Cloning of human PAK6 cDNA, preparation of anti-PAK6 polyclonal antibody and PAK6 expression in prostate cancer.
- Author:
Yu-jing HAN
1
;
Zheng-wen AN
;
Jin-xing YANG
;
Jun LI
;
Rong-cheng LUO
;
Hong-quan ZHANG
Author Information
- Publication Type:Journal Article
- MeSH: Aged; Animals; Antibodies, Monoclonal; immunology; isolation & purification; Cloning, Molecular; DNA, Complementary; chemistry; genetics; Electrophoresis, Polyacrylamide Gel; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Male; Polymerase Chain Reaction; Prostatic Neoplasms; enzymology; genetics; Rabbits; Recombinant Fusion Proteins; immunology; metabolism; Sequence Analysis, DNA; p21-Activated Kinases; genetics; immunology; metabolism
- From: Journal of Southern Medical University 2007;27(6):827-830
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo 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.
METHODSBased 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.
RESULTS AND CONCLUSIONWe 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.