Human immunodeficiency virus (HIV) continues to be a major public health problem throughout the world, with high levels of mortality and morbidity associated with AIDS. The urgency of designing an effective, safe, inexpensive and easily administrable vaccine to protect people from HIV and/or AIDS is absolute priority and it has turned out to be enormous challenge for the scientific community. Considerable efforts to develop an effective vaccine for HIV have been directed towards the generation of cellular, humoral, and mucosal immune responses that will result in enduring, broadly protective immunity, yet an efficacious vaccine remains elusive. DCs pulsed with antigen as a therapeutic DC vaccine for AIDS and delivered into body can induce strong cell-mediated immune response against HIV-1, which can control virus replication and delay disease progress to AIDS. We presumed DCs infected with recombinant Adenovirus expressing HIV-1 gene and delivered into body would control viral replication and prevent infection. In this thesis, we focused on the construction of recombinant Adenovirus expressing HIV-1 gp140, tat and gp140-tat, with GFP gene as a indicator which was included in the shuttle vector pTrack-CMV and transcriped under the control of the CMV promotor. Structural protein Env could induce neutralizing antibodies to prevent infection. Truncated gp140 was selected in this study, which have a deletion in COOH-terminal cytoplasmic for the toxicity of full Env (gp160) and transmembrane domain was retained to mimic native surface glycoprotein to elicite significantly broader immunity than soluble forms of envelope. Meanwhile, Tat-specific immune responses elicited by prophylactic vaccine can potentially have a critical impact on HIV transmission and replication. Humoral and cellular responses directed against Tat correlate with the non-progression to AIDS. We cloned gp140 and tat from plasmid pMET-HXB2-env and the genome of HIV-1IIIB respectively, and then recombine them into a DNA fragment by overlap PCR, which were inserted into the expressing vectors. The resulting recombinant plasmids, designated prAd-tat, prAd-gp140, prAd-gp140-tat, were digested by Pac I and then transfected into packaging cell lines 293 cells. The infectivity of recombinant Adenovirus was dictated by expression of the report gene of GFP. HIV-1 gp140, tat and gp140-tat gene could be detected in the supernatants of infected 293 cells by PCR. Their expression were detected in 293 cells by Western Blotting. Finally, HIV-1 genes expression was identified in Western Blooting except for recombinant gene gp140-tat. At the same time, we constructed an Adenovirus without inserted gene, which could be used as a control. The further works will include phenotype changes on the DC and analyzing function of DC infected with various recombinant Adenovirus, in addtion to their expression in the DC.
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