[보고서]곤충생체반응기를 이용한 동물백신 생산 및 분리정제 시스템 개발

보고서 정보

주관연구기관

동아대학교
Donga University

보고서유형

최종보고서

발행국가

대한민국

언어

한국어

발행년월

2011-02

과제시작연도

2007

주관부처

농촌진흥청
Rural Development Administration(RDA)

과제관리전문기관

농촌진흥청
Rural Development Administration

등록번호

TRKO201200000058

과제고유번호

1395011745

사업명

농업생명공학기술개발

DB 구축일자

2013-04-18

DOI

https://doi.org/10.23000/TRKO201200000058

표 SDS-PAGE analysis of the purified virus-like particles protein (A) and productionlevel of virus-like particles from silkworm larva (B).
표 Electron microscopy of purified VLPs. VLPs purified from PRRSV, larvae infectedwith BmNPV-M, BmNPV-GP5/M or BmNPV-ORF4/GP5/M. Bar indicates 20 µm.
표 Transfer vector for the production of recombinant BmNPV that expresses PRRSV GP5-GPI anchored protein in insect cells. Transfer vector pBmNPV-GP5-GPI. The GP5-GPI were introduced under the control of the polyhedrin (PPH) promoter. The arrow indicates the direction of transcription.
표 SDS-PAGE and western blot analyses of recombinant GP5-GPI in BmNPV-GP5-GPI-infected BmN cells. BmN cells were infected with the recombinant BmNPV-GP5-GPI at an MOI of 5 pfu per cell. Cells were collected at 3 dpi. Total protein (lane 2), soluble protein (Lane 3) and membrane protein (lane 4) were subjected to 12% SDS-PAGE (left), electroblotted, and incubated with anti-GP5 antibody (right). Molecular weight standards were used as a size marker (lane 1). The GP5-GP1 fusion protein is indicated by arrow.
표 Immunofluorescent staining of BmN cells. BmN cells were mock-infected (upper) or infected with the recombinant BmNPVGP5-GPI (lower). Cells were incubated with anti-GP5 antibody (red). Bar indicates 20 μm.
표 Transfer vector for the production of recombinant BmNPV that expresses recombinant polyhedra carrying PRRAV GP5Δ or MΔ. (A) The domain structure of PRRAV GP5, GP5Δ, M and MΔ. Epitope sites (ES) and N-glycosylation sites (N) are shown. (B) Transfer vector pBmNPV-GP5 /M . The BmNPV native Δ Δ polyhedrin gene and the fusion gene polh-GP5Δ/MΔ were introduced under the control of the p10 (Pp10) or the polyhedrin (PPH) promoter, respectively. The arrows indicate the direction of transcription.
표 Expression of recombinant polyhedra carrying GP5Δ or MΔ in BmNPV-GP5Δ/MΔ-infected BmN cells. BmN cells infected with either the wild-type BmNPV or the recombinant BmNPV-GP5Δ/MΔ were photographed at 3 dpi. Bar indicates 20 μm.
표 Scanning electron microscopy of wild-type BmNPV polyhedra (left), recombinantpolyhedra carrying PRRSV GP5Δ (middle) and PRRSV MΔ (right). Scale bar, 20 µm.
표 Immunofluorescent staining of wild-type BmNPV polyhedra (upper), recombinantpolyhedra carrying PRRSV GP5Δ (middle) and PRRSV MΔ (lower). The polyhedra were incubated with anti-polyhedrin antibody (green) or anti-PRRSV antibody (red).
표 Glycoprotein staining in recombinant polyhedra carrying PRRAV GP5Δ (A) or MΔ(B). The nonglycosylated protein soybean trypsin inhibitor (lane 1) and wild type polyhedrin protein (lane 3) were used as a negative control. The glycosylated protein horseradish peroxidase served as a positive control (lane 2). The recombinant polyhedra carrying PRRAV GP5Δ or MΔ were analyzed to lane 4. The polh-GP5Δ/MΔ fusion proteins are indicated by arrows.
표 SDS-PAGE and western blot analyses of recombinant polyhedra carrying PRRSV GP5Δ that were produced in BmNPV-GP5Δ-infected silkworm larvae. Recombinant polyhedra were subjected to 13% SDS-PAGE (A), electroblotted, and incubated with anti-polyhedrin antibody (B), anti-polh-GP5Δ or anti-PRRSV GP5Δ antibody (D). Molecular weight standards were used as size markers (lane M).
표 Productivity of the PRRSV GP5Δ antigen in BmNPV-GP5Δ-infected silkwormlarvae. SDS-PAGE of recombinant polyhedra carrying PRRSV GP5Δ that were produced in BmNPV-GP5Δ-infected silkworm larvae (A). On day 1 of the fifth instar larval stage, B. mori larvae were infected with recombinant BmNPV-GP5Δ at 1 × 105 pfu/larva. Hemolymph and recombinant polyhedra were collected at 3, 4, 5, and 6 dpi. SDS-PAGE of recombinant polyhedra carrying PRRSV GP5Δ, cleavage of GP5Δ by treatment of factor-Xa and the purified GP5Δ antigen (B). The productivity of GP5Δ and the fusion protein were measured at 6 dpi and expressed as ㎎ per ml of mg per larva (C).
표 SDS-PAGE and western blot analyses of recombinant polyhedra carrying PRRSV MΔ that were produced in BmNPV-MΔ-infected silkworm larvae. Recombinant polyhedra were subjected to 13% SDS-PAGE (A), electroblotted, and incubated with anti-polyhedrin antibody (B), anti-polh-MΔ or anti-PRRSV MΔ antibody (D). Molecular weight standards were used as size markers (lane M).
표 Productivity of the PRRSV M antigen in BmNPV-M Δ Δ-infected silkworm larvae. SDS-PAGE of recombinant polyhedra carrying PRRSV MΔ that were produced in BmNPV-MΔ-infected silkworm larvae (A). On day 1 of the fifth instar larval stage, B. mori larvae were infected with recombinant BmNPV-MΔ at 1 × 105 pfu/larva. Hemolymph and recombinant polyhedra were collected at 3, 4, 5, and 6 dpi. SDS-PAGE of recombinant polyhedra carrying PRRSV MΔ, cleavage of MΔ by treatment of factor-Xa and the purified MΔ antigen (B). The productivity of MΔ and the fusion protein were measured at 6 dpi and expressed as ㎎ per ml of mg per larva (C).
표 PRRSV GP5 (A) or M (B) antibody titers in the sera of mice immunized with 10 ㎍ granule form, 10 granule form with adjuvant and 10 ㎍ soluble form with adjuvant.
표 The domain structure of PRV gBΔ (A) and gCΔ (C). SDS-PAGE of recombinantpolyhedra carrying PRRSV gBΔ (B) or gCΔ (D) that were produced in BmNPV-gBΔ or gCΔ-infected BmN cell.
표 Expression of recombinant polyhedra carrying gBΔ or gCΔ in BmNPV-gBΔ/gCΔ-infected BmN cells. BmN cells infected with either the wild-type BmNPV or the recombinant BmNPV-gBΔ/gCΔ were photographed at 3 dpi. Bar indicates 20 μm.
표 Scanning electron microscopy of wild-type BmNPV polyhedra (left), recombinant polyhedra carrying PRV gB (middle) and PRV Δ gCΔ (right). Scale bar, 20 μm.
표 Immunofluorescent staining of wild-type BmNPV polyhedra (upper), recombinant polyhedra carrying PRRSV gBΔ (middle) and PRRSV gCΔ (lower). The polyhedra were incubated with anti-polyhedrin antibody (green) or anti-PRV antibody (red).
표 Glycoprotein staining in recombinant polyhedra carrying PRV gBΔ (A) or gCΔ (B). The nonglycosylated protein soybean trypsin inhibitor (lane 1) and wild type polyhedrin protein (lane 3) were used as a negative control. The glycosylated protein horseradish peroxidase served as a positive control (lane 2). The recombinant polyhedra carrying PRRAV gBΔ or gCΔ were analyzed to lane 4. The polh-gBΔ/gCΔ fusion proteins are indicated by arrows.
표 SDS-PAGE and western blot analyses of recombinant polyhedra carrying PRV gB Δ (A) or gCΔ (B). Recombinant polyhedra were subjected to SDS-PAGE, electroblotted, and incubated with anti-polyhedrin antibody, anti-polh-gBΔ/gCΔ or anti-PRV gBΔ/gCΔ antibody. Molecular weight standards were used as size markers (lane M).
표 Productivity of the PRV gBΔ (A) and PRV gCΔ (B) antigen in BmNPV-gBΔ/gCΔ -infected silkworm larvae. SDS-PAGE of recombinant polyhedra carrying PRV gBΔ/gCΔ that were produced in BmNPV-gBΔ/gCΔ-infected silkworm larvae. On day 1 of the fifth instar larval stage, B. mori larvae were infected with recombinant BmNPV-gBΔ/gCΔ at 1 × 105 pfu/larva. Hemolymph and recombinant polyhedra were collected at 3, 4, 5, and 6 dpi. The productivity of gBΔ/gCΔ and the fusion protein were measured at 6 dpi and expressed as ㎎ per ml of mg per larva.
표 Transfer vector for the production of recombinant BmNPV that expresses recombinant polyhedra carrying CSFV E2ΔC. (A) The domain structure of CSFV E2 and E2ΔC. Epitope sites (ES) and N-glycosylation sites (N) are shown. (B) Transfer vector pBmNPV-E2ΔC. The BmNPV native polyhedrin gene and the fusion gene polhΔC-E2ΔC were introduced under the control of the p10 (Pp10) or the polyhedrin (PPH) promoter, respectively. The arrows indicate the direction of transcription.
표 Expression of recombinant polyhedra carrying E2 ΔC in BmNPV-E2ΔC-infected BmN cells. (A) SDS-PAGE and western blot analyses of recombinant polyhedra carrying E2ΔC in BmNPV-E2ΔC-infected BmN cells. BmN cells were mock-infected or infected with either the wild-type BmNPV or the recombinant BmNPV-E2ΔC at an MOI of 5 pfu per cell. Cells were collected at 3 dpi. Total cellular lysates were subjected to 10% SDS-PAGE (left), electroblotted, andincubated with anti-polyhedrin antibody (middle) or anti-CSFV E2 antibody (right). Molecular weight standards were used as a size marker (lane M). The polhΔC-E2ΔC fusion protein and native polyhedrin are indicated by arrows. (B) Production of polyhedra in BmN cells. BmN cells infected with either the wild-type BmNPV or the recombinant BmNPV-E2ΔC were photographed at 3 dpi (500×). The polyhedra are indicated by arrows.
표 Expression of recombinant polyhedra carrying E2ΔC in BmNPV-E2ΔC-infected silkworm larvae. SDS-PAGE and western blot analyses of the hemolymph of BmNPV-E2Δ C-infected silkworm larvae. On day 1 of the fifth instar larval stage, B. mori larvae were infected with recombinant BmNPV-E2ΔC at 1 × 105 pfu/larva. Hemolymph was collected at 3, 4, 5, and 6 dpi. Hemolymph samples (1 ㎕ of hemolymph/lane) were subjected to 10% SDS-PAGE (left), electroblotted, and incubated with anti-polyhedrin antibody (middle) or anti-CSFV E2 antibody (right). Molecular weight standards were used as size markers (lane M). The polh C-E2 C fusion protein and native polyhedrin Δ Δ are indicated by arrows.
표 Expression of recombinant polyhedra carrying E2ΔC in BmNPV-E2ΔC-infected silkworm larvae. SDS-PAGE and western blot analyses of the hemolymph of BmNPV-E2Δ C-infected silkworm larvae. On day 1 of the fifth instar larval stage, B. mori larvae were infected with recombinant BmNPV-E2ΔC at 1 × 105 pfu/larva. Hemolymph was collected at 3, 4, 5, and 6 dpi. Hemolymph samples (1 ㎕ of hemolymph/lane) were subjected to 10% SDS-PAGE (left), electroblotted, and incubated with anti-polyhedrin antibody (middle) or anti-CSFV E2 antibody (right). Molecular weight standards were used as size markers (lane M). The polh C-E2 C fusion protein and native polyhedrin Δ Δ are indicated by arrows.
표 Production ofrecombinant polyhedra carrying E2ΔC in BmNPV-E2ΔC-infected silkworm larvae. (A) Scanning electron microscopy of wild-type BmNPV polyhedra (left) and recombinant polyhedra carrying CSFV E2ΔC (right). Scale bar, 5 μm. (B) SDS-PAGE and western blot analyses of recombinant polyhedra carrying CSFV E2ΔC that were produced in BmNPV-E2ΔC-infected silkworm larvae. Recombinant polyhedra were subjected to 10% SDS-PAGE (left), electroblotted, and incubated with anti-polyhedrin antibody (middle) or anti-CSFV E2 antibody (right). Molecular weight standards were used as size markers (lane M). The polhΔC-E2ΔC fusion protein and native polyhedrin are indicated by arrows. (C) Immunofluorescent staining of wild-type BmNPV polyhedra (upper) and recombinant polyhedra carrying CSFV E2ΔC (lower). The polyhedra were incubated with anti-polyhedrin antibody (green) or anti-CSFV E2 antibody (red). Scale bar, 5 μm. (D) Glycoprotein staining in recombinant polyhedra carrying CSFV E2ΔC (lane 3). The glycosylated protein horseradish peroxidase (5 μg) served as a positive control (lane 1). The nonglycosylated protein soybean trypsin inhibitor (5 μg) was used as a negative control (lane 2). The polhΔC-E2ΔC fusion protein is indicated by arrows.
표 Productivity of the CSFV E2ΔC antigen in BmNPV-E2ΔC-infected silkwormlarvae. The productivity of E2ΔC and the fusion protein were measured at 6 dpi and expressed as ㎎ per ml of hemolymph (A) and ㎎ per larva (B).
표 CSFV E2 antibody titers in the sera of mice immunized with purified E2ΔC,granule- or soluble-form recombinant polyhedra carrying E2ΔC. (A) Anti-E2 antibodies that were induced in mice via a prime-boost immunization. Arrows represent the prime and the boost immunization. The sera of the tested mice were harvested at 1-week intervals after the prime-boost immunization and diluted 1:100. CSFV E2 antibody titers are expressed as the mean ± SD (n = 3). (B) Anti-E2 antibodies that were induced in mice by the prime immunization. The arrow represents the prime immunization. The sera of the tested mice were harvested at 1-week intervals after the prime immunization and diluted 1:100. CSFV E2 antibody titers are expressed as the mean ± SD (n = 3).
표 Neutralizing antibody titers in the sera of immunized mice.
표 Blocking rate of anti-E2 antibodies induced in the sera of mice immunized withpurified E2ΔC, granule- or soluble-form recombinant polyhedra carrying E2ΔC. The levels of E2-specific antibodies in the sera were measured using a CSFV antibody test kit. The test samples were considered serologically positive when the blocking percentage was ≥ 40%. Blocking percentages are expressed as the mean ± SD (n = 3).
표 Schematic outline of the purification of recombinant polyhedra, solubilizedrecombinant polyhedra, and E2ΔC protein.
표 Transfer vector pBmNPV-gBΔ/gCΔ/GP5Δ/E2ΔC for the production of recombinant BmNPV that expresses recombinant polyhedra carrying PRV gBΔ (A), PRV gCΔ (B), PRRSV GP5Δ (C) or CSFV E2ΔC (D). The arrows indicate the direction of transcription.
표 Production of recombinant polyhedra carrying gBΔ in BmNPV-gBΔ-infectedsilkworm larvae. (A) Immunofluorescent staining of fusion-form antigen carrying PRV gB Δ. The recombinant fusion protein were incubated with anti-polyhedrin antibody (green) or anti-PRV gB antibody (red). Scale bar, 5 μm. (B) SDS-PAGE analyses of recombinant fusion protein carrying PRV gBΔ that were produced in BmNPV-gBΔ-infected silkworm larvae. On day 1 of the fifth instar larval stage, B. mori larvae were infected with recombinant BmNPV-gBΔ at 1 ×105 pfu/larva. Recombinant fusion protein were collected at 3, 4, 5, and 6 dpi. Molecular weight standards were used as size markers (lane M). (C) The polhΔC-gBΔ fusion protein is indicated by arrows. Productivity of the PRV gBΔ antigen in BmNPV-gBΔ-infected silkworm larvae.
표 Schematic outline of the purification of recombinant polyhedra, solubilizedrecombinant polyhedra, and E2ΔC protein.
표 Schematic construction map of the recombinant AcNPV genome, bEasyBac.
표 Construction map of the donor vector, pDualBac.
표 Construction map (A) and confirmation of the internal structure (B) of the donor vector,pDualBac-Polh, by restriction endonuclease digestion pattern.
표 Construction map of the donor vector, pDualBac-EGFP.
표 Construction map of the donor vector, pDualBac-Luc.
표 Construction map of the donor vector, pDualBac-PolhEKA.
표 Construction map of the transfer vector, pBac9-PolhEKA.
표 Construction map of the transfer vector, pBac9-PolhEKA-EGFP.
표 Construction map of the polyhedrin-fusion transfer vector, pOBⅠ.
표 Construction map of the transfer vectors, pOBⅠ-PRRSV'X' in which the PRRSV ORFs were C-terminally fused to polyhedrin gene. The capital 'X' of pOBⅠ-PRRSV'X' represents 2, 3, 4, 5, 6, and 7, indicating respective PRRSV ORF number.
표 Primers used for construction of baculovirus transfer vectors containing PRRSV ORFs
표 Primers used for construction of baculovirus transfer vectors containing PRRSV ORFs
표 Construction map of the donor vector, pDualBac-PolhEKA-PRRSV56.
표 Construction map of the transfer vectors, pDualBac-PolhEKA-E'X'K in which the SFVORFs were C-terminally fused to polyhedrin gene. The capital 'X' of pDualBac-PolhEKA-E'X'K represents 0, 1 and 2, indicating respective SFV ORF.
표 Primers used for construction of baculovirus transfer vectors containing SFV ORFs
표 Construction map of the transfer vectors, pDualBac-PolhEKA-g'X' in which the ADVORFs were C-terminally fused to polyhedrin gene. The capital 'X' of pDualBac-PolhEKA-g'X' represents C and D, indicating respective ADV ORF.
표 Primers used for construction of baculovirus transfer vectors containing ADV ORFs
표 Flow chart of high-throughput recombinant virus generation using EasyBac system.
표 Growth of Sf9 cells transfected with the recombinant AcNPV genome, bEasyBac, expressingBarnase under the control of CpBV ORF3005 promoter.
표 Detection of viral mRNAs from Sf9 cells transfected with the recombinant AcNPV genome,bEasyBac, along serial passage. Passage numbers are indicated as 'Pn', 'P' for passage and 'n' for number.
표 Production of polyhedra in Sf9 cells infected with the recombinant AcNPV, EasyBac-Polh.The EasyBac-Polh was generated by both in vitro site specific transposition and in vivo homologous recombination between the bEasyBac and the pDualBac-Polh.
표 SDS-GAGE analysis of the polyhedrin expressed by the recombinant AcNPV,EasyBac-Polh, along serial passage. The EasyBac-Polh generated by both in vitro transposition or in vivo homologous recombination. Passage numbers are indicated as 'Pn', 'P' for passage and 'n' for number.
표 in situ Assay of EGFP (A) and luciferase (B) in Sf9 cells infected with AcEasy-EGFP andAcEasy-Luc, respectively. Quantities of bEasyBac genome and donor vector used for transposition were indicated at right of each pannel.
표 Expression of EGFP in Sf9 cells infected with the recombinant AcNPVs along serialpassage. The AcEasy-EGFP and AcEGFP were constructed using bEasyBac and bAcGOZA as parent viral genome, respectively.
표 Verification of the genome structure of the recombinant viruses, AcEasy-EGFP ansAcEGFP, along serial passage. Passage numbers are indicated as 'Pn', 'P' for passage and 'n' for number.
표 Nucleotide and amino acid sequences of the linker and enterokinase cleavage sites inpBac9-PolhEKA vector.
표 Formation of fluorescent granules after infection of Sf9 cells with AcPolhEKA-EGFP. Sf9cells infected with AcEGFP and AcPolhEKA-EGFP were observed 3 days p. i. by fluorescent microscopy using an Axiophot Universal microscope (Zeiss, Germany) (Magnification: × 1,000).
표 SDS-PAGE analysis of Sf9 cells infected with AcPolhEKA-EGFP. At 3 days p. i., total cellular lysates were separated by 12% SDS-PAGE, and the gels were then stained with Coomassie Brilliant Blue. Lane M, protein molecular weight marker; 1, mock-infected Sf9 cells; 2, AcMNPV; 3, AcEGFP; 4, AcPolhEKA-EGFP. The arrow indicates the PolhEKA-EGFP fusion protein.
표 Localization of EGFP fluorescence during purification of the PolhEKA-EGFP fusion protein.Cells, cell-harvest step; Sonication, centrifugation after sonication; EK treatment, centrifugation after enterokinase digestion; SUP, supernatant; PPT, precipitated pellet. All EGFP photographs were taken on a UV-transilluminator in a dark room.
표 Distribution of EGFP fluorescence during purification of the PolhEKA-EGFP fusion protein.Cells, cell-harvest step; Sonication, centrifugation after sonication; EK treatment, centrifugation after enterokinase digestion; SUP, supernatant; PPT, precipitated pellet. All EGFP photographs were taken on a UV-transilluminator in a dark room. The lines indicate the shift of EGFP fluorescence from the pellet to the supernatant. The numbers in the columns indicate the relative fluorescence (%) at each purification step.
표 Protein profiles during purification of the PolhEKA-EGFP fusion protein. The proteins ateach step were separated by 12% SDS-PAGE, and the gels were then stained with Coomassie Brilliant Blue. Lane M, protein molecular weight marker; P, PPT S, SUP Cell, after cell harvest step; Soni, after sonication step EK, after enterokinase digestion step. The arrow indicates the purified EGFP protein.
표 Confirmation of the internal structure of transfer vectors expressing polyhedrin-PRRSVORFs fusion protein by restriction endonuclease digestion pattern. Lane: M, 1 Kb DNA Ladder; 1, DraⅠ; 2, HincⅡ; 3, HindⅢ; 4, SalⅠ.
표 Production of polyhedra in Sf9 cells infected with the recombinant AcMNPVs expressingpolyhedrin-PRRSV ORFs fusion protein.
표 SDS-PAGE analysis of the Sf9 cells infected with the recombinant AcMNPV, ApPRRSV7expressing polyhedrin-PRRSV ORF7 fusion protein. Lane: M, Protein molecular weight marker; 1, mock-infected Sf9 cells; 2, Sf9 cells infected with wtAcNPV; 3, Sf9 cells infected with ApPRRSV7.
표 SDS-PAGE analysis of the polyhedra produced by the recombinant AcMNPV, ApPRRSV7expressing polyhedrin-PRRSV ORF7 fusion protein. Lane: M, Protein molecular weight marker; 1, polyhedra produced by wtAcNPV; 2, polyhedra produced by ApPRRSV7.
표 SDS-PAGE analysis of the Sf9 cells infected with the recombinant AcMNPVs, ApPRRSV2,ApPRRSV4, ApPRRSV5 and ApPRRSV6 expressing polyhedrin-PRRSV ORFs fusion protein.
표 Protein hydrophobicity plot (A) and transmembrane helices prediction (B) of the PRRSVORF7.
표 Protein hydrophobicity plot (A), transmembrane helices prediction (B) and antibody epitopeprediction (C) of the PRRSV ORF5.
표 Protein hydrophobicity plot (A), transmembrane helices prediction (B) and antibody epitopeprediction (C) of the PRRSV ORF6.
표 Schematic diagram (A) and recursive PCR (B) of the hydrophobic regions from PRRSVORF5 and ORF6 amino acid sequences.
표 SDS-PAGE analysis of the Sf9 cells infected (A) and the polyhedra produced (B) with therecombinant AcMNPV, ApPolhEKA-PRRSV5 expressing polyhedrin-PRRSV ORF5 fusion protein.
표 SDS-PAGE analysis of the Sf9 cells infected (A) and the polyhedra produced (B) with the recombinant AcMNPV, ApPolhEKA-PRRSV6 expressing polyhedrin-PRRSV ORF6 fusion protein. Lane: M, Protein molecular weight marker; 1, mock-infected Sf9 cells; 2, Sf9 cells infected with AcMNPV; 3, Sf9 cells infected with ApPolhEKA-PRRSV6.
표 SDS-PAGE analysis of the Sf9 cells infected (A) and the polyhedra produced (B) with therecombinant AcMNPV, ApPolhEKA-PRRSV4△TmR expressing polyhedrin-PRRSV ORF4 fusion protein. Lane: M, Protein molecular weight marker; 1, mock-infected Sf9 cells; 2, Sf9 cells infected with AcMNPV; 3, Sf9 cells infected with ApPolhEKA-PRRSV4△TmR.
표 SDS-PAGE analysis of the Sf9 cells infected (A) and the polyhedra produced (B) with therecombinant AcMNPV, ApPolhEKA-PRRSV56 expressing polyhedrin-PRRSV ORF5-PRRSV ORF6 fusion protein. Lane: M, Protein molecular weight marker; 1, mock-infected Sf9 cells; 2, Sf9 cells infected with AcMNPV; 3, Sf9 cells infected with ApPolhEKA-PRRSV56.
표 SDS-PAGE analysis of the Sf9 cells infected (A) and the polyhedra produced (B) with therecombinant AcMNPV, ApPolhEKA-E0K expressing polyhedrin-SFV E0K fusion protein. Lane: M, Protein molecular weight marker; 1, mock-infected Sf9 cells; 2, Sf9 cells infected with AcMNPV; 3, Sf9 cells infected with ApPolhEKA-E0K.
표 SDS-PAGE analysis of the Sf9 cells infected (A) and the polyhedra produced (B) with therecombinant AcMNPV, ApPolhEKA-E1K expressing polyhedrin-SFV E1K fusion protein. Lane: M, Protein molecular weight marker; 1, mock-infected Sf9 cells; 2, Sf9 cells infected with AcMNPV; 3, Sf9 cells infected with ApPolhEKA-E1K.
표 SDS-PAGE analysis of the Sf9 cells infected (A) and the polyhedra produced (B) with therecombinant AcMNPV, ApPolhEKA-E2K expressing polyhedrin-SFV E2K fusion protein. Lane: M, Protein molecular weight marker; 1, mock-infected Sf9 cells; 2, Sf9 cells infected with AcMNPV; 3, Sf9 cells infected with ApPolhEKA-E2K.
표 SDS-PAGE analysis of the Sf9 cells infected (A) and the polyhedra produced (B) with therecombinant AcMNPV, ApPolhEKA-gC expressing polyhedrin-ADV gC fusion protein. Lane: M, Protein molecular weight marker; 1, mock-infected Sf9 cells; 2, Sf9 cells infected with AcMNPV; 3, Sf9 cells infected with ApPolhEKA-gC.
표 SDS-PAGE analysis of the Sf9 cells infected (A) and the polyhedra produced (B) with therecombinant AcMNPV, ApPolhEKA-gD expressing polyhedrin-ADV gD fusion protein. Lane: M, Protein molecular weight marker; 1, mock-infected Sf9 cells; 2, Sf9 cells infected with AcMNPV; 3, Sf9 cells infected with ApPolhEKA-gD.
표 SDS-PAGE analysis of the Sf9 cells infected with the recombinant AcMNPV,AcEasy-PolhEKA-PRRSV4△TmR expressing polyhedrin-PRRSV ORF4 fusion protein. Lane: M, Protein molecular weight marker; 1, mock-infected Sf9 cells; 2, Sf9 cells infected with AcMNPV; 3, Sf9 cells infected with AcEasy-PolhEKA-PRRSV4△TmR.
표 SDS-PAGE analysis of the Sf9 cells infected with the recombinant AcMNPV,AcEasy-PolhEKA-PRRSV5 expressing polyhedrin-PRRSV ORF5 fusion protein. Lane: M, Protein molecular weight marker; 1, mock-infected Sf9 cells; 2, Sf9 cells infected with AcMNPV; 3, Sf9 cells infected with AcEasy-PolhEKA-PRRSV5.
표 SDS-PAGE analysis of the Sf9 cells infected with the recombinant AcMNPV,AcEasy-PolhEKA-PRRSV6 expressing polyhedrin-PRRSV ORF6 fusion protein. Lane: M, Protein molecular weight marker; 1, mock-infected Sf9 cells; 2, Sf9 cells infected with AcMNPV; 3, Sf9 cells infected with AcEasy-PolhEKA-PRRSV6.
표 SDS-PAGE analysis of the Sf9 cells infected with the recombinant AcMNPV,AcEasy-PolhEKA-PRRSV56 expressing polyhedrin-PRRSV ORF5-PRRSV ORF6 fusion protein. Lane: M, Protein molecular weight marker; 1, mock-infected Sf9 cells; 2, Sf9 cells infected with AcMNPV; 3, Sf9 cells infected with AcEasy-PolhEKA-PRRSV56.
표 SDS-PAGE analysis of the Sf9 cells infected with the recombinant AcMNPV,AcEasy-PolhEKA-E0K expressing polyhedrin-SFV E0K fusion protein. Lane: M, Protein molecular weight marker; 1, mock-infected Sf9 cells; 2, Sf9 cells infected with AcMNPV; 3, Sf9 cells infected with AcEasy-PolhEKA-E0K.
표 SDS-PAGE analysis of the Sf9 cells infected with the recombinant AcMNPV,AcEasy-PolhEKA-E1K expressing polyhedrin-SFV E1K fusion protein. Lane: M, Protein molecular weight marker; 1, mock-infected Sf9 cells; 2, Sf9 cells infected with AcMNPV; 3, Sf9 cells infected with AcEasy-PolhEKA-E1K.
표 SDS-PAGE analysis of the Sf9 cells infected with the recombinant AcMNPV,AcEasy-PolhEKA-E2K expressing polyhedrin-SFV E2K fusion protein. Lane: M, Protein molecular weight marker; 1, mock-infected Sf9 cells; 2, Sf9 cells infected with AcMNPV; 3, Sf9 cells infected with AcEasy-PolhEKA-E2K.
표 SDS-PAGE analysis of the Sf9 cells infected with the recombinant AcMNPV,AcEasy-PolhEKA-gC expressing polyhedrin-ADV gC fusion protein. Lane: M, Protein molecular weight marker; 1, mock-infected Sf9 cells; 2, Sf9 cells infected with AcMNPV; 3, Sf9 cells infected with AcEasy-PolhEKA-gC.
표 SDS-PAGE analysis of the Sf9 cells infected with the recombinant AcMNPV,AcEasy-PolhEKA-gD expressing polyhedrin-ADV gD fusion protein. Lane: M, Protein molecular weight marker; 1, mock-infected Sf9 cells; 2, Sf9 cells infected with AcMNPV; 3, Sf9 cells infected with AcEasy-PolhEKA-gD.
표 SDS-PAGE analysis of the polyhedrin-PRRSV ORF5 fusion protein purified byelectro-elution. Lane: M, Protein molecular weight marker; 1, BSA 2.5 ㎍; 2, BSA 5.0 ㎍; 3, BSA 7.5 ㎍; 4, BSA 10.0 ㎍; 5, BSA 12.5 ㎍; 6, BSA 15.0 ㎍; 7, purified polyhedrin-PRRSV ORF5 fusion protein.
표 SDS-PAGE analysis of the polyhedrin-PRRSV ORF6 fusion protein purified byelectro-elution. Lane: M, Protein molecular weight marker; 1, BSA 2.5 ㎍; 2, BSA 5.0 ㎍; 3, BSA 7.5 ㎍; 4, BSA 10.0 ㎍; 5, BSA 12.5 ㎍; 6, BSA 15.0 ㎍; 7, purified polyhedrin-PRRSV ORF6 fusion protein.
표 SDS-PAGE analysis of the polyhedrin-PRRSV ORF5-PRRSV ORF6 fusion protein purifiedby electro-elution. Lane: M, Protein molecular weight marker; 1, BSA 2.5 ㎍; 2, BSA 5.0 ㎍; 3, BSA 7.5 ㎍; 4, BSA 10.0 ㎍; 5, BSA 12.5 ㎍; 6, BSA 15.0 ㎍; 7, purified polyhedrin-PRRSV ORF5-PRRSV ORF6 fusion protein.
표 Purification of the anti-PRRSV ORF5 antibody using Protein G column.
표 Purification of the anti-PRRSV ORF6 antibody using Protein G column.
표 Purification of the anti-PRRSV ORF5-PRRSV ORF6 antibody using Protein G column.
표 바이러스 주요 항원 유전자
표 각 바이러스 주요항원 유전자의 PCR 증폭용 primer 정보
표 각 항원유전자의 PCR 조건
표 PRRSV PCR 산물
표 HCV PCR 산물
표 클로닝 된 벡터의 명명
표 클로닝 된 벡터의 Map
표 PRRSV 항원 유전자 ORF2-6을 포함하는 전체염기서열 및 아미노산 서열
표 PRRSV 전체 염기서열내 추정 ORF 모식도
표 PRRSV의 염기서열 및 아미노산 상동성 비교
표 ADV 항원 유전자 gB의 염기서열 및 아미노산 서열
표 ADV 항원 유전자 gD의 염기서열 및 아미노산 서열
표 ADV의 염기서열 및 아미노산 상동성 비교
표 gB 유전자 서열을 토대로 한 계통수 비교
표 gC 유전자 서열을 토대로 한 계통수 비교
표 gD 유전자 서열을 토대로 한 계통수 비교
표 감염 후 4일 된 재조합 바이러스감염세포
표 재조합 바이러스에 대한 PCR 결과
표 PRRSV 각 항원 유전자를 가진 재조합 바이러스 감염세포에 대한 PCR 및 RT-PCR 결과
표 재조합 바이러스의 명명
표 재조합 바이러스에 감염된 Sf9 세포에서 단백질 발현 분석
표 재조합 바이러스에 감염된 Sf9 mimic 세포에서 단백질 발현 분석
표 재조합 바이러스에 감염된 Bm5 세포에서의 단백질 발현 분석
표 PRRSV 항원단백질 (ORF2 ∼ 7)의 시간별 발현 분석
표 Bm5 세포에서의 CSFV 항원단백질의 발현 분석
표 CSFV 항원단백질 E2의 시간별 발현 분석
표 ADV 주요 항원 유전자의 transmembrane domain 예측
표 Bm5 세포에서 ADV 남양주주 gD의 발현 분석
표 ADV 당단백질 gD의 glycosylation 부위 예측
표 Bm5 세포에서 Tunicamycin에 의한 ADV 당단백질 gD의 발현 분석
표 PRRSV 재조합 바이러스 조합접종에 의한 Sf9 mimic 세포에서의 단백질 발현 분석
표 PRRSV 재조합 바이러스 조합접종에 의한 Sf9 세포에서의 단백질 발현 분석
표 CSFV 재조합 바이러스 조합접종에 의한 Bm5 세포에서의 단백질 발현 분석
표 PRRSV ORF4-5-6의 융합을 위한 모식도 및 클로닝
표 rApRSV-ORF456에 감염된 Sf21 세포에서 융합단백질 발현 분석
표 주요 항원유전자 CSFV-E2, PRRSV-ORF5, ADV-gD의 융합을 위한 모식도 및 클로닝
표 Bm5 세포에서 다중 표면항원단백질 발현 분석
표 SUMO fusion에 의한 재조합 바이러스의 명명
표 SUMO fusion에 의한 Bm5 세포에서의 단백질 발현 분석
표 SUMO fusion에 의한 Bm5 세포에서의 단백질 발현 분석
표 Bm5 세포에서 Tunicamycin에 의한 단백질 발현 분석
표 SUMOstar fusion을 위한 모식도
표 SUMOstar fusion에 의한 재조합 바이러스의 명명
표 SUMOstar fusion에 의한 Bm5 세포에서의 단백질 발현 분석
표 ProFoldTM-ER1의 구조
표 분자 chaperon에 의한 재조합 바이러스의 명명
표 분자 chaperon을 공급하는 vector내 GFP 발현 확인
표 분자 chaperon에 의한 Sf21 세포에서의 단백질 발현 분석
표 분자 chaperon을 공급하는 vector내 GFP 발현 확인
표 SUMOstar와 분자 chaperon 동시 도입에 의한 ORF5 단백질의 발현 분석
표 B. mori larvae에서 SUMO fusion에 의한 주요 항원단백질 발현 분석
표 B. mori larvae의 fat body에서 분리한 주요 항원단백질 분석
표 B. mori larvae에서 SUMOstar fusion에 의한 주요 항원단백질 발현 분석
표 B. mori larvae에서 ADV 남양주주 gD의 발현 분석
표 다각체 단백질 일부와 CSFV의 E2K를 융합한 벡터 맵
표 다각체단백질일부와 CSFV E2-ΔTMR 단백질의 융합의 발현
표 SUMOstar fusion에 의한 재조합 바이러스의 명명
표 마우스에 재조합 표면항원 접종 방법
표 재조합 표면항원단백질 (CSFV E2)의 접종
표 용해형 재조합 표면항원 E2, 융합형 재조합 표면항원 E2 및 분리 정제한 E2로 면역유도한 동물 혈청의 CSFV E2에 대한 bloking 효과.
표 혈청의 바이러스 중화능 평가

과제명(ProjectTitle) :	-
연구책임자(Manager) :	-
과제기간(DetailSeriesProject) :	-
총연구비 (DetailSeriesProject) :	-
키워드(keyword) :	-
과제수행기간(LeadAgency) :	-
연구목표(Goal) :	-
연구내용(Abstract) :	-
기대효과(Effect) :	-

내보내기 구분	파일저장 인쇄 메일전송
구성항목	기본정보 상세정보 관리번호, 제목(한글), 저자명(한글), 발행일자, 전자원문, 초록(한글), 초록(영문) 관리번호, 제목(한글), 제목(영문), 저자명(한글), 저자명(영문), 주관연구기관(한글), 주관연구기관(영문), 발행일자, 총페이지수, 주관부처명, 과제시작일, 보고서번호, 과제종료일, 주제분류, 키워드(한글), 전자원문, 키워드(영문), 입수제어번호, 초록(한글), 초록(영문), 목차
저장형식	Text(ASCII format) Excel format
메일정보	받는사람 (필수) @ 보내는사람 (선택) @ 제목 내용 KISTI 검색결과 이메일 서비스
안내	총 건의 자료가 검색되었습니다. 다운받으실 자료의 인덱스를 입력하세요. (1-10,000) 검색결과의 순서대로 최대 10,000건 까지 다운로드가 가능합니다. 데이타가 많을 경우 속도가 느려질 수 있습니다.(최대 2~3분 소요) 다운로드 파일은 UTF-8 형태로 저장됩니다. 파일의 내용이 제대로 보이지 않을실 때는 웹브라우저 상단의 보기 -> 인코딩 -> 자동선택 여부를 확인하십시오. ~ Text(ASCII format) Excel format

연합인증

[국가R&D연구보고서] 곤충생체반응기를 이용한 동물백신 생산 및 분리정제 시스템 개발
vaccPirnoedsucutsioinngoafnhiingshelyct-efbfieocrteivaectoarniamnadl its purification system development 원문보기

초록

Abstract ▼

목차 Contents

표/그림 (160)

표/그림 (160)

참고문헌 (25)

연구과제 타임라인

관련 콘텐츠

원문 보기

이 보고서와 함께 이용한 콘텐츠

AI-Helper ※ AI-Helper는 오픈소스 모델을 사용합니다.

선택된 텍스트

연합인증

[국가R&D연구보고서] 곤충생체반응기를 이용한 동물백신 생산 및 분리정제 시스템 개발 vaccPirnoedsucutsioinngoafnhiingshelyct-efbfieocrteivaectoarniamnadl its purification system development 원문보기

초록

Abstract ▼

목차 Contents

표/그림 (160) 모든 표/그림 보기

표/그림 (160) 슬라이드로 보기

참고문헌 (25)

연구과제 타임라인

전체(0) 논문(0) 특허(0) 보고서(0)

전체(0) 논문(0) 특허(0) 보고서(0)

관련 콘텐츠

원문 보기

이 보고서와 함께 이용한 콘텐츠

AI-Helper ※ AI-Helper는 오픈소스 모델을 사용합니다.

선택된 텍스트

[국가R&D연구보고서] 곤충생체반응기를 이용한 동물백신 생산 및 분리정제 시스템 개발
vaccPirnoedsucutsioinngoafnhiingshelyct-efbfieocrteivaectoarniamnadl its purification system development 원문보기

표/그림 (160)

표/그림 (160)