Cloning and sequencing the plasmid encoding Toxoplasma gondii Microneme 3 protein

Toxoplasma gondii, an obligatory intracellular protozoan parasite, causing toxoplasmosis in human and animal with worldwide spread. Microneme 3 (MIC3) protein, a 90 kDa parasite factor attaching to the host cells in the beginning of the invasion, is secreted in all stages of parasite development (e.g. sporozoite, tachyzoite and bradyzoite) and also is considered as a potent antigen. Therefore, besides the immunogenicity and the candidacy for vaccine design, the protein is used for diagnostic purposes, as well. The aim of the present study was to transfer MIC3 gene into plasmid vector (PTZ57R/T) for subcloning in eukaryotic and prokaryotic plasmids.

Toxoplasmia genomic DNA extracted using phenol-chloroform method and MIC3 gene was then amplified by PCR with specific primers. Electrophoresis was performed by using agarose gel and PCR product was purified by T4 DNA ligase enzyme into a cloning vector. Finally, recombinant plasmid was transformed into E.coli (Top10 strain). The extracted clone was verified with PCR, digestion enzymes and sequencing.

The PCR product was seen as a 1052bp band in agarose gel (1%). The recombinant plasmids was restricted by HindIII and EcoRV enzymes and two obtained 2886 and 1052bp bands showed that the MIC3 gene was cloned in PTZ57R/T plasmid.

The results revealed that the cloning and transformation of MIC3 gene in pTZ57R/T was done successfully.