Prokaryotic expression, purification and antibody preparation of human fibronectin 1

Abstract

Objective To express human fibronectin 1 and generate specific human fibronectin 1 antibody in rabbit. Methods A 450 bp length fragment of human FN1 gene, containing the ISO amino acids of the C-terminal, was amplified by RT-PCR, and was cloned to pRSETA2 vector. The expression of FN1 was in BL21 (DE3) and the protein was purified by the Ni2 + -NTA resin. Purified FN1 was injected to the rabbit to raise antibody, and purified the antibody by affinity method. The specification of the antibody was detected through Western blot and immunohistochemistry. Results The pRSETA2 -FN1 vector was confirmed correctly through restriction enzyme digestion and sequencing. The fusion protein with 20 000 (FN1-His tag) was purified by Ni~(2+) -NTA column. The titer of generated FN1 antibody in rabbit was about 1: 10 000 by ELISA. The purified FN1 antibody by affinity was used to detect the FN1 in lung and lung cancer tissue by immunohistochemistry , and to detect FN1 in human plasma and HepG2 cell lysate by western blot. Conclusion The C-terminal of human FN1 was successfully prokaryotically expressed in this study. A specific FN1 antibody was generated by this recombinant FN1 protein. Key words: Fibronectin;  Gene expression;  Antibody;  Cancer