Prokaryotic Expression, Purification And Polyclonal Antibody Preparation Of Focal Adhesion Kinase

Focal adhesion kinase (FAK) is a monosubunit inreceptor PTK. And FAK participate to regulate cell growth, development, sticky, cytoskeleton recombination, inversion, diffusion and migration through much signal-path. In this study, the fragment of FAK gene encoding the last 273 amino acids in the C terminus was amplified from Flag-FAK plasmid by PCR. And this fragment was inserted into the prokaryotic expression vector to construct the recombinant plasmid pET28a(+)/FAK, and expressed in fusion protein. The plasmid was expressed in Ecoli.BL21(DE3), and the expression product was purified. Mice were immunized with the purified FAK protein and the antiserum was obtained. The results indicated that the plasmid pET28a(+)/FAK which contained the FAK C terminus functional structural domain was constructed. After induction with IPTG, a molecular weight of 33 000 fusion protein was obtained by Ni-NTA affinity chromatography. The anti-FAK antibody was obtained and purified .The results of ELISA and immunofluorescence indicated that the polyclonal antibody had high titration and specificity, and will be used in the further research on FAK mechanism in NLT.