Prokaryotic expression, purification and polyclonal antibody preparation of ferritin light chain / 第二军医大学学报

ABSTRACT

Objective: To construct prokaryotic expression vector pET30a-FTL, obtain purified FTL protein and to prepare anti-FTL polyclonal antibody, so as to provide evidence for studying the biological function of the antibody. Methods: Gene recombination technology was used to link FTL gene (PCR product) and prokaryotic expression vector pET30a(+); the recombinant plasmid was then transformed into E. coli BL21 and the positive clones were identified by restriction enzyme digestion and PCR. Expression FTL protein was induced with IPTG and the expression product was purified. The purified FTL protein was used to immunize rabbits to obtain the antiserum. The specificity of the antibodies was examined by Western blotting. Results: The prokaryotic expression vector pET30a-FTL was successfully constructed and a fusion protein, with a molecular weight of 25 800, was obtained after induction with IPTG and affinity chromatography. The anti-FTL antibody was obtained from the immunized rabbits. The results of Western blotting indicated that the polyclonal antibody had high specificity to FTL protein. Conclusion: We have successfully obtained the purified FTL protein and anti-FTL polyclonal antibody, which lays a foundation for further research on the biological function of FTL and expression of FTL in lung cancer tissues.