猪弓形虫感染血清学诊断中重组多表位肽抗原 MAG 的鉴定与评价。
Characterization and evaluation of a recombinant multiepitope peptide antigen MAG in the serological diagnosis of Toxoplasma gondii infection in pigs.
机构信息
Key Laboratory Preventive Veterinary of Hubei Province, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, 430070, Hubei, People's Republic of China.
State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, 430070, Hubei, People's Republic of China.
出版信息
Parasit Vectors. 2021 Aug 17;14(1):408. doi: 10.1186/s13071-021-04917-w.
BACKGROUND
Toxoplasmosis caused by Toxoplasma gondii is a serious disease threatening human and animal health. People can be infected with T. gondii by ingesting raw pork contaminated with cysts or oocysts. Serological test is a sensitive and specific method usually used for large-scale diagnosis of T. gondii infection in humans and animals (such as pigs). Commercial pig Toxoplasma antibody ELISA diagnostic kits are expensive, which limits their use; moreover, the wide antigen composition used in these diagnostic kits is still unclear and difficult to standardize. The multiepitope peptide antigen is a novel diagnostic marker, and it has potential to be developed into more accurate and inexpensive diagnostic kits.
METHODS
The synthetic multiepitope antigen (MAG) cDNA encoding a protein with epitopes from five T. gondii-dominant antigens (SAG1, GRA1, ROP2, GRA4, and MIC3) was designed, synthesized, and expressed in Escherichia coli BL21 (DE3) strain. The recombinant protein was detected through western blot with pig anti-T. gondii-positive and -negative serum, and then IgG enzyme-linked immunosorbent assay (ELISA) named MAG-ELISA was designed. The MAG-ELISA was evaluated in terms of specificity, sensitivity, and stability. The MAG-ELISA was also compared with a commercial PrioCHECK Toxoplasma Ab porcine ELISA (PrioCHECK ELISA). Finally, the trend of pig anti-T. gondii IgG levels after artificial infection with RH tachyzoites was evaluated using MAG-ELISA and two other ELISA methods (rMIC3-ELISA and PrioCHECK ELISA).
RESULTS
MAG antigen could be specifically recognized by pig anti-T. gondii-positive but not -negative serum. MAG-ELISA showed high diagnostic performance in terms of specificity (88.6%) and sensitivity (79.1%). MAG-ELISA could be used for detecting anti-T. gondii IgG in the early stage of T. gondii infection in pigs (at least 7 days after artificial infection).
CONCLUSIONS
Our results suggest that MAG antigen can be applied to specifically recognize anti-T. gondii IgG in pig, and MAG-ELISA has the potential for large-scale screening tests of T. gondii infection in pig farms and intensive industries.
背景
弓形虫引起的弓形体病是一种严重威胁人类和动物健康的疾病。人们可以通过摄入含有包囊或卵囊的生猪肉而感染弓形虫。血清学检测是一种用于人类和动物（如猪）大规模诊断弓形虫感染的敏感和特异方法。商业猪弓形虫抗体 ELISA 诊断试剂盒价格昂贵，限制了其应用；此外，这些诊断试剂盒中广泛使用的抗原组成仍不清楚，难以标准化。多表位肽抗原是一种新型诊断标志物，具有开发更准确、更廉价的诊断试剂盒的潜力。
方法
设计、合成并在大肠杆菌 BL21（DE3）菌株中表达编码来自弓形虫五个优势抗原（SAG1、GRA1、ROP2、GRA4 和 MIC3）的表位的合成多表位抗原（MAG）cDNA。用猪抗弓形虫阳性和阴性血清通过 Western blot 检测重组蛋白，然后设计 IgG 酶联免疫吸附试验（ELISA），命名为 MAG-ELISA。评价 MAG-ELISA 的特异性、敏感性和稳定性。还将 MAG-ELISA 与商业 PrioCHECK 弓形虫 Ab 猪 ELISA（PrioCHECK ELISA）进行比较。最后，用 MAG-ELISA 和另外两种 ELISA 方法（rMIC3-ELISA 和 PrioCHECK ELISA）评估人工感染 RH 速殖子后猪抗弓形虫 IgG 水平的趋势。
结果
MAG 抗原可被猪抗弓形虫阳性血清特异性识别，但不能被阴性血清识别。MAG-ELISA 在特异性（88.6%）和敏感性（79.1%）方面具有较高的诊断性能。MAG-ELISA 可用于检测猪弓形虫感染早期（人工感染后至少 7 天）的抗弓形虫 IgG。
结论
我们的结果表明，MAG 抗原可特异性识别猪抗弓形虫 IgG，MAG-ELISA 具有在猪场和集约化产业中大规模筛查弓形虫感染的潜力。
Zotero 插件