[Cloning of human PAK6 cDNA, preparation of anti-PAK6 polyclonal antibody and PAK6 expression in prostate cancer].
OBJECTIVE
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.


METHODS
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.


CONCLUSIONS
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.
Han
Yu-jing
YJ
Department of Oncology, Nanfang Hospital, Southern Medical University, Guangzhou, China. hanyuj2@yahoo.com.cn
An
Zheng-wen
ZW
Yang
Jin-xing
JX
Li
Jun
J
Luo
Rong-cheng
RC
Zhang
Hong-quan
HQ
D000368:Aged; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000302:isolation & purification; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D015971:Gene Expression Regulation, Enzymologic; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D016133:Polymerase Chain Reaction; D011471:Prostatic Neoplasms / Q000201:enzymology* / Q000235:genetics; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000276:immunology / Q000378:metabolism; D017422:Sequence Analysis, DNA; D054462:p21-Activated Kinases / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; C429007:PAK6 protein, human; D054462:p21-Activated Kinases