[Efficient soluble expression, purification and identification of the truncated SAG1 gene of Toxoplasma gondii in Escherichia coli].
OBJECTIVE
To express truncated SAG1 gene in Escherichia coli to obtain purified recombinant SAG1, and identify the immunoreactivity of the product.


METHODS
The plasmid pET-30a(+)-trSAG1 was constructed, which was cut by Nco I and HindIII to obtain truncated SAG1 gene and inserted into pET-32a(+) cut by the same two restriction enzymes. After identification by restriction enzymes, the plasmid pET-32a(+)-trSAG1 was transformed into E.coli BL21, and the soluble product induced by 0.1 mmol/L IPTG for 4 hour before purification with Ni-NTA agarose, followed by identification of the purified recombinant protein with SDS-PAGE, Western-blotting and ELISA.


RESULTS
Recombinant pET-32a(+)-trSAG1 was successfully constructed and high level of truncated SAG1 expression achieved in E.coli. SDS-PAGE showed that the expressed protein was approximately 40,000 in size and expressed in a soluble form that could be easily purified by Ni-NTA agarose. Western-blotting demonstrated that the purified product could be specifically recognized by sera from rabbits immunized with native antigen of T.gondii, and could also be recognized, as shown by ELISA, by sera from human with T.gondii infection.


CONCLUSIONS
The truncated SAG1 gene was successfully expressed in E.coli in a soluble form, and the recombinant protein may be of value in the diagnosis of toxoplasmosis.
Yan
Hui
H
Departmemt of Parasitology, First Military Medical University, Guangzhou 510515, China.
Li
Hua
H
Zhou
Xiao-hong
XH
Wu
Kun
K
Chen
Xiao-guang
XG
D000818:Animals; D000953:Antigens, Protozoan / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D015153:Blotting, Western; D004926:Escherichia coli / Q000235:genetics; D008297:Male; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D011817:Rabbits; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification
D000953:Antigens, Protozoan; D015800:Protozoan Proteins; D011994:Recombinant Proteins; C047168:SAG1 antigen, Toxoplasma