Cloning and Molecular Characterization of Toxoplasma Gondii Granular Antigen 4 Gene Expression Eukaryotic Plasmid pcDNA3

Toxoplasmosis, caused by an intracellular protozoan parasite, Toxoplasma gondii, is widespread throughout the world. Being a cause of congenital disease and abortion in humans and in domestic animals, the disease is of major medical and veterinary importance. In addition, it has increased importance due to toxoplasma encephalitis in AIDS patients. Therefore, developing a vaccine for toxoplasma is an important goal in the world. GRA4 was known as a major antigen of Toxoplasma gondii. Thus, it is considered as a vaccine candidate. GRA4 was secreted from bradyzoite and tachyzoite. This genome has unique version and without intron. In this study, GRA4was cloned in pTZ57R, afterwards, it was transformed into TG1 strain of E.coli Bacteria. The recombined Plasmid extracted from E.coli bacteria and amplified through PCR technique. Besides, pcDNA3 Plasmid for receiving and coloning of GRA4 segment was digested by KpnI & EcoRI enzymes. GRA4 was subcloned into pcDNA3 and the reaction liggation product was transformed for the above bacteria. The bacteria grew in LB culture with ampicilin. Recombined pcGRA4 plasmids were purified from E.coli by Plasmid extraction kit. The accuracy correctness was confirmed by using restriction enzymes and PCR methods. GRA4 was cloned into expression eukaryotic plasmid pcDNA3. The results showed that the cloning and transformation of fragment GRA4 in pcDNA3 was done properly.