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Published on: March 12, 2015
[Cloning, expression and immunoreactivity analysis of rhoptry protein 18 (rop18) from Toxoplasma gondii]
Ying Zhang1, Jin-Shun Zhang, Xiao-Hui Jia
1Institute of Pathogen Microbes and Immunology, Hebei North University, Zhangjiakou, 075000, China.
Objective:
To clone and express rhoptry protein 18 (ROP18) gene of Toxoplasma gondii, and analyze its immunoreactivity.
Methods:
The genomic DNA was extracted from T. gondii (RH strain) tachyzoites. TgROP18 gene was amplified by PCR, and cloned into pET30a (+) vector. The constructed pET30a (+)-TgROP18 was transformed into E. coli BL21(DE3) and followed by expression of the protein induced by IPTG. The recombinant protein was analyzed through SDS-PAGE, and identified by Western blotting with mouse anti-T. gondii serum.
Results:
The TgROP18 gene was about 1 665 bp in length and encoded for a protein of 544 amino acid residues and the former 47 amino acids consisted signal peptide sequences. PCR, enzyme digestion and DNA sequencing confirmed that the recombinant plasmid pET30a (+)-TgROP18 was constructed. Bacteria containing recombinant plasmid pET30a (+)-TgROP18 expressed a soluble protein of His-TgROP18 (M, 59 800) after being induced with IPTG. His-TgROP18 reacted positively with mouse anti-T. gondii serum by Western blotting analysis.
Conclusion:
The soluble His-TgROP18 protein shows certain immunoreactivity.
