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猪流行性腹泻病毒非结构蛋白8的表达与免疫原性

Expression and immunogenicity of non-structural protein 8 of porcine epidemic diarrhea virus.

作者信息

Chen Hong, Wan Jiawu, Wei Meihua, Liu Ping, Kong Lingbao, Xin Xiu

机构信息

Institute of Pathogenic Microbiology, College of Biological Science and Engineering, Jiangxi Agricultural University, Nanchang, China.

Nanchang Key Laboratory of Animal Virus and Genetic Engineering, College of Biological Science and Engineering, Jiangxi Agricultural University, Nanchang, China.

出版信息

Vet Res Forum. 2024;15(2):65-73. doi: 10.30466/vrf.2023.2009322.3977. Epub 2024 Feb 15.

DOI:10.30466/vrf.2023.2009322.3977
PMID:38465319
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC10924293/
Abstract

The non-structural protein (nsp) 8 of the porcine epidemic diarrhea virus (PEDV) is highly stable across different PEDV strains and plays an important role in PEDV virulence. In current study, nsp8 prokaryotic expression vectors were constructed based on parental vectors pMAL-c2x-maltose binding protein (MBP) and pET-28a (+). Subsequently, the optimization of expression conditions in , including induced temperature, time and isopropyl β-D-thiogalactopyranoside concentration were performed to obtain a stable expression of MBP-nsp8 and nsp8. The nsp8 fused with MBP increased the water solubility of the expressed products. Target proteins were further purified from culture and their immunogenicities were evaluated by mice. The antibody titers of serum from nsp8 immunized mice were up to 1:7,750,000 when measured by indirect enzyme-linked immunosorbent assay; meanwhile, the mice immunized with MBP-nsp8 gave an antibody titer reaching 1:1,000,000. In all, the expression and purification system of PEDV nsp8 and MBP-nsp8 were successfully established in this work and a strong immune response was elicited in mice by both purified nsp8 and MBP-nsp8, providing a basis for the study of the structure and function of PEDV nsp8.

摘要

猪流行性腹泻病毒（PEDV）的非结构蛋白（nsp）8在不同的PEDV毒株中高度稳定，并且在PEDV毒力方面发挥重要作用。在本研究中，基于亲本载体pMAL-c2x-麦芽糖结合蛋白（MBP）和pET-28a（+）构建了nsp8原核表达载体。随后，对包括诱导温度、时间和异丙基β-D-硫代半乳糖苷浓度在内的表达条件进行优化，以获得MBP-nsp8和nsp8的稳定表达。与MBP融合的nsp8提高了表达产物的水溶性。从培养物中进一步纯化目标蛋白，并通过小鼠评估其免疫原性。通过间接酶联免疫吸附测定法检测时，nsp8免疫小鼠血清的抗体效价高达1:7,750,000；同时，用MBP-nsp8免疫的小鼠抗体效价达到1:1,000,000。总之，本研究成功建立了PEDV nsp8和MBP-nsp8的表达和纯化系统，纯化的nsp8和MBP-nsp8均在小鼠中引发了强烈的免疫反应，为PEDV nsp8的结构和功能研究提供了基础。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1a5a/10924293/885fd4870b16/vrf-15-65-g008.jpg
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https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1a5a/10924293/97f9a644e1aa/vrf-15-65-g005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1a5a/10924293/604a90ddfe8b/vrf-15-65-g006.jpg
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https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1a5a/10924293/885fd4870b16/vrf-15-65-g008.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1a5a/10924293/c03f2163ce02/vrf-15-65-g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1a5a/10924293/f483a1e5dde9/vrf-15-65-g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1a5a/10924293/2f044635cc58/vrf-15-65-g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1a5a/10924293/7283d625e1b2/vrf-15-65-g004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1a5a/10924293/97f9a644e1aa/vrf-15-65-g005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1a5a/10924293/604a90ddfe8b/vrf-15-65-g006.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1a5a/10924293/8456f4d88a48/vrf-15-65-g007.jpg
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本文引用的文献

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Genome-wide analysis of protein-protein interactions and involvement of viral proteins in SARS-CoV-2 replication.SARS-CoV-2复制过程中蛋白质-蛋白质相互作用及病毒蛋白参与情况的全基因组分析。
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Crucial mutation in the exoribonuclease domain of nsp14 of PEDV leads to high genetic instability during viral replication.
猪流行性腹泻病毒nsp14外切核糖核酸酶结构域中的关键突变导致病毒复制过程中高度的基因不稳定。
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