Cloning, Expression And Identification Of The 14-3-3 Gene Of Toxoplasma Gondii

Toxoplasma gondii(T. gondii) is a harmful opportunistic protozoa. However. So far no practical antigens has been obtained. Currently the two issues of T. gondii are still in the search of the antigens of diagnosis with high sensitivity and specificity and of candidated molecular vaccine. The recombinant DNA technology is an available approach to screen the recombinant proteins. As known, an challenged field is the cell signal transduction between host immune system and parasites. So we manage to develop the signal transduction 14-3-3 protein which control some basic biochemical metabolism of T. gondii. 14-3-3 is an evolutionarily conserved protein expressed in all eukaryotic cells including plants, yeasts, protozoa, helminth, insects and mammalian animals. 14-3-3 proteins exhibit a remarkable degree of sequence conservation among species and share some essential biochemical properties. 14-3-3 proteins bind multitude of functionally diverse signaling protein, including kinases. phosphatases. transrnembrane receptors and become signal transduction and cell cycle regulation f mediator. Therefore, a pair of primers were designed and synthesized based on the . mRNA sequence cloned from the enteroepithelial stage of T. gondii (Beverley strain). Toxo 14-3-3 gene(RH strain) was amplified by RT-PCR. the PCR products were cloned into pGEM-T vector and the inserted DNA fragements w-ere confirmed by restriction -endonuciease digestion and PCR followed by DNA sequencing. A complete openreading frame(ORF) of 798bp was verified, which encodes 265 amino acid with theorial molecular weights of 31KDa and is identical to Toxol4-3-3 gene submitted to GenBank(Accession No ABO 12775). Then, the Toxo14-3-3 DNA fragments were subcloned into pBK-CMV expressing vector and transformed the recombinant plasmid into E.coli XL-1 blue. The positive recombinant pBK-CMV/Toxo 14-3-3 was induced by IPTG and the cell h sates were analysed with SDS-PAGE. which showed an obvious band of fusion protein between 31KDa?3KDa. The migration of the expression, however, is 5KDa slower than expected. The fusion protein can be recognized by anti-14-3-3 ?immunogen.