Inactivation Analysis of
HcNPV Cysteine Protease Gene and Chitinase Gene
GONG Cheng-Liang, KOBAYASHI Jun1, JIN Wei2,
WU Xiang-Fu3
( Gene Engineering Lab, School of Sericulture, Suzhou University, Suzhou 215151,
China； 1Department of Chemistry for Materials
Faculty of Engineering, Mie University Mie 514, Japan； 2Department of Sericulture Science,
Zhejiang University Hangzhou 310029, China； 3Shanghai Institute of Biochemistry, the
Chinese Academy of Science, Shanghai 200031, China )
Abstract The
fragment of Hyphantria cunea nuclear polyhedrosis virus (HcNPV), which
contains cysteine protease gene(CP) and chitinase gene (ChiA),was
inserted into plasmid PCRII to construct transfer vector pHcCVdel.
Recombinant transfer vector pHcCVpolh was constructed by inserting the
fragment of HcNPV polyhedrin gene (polh) into the EcoRI site
of the pHcCVdel. By contransfection of the pHcCVpolh and
HcNPV-PTTH+ DNA, which contained Bombyx mori
prothoracicotropic hormone gene (PTTH) into SPIM cells, the region
from +76 downstream of ChiA gene initiation codon to +20 downstream of
CP gene initiation codon was substituted by the polh gene,
and the recombinant virus HcNPVpolh+CP–ChiA–PTTH+
was produced. The recombinant virus, in which CP gene and ChiA
gene were inactive, could express PTTH gene and form polyhedra. SPIM
cells infected with the recombinant virus revealed that CP and ChiA
gene were not essential for viral replication and had no significant effect on
polyhedron formation, but the infected cells survived 2 days more than those
infected with HcNPV and HcNPVPTTH+. Therefore, the
expression time of foreign gene may be prolonged when CP gene and ChiA
gene were inactivated.
Key words Hyphantria cunea nuclear
polyhedrosis virus； cysteine protease； chitinase； gene inactivation
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