[Cloning and identification of ROP2 gene of Toxoplasma gondii].

ABSTRACT

OBJECTIVE: To amplify ROP2 from the genomic DNA of Toxoplasma gondii RH strain and construct eukaryotic expression plasmid pc-DNA3-ROP2. METHODS: Tachyzoites of T. gondii RH strain were collected and depurated to obtain genome. A pair of primers was designed and synthesized according ROP2 gene sequence. The gene fragment encoding ROP2 was amplified from the genomic DNA of T. gondii RH strain by means of PCR. It was then reclaimed and purified, and inserted into cloning vector pUCm-T. The recon was cut by EcoR I, Hind III, and the inserted ROP2 gene fragment was subcloned into pc-DNA3 eukaryotic expression vector using T4DNA ligase, followed by further PCR identification, double digestion via restrictive enzymes, and sequencing. RESULTS: The amplified specific gene fragment of ROP2 was about 1.7 kb in length. The gene fragment cloned and subcloned into pc-DNA3 was correct, and the eukaryotic expression plasmid contained ROP2 gene fragment was successfully constructed. CONCLUSION: The recombinant plasmid pc-DNA3-ROP2 was successfully constructed.