Expression And Functional Analysis Of DjTRAF3from Planarian Dugesia Japonica

Platyhelminthes is the first one to be bilateral symmetry and the threelayer groups in the evolution process, and Dujesia japonica planaria is therepresentative animals of platyhelminthes turbellaria, also Dujesia japonicaplanaria have strong regeneration ability, thus they have become model animalsfor researches of evolution, embryonic development and regeneration mechanism.At present,people have discovered the existence of TRAF3genes among avariety of organisms, such as Caenorhabditis elegans, Drosophila, Barchydaniorerio var, rat and huaman, in mammals TRAF3are widely distributed in variousorganizations in the body, for example heart, spleen, lung, liver and intestinal,combined with TNFR and TLRs in organisms immune, affecting cell survival,proliferation, differentiation, and death.As known, TRAF3can combine withTBK-1inducing the produced of interferons, participating in TLRs signalingpathways, they can also suppress the NF-κB signaling pathways through thenegative regulation of NIK, moreover they also play an important role in theprocess antiviral which independent of the TLRs signaling pathways.We got a genes whose code is12118-283from our laboratory cDNA library,its sequence is about746bp, the biggest ORF included for564bp, coding aprotein containing187amino acids for molecular weight about21.3KDa.Wefound that the protein contains a MATH (meprin homology and TRAF-C)superfamily domain also as TRAF domain, predict its substrates combinationregion is between98-159amino acids region by Protein sequence comparisonthroug NCBI BLAST.Compared with other animals TRAF3amino acidsequence, found that the protein estimated at23%similarity with the Drosophila,estimated at36%similarity with the Zebrafish, similarity with mammals as high as35-39%.For each species TRAF domains alignment, the results showed that TRAFdomain is highly conservative.Evolutionary analysis results also suggested thatDjTRAF3and Drosophila TRAF3clusted, located at the base of the evolutionarytree, belong to the more primitive types.Through the Northern blotting, determine whether DjTRAF3genes exist inDujesia japonica planaria, and detect its size.Northern blotting results showed thatit is about750bp whic in accordance with the existing cDNA.Results by using themethod of in situ hybridization in planaria showed that DjTRAF3genes disttibuted in gut of planaria.At the same time, in situ biopsy results showed that theDjTRAF3genes are on the whole bowel, presumably the genes involved inplanaria immune process. Throug Real-time PCR detection by using differentperiods planaria induced with LPS, we found that DjTRAF3gene expressionappared ups and downs after induced by LPS, suggested that planaria DjTRAF3genes involved in planaria immune process.Building PET28a-DjTRAF3and pGEX-DjTRAF3prokaryotic expressionvector, using IPTG and HB-PET auto-induction medium induced fusion proteinand western blotting identification, SDS-page electrophoresis showed that theyboth expressed a protein molecular weight about21kda, Western blotting resultsconfirmed this is DjTRAF3protein, but PET28a-DjTRAF3recombinant vectorhad a higher expression also more simple and convenient operation, thus furtherexpressed using PET28a-DjTRAF3.With protein obtained we did a bacteriostaticexperiment, results showed that DjTRAF3protein can inhibite bacteria at a certaindegree.