OBJECTIVETo clone PAK5-N terminal sequence for expression in E. coli to prepare its polyclonal antibody, and examine the role of PAK5 in dental germ cells.

METHODSBased on human PAK5 cDNA sequence, PCR primers were designed to amplify PAK5-N terminal sequence. The PCR product was cloned into the expression vector pGEX-4T-1 EcoRI/XhoI sites, and the recombinant plasmids were identified by agarose gel electrophoresis followed by DNA sequence analysis. The recombinant plasmids were transformed into E. coli BL21 and the expression of GST-fusion protein was induced by IPTG. Glutathione-Sepharose beads were used to purify GST-fusion PAK5-N-terminal fragment. Anti-PAK5 polyclonal antibody was obtained in immunizing rabbits with purified GST-PAK5 N-terminal fusion protein, and the antibodies were purified by protein A beads and used for detection of PAK5 expression in dental germ cells.

RESULTS AND CONCLUSIONSWe successfully cloned PAK5-N terminal gene fragment, and achieved protein expression, purification and production of PAK5-NT polyclonal antibody. The results of Western blotting indicated that PAK5 can be highly expressed in the dental germ cells, suggesting that PAK5 may play an important role in biological function of dental germ cells.

Animals ; Antibodies, Monoclonal ; biosynthesis ; genetics ; immunology ; Base Sequence ; Blotting, Western ; Cloning, Molecular ; Humans ; Molecular Sequence Data ; Rabbits ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Sequence Analysis, DNA ; Tooth Germ ; cytology ; embryology ; enzymology ; p21-Activated Kinases ; biosynthesis ; genetics ; immunology