[Cloning and expression of a bradyzoite-specific gene of Toxoplasma gondii and immunoreactive analysis on the recombinant antigen].
OBJECTIVE
To clone and express bradyzoite antigen 1 (BAG1) gene of T. gondii, and analyze the immunoreactivity of the recombinant product.


METHODS
The differentiation of T. gondii RH strain tachyzoites into bradyzoites was induced in vitro, and the coding sequence of BAG1 was amplified from bradyzoites by RT-PCR. The PCR product was analyzed by sequencing. The BAG1 coding sequence was further subcloned into the plasmid pET32a(+). The plasmid pET32a(+) -BAG1 was then transformed into BL21(DE3) to express after IPTG induction. The expression product was purified with Ni-NTA agarose and the purified BAG1 was further analyzed by Western blotting and ELISA.


RESULTS
BAG1 cDNA was amplified from bradyzoites. After IPTG induction, BAG1 was expressed in a fusional form in E. coli. Western blotting showed that the purified recombinant protein could be specifically recognized by sera from mice chronically infected by T. gondii B36 strain. ELISA showed that the positive rate of T. gondii IgG antibodies of 350 human sera detected by the recombinant BAG1 (17.4%) was higher than by recombinant SAGI (12.6%) (P < 0.05).


CONCLUSIONS
The expressed recombinant BAG1 shows a specific immunoreactivity.
Wang
Qiong
Q
Department of Pathogen Biology, The Southern Medical University, Guangzhou 510515, China.
Wu
Kun
K
Chen
Xiao-guang
XG
Hao
Li
L
Cheng
Lu
L
D000818:Animals; D000913:Antibodies, Protozoan / Q000097:blood; D015153:Blotting, Western; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006360:Heat-Shock Proteins / Q000235:genetics* / Q000276:immunology; D006801:Humans; D007106:Immune Sera / Q000276:immunology; D007074:Immunoglobulin G / Q000097:blood; D051379:Mice; D041681:NIH 3T3 Cells; D015800:Protozoan Proteins / Q000235:genetics* / Q000276:immunology; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014122:Toxoplasma / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D014124:Toxoplasmosis, Animal / Q000097:blood / Q000469:parasitology
D000913:Antibodies, Protozoan; C106179:BAG1 protein, Toxoplasma; D006360:Heat-Shock Proteins; D007106:Immune Sera; D007074:Immunoglobulin G; D015800:Protozoan Proteins; D011994:Recombinant Proteins