• Molecules and Cells
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• v.37 no.10
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• pp.727-733
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• 2014

Spinosyns A and D are potent ingredient for insect control with exceptional safety to non-target organisms. It consists of a 21-carbon tetracyclic lactone with forosamine and tri-Omethylated rhamnose which are derived from S-adenosyl-methionine. Although previous studies have revealed the involvement of metK1 (S-adenosylmethionine synthetase), rmbA (glucose-1-phosphate thymidylyltransferase), and rmbB (TDP-D-glucose-4, 6-dehydratase) in the biosynthesis of spinosad, expression of these genes into rational screened Saccharopolyspora spinosa (S. spinosa MUV) has not been elucidated till date. In the present study, S. spinosa MUV was developed to utilize for metabolic engineering. The yield of spinosyns A and D in S. spinosa MUV was $244mgL^{-1}$ and $129mgL^{-1}$, which was 4.88-fold and 4.77-fold higher than that in the wild-type ($50mgL^{-1}$ and $27mgL^{-1}$), respectively. To achieve the better production; positive regulator metK1-sp, rmbA and rmbB genes from Streptomyces peucetius, were expressed and co-expressed in S. spinosa MUV under the control of strong $ermE^*$ promoter, using an integration vector pSET152 and expression vector pIBR25, respectively. Here-with, the genetically engineered strain of S. spinosa MUV, produce spinosyns A and D up to $372/217mgL^{-1}$ that is 7.44/8.03-fold greater than that of wild type. This result demonstrates the use of metabolic engineering on rationally developed high producing natural variants for the production.

• Molecules and Cells
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• v.23 no.1
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• pp.108-114
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• 2007

The mitogen-activated protein kinase (MAPK) signaling cascade is critical for regulating plant defense systems against various kinds of pathogen and environmental stresses. One component of this cascade, the MAP kinase kinases (MAPKK), has not yet been shown to be induced in plants following biotic attacks, such as those by insects and fungi. We describe here a gene coding for a blast (Magnaporthe grisea)- and insect (Nilaparvata lugens)-responsive putative MAPK kinase, OmMKK1 (Oryza minuta MAPKK 1), which was identified in a library of O. minuta expressed sequence tags (ESTs). Two copies of OmMKK1 are present in the O. minuta genome. They encode a predicted protein with molecular mass 39 kDa and pI of 6.2. Transcript patterns following imbibition of plant hormones such as methyl jasmonic acid (MeJA), ethephone, salicylic acid (SA) and abscisic acid (ABA), as well as exposure to methyl viologen (MV), revealed that the expression of OmMKK1 is related to defense response signaling pathways. A comparative analysis of OmMKK1 and its O. sativa ortholog OsMKK1 showed that both were induced by stress-related hormones and biotic stresses, but that the kinetics of their responses differed despite their high amino acid sequence identity (96%).

• BMB Reports
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• v.38 no.2
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• pp.225-231
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• 2005

Acid-labile subunit (ALS) is a component of the 150-kDa insulin-like growth factor-binding protein-3 (IGFBP-3) complex, which, by sequestering the majority of IGFs-I and -II and thereby prolonging the half-life of them in plasma, serves as a circulating reservoir of IGFs in mammalian species. A pGEX-2T plasmid and a baculovirus expression constructs harboring a coding sequence for glutathione-S transferase (GST)-porcine ALS (pALS) fusion protein were expressed in BL21(DE3) E. coli and Sf9 insect cells, respectively. The expressed protein was purified by glutathione or Ni-NTN affinity chromatography, followed by cleavage of the fusion protein using Factor Xa. In addition, pALS and hIGFBP-3 were also produced in small amounts in the Xenopus oocyte expression system which does not require any purification procedure. A 65-kDa pALS polypeptide was obtained following the prokaryotic expression and the enzymatic digestion, but biochemical characterization of this polypeptide was precluded because of an extremely low expression efficiency. The baculovirus-as well as Xenopus-expressed pALS exhibited the expected molecular mass of 85 kDa which was reduced into 75 and 65 kDa following deglycosylation of Asn-linked carbohydrates by Endo-F glycosidase, indicating that the expressed pALS was properly glycosylated. Moreover, irrespective of the source of pALS, the recombinant pALS and hIGFBP-3 formed a 130-kDa binary complex which could be immunoprecipitated by anti-hIGFBP-3 antibodies. Collectively, results indicate that an authentic pALS protein can be produced by the current expression systems.

• Proceedings of the Korean Society of Plant Pathology Conference
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• 1997.06a
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• pp.5-21
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• 1997

Unique virus-like particles were associated with five eriophyid mite-borne plant diseases of unknown etiology; fig mosaic, redbud yellow ringspot, rose orsette, thistle mosaic, and high plains disease of corn and wheat. Quasi-spherical, double membrane-bound particles (DMPs), 120 - 200 nm in diameter, were observed in the cytoplasm of all cell types in symptomatic leaves of infected plants. No DMPs were observed in symptomless plants. The DMPs in symptomatic thistles were associated with two types of inclusions, electron-dense amorphous material and tubular aggregates. Similar amorphous inclusions were also found in corn and wheat with high plains disease, while tubular inclusions were observed in figs with mosaic symptoms. The particles and inclusions were similar in some aspects to immature particles associated with viroplasms of animal and insect poxviruses and also to the double-enveloped particles of tomato spotted wilt virus associated with viroplasms during early stages of infection, but were unique and unlike any known plant viruses. The DMPs and associated viroplasm-like inclusions in the high plains disease were specifically immunogold labeled in situ with the disease-specific antiserum. Thread-like structures, similar to tenuivirus particles, present in the partially purified virus preparations were also immunogold labeled with the antiserum. It is suggested that the thread-like structures are derived from the DMP. In many cells of symptomatic corn and wheat samples, DMPs occurred together with flexuous rod-shaped particles and cylindrical inclusions of wheat streak mosaic potyvirus (WSMV), suggesting that the disease is caused by a mixed infection of WSMV and the agent represented by the DMPs. Based on cytopathology, symptomatology and mite and/or graft-transmissibility, the five diseases described in this paper are potentially caused by virus(es) and the DMPs associated with these diseases may represent virus particles. If the DMPs are indeed viral in nature, they would comprise a new group of plant viruses.

• Parasites, Hosts and Diseases
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• v.55 no.2
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• pp.143-148
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• 2017

Toxoplasma gondii infections occur throughout the world, and efforts are needed to develop various vaccine candidates expressing recombinant protein antigens. In this study, influenza matrix protein (M1) virus-like particles (VLPs) consisting of T. gondii rhoptry antigen 4 (ROP4 protein) were generated using baculovirus (rBV) expression system. Recombinant ROP4 protein with influenza M1 were cloned and expressed in rBV. SF9 insect cells were coinfected with recombinant rBVs expressing T. gondii ROP4 and influenza M1. As the results, influenza M1 VLPs showed spherical shapes, and T. gondii ROP4 protein exhibited as spikes on VLP surface under transmission electron microscopy (TEM). The M1 VLPs resemble virions in morphology and size. We found that M1 VLPs reacted with antibody from T. gondii-infected mice by western blot and ELISA. This study demonstrated that T. gondii ROP4 protein can be expressed on the surface of influenza M1 VLPs and the M1 VLPs containing T. gondii ROP4 reacted with T. gondii-infected sera, indicating the possibility that M1 VLPs could be used as a coating antigen for diagnostic and/or vaccine candidate against T. gondii infection.

• Korean Journal of Pharmacognosy
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• v.37 no.2 s.145
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• pp.110-115
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• 2006

Melittia inouei (Yuri Nabang) larvae are used as a crude drug in East Asia for treating stomach cancer and inflammation, and currently reared as a pharmaceutical insect in Jejudo, Korea. This study evaluated the immuno-modulating activity of these extracts, by determining the level of, cytokine production from mouse splenocytes stimulated with the extracts. The Melittia inouei larvae extracts did not induce the splenocyte proliferation. On the other hand, they stimulated the splenocytes to produce cytokines such as $TNF-{\alpha}$, whereas they did not stimulate IL10, IL12 or $IFN-{\gamma}$. The aqueous portion of its plant (Tri-chosanthis kirilowii) extract (sap) was found to be a potent inducer of NO production from the CPAE cells. However, it showed weak inhibitory effects on vascular endothelial growth factor (VEGF) production from splenocytes. These data suggests that a Melittia inouei larvae extract immune modulatory activity in cytokine prodcutions such as $TNF-{\alpha}$ and VEGF which might be related its anticancer effect.

• 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 Pharmaceutical Investigation
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• v.31 no.3
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• pp.185-190
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• 2001

Human papillomavirus (HPV) infection is known to cause cervical cancers. Human papillomavirus-like particles (VLP) have been studied as preventive vaccines of cervical cancers. To develop VLP as a therapeutic gene carrier, we studied the method to encapsulate cytokine genes in virus-like particles. HPV type 16 capsid L1 genes were amplified by polymerase chain reaction and cloned into T vector. L1 gene was then inserted into baculovirus transfer vector. The clone of baculovirus encoding L1 gene was isolated and used to express L1 protein in Sf 21 insect cells. VLP were purified by CsCl density gradient and ultracentrifugation. VLP were disassembled to capsomer units by treatment of a reducing agent. Given that interleukin-2 (IL-2) genes have been used in anticancer gene therapy and as a molecular adjuvant, IL-2 cytokine plasmids were chosen as a model gene. IL-2 plasmids were incubated with the disassembled capsomer suspension. To reassemble the particles, the mixture of capsomers and cytokine plasmids was dialyzed. The disassembly and reassembly of VLP were confirmed by transmission electron microscopy. The entrapment of cytokine plasmids in reassembled VLP was tested by the stability of plasmids against DNase I. After treatment of reassembled virus-like particles with DNase I, discrete IL-2 DNA band was observed. Our results indicate that IL-2 cytokine plasmid (3.5 kb size) can be encapsulated in the virus-like particles, suggesting the potential of VLP as a gene delivery system. Moreover, VLP containing the adjuvant cytokine plasmids might function as more effective subunit vaccines.

• Journal of Microbiology and Biotechnology
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• v.25 no.3
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• pp.386-392
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• 2015

A novel recombinant bacmid, bEasyBm, that enables the easy and fast generation of pure recombinant baculovirus without any purification step was constructed. In bEasyBm, attR recombination sites were introduced to facilitate the generation of a recombinant viral genome by in vitro transposition. Moreover, the extracellular RNase gene from Bacillus amyloliquefaciens, barnase, was expressed under the control of the Cotesia plutellae bracovirus early promoter to negatively select against the nonrecombinant background. The bEasyBm bacmid could only replicate in host insect cells when the barnase gene was replaced with the gene of interest by in vitro transposition. When bEasyBm was transposed with pDualBac-EGFP, the resulting recombinant virus, EasyBm-EGFP, showed high levels of EGFP expression efficiency compared with that of non-purified recombinant virus BmGOZA-EGFP, which was constructed using the bBmGOZA system. In addition, nonrecombinant backgrounds were not detected in unpurified EasyBm-EGFP stocks. Based on these results, a high-throughput system for the generation of multiple recombinant viruses at a time was established.

• Journal of Microbiology and Biotechnology
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• v.17 no.11
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• pp.1833-1840
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• 2007

Viruses in garlic plants (Allium sativum L.) have accumulated and evolved over generations, resulting in serious consequences for the garlic trade around the world. These viral epidemics are also known to be caused by aphids and eriophyid mites (Aceria tulipae) carrying Potyviruses, Carlaviruses, and Allexiviruses. However, little is known about viral epidemics in garlic plants caused by eriophyid mites. Therefore, this study investigated the infection of garlic plants with Allexiviruses by eriophyid mites. When healthy garlic plants were cocultured with eriophyid mites, the leaves of the garlic plants developed yellow mosaic strips and became distorted. In extracts from the eriophyid mites, Allexiviruses were observed using immunosorbent electron microscopy (ISEM). From an immunoblot analysis, coat proteins against an Allexivirus garlic-virus antiserum were clearly identified in purified extracts from collected viral-infected garlic plants, eriophyid mites, and garlic plants infected by eriophyid mites. A new strain of GarV-B was isolated and named GarV-B Korea isolate 1 (GarV-B1). The ORF1 and ORF2 in GarV-B1 contained a typical viral helicase, RNA-directed RNA polymerase (RdRp), and triple gene block protein (TGBp) for viral movement between cells. The newly identified GarV-B1 was phylogenetically grouped with GarV-C and GarV-X in the Allexivirus genus. All the results in this study demonstrated that eriophyid mites are a transmitter insect species for Allexiviruses.