박테리오파지 E3의 Major Capsid Protein을 만드는 유전자의 Mapping 및 염기서열 분석

배수진,명희준,Bae, Soo-Jin,Myung, Hee-Joon 한국미생물학회 1999 미생물학회지 Vol.35 No.4

박테리오파지 E3가 만드는 plaque은 그 지름이 약 1㎝정도이고 대단히 빠르게 성장한다. 구조 단백질 중 가장 많은 copy를 가지는 major capsid 단백질을 발현하는 유전자를 조절하는 promoter가 가장 효율적일 것이라 생각되며, 이 promoter를 찾기 위하여 먼저 이 유전자를 mapping하였다. 정제한 파지 입자로부터 major capsid 단백질을 분리하여 그 N-terminal amino acid 서열을 확인하였고, 그에 해당하는 degenerate oligonucleotide probe를 이용하여 E3의 genomic library로부터 major capsid 단백질을 발현하는 유전자를 함유하는 clone을 찾았다. 이 clone의 DNA 서열 분석을 통하여 major capsid 단백질을 발현하는 유전자를 확인하였으며, 이는 E3 genome에서 약 72%에 mapping 되었다. 이 gene을 조절하는 promoter의 성질을 고찰하기 위하여 E3의 성장이 rifampicin에 의하여 영향을 받는지 확인한 결과 E3는 자기 고유의 RNA polymerase를 가지고 있음을 알 수 있었다. Bacteriophage E3 grows very rapidly and forms a large size plaque with a diameter of 1 cm. The promoter controlling the expression of the gene encoding the major capsid protein is thought to be most efficient. To find out this promoter, this gene was mapped in the genome according to the following procedure. The major capsid protein was purified from phage particle and the N-terminal amino acid sequence was revealed. Based on this sequence,a degernerate oligonucleotide probe was designed and used for screening of the genomic DNA fragments. From the DNA sequence of the selected clone, the gene encoding the major capsid protein was mapped at 70% of E3 genome. The expression of this gene was not sensitive to rifampicin which indicated the presence of E3's own RNA polymerase.

Baculovirus Vector System에 의해 발현된 재조합 Pseudorabies Virus Major Capsid Protein의 면역원성

전무형,안동준,장경수,조용성,박종현,송재영,현방훈,안수환 충남대학교 생물공학연구소 1997 생물공학연구지 Vol.5 No.-

The recombinant pseudorabies virus major capsid protein (rMCP) was produced by expression of the MCP gene in Sf-9 cell using baculovirus transfer vector system. Following evaluation of the immunochemical properties of the rMCP, the immunogenicity of the recombinant subunit protiens were investigated in guinea pig and swine to obtain the preliminary guide line for the subunit vaccine using rMCP and gP50. It was proved that ultrasonication and 30% ammonium sulfate was most efficient to concentrate and purify the protein. The rMCP was safe in mice, guinea pigs and piglets. In guinea pigs, rMCP mixed with various adjuvants induced substantial degree of serum neutralizing antibody titers, but revealed incomplete protecivity against challenge. In swine, the combination of rMCP and gP50 showed the higher serum neutralizing antibody titers and cellular immune responses than rMCP alone. However, the protectivity was lower in comparison with the commercial gI-deleted inactivated vaccine. We expect these results to contribute to characterization of MCP gene of Korean isolate of PRV and to ultilize as preliminary information for prodution and evaluation of PRV recombinant subunit vaccines.

DNA vaccine encoding myristoylated membrane protein (MMP) of rock bream iridovirus (RBIV) induces protective immunity in rock bream (<i>Oplegnathus fasciatus</i>)

Jung, Myung-Hwa,Nikapitiya, Chamilani,Jung, Sung-Ju Elsevier 2018 Vaccine Vol.36 No.6

<P><B>Abstract</B></P> <P>Rock bream iridovirus (RBIV) causes severe mass mortalities in rock bream (<I>Oplegnathus fasciatus</I>) in Korea. In this study, we investigated the potential of viral membrane protein to induce antiviral status protecting rock bream against RBIV infection. We found that fish administered with ORF008L (myristoylated membrane protein, MMP) vaccine exhibited significantly higher levels of survival compared to ORF007L (major capsid protein, MCP). Moreover, ORF008L-based DNA vaccinated fish showed significant protection at 4 and 8 weeks post vaccination (wpv) than non-vaccinated fish after infected with RBIV (6.7 × 10<SUP>5</SUP>) at 23 °C, with relative percent survival (RPS) of 73.36% and 46.72%, respectively. All of the survivors from the first RBIV infection were strongly protected (100% RPS) from re-infected with RBIV (1.1 × 10<SUP>7</SUP>) at 100 dpi. In addition, the MMP (ORF008L)-based DNA vaccine significantly induced the gene expression of TLR3 (14.2-fold), MyD88 (11.6-fold), Mx (84.7-fold), ISG15 (8.7-fold), PKR (25.6-fold), MHC class I (13.3-fold), Fas (6.7-fold), Fas ligand (6.7-fold), caspase9 (17.0-fold) and caspase3 (15.3-fold) at 7 days post vaccination in the muscle (vaccine injection site). Our results showed the induction of immune responses and suggest the possibility of developing preventive measures against RBIV using myristoylated membrane protein-based DNA vaccine.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Injection of ORF008L (myristoylated membrane protein, MMP) vaccine offered high protection. </LI> <LI> TLR3, MyD88, Mx, ISG15, PKR, MHC class I, Fas, Fas ligand, caspase9 and 3 expressed high in vaccine group. </LI> <LI> The inflammatory cytokines (IL1β, IL8 and TNFα) were not enhanced in the ORF008L vaccinated group. </LI> </UL> </P>

Baculovirus Vector System 에 의해 발현된 재조합 Pseudorabies Virus Major Capsid Protein의 면역원성

전무형,안동준,장경수,조용성,박종현,송재영,현방훈,안수환 대한바이러스학회 1996 Journal of Bacteriology and Virology Vol.26 No.2

The recombinant pseudorabies virus major capsid protein (rMCP) was produced by expression of the MCP gene in Sf-9 cell using baculovirus transfer vector system. Following evaluation of the immunochemical properties of the rMCP, the immunogenicity of the recombinant subunit protiens were investigated in guinea pig and swine to obtain the preliminary guide line for the subunit vaccine using rMCP and gPSO. It was proved that ultrasonication and 30% ammonium sulfate was most efficient to concentrate and purify the protein. The rMCP was safe in mice, guinea pigs and piglets. In guinea pigs, rMCP mixed with various adjuvants induced substantial degree of serum neutralizing antibody titers, but revealed incomplete protectivity against challenge. In swine, the combination of rMCP and gP50 showed the higher serum neutralizing antibody titers and cellular immune responses than rMCP alone. However, the protectivity was lower in comparison with the commercial gI-deleted inactivated vaccine. We expect these results to contribute to characterization of MCP gene of Korean isolate of PRV and to ultilize as preliminary information for prodution and evaluation of PRV recombinant subunit vaccines.

국내 어류에서 분리된 Megalocytivirus의 유전형 분류 및 상관관계 분석

이은선 ( Eun Sun Lee ),조미영 ( Miyoung Cho ),민은영 ( Eun Young Min ),정승희 ( Sung Hee Jung ),김광일 ( Kwang Il Kim ) 한국어병학회 2019 한국어병학회지 Vol.32 No.2

본 연구에서는 2012년부터 2018년 1월까지 국내어류에서 참돔이리도바이러스병 (red sea bream iridoviral disease; RSIVD) 으로 확정 진단된 주요분리주에 대하여 major capsid protein (MCP) 유전자의 염기서열을 바탕으로 Megalocytivirus의 유전적 관계를 명확히 했다. 또한, Megalocytivirus와 숙주 세포간 상호작용에 영향을 줄 수 있는 특이 유전자 2종, Vascular endothelial growth factor (VEGF) 및 Histidine triad motif인 HIT-like 단백질 (HIT)에 대한 염기서열 분석을 통해 유전적 상관관계를 고찰하였다. 국내 어류에서 분리된 39개의 megalocytiviruses는 2가지 유전형인 red sea bream iridovirus (RSIV)형과 turbot reddish body iridovirus(TRBIV)형으로 분류되었으며, RSIV형의 megalocytiviruses는 다시 유전아형인 RSIV-subgroup 1형과 2형으로 분류되었다. 그리고 VEGF 유전자 염기서열 분석 결과에서는 RSIV형의 바이러스에서 변이가 발생되었음을 확인할 수 있었으며, HIT-like 단백질 유전자는 RSIV형의 Megalocytivirus에서만 발견되는 것을 알 수 있었다. In this study, we collected 39 megalocytiviruses isolated from diseased fish in Korea from 2012 to 2018. Major capsid protein (MCP) gene, a part of vascular endothelial growth factor (VEGF) gene and histidine triad motif-like protein (HIT) genes of Megalocytivirus were targeted for PCR amplification and analysis of those DNA nucleotide sequences. Korean strains revealed two genotypes (red sea bream iridovirus and turbot reddish body iridovirus types) based on the phylogeny of MCP gene. The red sea bream iridovirus type (RSIV-type) megalocytiviruses were divided into RSIV­subgroup 1 and 2. From the phylogenetic analysis of the VEGF genes, a genotypic variant of RSIV-type Megalocytivirus was identified. The HIT-like protein gene was detected in RSIVs, but not in TBRIV and ISKNV, suggesting that HIT-like protein gene may be specific in RSIV.

Pseudorabies Virus의 Major Capsid Protein 유전자의 클론닝과 Baculovirus Vector System에 의한 발현

안동준,전무형,송재영,박종현,현방훈,장경수,안수환 충남대학교 생물공학연구소 1997 생물공학연구지 Vol.5 No.-

Pseudorabies is caused by Pseudorabies virus (PRV: Aujeszky's disease virus) of Herpesviridae that is characterized by 100 to 150nm in size with a linear double-stranded DNA molecule with of approximately 90 × 10 exp (6)Da. This disease affects most of domestic animals such as swine, cattle, dog, sheep, cat, chicken, etc. causing high mortality and economic losses. In swine, young piglets show high mortality and pregnant sows, reproductive failures. However the adult swine reveals no clinical signs in general. But they become a carrier state and play an importnat role for propagation of the disease. In this study, the nucleotide sequence of major casid protein gene of PRV, Yangsan strain isolated from the diseased swine in Korea was analyzed, and the recombinant MCP was produced by expression of the MCP gene in Sf-9 cell using baculovirus transfer vector system. As result, in BamHI digestion, MCP gene locus of PRV YS strain showed different from that of Indiana S strain. The patterns of enzyme mapping were also found to be unidentical each other. The sequence of the MCP gene partially analyzed showed 98.09% identity to Indiana S strain. The expression of MCP in Sf-9 cell cotransfected by pVLMCP-44 baculovirus expression vector was characterized by Southern blot hybridization, immunofluoresent and immunocytochemical tests, SDS-PAGE and Western blotting. The rMCP with M.W. 142kDa was most effectively expressed in Sf-9 cells at the 3-4th days post inoculation of the recombinant baculovirus by 2 moi.

The serodiagnosis of a Lymphocystis disease virus infection using an antibody raised against a recombinant major capsid protein

Ja Young Seo,Bong Jo Kang,Hyoung Jong Oh,Jae Il Lee,Tae Jung Kim 한국어병학회 2008 한국어병학회지 Vol.21 No.3

Pseudorabies Virus 의 Major Capsid Protein 유전자의 클론닝과 Baculovirus Vector System에 의한 발현

안동준,전무형,송재영,박종현,현방훈,장경수,안수환 대한바이러스학회 1996 Journal of Bacteriology and Virology Vol.26 No.2

Pseudorabies is caused by Pseudorabies virus (PRV: Aujeszky's disease virus) of Herpesviridae that is characterized by 100 to 150nm in size with a linear double-stranded DNA molecule with of approximately 90 X 10'Da. This disease affects most of domestic animals such as swine, cattle, dog, sheep, cat, chicken, etc. causing high mortality and economic losses. In swine, young piglets show high mortality and pregnant sows, reproductive failures. However the adult swine reveals no clinical signs in general. But they become a carrier state and play an important role for propagation of the disease. In this study, the nucleotide sequence of major casid protein gene of PRV, Yangsan strain isolated from the diseased swine in Korea was analyzed, and the recombinant MCP was produced by expression of the MCP gene in Sf-9 cell using baculovirus transfer vector system. As result, in BamHI digestion, MCP gene locus of PRV YS strain showed different from that of Indiana S strain. The patterns of enzyme mapping were also found to be unidentical each other. The sequence of the MCP gene partially analyzed showed 98.09% identity to Indiana S strain. 1he expression of MCP in Sf-9 cell cotransfected by pVLMCP-44 baculovirus expression vector was characterized by Southern blot hybridization, immunofluoresent and immunocytochemical tests, SDS-PAGE and Western blotting. The rMCP with M.W. 142kDa was most effectively expressed in Sf-9 cells at the 3-4th days post inoculation of the recombinant baculovirus by 2 moi.

Production of a Bacteria-like Particle Vaccine Targeting Rock Bream (Oplegnathus fasciatus) Iridovirus Using Nicotiana benthamiana

안경익,차준영,Lee Jeong Won,Park Gyeongran,신경임,Song Shi-Jian,Ryu Gyeongryul,황인환,김민갑,김외연 한국식물학회 2022 Journal of Plant Biology Vol.65 No.1

Viral diseases are extremely widespread infections that change constantly through mutations. To produce vaccines against viral diseases, transient expression systems are employed, and Nicotiana benthamiana (tobacco) plants are a rapidly expanding platform. In this study, we developed a recombinant protein vaccine targeting the major capsid protein (MCP) of iridovirus fused with the lysine motif (LysM) and coiled-coil domain of coronin 1 (ccCor1) for surface display using Lactococcus lactis. The protein was abundantly produced in N. benthamiana in its N-glycosylated form. Total soluble proteins isolated from infiltrated N. benthamiana leaves were treated sequentially with increasing ammonium sulfate solution, and recombinant MCP mainly precipitated at 40–60%. Additionally, affinity chromatography using Ni–NTA resin was applied for further purification. Native structure analysis using size exclusion chromatography showed that recombinant MCP existed in a large oligomeric form. A minimum OD600 value of 0.4 trichloroacetic acid (TCA)-treated L. lactis was required for efficient recombinant MCP display. Immunogenicity of recombinant MCP was assessed in a mouse model through enzyme-linked immunosorbent assay (ELISA) with serum-injected recombinant MCP-displaying L. lactis. In summary, we developed a plant-based recombinant vaccine production system combined with surface display on L. lactis.

First detection of ranavirus in a wild population of Dybowski's brown frog (Rana dybowskii) in South Korea

Park, Jaejin,Grajal-Puche, Alejandro,Roh, Nam-Ho,Park, Il-Kook,Ra, Nam-Yong,Park, Daesik The Ecological Society of Korea 2021 Journal of Ecology and Environment Vol.45 No.1

Background: Ranavirus is an emerging infectious disease which has been linked to mass mortality events in various amphibian species. In this study, we document the first mass mortality event of an adult population of Dybowski's brown frogs (Rana dybowskii), in 2017, within a mountain valley in South Korea. Results: We confirmed the presence of ranavirus from all collected frogs (n = 22) via PCR and obtained the 500 bp major capsid protein (MCP) sequence from 13 individuals. The identified MCP sequence highly resembled Frog virus 3 (FV3) and was the same haplotype of a previously identified viral sequence collected from Huanren brown frog (R. huanrenensis) tadpoles in South Korea. Human habitat alteration, by recent erosion control works, may be partially responsible for this mass mortality event. Conclusion: We document the first mass mortality event in a wild Korean population of R. dybowskii. We also suggest, to determine if ranavirus infection is a threat to amphibians, government officials and researchers should develop continuous, country-wide, ranavirus monitoring programs of Korean amphibian populations.