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Contraction and Identification of a Recombinant Adenovirus Carrying HSP70

Author: XuXiaoNa
Tutor: QuYan
School: Qingdao University
Course: Nursing
Keywords: HSP70 Adenovirus Expression vector People
CLC: R346
Type: Master's thesis
Year: 2008
Downloads: 43
Quote: 0
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Abstract


Objective To construct the human heat shock protein 70 ( heat shock protein70 HSP70) adenovirus expression vector , aimed at carrying exogenous gene HSP70 successfully constructed recombinant adenovirus infection of target cells , thereby further investigate the stress state of HSP70 on cell the protective effect of an experimental basis . Since after the heat shock treatment of human cervical carcinoma cell line Hela extracting total cellular RNA, after reverse transcription reaction , using synthetic primers for PCR amplification, to obtain the cDNA of the target gene of HSP70 . Exogenous cDNA subcloned into pAdTrack-CMV plasmid using the AdEasy system to complete the shuttle plasmid pAdTrack-CMV-HSP70 and the backbone plasmid pAdEasy-1 between the highly efficient homologous recombination in procaryotic cells E.coli BJ5183 constructed HSP70 recombinant adenovirus vector. Recombinant After screening , liposome method transfected human embryonic kidney 293 cells , packaging produce the recombinant adenovirus vAd - of HSP70 . Finally , the harvest the virus titer identification , transcriptional expression of the target gene in Hela cells was detected by RT-PCR method . Results are confirmed by PCR, sequencing , and restriction HSP70 gene inserted correctly adenovirus expression vector , and recombinant adenovirus was packaged in HEK293 cells ; observed GFP expression under a fluorescence microscope , harvest the virus titer of 3.2 × 10 12 IU / L, to obtain a higher viral titer recombinant adenovirus . The conclusions of this study to build pAd-HSP70 recombinant adenovirus packaging obtained the recombinant adenovirus vAd - of HSP70 in human embryonic kidney 293 cells proved recombinant adenovirus vAd-HSP70 in human cervical carcinoma line Hela cells The stable expression within a target gene and efficient transcription .

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CLC: > Medicine, health > Basic Medical > Human biochemistry, molecular biology
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