Ribeiro Patrícia A F, Souza Marilen Q, Dias Daniel S, Álvares Alice C M, Nogueira Laís M, Machado Juliana M, Dos Santos José C, Godoi Renato R, Nobrega Yanna K M, Campos-da-Paz Mariana, de Freitas Sonia M, Felipe Maria S S, Torres Fernando A G, Galdino Alexsandro S
Laboratorio de Biotecnologia de Microrganismos, Universidade Federal de Sao Joao Del-Rei, Divinopolis, MG, 35501-296, Brazil.
Departamento de Biologia Celular, Universidade de Brasilia, Brasília, DF, 70910-900, Brazil.
Recent Pat Biotechnol. 2019;13(4):316-328. doi: 10.2174/1872208313666190716093911.
The Human Cytomegalovirus (HCMV) has infected more than 90% of the world population and its prevalence can be related to the individuals geographical and socialeconomic status. Serological tests based on ELISA are pivotal for HCMV diagnosis. Due to the lack of standardization in the production/purification of antigens from viral preparations, ELISA tests are based on several recombinant proteins or peptides. As an alternative, multiepitope proteins may be employed.
In this work, we developed a recombinant multiepitope protein (rMEHCMV) for HCMV diagnosis based on conserved and immunodominant epitopes derived from tegument (pp150, pp65 and pp28), glycoprotein gB (pp38) and DNA polymerase subunit (pp52) of HCMV.
The rMEHCMV gene was synthesized de novo and overexpressed in Escherichia coli cells. The recombinant protein was purified to homogeneity using a Ni-NTA column. Biophysical analysis of recombinant protein was performed by circular dichroism. A preliminary biological activity test was performed using 12 positive human sera samples by using an in-house IgG ELISA. The following patents database were consulted: Espacenet, Google Patents and the National Institute of Intellectual Property (INPI, Brazil).
The recombinant multiepitope protein was successfully expressed in E. coli. The structural data obtained by circular dichroism spectroscopy showed that rMEHCMV is structurally disordered. An in-house IgG ELISA test with rMEHCMV was successfully used to recognized IgG from human serum samples.
Together, our results show that rMEHCMV should be considered as a potential antigenic target for HCMV diagnosis.
人类巨细胞病毒（HCMV）已感染全球90%以上的人口，其流行率可能与个体的地理和社会经济状况有关。基于酶联免疫吸附测定（ELISA）的血清学检测对于HCMV诊断至关重要。由于从病毒制剂中生产/纯化抗原缺乏标准化，ELISA检测基于几种重组蛋白或肽。作为替代方案，可以使用多表位蛋白。
在本研究中，我们基于HCMV包膜（pp150、pp65和pp28）、糖蛋白gB（pp38）和DNA聚合酶亚基（pp52）的保守和免疫显性表位，开发了一种用于HCMV诊断的重组多表位蛋白（rMEHCMV）。
从头合成rMEHCMV基因并在大肠杆菌细胞中过表达。使用镍-亚氨基二乙酸（Ni-NTA）柱将重组蛋白纯化至同质。通过圆二色性对重组蛋白进行生物物理分析。使用12份阳性人血清样本通过内部IgG ELISA进行初步生物活性测试。查阅了以下专利数据库：欧洲专利局专利检索数据库（Espacenet）、谷歌专利和巴西国家知识产权局（INPI）。
重组多表位蛋白在大肠杆菌中成功表达。通过圆二色光谱获得的结构数据表明rMEHCMV在结构上是无序的。使用rMEHCMV的内部IgG ELISA测试成功用于识别来自人血清样本的IgG。
总之，我们的结果表明rMEHCMV应被视为HCMV诊断的潜在抗原靶点。
