Expressão e purificação de uma proteína multiepítopo recombinante desenhada a partir de proteínas do vírus da hepatite C
Hepatitis C is considered one of the largest problems of public health in theworld, inclusively in Brazil, whereas a preventive vaccine none is available; the numberof asymptomatic infections is elevated and for control of the disease, there exist onlyimported kits. Considering, principally, the increase of preoccupation with the detectionof precocious hepatitis C and that the diagnostic methods of this infection are of thehighest clinical importance, the presence study proposes the expression and thepurification of one codified protein through a synthetic gene (MEHCV ? MultiepitopeHCV). To achieve this objective, the new recombinant protein multiepitope wasdesigned on the basis of epitopes linear, immunodominant and phylogeneticallyconserved from the virus of hepatitis C (HCV), including the immune dominatingsequences of the genotypes that are more prevalent in Brazil (1a e 3a) and a His-tag inC-terminal to facilitate the purification of the recombinant protein express in bacteria.These epitopes (core, NS3, NS4A, NS4B e NS5) are considered among the mostimportant for the diagnosis of the illness utilized for many HCV detection tests. TheMEHCV gene (~720pb) possesses restrictive of sites (NdeI e XhoI) in yours extremitiesthat permits the clonage in phase in the expression of vector bacterial pET-21a. To thesynthesize of this gene was made the optimization to the codon usage of E. coli. Theprotein of interest (~29kDa) was detected by SDS-PAGE and Western blot. Thepurification of the protein express was realized by centrifugal in resin Ni-NTA byaffinitive chromatography. Inasmuch as the purification was not total, a newpurification was made of this protein by means of chromatography of affinity in columnwith resin Ni-NTA. However, did not having this purification owe a presence of cellularproteins. In such case, in the trial of obtain a total purification of this proteinmultiepitope, would be interesting the utilization of the one new protocol, with theextraction of the inclusion body, wherein all the dissolvable proteins cellular would beremove of the solution.
Advisor:IRMTRAUT ARACI HOFFMANN PFRIMER; Fernando Araripe Gonçalves Torres
School:Universidade Católica de Goiás
Source Type:Master's Thesis
Keywords:proteína multiepítopo hepatite ciencias da saude
Date of Publication:03/23/2007