Focal Adhesion Kinase（FAK），局部趨化激?，是一種酪胺酸激?（protein tyrosine kinase; PTK），PTK表現量異常或是發生突變而導致其本身活化時，便可能擾亂細胞生長的調控，甚至與腫瘤的形成有關。 當FAK上的Integrin接受器與細胞間質蛋白產生訊息交互作用時，將會促使FAK酪胺酸磷酸化，引發FAK相關訊息傳遞分子，諸如：Src-family PTKs, p130Cas, Shc, Grb2, PI 3-kinase, 及 paxillin 進而影響細胞生長、存活、侵襲與轉移；於本研究亦發現肝癌組織中FAK有過度表達之現象。 FRNK （FAK-related non-kinase）是FAK C端之異構型突變種，不具催化激?。本研究首先利用DNA陽離子微脂粒 Lipofectamin將FRNK遞送進入肝癌細胞株，降低了FAK之磷酸化作用並抑制肝癌細胞株Hep3B細胞之增生，由於基因遞送效益僅20%，故建構腺病毒載体以提高效益。以往之腺病毒載体的建構，須將選殖的基因送入一個穿梭載體（shuttle vector），再將此穿梭載體和腺病毒部份基因体同時送入293細胞內進行同源重組。然而在哺乳動物細胞中同源重組的效率很低，所以我們利用E. coli系統進行同源重組。E. coli之生長速度較快，且重組之機率較高；用此系統我們可利用抗生素及PCR篩選得到產生重組的選殖株，再送入293細胞，用以產生重組腺病毒。; Focal adhesion kinase（FAK）is a protein tyrosine kinase involved in the control of cell adhesion, migration, cell cycle progression, and apoptosis. Initial analysis by western blot indicated that FAK was highly expressed in hepatocellular carcinoma cell lines and may play a role in liver cancer tumorogenesis. In this study, we determined whether FAK is a molecular target for cancer gene therapy by overexpressing the noncatalytic C-terminal domain of FAK, the FAK-related nonkinase（FRNK）, as a negative regulator of FAK activity. An adenoviral expressing plasmid carrying FRNK cDNA have been constructed. Transient expression study indicated that the FRNK protein could be produced as detected by western blot analysis with anti-FAK antibody. In comparison to the conventional construction of recombinant adenovirus via 293 mammalian cells with low yield, we adapted BJ5183 E coli cells for a better homologous recombination. The rAdFRNA demonstrated an affective inhibiton on cell proliferation, suggesting this recombianant molecule a good candidate for cancer gene therapy.
