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signal sequence is encoded — was deleted from the expression con-
struct. 6,16 It has also been reported that additional endogenous cleav-
age of 52 amino acids at the C-terminal is necessary for the assembly
of a VLP. 6,7 HEV-VLP appears as an empty particle of a slightly
smaller size than that of a mature HEV particle. 6,7 An HEV-VLP has
several advantages for studying virus formation or host recognition.
In our experience, large amounts can be easily obtained from standard
cultivation protocols compared with amounts of other VLPs
obtained. The amount of purified HEV-VLPs collected from culture
supernatant of 50 to 100
g/ml is more than 100 times greater than
that of other VLPs. It has recently been found that the VLPs elicit
strong immune responses when administrated orally into mice as same
to a natural infection route. 17 Moreover, it has been shown that oral
inoculation of cynomolgus monkeys with HEV-VLP prevents the
infection of native HEV by intravenous injection. 18 These findings
indicated that HEV-VLPs conserved original HEV construction to
enter the target cells. Conservation of the virus construct in VLPs is
very attractive for vaccines inducing the same type of immune
responses to virus infection.
µ
Chimeric HEV-VLP Carrying Foreign Epitope
pVL5480/7126, a baculovirus transfer vector that includes a portion of
the ORF2 from HEV (dORF2), was described previously. 6 To insert the
tag sequence within dORF2, oligonucleotides that encode the tag amino
acid sequence were synthesized as shown in Table 1, and described pre-
viously. 9 The restriction sites used for insertion sites 1 to 4 were Hind III,
Sac II, Bss HII, and Sac II sites at nucleotide positions 5679, 6245, 6664,
and 6773, respectively. For each site, oligonucleotide pairs of Htg5(0)
and Htg3(GA), Htg5(
+
1) and Htg3(0), Htg5(0) and Htg3(GG), and
1) and Htg3(0) were used, respectively. A C-terminal tag was
added at a position 52 amino acids upstream from the translational
terminal. This site was chosen because the last 52 amino acids at the
C-terminal of ORF2 are cleaved off during the formation of VLPs. The
nucleotide sequences around the inserted tag are schematically shown in
Fig. 1. The plasmid containing the chimeric dORF2 was co-transfected
Htg5(
+
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