Abstract:

[Abstract] Objective To construct eukaryotic Flag (DYKDDDDK) expressing recombinant plasmids, pCMV- N Flag-SND1-No1/2, which contain the coding sequence of human SND1-No1（from 1 st AUG）orSND1-No2(from 2 nd AUG), and perform the cellular localization analysis of Flag-tagged SND1-No1/2under stress condition to study the function of the two AUG in the SND1containing stress granules formation.Methods The gene fragments ofSND1-No1/2were amplified by PCR from the wholeSND1transcript and inserted into pCMV-N-Flag expressing vector through BamHI/EcoRI double en? zyme digestion and T4DNA Ligase connection. The recombinant pCMV-N-Flag-SND1-No1/2plasmids were transfected in? to HeLa cells and the expression of Flag-SND1-No1/2fusion proteins was examined by Western blotting assay. Immunofluo? rescence assay was performed to detect the co-localization of Flag-SND1-No1/2with endogenous SND1granule.Results The pCMV-N-Flag-SND1-No1/2were sequenced and digested correctly by restriction single/double enzyme. The Flag tagged SND1- No1/2fusion proteins were also detected in transfected HeLa cell by Western blotting assay. Both of them showed the co- localization with endogenous SND1 granule. Conclusion The recombinant eukaryotic plasmids of pCMV-N-Flag-SND1-No1/2were constructed successfully and expressed effectively. The depletion of1 st AUG failed to af? fect the formation of SND1containing stress granules.

Key words: SND1, AUG, fusion protein, stress granules