Expressão e purificação de uma proteína multiepítopo recombinante desenhada a partir de proteínas do vírus da hepatite C

Abstract

Hepatitis C is considered one of the largest problems of public health in the world, inclusively in Brazil, whereas a preventive vaccine none is available; the number of asymptomatic infections is elevated and for control of the disease, there exist only imported kits. Considering, principally, the increase of preoccupation with the detection of precocious hepatitis C and that the diagnostic methods of this infection are of the highest clinical importance, the presence study proposes the expression and the purification of one codified protein through a synthetic gene (MEHCV Multiepitope HCV). To achieve this objective, the new recombinant protein multiepitope was designed on the basis of epitopes linear, immunodominant and phylogenetically conserved from the virus of hepatitis C (HCV), including the immune dominating sequences of the genotypes that are more prevalent in Brazil (1a e 3a) and a His-tag in C-terminal to facilitate the purification of the recombinant protein express in bacteria. These epitopes (core, NS3, NS4A, NS4B e NS5) are considered among the most important for the diagnosis of the illness utilized for many HCV detection tests. The MEHCV gene (~720pb) possesses restrictive of sites (NdeI e XhoI) in yours extremities that permits the clonage in phase in the expression of vector bacterial pET-21a. To the synthesize of this gene was made the optimization to the codon usage of E. coli. The protein of interest (~29kDa) was detected by SDS-PAGE and Western blot. The purification of the protein express was realized by centrifugal in resin Ni-NTA by affinitive chromatography. Inasmuch as the purification was not total, a new purification was made of this protein by means of chromatography of affinity in column with resin Ni-NTA. However, did not having this purification owe a presence of cellular proteins. In such case, in the trial of obtain a total purification of this protein multiepitope, would be interesting the utilization of the one new protocol, with the extraction of the inclusion body, wherein all the dissolvable proteins cellular would be remove of the solution.