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Thus, HCV infection is a major global public health problem, and there is an urgent need to understand the virus better and to develop vaccines and more effective treatment.
Each mutant was transformed into DH5α competent cells (GIBCO/BRL) and selected on LB agar plates containing 100 μg/ml ampicillin (Sigma).
Several colonies were cultured in LB liquid containing ampicillin at 30°C for ≈18–20 h.
15) by using standard PCR procedures and/or synthesized oligonucleotides.
The mutated 3′ UTR was subcloned into a TA vector (Invitrogen), substituted for the entire 3′ UTR of H77C (15) contained in a p GEM vector (p H77-3′UTR), or substituted for the corresponding sequence of H77C.
The UTRs of HCV have highly conserved elements believed to be critical for RNA replication and packaging, as well as for polyprotein translation (5–8).