Abstract:

Abstract:Objective To clone the GRA1 gene and construct the prokaryotic expression vector PET-28a-GRA1,and to transform the expression vector  into E.coli BL21,then  induce and identiy,and to lay a foundation for the study on the biological characteristics and immune protection of GRA1.Methods A pair of primers were designed according to the sequence of GRA1 from GenBank.The GRA1 gene was amplified by PCR.The amplified GRA1 gene was inserted into prokaryotic expression vector PET-28a,and the constructed recombinant plasmid PET-28a-GRA1 was transformed to E.coli BL21 for expression under induction of IPTG.The expressed product was identified by SDS-PAGE and Western blotting.Results:The sequencing result showed the homology of 100% of the cloned GRA1 gene to that reported in GenBank.The positive recombinant plasmids PET-28a-GRA1 were identified by restriction endonuclease digestion and PCR,the objective gene fragment with the size of 573 bp was acquired,in accordance with the expected results,and the recombinant plasmid PET-28a-GRA1 was constructed successfully.The results of SDS-PAGE  revealed that the molecular  weight of recombinant protein PET-28a-GRA1 was 24 000 and Western blotting proved that the recombinant protein was recognized by murine antisera against Toxoplasma gondii.Conclusion The GRA1 gene has been successfully cloned,the recombinant
 plasmid PET-28a-GRA1 is generated and expressed highly in prokaryote.

Key words: Toxoplasma gondii；GRA1；cloning；gene expression