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- Title
Soluble Expression, Rapid Purification and Antiviral Activity of Recimbinant Bovine Interferon-α in Escherichia coli.
- Authors
Yu, H.-Y.; Liu, J.; He, Z.-Y.; Zhou, W.; Xia, B.-B.; Wang, M.; Chen, J.; Wang, M.-L.; Jiang, G.-T.; Zhao, J.
- Abstract
In this study, the cDNA of mature bovine interferon-α (BoIFN-α) gene was cloned and expressed in the Escherichia coli expression system. Total RNA was extracted from bovine liver cells and reverse transcribed into cDNA, and then BoIFN-α gene fragment was amplified with specific primers by PCR method before being ligated into the pET-32a (+) expression vector. Since the pET-32a (+) plasmid expresses the thioredoxin A (TrxA) tag protein, it can increase the solubility of the target protein. In order to express the target protein, the pET-32a-BoIFN-α recombinant plasmid was transformed into E. coli Rosetta (DE3) strain followed by IPTG induction. It was found that the recombinant fusion protein Trx-rBoIFN-α was finally expressed in a soluble form in the host cell, accounting for about 33.45% of the total cellular protein. The protein was purified by two-step chromatography, including Ni2+-chelating Sepharose affinity chromatography and DEAE anion exchange chromatography. The purity of the purified product reached 93.0%, and the cytopathic effects (CPE) inhibition method was adopted to determine the antiviral activity of the expressed fusion protein in vesicular stomatitis virus/ Madin-Darby bovine kidney cells titration system. The specific activity of the recombinant fusion protein Trx-rBoIFN-α was measured as (3.6 ± 0.25) × 106 U/mg. After cutting of the Trx tag protein by enterokinase digestion, the remaining solo protein was confirmed as rBoIFN-α protein by Western blot assay. These findings will enable us to produce a large number of bioactive rBoIFN-α protein for future application.
- Subjects
ANTISENSE DNA; DNA primers; ESCHERICHIA coli; CHIMERIC proteins; RECOMBINANT proteins; VESICULAR stomatitis; PROTEOLYSIS; TYPE I interferons
- Publication
Applied Biochemistry & Microbiology, 2020, Vol 56, Issue 2, p154
- ISSN
0003-6838
- Publication type
Article
- DOI
10.1134/S0003683820020143