22 research outputs found

    λˆ„μ—λ‚˜λ°©(Bombyx mori) ν•΅λ‹€κ°μ²΄λ³‘λ°”μ΄λŸ¬μŠ€λ₯Ό μ΄μš©ν•œ μƒˆλ‘œμš΄ λ² ν˜λ‘œλ°”μ΄λŸ¬μŠ€ λ°œν˜„μ‹œμŠ€ν…œ ꡬ좕과 νŠΉμ„± ꡬλͺ…

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    ν•™μœ„λ…Όλ¬Έ (박사)-- μ„œμšΈλŒ€ν•™κ΅ λŒ€ν•™μ› : 농생λͺ…곡학뢀(곀좩학전곡), 2015. 8. μ œμ—°ν˜Έ.A novel recombinant bacmid, bEasyBm, that enables the easy and fast generation of pure recombinant baculovirus without any purification step was constructed. In the bEasyBm, attR recombination sites were introduced to facilitate the generation of a recombinant viral genome by in vitro transposition. Moreover, an extracellular RNase gene from Bacillus amyloliquefaciens, barnase, was expressed under the control of the Cotesia plutellae bracovirus early promoter to negatively select against the non-recombinant background. The bEasyBm bacmid could be replicated only 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 to that of non-purified recombinant virus BmGOZA-EGFP, which was constructed using the bBmGOZA system. In addition, no non-recombinant backgrounds were 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. Also, an easy, fast and mass purification system was developed using baculovirus expression vector system (BEVS) in which Expression by translational fusion of the polyhedrin-enhanced green fluorescence protein (EGFP) led to the formation of granular structures and these fluorescent granules were easily precipitated by high-speed centrifugation. Cry1Ac foreign protein fused with the partial polyhedrin and EGFP gene at the C-terminus, including an enterokinase (EK) site between EGFP and Cry1Ac protein, was expressed in insect cells. Cells infected by BmPolh19EG-1Ac or BmPolh32EG-1Ac produced fluorescent granules. The Cry1Ac fusion protein was purified from granule-containing cells in three steps: cell harvest, sonication, and EK digestion. Through final enterokinase digestion, Cry1Ac presented mainly in the supernatant, and this supernatant fraction also showed a pure Cry1Ac band. These results suggest that the combined procedure of polyhedrin fusion expression and enterokinase digestion can be used for rapid and easy purification of other proteins.ABSTRACT ----------------------------------------------------------------------------------------- i TABLE OF CONTENTS ------------------------------------------------------------------------ iv LIST OF TABLES ------------------------------------------------------------------------------- vii LIST OF FIGURES ---------------------------------------------------------------------------- viii INTRODUCTION --------------------------------------------------------------------------------- 1 LITERATURE REVIEW ------------------------------------------------------------------------ 5 MATERIALS AND METHODS -------------------------------------------------------------- 23 1. Insects, insect cells and baculoviruses ---------------------------------------------------- 23 2. Plasmid, bacmid, Escherichia coli and cloning ----------------------------------------- 23 3. PCR and primers ----------------------------------------------------------------------------- 24 4. Construction of plasmid vectors ----------------------------------------------------------- 24 5. In vitro transposition ------------------------------------------------------------------------ 25 6. Transfection ---------------------------------------------------------------------------------- 26 7. RNA and RT-PCR --------------------------------------------------------------------------- 26 8. Infection of cells with baculoviruses ------------------------------------------------------ 28 9. Quantification of EGFP --------------------------------------------------------------------- 28 10. Luciferase activity assay ------------------------------------------------------------------ 29 11. Expression and purification of polyhedrin-EGFP fusion protein ----------------- 29 12. SDS-PAGE and measurement of EGFP ------------------------------------------------- 30 13. Bioassay ------------------------------------------------------------------------------------- 30 RESULTS ------------------------------------------------------------------------------------------ 32 1. Construction of EasyBm system for high-throughput recombinant virus generation ----------------------------------------------------------------------------- 32 1) Construction of recombinant virus, vBpLacZ --------------------------------------- 32 2) Construction of transfer vector, pBm101-MF --------------------------------------- 32 3) Construction of recombinant bacmid, bBmTenLacZ ------------------------------- 38 4) Construction of recombinant bacmid, bEasyBm ------------------------------------ 45 2. Activities and efficiency of bEasyBm system --------------------------------------- 45 1) Cytopathic effect of barnase gene ------------------------------------------------------ 45 2) Cytotoxic effect of barnase on baculovirus genome --------------------------------- 50 3) Recombination efficiency of recombinant bacmid, bEasyBm, through in vitro transposition -------------------------------------------------------- 50 4) Expression efficiency of foreign genes using bEasyBm through in vitro transposition --------------------------------------------------------------------------- 55 5) Generation efficiency of foreign genes using in vivo system, B. mori ------------ 55 3. Construction of easy purification system using polyhedrin-EGFP fusion protein -- 58 1) Construction of transfer vector pPolh19-110-EGEK -------------------------------- 58 2) Construction of transfer vector pGT-Polh32-110-EGEK(+) ------------------------ 63 3) Purification efficiency of foreign protein in in vitro system using insect cells -- 67 4. Production of insecticidal protein and its insecticidal activity ------------------------ 78 DISCUSSION ----------------------------------------------------------------------------------- 88 LITERATURE CITED ------------------------------------------------------------------------ 93 ABSTRACT IN KOREAN ------------------------------------------------------------------- 107 ACKNOWLEDGEMENTS ------------------------------------------------------------------ 110Docto

    μž„ν”Œλž€νŠΈ νŒŒμ ˆμ— κ΄€ν•œ μœ ν•œμš”μ†Œλ²•μ  연ꡬ

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    Thesis(doctoral)--μ„œμšΈλŒ€ν•™κ΅ λŒ€ν•™μ› :μΉ˜μ˜ν•™κ³Ό μΉ˜κ³Όλ³΄μ² ν•™μ „κ³΅,2006.Docto

    Jaw movement analysis in subjects with implant-supported prosthesis

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    Thesis (master`s)--μ„œμšΈλŒ€ν•™κ΅ λŒ€ν•™μ› :μΉ˜μ˜ν•™κ³Ό μΉ˜κ³Όλ³΄μ² ν•™ 전곡,1999.Maste

    Postantibiotic effect and concentration-dependent bacterial killing rate of vancomycin for constructing proper dosage regimen

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    ν•™μœ„λ…Όλ¬Έ(박사)--μ„œμšΈλŒ€ν•™κ΅ λŒ€ν•™μ› :μ˜ν•™κ³Ό 약리학전곡,1998.Docto

    Bacillus thuringiensis cry1Ac μœ μ „μžμ˜ mutant μ œμž‘ 및 μ‚΄μΆ©μ„± κ²€μ •

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    ν•™μœ„λ…Όλ¬Έ(석사) --μ„œμšΈλŒ€ν•™κ΅ λŒ€ν•™μ› :농생λͺ…곡학뢀,2007.Maste

    A study on the problem of the intellectual property law of the parody

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    ν•™μœ„λ…Όλ¬Έ(박사) --μ„œμšΈλŒ€ν•™κ΅ λŒ€ν•™μ› :법학과,2007.Docto

    특수ꡐ윑의 관점과 정책논리

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    ν•™μœ„λ…Όλ¬Έ(석사)--μ„œμšΈλŒ€ν•™κ΅ λŒ€ν•™μ› :κ΅μœ‘ν•™κ³Ό κ΅μœ‘ν–‰μ •μ „κ³΅,2000.Maste

    둜마 곡화정 ν›„κΈ°, μ œμ •μ΄ˆμ˜ κ°€λΆ€μž₯ꢌ : κ°€μ‘± 관계λ₯Ό μ€‘μ‹¬μœΌλ‘œ

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    ν•™μœ„λ…Όλ¬Έ(석사)--μ„œμšΈε€§ε­Έζ ‘ 倧學陒 :η€Ύζœƒζ•Žθ‚²η§‘ ο¦Œε²ε°ˆζ”»,1995.Maste

    ν”„λ‘œκ·Έλž¨ λ©”λ‰΄ζ§‹ι€ μ˜ θ‘—δ½œη‰©ζ€§

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    ν•™μœ„λ…Όλ¬Έ(석사)--μ„œμšΈλŒ€ν•™κ΅ λŒ€ν•™μ› :법학과 μ§€μ μ†Œμœ κΆŒ 전곡,2000.Maste
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