Construction and identification of a recombinant adenovirus expressing HPV16 E7 gene
Objective To construct a recombinant adenovirus expressing the HPV16 E7 gene.Methods Two primer pairs,with and without respectively the insertion restriction site,were designed to amplify the target fragment of ccdBKan gene.Each of the ccdBKan products were transferred with the plasmid pBR322-Ad4-eGFP into electrocompetent strain of GB08red-gyr462 to construct two recombinant Ad4 plasmids expressing ccdBKan gene.The ccdB positive/negative selection method and ExoCET/Red recombination technology were used to insert the HPV16E7 gene into the E1 A region of Ad4 and constructed a recombinant adenovirus plasmid pBR322-Ad4-E1 Amut-C16E7P that can express HPV16 E7 gene.The recombinant adenovirus plasmid was confirmed by enzyme digestion and sequencing.The plasmid pBR322-Ad4-EIAmut-C16E7P was isolated to release the genome.The genome was used in the transfection of 293T cells for virus package and amplification.The viruses were used to infect 293T cells for the transcription level and protein expression of HPV16 E7 gene in the cells.The recombinant virus was inoculated into mice by intramuscular injection.The mouse serum was collected for virus neutralization assays.Results The recombinant adenovirus plasmid expressing the HPV16 E7 gene was constructed successfully and the recombinant virus Ad4-HPV16E7 was packaged in 293T cells.The qRT-PCR and western blotting results confirmed the successful expression of the HPV16 E7 gene of the recombinant adenovirus Ad4-HPV16E7 in 293T cells.Virus neutralization assay indicated that the serum from mice immunized with Ad4-HPV16E7 can neutralize the activity of Ad4-HPV16E7.Conclusions A recombinant adenovirus strain expressing the HPV16 E7 gene was successfully generated that provided new perspectives and a foundation for further investigating the role of HPV16 E7 in cancer pathogenesis and potential therapeutic strategies for HPV-induced cancers.Additionally,this work may also provide a potential strategy for exploring therapeutic HPV vaccines.
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