A Preliminary Study Of 14-3-3 Proteins In Entamoeba Histolytica

Objective:To predict the structure and function of Eh14-3-3proteins,clone and characterize the Eh14-3-3 gene,purify the recombinant Eh14-3-3 proteins,analyse the immunologic properties of purified products,prepare the anti-Eh14-3-3 sera,compare the Eh14-3-3 sequences from different strains of Entamoeba,and to see whether 14-3-3 proteins can be used as a phylogenetic marker.Methods:The bioinformatics prediction of the Eh14-3-3 gene was performed by employing tools at online bioinformatics websites and software packages such as Vector NTI suite.Specific primers were designed,and the Eh14-3-3 sequences were amplified based on both the cDNA cloned from the total RNA of Entamoeba histolytica at the stage of trophozoite and the genomic DNA sequence,and then cloned into the pET19b vector.The inserted fragments were confirmed by both restriction endonuclease digestion and sequencing.The Eh14-3-3 gene was then introduced into E.coli BL21(DE3) pLysS bacteria and induced to expression.Each Eh14-3-3 protein was identified by SDS-PAGE.The recombinant Eh14-3-3 proteins were purified by resin chromatography.And their immunogenic properties were identified by ELISA assay to verify its reactivity to the serum of healthy people and patients.Then,a mouse-anti-recombinant Eh14-3-3 protein serum was obtained.Finally,the 14-3-3 sequences of different Entamoeba strains were compared.Results:There are three 14-3-3 protein isoforms in Entamoeba histolytica,which were verified and identical to the sequences submitted to Genebank(Accesion No.EHU13418,EHU13419 and EHU13420).The Eh 14-3-3 protein isoforms were induced to express in E.coli.After resin chromatography,we obtained purified products with a 30kDa molecular mass.The result of ELISA assay indicated the purified protein had good reactivity to the serum of chronic patients and AC patients, but not to the serum of acute ALA patients,cyst carriers and healthy individuals. The anti-recombinant Eh14-3-3 protein serum reacts with crude antigen at a titer of 1:200.However,result of the following subcellular localization experiment turned out to be nagetive.The sequence comparison between different Entamoeba strains indicated that the 14-3-3 gene is highly conserved in Entamoeba.The bioinformatics analysis also indicated that the 14-3-3 protein can be as a phylogenetic marker.Conelusions:The 14-3-3 genes are highly conserved in Entamoeba.The 14-3-3 proteins may play an important role in the encystations of Entamoeba.The 14-3-3 protein can be used as a phygenetic marker.