Construction And Identification Of Recombinant Adenoviral Vector Encoding Foxp3 Gene

The transcription factor forkhead box P3 (Foxp3) is a new P subfamily member of Forkhead transcription factor, which is specifically expressed in natural arising CD4+CD25+ regulatory T cells. More recent studies have shown that Foxp3 is not only a key intracellular marker but also a crucial developmental and functional factor for CD4+CD25+ regulatory T cells. The active suppression by regulatory T cells has been considered to be a fifth mechanism of peripheral tolerance, except for clonal anergy, split tolerance, clonal ignorance and AICD. Foxp3 contained several conservative domains, a Forkhead box domain in C terminal, a C2H2 zinc finger, and a Leucine zipper domain, which controlling some genes' expression by connecting with their promoters or some transcription factors.Increasing researches on the potential mechanism of Foxp3 gene and its therapeutic manipulation have been carried out by constructing recombinant retrovirus vector or transgenic models. Foxp3 may induce generation of regulatory T cells in vitro, which has a similar phenotype and action to natural arising CD4+CD25+ regulatory T cells. In vivo experiments, Foxp3 could induce relative immune tolerance in local environment by directly or indirectly suppressing theproliferation, differentiation and function of some immune cells, such as active T lymphocytes, B lymphocytes and some antigenic presenting cells. Therefore, novel therapeutic strategies in autoimmune disease and transplantation rejection based on Foxp3 gene and regulatory T cells have been arisen. Recent evidence has demonstrated the expression of Foxp3 in some tumor tissues or draining lymph nodes, which means that regulatory-T-cell-mediated immunosuppression is one of the crucial tumour immune-evasion mechanisms and the main obstacle of successful anti-tumour immunotherapy.In this experiment, we choose AdEasy? XL system to construct the replication-deficient recombinant adenovirus-Ad-Foxp3, by efficient homologous recombination in bacteria. Methods: The Foxp3 cDNA was obtained from mouse thymus by RT-PCR, and the recombinant shuttle plasmid was established by inserting Foxp3 gene into MCS of pShuttle-IRES-hrGFP-1. After that, the Pme I linearized shuttle plasmid was transformed into E.coli BJ5183-AD-1 to obtain the recombinant adenoviral plasmid-pAd-Foxp3 by efficient homologous recombination. This adenoviral plasmid carried both Foxp3 and a humanized recombinant green fluorescent protein (hrGFP) as reporter gene. Then the recombinant adenovirus-Ad-Foxp3 was obtained by packaging Pac I linearized pAd-Foxp3 in AD293 cells. The mRNA and protein expression of Foxp3 in Ad-Foxp3 infected AD293 cells were detected by RT-PCR and Western blot, respectively. The titer was determined by TCID50( tissue culture infectious dose 50)after three cycles' proliferation. At last, HeLa cells were infected by recombinant adenovirus to detect RCA (replication-competent adenovirus) for quality control. Results: The recombinant plasmid pAd-Foxp3 was successfully generated by homologous recombination, and the primary Ad-Foxp3 was obtained by packaging pAd-Foxp3 in AD293 cells . hrGFP expression could be detected 3 days after infection and CPE could be observed 6 to7 days after infection. Ad-Foxp3 was titered at 2 X108pfuAnl after three cycles'proliferation. Quality control shows no detection of RCA, which indicated no mutation of wild type adenovirus. Conclusions: Recombinant adenovirus Ad-Foxp3 can be constructed successfully by AdEasy? XL system. The recombinant Ad-Foxp3 may serve as a new strategy for Foxp3 signaling pathway, immunotolerance induction, and gene therapy as well.