Abstract:
Objective This study was conducted to explore a highly efficient chicken gene engineering antiviral agent to prevent and control the chicken viral disease. A recombinant plasmid pET28a-rChIFNα-IL18 was constructed, which was expressed inEscherichia coli, purified by Ni-NTA and tested for antiviral bioactivitiy.
Method This study introduced a novel method for constructing vectors by fusion PCR, which generated PCR products with overlapping chain using Primers complementary to the end. Through the extension of PCR products, ChIFN-α and ChIL-18 constituted the ChIFN-α-ChIL-18 fusion gene when cloned into the vector pET-28a, induced the expression inE. coli strain BL21 (DE3). This protein was purified by Ni-NTA and detected by SDS-P AGE and Western-blot. Cytopathic effects(CPE)were applied to examine the potency of recombination rChIFN-α-ChIL-18 against vesicular stomatitis virus(VSV)and newcastle disease virus(NDV)proliferation.
Result and conclusion The fusion gene of ChIFN-α-ChIL-18 was successfully constructed and cloned into pET-28a vector. The rChIFN-α-ChIL-18 protein was expressed inE. coli and successfully purified with a molecular mass of about 38 000 and more than 90% on SDS-PAGE, which indicated that the correct rChIFN-α-ChIL-18 fusion protein had been obtained.The antiviral activity units of rChIFN-α-ChIL-18 protein inhibiting the reproduction of VSV and NDV on chicken embryo fibroblast (CEF) cells were much higher than those of the recombinant rChIFN-α, rChIL-18 protein.
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