热休克蛋白70重组腺病毒载体的构建及鉴定

发布时间：2018-01-10 11:10

本文关键词：热休克蛋白70重组腺病毒载体的构建及鉴定　出处：《青岛大学》2008年硕士论文　论文类型：学位论文

【摘要】： 目的构建人热休克蛋70(heat shock protein70,HSP70)的腺病毒表达载体,旨在将成功构建的携带外源基因HSP70的重组腺病毒感染靶细胞,从而为进一步探讨HSP70对应激状态下细胞的保护作用奠定了实验基础。方法自热休克处理后的人宫颈癌细胞系Hela中提取细胞总RNA,经逆转录反应后,利用人工合成的引物行PCR扩增,获得目的基因HSP70的cDNA。将外源cDNA定向亚克隆到pAdTrack-CMV质粒上,采用AdEasy系统在原核细胞E.coli BJ5183中完成穿梭质粒pAdTrack-CMV-HSP70与骨架质粒pAdEasy-1之间的高效同源重组,构建HSP70重组腺病毒载体。重组体经筛选后,脂质体法转染人胚肾293细胞,包装产生重组腺病毒vAd-HSP70。最后,收获病毒进行滴度鉴定,用RT-PCR方法检测目的基因在人宫颈癌Hela细胞的转录表达。结果通过PCR、序列测定以及限制性酶切证实HSP70基因能够正确插入腺病毒表达载体,并在HEK293细胞中包装出重组腺病毒;荧光显微镜下可观察到GFP的表达,收获病毒的滴度为3.2×10~(12)IU/L,获得了病毒滴度较高的重组腺病毒。结论本研究成功的构建了pAd-HSP70重组腺病毒表达载体,并在人胚肾293细胞中包装获得重组腺病毒vAd-HSP70,且证明了重组腺病毒vAd-HSP70可在人宫颈癌系Hela细胞内稳定表达目的基因并有效转录。
[Abstract]:Objective to construct the adenovirus expression vector of heat shock protein 70 (HSP70). The aim of this study was to infect the target cells with recombinant adenovirus carrying exogenous gene HSP70, thus laying an experimental foundation for the further study of the protective effect of HSP70 on the cells under stress. Methods Total RNAs were extracted from human cervical cancer cell line Hela after self-heat shock. After reverse transcription, PCR amplification was performed with synthetic primers. The cDNA of the target gene HSP70 was obtained. The exogenous cDNA was subcloned into the pAdTrack-CMV plasmid. Application of AdEasy system in E.coli of prokaryotic cells. The efficient homologous recombination of shuttle plasmid pAdTrack-CMV-HSP70 and skeleton plasmid pAdEasy-1 was completed in BJ5183. The recombinant adenovirus vector of HSP70 was constructed. After screening, the recombinant adenovirus vAd-HSP70was transfected into human embryonic kidney 293 cells by liposome method. Finally, the recombinant adenovirus vAd-HSP70was produced. The expression of target gene in human cervical cancer Hela cells was detected by RT-PCR assay. Results HSP70 gene was correctly inserted into adenovirus expression vector by PCR sequencing and restriction endonuclease digestion and the recombinant adenovirus was packaged in HEK293 cells. The expression of GFP was observed under fluorescence microscope. The titer of the virus was 3.2 脳 10 ~ (-1) ~ (12) IUU / L, and the recombinant adenovirus with high titer was obtained. Conclusion the recombinant adenovirus expression vector of pAd-HSP70 was successfully constructed and the recombinant adenovirus vAd-HSP70 was obtained by packaging the recombinant adenovirus in 293 cells of human embryonic kidney. It was proved that recombinant adenovirus vAd-HSP70 could stably express the target gene and transcribe effectively in human cervical cancer Hela cells.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R346

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