[Prokaryotic expression and polyclonal antibodies preparation and identification of human focal adhesion kinase (FAK)].

Abstract

Objective To Clone, express, and purify the focal adhesion kinase (FAK) gene C-terminal focal adhesion location sequence (aa798-aa1041), and to prepare and identify the rabbit anti-FAK polyclonal antibodies. Methods The C-terminal (2671 bp-3402 bp) gene of the FAK gene was amplified by PCR in vitro and cloned into pCZN1 vector to construct a pCZN1-FAK recombinant expression vector. The recombinant expression vector was transformed into E. coli expression strain BL21 (DE3) competent cells, and then induced by isopropy-β-D-thiogalactoside (IPTG). The protein was purified by affinity chromatography resin Ni-NTA and immunized with New Zealand white Rabbit to prepare polyclonal antibodies. The antibody titer was detected by indirect ELISA and the specificity was identified by Western blot analysis. Results The pCZN1-FAK recombinant expression vector was successfully constructed. The FAK protein was mainly expressed in the form of inclusion bodies. After purification of the target protein, the prepared rabbit anti-FAK polyclonal antibody showed a titer of 1:512 000, and could specifically react with exogenous and endogenous FAK proteins. Conclusion The FAK protein is successfully cloned, expressed and purified, and a rabbit anti-FAK polyclonal antibody is prepared, which can be used for the specific detection of endogenous FAK protein.