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[Cloning of Human PAK6 cDNA, Preparation of anti-PAK6 Polyclonal Antibody and PAK6 Expression in Prostate Cancer]

[Cloning of Human PAK6 cDNA, Preparation of anti-PAK6 Polyclonal Antibody and PAK6 Expression in Prostate Cancer]

Yu-jing Han, Zheng-wen An, Jin-xing Yang, Jun Li, Rong-cheng Luo, Hong-quan Zhang

Nan Fang Yi Ke Da Xue Xue Bao. 2007 Jun;27(6):827-30.

PMID: 17584649

Abstract:

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.
Results and conclusion:
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.

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