| Spodoptera litura multicapsid NucleopolyhedrovirusⅡ(SpltMNPVⅡ) is a novel baculovirus strain with high reproduction rate and virulence, whose genome sequence has been determined for the present. It contains 149 ORFs. Some of these genes'function is to be clarified, but there are manys whose function is not clear yet. ORF56 is one of the important unknown genes. In this study, the gene was characterized from the transcription, expression, localization and knockout on the basis of sequence analysis, cloning, expression and polyclonal antibody preparation. The object of this study is to enhance our understanding of baculovirus molecular biology. The main complishments are as follows:1. The sequence of SpltMNPVⅡO RF56 gene was analysised by the use of bioinformatics software. ORF56 is located at 54166～54840 bp of SpltMNPVⅡgenome, 675 bp in length, encoding a putative protein of 224 aa with a predicted size of 25.9 kD. Sequence analysis indicated that the late transcription motif of baculoviridae, GTAAG was located at the upstream of ATG. and the transcription terminal signal, AATAAA was found at the downstream of the terminal codon TAA. 13 putative phosphorylation sites, 3 N-linked glycosylation sites a bromodomain, and a TFS2N motif are found in the predicted ORF56 protein. Homology comparison showed that ORF56 is a conserved gene, whose homologues can be found in 47 baculovirus.2. The ORF56 fusion protein was expressed using the E.coli expression system and its polyclonal antibody was harvested. The primer was designed as the SpltMNPVⅡORF56 gene sequence. The coding region of ORF56 was amplified from SpltMNPVⅡgenome DNA by PCR, cloned into the expression vector pET-32a(+), and then expressed in the bacterium strain BL21. The fusion protein was retrieved from the SDS-PAGE gel and used to immunize the New Zealand white rabbits to raise the antibody against ORF56.3. The RT-PCR and Real-time PCR results showed that ORF56 gene infected cells from 12 h to 72 h, indicating that it may be a late gene. The expression phase analysis showed that that ORF56 was detected in the infected cells from 24 to 72 h, which further suggesting that ORF56 is a late gene. The detected ORF56 had a molecular weight of 26 kD, same as the predicted one, which suggesting that ORF56 have no obvious post-transcription modification. Western blot analysis showed ORF56 was detected both in budded viruses and occlusion-derived virus.4. The recombinant virus marked by egfp was constructed, ORF56 was knocked out. The fragment of pph-egfp was inserted into this vector between the 5'end and 3'end-flanking fragments of SpltMNPVⅡO RF56 gene tandem linked into pUC19. The Spli cells were cotransfected with p△ORF56-egfp and the wild SpltMNPVⅡgenome DNA. The recombinant virus SpltMNPVⅡ-△ORF56-egfp containing egfp was selected with the method of homologous recombination and plaque screening. The results showed that the deletion of ORF56 did not affect both the reproduction and the replication in the cell level, which showed that ORF56 was a non-essential gene for virus replication.These results can be as a basis for further researching the biological function of ORF56 in the SpltMNPVⅡ-infected S.litura larvae.
|
PDF Full Text Request