Bashiri Sareh, Nemati Mansoor Fahimeh, Valadkhani Zarrintaj
Department of Biotechnology, Faculty of Advanced Sciences and Technology, Tehran Medical Sciences, Islamic Azad University, Tehran, Iran.
Department of Parasitology, Pasteur Institute of Iran, Tehran, Iran.
Iran J Basic Med Sci. 2019 Feb;22(2):134-139. doi: 10.22038/ijbms.2018.29024.7021.
Hydatidosis is a zoonotic infection and endemic in Iran. Due to the serological cross-reactivity (of sera) with other parasitic infection, diagnosis of hydatid cyst is considered to be problematic. In this regard, application of recombinant antigens improves serological diagnosis for human hydatidosis. Here, we present an ELISA test based on B8/2 recombinant antigen of with particular regard to its capability to diagnose human hydatidosis.
The synthesized B8/2 (EgB8/2) gene was sub-cloned into pET28b (+) plasmid. Nde1 and Hind3 restriction enzymes were used to confirm the recombinant plasmid extraction. Cloning was verified by colony PCR, digestion enzymes, and sequence determination methods. To express rtEgB8/2, strains of BL21 (DE3) pLysS and Rosetta (DE3) were induced with isopropyl β-D-1-thiogalactopyranoside (IPTG). A Ni-NTA column was used for purification, and the expressed protein was analyzed by SDS-PAGE as well as western blotting. ELISA test was used to identify the antigenicity of produced protein.
The presence of EgB8/2 gene fragment in the recombinant plasmid was confirmed. SDS-PAGE showed that the BL21 (DE3) pLysS strain had the highest level of expression and a protein band of 11 kDa was observed in induced bacteria. Western blotting approved the purity of rtEgB8/2 protein, and ELISA test measured sensitivity and specificity as 95% and 97.5%, respectively.
metacestode contains a high amount of antigen B protein. These results confirm the reproducibility of high-quality rtEgB8/2 recombinant antigen as a reliable candidate in serological test.
包虫病是一种人畜共患感染病，在伊朗呈地方性流行。由于血清与其他寄生虫感染存在血清学交叉反应，包虫囊肿的诊断被认为存在问题。在这方面，重组抗原的应用改善了人类包虫病的血清学诊断。在此，我们提出一种基于B8/2重组抗原的酶联免疫吸附测定（ELISA）试验，特别关注其诊断人类包虫病的能力。
将合成的B8/2（EgB8/2）基因亚克隆到pET28b（+）质粒中。使用Nde1和Hind3限制性内切酶确认重组质粒提取。通过菌落聚合酶链反应（PCR）、消化酶和测序方法验证克隆。为表达重组EgB8/2（rtEgB8/2），用异丙基-β-D-1-硫代半乳糖苷（IPTG）诱导BL21（DE3）pLysS和Rosetta（DE3）菌株。使用镍-亚氨基二乙酸（Ni-NTA）柱进行纯化，并通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）以及蛋白质免疫印迹法分析表达的蛋白质。采用ELISA试验鉴定所产生蛋白质的抗原性。
证实重组质粒中存在EgB8/2基因片段。SDS-PAGE显示BL21（DE3）pLysS菌株表达水平最高，在诱导细菌中观察到一条11千道尔顿（kDa）的蛋白条带。蛋白质免疫印迹法证实了rtEgB8/2蛋白的纯度，ELISA试验测得敏感性和特异性分别为95%和97.5%。
包虫囊尾蚴含有大量抗原B蛋白。这些结果证实了高质量rtEgB8/2重组抗原在血清学检测中作为可靠候选物的可重复性。
