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非洲猪瘟病毒（ASFV）p72蛋白截短形式的克隆与表达及其在高效间接酶联免疫吸附测定（ELISA）系统中的应用

Cloning and Expression of a Truncated Form of the p72 Protein of the African Swine Fever Virus (ASFV) for Application in an Efficient Indirect ELISA System.

作者信息

Cuevas-Romero Julieta Sandra, Zavala-Ocampo Perla Lucero, Pina-Pedrero Sonia, Ganges Llilianne, Muñoz-Aguilera Adriana, García-Cambrón José Bryan, Rodriguez Fernando, Ambagala Aruna, Cerriteño-Sánchez José Luis

机构信息

Centro Nacional de Investigación Disciplinaria en Salud Animal e Inocuidad, Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias, Cuajimalpa de Morelos, Mexico City 05110, Mexico.

Posgrado en Ciencias de la Producción y de la Salud Animal, Facultad de Medicina Veterinaria, Universidad Nacional Autónoma de México. Av. Universidad 3004, Copilco Universidad, Mexico City 04510, Mexico.

出版信息

Pathogens. 2025 May 29;14(6):542. doi: 10.3390/pathogens14060542.

DOI:10.3390/pathogens14060542
PMID:40559550
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC12196084/
Abstract

African swine fever (ASF) is a disease that affects both domestic and wild swine. It was recently reported in the Dominican Republic and Haiti (2021), representing a substantial risk to America. The goal of this study was to produce a truncated form of the ASF-p72 recombinant protein based on the ASF strain genotype II (Georgia 2017) as well as to develop and validate a sensitive and specific ASF indirect-ELISA (iELISA) for early detection of ASF. The truncated ASF-p72 recombinant protein was successfully expressed in BL21/DE3 cells using the pET-SUMO plasmid. Bioinformatics analysis showed 100% homology among the new isolates of ASFV from genotype II. The ASF-p72-truncated protein was used to develop an iELISA, which had a high sensitivity (88%) and strong specificity (97%); the concordance index kappa was K = 0.872, indicating nearly perfect agreement compared to the WOAH confirmatory immunoperoxidase test. The validation results utilizing the reference sera panel from the OIE-ASF Reference Laboratory show the excellent detection capabilities of ASF antibodies up to a 1:1000 serum dilution. The inter-assay coefficient of variation (CV 10.4%) and intra-assay CV (2.8%) data show that the assay is precise and reproducible. This biotechnology advancement can be used to conduct future epidemiological research for ASF surveillance in ASF-free American countries.

摘要

非洲猪瘟（ASF）是一种影响家猪和野猪的疾病。最近在多米尼加共和国和海地（2021年）有相关报道，这对美洲构成了重大风险。本研究的目的是基于ASF II型毒株（格鲁吉亚2017）生产截短形式的ASF-p72重组蛋白，并开发和验证一种灵敏且特异的ASF间接ELISA（iELISA）用于ASF的早期检测。截短的ASF-p72重组蛋白利用pET-SUMO质粒在BL21/DE3细胞中成功表达。生物信息学分析显示来自II型ASFV新分离株之间具有100%的同源性。ASF-p72截短蛋白用于开发一种iELISA,其具有高灵敏度（88%）和强特异性（97%）；一致性指数kappa为K = 0.872，与世界动物卫生组织（WOAH）确证免疫过氧化物酶试验相比表明几乎完全一致。利用来自OIE-ASF参考实验室参考血清组的验证结果显示该方法在血清稀释至1:1000时对ASF抗体具有出色检测能力。批间变异系数（CV 10.4%）和批内CV（2.8%）数据表明该检测方法精确且可重复。这一生物技术进展可用于未来在美洲无ASF国家开展ASF监测的流行病学研究。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3dd7/12196084/5101e391fd37/pathogens-14-00542-g010.jpg
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https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3dd7/12196084/8d3a43991d4d/pathogens-14-00542-g005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3dd7/12196084/e14274e28c2f/pathogens-14-00542-g006.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3dd7/12196084/03d59c233b06/pathogens-14-00542-g007.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3dd7/12196084/64bd4dd32c0b/pathogens-14-00542-g008.jpg
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Overview of Modern Commercial Kits for Laboratory Diagnosis of African Swine Fever and Swine Influenza A Viruses.用于非洲猪瘟和甲型猪流感病毒实验室诊断的现代商业试剂盒概述。
Viruses. 2024 Mar 26;16(4):505. doi: 10.3390/v16040505.
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Nanobodies against African swine fever virus p72 and CD2v proteins as reagents for developing two cELISAs to detect viral antibodies.
抗非洲猪瘟病毒 p72 和 CD2v 蛋白的纳米抗体作为开发两种 cELISA 检测病毒抗体的试剂。
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A Re-Evaluation of African Swine Fever Genotypes Based on p72 Sequences Reveals the Existence of Only Six Distinct p72 Groups.基于 p72 序列对非洲猪瘟基因型的重新评估显示，仅存在六个不同的 p72 群。
Viruses. 2023 Nov 11;15(11):2246. doi: 10.3390/v15112246.
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Establishment of a Dual-Antigen Indirect ELISA Based on p30 and pB602L to Detect Antibodies against African Swine Fever Virus.建立基于 p30 和 pB602L 的双抗原间接 ELISA 检测非洲猪瘟病毒抗体。
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