Studies On Identification, Expression Of 14-3-3 Gene Family Of Taenia Solium And Tissue Location Of Ts14-3-3.3 Protein

Objective: To analyze and predict the sequence,structure and phylogenetic of Ts14-3-3 gene family and its encoded protein of Taenia solium by bioinformatics technology.To analyze the expression difference of Ts14-3-3 gene family at the stage of adult and cysticercus of Taenia solium.To express Ts14-3-3.3 gene in Escherichia coli(E.coli)and prepare Ts14-3-3.3 polyclonal antibody.To analyze the tissue location of Ts14-3-3.3 protein.These results would lay the foundation for the further study of Ts14-3-3.3 protein.Methods: 1.From the whole genome data of Taenia solium were searched in the Gene DB database.The gene structure,physical-chemical characteristics,secondary structure,subcellular localization,three-dimensional structure and phylogenetic tree of Ts14-3-3 gene family of Taenia solium were analyzed and predicted by bioinformatics analysis tools and various software packages.2.Taenia solium adult was collected from epidemic patients with taeniasis solium.A pig model with cysticercosis was established,cysticercus cellulosae was collected.The relative expression quantity of Ts14-3-3 gene family at the stage of adult and cysticercus of Taenia solium were detected by Real-time fluorescent quantitative PCR(RT-q PCR).3.Ts14-3-3.3 gene was cloned into the prokaryotic expression vector p CznⅠ and then expressed in Arctic Express with Isopropyl-1-thio-β-galactopyranoside(IPTG)induction.Ts14-3-3.3 recombinant protein was detected by SDS-PAGE and purified by His Link affinity chromatography.Ts14-3-3.3 recombinant protein was identified by Western blot.4.New Zealand rabbits were immunized with the purified expression products of Ts14-3-3.3 recombinant protein and Ts14-3-3.3 rabbit anti-serum was prepared.The tissue location of Ts14-3-3.3 nature protein at the stage of adult and cysticercus were analyzed by Western blot and immunohistochemistry.Results: 1.Six members of Ts14-3-3 gene family of Taenia solium were obtained and the structure of all genes contains exons and introns.The relative molecular weight of all coded proteins were ranged from 26.88 kilodalton to 29.33 kilodalton.All coded proteins presented in the form of dimers which were localized on four different clades of the phylogenetic tree.2.The result of RT-q PCR showed that six Ts14-3-3 genes at the stage of adult and cysticercus of Taenia solium were expressed.The expression quantity of Ts14-3-3.1,Ts14-3-3.2,Ts14-3-3.3 and Ts14-3-3.4 were higher at the stage of cysticercus,while the expression quantity of Ts14-3-3.5 and Ts14-3-3.6 were higher at the stage of adult.3.The recombinant expression plasmid p CznⅠ-Ts14-3-3.3 was successfully constructed.After inducing with IPTG,Ts14-3-3.3recombinant protein was obtained.The relative molecular weight was about 29.31 kilodalton and the protein was soluble.The purified Ts14-3-3.3 recombinant protein with His-tag was recognized by anti-serum.4.New Zealand rabbits were immunized with the purified Ts14-3-3.3 recombinant protein and a high-titer Ts14-3-3.3polyclonal antibody was obtained.The results of Western blot and immunohistochemical analysis showed that Ts14-3-3.3 nature protein was expressed at the stage of adult and cysticercus of Taenia solium.Conclusion:1.Six members of Ts14-3-3 gene family of Taenia solium were obtained.All Ts14-3-3 encoded proteins contained typical 14-3-3 protein domain and had a high conservation.2.Six members of Ts14-3-3 gene family of Taenia solium were expressed at the stage of both adult and cysticercus.3.The prokaryotic expression plasmid p CznⅠ-Ts14-3-3.3 was successfully constructed.Ts14-3-3.3 recombinant protein could be highly expressed in soluble form in E.coli.A high-titer polyclonal antibody of Ts14-3-3.3 was successfully prepared.4.Ts14-3-3.3 nature protein was expressed at the stage of both adult and cysticercus of Taenia solium.