重组牛α干扰素的分泌表达及抗病毒活性分析

doi:10.16431/j.cnki.1671-7236.2021.04.033

Abstract

Abstract: The purpose of this study was to use Lactococcus lactis to construct a secretory expression system for bovine interferon-α (BoIFN-α) and analyze its antiviral activity.According to the preference of Lactococcus lactis MG1363 codon usage,the BoIFN-α gene was optimized and synthesized.The target gene and the Lactococcus lactis expression vector plasmid pAMJ399 were digested with SalⅠ and BglⅡ,respectively,and the gel recovered products were ligated and transformed into E.coli TG1 competent cells.The positive plasmid was extracted and electrotransformed into Lactococcus lactis.SDS-PAGE and Western blotting methods were used to analyze and identify the expressed protein in the mature cells.At the same time,the recombinant Lactococcus lactis was cultured for 28 h and then the supernatant was taken and concentrated with PEG20000 about 10 times for in vitro antiviral test.The results showed that the recombinant Lactococcus lactis pAMJ399-BoIFN-α/MG1363 was constructed successfully in this experiment.The supernatant and precipitate of the recombinant bacteria showed a target band of about 20 ku,indicating that the recombinant protein was secreted in both the supernatant and the precipitation.The concentrated rBoIFN-α supernatant acted simultaneously with Vesicular stomatitis virus (VSV).The titer of rBoIFN-α was determined to be 1.02×10⁶ U/L by the MDBK/VSV system by the microcytopathic inhibition method.The Alamer Blue method analysis result showed that the addition of rBoIFN-α had little effect on cell activity.Indirect immunofluorescence method showed that the cells had no green fluorescence after adding rBoIFN-α,indicating that rBoIFN-α had a good antiviral effect.Real-time PCR results further confirmed that rBoIFN-α had a certain antiviral effect.In this experiment,Lactococcus lactis could secrete and express rBoIFN-α was constructed.A series of in vitro antiviral tests proved that rBoIFN-α had good antiviral activity.The test results provided a new way for the development of rBoIFN-α as antiviral drugs,immune enhancers,peptide drugs and clinical applications.

Key words: BoIFN-α; Lactococcus lactis; secretory expression; antiviral activity

CLC Number:

S852.4

LIU Zengsu, WANG Zhaorui, WANG Xueying, JIANG Yanping, CUI Wen, WANG Li, QIAO Xinyuan, ZHOU Han, XU Yigang, LI Yijing, TANG Lijie. Secretory Expression and Antiviral Activity Analysis of Recombinant Bovine Interferon-α[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(4): 1449-1456.

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Secretory Expression and Antiviral Activity Analysis of Recombinant Bovine Interferon-α

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