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Nested PCR을 이용한 Cowpea chlorotic mottle virus 정밀 진단 시스템 개발
민병대(Byung-Dae Min),김영석(Young-Suk Kim),이시원(Siwon Lee),이수헌(Su-Heon Lee) 충남대학교 농업과학연구소 2014 농업과학연구 Vol.41 No.4
Cowpea chlorotic mottle virus (CCMV)는 Group IV positive sense single strand RNA virus, Bromoviridae과, Bromovirus속으로 분류하는 식물병원성 바이러스로, 강낭콩(Phaseolus vulgaris), 나비완두(Clitoria ternatea), 담배(Nicotiana tabaccum), 대두(Glycine max), 동부(Vigna unguiculata, Vigna siensis) 및 땅콩(Arachis hypogaea)이 국내로 수입될 경우, 검사를 수행하는 관리급 검역바이러스이다. 본 연구에서는, RT-PCR, nested PCR 및 유전자-삽입 양성대조구를 개발하여, CCMV를 현장에서 신속, 정확하게 진단할 수 있는 정밀검정 시스템을 구현하였다. 본 연구에서 개발한 방법은 지속적으로 현장에서 활용되어 식물검역에 기여할 것이라고 기대된다. Cowper chlorotic mottle virus (CCMV) is the ‘controlled’ quarantine virus as plant pathogenic virus that are classed as group Ⅵ (+) ssRNA virus that belongs to the genus Bromovirus and family Bromoviridae, When plants that are Phaseolus vulgaris, Clitoria ternatea, Nicotiana tabaccum, Glycine max, Vigna unguiculata and Vigna siensis, and Arachis hypogaea is imported in domestic. In this study, inspection system is implemented to analyze CCMV accurately and rapidly by developing RT-PCR, nested PCR, and gene insertion positive control. It is expected that the method developed in this study will contribute to the plant quarantine to be consistently utilized in the field.
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Nested PCR을 이용한 Cowpea chlorotic mottle virus 정밀 진단 시스템 개발
민병대(Byung-Dae Min),김영석(Young-Suk Kim),이시원(Siwon Lee),이수헌(Su-Heon Lee) 충남대학교 농업과학연구소 2014 Korean Journal of Agricultural Science Vol.41 No.4
Cowpea chlorotic mottle virus (CCMV)는 Group IV positive sense single strand RNA virus, Bromoviridae과, Bromovirus속으로 분류하는 식물병원성 바이러스로, 강낭콩(Phaseolus vulgaris), 나비완두(Clitoria ternatea), 담배(Nicotiana tabaccum), 대두(Glycine max), 동부(Vigna unguiculata, Vigna siensis) 및 땅콩(Arachis hypogaea)이 국내로 수입될 경우, 검사를 수행하는 관리급 검역바이러스이다. 본 연구에서는, RT-PCR, nested PCR 및 유전자-삽입 양성대조구를 개발하여, CCMV를 현장에서 신속, 정확하게 진단할 수 있는 정밀검정 시스템을 구현하였다. 본 연구에서 개발한 방법은 지속적으로 현장에서 활용되어 식물검역에 기여할 것이라고 기대된다. Cowper chlorotic mottle virus (CCMV) is the ‘controlled’ quarantine virus as plant pathogenic virus that are classed as group Ⅵ (+) ssRNA virus that belongs to the genus Bromovirus and family Bromoviridae, When plants that are Phaseolus vulgaris, Clitoria ternatea, Nicotiana tabaccum, Glycine max, Vigna unguiculata and Vigna siensis, and Arachis hypogaea is imported in domestic. In this study, inspection system is implemented to analyze CCMV accurately and rapidly by developing RT-PCR, nested PCR, and gene insertion positive control. It is expected that the method developed in this study will contribute to the plant quarantine to be consistently utilized in the field.
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Yuanzheng Wu,Hetong Yang,전영진,이민영,Jishun Li,신현재 한국생물공학회 2014 Biotechnology and Bioprocess Engineering Vol.19 No.4
A copper(I)-catalyzed azide-alkyne cycloaddition(CuAAC) reaction was exploited for the surface modificationof cowpea chlorotic mottle virus (CCMV). The exposedcarboxyl residues of the CCMV capsids were modifiedwith an alkyne and then further modified with an azide,using a triazole connection in the presence of CuSO4,tris(2-carboxyethyl)phosphine hydrochloride (TCEP), anda bathocuproin disulfonic acid disodium salt (BCDS). Fluorogenic coumarin was successfully grafted onto theCCMV capsids and monitored by fast protein liquidchromatography (FPLC) and UV-irradiated SDS-PAGE. An oligo-ethylene glycol (OEG) short chain and an Arg-Gly-Asp (RGD) peptide were also connected to the CCMVcapsids via the CuAAC reaction. Size-exclusion FPLC,transmission electron microscopy (TEM), and dynamiclight scattering (DLS) analyses confirmed the modificationand integrity of the viral capsids. Interestingly, OEG-CCMVdisplayed a unique phenomenon of connected bridges withthe intact capsids crosslinked to each other. Coumarin-CCMV, OEG-CCMV, and RGD-CCMV were absorbedonto APTES slides for cell binding with HeLa cells. Theopposite adhesion behavior of OEG-CCMV and RGDCCMVindicated the inhibition effect of OEG and thepromotion effect of RGD for cell attachment. This providesa generalized method for chemical modification of thesurface of virus capsids with multivalent ligands, whichdemonstrates the potential applications in bioimaging, tissueengineering, and drug delivery.
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Yuanzheng Wu,Jishun Li,Hetong Yang,성지현,임호동,김근중,신현재 한국생물공학회 2017 Biotechnology and Bioprocess Engineering Vol.22 No.6
Our aim was to devise targeted drug delivery systems using genetically modified cowpea chlorotic mottle virus (CCMV) capsids by fusion expression with tumorhoming peptide F3 for efficient delivery of therapeutic substances into tumor cells. The RNA-binding domain at the N terminus (amino acid residues 1–25) of CCMV capsid protein (CP) was selectively deleted, and F3 was inserted for the expression in Pichia pastoris. After chromatographic purification, F3-CCMV capsids were obtained via selfassembly of the F3-CP fusion protein and then analyzed by transmission electron microscopy and dynamic light scattering analysis, which revealed spherical nanoparticles (NPs) ca. 18 nm in diameter with regular monodispersity. Near-infrared fluorescent dye IR780 iodide, which has been applied for cancer imaging, photodynamic therapy, and photothermal therapy, was encapsulated in F3-CCMV NPs. The resultant F3-CCMV-IR780 NPs showed excellent molecular targeting to nucleolin receptor overexpressed on the surface of MCF-7 tumor cells. Furthermore, the in vitro cellular uptake and cell viability assay proved a photothermal effect by a single dose of near-infrared laser irradiation. The present system may offer a programmable nanoscaffoldbased drug delivery system vehicle for fabrication of promising therapeutic substances for cancer therapy.
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