Clonagem in silico das proteínas do vírus Chikungunya

Abstract

Chikungunya fever (CHIKF) is currently among the diseases that cause worldwide concern, threatening public health. The disease is an arbovirus caused by the Chikungunya virus (CHIKV), and transmitted by the mosquitoes Aedes aegypti and A. albopictus. Different of other arboviruses, a CHIKF is symptomatic with the name fever, myalgia, rashes and suckers of joint pain, a marked symptom of the disease, and most of the morbidity rate associated with the infection. In Brazil, the first reports of the disease occurred in 2014 and since then there have been little more than half a million suspected cases, resulting in approximately 300 deaths. To date there are no vaccines that guarantee immunity against the virus or rapid diagnostic kits, which accurately and securely identify the infection. Thus, the objective of the work was the construction and in silico analysis of vectors carrying the genes of the CHIKV proteins in order to make possible the production of recombinant proteins in the in vitro and in vivo stages that can be used in the development of antibodies against the virus and consequently in the production of a diagnostic kit and / or in the formulation of a vaccine. For this, the sequences of the nine CHIKV proteins were selected in the GenBank database. Subsequently, restriction sites for the HindIII, BamHI and EcoRI enzymes were added at both ends of each gene, in addition to a recombination sequence for the Gateway® cloning system. The vector chosen to simulate the cloning in the bacterial platform was pET-28a(+), while for the vegetal platform the plasmids pEAQ-HT-DEST2 and pEAQ-HT-DEST3 were selected. Using the SnapGene program, the clones were then simulated initially by treating the plasmid pET and the genes of the proteins with the restriction enzymes, promoting their binding soon after. Subsequently, the binding of the inserts to the pEAQ vectors was simulated by recombination using the same software. As a result, in all simulations, the cloned genes were successfully obtained, in their appropriate open reading frames, in the future, making possible the expression of the virus proteins in both prokaryotic and eukaryotic systems.