Preparation and Preliminary Application of Recombinant Adenoviruses Based on Accessory Genes Orfa and Orfc of Walleye Dermal Sarcoma Virus(wdsv)

【Objective】 This study aimed to amplify the accessory genes orfA and orfC of Walleye Dermal Sarcoma Virus(WDSV),and prepare the recombinant adenovirus based on amplified genes to detect their effects on Hela229 and SPC-A-1 cells.【Method】 WDSV accessory genes orfA and orfC from pWDSV were amplified by PCR technology before being inserted into adenovirus vector(pAdTrack) to generate pAdTrack-orfA and pAdTrack-orfC.Then they were recombined with pAdEasy in BJ5183 to obtain the pAdEasy-orfA and pAdEasy-orfC,and the control pAdEasy-CK was also generated.Then pAdEasy-orfA,pAdEasy-orfC,and the control pAdEasy-CK were transfected into HEK293 with Lipofectamine2000 to get the recombinant adenovirus particles Ad-orfA,Ad-orfC and Ad-CK.The titers of the recombinant adenovirus were validated through the green fluorescent protein methods.Ad-orfA,Ad-orfC and Ad-CK were transfected to human tumor cells Hela229 and SPC-A-1,respectively.The expressions of the orfA and orfC genes were detected through western blot and effects of Ad-orfA and Ad-orfC on Hela229 and SPC-A-1 were measured by MTT assay.【Result】 We amplified the 891 bp orfA and 360 bp orfC by PCR and got the shuttle vectors pAdtrack-orfA and pAdtrack-orfC.Recombination of these shuttle vectors in BJ5183 with pAd-easy produced pAdEasy-orfA and pAdEasy-orfC.pAdEasy-orfA,pAdEasy-orfC and control vector were packaged in HEK293 to get Ad-orfA,Ad-orfC and Ad-CK.Titers of adenovirus measured in Hela229 cells were 5.67×109,2.00×109 and 7.33×108 GFU/mL,respectively.Ad-orfA,Ad-orfC and Ad-CK infected tumor cells Hela229 and SPC-A-1.The expression of orfA and orfC showed inhibiting effects on tumor cell growth.【Conclusion】 The results showed that orfA and orfC genes inhibited the growth of human tumor cells Hela229 and SPC-A-1.This work is a good start for further studies on the WDSV auxiliary genes in human tumors and the molecular mechanism of tumor shrinkage.