Screening Of Tap Protein Interacting Factors In Drosophila Melanoganster

Objective:Screening of Tap interacting protein of Drosophila melanogaster by co-immunoprecipitation and mass spectrometry to explore the regulatory mechanism of tap Gene in drosophila neural developmentMethod:(1)HA tags expression in tap transgenic Drosophila drosophila strains were detected by immunofluorescence and Western blotThe expression pattern of HA tag in Drosophila melanogaster was observed by immunofluorescence staining.Western blot was used to detect the correct size of TapHA protein and determine whether the HA tag could function normally.(2)The expression of Tap-HA labeled target protein was detected by immunoprecipitation and Western blotThe protein interacting with Tap-HA in the whole embryo protein of Drosophila melanogaster was identified and pulled by using HA antibody of IP level,and then the tag target protein after Co-IP was detected by HA tag antibody again.(3)Identification and screening of interaction factors with Tap-HA target protein by mass spectrometryMass spectrometer LC-MS /MS was used to identify Tap-HA interacting protein liquid components obtained through Co-IP.Then,through database analysis,the proteins interacting with the labeled target protein tap-ha were retrieved,and the proteins were further screened according to cell localization,biological process,molecular function and other annotationsResult:(1)The green fluorescent signal of Tap-HA label was found in the abdominal nerve cord of Drosophila melanogaster embryo at stage 13 by immunofluorescence staining of HA label.Through Western blot detection,a clear target protein band was observed at approximately 45 KDa.(2)The results of electrophoretic silver staining after co-immunoprecipitation showed that the IP group had significant protein enrichment with multiple protein separation bands,and a clear band at 45 KDa was observed by naked eyes.The Ig G control group had several protein separation bands,with the largest Input histone band,and a clear band at 45 KDa was observed.The results showed that Co-IP successfully enriched the proteins interacting with the target proteins.(3)Identification results of mass spectrometer LC-MS/MS : 535 proteins were identified in the IP group and 458 in the Ig G group.Among them,255 identical proteins were identified in the two groups at the same time,and 203 and 280 differentially specific proteins were identified in the Ig G group and the IP group,respectively.Through bioinformatics software analysis,and screens out candidate factors which interact with the target protein :Nerfin-1,Da,Beag,Nab2,Pnuts,Taf6,If.Conclusion:Co-immunoprecipitation successfully enriched for proteins that interact with the target protein.co-immunoprecipitation combined with mass spectrometry identifies280 proteins that interact directly or indirectly with target proteins.Based on functional annotation of candidate proteins and analysis of bioinformatics software,preliminary screening of Nerfin-1,Da,Beag,Nab2,Pnuts,Taf6,and if may be involved in the transcription and regulation of tap.It lays a solid foundation for the next step to identify the interaction mechanism between the interaction protein and Tap.