[Cloning, expression and antigenicity analysis of phosphoglycerate mutase 2 gene of Toxoplasma gondii].
OBJECTIVE
To clone and express the phosphoglycerate mutase 2 (PGAM2) gene of Toxoplasma gondii, and analyze the antigenicity of the recombinant protein.


METHODS
Total RNA was extracted from T. gondii tachyzoites of RH strain and reversely transcribed into cDNA. TgPGAM2 gene was amplified by PCR and cloned into pET30a(+) vector. The constructed pET30a(+)-TgPGAM2 was transformed into E. coli DH5alpha first and selected through the colony-PCR and confirmed by the double restriction enzyme digestion and sequencing. The correct plasmid was transformed into E. coli BL21 for expression induced by IPTG and the recombinant protein was further analyzed through SDS-PAGE followed by Coomassie brilliant blue staining. Western blotting assay with rabbit anti-T. gondii serum was used to analyze its antigenicity.


RESULTS
The length of PCR product was about 750 bp and the recombinant plasmid pET30a(+)-TgPGAM2 was successfully constructed. The results of SDS-PAGE and Western blotting revealed that the relative molecular weight (Mr) of the soluble recombinant protein was approximately 30 000 and could be recognized by rabbit anti-T. gondii serum.


CONCLUSIONS
The soluble TgPGAM2 protein has been expressed in the prokaryotic expression system and maintains its antigenicity.
Department of Physiology, Key Laboratory of Cellular Physiology Co-constructed by Province and Ministry of Education, Shanxi Medical University, Taiyuan 030001, China.
Li-Tian
LT
Yin
Fen
F
Wang
Xiao-Li
XL
Meng
Hai-Long
HL
Wang
Hong-Li
HL
Liu
Jin-Yan
JY
Shen
Guo-Rong
GR
Yin
D000953:Antigens, Protozoan / Q000235:genetics / Q000276:immunology*; D003001:Cloning, Molecular; D015870:Gene Expression; D010736:Phosphoglycerate Mutase / Q000235:genetics* / Q000276:immunology*; D010957:Plasmids; D015800:Protozoan Proteins / Q000235:genetics / Q000276:immunology; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D014122:Toxoplasma / Q000201:enzymology / Q000235:genetics* / Q000276:immunology*
D000953:Antigens, Protozoan; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D010736:Phosphoglycerate Mutase