WANG Xuelong, SHEN Jilong and
JIANG Zuojun
Department of Microbiology
and Parasitology, Anhui Medical University, Hefei, 230032 P.R.
China
14-3-3 protein is a family of conserved regulatory molecules expressed in all eukaryotic cells including plants, protozoa, and mammalian animals. A striking feature of the 14-3-3 proteins is their ability to bind a multitude of functionally diverse signaling proteins, regulate the activities of enzymes including protein kinase C, activate tyrosine and tryptophan hydroxylase, and stimulate Ca2+-dependent exocytosis. This plethora of interacting proteins allows 14-3-3 to play important roles in a wide range of vital regulatory processes, such as mitogenic signal transduction, apoptotic cell death, and cell cycle control. The present work is a novel approach to study of the potential role of Sj14-3-3 in immuno-prevention and immunodiagnosis of schistosomiasis by its double blockage of signal transduction and immunoreaction. S.japonicum (Chinese strain) adult worms were harvested from infected rabbits by hepatoportal perfusion. Schistosome total RNA was isolated and the first strand of cDNA was synthesized. A pair of primers were generated for amplification of Sj14-3-3 gene based on the open reading frame of GenBank database: P1: EcoR I forward: 5'-TAGGAATTCCATGAGGGATTCGTTC-3' and P2: Xho I reverse:5'-TAGCTCGAGTCAGCCATCATTTCCG-3'. PCR products were purified and ligated to the TA cloning vector pGEM-T (Promega) with T4 DNA ligase. Transformation was performed with E.coli XL-1blue strain by DMSO. The recombinant plasmids were verified by restriction digestion followed by agarose electrophoresis. Sj14-3-3 gene was sequenced by automated DNA sequencer(ABI 377). pGEM-T/Sj14-3-3 was digested with EcoR I and Xho I and the target fragments ,with an open reading frame of 765 bp were inserted to plasmid pBK-CMV for prokaryotic expression. The ß-galactosidase Sj14-3-3 fusion protein was induced by IPTG and confirmed by SDS-PAGE followed by Western blot. The recombinant fusion was identified as a predominant band corresponding to a mw. of 30 kDa. The effect of pBK-CMV/Sj14-3-3 DNA vaccine on the challenge of S. japonicum cercaria is under investigation. Monoclonal antibody to recombinant Sj14-3-3 is in preparation.
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created 21/12/00