• Journal of Microbiology and Biotechnology
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• 제27권6호
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• pp.1098-1105
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• 2017

Vesicular stomatitis virus G glycoprotein (VSV-G) has been widely used for pseudotyping retroviral, lentiviral, and artificial viral vectors. The objective of this study was to establish a potential approach for large-scale production of VSV-G. To this end, VSV-G was cloned with an N-terminal His-tag into Pichia pastoris expression vector pPIC3.5K. Three clones ($Mut^s$) containing the VSV-G expression cassette were identified by PCR. All clones proliferated normally in expansion medium, whereas the proliferation was reduced significantly under induction conditions. VSV-G protein was detected in cell lysates by western blot analysis, and the highest expression level was observed at 96 h post induction. VSV-G could also be obtained from the condition medium of yeast protoplasts. Furthermore, VSV-G could be incorporated into Ad293 cells and was able to induce cell fusion, leading to the transfer of cytoplasmic protein. Finally, VSV-G-mediated DNA transfection was assayed by flow cytometry and luciferase measurement. Incubation of VSV-G lysate with the pGL3-control DNA complex increased the luciferase activity in Ad293 and HeLa cells by about 3-fold. Likewise, incubation of VSV-G lysate with the pCMV-DsRed DNA complex improved the transfection efficiency into Ad293 by 10% and into HeLa cells by about 1-fold. In conclusion, these results demonstrate that VSV-G could be produced from P. pastoris with biofunctionalities, demonstrating that large-scale production of the viral glycoprotein is feasible.

• 한국방사선학회논문지
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• 제13권3호
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• pp.359-369
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• 2019

관상동맥 스텐트 혈전증 치료에 있어서 혈소판 당단백 IIb/IIIa 수용체 차단제와 카테터를 이용한 혈전 흡입술을 동시에 시행한 경우의 효과에 대하여 알아보고자 하였다. 2008년 07월부터 2017년 07월까지의 일대학교병원 심혈관센터에서 경피적 관상동맥 스텐트 삽입술 후 관상동맥 조영술에서 스텐트 혈전증이 발생한 환자 267명($64.6{\pm}12.1$년, 187남자) 그룹 I(혈전용해술과 혈전흡입술 동시 시행한 경우, n=32명), 그룹 II(혈전용해술이나 혈전흡입술 한 가지만 시행한 경우 혹은 둘 다 시행하지 않은 경우, n=235명)로 분류하여 주요심장사건, 사망 발생률, 표적병변 재개통술 그리고 스텐트 혈전 등을 1 년동안 추적 관찰하였다. 두 군간에 임상 특성에서 연령(그룹I: $60.8{\pm}12.9$ vs. 그룹II: $65.1{\pm}11.9$, p=0.603), 남성(그룹I: 75.0% vs. 그룹II: 69.4%, p=0.681), 좌심실구혈율(그룹I: $58.1{\pm}9.0$ vs. 그룹II: $59.5{\pm}11.9$, p=0.127)등 양군 간에 차이는 없었다. 주요 심장사건은 두 군간에 차이를 보이지 않았으나(그룹I: 12.5% vs. 그룹II: 23.8%, p=0.180), 세부적으로 살펴보면 사망발생률(그룹I: 0% vs. 그룹II: 13.2%, p=0.034), 표적병변 재개통술(그룹I: 9.4% vs. 그룹II: 6.4%, p=0.461) 그리고 스텐트 혈전증(그룹I: 3.1% vs. 그룹II: 4.7%, p=1000)으로, 사망발생률은 혈소판 당단백 IIb/IIIa 수용체 차단제와 혈전흡입술을 동시에 시행한 군에서 유의하게 낮았다. 결론적으로, 관상동맥 스텐트 혈전증 치료에 있어 혈전용해술과 혈전흡입술을 동시 시행하는 경우에 비교군보다 사망 발생율을 감소시켰다.

• 대한수의학회지
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• 제38권3호
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• pp.581-588
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• 1998

Eight monoclonal antibodies(MAbs) against bovine coronavirus(BCV) were produced and characterized. Three MAbs(1G9, 4H12, 5C1) specific to the S glycoprotein and two HE glycoprotein-specific MAbs(2A5, 5G4) were found to neutralize the BCV in fluorescence focus neutralization(FFN) test. Two HE-specific MAbs from the neutralizing MAbs inhibited the hemagglutinating activity of the BCV. None of the N protein-specific MAbs(1C1, 5A12, 6H1) neutralized the virus infectivity. Bovine coronavirus and mouse hepatitis virus, which belong to group II coronaviruses, were differentiated from other groups of coronaviruses(porcine transmissible gastroenteritis virus, porcine epidemic diarrhea virus, canine coronavirus) by all MAbs in fluorescence antibody test(FA), but not in FFN test.

• Bulletin of the Korean Chemical Society
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• 제33권2호
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• pp.651-656
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• 2012

Cationic immunoliposomes were prepared by conjugation of Fab' fragments of the recombinant humanized monoclonal antibody (HuCC49) against tumor-associated glycoprotein (TAG)-72 to sterically unilamella liposomes. The cationic immunoliposomes are composed of cationic lipid (O,O'-dimyristyl-N-lysyl aspartate, DMKD), cholesterol, and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethyleneglycol)$_{2000}$] (DPPE-PEG-maleimide) with a molar ratio of 0.5:0.47:0.03. Plasmid DNA was effectively condensed by addition of transferrin (Tf) during the formation of anti-TAG-72 PEG-immunolipoplexes (PILPs). These anti-TAG-72 PILPs were able to adhere to the surface of TAG-72-overexepressing LS174T human colon cancer cells more effectively than conventional liposomes, thereby facilitating gene delivery in vitro. Furthermore, intravenous administration of the anti-TAG-72 PILPs into the tumor-carrying mice exhibited efficient localization of the reporter gene in the tumor tissues.

• 대한수의학회지
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• 제32권2호
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• pp.195-205
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• 1992

The soluble antigen profiles and antigenic specificities of Leptospira interrogans serovars icterhaemorrhagiae, canicola, pomona and hardjo were examined by SDS-polyacrylamide gel electrophoresis, crossed immunoeletrophoresis and immunoblotting. The profiles of protein, glycoprotein and fraction containing N-acetylglucosamine of 4 serovars were compared. The protein profiles of 4 serovars were very similar except the range of 14,400 to 30,000 daltons. Molecular weight of glycoprotein of L, pomona was lower than other serovars. L canicola showed extra N-acetylglucosamine bands having molecular weight of 82,000 and 90,000 daltons. In crossed immunoelectrophoresis, a close antigenic relationship was found between L icterohaemorrhagiae and L canicola. In immunoblottings conducted with soluble antigens and rabbit antisera of 4 serovars, Leptospira interrogans serovars possessed cross-reactive antigens and serovar-specific antigens. The molecular weights of serovar-specific antigens were 45,000, 82,000 and 90,000, 31,000 and 24,000 daltons in L icterohaemorrhagiae, L canicola, L pomona, and L hardjo, respectively.

• 한국어병학회지
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• 제16권1호
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• pp.1-12
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• 2003

RT-PCR method was applied to detect and clone the nucleocapsid protein (N) gene and glycoprotein (G) gene for sequencing 5 Korean VHSV isolates from cultured olive flounder, Paralichthys olivaceus. Phylogenetic analysis was performed to investigate their relationship with the VHSV strains described previously and isolated from different geographical area. Generally, VHSV strains were separated phylogenetically according to the major geographical area of isolation: Genogroup I (American type), Genogroup Il (British Isles) and Genogroup ill (European type). This study revealed that all 5 Korean VHSV isolates were belonged to Genogroup I and closely related to Japanese Obama25 type.

• Journal of Microbiology and Biotechnology
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• 제24권12호
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• pp.1719-1727
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• 2014

In the present study, whispovirus immediate early 1 promoter (ie-1) was used to initiate surface expression of the hemagglutinin (HA) protein of Egyptian H5N1 avian influenza virus (AIV) by using the baculovirus expression vector system. The HA gene and whispovirus ie-1 promoter sequence were synthesized as a fused expression cassette (ie1-HA) and successfully cloned into the pFastBac-1 transfer vector. The recombinant vector was transformed into DH10Bac competent cells, and the recombinant bacmid was generated via site-specific transposition. The recombinant bacmid was used for transfection of Spodoptera frugiperda (Sf-9) insect cells to construct the recombinant baculovirus and to induce expression of the HA protein of H5N1 AIV. The recombinant glycoprotein expressed in Sf-9 cells showed hemadsorption activity. Hemagglutination activity was also detected in both extra- and intracellular recombinant HAs. Both the HA and hemadsorption activities were inhibited by reference polyclonal anti-H5 sera. Significant expression of the recombinant protein was observed on the surface of infected insect cells by using immunofluorescence. SDS-PAGE analysis of the expressed protein revealed the presence of a visually distinguishable band of ~63 kDa in size, which was absent in the non-infected cell control. Western blot analysis confirmed that the distinct 63 kDa band corresponded to the recombinant HA glycoprotein of H5N1 AIV. This study reports the successful expression of the HA protein of H5N1 AIV. The expressed protein was displayed on the plasma membrane of infected insect cells under the control of whispovirus ie-1 promoter by using the baculovirus expression vector system.

• 한국생물물리학회:학술대회논문집
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• 한국생물물리학회 2003년도 정기총회 및 학술발표회
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• pp.58-58
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• 2003

The envelope glycoprotein of HIV, gp41, mediates the membrane fusion with human cells. The extracellular domain of gp41 has two helical regions. The N-terminus helical region (N-helix) forms trimeric coiled coil, interacts with the C-terminus helical region (C-helix) of gp41 to form a stable helical bundle structure. In this study, we have shown that the N-helix of gp41 has membrane interacting and disrupting abilities. It was localized into the interface of the lipidic phase and head group of the membrane. In contrast, the N-helix region with membrane fusion defective mutations could not bind to membrane. In addition, the N-helix bound on the membrane was released from the membrane by the C-helix, and the complex of the N- and C-helix did not interact with membrane. These results suggested that the membrane binding ability of the N-helix is necessary for the fusion activity of gp41, and such property is possibly controlled by the C-helm.

• Mass Spectrometry Letters
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• 제9권3호
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• pp.67-72
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• 2018

Alkaline phosphatase (AP) is a membrane-bound glycoprotein that is widely distributed in the plasma membrane of cells of various organs and also found in many organisms from bacteria to humans. The complete amino acid sequence and three-dimensional structure of human placental alkaline phosphatase have been reported. Based on the literature data, AP consists of two presumptive glycosylation sites, at Asn-144 and Asn-271. However, it only contains a single occupied N-linked glycosylation site and no occupied O-linked glycosylation sites. Hydrophilic interaction chromatography (HILIC) has been primarily employed for the characterization of the glycan structures derived from glycoproteins. N-glycan structures from human placental alkaline phosphatase (PLAP) were investigated using HILIC-Orbitrap MS, and subsequent data processing and glycan assignment software. 16 structures including 10 sialylated N-glycans were identified from PLAP.

• 한국식품과학회지
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• 제31권2호
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• pp.482-487
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• 1999

과요오드산으로 산화시킨 당을 유리아미노산과 반응시켜서 ${\alpha}-NH_2$기와 펩티딜리신의 ${\varepsilon}-NH_2$기와 반응하여 결합한 것을 확인하였다. 그래서, 제조한 과요드산-산화당은 단백질 분자 표면의 리신의 ${\varepsilon}-NH_2$기와 Schiff 염기 반응으로 공유결합하여 당단백질을 만드는 것으로 볼 수 있다. 과요드산 산화당으로 변형한 고구마 ${\beta}$-아밀라아제는 C, H는 증가, N은 감소하였다. N말단의 ${\alpha}-NH_2$기는 70% (pentamer), 73% (monomer), ${\varepsilon}-NH_2$기는 33% (pentamer), 26% (monomer)가 산화당과 반응하였다. 페놀-황산법에 의한 총당 정량과 DNP 법으로 분석한 결과, $IO_4$-산화말토헥사오스는 고구마 ${\beta}$-아밀라아제 1몰당 6몰이 결합된 것으로 나타났고, 고구마 ${\beta}$-아밀라아제에 결합된 산화가용성 전분은 효소단백질 대비 13.2% (monomer), 13.5% (pentamer), 산화말토헥사오스는 9.7% (pentamer), 9.3% (monomer)로 나타났다.