發(fā)布時間：2018-04-09 21:40

  本文選題：eIF4E　切入點：pLVTHM　出處：《青島大學(xué)》2011年碩士論文

【摘要】：目的利用RNA干擾技術(shù),以人eIF4E基因為靶基因設(shè)計特異性的小干擾RNA(small interference RNA, siRNA),構(gòu)建人eIF4E基因短發(fā)夾RNA (short hairpin RNA, shRNA)重組慢病毒質(zhì)粒表達載體,并進行PCR和測序鑒定；利用重組成功的慢病毒表達載體轉(zhuǎn)染包裝細胞293T,初步觀察慢病毒顆粒對宮頸癌Hela細胞的感染情況,為進一步研究該載體對目的基因eIF4E的沉默效果以及探索宮頸癌基因治療的新方法奠定基礎(chǔ)。 方法設(shè)計并合成人eIF4E基因特異性干擾片段,連接到經(jīng)雙酶切線性化的PLVTHM載體質(zhì)粒,轉(zhuǎn)化大腸桿菌(escherichia coli,E.coli) TOP10感受態(tài)細胞,篩選陽性菌落,擴增后提取質(zhì)粒,進行PCR和測序鑒定；將成功插入干擾片段的慢病毒質(zhì)粒PLVTHM、包裝質(zhì)粒pMDLg-pRRE、pRSV-REV、包膜質(zhì)粒PMD2G按照一定比例混合,與轉(zhuǎn)染試劑LipoD293TM DNA In Vitro共同轉(zhuǎn)染包裝細胞293T,收集含有慢病毒顆粒的培養(yǎng)基上清液,濃縮病毒顆粒并測定滴度,將慢病毒顆粒感染Hela細胞,初步觀察Hela細胞感染情況。 結(jié)果經(jīng)過PCR鑒定和測序分析,成功構(gòu)建出4個人eIF4E基因shRNA重組慢病毒表達載體；慢病毒質(zhì)粒轉(zhuǎn)染293T細胞包裝生產(chǎn)慢病毒顆粒,收集濃縮后病毒滴度達4×108TU/ml;感染Hela細胞后,初步觀察發(fā)現(xiàn)強弱不等的熒光信號。 結(jié)論成功構(gòu)建了4個人eIF4e基因特異性shRNA重組慢病毒表達載體,并包裝出慢病毒顆粒,成功感染了宮頸癌Hela細胞,為進一步研究宮頸癌靶向基因治療奠定基礎(chǔ)。
[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.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R346

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