The role of scavenger receptor BI in hepatitis - eTheses Repository ...

2.7 Cloning and synthesis of JFH-1 wt and G451R soluble E2.
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Many early studies into HCV attachment and entry were performed using
truncated soluble forms of the E2 glycoprotein (99, 100). These recombinant
proteins contain amino acids 384-661 of the HCV polyprotein, a region
encompassing all of E2 with the exception of the trans-membrane domain,
thus rendering the proteins soluble (99, 100). Our study required the direct
examination of JFH-1 wt and G451R glycoprotein–receptor interactions, to
achieve this it was necessary to engineer plasmids encoding sE2 for each
virus.
Our cloning strategy used an existing PCDNA3.1 plasmid that encodes a
strain H sE2 downstream of a tissue plasminogen activator (tpa) leader
sequence. When introduced into 293T cells, the tpa leader directs the
secretion of synthesised sE2 into the culture media and is therefore an
essential element of the construct. By insertion of a silent and unique
restriction site at the interface between the tpa leader and sE2 ORF we were
able to excise the strain H sE2 sequence and transfer clone JFH-1 sE2 PCR
products into the plasmid background. We also introduced a C-terminal
epitope tag to the JFH-1 sE2s via an extended reverse primer. This work was
carried out partly by Dr Peter Balfe.
1. A QuikChange II (Stratagene) site directed mutagenesis kit was used
to insert a synonymous mutation in the strain H sE2 plasmid at the 3’
end of the tpa leader sequence. This alteration created a unique SacII
restriction site directly upstream of the H sE2 coding sequence, which