• Asian-Australasian Journal of Animal Sciences
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• 제17권1호
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• pp.27-32
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• 2004

Using MLV (murine leukemia virus)-based retrovirus vectors encapsidated with VSV-G (vesicular stomatitis virus G glycoprotein), we tried to make transgenic chickens carrying the transferred genes in their chromosomes. Twenty one days after virus injection beneath the blastoderms of unincubated chicken embryos (stage Ⅹ, at laying), DNA isolated from the hatched chicks were analyzed by PCR with two sets of primers specific for EGFP (enhanced green fluorescence protein) gene or $Neo^R$ (E. coli neomycin resistant) gene. Among sixty-seven embryos injected with retrovirus, four of them were identified to carry the EGFP genes in their genomes. Remarkably, one transgenic chick showed presence of the retrovirus vector sequences in all organs differentiated from one of endoderm, mesoderm, and ectoderm. Expression of EGFP gene was not detected, however, the stable germ line transmission of transgene was verified in spermatozoa from the founder chicken and 50% of $F_1$ progenies.

• 대한핵의학회지
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• 제28권1호
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• pp.112-123
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• 1994

${\gamma}$-Glutamyltransferase (GGT: E.C. 2.3.2.2.) is a glycoprotein enzyme which is involved in glutathione metabolism and amino acid transport through the plasma membrane. It is distributed widely in several organs including liver and kidney. Several isozymes of GGT have been reported and some of the isozymes may be associated with hepatocarcinogenesis. We have produced six monoclnal antibodies (mAbs) against GGT purified from the liver of 2-acetamidofluorene (AAF) treated rats. All of the six mAbs were obtained by immunizing mice with liver GGT Six hybridomas which produced anti-GGT Abs were extensively subcloned and injected into the peritoneal cavity of BALB/c mice to obtain large quantities of Abs. These mAbs were purified from ascites by ammonium sulfate precipitation and protein A sepharose CL-4B column chromatography. Using these mAbs we preformed enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunohistochemistry (IHC), and autoradiography (ARG) to study the distribution of GGT isozyme in tissue. The results indicate that GGT-mAb 1 is specific for the AAF treated liver GGT, GGT-mAb 5 for the normal liver GGT, and GGT-mAb 6 for the normal kindey GGT. These mAbs may be used to evaluate the distribution of GGT isozymes in different tissues.

• Journal of Periodontal and Implant Science
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• 제38권sup2호
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• pp.395-404
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• 2008

Purpose: Osteoprotegerin (OPG) is a secreted glycoprotein and a member of the tumor necrosis factor (TNF) receptor family that inhibits bone resorption by suppressing osteoclastogenesis. Gingival fibroblasts (GF) play a role in periodontal disease progression, and the purpose of this experiment was to evaluate influence of osteotropic factors on the expression of osteoprotegerin mRNA in these cells. Materials and Methods: In this experiment, the influence of osteoclastogenic factors, interleukin-1 beta (IL-$1{\beta}$), TNF-$\alpha$, prostanglandin E2 ($PEG_2$). parathyroid hormone (PTH) and 1$\alpha$, 25-dihydroxyvitamin $D_3$ on the expression of osteoprotegerin mRNA in GF was studied by Northern blot hybridization. Results: As expected, $PEG_2$ tended to inhibit OPG levels and this was most prominent at 24 hours of culture with $10^{-7}M$ of $PEG_2$. TNF-$\alpha$ at 10ng/ml and also at 25ng/ml decreased OPG levels to almost 30% of the control at 24 hours. This contrasts with reports of increased OPG levels from osteoblast/stromal cells and gingival fibroblasts stimulated by TNF-$\alpha$. Decrease of OPG levels with $PEG_2$ and TNF-$\alpha$ suggests a pathway whereby these mediators exert their resorptive effects. However, OPG levels were increased almost 3-fold at 24 hours with IL-1$\beta$(1 to 15ng/ml) and increased 1.4 fold with 24-hour treatment of $10^{-7}M$ PTH. Conclusion: Increase of OPG levels suggests that these 'osteoclastogenic' factors act in more complex ways and may act to inhibit bone resorption in inflammatory periodontitis. This result supports the role of OPG as a negative feedback mechanism in osteoclastic activity.

• 한국해양바이오학회지
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• 제1권3호
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• pp.156-162
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• 2006

The halophilic enterobacteria, Enterobacteria cancerogenus, was isolated from the intestines of the fusiform fish (Trachurus japonicus) to yield a protein-like material termed PLM-f74. PLM-f74 was characterized by strong inhibition ratios to angiogenesis (82.8% at the concentration of $18.5{\mu}g/mL$) and elevated antioxidative capacities with low toxicity. The PLM-f74 is a glycoprotein comprised of saccharides and amino acids. PLM-f74 inhibited non-activated U937 monocytic cell adhesion to HUVECs activated with IL-$1{\beta}$ by 78.0%, and the adherence of U937 cells treated with the PLM-f74 and stimulated with IL-$1{\beta}$ to unstimulated HUVECs decreased by 102%. When both cell types were pretreated with PLM-f74, the adhesion of U937 cells to IL-$1{\beta}$ stimulated HUVECs was completely suppressed by 121% at a concentration of 18.5 ug/mL. PLM-f74 blocked signal pathways from VEGFR2, PI3K, ${\beta}$-catenin and VE-cadherin to NF-kB based on western bolt analysis. And also inhibited IL-1-stimulated HUVEC expression of the adhesion molecules, ICAM-1 by 40%, VCAM-1 by 60%, and E-selectin by 70% at the same concentration noted above. New anti-angiogenic and anti-cell adhesion materials showing elevated antioxidative capacities and non-toxicity may be expected from these results.

• Journal of Microbiology and Biotechnology
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• 제16권10호
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• pp.1544-1553
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• 2006

The halophilic enterobacteria, Enterobacteria cancerogenus, was isolated from the intestines of the fusiform fish (Trachurus japonicus) to yield a protein-like material termed PLM-f74. PLM-f74 was characterized by strong inhibition ratios to angiogenesis (82.8% at the concentration of $18.5{\mu}g/ml$) and elevated antioxidative capacities with low toxicity. The PLM-f74 is a glycoprotein comprised of saccharides and amino acids. PLM-f74 inhibited cell adhesion that non-activated U937 monocytic cell adhesion to HUVECs activated with $IL-1{\beta}$ by 78.0%, and the adherence of U937 cells treated with the PLM-f74 and stimulated with $IL-1{\beta}$ to unstimulated HUVECs decreased by 102%. When both cell types were pretreated with PLM-f74, the adhesion of U937 cells to $IL-1{\beta}$-stimulated HUVECs was completely suppressed by 121% at a concentration of $18.5{\mu}g/ml$. PLM-f74 blocked signal pathways from VEGFR2, PI3K, ${\beta}$-catenin, and VE-cadherin to NF-kB, based on western bolt analysis. It also inhibited IL-l-stimulated HUVEC expression of the adhesion molecules, ICAM-l by 40%, VCAM-l by 60%, and E-selectin by 70% at the same concentration noted above. New anti-angiogenic and anti-cell adhesion materials showing elevated antioxidative capacities, and non-toxicity may be expected from these results.

• 한국식물병리학회:학술대회논문집
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• 한국식물병리학회 2003년도 정기총회 및 추계학술발표회
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• pp.65.2-65
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• 2003

To protect the plant against several soil-borne pathogens, we are currently constructing disease-resistant transgenic root stock for the growth of cucurbitaceae vegetable plants, watermelon and gourd. We made a watermelon cDNA library from Cladosporium cucumerinum-Infected leaves for substractive hybriazation and differential screening. We isolated the several pathogen inducible cDNA clones, such as caffeoyl-CoA-methyltransferase, LAA induced protein, receptor-like kinase homolog, hydroxyproline-rich glycoprotein, catalase, calmodulin binding protein, mitochondrial ATPase beta subunit, methyl tRNA synthetase and WRKY transcription factors. We previously obtained CaMADS in pepper and galactinol synthase ( CsGolS) in cucumber that were confirmed to be related with disease-resistance. CaMADS and CsGolS2 were transformed into the inbred line 'GO701-2' gourd, the inbred line '6-2-2' watermelon and the Kong-dye watermelon by Agrobacterium tumerfaciens LBA4404. Plant growth regulators (zeatin, BAP and IAA) were used for shoot regeneration and root induction for optimal condition. Putative transgenic plants were selected in medium containing 100mg/L kanamycin and integration of the CaMADS and CsGO/S2 into the genomic DNA were demonstrated by the PCR analysis. We isolated major soil-borne pathogens, such as Monosporascus cannonballus, Didymella bryoniae, Cladosporium cuvumerinum from the cultivation area of watermelon or root stock, and successfully established artificial inoculation method for each pathogen. This work was supported by a grant from BioGreen 21 program, Rural Development Administration, Republic of Korea.

• 한국미생물학회:학술대회논문집
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• 한국미생물학회 2000년도 추계학술발표대회
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• pp.78-82
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• 2000

The AIDS epidemic continues unabated in many part of the world. After near two decades, no vaccine is available to combat the spread of this deadly disease. Much of the HIV -1 vaccine effort during the past decade has focused on the viral envelope glycoprotein, largely because it is the only protein that can elicit neutralizing antibodies (Nabs). Eliciting broadly cross-reactive Nabs has been a primary goal. The intrinsic genetic diversity of the viral envelope, however, has been one of the major impediments in vaccine development. We have recently completed a comprehensive study examining whether it is possible to elicit broadly acting Nabs by immunizing monkeys with mixtures of envelope proteins from multiple HIV -1 isolates. We compared the humoral immune responses elicited by vaccination with either single or multiple envelope proteins and evaluated the importance of humoral and non-humoral immune response in protection against a challenge virus with a homologous or heterologous envelope protein. Our results show that (1) Nab is the correlate of sterilizing immunity, (2) Nabs against primary HIV -1 isolates can be elicited by the live vector-prime/protein boost approach, and (3) polyvalent envelope vaccines elicit broader Nab response than monovalent vaccines. Nonetheless, our findings clearly indicate that the increased breadth of Nab response is by and large limited to strains included in the vaccine mixture and does not extend to heterologous non-vaccine strains. Our study strongly demonstrates how difficult it may be to elicit broadly reactive Nabs using envelope proteins and sadly predicts a similar fate for many of the vaccine candidates currently being evaluated in clinical trials. We have started to evaluate other vaccine candidates (e.g. genetically modified envelope proteins) that might elicit broadly reactive Nabs. We are also exploring other vaccine strategies to elicit potent cytotoxic T lymphocyte responses. Preliminary results from some of these experiments will be discussed.

• 미생물학회지
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• 제51권3호
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• pp.271-279
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• 2015

락토페린(LF)는 철이온과 결합하는 당 단백질로서 항균, 항바이러스, 항진균 등의 기능을 가지고 있으며, 생체의 각종 체액으로부터 분비되는 다기능성 단백질이다. 본 연구에서는 사람의 락토페린(hLF)으로부터 유래된 N-lobe의 유전자를 분리하고 산업용 균주로서 많이 사용되는 메탄올자화 효모인 Pichia pastoris에서 발현시켰다. 재조합 사람 락토페린 N-lobe (rhLF-N)는 배양액으로 분비 발현되었으며, 3L 발효조에서 약 $458{\mu}g/ml$이 수준으로 생성되었다. rhLF-N을 정제한 다음 SDS-PAGE와 western blot으로 분석하여 분자량 35 kDa 단백질을 확인하였으며, hLF에 대한 항체를 이용하여 면역확산법으로 면역성을 확인하였다. rhLF-N의 mRNA 발현양상을 qRT-PCR로 분석한 결과 메탄올 첨가에 의한 발현 유도 후 2-3일째에 발현율이 가장 높았으며, 4일째에는 점차적으로 감소하였다. 정제한 rhLF-N을 이용하여 항균활성을 조사한 결과 Staphylococcus aureus, E. coli, Pseudomonas aeruginosa, Burkholderia cepacia, Salmonella typhimurium과 같은 병원성 균에 대해 광범위한 항균활성을 보였으나, LF유래 항균 peptide들과 항균활성을 비교하였을 때, 항균력이 상대적으로 매우 떨어지는 것으로 나타났다. 비록 본 연구에서 발현한 rhLF-N은 항균력은 떨어지나, hLF에 비해 그 크기가 작고 배양조건 연구로 P. pastoris에서 대량 생산이 가능하며, 배양액으로 분비시킬 수 있기 때문에 정제 비용 등을 고려 할 때 산업적 응용에는 보다 유리할 것으로 사료된다.

• Journal of Microbiology
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• 제42권1호
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• pp.25-31
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• 2004

Sf9 cells have obvious advantages for the conventional production technology of vaccine. They are useful tools for high concentration and large-scale cultures. Sf9 cells were grown to maximal concentration, 8${\times}$l0$\^$6/ cells/$m\ell$ in a 500$m\ell$ spinner flask, with a doubling time at the exponentially growing phase of 24.5 hours, using serum-free media. To explore the ability of Sf9 cells to be infected by the Japanese encephalitis (JE) virus Beijing-l strain, Sf9 cells were infected with the virus. By 4-5 days post-infection, 10-15 % of the Sf9 cells showed cytopathic effect (CPE), from granularity to the formation of syncytia and multinucleated giant cells continuously observed over a period of 35 days. Positive fluorescent reactions were detected in 30-40% of cells infected with the JE virus Beijing-l strain, and the uninfected Sf9 cells were completely negative. Virus particles, propagated in Sf9 and Vero cells, were concentrated by sedimentation on 40% trehalose cushions by ultracentrifugation, and showed identical patterns of viral morphogenesis. Complete virus particles, 40 to 50 nm in diameter, were observed, and JE virus envelope (E) proteins, at 53 kDa, were found in the western blot analysis to the anti-JE virus E protein monoclonal antibody and reacted as a magenta band in the same position to the glycoprotein staining. To evaluate whether the infectious virus was produced in Sf9 cells inoculated with the JE virus Beijing-l stain, Sf9 cells were inoculated with the virus, and sample harvested every 5 days. The titers of the JE virus Beijing-l strain rose from 1.0${\times}$l0$\^$5/ to 1.5${\times}$l0$\^$6/ pfu/$m\ell$. The infected Sf9 cells could be subcultured in serum-free medium, with no change in the plaque sizes formed by the JE virus Beijing-l strain in the plaque assay. It is suggested that the ability of the JE virus Beijing-l strain to infect Sf9 cells in serum-free media will provide a useful insect cell system, where the JE virus replication, cytopathogenicity and vaccine immunogen can be studied.

• 생명과학회지
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• 제20권8호
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• pp.1249-1253
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• 2010

Sericin은 실크를 싸고 있는 고분자 수용성 당단백질로서 세포배양에 사용되며 세포분화를 촉진한다. 본 연구의 목적은 갑상선세포(FRTL-5)에서 thyroglobulin (Tg)의 분비에 sericin이 영향을 주는지를 알려고 한다. Sericin에 의해서 Tg의 분비가 촉진되었지만 Tg-mRNA의 발현은 촉진되지 않았다. 이런 상태에서 소포체 샤페론(Bip & calreticulin)과 소포체 막 단백질(IRE1, PERK & ATF6)의 발현이 증가한 것이 확인되었다. 한편 IRE1의 하부 신호전달자인 XBP1의 mRNA splicing 이 약하게 확인되었지만 PERK의 하부 신호전달자인 $eIF2{\alpha}$의 인산화는 일어나지 않았다. 그리고 sericin은 MTT assay 결과 cell viability을 촉진시키는 것도 확인되었다. 위의 결과는 sericin은 재조합 단백질생산에 유용하게 이용될 수 있는 새로운 생체물질로 증명되었다.