EBV即刻早期基因BZLF1腺病毒載體的構(gòu)建和表達
發(fā)布時間：2018-07-23 15:02
【摘要】：目的 構(gòu)建EBV即刻早期基因BZLF1的非復制型重組腺病毒載體,293細胞包裝腺病毒,并檢測BZLF1在293細胞中的表達,為進一步研究腺病毒介導BZLF1進行EBV相關(guān)腫瘤的基因治療奠定基礎。 方法 RT-PCR擴增EBV BZLF1基因片段,經(jīng)雙酶切后亞克隆到腺病毒穿梭質(zhì)粒pAdTrack-CMV上,用TSS兩步法與腺病毒骨架質(zhì)粒pAdEasy-1共轉(zhuǎn)染大腸桿菌BJ5183,經(jīng)細菌內(nèi)同源重組產(chǎn)生攜帶BZLF1基因的重組腺病毒載體pAd-BZ,脂質(zhì)體法轉(zhuǎn)染人胚腎293細胞,包裝產(chǎn)生重組腺病毒vAd-BZ。采用RT-PCR和Western blotting檢測BZLF1基因在293細胞中的表達。 結(jié)果 通過PCR、序列測定、雙酶切等技術(shù)鑒定BZLF1基因已正確插入穿梭質(zhì)粒中,并與病毒骨架質(zhì)粒pAdEasy-1重組,成功構(gòu)建了表達BZLF1基因的重組腺病毒。熒光顯微鏡觀察到綠色熒光,證實在293細胞中包裝出vAd-BZ,PCR和Western結(jié)果表明轉(zhuǎn)染pAd-BZ的293細胞中有BZLF1基因的表達。 結(jié)論 成功構(gòu)建了表達EBV BZLF1重組腺病毒vAd-BZ,為進一步探討B(tài)ZLF1基因表達誘導EBV陽性腫瘤細胞中潛伏感染狀態(tài)病毒進入裂解期,進而殺傷EBV陽性腫瘤細胞奠定了實驗基礎。
[Abstract]:Objective to construct the non-replicating recombinant adenovirus vector of EBV immediate early gene BZLF1, and to detect the expression of BZLF1 in 293cells, so as to lay a foundation for the further study of adenovirus-mediated BZLF1 gene therapy for EBV related tumors. Methods the EBV BZLF1 gene fragment was amplified by RT-PCR and subcloned into adenovirus shuttle plasmid pAdTrack-CMV after double enzyme digestion. E. coli BJ5183 was co-transfected by TSS two-step method with adenovirus skeleton plasmid pAdEasy-1. Recombinant adenovirus vector pAd-BZwith BZLF1 gene was produced by homologous recombination in bacteria. The recombinant adenovirus vAd-BZwas packaged and transfected into human embryonic kidney 293 cells by liposome method. The expression of BZLF1 gene in 293 cells was detected by RT-PCR and Western blotting. Results the BZLF1 gene was correctly inserted into the shuttle plasmid by PCR sequencing and double enzyme digestion. The recombinant adenovirus expressing BZLF1 gene was successfully constructed by recombination with the viral skeleton plasmid pAdEasy-1. Green fluorescence was observed by fluorescence microscope. The results of vAd-BZN PCR and Western showed that the BZLF1 gene was expressed in 293 cells transfected with pAd-BZ. Conclusion the expression of EBV BZLF1 recombinant adenovirus vAd-BZwas successfully constructed, which laid an experimental foundation for further study on the induction of latent infection virus in EBV positive tumor cells into lytic phase by BZLF1 gene expression and the killing of EBV positive tumor cells.
【學位授予單位】：青島大學
【學位級別】：碩士
【學位授予年份】：2005
【分類號】：R346
本文編號：2139769
[Abstract]:Objective to construct the non-replicating recombinant adenovirus vector of EBV immediate early gene BZLF1, and to detect the expression of BZLF1 in 293cells, so as to lay a foundation for the further study of adenovirus-mediated BZLF1 gene therapy for EBV related tumors. Methods the EBV BZLF1 gene fragment was amplified by RT-PCR and subcloned into adenovirus shuttle plasmid pAdTrack-CMV after double enzyme digestion. E. coli BJ5183 was co-transfected by TSS two-step method with adenovirus skeleton plasmid pAdEasy-1. Recombinant adenovirus vector pAd-BZwith BZLF1 gene was produced by homologous recombination in bacteria. The recombinant adenovirus vAd-BZwas packaged and transfected into human embryonic kidney 293 cells by liposome method. The expression of BZLF1 gene in 293 cells was detected by RT-PCR and Western blotting. Results the BZLF1 gene was correctly inserted into the shuttle plasmid by PCR sequencing and double enzyme digestion. The recombinant adenovirus expressing BZLF1 gene was successfully constructed by recombination with the viral skeleton plasmid pAdEasy-1. Green fluorescence was observed by fluorescence microscope. The results of vAd-BZN PCR and Western showed that the BZLF1 gene was expressed in 293 cells transfected with pAd-BZ. Conclusion the expression of EBV BZLF1 recombinant adenovirus vAd-BZwas successfully constructed, which laid an experimental foundation for further study on the induction of latent infection virus in EBV positive tumor cells into lytic phase by BZLF1 gene expression and the killing of EBV positive tumor cells.
【學位授予單位】：青島大學
【學位級別】：碩士
【學位授予年份】：2005
【分類號】：R346
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