Prokaryotic Expression,Purification and Antiserum Preparation of Recombinant EDF-1 of Bufo gargarizans

doi:10.13560/j.cnki.biotech.bull.1985.2018-0291

Abstract

Abstract: This study aims to acquire the recombinant protein of endothelial differentiation related factor-1（EDF-1）of Bufo gargarizans and high-titer antiserum. The open reading frame（ORF）of EDF-1 of B. gargarizans has been cloned（GenBank accession number：K F769459）in previous studies. However,the recombinant protein using original ORF was not expressed in prokaryotic system due to the codon bias that might make the codons of B. gargarizans difficult to be recognized in prokaryotic cells. The codon of EDF-1 ORF of B. gargarizans was optimized,synthesized chemically,and inserted into prokaryotic expression vector,thus the recombinant plasmid pET-28b-EDF-1-Bufo for prokaryotic expression was constructed. The recombinant plasmid was transformed into Escherichia coli competent cells BL21（DE3）. The expression of recombinant EDF-1 of B. gargarizans was induced by IPTG,and the EDF-1 was purified with cobalt agarose. Then mice were immunized with the purified recombinant protein to prepare the antiserum against EDF-1. The specificity and titer of the antiserum was determined by Western blot and ELISA,respectively. As results,the recombinant EDF-1 of B. gargarizans was well expressed and the antiserum was highly specific,and the titer of the antiserum against recombinant EDF-1 of B. gargarizans was 1∶8 000. These results lay a foundation for the further study of biological function of EDF-1 and provide important reference for development of antitumor drugs for inhibiting endothelia cell differentiation.

Key words: Bufo gargarizans, EDF-1, recombinant protein expression, codon optimization

LIU Yi-jun, JIA Yu-kun, WANG Ling-fang, LIU Hong-xing, YANG Xian-yu. Prokaryotic Expression,Purification and Antiserum Preparation of Recombinant EDF-1 of Bufo gargarizans[J]. Biotechnology Bulletin, 2018, 34(10): 129-134.

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