| 其他摘要 | Two distinct projects involving HIV-1/AIDS were discussed in this thesis: (1) molecular evolutionary of TRIM5α, a HIV-1 infection restrictive factor in Old World monkey; (2) the construction of rAdenovirus experssing HSP 70，HIV-1 gp120, gp41, the aim is by infecting dendritic cell (DC) to stimulate DC with HIV-1 antigen and increase the processing capacity of DC, as the base of AIDS immunotherapy with DC. Most of the Old World monkeys are restrictive to HIV-1 infection, while pig-tailed monkey is permissive to both of HIV-1 and SIV infection, what is more this kind of monkey takes on more serious symptom when infected by SIV than other kinds of Old world monkey do. The cellular TRIM5α protein, a potential innate immunity factor in many kinds primate which can restrict retrovirus was recently identified as underlying HIV-1 restriction in Old World monkey cells. TRIM5α belongs to a large family of proteins containing a tripartite motif (TRIM) comprising a RING domain, one or two B-box domains, and a coiled-coil domain, TRIM5α also contains a characteristic SPRY domain at its carboxy terminus critical for determining the species-specific restriction of HIV-1 by Old World monkeys. Here by analyzing its evolutionary history, we find strong evidence for ancient positive selection in the primate TRIM5α gene, especially in the SPRY domain which could recognize the capsid of the invading retroviruses. This history suggests that TRIM5α evolution has been driven by antagonistic interactions with a variety of viruses and endogenous retroviruses that predate the origin of primate lentiviruses. Our results provide more evidence to identify TRIM5α as a widespread innate immunity factor. TRIM5α gene encoding sequences from different primate vary greatly which interpret primate represent diverse symptom after infected by HIV-1. Neutral test based on the single codon reveals amino acids in the position of 347/354/552 show strong positive selection, which could prove those amino acid positions had played an important role in the restriction of retroviruses. In the genome of owl monkey, TRIM5α gene fuse with CypA gene. In the genome of pig-tailed monkey, we found both of them had fused together in a new way, and the amino acids related close with its function had changed, which could be the main reason that pig-tailed monkey can be infected by HIV-1, and show the most serious symptom when infected by SIV. The second project focused on the construction of rAdenovirus expressing HSP 70, HIV-1 gp120, gp41, with GFP gene as indicator. After transported back to the body, DC pulsed with HIV-1 antigen can induce strong immunity against HIV-1, which can cure AIDS theoretically and cooperate with the HARRT. HSP70 protein can boost the ability of DC to process and present antigen. To verify the therapy effect of the immunity induced by DC pulsed with HIV-1 antigen to AIDS, test the ability of HSP70 protein and find out the problems in this way to treat AIDS. We initially cloned gp120 and gp41 from the genome of HIV-1IIIB, and cloned the HSP70 gene from a plasmid. The resorts of sequencing and sequence analysis prove no mutation has occurred which can affect the genes to express. then we inserted the genes to the expressing vectors, and constructed the rAdenoviruses. Expression of the report gene of GFP testified rAdenoviruses had been constructed successfully, and the resorts of PCR amplification also proved rAdenoviruses took the genes separately. At the same time we have constructed a rAdenovirus expressing no inserted gene, which will be used as a control.The further works will include the test of protein expression, rAdenoviruses infecting DC and analyzing function of DC infected by rAdenovirus etc. |
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