Preparation and Detection of Polyclonal Antibody of GAPDH Protein from Bombyx mori

doi:10.13560/j.cnki.biotech.bull.1985.2017-0104

Abstract

Abstract: This work is to prepare and detect the polyclonal antibody of GAPDH protein in Bombyx mori. GAPDH gene was cloned from silkworm by PCR，then prokaryotic expression vector was constructed and transformed into Escherichia coli competent cell. The recombinant protein was induced and purified. The purified protein was used as antigen to prepare the polyclonal antibody by immunizing New Zealand purebred rabbit. The titer and specificity of the polyclonal antibody was detected by enzyme-linked immune sorbent assay（ELISA）and Western blot. As results，GAPDH/pET-28a vector was constructed successfully，and recombinant GAPDH fusion protein in high purity was obtained. SDS-PAGE and anti-His monoclonal antibody detection showed that the molecular weight of the purified protein was consistent with that predicted. GAPDH polyclonal antiserum was obtained by immunized New Zealand purebred rabbit for four times using the purified protein as an antigen. The titer of GAPDH polyclonal antibody was 1：8 000 by ELISA，and the antiserum was able to bind specifically with the total protein of B mori. Conclusively，the polyclonal antibody of GAPDH is successfully prepared，which lays a solid foundation for further studying the physiological function of different proteins in B. mori.

Key words: Bombyx mori, glyceraldehyde-3-phosphate dehydrogenase, polyclonal antibody

CHEN Ying, WANG Yuna-zhuo, YANG Juan-juan, GUO Xing-guo, QIAO Hui-li. Preparation and Detection of Polyclonal Antibody of GAPDH Protein from Bombyx mori[J]. Biotechnology Bulletin, 2017, 33(7): 138-144.

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