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Prep Biochem Biotechnol. 2019 Feb 8:1-10. doi: 10.1080/10826068.2019.1566150. [Epub ahead of print]

Soluble expression, rapid purification, biological identification of chicken interferon-alpha using a thioredoxin fusion system in E. coli and its antiviral effects to H9N2 avian influenza virus.

Zhao J1,2,3,4, Yu HY1, Zhao Y2, Li FH5, Zhou W2, Xia BB4, He ZY4, Chen J1,6, Jiang GT5, Wang ML1,2,3,4.

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a Department of Microbiology , Anhui Medical University , Hefei , Anhui , P.R. China.
b Anhui JiuChuan Biotech Co., Ltd , Wuhu , Anhui , P.R. China.
c Wuhu Overseas Students Pioneer Park , Wuhu , Anhui , P.R. China.
d Wuhu Interferon Bio-products Industry Research Institute Co., Ltd , Wuhu , Anhui , P.R. China.
e Dalian SanYi animal medicine Co., Ltd , Dalian , Liaoning , P.R. China.
f Department of Pathology and Cell Biology , Columbia University , New York , USA.


In this paper, we report a soluble expression based on Escherichia coli and two-step purification of a novel thioredoxin-tagged chicken interferon-α fusion protein (Trx-rChIFN-α) by using pET32a(+) expression system. The mature ChIFN-α gene was amplified by Reverse transcriptase-polymerase chain reaction (RT-PCR) and subcloned into pET-32a (+) vector prior to transformation into Rosetta (DE3) competent cells. After IPTG induction, the recombinant fusion protein was expressed efficiently in the soluble fraction. The protein purification was performed by nickel affinity chromatography and DEAE anion exchange chromatography. The purified product has a purity of 95% with a yield of 47.3 mg/L of culture. The specific activity of the fusion protein reaches to 2.0 × 107 IU/mg as determined in the CEF/VSV titration system. After excision of the Trx tag by enterokinase, the remaining solo protein was confirmed as rChIFN-α protein by SDS-PAGE, N-terminal sequencing and mass spectrometry. The effects of this Trx-rChIFN-α fusion protein against H9N2 influenza virus infection were also evaluated in ovo. The results showed that the Trx-rChIFN-α protein could significantly reduce the hemagglutination titer of H9N2 virus, and the H9N2 viruses HA gene copy numbers. These findings will enable us to produce large amount and bio-active rChIFN-α protein for future applications.


Avian influenza virus; chicken interferon-α; ; heterologous expression; recombinant protein; thioredoxin fusion system

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