Contraction And Identification Of A Recombinant Adenovirus Carrying HSP70

Objective To construct recombinant adenovirus expression vector containning human HSP70 and as the base of investigating the protective role of HSP70 on the cell which are exposed to various kinds of stress.Methods The total RNA was extracted from Hela cells which were treated by heat shock,and was used for reverse transcription into complementary DNA.To get sufficient cDNA of aim gene,the PCR amplification was performed under certain conditions.The recombinant adenovirus vector pAd-HSP70,carrying HSP70 was constructed with AdEasy system and then transfected 293 cells to produce recombinant adenovirus vAd-HSP70.The viral titer was tested.The expression of target gene in Hela were tested by RT-PCR.Results By sequencing,it was confirmed that the PCR product was a full—length cDNA of HSP70,By the restriction endonuclease analysis of pAd—HSP70,a long fragment of about 30kb and a marked strap of 4.5kb could be got and by PCR,the aim gene was found.Above these would indicate that the target gene was cloned correctly to the shuttle plasmid.The expression of GFP could be observed by fluorescence microscope.The viral titer was 3.2×10¹²IU/ml,and RT-PCR indicated that HSP70 could effectively express in Hela.Conclusion Recombinant adenovirus vector was constructed successfully and was packed in 293 cells.Recombinant adenovirus vAd-HSP70 can be transcribed efficiently and expressed continuely.