[Expression of truncated Toxoplasma gondii GRA8 in the prokaryotic expression plasmids].
OBJECTIVE
To construct the prokaryotic recombinant expression plasmids of Toxoplasma gondii GRA8 and analyze their expression in E. coli containing the prokaryotic recombinant plasmids.


METHODS
The full gene and its truncated fragment of GRA8 were amplified by PCR from T. gondii genomic DNA, and cloned into pMD18-T vector. The right genes in positive clones sequenced with ABI PRISMTM 377XL DNA Sequencer were digested with restrictive endonucleases and subcloned into pGEX-4T-2. The recombinant plasmids were transformed into E. coli JM109. The recombinant clones were characterized by PCR and digested with restriction endonucleases. Positive clones were induced with IPTG to express target protein and characterized by SDS-PAGE. The recombinant protein was purified from E. coli lysate by affinity chromatography and SDS-PAGE. The immunological activity of this protein was analyzed by Western blotting.


RESULTS
The gene fragments encoding GRA8 were amplified by PCR from T. gondii genomic DNA. The inserts of GRA8 in positive clones were coincident with the original sequence of GRA8 gene from GenBank. The recombinant expression plasmids were constructed through subcloning the right inserts of GRA8 into pGEX-4T-2. The expression level of GRA8 from the recombinant expression plasmids in E. coli was low, and there was almost no full-length GRA8 expressed in E. coli. The purified protein reacted with the sera from rabbits infected with T. gondii RH strain.


CONCLUSIONS
The expression level of GRA8 from the recombinant expression plasmids in E. coli is low and the purified truncated GRA8 shows certain antigenicity.
Yuan
Shi-Shan
SS
Research Base in Shenzhen Center for Disease Control and Prevention, Postdoctoral Mobile Station of Basic Medicine, Sun Yat-sen University, Shenzhen 518020, China.
Wu
Shao-Ting
ST
Zhang
Ren-Li
RL
Gao
Shi-Tong
ST
Huang
Da-Na
DN
Yu
Xin-Bing
XB
D000818:Animals; D000953:Antigens, Protozoan / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D010957:Plasmids / Q000235:genetics*; D015800:Protozoan Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology; D014122:Toxoplasma / Q000235:genetics* / Q000276:immunology
D000953:Antigens, Protozoan; C403417:GRA8 protein, Toxoplasma gondii; D015800:Protozoan Proteins; D011994:Recombinant Proteins