Ali Amjad, Nisar Muhammad, Idrees Muhammad, Rafique Shazia, Iqbal Muhammad
Department of Biotechnology University of Malakand Chakdara, Dir (lower) Khyber Pakhtunkhwa, Pakistan.
Department of Botany University of Malakand Chakdara Dir (lower), Khyber Pakhtunkhwa, Pakistan.
Int J Infect Dis. 2015 May;34:84-9. doi: 10.1016/j.ijid.2015.03.010. Epub 2015 Mar 18.
Early diagnosis of HCV infection is based on detection of antibodies against HCV proteins using recombinant viral antigens. The present study was designed to select, clone and express the antigenic regions of Core and E2 genes from local HCV-3a genotype and to utilize the antigenic recombinant proteins (Core & E2) to develop highly sensitive, specific and economical diagnostic assays for detection of HCV infection.
The antigenic sites were determined within Core and E2 genes and were then cloned in pET-28a expression vector. The right orientation of the desired inserted fragments of Core and E2 were confirmed via sequencing prior to expression and were then transformed in BL21 (DE3) pLysS strains of E. coli and induced with 0.5mM Isopropyl-b-D-thiogalactopyranoside (IPTG) for the production of antigenic recombinant proteins. The produced truncated antigens were then purified by Nickel affinity chromatography and were confirmed by western blotting, immunoblotting and enzyme-linked immunosorbent assay (ELISA).
The expressed Core and E2 recombinant antigens were used to develop immunoblotting assay for the detection of anti-HCV antibodies in sera. With immunoblotting, a total of 93-HCV infected sera and 35-HCV negative individuals were tested for the presence of anti-HCV antibodies to the Core and E2 antigens. Recombinant antigen showed 100% reactivity against HCV infected sera, with no cross reactivity against HCV-negative sera. The immunoblot assay mixture of recombinant antigens (Core+E2) showed a strong reaction intensity in the test area (TA) as compared to the individual truncated Core and E2 recombinant antigens. In the in-house ELISA assay, mixed Core and E2 recombinant antigens showed 100% reactivity against a standardized panel of 150-HCV-positive sera and non reactivity against a standardized panel of 150 HCV-negative sera while also being non reactive to sera positive for other viral infections. The antigenic recombinant antigens also were tested for the 30 sera of known genotypes. The antigens did not detect antibodies to genotype-3a, but detected antibodies to all genotypes and did not discriminate them genotype wise. A panel of 175 of HCV-suspected serum samples was subjected to comparative analysis with our in-house ELISA assay and with commercial HCV screening assays. After subjecting the results to the formulas for determining the quality parameters, immunoblot assay had 100% sensitivity and specificity, while the ELISA assay had 100% sensitivity and 98.8% specificity as compared to commercially available assays.
This study indicates that a mixture of Core and E2 antigens are potentially valuable antigens and there is the possibility of developing serological assays for monitoring HCV infection.
丙型肝炎病毒（HCV）感染的早期诊断基于使用重组病毒抗原检测抗HCV蛋白抗体。本研究旨在从本地HCV - 3a基因型中选择、克隆和表达核心（Core）基因和E2基因的抗原区域，并利用抗原重组蛋白（Core和E2）开发用于检测HCV感染的高灵敏度、特异性和经济的诊断方法。
确定Core和E2基因内的抗原位点，然后克隆到pET - 28a表达载体中。在表达之前，通过测序确认Core和E2所需插入片段的正确方向，然后转化到大肠杆菌的BL21（DE3）pLysS菌株中，并用0.5mM异丙基 - β - D - 硫代半乳糖苷（IPTG）诱导产生抗原重组蛋白。然后通过镍亲和层析纯化产生的截短抗原，并通过蛋白质免疫印迹法、免疫印迹法和酶联免疫吸附测定（ELISA）进行确认。
表达的Core和E2重组抗原用于开发免疫印迹法检测血清中的抗HCV抗体。通过免疫印迹法，共检测了93份HCV感染血清和35份HCV阴性个体血清中针对Core和E2抗原的抗HCV抗体。重组抗原对HCV感染血清显示100%的反应性，对HCV阴性血清无交叉反应性。与单独的截短Core和E2重组抗原相比，重组抗原（Core + E2）的免疫印迹检测混合物在检测区域（TA）显示出强烈的反应强度。在内部ELISA检测中，混合的Core和E2重组抗原对150份标准化HCV阳性血清显示100%的反应性，对150份标准化HCV阴性血清无反应性，同时对其他病毒感染阳性血清也无反应性。还对30份已知基因型的血清进行了抗原重组抗原检测。这些抗原未检测到针对3a基因型的抗体，但检测到了所有基因型的抗体，且无法按基因型区分。对175份疑似HCV血清样本进行了比较分析，将我们的内部ELISA检测与商业HCV筛查检测进行对比。将结果代入确定质量参数的公式后，免疫印迹法的灵敏度和特异性均为100%，而ELISA检测与市售检测相比，灵敏度为100%，特异性为98.8%。
本研究表明Core和E2抗原混合物是潜在有价值的抗原，并且有可能开发用于监测HCV感染的血清学检测方法。
