[Expression and purification of Toxoplasma gondii GRA4 gene in prokaryotic system].
OBJECTIVE
To construct a prokaryotic expression system containing the dense granule protein 4 (GRA4) of Toxoplasma gondii, purify the expressed protein and detect its immunogenicity.


METHODS
The specific fragment of GRA4 gene was amplified by PCR. After subcloning the prokaryotic expression recombinant pET-GRA4, the expressed product was purified with His Bind affinity chromatography and analyzed by Western blot. BALB/c mice were immunized with the GRA4 recombinant protein, and the antibody IgG titer was detected by ELISA.


RESULTS
The pET-GRA4 prokaryotic expression system was obtained. The MW of the expressed protein was Mr 40,000 and formed in inclusion body. After purification, the recombinant protein could be specifically recognized by the T. gondii infected rabbit serum. Mice immunized with the purified recombinant protein elicited high titer of IgG antibody.


CONCLUSIONS
The pET-GRA4 recombinant protein was successfully expressed and purified, which shows the immunogenicity.
Shenzhen Center for Disease Control and Prevention, Shenzhen 518020, China.
Qi-ping
QP
Lin
Shao-ting
ST
Wu
Ya-biao
YB
Weng
Ming-jun
MJ
Lei
Hui-rong
HR
Pan
Shi-shan
SS
Yuan
Jian-xiang
JX
Wen
Li
L
Qin
Da-na
DN
Huang
Ren-li
RL
Zhang
Shi-tong
ST
Gao
D000818:Animals; D000913:Antibodies, Protozoan / Q000097:blood; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D015870:Gene Expression; D007074:Immunoglobulin G / Q000097:blood; D051379:Mice; D008807:Mice, Inbred BALB C; D015800:Protozoan Proteins / Q000096:biosynthesis* / Q000276:immunology / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000276:immunology / Q000302:isolation & purification; D014122:Toxoplasma / Q000235:genetics / Q000276:immunology*
D000913:Antibodies, Protozoan; C079248:GRA4 protein, Toxoplasma gondii; D007074:Immunoglobulin G; D015800:Protozoan Proteins; D011994:Recombinant Proteins