Clonagem e expressão da proteína do capsídeo do vírus Chikungunya para produção de antígeno recombinante: Produção de antígeno recombinante através de gene sintético do vírus Chikungunya

Abstract

The Chikungunya virus (CHIKV) is an RNA virus (family Togaviridae, genus Alphavirus) transmitted to humans through the bite of female mosquitoes Aedes aegypti and Aedes albopictus. The clinical onset in CHIKV infection is most often characterized by fever and joint pain, and so far there is no specific antiviral therapy or vaccine for the treatment of the infection. The diagnosis of CHIKV infection is based on clinical and laboratory findings, with the latter being performed by virus isolation, reverse transcription-polymerase chain reaction (RT-PCR), serology, and rapid tests: To produce a recombinant antigen through the cloning and expression of the capsid protein (C) of CHIKV in order to detect anti-CHIKV antibodies in human serum samples by immunoenzymatic assay. A synthetic gene (gBlock), specifically designed for this study, corresponding to the protein C (400 base pair) of Chikungunya virus, was amplified by polymerase chain reaction (PCR) and then cloned into pGEM-T Easy system-Escherichia coli. The transformant clones were sequenced, and recombinant products were digested using the restriction endonucleases EcoRI and BamHI, and then subcloned and expressed in the vector pET-23a+ Escherichia coli BL21 (DE3). The recombinant protein C expression and the molecular weight were determined by SDS-PAGE and Dot Blot and purified by affinity chromatography using nickel column. A immunoenzymatic assay was performed using the recombinant antigen to detect IgM and IgG antibodies in sera from patients with CHIKV infection confirmed by the National Reference Laboratory of the Ministry of Health, as well as sera from patients tested positive for Mayaro virus, Dengue virus and Cytomegalovirus infection. The derived recombinant protein showed size and antigenicity compatible with the native protein C from the CHIKV; a concentration of 0.342 ng/mL of recombinant protein C was obtained using the pET-23a+ E. coli BL21 (DE3); the affinity chromatography using nickel column was effective to obtain the soluble protein C, confirmed by the Bradford method; the immunoenzymatic assay using the recombinant antigen showed cross-reactivity to others Alphavirus pathogens. The results indicate that the expression system pET-23a+ E. coli BL21 (DE3) was effective to produce the recombinant protein C of CHIKV, however the antigen was not sensitive enough to detect only the CHIKV infection.