• The Journal of Korean Society of Virology
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• v.29 no.3
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• pp.145-153
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• 1999

We have now constructed a novel recombinant baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) producing polyhedra with Bacillus thuringiensis (Bt) CryIAc crystal protein. The recombinant polyhedra produced by the recombinant baculovirus, Btrus, in insect cells was characterized. The recombinant baculovirus has two independent transcription units in opposite orientations with two promoters, p10 or polyhedrin gene promoter each initiating transcription of either native polyhedrin or fusion protein with polyhedrin and Bt Cry1Ac crystal protein. Surprisingly, this recombinant baculovirus stably produced recombinant polyhedra which were nearly similar to those of wild-type AcNPV. The immunogold staining experiment showed that the recombinant polyhedra were assembled with polyhedrin and Bt Cry1Ac crystal protein, and contained virus particles. Insecticidal toxicity of recombinant polyhedra of Btrus to the fall webworm, Hyphantria cunea, was strikingly improved in comparison with the wild-type AcNPV.

• Horticultural Science & Technology
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• v.32 no.4
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• pp.544-549
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• 2014

Lily symptomless virus (LSV), Lily mottle virus (LMoV), and Cucumber mosaic virus (CMV) are the most prevalent viruses infecting lilies in Korea. Leaf and bulb samples showing characteristic symptoms of virus infection were collected in 2012, and 80 field samples were analyzed by reverse transcription polymerase chain reaction (RT-PCR). The infection frequencies were 79% for LMoV, 5% for LSV, and 3% for CMV. The LMoV coat protein gene was amplified and cloned into the pET21d(+) expression vector to develop serological diagnostic tools to detect LMoV. The resulting carboxy-terminal His-tagged coat proteins were expressed in Escherichia coli strain BL21 (DE3) by induction with IPTG. The recombinant proteins were purified using Ni-NTA agarose beads and used as an antigen to produce polyclonal antibodies in laying hens. The resulting egg yolk immunoglobulin (IgY) specifically recognized LMoV from infected plant tissues in immunoblotting assays and had comparable sensitivity to that of a mammalian antibody. In addition, method of immunocapture RT-PCR using this IgY was developed for sensitive, efficient, and rapid detection of LMoV. Based on these results, large-scale bulb tests and detection of LMoV in epidemiological studies can be performed routinely using this IgY. This is the first report of production of a polyclonal IgY against a plant virus and its use for diagnosis.

• IMMUNE NETWORK
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• v.9 no.6
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• pp.265-273
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• 2009

Foot-and-mouth disease virus (FMDV) is a small single-stranded RNA virus which belongs to the family Picornaviridae, genus Apthovirus. It is a principal cause of FMD which is highly contagious in livestock. In a wild type virus infection, infected animals usually elicit antibodies against structural and non-structural protein of FMDV. A structural protein, VP1, is involved in neutralization of virus particle, and has both B and T cell epitopes. A RNA-dependent RNA polymerase, 3D, is highly conserved among other serotypes and strongly immunogenic, therefore, we selected VP1 and 3D as vaccine targets. VP1 and 3D genes were codon-optimized to enhance protein expression level and cloned into mammalian expression vector. To produce recombinant protein, VP1 and 3D genes were also cloned into pET vector. The VP1 and 3D DNA or proteins were co-immunized into 5 weeks old BALB/C mice. Antigen-specific serum antibody (Ab) responses were detected by Ab ELISA. Cellular immune response against VP1 and 3D was confirmed by ELISpot assay. The results showed that all DNA- and protein-immunized groups induced cellular immune responses, suggesting that both DNA and recombinant protein vaccine administration efficiently induced Ag-specific humoral and cellular immune responses.

• Journal of Microbiology and Biotechnology
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• v.22 no.11
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• pp.1580-1587
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• 2012

Japanese encephalitis virus (JEV) envelope (E) protein holds great promise for use in the development of a recombinant vaccine. Purified recombinant E (rE) protein may be useful for numerous clinical applications; however, there are limitations in using the Escherichia coli expression system for producing high-quality rE protein. Therefore, in this study, the yeast expression system was used to generate the rE protein. For protein production using the yeast system, the full-length JEV E gene was cloned into Pichia pastoris. SDS-PAGE and immunoblotting analysis demonstrated that the rE protein had a molecular mass of 58 kDa and was glycosylated. The predicted size of the mature unmodified E protein is 53 kDa, suggesting that post-translational modifications resulted in the higher molecular mass. The rE protein was purified to greater than 95% purity using combined ammonium sulfate precipitation and a SP-Sepharose Fast Flow column. This purified rE protein was evaluated for immunogenicity and protective efficacy in mice. The survival rates of mice immunized with the rE protein were significantly increased over that of Hyphantria cunea nuclear polyhedrosis virus E protein (HcE). Our results indicate that the rE protein expressed in the P. pastoris expression system holds great promise for use in the development of a subunit vaccine against JEV.

• The Journal of Korean Society of Virology
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• v.27 no.2
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• pp.239-255
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• 1997

Expression of the cDNA of the VP1 gene on the genome RNA B segment of infectious pancreatic necrosis virus (IPNV) DRT strain in E. coli and a recombinant baculovirus were carried out. The VP1 gene in the pMal-pol clone (Lee et al. 1995) was cleaved with XbaI and transferred into baculovirus transfer vector, pBacPAK9 and it was named pBacVP1 clone. The VP1 gene in the pBacVP1 clone was double-digested with SacI and PstI and then inserted just behind T5 phage promoter and the $6{\times}His$ region of the pQE-3D expression vector, and it was called pQEVPl. Again, the $6{\times}$His-tagged VP1 DNA fragment in the pQEVP1 was cleaved with EcoRI and transferred into the VP1 site of the pBacVP1, resulting pBacHis-VP1 recombinant. The pBacHis-VP1 DNA was cotransfected with LacZ-Hyphantria cunea nuclear polyhedrosis virus (LacZ-HcNPV) DNA digested with Bsu361 onto S. frugiperda cells to make a recombinant virus. One VP1-gene inserted recombinant virus was selected by plaque assay. The recombinant virus was named VP1-HcNPV-1. The $6{\times}$His-tagged VP1 protein produced by the pQEVP1 was purified with Ni-NTA resin chromatography and analyzed by SDS-PAGE and Western blot analysis. The molecular weight of the VP1 protein was 94 kDa. The recombinant virus, VP1-HcNPV-1 did not form polyhedral inclusion bodies and expressed VP1 protein with 95 kDa in the infected S. frugiperda cells, which was detected by Western blot. The titer of the VP1-HcNPV-1 in the first infected cells was $2.0{\times}10^5\;pfu/ml$ at 7 days postinfection.

• Korean Journal of Veterinary Research
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• v.32 no.3
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• pp.389-398
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• 1992

Molecular cloning was carried out on the Danish strain of bovine viral diarrhea virus(BVDV) to construct strategy for the diagnostic tools and effective vaccine of BVD afterwards. A recombinant DNA clone(No. 29) was established successfully from cDNA for viral RNA tailed with adenine homopolymer at 3'-end. $^{32}P$-labeled DNA probes of 300~1,800bp fragments, originating from the clone 29, directed specific DNA-RNA hybridization results with BVDV RNA. Recombinant DNA of the clone 29 was about 5,200bp representing 41.6% of the full length of Danish strain's RNA, and restriction sites were recognized for EcoR I, Sst I, Hin d III and Pst I restriction enzymes in the DNA fragment.

• International Journal of Industrial Entomology and Biomaterials
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• v.2 no.2
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• pp.155-160
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• 2001

We have generated a novel baculovirus genome which can be maintained in Escherichia coli that facilitates the rapid and efficient generation of recombinant baculovirus expression vectors. To make $Occ^{+}$ recombinant expression vectors, polyhedrin gene under the control of p10 promoter was inserted to bAcGOZA and this genome was designated bApGOZA. As in bAcGOZA, bApGOZA lacks a portion of the essential ORF1629 gene, but includes a mini-F replicon and selectable kanamycin-resistance marker, This occasion-producing activity of bApGOZA can be used very conveniently for its oral infectivity to insect larvae in mass production of foreign protein and insecticides.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1993.04a
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• pp.80-80
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• 1993

(목적) 본 연구에서 사용된 바이러스는 HIV-1 nef유전자가 일부 삭제되고 대신 Chloramphenicol acetyltransferase(CAT)가 pSVCAT recombinant 바이러스다. 이러한 recombinant 바이러스를 사용하는 이유는 첫째, CAT activity가 매우 민감하므로 바이러스의 복제억제 정도를 정확하게 측정 할 수 있고 둘째, simian immunodeficiency virus(SIV)의 경우 nef 유전가 in vivo에서는 바이러스의 복제에 필수적이므로 HIV가 SIV와 유사한 것으로 미루어 본 연구에서 사용되는 recombinant SVCAT 바이러스가 안전한 것으로 고려되기 때문이다. (방법) 특히 화합물이 HIV-1의 복제에 얼마나 영향이 있는가는 1) 어느정 도의 virus inoculm을 넣었는지 2) 사용하는 cell line 3) 사용한 cell line의 infection kinetics 4) 실험의 지속기간 5) 테스트하는 assay의 sensitivity에 의존한다. 따라서 $10^{5}$ cell의 H9과 sup T1을 24 well plate에 넣고 sup T1 cell line의 경우 3일 후 항 화합물에 의한 syncytia 형성 및 CAT activity의 억제정도를 현재 AIDS drug으로 쓰이고 있는 Zidovudine을 control로 비교 관찰하였다. H9 cell line의 경우 3일 간격으로 media의 3/4을 fresh media로 바꾸어 주고 9일 후 CAT assay를 하였다. 이러한 assay에서 activity를 보이는 화합물을 reverse transcriptase와 P24 ELISA assay를 재확인하였다.다.

• IMMUNE NETWORK
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• v.12 no.1
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• pp.8-17
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• 2012

Background: Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract diseases in infancy and early childhood. Despite its importance as a pathogen, there is no licensed vaccine against RSV yet. The attachment glycoprotein (G) of RSV is a potentially important target for protective antiviral immune responses. Recombinant baculovirus has been recently emerged as a new vaccine vector, since it has intrinsic immunostimulatory properties and good bio-safety profile. Methods: We have constructed a recombinant baculovirus-based RSV vaccine, Bac-RSV/G, displaying G glycoprotein, and evaluated immunogenicity and protective efficacy by intranasal immunization of BALB/c mice with Bac-RSV/G. Results: Bac-RSV/G efficiently provides protective immunity against RSV challenge. Strong serum IgG and mucosal IgA responses were induced by intranasal immunization with Bac-RSV/G. In addition to humoral immunity, G-specific Th17- as well as Th1-type T-cell responses were detected in the lungs of Bac-RSV/G-immune mice upon RSV challenge. Neither lung eosinophilia nor vaccine-induced weight loss was observed upon Bac-RSV/G immunization and subsequent RSV infection. Conclusion: Our data demonstrate that intranasal administration of baculovirus-based Bac-RSV/G vaccine is efficient for the induction of protection against RSV and represents a promising prophylactic vaccination regimen.

• Journal of Sericultural and Entomological Science
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• v.39 no.2
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• pp.180-185
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• 1997

To develope the novel baculovirus transfer vector, the p10 gene was cloned from the Bombyx mori nuclear polygedrosis virus (BmNPV) vB2 strain isolated from the B. mori larvae of sericultural farms. The novel transfer vector was constructed by using the p10 gene of BmNPV vB2 strain was 210 bp. The TAAG sequence at the -71 bp of upstream from translation initiator ATG and two polyadenylation signal site at the downstream from terminator TAA were also detected in the p10 gene. The 5' and 3' flanking region of the p10 gene amplified by PCR was cloned into pBluescriptII SK(+) and then transfer vector pBm10 was construceted. The 7.9 kb pBm10 was analysed by restriction enzymes and the map was confirmed. In order to determine the expression of foreign gene of pBm10, $\beta$-galactosidase gene was inserted in the SmaI site of foreign gene cloning site of pBm10. The pBm10 containing $\beta$-galactosidase gene was cotranfected wth genomic DNA of BmNPV vB2 into BmN-4 cells. The recombinant baculovirus expressing $\beta$-galactosidase was also produced polygedra in the infected cells. The results indicated that pBm10 is functional, suggesting that in the baculovirus expression vector system, the recombinant virus produced by pBm10 was effective by oral infection for the producing recombinant proteins in in vivo expression.