09 21:40

本文选题：eIF4E　切入点：pLVTHM　出处：《青岛大学》2011年硕士论文

【摘要】：目的利用RNA干扰技术,以人eIF4E基因为靶基因设计特异性的小干扰RNA(small interference RNA, siRNA),构建人eIF4E基因短发夹RNA (short hairpin RNA, shRNA)重组慢病毒质粒表达载体,并进行PCR和测序鉴定；利用重组成功的慢病毒表达载体转染包装细胞293T,初步观察慢病毒颗粒对宫颈癌Hela细胞的感染情况,为进一步研究该载体对目的基因eIF4E的沉默效果以及探索宫颈癌基因治疗的新方法奠定基础。方法设计并合成人eIF4E基因特异性干扰片段,连接到经双酶切线性化的PLVTHM载体质粒,转化大肠杆菌(escherichia coli,E.coli) TOP10感受态细胞,筛选阳性菌落,扩增后提取质粒,进行PCR和测序鉴定；将成功插入干扰片段的慢病毒质粒PLVTHM、包装质粒pMDLg-pRRE、pRSV-REV、包膜质粒PMD2G按照一定比例混合,与转染试剂LipoD293TM DNA In Vitro共同转染包装细胞293T,收集含有慢病毒颗粒的培养基上清液,浓缩病毒颗粒并测定滴度,将慢病毒颗粒感染Hela细胞,初步观察Hela细胞感染情况。结果经过PCR鉴定和测序分析,成功构建出4个人eIF4E基因shRNA重组慢病毒表达载体；慢病毒质粒转染293T细胞包装生产慢病毒颗粒,收集浓缩后病毒滴度达4×108TU/ml;感染Hela细胞后,初步观察发现强弱不等的荧光信号。结论成功构建了4个人eIF4e基因特异性shRNA重组慢病毒表达载体,并包装出慢病毒颗粒,成功感染了宫颈癌Hela细胞,为进一步研究宫颈癌靶向基因治疗奠定基础。
[Abstract]:Objective to construct a recombinant lentivirus plasmid expressing human eIF4E gene short hairpin RNA short hairpin (shRNAs) using human eIF4E gene as target gene and design small interfering RNA(small interference RNAs (siRNAs) with human eIF4E gene as target gene, and to identify the expression vector by PCR and sequencing.The recombinant lentivirus expression vector was used to transfect the packaging cell line 293T, and the infection of lentivirus particles to cervical cancer Hela cells was preliminarily observed.It provides a basis for further studying the silencing effect of the vector on the target gene eIF4E and exploring a new method of gene therapy for cervical cancer.Methods the specific interference fragment of human eIF4E gene was designed and synthesized, and ligated to the PLVTHM vector plasmid with double enzyme digestion. The plasmid was transformed into E. coli (E. coli) cells, and the positive colony was screened. The plasmid was amplified and identified by PCR and sequencing.The lentivirus plasmid PLVTHM was successfully inserted into the interfering fragment, and the packaging plasmid pMDLg-pRREP pRSV-REV.The encapsulated plasmid PMD2G was mixed in a certain proportion and co-transfected with the transfection reagent LipoD293TM DNA in Vitro into the packaging cell 293T, and the supernatant of the medium containing lentivirus particles was collected.The virus particles were concentrated and the titer was measured. The lentivirus particles were infected with Hela cells and the infection of Hela cells was observed.Results four recombinant lentivirus expression vectors of eIF4E gene shRNA were successfully constructed by PCR identification and sequencing analysis. Lentivirus particles were produced by packaging of lentivirus plasmid transfected into 293T cells, and the titer of concentrated virus was 4 脳 108 TU / ml. After infection with Hela cells, 4 脳 108TU / ml was collected.Preliminary observation showed that the intensity of fluorescence signal was different.Conclusion four human eIF4e gene-specific shRNA recombinant lentivirus expression vectors were successfully constructed, and lentivirus particles were packaged, and successfully infected with cervical cancer Hela cells, which laid a foundation for further research on cervical cancer targeted gene therapy.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R346

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