Prokaryotic Expression,Purification and Polyclonal Antibody Preparation of Neurospora crassa ERG-11 Protein Antigen

doi:10.13560/j.cnki.biotech.bull.1985.2017-0258

Abstract

Abstract: This work aims to construct the recombinant plasmid pET-28a（+）-ERG-11 and express the 6 × His-ERG-11 fusion protein for preparing ERG-11 polyclonal antibody. The target fragment amplified by PCR was inserted into pET-28a（+）prokaryotic expression vector,and then transformed into the competent cells of Escherichia coli BL21（DE3）for expressing the fusion protein. Further,the fusion protein was purified by affinity purification and gel filtration chromatography,and then immunized to New Zealand white rabbit for preparing polyclonal antibody. Taking serum,the titer and specificity of the polyclonal antibody were detected through indirect ELISA and Western blot,respectively. As results,the pET-28a（+）-ERG-11 expression vector was successfully constructed. SDS-PAGE showed that the 6 × His-ERG-11 fusion protein was induced successfully in the inclusion form. After two-step purification,the antigen with high purity was obtained. ELISA showed that the polyclonal antibody titer reached 1∶512 000,and Western blot confirmed its high specificity. Conclusively,the prokaryotic expression of ERG-11 gene was successfully conducted,and a polyclonal antibody against Neurospora crassa ERG-11 was prepared,providing a foundation for further study on the genetic structure and function of ERG-11 in N. crassa.

Key words: Neurospora crassa ERG-11, cloning, protein expression and purification, polyclonal antibody

LIU Yan-xia,LI Shao-jie,FAN Zhen-chuan. Prokaryotic Expression,Purification and Polyclonal Antibody Preparation of Neurospora crassa ERG-11 Protein Antigen[J]. Biotechnology Bulletin, 2017, 33(9): 216-222.

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