EXPRESSION AND IDENTIFICATION OF 14-3-3 SIGNAL TRANSDUCTION PROTEIN EPSILON ISOFORMS GENE FROM SCHISTOSOMA JAPONICUM(CHINESE STRAIN)

Objective To construct an eukaryotic expression plasmid pBK-Sj14-3-3 of 14-3-3 signal transduction protein(epsilon isoforms) gene from Schistosoma japonicum (Chinese strain) and to express and identify the fusion protein, so as to provide conditions for further studies on protective immunity. Methods A pair of primers were designed and synthesized according to the known nucleotide sequence of Sj14-3-3 protein. Using adult worms total RNA of S. japonicum as template, a cDNA first strand synthesis was driven by RT-PCR technique. The product was cloned into pGEM-T vector and identified by double endonuclease digestion and PCR. The fragment was subcloned into an eukaryotic expression vector pBK-CMV again, a recombinant pBK-Sj14-3-3 was constructed and transferred into E. coli BL21, after identification expressed in E. coli BL21. Results The same specific gene fragment was obtained by RT-PCR, endonuclease digestion of pGEM-T-Sj14-3-3 and pBK-Sj14-3-3, SDS-PAGE revealed that the molecular weight of this expressed product was around 32 ku. Western-blot showed that the recombinant protein was identified by 14-3-3 polyclonal antibody. Conclusion Using an eukaryotic expression plasmid pBK-Sj14-3-3, a 32 ku fusion protein was expressed in E. coli BL21 and identified by 14-3-3e polyclonal antibody.