Abstract:

【Abstract】 Objective  To construct a recombinant eukaryotic expression plasmid containing ROP18-ROP12  (encoding rhoptry protein 18 and 12) complex gene of Toxoplasma gondii, and examine its expression in eukaryotic cells. Methods Recombinant plasmids pVAX1-ROP18 and pVAX1-ROP12 were digested by restriction enzymes BamHⅠ and XbaⅠ. ROP12 gene was cloned into pVAX1-ROP18 to construct the eukaryotic expression plasmid pVAX1-ROP18-ROP12. After colony PCR,  enzyme digestion and sequencing, the correct recombinant plasmid pVAX1-ROP18-ROP12 was transfected into HeLa cells. Along with it were groups of empty plasmid, pVAX1-ROP18 and pVAX1-ROP12. Total RNA was extracted from HeLa cells and reverse-transcribed to cDNA. RT-PCR was performed to evaluate mRNA expression of the housekeeping gene β-actin and ROP18-ROP12 complex gene. Immunofluorescence assay and Western blotting were performed to determine the protein levels of ROP18-ROP12 fusion protein. Results Colony PCR in recombinant plasmid pVAX1-ROP18-ROP12 showed a specific band at about 2 373 bp, consistent with expectation. The extracted recombinant plasmids were confirmed by HindⅢ, BamHⅠ and XbaⅠ digestion. Sequencing results showed that the sequence of pVAX1-ROP18-ROP12 was 100% identical to that of T. gondii RH strain ROP18 gene  (Accession No. AM075204.1) and 99% identical to that of T. gondii RH strain ROP12 gene (Accession No. DQ096559.1). Further, RT-PCR showed amplification products at 613 bp for β-actin in all the groups, while only the pVAX1-ROP18-ROP12 transfection group showed amplification products for the ROP18-ROP12 complex at 2 373 bp. In addition, the indirect immunofluorescence assay showed yellow-green fluorescence in HeLa cells transfected with pVAX1-ROP18-ROP12, but not in control cells. Western blotting showed that the ROP18-ROP12 fusion protein was expressed in HeLa cells transfected with recombinant plasmid pVAX1-ROP18-ROP12. Conclusions The recombinant eukaryotic plasmid pVAX1-ROP18-ROP12 is constructed and can be expressed in eukaryotic system.

Key words: Toxoplasma gondii, Rhoptry protein 18, Rhoptry protein 12, Complex recombinant plasmid, Eukaryotic expression