• 미생물학회지
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• 제40권4호
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• pp.348-354
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• 2004

사람의 모유에 많이 함유된 human lactoferrin(hLF)은 항균 및 항 바이러스 작용이 있는 것으로 보고되고 있다. 본 연구에서는 hLf를 메탄올자화 효모인 Pichia pastoris에 cloning하고 그 발현을 RT-PCR, Northern blotting, SDS-PAGE및 Western blotting으로 확인하였다. 그 결과 2.1 kb의 hLf 유전자가 P.pastoris의 염색체 DNA로 끼어들어가 안정적으로 hLf를 발현하였다. 이 재조합 P.pastotis로부터 hLf를 포함하는 세포 추출액을 얻어 항균 작용을 연구하였다. 발현된 재조합 hLf는 Staphylococcus aureus, Micrococcus flavus 등의 그람 양성균에 대해 강력한 항균작용을 보일 뿐만 아니라 그람 음성 동물성 병원균인 Pseudomonas fluorescens ID 9631, E. coli ATCC8739, 25922,35 등과 Salmonella typhimurium 114,115 등 다양한 균에 대해서도 강력한 항균작용을 보였다. 이는 재조합 hLf가 생물학적 활성이 있다는 것을 보여준다.

• 미생물학회지
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• 제44권4호
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• pp.277-281
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• 2008

HIV에 감염된 환자의 경우 다양한 종류의 암이 발생하는 것으로 알려져 있다. 이러한 암종의 높은 발생률의 원인으로, 감염에 의한 면역세포의 감소 및 결핍과 같은 간접적인 이유 뿐 아니라, HIV 바이러스 단백질의 발현이 직접적으로 병의 발생에 관여한다는 보고가 있다. 본 연구에서는 HIV 환자에서 높게 나타나는 암의 발생에 대한 기전을 이해하기 위하여 HIV-1 Tat 유전자와, 다수의 암 발생과 관련이 있는 세포막단백 CD99와의 관계를 규명하였다. 먼저 CD99의 발현에 미치는 Tat의 영향을 알아보기 위하여 HIV-1 Tat 발현 안정화 세포주를 확립하고 Tat 단백에 의한 CD99 유전자의 발현 양상 변화를 분석하였다. 실험결과 Tat의 발현에 의하여 CD99 유전자의 발현이 활성화되는 것이 관찰되었으며 이와 반대로 STAT3의 발현은 낮아졌다. CD99 프로모터는 CpG 함량이 높기 때문에 Tat 단백이 DNA 메칠화를 통해서 CD99 유전자의 발현을 조절하는지 확인하기 위하여 methylation specific PCR을 수행하였고 Tat의 발현이 높은 곳에서 특이적으로 CD99 프로모터 부위가 탈메칠화되는 것을 발견하였다. Tat 발현 세포에서만 특이적인 발현 차이를 보이는 유전자 분석을 위한 Differentially Expressed Gene keratin 17과 collagen, type IV 증가됨이 확인되었다. 위의 결과는 HIV Tat 단백이 직접 세포 단백들을 조절하여 암을 발생시킬 수 있다는 보고를 뒷받침한다.

• 한국동물위생학회지
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• 제25권3호
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• pp.245-257
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• 2002

The gene encoding the HN protein from the CBP-1 strain, a heat stable Newcastle disease virus (NDV) isolated from diseased pheasants in Korea, was characterized by reverse transcriptase- polymerase chain reaction(RT-PCR) and the nucleotide and amino acid sequences were analyzed following cloning of the HN gene. In all of the NDV strains studied, a 1.75 kb size cDNA fragment for the HN gene was generated by RT-PCR and smaller specific band sizes harboring the internal portions of the HN gene were also detected by using four pairs of primers. The RT-PCR was sensitive enough to detect viral transcripts when the virus titer was above 25 hemagglutination units. The amplified 1.75 kb cDNA was cloned into a BamHI site of the pVL1393 Baculo transfer vector. The nucleotide sequences of the 1,758 bp HN gene from the CBP-1 strain were determined by the dye terminator cyclic sequencing method. The gene sequences were compared among the strains of CBP-1, Texas GB, Beaudette C, LaSota, B1 and Ulster. The homology of the CBP-1 HN gene to other HN variants was 97.8% to Texas GB, 98.4% to Beaudette C, 95.4% to LaSota, 95.6% to B1 and 90.2% to Ulster. As the deduced 577 amino acid sequences were compared among the strains, the homology for CBP-1 HN appeared to be 96.7% to Texas GB, 97.9% to Beaudette C, 95.5% to LaSota, 95.5% to B1 and 92.7% to Ulster. It was evident that the amino acid sequences included 5 sites for N-asparagine linked glycosylation and 12 cysteine residues. The three conserved leucine residues within the predicted transmembrane domain of the HN protein are amino acid 30, 37 and 44. The three antigenic sites on the HN protein of NDV are amino acids 347(Glu), 481(Asn) and 495(Glu). These data indicate that the genotype of the CBP-1 strain is more closely associated with the strains of Texas GB and Beaudette C than it is for the LaSota, B1 and Ulster strains.

• 대한수의학회지
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• 제44권4호
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• pp.655-662
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• 2004

The aim of this work was the validation of a rapid real-time PCR assay based on TaqMan technology for the unequivocal identification of infectious canine hepatitis (ICH) virus, to be used directly on DNA purified from blood specimens. A real-time PCR system targeting at the E3 ORFA gene sequence of canine adenovirus type 1 was optimized and validated through comparative analysis of samples using conventional PCR system. The real-time PCR assay based on TaqMan technology could disclose 23 (37.7%) out of 61 samples as PCR positive. In contrast, 18 (29.5%) samples were found PCR positive when conventional PCR was applied on these samples. The use of the ABI Prism 7700 sequence detection system allowed the efficient determination of the amplified product accumulation through a fluorogenic probe. The entire real-time TaqMan PCR assay, including DNA extraction, amplification, and detection could be completed within 3 hours. The detection method of real-time TaqMan PCR assay was 1,000 times more sensitive than conventional PCR. Real-time TaqMan probe and primer set developed and optimized in this study is a sensitive, rapid and accurate method for detection of ICH virus and can be effective screening tool for the detection of ICH in a diagnostic laboratory routines.

• Journal of Korean Neurosurgical Society
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• 제29권4호
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• pp.471-476
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• 2000

Objective : p16/INK4a, a kind of tumor suppressor genes, encodes a specific inhibitor of the cyclin D-dependent kinases CDK4 and CDK6. This prevents the association of CDK4 with cyclin D1, and subsequently inhibits phosphorylation of retinoblastoma tumor suppressor protein(pRb), thus preventing exit from the G1 phase. According to previous reports, over 50% of glioma tissue and 80% of glioma cell lines have been demonstrated inactivation of p16/INK4a gene. The purpose of this study was to determine whether recombinant adenovirus-p16 virus is a suitable candidate for gene replacement therapy in cases of glioma. Methods : Three human glioma cell lines(U251MG, U87MG and U373MG) that express mutant p16 protein were used. Replication-deficient adenovirus was utilized as an expression vector to transfer exogenous p16 cDNA into the cells ; control cells were infected with the Ad-${\beta}$-gal expressing ${\beta}$-galactosidase. To monitor gene transfer and the expression of exogenous genes, we used Western Blotting analysis. Flow cytometry studies of cellular DNA content were performed to determine the cell cycle phenotype of the glioma cells before and after treatment. Results : We showed here that restoration of p16/INK4a expression in p16 negative U87MG, U251MG and partially deleted U373MG by Ad-CMV-p16 induced growth suppression in vitro. Flow cytometric study revealed that Ad-CMV-p16 infected U87MG cells were arrested during the G0-G1 phase of the cell cycle. Expression of p16 transferred by Ad-CMV-p16 in glioma cells was highly efficient and maintained for more than seven days. Conclusions : Our results suggest that Ad-CMV-p16 gene therapy strategy is potentially useful and warrants further clinical investigation for the treatment of gliomas.

• 한국잠사곤충학회지
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• 제34권2호
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• pp.20-25
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• 1992

유용물질을 생산할 수 있는 곤충 baculovirus expression vector system의 국내 개발을 위하여, Bm-NPV의 다각체 단백질 유전자를 탐색, 클로닝 하고 그 구조를 분석한 결과는 다음과 같다. 1. AcNPV의 다각체 단백질 유전자를 포함하고 있는 EcoRI-Ⅰ fragment내의 0.93kb를 probe로 하여 southern 분석한 결과, BmNPV의 다각체 단백질 유전자는 PstⅠ-F fragment(7.4Kb)에 위치하였다. 2. BmNPV의 다각체 단백질 유전자를 포함하는 PstⅠ-F fragment를 E. coli에 클로닝시켜서 pBmP-F라 명명하고, 다시 southern분석을 통해 subcloning하여 pBmP-H라 명명하였으며 pBmP-H의 제한효소 지도를 작성하였다. 3. pBmP-H의 염기서열분석결과 구조유전자를 포함한 1,259 bp의 염기서열이 결정되었다. Iatrou 등이 보고한 BmNPV 다각체 단백질 유전자의 염기서열과 비교한 결과 10개의 염기서열에서 차이를 보였으며, 74 Val이 Ile로, 76 Asn이 Ser으로 155 Met이 Val로 아미노산 변경된 결과를 보였다.

• 한국미생물·생명공학회지
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• 제44권4호
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• pp.493-496
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• 2016

Peach rosette mosaic virus (PRMV)는 1933년 복숭아에서 처음 보고되었으며, 복숭아, 자두, 블루베리, 민들레, 벚나무 등에 감염되는 식물바이러스이다. PRMV는 한국에서 보고된 적이 없으나, 식물검역에서 관리병(control viruses)으로 지정되어 있다. 이번 연구에서는 PRMV를 더욱 신속하고 특이적으로 진단하기 위하여 Loop-mediated isothermal amplification 분석법을 적용한 진단법을 개발하였다. LAMP 방법은 기존의 PCR 방법(RT-PCR 및 nested PCR)과 같은 검출 강도를 가지고 있다. 또한 LAMP 반응을 확인하기 위해 PRMV cDNA을 outer primer sets (Product size 264 bp)로 PCR 한 뒤, Pvu II (CAG/CTG) 제한효소를 처리하였다. 제한효소 처리 결과 2개의 digestion fragments (207 + 57 bp)가 확인되었다. PRMV의 LAMP 진단 방법은 관련 식물로부터 더욱 신속한 모니터링이 가능할 것으로 기대된다.

• 미생물학회지
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• 제39권2호
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• pp.118-122
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• 2003

바이러스 조립 과정 기작 연구를 위한 좋은 재료인 박데리오파아지 P2-P4시스템에 이용될 벡터 플라스미드를 개발하기 위하여, P4 ash8 sid71을 출발 물질로 삼아 새로운 P4 유도체 벡터를 조성하였다. 유전자 재조합 기법을 써서 쉽게 선택 가능한 tetracyclin내성 유전자(tetR)를 도입하고 플라스미드P 4의 크기를 조절하였다. 이를 통해 얻어진 P4 ash8(sid7l) tetR은 12.09 kb의 크기를 가지며, 필요할 때 P2로 induction하면 생물학적 활성을 가지는 박데리오파아지로 전환 가능하였다. 전환된 파아지의 burst size를 결정하고, CsCi 부양균등밀도 편차실험을 수행하였다. 균등밀도 실험 분포도에서 P2크기 파아지 머리의 packaging 상한을 추정할 수 있었다.

• 한국연초학회지
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• 제28권2호
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• pp.100-110
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• 2006

A mutant of Potato vims Y (PVY) was occurred in PVY resistance flue-cured tobacco breeding line KF0402 $(TC1146{\times}KF117)$ showing vein necrosis at Suwon in Korea. This isolate, PVY-SWM, was differentiated from other PVY based on biological properties and nucleotide sequence analyses of coat protein gene. PVY-SWM caused typical symptoms on 21 indicator plants as compared to the PVY-TOJC37. Remarkably, the PVY-SWM induced distinctly different symptom of systemic vein necrosis on tobacco cultivars V.SCR, PBD6, TN86, TN90, Virgin A Mutant (VAM), Wislica, NC744, KB108 and KB111, which were reported to have the recessive potyvirus resistance gene va. In RT-PCR assays with specific primers for detection of PVY, a single band of about 800bp in length was produced. The amplified DNA was cloned and the nucleotide sequence was determined. The coat protein gene of PVY-SWM showed 88.4%-99.0% and 92.5%-98.5% identities to the 12 different PVY isolates of Genbank Database at the nucleotide and amino acidi respectively. Multiple alignments as well as cluster dendrograms of PVY-SWM isolate revealed close phylogenetic relationship to the $PVY^{NTN}$ subgroup.

• 한국가금학회지
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• 제35권1호
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• pp.39-55
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• 2008

Like the other herpesviruses, the virion of MDV consists of an envelope, which surrounds an amorphous tegument. Within the tegument, and icosahedral capsid encloses a linear double-stranded DNA core. Although the genome structure of MDV indicates that it is an ${\alpha}-herpesvirus$ like herpes simplex and varicella-zoster viruses, biological properties indicate MDV is more akin to the ${\gamma}-herpesvirus$ group, which includes Epstein-Barr and Kaposi's sarcoma herpesviruses. These herpesviruses replicate lytically in lymphocytes, epithelial and fibroblastic cells, and persist in lymphoblastoid cells. MDV has a complex life cycle and uses two means of replication, productive and non-productive, to exist and propagate. The method of reproduction changes according to a defined pattern depending on changes in virus-cell interactions at different stages of the disease, and in different tissues. Productive (lytic) interactions involve active invasion and take-over of the host cell, resulting in the production of infectious progeny virions. However, some herpesviruses, including MDV, can also establish a non-productive (abortive) infection in certain cell types, resulting in production of cell-associated progeny virus. Non-productive interactions represent persistent infection, in which the viral genome is present but gene expression is limited, there is no structural or regulatory gene translation, no replication, no release of progeny virions and no cell death. Reactivation of the virus is rare, and usually the infectious virus can be re-isolated only after cultivation in vitro. MDV establishes latency in lymphoid cells, some of which are subsequently transformed. In this review article, recent knowledges of the pathogenesis mechanisms followed by MDV infection to sensitive cells and chickens are discussed precisely.