Screening And Identification Of Proteins Interacting With Drosophila Jumeau

Drosophila melanogaster, as a typical model organism, plays an important role in innate immunity studies of insects and humans. Nowadays, many findings about the innate immunity are from the research of Drosophila. Drosophila Schneider2(S2) Cells was derived from a primary culture of late stage (20-24hours old) Drosophila melanogaster embryos, many features of the S2cell line suggest that it is derived from a macrophage-like lineage. S2cell line that culture is simple and efficient, while having excellent characteristics both of insect expression system and mammalian expression system, is a highly efficient expression system. Jumeau (Jumu) protein is an important transcription factor in Drosophila, containing719amino acids. Jumu protein to be found as the first FKH/WH family proteins with dual function, can cause large-scale modification of heterochromatin. But, now, the function of Jumu protein in innate immunity and mechanism of transcriptional regulation is not very clear in Drosophila.In this study, we based on the results that Drosophila Jumu protein has potential interactions with Cos, CG5375. and L(1)sc proteins which were found by L.Giot and KG. Guruharsha et al. We builded up the recombinant plasmids with GFP-tagged protein, prepared GFP polyclonal antibody with high efficiency, used Drosophila S2cells protein expression system and protein co-immunoprecipitation technique to verify the protein interactions between Jumu and Cos, CG5375, L(1)sc. The results show that Drosophila Jumu protein with Cos, CG5375and L(1)sc do not exist protein interactions. To this end, we transfected empty vector plasmid pMK33-Flag as negative control and recombinant plasmid pMK33-Flag-Jumu full length into Drosophila S2cells and screened stable cell lines. Using both these stable cell lines and protein co-immunoprecipitation and two-dimensional electrophoresis techniques, we screened the proteins interacting with Jumu protein. We used the corresponding Software to analysis the two-dimensional electrophoresis gel images. The results showed the different sample points total of seven which have3or more than3times difference and relative molecular weights are141KDa.82KDa.69KDa.44KDa,38KDa,27KDa and26KDa. Based on these findings, we deal with these different points through deduction, digestion and mass spectrometry analysis. In the end. we get the specific amino acid sequences which belong to such proteins as Act5C, Hsc70-3, Rpn6, betaTub56D and CG10306. And then again, we would use co-immunoprecipitation technique to verify these protein interactions.