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Soluble Expression, Rapid Purification and Antiviral Activity of Recimbinant Bovine Interferon-α in Escherichia coli
Applied Biochemistry and Microbiology ( IF 1.0 ) Pub Date : 2020-03-30 , DOI: 10.1134/s0003683820020143 H.-Y. Yu , J. Liu , Z.-Y. He , W. Zhou , B.-B. Xia , M. Wang , J. Chen , M.-L. Wang , G.-T. Jiang , J. Zhao
中文翻译:
重组牛干扰素-α在大肠杆菌中的可溶性表达,快速纯化和抗病毒活性
更新日期:2020-03-30
Applied Biochemistry and Microbiology ( IF 1.0 ) Pub Date : 2020-03-30 , DOI: 10.1134/s0003683820020143 H.-Y. Yu , J. Liu , Z.-Y. He , W. Zhou , B.-B. Xia , M. Wang , J. Chen , M.-L. Wang , G.-T. Jiang , J. Zhao
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.中文翻译:
重组牛干扰素-α在大肠杆菌中的可溶性表达,快速纯化和抗病毒活性