| Summary: | 碩士 === 國立陽明大學 === 熱帶醫學研究所 === 94 === In mammal immunity, T cell plays a critical role to defend pathogens. ZAP-70 was verified as a ζ chain binding protein of the T cell receptor (TCR). It plays an important role in mediating T cell activation, which triggers complicate signaling cascades leading to transcriptional enhancement, cell proliferation and differentiation. In T cells, it has been demonstrated that Grb2 is a ZAP-70 associated protein. At present, T cells have not been identified in insects. However, a mosquito ZAP-like cDNA was first isolated in our laboratory. Therefore, investigation of AaZAP involved in immune signaling is an important issue to look insight the immune regulation in mosquitoes. In this study, we have demonstrated that AaZAP is inducible after bacterial challenge. An Aedes aegypti Grb2 (AaGrb2) cDNA was also isolated and sequenced. It has 2,036 bp endoding an open reading frame of 211 amino acids containing one SH2 and two SH3 domains. In addition, a partial sequence of AaGrb2 was over-expressed for polyclone antibody production. A nuclear localization signal (NLS) was found in AaZAP sequence. The function of the NLS was verified by transfecting a construct composing the NLS and a red fluorescent protein into culture cells. A western blot of nuclear protein extracts from Aag2 cells revealed a strong signal of AaZAP. Our results indicate that AaZAP protein contains an active nuclear localization signal. AaZAP were detected in both cytoplasm and nucleus before bacterial stimulation, indicating that AaZAP will translocate to nucleus. However, AaZAP could not be detected in the nucleus after bacteria challenge. These resuls coincide with findings in mammals. The cellular localization of AaZAP was analyzed by immunofluorescent assay in a mosquito cell line Ap61. After LPS (lipopolysaccharide) challenge, induction and membrane localization of AaZAP were observed by confocal microscopy. The expressions of Aedes aegypti antibacterial peptides, defensin and cecropin, were verified to be regulated by AaZAP and AaGrb2 using double strands RNA interference (dsRNAi). The interaction of AaZAP and AaGrb2 was also demonstrated by pull-down assay in a mosquito system for the first time.
|
|---|
