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( Bit Na Rae Yun ),( Sung Min Bae ),( Jun Beom Lee ),( Hee Jung Kim ),( Soo Dong Woo ) 한국잠사학회 2011 International Journal of Industrial Entomology Vol.23 No.1
The Pseudorabies (PR), also called Aujeszky`s disease (AD), 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. Pseudorabies virus (PRV) virions contain several envelope glycoproteins. Among them, gB, gC and gD are regarded as the major immunogenic proteins. We expressed these glycoproteins using the bacterial expression system and analyzed recombinant proteins. Expression of glycoproteins gC and gD were observed on SDS-PAGE or Western blot analysis, but gB was not. Optimal concentration of IPTG and inducing time were determined as 1.0 mM and 4 h, respectively, for the expression of both gC and gD in E. coli. A sodium dodecyl sulfate (SDS) was the most efficient detergent in solubilizing insoluble recombinant protein.
Bit Na Rae Yun,Sung Min Bae,Jae Bang Choi,Jun Beom Lee,Hee Jung Kim,Yeon Ho Je,Byung Rae Jin,Soo Dong Woo 한국응용곤충학회 2011 한국응용곤충학회 학술대회논문집 Vol.2011 No.05
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.
Expression of Pseudorabies Virus Glycoproteins gB, gC and gD using Insect Expression System
Yun, Bit Na Rae,Gwak, Won Seok,Lee, Ji Hoon,Woo, Soo Dong Korean Society of Sericultural Science 2017 International Journal of Industrial Entomology Vol.35 No.2
Porcine pseudorabies virus (PRV) causes the Aujeszky's disease (AD) which is economically important disease in the swine industry worldwide. Killed or live vaccines have been used to control this disease, but their efficacy and side effects remain problems to be solved. To solve these problems, in this study, production of recombinant PRV glycoprotein gB, gC and gD was investigated in insect expression system. Glycoprotein gB, gC and gD are regarded as the major immunogenic antigens in PRV. Abundant production and immunogenicity of glycoprotein gB, gC and gD were confirmed by SDS-PAGE and Western blot analysis, respectively. Optimal infection dose and time were also determined for the production of each recombinant PRV glycoprotein. Confirmation of glycosylation of recombinant gB, gC and gD suggested their usefulness as antigens for the development of diagnosis kit or vaccines for Aujeszky's disease.
Expression of Pseudorabies Virus (PRV) Glycoproteins gB, gC and gD using Bacterial Expression System
Yun, Bit-Na-Rae,Bae, Sung-Min,Lee, Jun-Beom,Kim, Hee-Jung,Woo, Soo-Dong Korean Society of Sericultural Science 2011 International Journal of Industrial Entomology Vol.23 No.1
The Pseudorabies (PR), also called Aujeszky's disease (AD), 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. Pseudorabies virus (PRV) virions contain several envelope glycoproteins. Among them, gB, gC and gD are regarded as the major immunogenic proteins. We expressed these glycoproteins using the bacterial expression system and analyzed recombinant proteins. Expression of glycoproteins gC and gD were observed on SDS-PAGE or Western blot analysis, but gB was not. Optimal concentration of IPTG and inducing time were determined as 1.0 mM and 4 h, respectively, for the expression of both gC and gD in E. coli. A sodium dodecyl sulfate (SDS) was the most efficient detergent in solubilizing insoluble recombinant protein.
Expression of Pseudorabies Virus Glycoproteins gB, gC and gD using Insect Expression System
( Bit Na Rae Yun ),( Won Seok Gwak ),( Ji Hoon Lee ),( Soo Dong Woo ) 한국잠사학회 2017 International Journal of Industrial Entomology Vol.35 No.2
Porcine pseudorabies virus (PRV) causes the Aujeszky's disease (AD) which is economically important disease in the swine industry worldwide. Killed or live vaccines have been used to control this disease, but their efficacy and side effects remain problems to be solved. To solve these problems, in this study, production of recombinant PRV glycoprotein gB, gC and gD was investigated in insect expression system. Glycoprotein gB, gC and gD are regarded as the major immunogenic antigens in PRV. Abundant production and immunogenicity of glycoprotein gB, gC and gD were confirmed by SDS-PAGE and Western blot analysis, respectively. Optimal infection dose and time were also determined for the production of each recombinant PRV glycoprotein. Confirmation of glycosylation of recombinant gB, gC and gD suggested their usefulness as antigens for the development of diagnosis kit or vaccines for Aujeszky’s disease. ⓒ 2017 The Korean Society of Sericultural Sciences Int. J. Indust. Entomol. 35(2), 118-122 (2017)
Sung Min Bae,Bit Na Rae Yun,Tae Young Shin,Jae Bang Choi,Yeon Ho Je,Byung Rae Jin,Soo Dong Woo 한국응용곤충학회 2010 한국응용곤충학회 학술대회논문집 Vol.2010 No.10
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.
Partial Polyhedrin Fusion Expression Enhance the Production of Recombinant Protein in Baculovirus
Sung Min Bae,Bit Na Rae Yun,Jun Beom Lee,Hee Jung Kim,Tae Young Shin,Jae Bang Choi,Yeon Ho Je,Byung Rae Jin,Soo Dong Woo 한국응용곤충학회 2011 한국응용곤충학회 학술대회논문집 Vol.2011 No.05
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.
Enhanced Expression of the ORF2 Protein of Porcine Circovir Type2 using Baculovirus
Jun Beom Lee,Sung Min Bae,Bit Na Rae Yun,Hee Jung Kim,Jae Bang Choi,Won Il Heo,Tae Young Shin,Yeon Ho Je,Byung Rae Jin,Soo Dong Woo 한국응용곤충학회 2011 한국응용곤충학회 학술대회논문집 Vol.2011 No.05
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.
Hyun Na Koo,Sung Min Bae,Tae Young Shin,Jae Bang Choi,Bit Na Rae Yun,Jae Young Choi,Kwang Sik Lee,Jong Yul Roh,Yeon Ho Je,Byung Rae Jin,Soo Dong Woo 한국응용곤충학회 2010 한국응용곤충학회 학술대회논문집 Vol.2010 No.05
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.
구현나,배성민,Jae Bang Choi,신태영,Bit Na Rae Yun,최재영,이광식,노종열,제연호,진병래,Sung Sik Yoo,김재수,In Joong Yoon,우수동,Young In Kim 한국응용곤충학회 2011 Journal of Asia-Pacific Entomology Vol.14 No.1
To characterize the NYJ strain of pseudorabies virus (PRV; Alphaherpesvirus of swine) isolated from the serum of an infected swine in Korea, the nucleotide sequence of three major glycoproteins (gB, gC, and gD)was analyzed. The expression of most potent immunogenic glycoprotein (gD) was also investigated using a Bombyx mori nucleopolyhedrovirus (BmNPV) expression system. The length of the glycoprotein genes corresponding to gB, gC, and gD of the NYJ strain were 2751 bp, 1443 bp, and 1203, respectively, and their identity ranged from 94.2% to 99.8% when compared with other strains. Phylogenetic analyses using these sequences showed that the NYJ strain forms a distinct branch with high bootstrap support. A novel transfer vector (pBmKSK4) was engineered with the polyhedrin promoter of BmNPV and a 6xHis tag to express glycoprotein gD in Bm5 cells and silkworm, B. mori, larvae. The immunogenicity of recombinant gD was demonstrated by its specific detection in both Bm5 cells and silkworm larvae by porcine anti-PRV antibody. The results of this study have implications both for the taxonomy of Korean PRV strains and vaccine development.