Amplification,cloning and identification of the CRA1 gene from Toxoplasma gondii in vitro

Aim Amplification of the GRA1gene from Toxoplasma gondii RH strain,and construction of recombinant eukaryotic expression plasmid of pEGFP N3-GRA1 Methods The tachyzoites had been harvested from ascites of mice infected by inoculating RH strain through the intraperitonea,purifying tachyzoites and preparing genomic DNA According to the cDNA sequence of GRA1 gene,a pair of primers were designed and synthesized Using PCR technique,a specific fragment of GRA1 gene was obtained by amplification from the genomic DNA of tachyzoites After having been digested by EcoRⅠ/BamHⅠ,the fragments of PCR products were cloned into a high level expression vector pEGFP N3,then transferred into Escherichia coli(E coli) DH5α,a recombinant pEGFPN3-GRA1 was constructed,it was identified by PCR and EcoRⅠ/BamHⅠdigesting methods Results The size of the amplification GRA1 gene fragment was in accord with the expected one matelly The recombinant pEGFP N3-GRA1 was successfully constructed Conclusions The gene encoding GRA1 was amplified from the DNA of RH strain of Toxoplasma gondii by PCR,and the recombinant plasmid PEGFPN3-GRA1 is constructed The expression of the GRA1 gene of Toxoplasma gondii RH strain will be further investigated