Clonagem e expressão heteróloga de proteínas estruturais (C, prM, E) de Zika Virus (ZIKV)
Abstract
Viruses are organisms which depend on the host cellular machinery to replicate,
and are relevant to human, livestock and plant health. The modern threat of emerging
pandemics is pressing and, in this scenario, Zika Virus (ZIKV) is an important recent
example, due to recent epidemics and confirmation of its involvement in congenital
development and neurological manifestations. The ability to produce recombinant virus
like particles (VLPs) and recombinant proteins can lead to the understanding of its
structure and the development of new therapeutic approaches, as well as the
understanding of interaction mechanisms between the virion and host cell. This study
aims to achieve heterologous production of ZIKV VLPs as well as ZIKV Capsid protein
for future structural analysis and to make them available to the research community. A
recombinant construct containing the three structural proteins of ZIKV (C, prM and E)
cloned into pPICZα vector was integrated in Pichia pastoris genome for AOX1 promoter
induced expression. C protein coding region was cloned into Escherichia coli expression
vector for separate expression and purification by affinity chromatography using a 6xHis
tag construct. Integration of the coding region for the proteins C, prM and E in the P.
pastoris genome was confirmed via PCR and sequencing. Results showed the possibility
of obtaining soluble secreted recombinant product. Production of isolated C protein was
achieved in E. coli and its purification was performed through affinity chromatography.