• Journal of Microbiology and Biotechnology
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• v.9 no.3
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• pp.318-322
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• 1999

A new baculovirus transfer vector (pPGP404) was constructed to increase the expression level of human thrombopoietin (hTPO) in insect cells. In pPGP404, hTPO was cloned next to the AcNPV polyhedrin-gp64 dual promoter and the leader sequence of hTPO was substituted with that of gp64. A recombinant baculovirus, AcPGP404, was constructed by using pPGP404 as a transfer vector. hTPO was expressed in AcPGP404-infected TN5 cells and it was observed that the expression levels of hTPO in TN5 cells increased three-fold ($6.0 {\mu}g/ml^{-1}$) compared to the level expressed under the control of the polyhedrin single promoter. These results indicate that the polyhedrin-gp64 dual promoter system would be useful for expression in large quantities of recombinant proteins in insect cells.

• 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.41 no.2
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• pp.102-107
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• 1999

To analysis a promoter strength of Atographa californica nucler polyhedrosis virus (AcNPV) IE1 gene, an immediate viral gene, ${\beta}$-glactosidase gene as a reporter gene was introduced under the control of the IE1 promoter. The restriction fragment containing IE1 promoter and ${\beta}$-galctosidase gene from pAcIE1-gal were inserter into pBacPAK9 to yield transfer vector pAcNPV-IE1-gal. The pAcNPV-IE1-gal was cotransfected with AcNPV genomic DNA BacPAK6 into Sf9 cells to produce recombinant baculovirus AcNPV-IE1-gal. In addition, recombinant bacvulovirus AcNPV-gal, which express ${\beta}$-galac-tosidase under the control of the polyhedrin promoter, was constrer, was constructed to compared with AcNPV-IE1-gal. The recombinant viruses were respectively infected into Sf9 cells and characterized by the virus titer and expression of ${\beta}$-galactoxidase in Sf9 cells. The promoter strength of IE1 and polyhedrin promoters was determined by the amount of ${\beta}$-galactosidase secreted into medium by viral infection. The titer of AcNPV-IE1-Gal determined by plaque assays in Sf9 cells was similar to that of AcNPV-gal. However, expression level of ${\beta}$-galactosidase by AcNPV-IE1-gal was significantly lower than that by AcNPV-gal. In conclusion, promoter strength of IE1 was approximately 25-fold lower than that of polyhedrin.

• 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.

• International Journal of Industrial Entomology and Biomaterials
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• v.15 no.2
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• pp.131-135
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• 2007

Open reading frame 4 of Bombyx mori nucleopolyhedrovirus (BmNPV), designated as Bm4, is a gene whose function is completely unknown. With the recently developed BmNPV bacmid and a modified pFastBac1 whose polyhedrin promoter was replaced with ie1 promoter, a recombinant bacmid expressing Bm4-EGFP fusion protein under the control of ie1 promoter in BmN cells was successfully constructed. The result not only showed that the polyhedrin promoter can be replaced efficiently with other promoters to direct the expression of foreign gene in BmN cells by using Bac-to-Bac/BmNPV baculovirus expression system but also laid the foundation for rescue experiment of Bm4 deletion mutant due to the ability of ie1 promoter to direct gene expression throughout the infection cycle.

• Journal of Microbiology and Biotechnology
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• v.11 no.4
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• pp.585-591
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• 2001

To compare the p10 promoter activity of Bombyx mori nucleopolyhedrovirus (BmNPV)K1 and K4, recombinant viruses Bm101-LacZ and Bm104-LacZ with a lacZ gene under the control of each p10 promoter were constructed. The $\beta$-galactosidase activity due to Bm101-LacZ was about 5.5- and 1.1-fold higher than that due to Bm104-LacZ and BmK1-LacZ, respectively. expressing ${\beta}$-galactosidase under the control of a polyhedrin promoter. The recombinant virus BmK1-104LacZ with the same genome structure as Bm101-LacZ, except for a p10 promoter region, produced a similar ${\beta}$-galactosidase activity to that due to Bm104-LacZ and 5.5-fold lower than that due to Bm101-LacZ. The virus yield, expression level of polyhedrin, and polyhedra productivity for each recombinant virus was almost similar. These results suggested that the difference in the expression level of ${\beta}$-galactosidase resulted from a difference in the p10 promoter regions, and that an expression vector using the p10 promoter of BmNPV-K1 could be usefully exploited in the mass production of foreign proteins with silkworm larvae by means of oral ingestion.

• 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.

• 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.

• 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.

• 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.