Cloning of human PAK6 cDNA, preparation of anti-PAK6 polyclonal antibody and PAK6 expression in prostate cancer / 南方医科大学学报
Journal of Southern Medical University
;
(12): 827-830, 2007.
Article
in Chinese
| WPRIM
| ID: wpr-337374
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>
Full text:
Available
Index:
WPRIM (Western Pacific)
Main subject:
Prostatic Neoplasms
/
Recombinant Fusion Proteins
/
Immunohistochemistry
/
Gene Expression Regulation, Enzymologic
/
Gene Expression Regulation, Neoplastic
/
Chemistry
/
Polymerase Chain Reaction
/
Cloning, Molecular
/
Sequence Analysis, DNA
/
DNA, Complementary
Limits:
Animals
/
Humans
/
Male
Language:
Chinese
Journal:
Journal of Southern Medical University
Year:
2007
Type:
Article
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