• Journal of fish pathology
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• v.8 no.1
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• pp.37-46
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• 1995

The amino acid analysis of polyhedrin protein and nucleotide sequence of polyhedrin gene in H. cunea nuclear polyhedrosis virus (HcNPV) genome have been studied. Polyhedrin had three polypeptide bands in SDS - polyactylamide gel electrophoresis. The major polypeptide had a molecular weight of 25 kd. The polyhedrin was composed of 17 different amino acids. HcNPV DNA was digested with EcoRI restriction enzyme and hybridized with ($\alpha^{32}P$) -labelled AcNPV polyhedrin gene cDNA. The polyhedrin gene was located on the fragment of EcoRI-H. The EcoRI - H fragment containing polyhedrin gene was cloned into the EcoRI site of pUC8 vector which was confirmed with southern blotting, and the recombinant plasmid containg polyhedrin gene was designated as hPE-H. The promoter region of polyhedrin genomic DNA was sequenced. The sequences identified as the TATA box was found at the 5' flanking region of the polyhedrin genomic DNA approximately -79 bp upstream from the transcriptional start site. But CAAT-like box was not shown near the TATA-like box in the polyhedrin gene. Four tandem repeats with the sequence 5' -CTAATAT-3' and 5'-TAAATAA-3' were found between -141 and -108 or -83 upstream and -52 bp downstream from the translation start site. About -141 bp region upstream from the translational start site was highly AT (78%) rich. The coding region for the polyhedrin starts and ends with ATG and TAA, respectively.

• Journal of Sericultural and Entomological Science
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• v.34 no.2
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• pp.20-25
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• 1992

The location of the polyhedrin gene of Bmbyx mori nuclear polyhedrosis virus(BmNPV) was determined by using a cloned polyhedrin gene from the Autographa californica nuclear polyhedrosis virus(AcNPV) as a hybridization probe. The 7.4 Kb PstⅠ fragment DNA of Bm-NPV was cloned to plasmid pUC19 vector. A fragment containing this gene was mapped and sequenced in its entire polyhedrin reading frame. Nucleotide sequences comparison of the polyhedrin of the BmNPV to that of previously reported by Ⅰatrou(1985) revealed that the sequence varied in 10 base, Comparison of the amino acid sequence of the two structured gene revealed that coding sequence varied 74 valine to isoleucine, 76 aspargine to serine and 155 methionine to valine.

• International Journal of Industrial Entomology and Biomaterials
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• v.3 no.1
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• pp.75-81
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• 2001

We have constructed a novel recombinant Bombyx mori nuclear polyhedrosis virus (BmNPV) producing the green fluorescent polyhedra. For the production of the fluorescent polyhedra, partial polyhedrin gene containing KRKK as nuclear localization site from the BmNPV polyhedrin gene and the green fluorescent protein (gfp) gene were introduced under the control of p10 promoter of BmNPV. The recombinant BmNPV was stably produced fluorescent polyhedra in the infected Bm5 cells and the morphology of the fluorescent polyhedra was similar to that of wild-type BmNPV. The fluorescent polyhedra had 32 kDa native polyhedrin and 41 kDa fusion protein. From these data, we have further developed a novel BmNPV p10-based transfer vector producing recombinant polyhedra with foreign gene Product. The novel BmNPV P10-based transfer vector is composed of partial polyhedrin gene, factor Xa, and multiple cloning sites.

• Korean journal of applied entomology
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• v.36 no.4
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• pp.341-350
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• 1997

We have now constructed a novel recombinant baculovirus producing fusion protein with Autographa californica nuclear polyhedrosis virus (AcNPV) polyhedrin and Bacillus thuringiensis(Bt) cryIA(c) crystal protein. The fusion protein expressed by the recombinant baculovirus in insect cells was characterized. The N-terminal of cryIA(c) gene of Bt subsp. kurstaki HD-73 was introduced under the control of polyhedrin gene promoter of AcNPV, by fusion in the front of intact polyhedrin gene or by insertion into the HindIII site in polyhedrin gene. The recombinant baculoviruses were named as BtrusI or BtrusII, respectively. Although single transcript from the fusion protein gene was apparently observed. BtrusI was produced the two proteins, 92 kDa fusion protein and only polyhedrin. In addition, fusion protein produced by BtrusI did not form polyhedra. Interestingly, however, the cells infected with BtrusII did not show a 33 kDa polyhedrin band as a cells infected with BtrusI. Cells infected with BtrusII were only produced fusion protein, but the polyhedra formed by fusion protein was not observed. To determine the insecticidal toxicity of fusion protein, therefore, Sf9 cells infected with BtrusI were inoculated to Bombyx mori larvae. Sf9 cells infected with BtrusI that expressed the fusion protein caused larval mortality although the insecticidal toxicity was low. In conclusion, our results clearly demonstrated that the fusion protein with polyhedrin and Bt cryIA(c) crystal protein have a insecticida toxicity.

• Journal of Sericultural and Entomological Science
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• v.38 no.2
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• pp.144-149
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• 1996

To develope the baculovirus expression vector system (BEVS) using Spodoptera exigua nuclear polyhedrosis virus (SeNPV), we characterized the polyhedrin of SeNPV. The SeNPV polyhedra was irregular and composed of the major protein molecular weight of 30 kDa determined by electronmicroscopy and SDS-AGE analysis, respectively. The nucleotid suquences of 876 bases including the coding region of polyhedrin gene was determined and it was revealed that the polyhedrin gene is located within Xho I 3.0Kb and Nco I 6.0 Kb by Southern blot analysis, respectively. Also, the Xho I 3.0 Kb and the Nco I 6.0 Kb fragments were cloned and restriction enzyme map of these clones were determined.

• International Journal of Industrial Entomology and Biomaterials
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• v.1 no.1
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• pp.19-23
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• 2000

A novel recombinant baculovirus Autographa californica nuclear polyhedrosis virus (ACNPV) producing the green fluorescent polyhedra was constructed and characterized. The recombinant virus was stably produced fluorescent polyhedra in the infected cells and the morphology of the polyhedra was nearly similar to that of wild-type AcNPV. For the production of the fluorescent polyhedral the green fluorescent protein (GFP) gene was introduced under the control of polyhedrin gene promoter of AcNPV by translational fusion in the front and back of intact polyhedrin gene. The recombinant baculovirus was named as CXEP, As expected, the 93 kDa fusion protein was expressed in the CXEP-infected cells. Interestingly, however, the cells infected with CXEP also showed a 33 kDa protein band as cells infected with wild-type AcNPV. The results of Southern blot analysis and plaque assay suggested that two types of baculoviruses expressing the GFP fusion protein or only native polyhedrin were formed through homologous recombination between two polyhedrin genes in the same orientation. Thus, this system can be applied for the production of recombinant polyhedra with foreign gene product of diverse interest.

• International Journal of Industrial Entomology and Biomaterials
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• v.22 no.2
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• pp.75-82
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• 2011

To elucidate the novelty of Hyphantria cunea nucleopolyhedrovirus (HcNPV) isolated in Korea, polyhedrin and inhibitor of apoptosis (iap) gene structures were determined and analyzed. The analysis of HcNPV polyhedrin showed a little difference with 97.6% at the nucleotide level but no difference at the amino acid level when compared with that of previously reported H. cunea NPV (HycuNPV). On the other hand, iap genes showed variable differences with 89.0-99.6% nucleotide and 90.0-99.6% amino acid sequence identities. Especially, the 5' and 3' non-coding flanking sequences of iap1 gene had lower degree of identity with those of HycuNPV. Although the phylogenetic analyses using polyhedrin and iap genes showed that HcNPV is closely related with HycuNPV, we could provide that HcNPV is a novel isolate having novel gene structures.

• Journal of Microbiology and Biotechnology
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• v.15 no.4
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• pp.710-715
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• 2005

A novel recombinant baculovirus, Bactrus, was constructed by the insertion of the Bacillus thuringiensis cry1Ac gene between two polyhedrin genes of Autographa californica nucleopolyhedrovirus (AcNPV) under the control of the polyhedrin gene promoter. Polyhedra produced by Bactrus in insect cells were incorporated with 130 kDa of polyhedrin-Cry1Ac-polyhedrin fusion protein, and 30 kDa of intact polyhedrin, resulting from a homologous recombination between two polyhedrin genes, was also expressed. The insecticidal activity of Bactrus against Spodoptera exigua larvae was similar to that of AcNPV, but it showed significantly higher toxicity towards Plutella xylostella larvae in comparison with that of AcNPV. The expression level of fusion protein and the insecticidal activity of recombinant polyhedra produced by the Bactrus against P. xylostella larvae were decreased after serial passages. In conclusion, the Bactrus had improved insecticidal activity and returned to wild-type AcNPV after several passages.

• Journal of Sericultural and Entomological Science
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• v.37 no.1
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• pp.46-51
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• 1995

In order to develope baculovirus expression vector system, we constructed new transfer vector of nuclear polyhedrosis virus of the silkworm, Bombyx mori. The promoter region containing only adenine of translation start codon of polyhedrin gene was cloned by polymerase chain reaction technique. And the 5' and 3' leader regions of polyhedrin gene was sequentially cloned. The polyhedrin coding gene was deleted from the ＋2 to the ＋597 position. As the result, we constructed new transfer vector which has EcoRI, SacI and KpnI sites for the cloning sites of foreign gene. New transfer vector was named as pBmKSKl. Escherichia coli $\beta$-galactosidase gene as foreign gene was inserted into pBmKSKl, under the control of the polyhedrin promoter and expressed in B. mori cells. The result showed that the new transfer vector pBmKSK1 is functional.

• Korean journal of applied entomology
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• v.38 no.2
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• pp.139-144
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• 1999

We have now constructed a novel recombinant baculovirus producing fusion protein with Autogrqha c.uliforrzica nuclear polyhedrosis virus (AcNPV) polyhedrin and green fluorescent protein (GFP). The fusion protein expressed by the recombinant baculovirus in insect cells was characterized. The GFP gene was introduced under the control of polyhedrin gene promoter of AcNPV, by fusion in the front or back of intact polyhedrin gene. The recombinant baculoviruses were named as Ac-GFPPOL or Ac-POLGFP. respectively. As expected, the 56 kDa fusion protein was expressed in the recombinant virus-infected cells. Interestingly. however, the fluorescence of GFP in the cells infected with Ac- POLGFP was only detected within the nuclei. and that was observed as polyhedra-like granular particles. In the microscopy of cells infected with Ac-GFPPOL, furthermore, GFP was detected in both cytoplasm and nuclei although most of GFP were present within the nuclei. However, fusion protein produced by recombinant virus did not form polyhedra although the fusion protein was fused with polyhedrin and GFP. It is suggested that difference of GFP location in the infected cells appear to be involved in the region of polyhedrin in the fusion protein, and the polyhedrin in the fusion protein might be responsible for the polyhedra-like granular particles present within nuclei.