[Preparation and application of anti-serum against mouse Foxp3].

ABSTRACT

AIM: To prepare the high titer and specific anti-serum against mouse Foxp3 and then use them to identify Foxp3 expression in normal mouse tissues. METHODS: The Foxp3 fragment was amplified by polymerase chain reactions(PCR) and cloned into the prokaryotic expression vector pGEX-6p-2. The recombinant plasmid pGEX-6p-2/Foxp3 was transformed into E.coli BL21(DE3) to be expressed. The expressed fusion protein GST-Foxp3 was analyzed by SDS-PAGE and Western blot. The polyclonal clanti-serum was obtained by immunizing New Zealand rabbits with the purified fusion protein as antigen. The Foxp3 expression in normal mouse tissues was detected by Western blot with the anti-serum. RESULTS: The expressed products were analyzed by SDS-PAGE and Western blot. The results showed that the molecular weight of the expressed protein was about 45 000. The titer of the anti-serum was above 112 800. We observed that the anti-serum could recognize Foxp3 protein expressed in NIH3T3 cells by immunoblotting. Western blot results showed that the Foxp3 protein was expressed highly in spleen, thymus and lymph nodes, less expressed in stomach, but not expressed in skeletal muscle and adipose tissue. CONCLUSION: High titer antiserum against mouse Foxp3 is produced and can be used to identify the Foxp3 expression in normal mouse tissues.