Galdino Alexsandro S, Santos José C, Souza Marilen Q, Nóbrega Yanna K M, Xavier Mary-Ann E, Felipe Maria S S, Freitas Sonia M, Torres Fernando A G
Laboratório de Biotecnologia de Microrganismos, Universidade Federal de São João Del-Rei, 35501-296 Divinópolis, MG, Brazil.
Departamento de Biologia Celular, Universidade de Brasília, 70910-900 Brasília, DF, Brazil.
Hepat Res Treat. 2016;2016:6592143. doi: 10.1155/2016/6592143. Epub 2016 Jan 28.
Hepatitis C virus (HCV) has emerged as the major pathogen of liver diseases in recent years leading to worldwide blood-transmitted chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Accurate diagnosis for differentiation of hepatitis C from other viruses is thus of pivotal importance for proper treatment. In this work we developed a recombinant multiepitope protein (rMEHCV) for hepatitis C diagnostic purposes based on conserved and immunodominant epitopes from core, NS3, NS4A, NS4B, and NS5 regions of the virus polyprotein of genotypes 1a, 1b, and 3a, the most prevalent genotypes in South America (especially in Brazil). A synthetic gene was designed to encode eight epitopes in tandem separated by a flexible linker and bearing a his-tag at the C-terminal end. The recombinant protein was produced in Escherichia coli and purified in a single affinity chromatographic step with >95% purity. Purified rMEHCV was used to perform an ELISA which showed that the recombinant protein was recognized by IgG and IgM from human serum samples. The structural data obtained by circular dichroism (CD) spectroscopy showed that rMEHCV is a highly thermal stable protein at neutral and alkaline conditions. Together, these results show that rMEHCV should be considered an alternative antigen for hepatitis C diagnosis.
丙型肝炎病毒（HCV）近年来已成为肝脏疾病的主要病原体，导致全球范围内因血液传播引起的慢性肝炎、肝硬化和肝细胞癌。因此，准确诊断丙型肝炎以与其他病毒相区分对于恰当治疗至关重要。在这项研究中，我们基于1a、1b和3a基因型病毒多聚蛋白的核心区、NS3、NS4A、NS4B和NS5区的保守且免疫显性表位，开发了一种用于丙型肝炎诊断的重组多表位蛋白（rMEHCV），这几种基因型是南美洲（尤其是巴西）最常见的基因型。设计了一个合成基因，用于串联编码八个表位，表位之间由一个柔性接头隔开，且在C末端带有一个组氨酸标签。重组蛋白在大肠杆菌中产生，并通过一步亲和层析纯化，纯度>95%。使用纯化的rMEHCV进行酶联免疫吸附测定（ELISA），结果表明该重组蛋白能被人血清样本中的IgG和IgM识别。通过圆二色性（CD）光谱获得的结构数据表明，rMEHCV在中性和碱性条件下是一种热稳定性很高的蛋白。总之，这些结果表明rMEHCV应被视为丙型肝炎诊断的替代抗原。
