基于 ASFV 抗原表位相关重组蛋白建立并鉴定一种新型间接 ELISA 方法。

Establishment and characterization of a novel indirect ELISA method based on ASFV antigenic epitope-associated recombinant protein.

机构信息

College of Veterinary Medicine, Henan Agricultural University, Zhengzhou, China; International Joint Research Center of National Animal Immunology, College of Veterinary Medicine, Henan Agricultural University, Zhengzhou, China.

School of Agricultural Sciences, Zhengzhou University, Zhengzhou, Henan, China.

出版信息

Int J Biol Macromol. 2023 Dec 31;253(Pt 7):127311. doi: 10.1016/j.ijbiomac.2023.127311. Epub 2023 Oct 20.

DOI:10.1016/j.ijbiomac.2023.127311

PMID:37865977

Abstract

African Swine Fever (ASF) is an acute and highly lethal disease in pigs caused by African Swine Fever Virus (ASFV). Viral proteins have been commonly used as antigenic targets for the development of ASF diagnostic methods. However, the prokaryotic expression of viral proteins has deficiencies such as instability, insolubility, and high cost in eukaryotic situations. This study screened and verified ASFV-encoded p72, p54, and p30 protein antigenic epitopes. Subsequently, a novel antigenic epitope-associated recombinant protein was designed based on an ideal structural protein and expressed in Escherichia coli (E. coli). Western blot analysis indicated that the recombinant protein could specifically react with the monoclonal antibody (mAb) of p72 and polyclonal antibodies of p54 and p30, respectively. Next, an ASF indirect ELISA (iELISA) method was established based on the recombinant protein, which has no specific reaction with sera of other important pig viral diseases. Meanwhile, it shows a sensitivity to detecting dilutions of ASF-positive reference serum up to 1:6400. The clinical sample detection results showed a high coincidence rate of 98 % with a commercial competition ELISA kit. In conclusion, we established a novel specific, and sensitive ASF serologic detection method that opens new avenues for ASF serodiagnostic method development.

摘要

非洲猪瘟（ASF）是一种由非洲猪瘟病毒（ASFV）引起的猪的急性、高致死性疾病。病毒蛋白通常被用作开发 ASF 诊断方法的抗原性靶标。然而，病毒蛋白的原核表达在真核情况下存在不稳定性、不溶性和高成本等缺陷。本研究筛选和验证了 ASFV 编码的 p72、p54 和 p30 蛋白的抗原表位。随后，根据理想的结构蛋白设计并在大肠杆菌（E. coli）中表达了一种新型的抗原表位相关重组蛋白。Western blot 分析表明，该重组蛋白可分别与 p72 的单克隆抗体（mAb）和 p54 和 p30 的多克隆抗体特异性反应。接下来，基于该重组蛋白建立了一种 ASF 间接 ELISA（iELISA）方法，该方法与其他重要猪病毒性疾病的血清无特异性反应。同时，它对检测 ASF 阳性参考血清的稀释度可达 1:6400。临床样本检测结果表明，与商业竞争 ELISA 试剂盒的符合率高达 98%。总之，我们建立了一种新型的特异性和敏感性 ASF 血清学检测方法，为 ASF 血清学诊断方法的发展开辟了新的途径。