Production of recombinant Zika virus NS1 protein and its potential use in a diagnostic test

ROLDAN, J. S.; CASTILLO DS; WOJCZUK F; CASSOLA A

Mendoza

Simposio; Second International Symposium on Traslational Medicine; 2019

Zika virus (ZIKV) recent outbreak has demonstrated an urgent need to develop rapid diagnostics tests to detect and distinguish acute viral infections to improve patient care. Non-structural protein 1 (NS1) is an important biomarker for early detection of the virus because of its release from infected cells and its accumulation at high levels in the patients serum during the acute phase. Intracellular NS1 is a dimer that has been described to play a role in the replication process, while extracellular secreted NS1 has a hexameric conformation and has been suggested to participate in immune evasion. The aim of this study was to express and purify a hexameric recombinant ZIKV NS1 (ZNS1) protein from the supernatant of HEK-293T ZNS1 stable human cells. For this purpose, HEK-293T cells were transduced with a lentiviral vector containing the ZNS1 gene and a 6x His tag. Flow cytometry assays revealed stable expression of the ZNS1-his protein in 43% of the cell population. With the aim to achieve a higher percentage of positive ZNS1-expressing cells, cells were cloned twice by limited dilution. Two positive clones were obtained, 1E4-C9 and 1E4-D11, with 90% and 88% of positive ZNS1-expressing cells respectively. Despite the similar percentages observed by flow cytometry, we continued working with 1E4-C9 cells for their higher intracellular and secreted ZNS1 protein levels assessed by Western Blot assays. Optimization of ZNS1 secretion was obtained by incubating 1E4-C9 cells for 9 days in serum-free media. Recombinant ZNS1-his protein was purified by nickel affinity chromatography with purity >95%. Finally, Western Blot assays under non-denaturing conditions confirmed the hexameric conformation of the purified ZNS1-protein. In conclusion, the obtained recombinant ZNS1 hexamer is a reliable biological tool that can be potentially used in the development of diagnostic tests to detect ZIKV infection.