[Protein interaction between aldolase and actin of Toxoplasma gondii by GST pull-down].
OBJECTIVE
To identify the protein-protein interaction between aldolase and actin of Toxoplasma gondii by GST pull-down.


METHODS
The aldolase and actin genes were obtained from cDNA library by PCR amplification, and subcloned respectively into pGEX-4T-1 and pET30a. The fusion protein GST-Aldolase and Actin-His6 were expressed in E. coli upon induction by 1 mmol/L IPTG and then purified with affinity chromatography. Fifteen rats were immunized intradermally with 200 microg Actin-His6 protein per rat at first time to produce the polyclonal antibodies. Then 100 microg Actin-His6 protein per rat on the 2nd-4th immunizations. Rats were immunized for 4 times with 7 days interval. The serum of rats was collected from heart at the fifth day after the final immunization. Glutathione sepharose beads were incubated with GST-Aldolase protein, then incubated with Actin-His6, and bound proteins were eluted using sample buffer. Eluants were resolved by SDS-PAGE and Western blotting.


RESULTS
The aldolase and actin genes were obtained, and the recombinant plasmid aldolase/pGEX-4T-1, actin/pET30a were successfully constructed. Protein GST-Aldolase and Actin-His6 were expressed and purified in vitro. Serum samples were prepared from rats immunized with protein Actin-His6, and polyclonal antibody was purified with affinity chromatography. SDS-PAGE and Western blotting analysis of products from GST pull-down experiment showed that the protein bands on NC membrane were specifically recognized by anti-Aldolase-His6 and anti-Actin-His6 antibody.


CONCLUSIONS
Aldolase interacts with Actin of Toxoplasma gondii.
Bin
B
Zheng
Zhi-Kui
ZK
Yin
Ai
A
He
Zhuo-Ya
ZY
Li
Xi-Mei
XM
Zhan
D000199:Actins / Q000378:metabolism*; D000818:Animals; D005634:Fructose-Bisphosphate Aldolase / Q000378:metabolism*; D008297:Male; D025941:Protein Interaction Mapping; D015800:Protozoan Proteins / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D014122:Toxoplasma / Q000276:immunology / Q000378:metabolism*
D000199:Actins; D015800:Protozoan Proteins; D005634:Fructose-Bisphosphate Aldolase