The baculovirus-insect cell expression system has been widely used method for the recombinant protein expression. The present study has several limitation. In this study, we constructed vectors consisting of transcriptional enhanced factor and promoter that improve the expression level. To confirm the usefulness of these vector system, Human papillomavirus (HPV) VLPs have been expressed by baculovirus hyper expression system. HPV VLPs were purified using a CaptoTM Core 700 (GE Healthcare Life Sciences) chromatography approach. Baculovirus hyper expression system production efficiency was influenced by the HPV VLPs production. HPV VLPs vaccination to BALB/c mice induced the generation of antibody confirmed by ELISA. This study could provide improvements on the vaccine production for the development of VLP vaccines high expression of useful heterologous proteins.

강원도농업기술원에서는 2003년 무름병에 비교적 강하고 화색과 화형이 우수하고 장미 분홍색을 가진 Zantedeschia rehmanni × hybrid ‘Super Gem’과 연한 노랑색 품종 Z. × hybrid ‘Black Magic’을 각각 모본과 부본으로 하여 인공 교배 하였다. 2006년에 개화특성을 검정하여 화색과 화형이 좋은 ‘GZ0616’를 선발하였으며, 2007년에 포장 재배하여 자구 증식률, 초장과 초세 등 1차 특성검정 후 2차 선발하였다. 2013부터 2015년까지 특성검정과 재배시험을 통하여 균일성과 안정성이 인정되어 ‘강교C4-6호’로 최종 선발되었으며, 2017년 2월에 ‘립스마일(Lip Smile)’로 품종등록 되었다. 화포 외부의 주 색은 연노랑바탕 적자색(Y2C+RP79C)이며, 화포 높이는 8.5cm, 폭은 6.2cm로 대형화이다. 개화소요일수는 64.3일, 초장은 66.0cm, 괴경은 80.0g이다. 기호도 평가에서도 ‘Captain Rosette’와 유사하였으며, 절화용으로 이용 가능하다.

MA Al alloys are examined to determine the effects of alloying of Mg and Cu and rolling on tensile deformation behavior at 748 K over a wide strain rate range(10−4-103/s). A powder metallurgy aluminum alloy produced from mechanically alloyed pure Al powder exhibits only a small elongation-to-failure(εf < ~50%) in high temperature(748 K) tensile deformation at high strain rates( = 1-102/s). εf in MA Al-0.5~4.0Mg alloys increases slightly with Mg content(εf = ~140% at 4 mass%). Combined addition of Mg and Cu(MA Al-1.5%Mg-4.0%Cu) is very effective for the occurrence of superplasticity(εf > 500%). Warm-rolling(at 393-492 K) tends to raise εf. Lowering the rolling-temperature is effective for increasing the ductility. The effect is rather weak in MA pure Al and MA Al-Mg alloys, but much larger in the MA Al-1.5%Mg-4.0%Cu alloy. Additions of Mg and Cu and warm-rolling of the alloy cause a remarkable reduction in the logarithm of the peak flow stress at low strain rates ( < ~1/s) and sharpening of microstructure and smoothening of grain boundaries. Additions of Mg and Cu make the strain rate sensitivity(the m value) larger at high strain rates, and the warm-rolling may make the grain boundary sliding easier with less cavitation. Grain boundary facets are observed on the fracture surface when εf is large, indicating the operation of grain boundary sliding to a large extent during superplastic deformation.

Binary Ti-Al alloys below 51.0 mass%Al content exhibit a breakaway, transferring from parabolic to linear rate law. The second Al2O3 layer might have some protectiveness before breakaway. Ti-63.1 mass%Al oxidized at 1173 K under parabolic law. Breakaway oxidation is observed in every alloy, except for Ti-63.1 mass%Al. After breakaway, oxidation rates of the binary TiAl alloys below 34.5 mass%Al obey almost linear kinetics. The corrosion rate of Ti-63.1 mass%Al appears to be almost parabolic. As content greater than 63.0 mass% is found to be necessary to form a protective alumina film. Addition of Mo improves the oxidation resistance dramatically. No breakaway is observed at 1123 K, and breakaway is delayed by Mo addition at 1173 K. At 1123 K, no breakaway, but a parabolic increase in mass gain, are observed in the Mo-added TiAl alloys. The binary Ti-34.5 mass%Al exhibits a transfer from parabolic to linear kinetics. At 1173 K, the binary alloys show vary fast linear oxidation and even the Mo-added alloys exhibit breakaway oxidation. The 2.0 mass%Mo-added TiAl exhibits a slope between linear and parabolic. At values of 4.0 and 6.0 mass% added TiAl alloys, slightly larger rates are observed than those for the parabolic rate law, even after breakaway. On those alloys, the second Al2O3 layer appears to be persistently continuous. Oxidation resistance is considerably degraded by the addition of Mn. Mn appears to have the effect of breaking the continuity of the second Al2O3 layer.

오늘날 물류 서비스 산업에서는 수요예측을 통해 불확실성을 줄여나가는 것이 경영상 매우 중요한 이슈로 제기되고 있다. 비교적 시장 점유율이 견고하게 유지되는 제조산업과는 달리 물류 서비스 산업은 매우 빠른 속도로 시장이 성장하고 변화하기 때문에, 시장 환경의 변화를 반영하여 정확한 수요 예측에 기반한 적절한 물류 서비스 공급을 위한 운송 및 인력 공급 계획을 수립하여 운용하여야 한다. 본 연구는 물류 서비스 업계를 위하여 추세 요소, 계절 요소 등 수요에 영향을 미치는 요소를 분석하고, 영향 효과를 산출하여 물류 서비스 산업을 위한 수요예측 시스템에 반영하는 방안을 연구하였다. 특히 지역 특산물, 기상효과, 음력으로 발생하는 명절 효과 등 수요를 크게 변화시키는 중요 영향 요소에 의한 수요 변동을 수요 예측에 활용하는 방안을 도출함으로써, 물류 서비스 산업계의 수요 예측을 위하여 분해법(Decomposition Method)을 제시하였다.

Polyhedrin is the major component of the nuclear viral occlusions produced during replication of the baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV). To enhance the production efficiency of foreign protein in baculovirus expression system, the effects of various polyhedrin fragments were investigated by fusion expressing them with the enhanced green fluorescent protein (EGFP). Recombinant viruses were generated to express EGFP fused with polyhedrin fragments based on the previously reported minimal region for self-assembly and the KRKK nuclear localization signal (NLS). The marked increase of EGFP by these fusion expressions was confirmed through protein and fluorescence intensity analyses. Among the fusion-expressed protein in nucleus and cytoplasm, the most hyper-expression was observed in the fusion of amino acids 19 to 110 and 32 to 59 of polyhedrin. Also these fragments, some degradation of only the fused polyhedrin was observed in the fusion of amino acids 19 to 85 and 32 to 85. The production of E2 protein, which is a major antigen of classical swine fever virus, was dramatically increased by fusion expression with polyhedrin amino acids 19 to 110, and its preliminary immunogenicity was verified using experimental guinea pigs. The production of luciferase was approximately two folds increased by fusion expression with polyhedrin amino acids 32 to 59, and its activity was measured using Luminometer. This study suggests a new option for higher expression of useful foreign recombinant protein using the partial polyhedrin fusion expression in baculovirus.

To enhance the production efficiency of foreign protein in baculovirus expression system, the effects of polyhedrin fragments were investigated by fusion expression them with the enhanced green fluorescence protein (EGFP). Recombinant viruses were generated to express EGFP fused with polyhedrin fragments based on the minimal region for self-assembly and the KRKK nuclear localization signal (NLS). The increase of EGFP production by fusion expressions was confirmed through protein and fluorescence intensity analyses. The importance of nuclear localization for enhanced production of EGFP was shown by the mutation of the NLS within the fused polyhedrin fragment. Among the fusion expressed protein in cytoplasm, the most hyper-expression was observed in the fusion of amino acids 32 to 59 of polyhedrin. Polyhedrin fragment fusion expression with classical swine fever virus E2 protein also resulted hyper-enhanced expression of E2 protein. However, the fusion expression of porcine circovirus ORF2 with polyhedrin fragment did not show significant enhance of ORF2 production. These results suggested that the enhancement of foreign protein production when fused with polyhedrin is caused by the enhanced stability of expressed protein.

Porcine Circovirus Type2 (PCV2), a single-stranded DNA virus associated with Postweaning multisystemic wasting syndrome(PMWS) of swine, has two major open reading frames, ORF1 and ORF2. The genomic size and molecular weight of ORF2 is respectively 699bp, 28kDa. ORF2 encodes the capsid protein (structural protein) that has type-specific epitopes and is very immunogenic and associated with the induction of neutralizing antibodies, suggesting its potential use in diagnostic assays as well as vaccine development. For efficient production of the capsid proteins, we expressed the PCV2 ORF2 gene with baculovirus in the insect cells. In this study, PCV2 ORF2 was appropriately ligated into the baculovirus transfer vector, pBacPAK9 and pB9-Acpol19-110-EK. Sf21 cells were transfected with a mixture of the purified recombinant transfer vector and bAcGOZA. We generated and purified recombinant viruses containing PCV2 ORF2, and named rAc-B9-PCV2ORF2 and rAc-B9-19-110-EK-PCV2ORF2, respectively. Expression levels of capsid fusion proteins with a partial polyhedrin region of AcNPV more increased than recombinant proteins from non-fusion expressed. Also, expression efficiency increased over time and differed at MOI. As a results, fusion expression of porcine circovirus type2 ORF2 using baculovirus could be utilized as an alternative expression method to produce recombinant antigen against PCV2 infection and is worthy of further investigation.

Aujeszky's disease (AD), also called pseudorabies, is an infectious viral disease caused by an alpha herpes virus and has domestic and wild pigs, as well as a wide range of domestic and wild animals, as the natural host. Aujeszky's disease virus (ADV) virions contain several envelope glycoproteins. Among them, glycoproteins gB, gC and gD are regarded as the major immunogenicity proteins and the antibodies induced by them can neutralize virus in vitro or in vivo. In this study, we investigated expression of these glycoproteins using the bacterial and baculovirus expressionn system. Successful expression of ADV glycoproteins in E. coli was confirmed by SDS-PAGE and Western blot analysis and their optimal expression condition was determined. However, the recombinant proteins generated in the bacterial expression system which lacks glycosylation process frequently lose their biological activity. We tried to express the ADV glycoproteins using the baculovirus expression vector system. The recombinant gB, gC and gD were detected at approximately 100, 60 and 50 kDa on SDS-PAGE and Western blotting, respectively. The optimal expression conditions were determined for MOI(multiplicity of infection) and post-infection days. One MOI and 4 or 5 days post-infection were the best conditions for the expression of the ADV glycoproteins in Sf21 cells. We are currently investigating the antigenicity of recombinant proteins using experimental animals.

Polyhedrin is the major component of the nuclear viral occlusions produced during replication of the baculovirus Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV). To enhance the expression level of baculovirus vector system, we constructed several fusion vectors using various fragments of the polyhedrin. The polyhedrin fragments were genetically fused to the enhanced green fluorescent protein (eGFP) under the control of polyhedrin promoter, and their expressions were analyzed in Sf21 insect cells. Expression of the fusion protein was identified by SDS-PAGE and Western blot analysis using anti-GFP and anti-Polyhedrin. The expression level of eGFP was markedly increased by the fusion of partial polyhedrin. Also, the fluorescence intensity of fusion proteins was higher than that of non-fusion protein. Confocal laser scanning microscopy demonstrated that fusion proteins were localized to the cytosol or nucleus of insect cells. In additional, the glycoprotein E2 (gE2) of classical swine fever virus (CSFV) expressed by the these vectors was dramatically increased and its immunogenicity was proofed using experimental animal guinea pigs that were immunized with the partial polyhedrin containing gE2. This study provides a new option for the higher expression of useful foreign recombinant protein by using the partial polyhedrin in BEVS.

The Classical Swine Fever Virus (CSFV) is a member of the Pestivirus genus of the Flaviviridae. The polyprotein composed of eight nonstructural and four structural proteins (nucleocapsid protein C and three envelope glycoprotein E0, E1 and E2). E2, the most immunogenic of the CSFV glycoproteins, induces a protective immune response in swine. The objective of this study was to enhance production of E2 protein by fusion with partial polyhedrin of nucleopolyhedrovirus in insect cells. We generated various E2 form by fusion with different combinations of the partial polyhedrin and deletion of the C-terminal transmembrane region (TMR). Expression of the E2 protein was identified by SDS-PAGE and Western blot analysis using anti-CSFV E2 monoclonal antibodies. The fusion expression of an E2 protein with the partial polyhedrin markedly increased expression levels. Also, expression of E2 proteinlacking TMR region was higher than that of intact E2 protein. As a result, the fusion expression of E2 protein lacking the C-terminal TMR with partial polyhedrin was significantly increased in insect cells. These suggest that the fusion of target foreign protein with partial polyhedrin could enhance significantly the production of target protein.

The porcine reproductive and respiratory syndrome virus (PRRSV) has three major structural proteins which designated as GP4, GP5, and M. They have been considered very important to arouse the humoral and cellular immune responses against PRRSV infection and proposed to be the excellent candidate proteins in the design of PRRS bioengineering vaccine. However, the PRRSV structural proteins are produced in low levels in the infected cells because it forms insoluble protein and possesses several transmembrane regions. To overcome this problem, we fused the GP4, GP5, and M with SUMO (Small ubiquitin-related modifier), and expressed the fused gene in Bm5 cells and silkworm larvae. Expression of the proteins were analyzed by 12% SDS-PAGE and western blotting using 6xHis tag and porcine anti-PRRSV antibodies. In results, SUMO fused proteins were expressed at a high level in Bm5 cells. The levels of protein using the silkworm larvae is higher than that using Bm5 cells. The fused protein was purified by Ni-NTA affinity chromatography. This study demonstrated that SUMO, when fused with PRRSV structural proteins, was able to promote its soluble expression. This may be a better method to produce PRRSV structural proteins for vaccine development.

The Classical Swine Fever Virus (CSFV) is a member of the Pestivirus genus of the Flaviviridae. The genome of CSFV is a positive single-stranded RNA molecule 12.3 kb and contains a single large open reading frame (ORF). The polyprotein composed of eight nonstructural and four structural proteins (nucleocapsid protein C and three envelope glycoprotein E0, E1 and E2). E2, the most immunogenic of the CSFV glycoproteins, induces a protective immune response in swine. To determine the characteristics of the CSFV, LOM strain, we investigated the nucleotide sequence of the glycoprotein E0, E1 and E2. Comparison of the LOM with the other strains revealed nucleotide sequence identity ranging from 97 to 98%. Expression of the glycoprotein E2 was identified by SDS-PAGE and Western blot analysis using anti-CSFV E2 monoclonal antibodies in Sf21 cells. The expression levels of glycoprotein E2 were observed from day 3 and 5 days maximum. In addition, its expression efficiency by media and cell line was investigated. The result showed that High-Five cells and Grace’s insect media for Sf21 were the best conditions for the expression of the glycoprotein E2.

Background : Codonopsis lanceolata is a perennial plant of Campanulaceae and mainly distributed in East Asia such as Korea, China, and Japan. C. lanceolata has a unique taste and aroma, and it is rich in minerals such as phosphorus and calcium, and vitamin B1 and B2, so our ancestors used the plant as medicinal herb and edible vegetable. However, systematic cultivation and development of varieties have not been achieved compared to demand or high added value. The genetic diversity and relationship analysis of the plants help to increase the efficiency of breeding through genetic variation. Methods and Results : Ten species of Codonopsis plants were used as materials and DNA was extracted from each 4 individuals per species and quantified at a concentration of 10 ng /㎕. The extracted DNA was pooled by species and PCR was performed using the EST-SSR marker developed based on C. lanceolata in the previous study. PCR amplification was carried out using a denaturation at 94℃ for 30 sec, annealing at 58℃ for 30 sec and extension at 72℃ for 30 sec, repeated for 35 total cycles. The PCR products were separated in a 4% agarose gell at 100 V for 40 min. Conclusion : In this study, C. lanceolata collections was determined among several Codonopsis species using these molecular marker. It is expected that the data of this study can be used as reference for genetic polymorphism analysis and related gene studies of Codonopsis species.

Background : Codonopsis lanceolata is a perennial plant of Campanulaceae with characteristic flavor and aroma and this plant has saponin, flavonoid, and inulin, which are reported to have physiological activity and antioxidant activity. In contrast, breeding or study of C. lanceolata varieties had not been done for a long time. Genetic polymorphism and phylogenetic relationship analysis of the plants by region of the crops can help the collection of genetic backgroud data for variety development. Methods and Results : In this study, we collected 26 C. lanceolata lines (95 individual plants) from 26 regions in Korea. We genotyped the collected lines using SSR markers developed in the previous study and analyzed the population structure based on the results. Population structures were analyzed using model-based STRUCTURE software (version 2.3.4) using the following parameters: Number of clusters (K) set = 1 to 12; Number of Iterations = 5; Length of Burning Period = 100,000; Number of MCMC (Markov Chain Monte Carlo) Reps after Burnin = 100,000. As a result, Of the 26 collections, were genetically grouped into 6 or 7 groups. Conclusion : The 26 C. lanceolata collections (95 individual plants) were genetically grouped but not grouped by collected regions. These results suggest that C. lanceolata has diverse genetic backgrounds and this data could be used as a basis for genetic polymorphism analysis of Codonopsis species.