Objective: To construct and express the eukaryotic expression vector of 14-3-3 protein of Toxoplasma gondii RH stain.
Methods: The structure and physicochemical property of 14-3-3 protein were predicted by bioinformatics analysis tools. The desired gene fragment was amplified from total RNA in T. gondii RH strain by RT-PCR, and sub-cloned into pcDNA3.0 to construct recombinant plasmid pcDNA3.0/14-3-3. After PCR confirming, double restriction enzyme digestion and DNA sequencing, the eukaryotic expression vector pcDNA3.0/14-3-3 was transfected into HeLa cells and the target protein was detected by Western blotting.
Results: The prediction of its gene sequence and amino acid sequence suggested that the 14-3-3 protein was acid soluble protein with five conserved regions, existing as homo- or hetero-dimers. The amplified gene fragment was about 800 bp, and the inserted fragment in pcDNA3.0/14-3-3 was 801 bp by sequencing, which had 99% homology to the 14-3-3 gene sequence of T. gondii in GenBank (Accession No. AB012775.1). Western blotting showed that there was more 14-3-3 protein expressed in the pcDNA3.0/14-3-3-transfected HeLa cells than not-transfected and mock transfected cells. Its relative molecular mass (Air) was about 30 000.
Conclusion: The eukaryotic expression vector pcDNA3.0/14-3-3 is constructed and expressed in eukaryotic cells.
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