Produção das proteínas recombinantes Core, Ns3, Ns4b, Ns5a do vírus da Hepatite C e caracterização da resposta imune humoral

Abstract

Introduction: Liver infections caused by viruses are among the most serious ones, with worldwide prevalence. According to WHO data, the Hepatitis C Virus (HCV) affects approximately 170 million people around the world, representing approximately 3% of world population. Of this total, the majority of individuals (80%) did not develop sustained virologic response, progressing to chronic infection. Objective: Production, expression and purification of HCV proteins CORE, NS3, NS4b and NS5a in E. coli and analysis of its reactivity with serum of chronic hepatitis C patients. Methods: Recombinant genes of CORE (594pb), NS3 (897pb), NS4b (804pb), NS5a F1 (678pb) and NS5a F2 proteins were constructed (692pb) for optimized expression in E. coli BL21 (DE3) pLysS. Restriction enzymes sites were added to the sequences for subcloning in pRSET, which include a 6xHIS tagged in the recombinant protein amino acid sequence. Sera of patients with chronic hepatitis C obtained in previous study were used in western blot to evaluate its reactivity with recombinant proteins produced. Results and conclusion: The recombinant proteins were expressed in E. coli showed a higher concentration in the insoluble portion. Each protein was purified in native and denatured condition using nickel column. CORE and NS5a F2 proteins were reactive to antibodies present in the sera of patients, and did not reacted with negative patients sera. Any reactivity was observed against the N-terminal portion of NS5a. Further studies will try to analyze individual reactivity of a large number of patients against each recombinant protein.