Department of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran.
Infectious Disease and Tropical Medicine Research Center, Research Institute of Cellular and Molecular Sciences in Infectious Diseases, Zahedan University of Medical Sciences, Zahedan, Iran.
Eur J Med Res. 2021 Nov 25;26(1):134. doi: 10.1186/s40001-021-00607-6.
Circumsporozoite protein (CSP) has a central immune domain that includes short regions of repeating amino acid sequences. This immunodynamic region is an epitope of B cells that can elicit an immune response in human and laboratory animals. The aim of the present study was to express the recombinant PvCSP-VK210 antigen and evaluate it for assaying antibodies obtained during human P. vivax infection by Western blotting and indirect ELISA (enzyme-linked immunosorbent assay).
Genomic DNA of P. vivax was isolated from a blood sample of an Iranian person with vivax malaria, and by PCR, the fragment of the PvCSP-VK210 gene was amplified. The gene fragment was cut after gel purification by BamHI and HindIII enzymes and then cloned into pET28a expression vector. Finally, the recombinant pET28a was transformed into the E. coli BL21 (DE3) as the expression host. In order to produce His-tagged protein, the expression host was cultured in LB medium. The protein was purified by Ni-NTA columns and immobilized metal affinity chromatography, and after confirmation by Western blotting technique, was used as the antigen in the indirect ELISA test.
The recombinant protein was expressed and purified as a 32-kDa protein. The sensitivity and specificity of the indirect ELISA test with the recombinant PvCSP-VK210 antigen were 61.42% and 97.14%, respectively, based on OD = 0.313. Between the results of the microscopic test and the indirect ELISA test with the recombinant PvCSP-VK210 antigen there was a Kappa coefficient of 0.586. The positive and negative predictive value and validity of the ELISA test with the recombinant PvCSP-VK210 antigen were 95.55%, 71.57%, 79.28%, respectively.
The sensitivity of the indirect ELISA method with the recombinant PvCSP-VK210 antigen was 61.42%, which is the first report from Iran.
环子孢子蛋白（CSP）具有一个中央免疫结构域，其中包括重复氨基酸序列的短区域。这个免疫动力区是 B 细胞的表位，可以在人类和实验动物中引发免疫反应。本研究的目的是表达重组 PvCSP-VK210 抗原，并通过 Western blot 和间接 ELISA（酶联免疫吸附试验）评估其检测人类间日疟原虫感染获得的抗体。
从一名伊朗间日疟患者的血液样本中分离出间日疟原虫基因组 DNA，通过 PCR 扩增 PvCSP-VK210 基因片段。用 BamHI 和 HindIII 酶切凝胶纯化后的基因片段，然后克隆到 pET28a 表达载体中。最后，将重组 pET28a 转化到 E. coli BL21（DE3）作为表达宿主。为了生产 His 标记的蛋白，表达宿主在 LB 培养基中培养。用 Ni-NTA 柱和固定化金属亲和层析法纯化蛋白，用 Western blot 技术确认后，将其作为间接 ELISA 试验的抗原。
重组蛋白表达并纯化为 32kDa 的蛋白。用重组 PvCSP-VK210 抗原进行间接 ELISA 试验的灵敏度和特异性分别为 61.42%和 97.14%，OD 值为 0.313。间接 ELISA 试验与重组 PvCSP-VK210 抗原的显微镜检测结果之间的 Kappa 系数为 0.586。用重组 PvCSP-VK210 抗原进行 ELISA 试验的阳性预测值、阴性预测值和有效性分别为 95.55%、71.57%、79.28%。
用重组 PvCSP-VK210 抗原进行间接 ELISA 方法的灵敏度为 61.42%，这是伊朗的首次报道。
