Construction And Characterization Of A Codon Optimized HIV-Ⅰ Gp120 Gene Recombinant Adenovirus Vector

Objective:To generate recombinant adenoviral vector containing mgp120 gene,and through DNA and protein levels of testing,confirmed mgp120 protein highly expressed in vitro,to provide the foundation for the development of new preventive HIV-I vaccine.Methods:The mgp120 gene was amplified by PCR and cloned into the pENTR/D-TOPO transfer vector to get recombinant shuttle vector,Adenoviral expression vector containing mgp120 gene was constructed by homologous recombinantion.The linearized DNA plasmid of the recombinant adenoviral vector was transfected human embryo kidney(HEK)293A cells to package and amplify recombinant adenovirus,to get the replication defective recombinant adenovirus rAden-mgp120.The recombinant adenovirus titer was characterized by using the End-dilution assay.The Expression of the mgp120 protein in 293A cells was detected by Western blot.Results:The recombinant shuttle vector plasmid was identified by PCR indicated that the target gene was inserted correctly;,the sequencing showed that it was consistent between gp120 gene and Genbank gene sequence.The recombinant adenovirus expression vector plasmid was identified by PCR showed that mgp120 was inserted correctly too.The adenovirus vector transfected into HEK293A cells,the cytopathic effect(CPE)appeared after 10 days,the typical CPE:cell swelling,turned round,shedding,a grape cluster deposition.The titer of Aden-mgp120 was characterized as 6.8×1010pfu/mL.The Western blot showed that 52.8KD specific band corresponding to the position but control cells were not after infecting HEK293A cells,thus it proved that rAd5-mgp120 genes can be effectively expressed in vitro.Conclusions:The mgp120 gene recombinant replication-defective adenovirus expression vector was constructed successfully and detected the expression of mgp120 protein in HEK293A cells,and this study provided a foundation for developement of HIV-Ⅰ vaccine.