The Preliminary Study On The Interaction Of Human EBI3 Protein And Its Mutants With Sedlin Protein

Objective Using bioinformatics methods analysed the protein structure of EBI3(Epstein-Barr virus-induced gene 3),and using several experiment techniques such as plasmid construct, GST pulldown, confocal immunofluorescence and immuneprecipitation to study different expressions and location of the human EBI3 protein and its mutants in mammalian cells,study the interaction between them. Understand the impact of the signal peptide to cellular localization of EBI3 protein.And explore the specific domain that EBI3 interacts with Sedlin occurred, and explore the impact on the interaction between EBI3 Asp210 and Sedlin protein.Methods We analyzed domain and signal peptides of EBI3 based on the amino acid sequence in NCBI database. Adopt the method of PCR, useing the plasmid p CDGFP-EBI3 containing full-length c DNA sequence of EBI3 as template,amplified sequence of wild EBI3, EBI3(1-135) and EBI3(125-230),respectively.After enzyme digestion gel electrophoresis recycling purpose fragment, inserted into the yeast expression vector p GADT7, p GBKT7 EBI3 to construct human EBI3 and its mutants yeast expression vector plasmid;Using the same method to construct eukaryotic expression vector plasmid pc DNA3.1 EBI3- FLAG, pc DNA3.1 EBI3(1-135)-FLAG,pc DNA3.1-EBI3(125-230)-FLAG and p CDGFP-EBI3(1-135) 、 p CDGFP-EBI3(125-230).Designed primer containing specific locus mutation,using the method of PCR, p CDNA3.1- EBI3- FLAG as the template, construct EBI3 Asp210 mutation eukaryotic expression plasmid p CDNA3-EBI3-D210A-FLAG. Respectively taked plasmids p CDNA3.1- EBI3 FLAG, p CDNA3.1 EBI3(1-135)- FLAG, p CDNA3.1- EBI3(125-230)- FLAG and p CDNA3- EBI3-D210A- FLAG with downstream FLAG label single or double with the p CDGFP-Sedlin transient transfection into COS7 cells, detected cellular localizations of EBI3 protein and its mutant, using fluorescent confocal microscopy, as well as with Sedlin proteins within the cells of positioning, compare their expression of positioning differences. Using of GST-Sedlin fusion protein and GST pulldown technology testing EBI3 protein technology and its lack of interaction between mutant and Sedlin protein in vitro. With FLAG label EBI3 and mutants of eukaryotic expression plasmid and p CDGFP- Sedlin common transient transfection into HEK 293 t cells, the detection of immune down experiment technology in mammalian cells wife EBI3 Sedlin and its mutant protein and protein interactions. The endogenous immune coprecipitation technology, detect Sedlin EBI3 and protein in human placenta tissue under the condition of natural interaction happens.Results To predict EBI3 FN3 domain structure and location base on NCBI database; EBI3 protein was predicted using Signal P 4.1 Server signal peptide and signal peptide cutting locus; Enzyme digestion and sequencing results certified plasmids were constructed correctly;Western blotting showed recombinant proteins with FLAG label can stably express in HEK293 T cells; GST Pulldown experimental proof of wild type EBI3 protein and mutants interacted with Sedlin protein,except mutant EBI3(125-230) with carboxyl end.Immunofluorescence showed wild type EBI3 protein and its mutants in COS7 cells expressed in the cytoplasm, cell nucleus and nuclear membrane, but there are differences, revealed wild type EBI3 and mutants positioning in COS7 cells has obvious changed, and the EBI3 Asp210 mutant cellular localization was similar to that of wild type.Immuno-colocaliza tion experiments showed EBI3 and mutants were co-exist with Sedlin; Co-Immunoprecipitation(Co- IP) experiments showed wild type EBI3 and its mutants interacted with Sedlin protein; Endogenous Co- IP also proved wild type EBI3 interacted with Sedlin protein.Conclusion In COS7 cells,wild type EBI3 and its mutants could co-location with Sedlin; there is of little effect on subcellular localization of EBI3 Asp210 mutant; GST pulldown experiments showed that the wild-type human EBI3 protein and its mutants could interact with Sedlin except EBI3(125-230).The result of Co-immunoprecipitation experiments are not exactly according to GST pulldown experiment results, which revealed that intracellular protein modification could influence the interaction of proteins.Endogenous Co-IP experiments further confirmed the presence of interaction between wild EBI3 and Sedlin.