猪IFN-δ5的表达纯化及其抗PEDV感染的作用分析

doi:10.16431/j.cnki.1671-7236.2022.08.033

Abstract

Abstract: 【Objective】 The purpose of this study was to establish a method for expression and purification of large amount of porcine interferon-δ5 (pIFN-δ5),and analyze the effect of pIFN-δ5 against Porcine epidemic diarrhea virus (PEDV) infection.【Method】 According to the sequence of pIFN-δ5 in GenBank(accession No.:NM_001164854.1),primers were designed and PCR amplification was carried out with cDNA obtained from porcine liver tissue as a template.The target gene was ligated into the linearized pET-32a(+) vector digested with EcoRⅤ and Hind Ⅲ,the recombinant plasmid was transformed into E.coli BL21(DE3) competent cells,the induction expression conditions were optimized,and detected by SDS-PAGE and Western blotting.pIFN-δ5 protein was abundantly expressed,purified using a nickel ion affinity chromatography column,and determined the protein purity.The interferon titer of pIFN-δ5 was detected by cytopathic inhibition method,the cytotoxicity of pIFN-δ5 was detected by CCK8 method,and the ability of against PEDV infection was further determined.【Result】 The recombinant expression plasmid pET-pIFNδ5 was successfully constructed.After exploring the induction conditions,it was found that when the D_{600 nm} value was 0.5 to 0.6,the IPTG concentration was 0.8 mmol/L,and the temperature was 37 ℃,the target protein pIFN-δ5 was mainly expressed in the supernatant of cell lysis.After massive expression and purification,pIFN-δ5 protein with purity of 95% could be obtained.The antiviral activity was determined by the VSV/MDCK titration system,and the specific activity was 5×10⁴ U/mg.CCK8 assays indicated that pIFN-δ5 had less effect on cell activity.Real-time quantitative PCR,Western blotting and indirect immunofluorescence results indicated that pIFN-δ5 exhibited significant resistance to PEDV infection.【Conclusion】 This study established a method for expression and purification of pIFN-δ5,and demonstrated that pIFN-δ5 had good activity against PEDV infection through a series of in vitro antiviral assays,which laid a foundation for the use of pIFN-δ5 as an antiviral agent and its clinical application.

Key words: porcine interferon-δ5(pIFN-δ5); expression; purification; Porcine epidemic diarrhea virus (PEDV); antiviral activity

CLC Number:

S852.65⁺9.6

SONG Shiying, GUO Weilu, XIA Xuefeng, ZHANG Xue, BI Zhenwei, ZHANG Xuehan, FAN Baochao, DONG Hailong, LI Bin. Expression and Purification of Porcine IFN-δ5 and Its Effect Analysis Against PEDV Infection[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(8): 3171-3179.

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Expression and Purification of Porcine IFN-δ5 and Its Effect Analysis Against PEDV Infection

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