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

摘要/Abstract

摘要： 【目的】试验旨在建立大量表达及纯化猪δ5干扰素(pIFN-δ5)的方法,并对其抗猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)感染的作用进行分析。【方法】根据GenBank中pIFN-δ5序列(登录号:NM_001164854.1)设计引物,以猪肝脏组织cDNA为模板进行PCR扩增;将目的基因连接入经EcoRⅤ和Hind Ⅲ双酶切的线性化pET-32a (+)载体,转化大肠杆菌BL21(DE3)感受态细胞,优化诱导表达条件,并进行SDS-PAGE和Western blotting检测;pIFN-δ5蛋白大量表达后使用镍离子亲和层析柱纯化,并测定蛋白纯度;采用细胞病变抑制法检测pIFN-δ5的干扰素效价,采用CCK8方法检测pIFN-δ5的细胞毒性,进一步测定其抗PEDV的感染能力。【结果】试验成功构建了重组表达质粒pET-pIFNδ5,经诱导条件摸索发现,在D_{600 nm}值为0.5~0.6、IPTG浓度为0.8 mmol/L、37 ℃诱导条件下,目的蛋白pIFN-δ5主要表达于菌体裂解上清中;经大量表达并纯化后可获得纯度＞95%的pIFN-δ5蛋白。使用VSV/MDCK细胞滴定系统检测pIFN-δ5的比活性为5×10⁴U/mg;CCK8检测表明pIFN-δ5的细胞毒性较小。实时荧光定量PCR、Western blotting和间接免疫荧光检测结果表明,pIFN-δ5具有显著抗PEDV感染能力。【结论】本试验建立了表达和纯化pIFN-δ5的方法,通过一系列的体外抗病毒试验证实pIFN-δ5具有良好的抗PEDV感染的活性,为将pIFN-δ5作为抗病毒药物及临床应用奠定了基础。

关键词: 猪干扰素-δ5(pIFN-δ5); 表达; 纯化; 猪流行性腹泻病毒(PEDV); 抗病毒活性

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

中图分类号:

S852.65⁺9.6

宋诗莹, 郭玮璐, 夏学峰, 张雪, 毕振威, 张雪寒, 范宝超, 董海龙, 李彬. 猪IFN-δ5的表达纯化及其抗PEDV感染的作用分析[J]. 中国畜牧兽医, 2022, 49(8): 3171-3179.

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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