돌돔, Oplegnathus fasciatus의 Cyclooxygenase-2 유전자의 cloning 및 발현분석

진지웅 ( Ji Woong Jin ),김도형 ( Do Hyung Kim ),김영철 ( Young Chul Kim ),정현도 ( Hyun Do Jeong ) 한국어병학회 2013 한국어병학회지 Vol.26 No.1

Megalocytivirus는 우리나라를 포함한 아시아 각국의 양식현장에서 고위험성 병원체이다. 그럼에도 불구하고 어류의 면역체계와 megalocytivirus의 상호관계에 관한 연구는 아직 부족한 실정이다. 다양한 연구에서 cyclooxygenase isoform중 COX-1 유전자는 constitutive 하게 발현되며, COX-2 enzyme은 면역반응에 중요한 역할을 한다고 알려져 있다. 또한 COX-2유전자는 LPS (lipopolysaccharide) 또는 병원체의 감염과 같은 염증반응 시 그 발현이 증가한다고 알려져있다. 본 연구는 다른 어종의 COX-2 유전자를 바탕으로 제작된 degenerated primer와 5``- 그리고 3``-end RACE-PCR을 이용하여 돌돔에서 COX-2 유전자의 전체 염기서열을 밝혔으며, 그 결과 rbCOX-2 (rock bream COX-2)유전자 cDNA의 전체 길이는 2655 bp 였으며, 609개의 아미노산으로 구성되어있었다. rbCOX-2 유전자의 genomic organization은 9개의 intron과 10개의 exon으로 구성되어 있었다. 또한 본 연구에서는 돌돔에 megalocytivirus의 인위감염 시 COX-2 유전자의 발현을 조사하였다. LPS 접종 시 rbCOX-2 유전자는 접종1일 후 대조구와 비교하여 13.10배 증가하여 최고 발현을 보였으나, megalocytivirus 접종 시 대조구와의 비교에서 유의적인 발현을 확인할 수 없었다. 돌돔에서 COX-2 유전자의 염기서열의 분석과 발현 분석은 바이러스 감염 시 어류의 방어기작을 이해하는데 도움이 될 것이며, 바이러스 백신개발 및 치료제 개발의 기초자료로 활용될 것이다. Megalocytivirus is a major fish pathogen in marine aquaculture of Asian countries including Korea. Despite of many species affected by this pathogen, little is known interaction between megalocytivirus and the fish immune system. One of the cyclooxygenase isoforms, named COX-2, is playing an important role in immune regulation, and distinct from COX-1 isoform of constitutive activity. COX-2 enzyme is induced by various inflammatory signals, including injection of lipopolysaccharide or infection by pathogenic agents. We cloned COX-2 gene in rock bream using degenerated primers designed from reported sequences of other fish species in PCR followed with 5``- and 3``-end RACE-PCR. The full length of cDNA of rbCOX2 (rock bream COX-2) gene are 2655 bp and that translates into 609 amino acids. The rbCOX-2 genomic organization are found to span 10 exons separated by 9 introns. We also studied if the experimental infection of rock bream with megalocytivirus could affect the expression of COX-2 gene. When injected with LPS, expression of the COX-2 gene was reached peak level at 1 day post injection and showed b13.10 fold increased level compared with that of control. While, when injected with megalocytivirus, we were not able to find significantly increased COX-2 gene expression different from that of control. Cloned and analyzed COX-2 gene in rock bream will help to understand defence mechanisms in fish after viral infection and will also support the development of the measures for treatment and prevention of viral infection.

관상어로부터 분리한 Megalocytiviruses에서 나타나는 ORF25 유전자 부위의 반복서열 특성 분석

진지웅 ( Ji Woong Jin ),남정희 ( Jeong Hee Nam ),김광일 ( Kwang Il Kim ),홍수희 ( Su Hee Hong ),변주영 ( Ju Young Byon ),정현도 ( Hyun Do Jeong ) 한국수산과학회(구 한국수산학회) 2011 한국수산과학회지 Vol.44 No.4

The presence of ISKNV-like viruses in various freshwater ornamental fish species imported from Asia was confirmed by polymerase chain reaction(PCR) amplification of the ATPase(adenosine triphosphatase) gene. Interestingly, molecular analyses of the Open Reading Frame 25(ORF25) region of these isolates based on the ISKNV(Infectious spleen and kidney necrosis virus) genome revealed the presence of various repetitive sequences. ORF25 repeat sequence length had no effect on cumulative mortality of rock bream Oplegnathus fasciatus challenged with tissue homogenates of infected pearl gourami, Trichogaster leeri; silver gourami, Trichogaster microlepis; blue gourami, or Trichogaster trichopterus. All isolates induce cumulative mortalities after 12 days of infection, confirming that ORF25 polymorphism did not affect the pathogenicity of ornamental fish megalocytiviruses that cross infect rock bream, a seawater fish. Also, no statistically significant differences in spleen index or viral copy number in infected tissues was detected between isolates with varying ORF25 repeat sequence lengths. However, further studies are necessary to fully characterize the functional characteristics of these polymorphisms in megalocytivirus disease in ornamental fishes.

Megalocytivirus 감염 해산 어류에서 나타나는 임상증상의 정량적 변화 분석

진지웅 ( Ji Woong Jin ),조혜진 ( Hye Jin Cho ),김광일 ( Kwang Il Kim ),정준범 ( Joon Bum Jeong ),박경현 ( Gyeong Hyun Park ),정현도 ( Hyun Do Jeong ) 한국어병학회 2011 한국어병학회지 Vol.24 No.2

Megalocytivirus 감염 시 나타나는 임상적 증상의 정량적인 분석을 위하여, 돌돔에서 분리된 megalocytivirus IVS-1을 돌돔성어와 치어에 인위 감염시켰다. 그 결과 spleen index가 각각 4.49±1.13 와 4.85±1.06로 나타났으며, 이것은 정상어에 비해 3배 이상 증가한 값이었다. 폐사율은 돌돔성어와 치어에서 모두 100% 폐사한 반면 참돔치어는 30일이 지나도 60%의 폐사만 나타나 돌돔폐사율에 비해 낮은 폐사율을 나타냈다. 또한 IVS-1을 감염시킨 빈사상태의 참돔치어는 spleen index가 동일 크기의 돌돔치어 보다 낮은 1.47±0.87을 보였다. Real-time PCR을 이용하여 감염의 진행에 따른 바이러스 농도를 측정 한 결과, 돌돔치어와 성어의 감염 조직 내 바이러스 최고 농도는 각각 2.03×10(7) copies/mg과 2.40×10(7) copies/mg으로 비슷하였으나 돌돔성어에서 먼저 최고치에 도달하였다. 더구나 비장의 비정형비대세포의 수 역시 돌돔치어의 경우 지속적으로 증가한 반면 돌돔성어의 경우 최고치에 도달 후 오히려 감소하는 경향을 보였다. 본 연구에서는 megalocytivirus 감염 시 조직의 형태적 변화와 조직 내의 바이러스 농도와 같은 임상적인 증상을 정량적인 분석을 통해 나타내었고 이러한 정량적인 수치와 megalocytivirus 감염의 진행 정도와의 상관관계에 대해 조사하였다. In quantitative studies of clinical signs, rock bream of adults and juveniles infected with Megalocytivirus IVS-1 isolated from rock bream (Oplegnathus fasciatus) in Korea showed average 4.49±1.13 and 4.85±1.06 of spleen index respectively. In challenge experiments, Megalocytivirus IVS-1 induced 100% cumulative mortality in both adult and juvenile rock bream. However we found 60% cumulative mortality in juvenile red sea bream (Pagrus major) even after 30 days of injection, which contradicted with the results of other laboratories. Interestingly, IVS-1 infected red sea bream of the same juvenile size with rock bream showed lower spleen index compared to that of rock bream. In real-time PCR, there was continuous increasing of the numbers of viral copies (2.03×10(7) copies/mg) in the spleen of juvenile rock bream infected, which were different from those in adult rock bream showing plateau level after reaching to the peak level. Moreover, enlarged cell numbers in the infected spleen were also increased continuously in the juvenile but not in adult of rock bream, even decreased after reaching to peak level. Consequently, significant differences in clinical signs: cumulative mortality. spleen index and viral copy number were found between rock bream and red sea bream, but not between adult and juvenile rock bream. Certainly quantitative expression of clinical sign as in this study may be a way to compare the progression of megalocitiviral disease more accurately in different species or physiological conditions.

다양한 PCR용 DNA 추출법에 의한 패류 내 Megalocytivirus의 검출

김진우 ( Jin Woo Kim ),조미영 ( Mi Young Cho ),진지웅 ( Ji Woong Jin ),김기홍 ( Ki Hong Kim ),정현도 ( Hyun Do Jeong ),김광일 ( Kwang Il Kim ) 한국어병학회 2011 한국어병학회지 Vol.24 No.2

패류 내에 오염되어 있는 바이러스의 검출에 있어서 정성·정량적 분석이 가능하며 신속하고 간편한 방법의 개발을 이루고자 하였다. 5g의 굴 중장선 조직을 Glycine buffer 및 PEG 8000 용액을 사용하여 제조한 농축 시료 (T5g-D)와 50mg의 굴 중장선 조직으로부터 직접적으로 제조한 시료 (sT50mg-D)를 대상으로 2-step PCR을 실시하여 검출감도를 비교하였다. 동일한 1㎕의 DNA template T5g-D와 sT50mg-D를 사용하였을 때, 35cycles의 1-step PCR에서 양성의 결과를 얻을 수 있었다. 인위적으로 sT50mg-D 혼합시료를 사용하여 2-step PCR (35cycles)을 실시하였을 때 T5g-D를 사용한 것과 비교하여 뚜렷한 차이를 나타내지 않았다. 또한 megalocytivirus에 오염된 양성시료 0.5~50mg을 사용하여 조제한 각각 50㎕의 template DNA 1㎕를 사용하여 qPCR을 하였을 때 6.14E+00~1.2E+02/㎕의 농도를 함유하고 있는 것으로 나타났다. 조제한 template DNA에 6.14E+00 copies/㎕이하의 농도가 있을 때는 qPCR에서 positive의 결과를 얻을 수 없었다. 결론적으로, 패류 내 megalocytivirus의 오염 유무 판단을 위한 2-step PCR 및 qPCR에서 50mg의 중장선을 사용하는 것은 i) 굴의 조직으로부터 오염된 megalocytivirus의 정성 및 정량을 위한 최소 검출 한계에 비하여 충분한 양이었으며 ⅱ) 개체별 분석이 가능하다는 장점이 있었으며 ⅲ) 이를 토대로 국내에서 서식하고 있는 굴에서 megalocytivirus의 오염을 확인할 수 있었다. In analysis of DNA viruses from the contaminated shellfish using PCR, preparation method of template DNA is an important factor to get enough copy number of viruses. In this study, we evaluated the efficiency of PCR template of Megalocytivirus (sT50mg-D) DNA obtained from 50 mg digestive gland homogenate of oyster using commercial method, and compared with that obtained from 5 g of the same tissues (T5g-D) after PEG precipitation procedures of virus. Both templates DNA suspended in the same volume of distilled water showed positive results by primary PCR with 35 cycles, and the presence of Megalocytivirus was confirmed in oysters collected from cultured farms in Korea. Moreover, PCR with sT50mg-D allowed us to discriminate the contaminated oyster individually, that can not be done in PCR with T5g-D prepared from the mixture of three different individual oyster to get 5 g digestive gland homogenate. In quantitative analysis with real time PCR, Megalocytivirus concentrations in 50 ㎕ templates prepared using 0.5~50 mg of one positive sample were appeared in the range 6.14E+00~1.2E+02/㎕. We were not able to get positive result using template DNA contained less than 6.14E+00 copies. Consequently, 2-step PCR performed with DNA extracts from oyster homogenate of small amount (sT50mg-D) i) was enough to detect the contaminated Megalocytivirus in shellfish, ii) allowed us to do the analysis for individual shellfish rather than mixture of several shellfish and iii) showed the presence of Megalocytivirus in oyster from Korea.

부산지역 노로바이러스의 유전적 분석

김광일 ( Kwang Il Kim ),진지웅 ( Ji Woong Jin ),정현도 ( Hyun Do Jeong ) 한국어병학회 2011 한국어병학회지 Vol.24 No.3

For detection of noroviruses (NVs), we compared various PCR primer sets based on reverse transcription nested PCR (RT-nested PCR) in the water samples from Dong brook in Busan, South Korea. We designed various new primer sets based on the most conserved sequences of the capsid protein gene that react with diverse NVs found in Korea. Designed primer sets (KG1F/KG1R and KG2F/KG2R, named as PNK) for the respective genogroups of NVs, genogroup I and II (GI and GII), were applied to detect NVs in the water samples from Dong brook concentrated with ultracentrifugation. In the application to the water samples, proportion of GI (76.47%) and GII (70.59%) in water samples of Dong brook in RT-nested PCR with the primer sets of this study. However, no significant differences of the proportion of the positive samples were not found between RT-nested PCRs with reported and newly designed primer sets. From the nucleotide sequencing, GI and GII of NVs present in Dong brook were appeared to be the members of 1/2/4/5/9/10 genotypes, and 3/4/5/11/13 genotypes respectively. Appeared genotype 4 of GII known as an one of main genotype found in patients of many Asian countries warned us to consider the risks of norovirus in aquatic environments in southern part of Korea.

Genetic Variations of Outer Membrane Protein Genes of Vibrio harveyi Isolated in Korea and Immunogenicity of OmpW in Olive Flounder, Paralichthys olivaceus

Myoung-Sug KIM(김명석),Ji-Woong JIN(진지웅),Sung-Hee JUNG(정승희),Jung-Soo SEO(서정수),Suhee HONG(홍수희) 한국수산해양교육학회 2015 水産海洋敎育硏究 Vol.27 No.5

HRM 분석법을 이용한 패류 내 Megalocytiviruses의 럼출과 유전적 분석

김광일 ( Kwang Il Kim ),진지웅 ( Ji Woong Jin ),김영철 ( Young Chul Kim ),정현도 ( Hyun Do Jeong ) 한국수산과학회 2014 한국수산과학회지 Vol.47 No.3

Viruses in the genus Megalocytivirus have been subdivided into four subgroups. Among these subgroups 2 and 4, represented by the red sea bream iridovirus (RBIV) and the olive flounder iridovirus (FLIV) respectively, are non-exotic. subgroups 1 and 3, represented by the red sea bream iridovirus (RSIV) and the infectious spleen and kidney necrosis virus (ISKNV), respectively, have not been detected in Korea and are known as exotic. Shellfish are filter-­feeders, and can thus filter and accumulate Megalocytivirus in their digestive glands, allowing us to track viral con­-tamination in surrounding aquatic environment. In this study, we developed a high-resolution melting (HRM) analy­-sis to differentiate among subgroups of Megalocytivirus accumulated in shellfish, and confirmed the convenience and efficiency of this method. More than two subgroups of Megalocytivirus were found in the digestive gland of a single shellfish. We classified all Megalocytivirus viruses from shellfish in Korea into subgroups 2 and 4, although proportions of subgroups were different among regions. Compared to nucleotide sequencing analysis, HRM analysis is a simple and rapid method for differentiating of Megalocytiviru subgroups.

수입산 담수관상어 및 양식 해산어의 장내세균에서 나타나는 내성균 위험성 비교

윤소혜 ( So Hye Yoon ),전려진 ( Lyu Jin Jun ),김영진 ( Young Jin Kim ),진지웅 ( Ji Woong Jin ),정현도 ( Hyun Do Jeong ) 한국어병학회 2012 한국어병학회지 Vol.25 No.2

Various antibiotics, that could induce the appearance of resistant microorganisms, have been used for treatment or prevention of bacterial diseases in marine and ornamental fish. We determined and characterized the level of antibiotic-resistant bacteria and proportion of multi-drug resistant bacteria in intestinal microflora of both marine fish cultured in Korea and imported ornamental freshwater fish. For this the bacterial species and resistance to antibiotics were investigated in intestine of rock bream Oplegnathus fasciatus cultured in Korea and pearl gourami Trichogaster leeri imported from Singapore to characterise. Although the bacterial species were different, proportions of resistant bacteria to single antibiotics or multi-drug were higher in intestinal microflora of pearl gourami Trichogaster leeri imported from Singapore than in rock bream Oplegnathus fasciatus cultured in Korea. These results indicate that various antibiotics have been being used before trading without measures in the market of asian ornamental fishes, providing high risks for the emergence of multi-drug resistant bacteria.

수입산 관상어로부터 분리된 tetracycline 내성 Aeromonas spp에 tet (M) 및 tet (G) 유전자의 특성 분석

박신후 ( Shin Hoo Park ),전려진 ( Lyu Jin Jun ),조기택 ( Ki Taek Cho ),진지웅 ( Ji Woong Jin ),정현도 ( Hyun Do Jeong ) 한국수산과학회 2012 한국수산과학회지 Vol.45 No.3

In this study, the molecular structures of tet(M) and tet(G) carried by tetracycline (Tc) resistant bacteria in intestinal microflora from the imported ornamental fish were characterized and compared with each other depend on the imported countries. Of the total isolates, approximately 8.9% of the Ent-lac+ (lactose fermentative bacteria on coliform media) Tc resistant isolates in fish from three different countries, Singapore, Taiwan and Brazil, were appeared to contain tet (M). Three representative isolates of different countries, Aeromonas spp. JSM-1 (Singapore), JTM-1 (Taiwan) and JBM-1 (Brazil), were isolated and analyzed the molecular structures of tet (M) gene. Interestingly, partial sequence tet (M) genes (1099 bp) in JBM-1 (Brazil) showed 99.5% homology with the tet (M) found in the Vibrio spp. RV16 isolate, obtained from marine fish in Korea and known to carry Tn1545 parent type of tet (M). In contrast, tet (M) gene JSM-1 and JTM-1 showed mosaic structure of Tn1545 and Tn916, and 100% homology with each other. It may suggest the presence of various characteristics in terms of tet (M) gene structure. The determined sequence of the tet(G) from Aeromonas spp. JSG-1 and JBG-1 isolated from Singapore and Indonesia ornamental fish respectively showed similar nucleotide sequence homology but revealed a few nucleotide changes in comparison with the sequence of the prototype tet (G) gene (S52437 in GenBank).

전남 갯벌 해역 낙지 인공 산란장 조성 및 적지 기준에 관한 연구

곽석남(Seok-Nam KWAK),김성수(Sung Soo KIM),강진묵(Jin Muk KANG),최옥인(Ok In CHOI),진지웅(Ji Woong JIN),김기동(Gi Dong KIM) 한국수산해양교육학회 2021 水産海洋敎育硏究 Vol.33 No.3