• Title/Summary/Keyword: Baculovirus vector

검색결과 87건 처리시간 0.03초

Investigation of post-translational modification of the secreted protein expressed in insect cell lines using baculovirus expression vector system(BEVS)

  • Yun, Eun-Young;Goo, Tae-Won;Kim, Sung-Wan;Park, Kwang-Ho;Hwang, Jae-Sam;Kang, Seok-Woo;Kwon, O-Yu
    • Proceedings of the Korean Society of Sericultural Science Conference
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    • 한국잠사학회 2003년도 제46회 춘계 학술연구 발표회
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    • pp.82-83
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    • 2003
  • In previous experiment, we reported when the heterologous protein is expressed by using baculovirus expression vector system (BEVS), although the amount of intracellular protein is abundant, the amount of extracellular Protein is poor. As the link in the chain of the research, we investigated the secretory pathway, important in case of the secretory protein, of the protein expressed in insect cells using BEVS. (omitted)

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Rabbit Hemorrhagic Disease Virus Variant Recombinant VP60 Protein Induces Protective Immunogenicity

  • Yang, Dong-Kun;Kim, Ha-Hyun;Nah, Jin-Ju;Song, Jae-Young
    • Journal of Microbiology and Biotechnology
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    • 제25권11호
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    • pp.1960-1965
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    • 2015
  • Rabbit hemorrhagic disease virus (RHDV) is highly contagious and often causes fatal disease that affects both wild and domestic rabbits of the species Oryctolagus cuniculus. A highly pathogenic RHDV variant (RHDVa) has been circulation in the Korean rabbit population since 2007 and has a devastating effect on the rabbit industry in Korea. A highly pathogenic RHDVa was isolated from naturally infected rabbits, and the gene encoding the VP60 protein was cloned into a baculovirus transfer vector and expressed in insect cells. The hemagglutination titer of the Sf-9 cell lysate infected with recombinant VP60 baculovirus was 131,072 units/50 μl and of the supernatant 4,096 units/50 μl. Guinea pigs immunized twice intramuscularly with a trial inactivated RHDVa vaccine containing recombinant VP60 contained 2,152 hemagglutination inhibition (HI) geometric mean titers. The 8-week-old white rabbits inoculated with one vaccine dose were challenged with a lethal RHDVa 21 days later and showed 100% survival rates. The recombinant VP60 protein expressed in a baculovirus system induced high HI titers in guinea pigs and rendered complete protection, which led to the development of a novel inactivated RHDVa vaccine.

Construction and Transfection of Recombinant Baculovirus Vectors (재조합 베큘로바이러스 벡터의 제조와 감염)

  • Sa, Young Hee;Lee, Ki Hwan;Hong, Seong-Karp
    • Proceedings of the Korean Institute of Information and Commucation Sciences Conference
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    • 한국정보통신학회 2017년도 춘계학술대회
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    • pp.700-703
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    • 2017
  • Baculovirus vectors were recombined using uroplakin II promoter, polyhedron promoter, vesicular stomatitis virus G (VSVG), enhanced green fluorescent protein (EGFP), protein transduction domain (PTD) gene and so on. These novel recombinant vectors were infected into various cell lines. We performed and analyzed gene transfer and gene expression of these recombinant vectors comparison with other control vectors. From this result, we identified that these recombinant vectors have higher efficient gene transfer and expression of than control vector.

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Recombinant human BMP-2/-7 heterodimer protein expression for bone tissue engineering using recombinant baculovirus expression system

  • Park, Seung-Won;Goo, Tae-Won;Kim, Seong Ryul;Choi, Kwang-Ho
    • International Journal of Industrial Entomology and Biomaterials
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    • 제32권2호
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    • pp.49-53
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    • 2016
  • Bone morphogenetic proteins (BMPs) are essential growth factors for bone formation, skeletal development and bone regeneration. The BMP-2/7 heterodimer is known to have remarkable effects on osteogenic induction that are even stronger than the BMP-2 or BMP-7 homodimers. We designed a recombinant human BMP-2/7 (rhBMP-2/7) heterodimer protein with four glycine residues between BMP-2 and BMP-7 protein to facilitate free bond rotation of domains. The Baculovirus Expression Vector System (BEVS) is routinely used to produce recombinant proteins in the milligram scale. In this study, the BEVS was used to express the rhBMP-2/7 protein whrer the recombinant baculovirus was recovered in the host Sf9 cells. To confirm the biological activity of rhBMP-2/7 protein secreted from the BEVS as an osteogenic differentiation and induction factor, we measured the BMP-induced ALP activity. rhBMP-2/7 could be used as an alternative to BMPs to overcome limitations like short half-life and requirement for high concentrations. Furthermore, rhBMP-2/7 may be an efficient tool for various application studies such as bone regeneration and skeletal development.

Construction of Transfer Vector for Production of Baculovirus Occlusion Bodies that Contain Novel Recombinant Crystal Protein

  • Shim, Hee-Jin;Choi, Jae-Young;Roh, Jong-Yul;Li, Ming Shun;Je, Yeon Ho
    • Proceedings of the Korean Society of Sericultural Science Conference
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    • 한국잠사학회 2003년도 International Symposium of Silkworm/Insect Biotechnology and Annual Meeting of Korea Society of Sericultural Science
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    • pp.118-119
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    • 2003
  • Baculovirus occlusion bodies have been recently engineered to incorporate foreign protein such as the Bacillus thuringiensis (Bt) CrylAc protein for improvement of insecticidal activity. In this study, polyhedrin, cylAc, egfp and crylCa genes were fused to produce occlusion bodies that contain novel recombinant crystal protein by homologous recombination between cylAc and crylCa genes in insect cells. (omitted)

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Substrate Specificity of the Human Flavin-containing Monooxygenase for Organic Selenium Compounds (사람 Flavin-containing Monooxygenase의 셀레니움화합물에 대한 기질 특이성에 관한 연구)

  • Kim, Young-Mi
    • Environmental Analysis Health and Toxicology
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    • 제15권4호
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    • pp.139-145
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    • 2000
  • The Flavin-containing monooxygenase (FMOs) (EC1.14.13.8) are NADPH-dependent flavoenzymes that catalyze oxidation of soft nucleophilic heteroatom centers in a range of structurally diverse compounds, including foods, drugs, pesticides, and other xenobiotics. In humans, FMO3 is quantitatively a major human liver monooxygenase. In the present study, the baculovirus expression vector system was used to overexpress human FMO3 in insect cells for catalytic studies. Six commercially available organic selenium compounds were examined for substrate activity with microsomes isolated from Spodoptera frugiperda (Sf)9 cells infected with human FMO3 recombinant baculovirus. While none of the aromatic heterocyclic selenides tested showed detectable activity, all dialkyl- and alkylaryl-selenides free from ionic groups catalyzed the NADPH- and O$_2$-dependent oxidation. Kinetic constants demonstrate that (based on Km) dialkyl-and alkylaryl- selenides are better substrates for human FMO3 than analogous nitrogen or sulfur compounds .

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Effect of Substrate Size on Activities of Thiocarbamides with the Human Flavin-containing Monooxygenase 3 (사람 Flavin-containing Monooxygenase 3의 Thiocarbamide 화합물의 기질 크기에 따른 효소활성에 관한 연구)

  • 김영미
    • Environmental Analysis Health and Toxicology
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    • 제16권2호
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    • pp.97-102
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    • 2001
  • The flavin-containing monooxygenases(FMOs) (EC1.14.13.8) are NADPH0dependent flavoenzymes that catalyze oxidation of soft nucleophilic heteroatom centers in a range of structurally diverse compounds, including foods, drugs, pesticides, and other xenobiotics. In humans, FMO3 is quantitatively a major human liver monooxygenase. In the present study, the baculovirus expression vector system was used to overexpress human FMO3 in sect cells for stalytic studies. Microsomes isolated from Spodoptera frugiperda(Sf)9 cells infected with human FMO3 recombinant baculovirus catalyzed the NADPH-and O$_2$-dependent oxidation of methimazole, thiourea, and phenylthiourea. However there was no detectable activity with 1, 3-diphenylthiourea or larger thiocarbamides. Microsomes from control Sf9 cells were devoid of methimazole or thiourea S-oxygenase activity. 1, 3-diphenylthiourea is apparently completely excluded from the catalytic site, these amines drugs are probably approaching the upper size limits of xenobiotics accepted by human FMO3. The substrate specificity of this iosform in humans appears considerably more restriceted than that of pig, guinea pig, rat or rabbit FMO3.

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Expression of the Gene Encoding Firefly Luciferase Using Bombyx mori Nucleopolyhedrovirus Vector

  • Woo, Soo-Dong;Cho, Kook-Ho;Jin, Byung-Rae;Boo, Kyung-Saeng;Kang, Seok-Kwon
    • International Journal of Industrial Entomology and Biomaterials
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    • 제1권1호
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    • pp.53-58
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    • 2000
  • A cDNA encoding the luciferase of firefly Luciola lateralis was cloned downstream from the polyhedrin gene promoter of Bombyx mori nucleopolyhedrovirus and expressed in B. mori cells (BmN-4). The coding soquence for luciferase was inserted into pBmKSK2 rectors) which was reconstructed from the polyhedrin-based transfer vector pBmKSKl by modifying cloning sites. Recombinant virus, BmK2-LUCDF, containing the luciferase gene was selected and purified in BmN-4 cells. The emission of luminescence by luciferase was only detected in BmK2-LUCDF-infected cell extracts. This result indicates that the cloned new luciferase gene of firefly L. lateralis can be expressed efficiently in baculovirus expression system and used as a useful reporter gene.

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Baculovirus Expression and Biochemical Characterization of the Bombyx mori Protein Disulfide Isomerase (bPDI)

  • Goo, Tae-Won;Yun, Eun-Young;Kim, Sung-Wan;Park, Kwang-Ho;Hwang, Jae-Sam;Kwon, O-Yu;Kang, Seok-Woo
    • International Journal of Industrial Entomology and Biomaterials
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    • 제7권2호
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    • pp.127-131
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    • 2003
  • Protein disulfide isomerase (PDI) found in the endoplasmic reticulum (ER) catalyzes disulfide bond exchange and assists in protein folding of newly synthesized proteins. PDI also functions as a molecular chaperone and has been found to be associated with proteins in the ER. In addition, PDI functions as a subunit of two more complex enzyme systems: the prolyl-4-hydroxylase and the triacylglycerol transfer proteins. A cDNA that encodes protein disulfide isomerase was previously isolated from Bombyx mori (bPDI), in which open reading frame of 494 amino acids contained two PDI-typical thioredoxin active site of WCGHCK and an ER retention signal of the KDEL motif at its C-terminal, and we report its functional characterization here. This putative bPDI cDNA is expressed in insect Sf9 cells as a recombinant proteins using baculovirus expression vector system. The bPDI recombinant proteins are successfully recognized by antirat PDI antibody, and shown to be biologically active in vitro by mediating the oxidative refolding of reduced and scrambled RNase. This suggests that bPDI may play an important role in protein folding mechanism of insects.

Antiviral Treatment Reveals a Cooperative Pathogenicity of Baculovirus and Iflavirus in Spodoptera exigua, a Lepidopteran Insect

  • Roy, Miltan Chandra;Ahmed, Shabbir;Mollah, Md. Mahi Imam;Kim, Yonggyun
    • Journal of Microbiology and Biotechnology
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    • 제31권4호
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    • pp.529-539
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    • 2021
  • The beet armyworm, Spodoptera exigua, is a serious insect pest infesting various vegetable crops. Two infectious insect viruses, baculovirus and iflavirus, are known to induce epizootics in S. exigua populations. Indeed, some laboratory colonies have appeared to be covertly infected by these viruses. Diagnostic PCR tests detected two different viruses: Spodoptera exigua multiple nucleopolyhedrosis virus (SeMNPV) and iflaviruses (SeIfV1 and SeIfV2). Viral extract from dead larvae of S. exigua could infect Sf9 cells and produce occlusion bodies (OBs). Feeding OBs to asymptomatic larvae of S. exigua caused significant viral disease. Interestingly, both SeIfV1 and SeIfV2 increased their titers at late larval stages. Sterilization of laid eggs with 1% sodium hypochloride significantly reduced SeMNPV titers and increased larval survival rate. Double-stranded RNA (dsRNA) specific to SeIfV1 or SeIfV2 significantly reduced viral titers and increased larval survival rate. To continuously feed dsRNA, a recombinant Escherichia coli HT115 expressing SeIfV1-dsRNA was constructed with an L4440 expression vector. Adding this recombinant E. coli to the artificial diet significantly reduced the SeIfV1 titer and increased larval survival. These results indicate that laboratory colony collapse of S. exigua is induced by multiple viral infections. In addition, either suppression of SeMNPV or SeIfV infection significantly increased larval survival, suggesting a cooperative pathogenicity between baculovirus and iflavirus against S. exigua.