ABSTRACT
<p><b>OBJECTIVE</b>To study the role of PAK6 in prostate cancer by cloning PAK6-N terminal sequence into E.coli and preparing its polyclonal rabbit antibody to detect PAK6 expression in prostate cancer.</p><p><b>METHODS</b>Based on human PAK6 cDNA sequence, we designed a pair of primers to amplify the PAK6-N terminal sequence by PCR. The PCR product was subcloned into the bacterial expression vector pGEX-4T-1 via EcoRI/XhoI sites, and the recombinant plasmids were identified by enzymatic cleavage followed by DNA sequence analysis. By transforming the expression vector into component E.coli BL21 cells, the GST-PAK6-N fusion protein was expressed with IPTG induction. Glutathione-Sepharose beads were used to purify GST- PAK6-N fusion protein. Anti-PAK6 polyclonal antibody was produced by immunizing rabbits with purified GST-PAK6 N-terminal fusion protein. Anti-PAK6 polyclonal antibody was purified by protein A beads and used for detection of PAK6 expression in 3 prostate cancer specimens.</p><p><b>RESULTS AND CONCLUSION</b>We cloned PAK6-N terminal gene fragment successfully, purified GST-PAK6 N-terminal fusion protein, and obtained polyclonal rabbit PAK6 antibody. Immunohistochemistry indicated that PAK6 expressed in the stroma instead of the cancer cells in prostate cancer. All of the 3 prostate cancer specimens showed positive staining in the stroma, suggesting that PAK6 may participate in the stroma-cancer cell interaction in prostate cancer.</p>
Subject(s)
Aged , Animals , Humans , Male , Rabbits , Antibodies, Monoclonal , Allergy and Immunology , Cloning, Molecular , DNA, Complementary , Chemistry , Genetics , Electrophoresis, Polyacrylamide Gel , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Neoplastic , Immunohistochemistry , Polymerase Chain Reaction , Prostatic Neoplasms , Genetics , Recombinant Fusion Proteins , Allergy and Immunology , Metabolism , Sequence Analysis, DNA , p21-Activated Kinases , Genetics , Allergy and Immunology , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>Based 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.</p><p><b>RESULTS AND CONCLUSIONS</b>We 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.</p>