猪流行性腹泻病毒S1蛋白的真核表达及中和性单克隆抗体制备

doi:10.16431/j.cnki.1671-7236.2025.10.030

Abstract

Abstract: 【Objective】 The aim of this study was to prepare the S1 recombinant protein of Porcine epidemic diarrhea virus (PEDV) and its neutralizing monoclonal antibodies (mAbs),and provide critical materials for the development of PEDV diagnostic reagents and novel vaccines.【Method】 A recombinant plasmid that expressed S1 protein was constructed and transfected into HEK293F cells.Nickel affinity chromatography and gel filtration chromatography were used to purify the expressed protein. PEDV CH/Hubei/2016 strain was cultured in Vero cells for propagation.The viral suspension was subsequently concentrated and purified via gel filtration chromatography.The purified PEDV was characterized by SDS-PAGE and Western blotting.BALB/c mice were then immunized with the purified antigen to generate hybridoma cells.Positive hybridoma cells were screened by indirect ELISA and immunoperoxidase monolayel assay (IPMA).The neutralizing monoclonal antibodies against PEDV S1 protein were screened and identified using virus neutralization assay.【Result】 SDS-PAGE and Western blotting analysis demonstrated that S1 recombinant protein was efficiently expressed in HEK293F cell culture supernatant.Purified PEDV with TCID₅₀ of 1 ×10^-6.5/0.1 mL was obtained.Five hybridoma cell lines stably secreting anti-S1 mAbs were obtained and the IPMA and ELISA titers of the ascitics ranged from 6.4×10^-4 to 1.28×10^-5 and 6.4×10^-5 to 1.024×10^-7,respectively.Virus neutralization assay revealed that mAbs 2E1E10 and 4A8C8 exhibited high neutralizing activity,of which the inhibitory concentration 50% (IC₅₀) values were calculated as 1.212 and 1.203 μg/mL for strain DR13,and 0.948 and 1.712 μg/mL for strain CHHB,respectively.【Conclusion】 PEDV S1 recombinant protein with high antigenic activity was obtained,and neutralizing monoclonal antibodies against S1 protein were produced,which providing a good research foundation for the development of PEDV diagnostic reagents and vaccines.

Key words: Porcine epidemic diarrhea virus (PEDV); S1 protein; monoclonal antibodies; neutralizing activity; broad-spectrum reactivity

CLC Number:

S852.65⁺9.6

LI Chunzhen, LI Qingmei, ZOU Wanying, MENG Zekun, SUN Yaning, YANG Suzhen, GUO Junqing, ZHANG Gaiping. Eukaryotic Expression of S1 Protein of Porcine Epidemic Diarrhea Virus and Preparation of Its Neutralizing Monoclonal Antibody[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(10): 4854-4863.

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Eukaryotic Expression of S1 Protein of Porcine Epidemic Diarrhea Virus and Preparation of Its Neutralizing Monoclonal Antibody

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