• International Journal of Industrial Entomology and Biomaterials
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• 제29권2호
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• pp.207-213
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• 2014

The structural proteins of classical swine fever virus (CSFV) consist of nucleocapsid protein C and envelope glycoprotein $E^{rns}$ (E0), E1 and E2. Among them, E2, the most immunogenic of the CSFV glycoproteins, induces a protective immune response in swine. In this study, to determine the optimal expression conditions of glycoprotein E2 using baculovirus system, we investigated the influence of insect cells and media to the expression of recombinant E2. Recombinant virus containing glycoprotein E2 coding gene was constructed with bApGOZA DNA. Expression of the glycoprotein E2 was analyzed by SDS-PAGE and Western blot analysis using anti-CSFV E2 monoclonal antibodies. Expression of glycoprotein E2 in Sf21 cells was first observed after 3 days and reached a maximum on the 5th day after infection. Furthermore, the highest levels of glycoprotein E2 expression were observed at multiplicity of infection (MOI) of 5. When three different insect cell lines (Sf21, High-Five and Se301) were tested, High-Five cells showed the highest production. In addition, four different serum-free and serum-supplemented media, respectively, were tested for the expression of glycoprotein E2 and the budded virus (BV) titers. As a result, serum-supplemented medium provided the best conditions for protein production and the BV yield.

• 한국식품과학회지
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• 제41권4호
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• pp.405-412
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• 2009

본 연구는 꾸지뽕 나무 열매로부터 75 kDa의 당단백질(꾸지뽕 당단백질)을 추출한 후 꾸지뽕 당단백질의 첨가에 따른 알레르기성 염증 인자인 histamine 유리 억제능력 및 COX-2의 활성 억제 효과를 평가하였다. RBL-2H3세포를 22시간 동안 IgE로 감작시킨 후, HSA를 처리하여 histamine의 유리양을 측정한 결과 꾸지뽕 당단백질을 처리한 농도가 증가함에 따라 histamine의 유리와 COX-2의 활성 억제율은 증가하였다. 또한 꾸지뽕 당단백질의 처리는 HSA에 의해 유도된 세포내 ROS 생성량을 농도에 의존적으로 억제하였다. 한편 꾸지뽕 당단백질을 농도별로 처리하여 세포내 단백질을 추출하여 western blot을 실시한 결과 100 ${\mu}g$/mL 농도의 꾸지뽕 당단백질을 처리한 그룹에서 ERK1/2, AP-1과 COX-2의 활성 수준은 현저히 억제 되었다(p<0.05). 따라서 이러 한 결과에 미루어볼 때, 꾸지뽕 당단백질은 세포내 해독효소의 활성을 증가시킴으로써 ROS 수준을 감소시켰으므로 꾸지뽕 당단백질의 역할이 다른 천연물 유래의 당단백질과 마찬가지로 특이적인 항산화 능력을 지니고 있음을 나타내며 histamine의 유리 억제와 COX-2의 활성이 억제되었을 것으로 생각된다. 이는 꾸지뽕 당단백질이 항 알레르기 효능을 갖는 물질로써 알레르기성 비염, 아토피 등과 같은 알레르기 관련 질환의 예방 및 치료제로 사용될 수 있을 것 사료된다.

• 한국미생물·생명공학회지
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• 제43권1호
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• pp.22-30
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• 2015

CSF is a major concern for the swine industry, representing currently the most epizootically dangerous disease to the species. Numerous CSFV isolates with various degrees of virulence have already been isolated worldwide, ranging from low virulent strains that do not result in any apparent clinical signs to highly virulent strains that cause a severe per acute hemorrhagic fever with very high mortality. The molecular epidemiology of CSFVs has proven to be an essential tool for effective disease control and the development of safe and effective vaccines. Therefore, this study cloned and sequenced local CSFV isolates, and conducted a phylogenetic analysis based on the E2 glycoprotein encoding sequences.The RNA was extracted from PK15 cell culture passaged CSFV isolates, the cDNA prepared, and the complete E2 gene amplified with a product size of 1186 bp. The gelpurified PCR product was cloned into a pGEMT easy vector and the positive clone commercially sequenced. Aligning the nucleotide (1119 bp) and amino acid (373) sequences with 29 reference strains revealed nucleotide and amino acid sequence identities of 82.60-97.80% and 88.70-98.70%, respectively, indicating a higher mutation rate of the field CSFV strains. The phylogenetic analysis based on the complete E2 amino acid sequences also revealed a reliable differentiation of all the analyzed strains into specific genetic groups and subgroups, plus the local isolate (CSFV-E2) was found to cluster with the CSFV subgroup 2.2. Thus, the full-length E2 cds proved to be most suitable for a reliable and statistically significant phylogenetic analysis of CSFV isolates.

• 한국식품저장유통학회지
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• 제21권5호
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• pp.735-739
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• 2014

Newcastle disease virus(NDV) 감염된 baby hamster kidney(BHK) 세포에서 syncytium(합포체) 형성은 세포막 표면으로의 수송된 바이러스 당단백질 hemagglutinin-neuramidase(HN)에 의해 일어난다. HAU 값은 추출물의 농도가 25과 3.2 ug/mL 사이에서는 현저하게 감소하였으나, NDV 감염된 HAD(%)는 25 ug/mL 농도에서 광범위한 흡착능의 감소를 나타내 바이러스 당단백질의 세포내 생합성은 저해되지 않았다. 그러므로, 약용식물인 마황 메탄올 추출물이 바이러스 당단백질의 세포막으로의 수송과 함께 합포체 형성을 저해하여 항바이러스 작용을 하였다. 또한 마황 추출물의 저해활성을 조사한 결과 ${\alpha}$-glucosidase에 대한 추출물의 $IC_{50}$은 $18{\mu}g/mL$이었으며, ${\beta}$-glucosidase, ${\alpha}$-mannosidase, ${\beta}$-mannosidase에 대한 마황 추출물의 $IC_{50}$은 각각 60, 40, $150{\mu}g/mL$로 나타나 ${\beta}$-type glycosidases 보다 ${\alpha}$-type glycosidase에 대한 효소활성 저해능이 우수하였다. 따라서 $IC_{50}$농도에서는 세포내에서 당단백질 생합성은 저해되지 않으며 당단백질의 수송을 저해하는 것으로 판단되었으며 향후 항바이러스 관련 작용기작의 연구가 필요하다고 판단된다.

• 한국어병학회지
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• 제33권2호
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• pp.103-109
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• 2020

In order to use nervous necrosis virus (NNV) virus-like particles (VLPs) as a delivery tool for heterologous antigens or plasmids, we attempted to produce red-spotted grouper nervous necrosis virus (RGNNV) VLPs displaying a partial region of viral hemorrhagic septicemia virus (VHSV) glycoprotein at the surface and VLPs that are harboring DNA vaccine plasmids within the VLP. A peptide encoding 105 amino acids of VHSV glycoprotein was genetically inserted in the loop region of NNV capsid gene, and VLPs expressing the partial part of VHSV glycoprotein were successfully produced. However, in the transmission electron microscope analysis, the shape and size of the partial VHSV glycoprotein-expressing NNV VLPs were irregular and variable, respectively, indicating that the normal assembly of capsid proteins was inhibited by the relatively long foreign peptide (105 aa) on the loop region. To encapsulate by simultaneous transformation with both NNV capsid gene expressing plasmids and DNA vaccine plasmids (having an eGFP expressing cassette under the CMV promoter), NNV VLPs containing plasmids were produced. The encapsulation of plasmids in the NNV VLPs was demonstrated by PCR and cells exposed to the VLPs encapsulating DNA vaccine plasmids showed fluorescence. These results suggest that the encapsulation of plasmids in NNV VLPs can be done with a simple one-step process, excluding the process of disassembly-reassembly of VLPs, and NNV VLPs can be used as a delivery tool for DNA vaccine vectors.

• Journal of Microbiology and Biotechnology
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• 제2권3호
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• pp.174-182
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• 1992

The hemagglutinin-esterase glycoprotein (HE) gene of bovine coronavirus, coupled with a simian virus 40 early promoter and polyadenylation signal, was inserted into a human adenovirus transfer vector. The transfer vector was used to co-transfect 293 cells along with adenovirus genomic DNA. The hemagglutinin-esterase transcription unit was rescued into the adenovirus genome by homologous in vivo DNA recombination between the vector plasmid DNA and the adenovirus genomic DNA, and a recombinant adenovirus was isolated by several rounds of plaque assays. Thus the recombinant adenovirus carries the hemagglutinin-esterase gene in the early transcription region 3 (E3) of the adenovirus genome in the parallel orientation to the E3 transcription. The recombinant adenovirus synthesized the HE polypeptide in HeLa cells as demonstrated by immunoprecipitation with anti-coronavirus rabbit antisera. The recombinant HE polypeptide could be labelled by $[^3H]$glucosamine, demonstrating that the recombinant HE was glycosylated. Cells expressing the HE polypeptide exhibited hemadsorption activity when incubated with mouse erythrocytes. The HE was transported to the plasma membrane as shown by the cell surface immunofluorescence, indicating that the recombinant HE polypeptide retained its biological activities. Potential for the use of infectious recombinant adenovirus as a live virus-vectored vaccine candidate for bovine coronavirus disease is discussed.

• 한국동물학회:학술대회논문집
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• 한국동물학회 1994년도 한국생물과학협회 학술발표대회
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• pp.154.1-154
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• 1994

No Abstract, See Full Text

• The Plant Pathology Journal
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• 제15권6호
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• pp.313-318
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• 1999

Cloning of the 5'untranslated region (5' UTR) and Nterminus of the glycoprotein precursor (G2G1) open reading frame of tomato spotted wilt virus has been problematic, possibly because of the toxicity of a signal peptide at the beginning of th G2G1 protein precursor. The toxicity of the signal peptide to bacterial growth and the reason for the expression of the peptide gene in Escherichia coli were investigated by cloning the 5' UTR and the signal peptide sequence separately. Cells transformed with the plasmid containing both the first 30 amino acids of the glycoprotein and the 5' UTR showed a severe growth inhibition whereas transformants harboring either the plasmid with the signal sequence or the 5'UTR alone did not show any ingibition. An E. coli promoter-like sequence was found in the 5'UTR and tis promoter acivity was confirmed with a promoter-less GUS gene cloned downstream of the 5'UTR. In the cloning of the Tomato spotted wilt virus (TSWV) glycoprotein G2G1 open reading frame all the recovered plasmids contained stop codons in the signal sequence region. However, clones containing no stop codon were recovered when the signal sequence and the 5'UTR were cloned separately.

• 한국식품영양과학회지
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• 제28권2호
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• pp.285-291
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• 1999

To confirm the food quality of Urechis unicinctus which have been favored as a special raw seafood in southern area in Korea, the chemical composition of Urechis unicinctus and those glycoprotein were studied. Freeze dried Urechis unicinctus was composed of more than 70% of crude protein and 15% of total carbohydrate. The amino acids such as glycine(18.69%), glutamic acid(12.50%) and aspartic acid(9.37%) were noted as major components of total amino acids. The predominant free amino acids were alanine(32.98%), glycine(27.50%), asparagine(19.65%) and taurine(8.29%), and the sum of them were more than 88% to total free amino acids, so they may cause unique taste of Urechis unicinctus with sweetness. As the basis of sugar composition analysis, 56.35% of glucose and 30.49% of N acetylglucosamine were contained respectively, and they might also play an important role in a sweet taste. The leading carbohydrate moiety of glycoprotein from Urechis unicinctus was identified as glucose and N acetylglucosamine similarly to raw muscle, and they occupied more than 50% of total sugar content. Fucose(18.32%) and D glucuronic acid(12.31%) also detected in higher levels com pared to raw muscle. The total amino acid profile of glycoprotein showed a similar trend to raw muscle protein but glycine was detected a significantly lower than that in raw muscle. The glycoprotein from Urechis unicinctus was composed of 4 kinds of subunits, i.e. 25kDa, 20kDa, 18kDa and 12.5kDa of molecular weights through the SDS polyacrylamide gel electrophoresis. On the basis of the IR spectrum of absorptions appeared in 1,240cm-1 and 850cm-1, the glycoprotein had sulfate groups.

• Journal of Veterinary Science
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• 제22권5호
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• pp.61.1-61.13
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• 2021

Background: Hepatocellular carcinoma is the most common primary hepatic malignancy in humans and dogs. Several differentially expressed molecules have been studied and reported in human hepatocellular carcinoma and non-neoplastic liver lesions. However, studies on the features of canine hepatocellular carcinoma are limited, especially related to the differential characteristics of neoplastic and non-neoplastic lesions. Objectives: The study's objective was 1) to examine and evaluate the expression of arginase-1, P-glycoprotein, and cytokeratin 19 in canine liver tissues and 2) to investigate the differential features of hepatocellular carcinomas, liver tissue with non-neoplastic lesions, and paracancerous liver tissues in dogs. Methods: The expression levels of three markers underwent immunohistochemical analysis in 40 non-neoplastic liver tissues, 32 hepatocellular carcinoma tissues, and 11 paracancerous liver tissues. Scoring of each marker was performed semi-quantitatively. Results: Arginase-1 and P-glycoprotein were significantly downregulated in hepatocellular carcinoma, compared with hepatic tissues with non-neoplastic diseases (p < 0.001). Expression levels of arginase-1 and P-glycoprotein were also significantly lower in hepatocellular carcinoma than in paracancerous liver tissues (arginase-1, p = 0.0195; P-glycoprotein, p = 0.047). Few cytokeratin 19-positive hepatocytes were detected and only in one hepatocellular carcinoma and one cirrhotic liver sample. Conclusions: The results of this study suggest that downregulation of arginase-1 and P-glycoprotein is a feature of canine hepatocellular carcinoma; thus, those markers are potential candidates for use in differentiating hepatocellular carcinomas from non-neoplastic liver lesions in dogs.