| Summary: | 碩士 === 國立中興大學 === 昆蟲學系 === 83 === The Bombyx mori nuclear polyhedrosis virus (BmNPV) genome and
the Autographa californica nuclear polyhedrosis virus (AcMNPV)
transfer vectors containing the complete carp gonadotropin
(GTH) gene were simultaneously cotransfected into BmN cells.
The insertion of carp GTH gene into th BmNPV genome was
achieved. Monolayers of BmN cells were infected with
recombinant virus. Five days post-infection, the carp GTH a or
b-subunit was synthesized and entirely secreted from BmN cells
into media. Subsequently, determination of the virus titer
suffered from failure in forming single plaques. The
cotransfection was repeated and no more recombinant virus could
be obtained. In order to improve the secretion of hepatitis B
surface antigen (HBsAg) that was synthesized in BmN cells
infected with recombinant BmNPV, the carp GTH a-signal peptide
was constructed inframe upstream of the HBsAg pre-S2 gene. The
chimeric gene was inserted into the BmNPV transfer vector,
pBmp10MZ, downstream of the p10 promoter. From 4 cotransfection
experiments, 158 occlusion-positive and b-galactosidase-
negative recombinant virus were obtained via plaque assay (9
times) or end-point dilution (9 times) experiments. Polymerase
chain reactions (PCR) were conducted to examine the recombinant
virus for the presence of the chimeric HBsAg. The results
indicated that despite of their lack of b-galactosidase
activity, no HBsAg gene was detectable in thses recombinant
viruses. These results suggested that selection for the
replacement of the lacZ gene carried on the parent virus could
be technically difficult. The DNA fragment upstream of the
BmNPV p10 gene was cloned and sequenced, so that it could be
compared with the nucleotide sequence upstream of the AcMNPV
p10 gene. An open reading frame (ORF) encoding a polypeptide of
240 amino acids was detected. It shares 95.8% amino acid
sequence similarity with that of AcMNPV p26 protien.Analysis of
the BmNPV p26 amino acid sequence did not reveal any structural
motifs.
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