• Asian-Australasian Journal of Animal Sciences
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• 제20권4호
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• pp.482-486
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• 2007

Basic objective of this research was to compare the milk yield and composition of New Zealand White transgenic rabbit females expressing recombinant human factor VIII (hFVIII) in mammary gland during lactation with that of non-transgenic rabbit females of the same age during 30 days of lactation. Transgenic founders were generated by the microinjection of foreign DNA (mWAP-hFVIII gene construct) into the egg. F1, F2 and F3 generations of transgenic rabbits were obtained after mating of transgenic founder rabbits with non-transgenic rabbits. The amount of milk rejected was measured by weight-suckle-weight method at $10^{th}$, $20^{th}$and $30^{th}$ day of lactation. Quality of milk (content of fat, protein, lactose, dry ash, and some minerals) from transgenic and non-transgenic rabbit was also determined. Comparison of milk yield, determined by weight-suckle-weight method, showed significantly higher (p<0.05) milk production at day 20 of first lactation in non-transgenic females, but on the same day of second lactation higher milk yield was measured in transgenic ones. Significantly higher (p<0.05) content of milk fat and protein was determined in transgenic milk whilst higher content of lactose was found in non-transgenic milk. The content of minerals (calcium, phosphorus, magnesium and sodium) did not differ in both experimental and control groups. Our results showed that milk yield and composition of transgenic rabbit females (mammary specific transgenic over-expression of hFVIII) over several generations is only slightly and transiently different from milk yield of non-transgenic females, which had no significant consequence on the litter size and viability.

• 대한의생명과학회지
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• 제15권1호
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• pp.37-45
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• 2009

Insect cell cultures have become important tools in the production of biological substances for use in a variety of research, human and veterinary medicine, and pest control applications. These applications often require the introduction of foreign DNA into the cells and have generally used methods originally developed for use with human and other mammalian cell cultures. While these methods can be successfully employed, they are often less efficient with insect cells and frequently involve complex procedures or require specialized equipment. Even when they do work, they may require substantial modification because of differences in the culture medium or growth patterns of insect cells. In this study, We have optimized transfection conditions of Sf9 cell line using insect expression vector pIZT/V5-His which expresses green fluorescent protein effectively. Human stem cell factor (hSCF) is a glycoprotein that plays a key role in hematopoiesis acting both as a positive and negative regulator, often in synergy with other cytokines. It also plays a key role in mast cell development, gametogenesis, and melanogenesis. It can exist in membrane-bound form and in proteolytically released soluble form. As determined by an enzyme-linked immunosorbent assay performed, hSCF level in supernatant averaged 995ng/ml. The human hSCF was partially purified by immunoaffinity chromatography and analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. The results show that the hSCF has N-linked carbohydrate and corresponds to the soluble form, at or about 223 amino acids in length. The findings suggest functional importance for soluble hSCF in cells.

• Animal Bioscience
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• 제34권8호
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• pp.1321-1330
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• 2021

Objective: Transgenic hens hold a great promise to produce various valuable proteins. Through virus transduction into stage X embryo, the transgene expression under the control of constructed chicken ovalbumin promoters has been successfully achieved. However, a validation system that can evaluate differently developed ovalbumin promoters in in vitro, remains to be developed. Methods: In the present study, chicken oviduct epithelial cells (cOECs) were isolated from oviduct tissue and shortly cultured with keratinocyte complete medium supplemented with chicken serum. The isolated cells were characterized with immunofluorescence, western blot, and flow cytometry using oviduct-specific marker. Chicken mutated ovalbumin promoter (Mut-4.4-kb-pOV) was validated in these cells using luciferase reporter analysis. Results: The isolated cOECs revealed that the oviduct-specific marker, ovalbumin protein, was clearly detected by immunofluorescence, western blot, and flow cytometry analysis revealed that approximately 79.40% of the cells contained this protein. Also, luciferase reporter analysis showed that the constructed Mut-4.4-kb-pOV exhibited 7.1-fold (p<0.001) higher activity in the cOECs. Conclusion: Collectively, these results demonstrate the efficient isolation and characterization of cOECs and validate the activity of the constructed ovalbumin promoter in the cultured cOECs. The in vitro validation of the recombinant promoter activity in cOECs can facilitate the production of efficient transgenic chickens for potential use as bioreactors.

• 한국어병학회지
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• 제18권1호
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• pp.67-80
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• 2005

미꾸라지 (mudloach, Misgunus mizolepis)의 간으로부터 클로닝한 phosphoinositide-specific phospholipase C$\delta$ (ML-PLC$\delta$)를 대장균 (E. coli)에서 과발현시켜 만든 재조합 ML-PLC$\delta$와 미꾸라지 간 조직으로부터 직접 정제한 ML-PLC$\delta$의 생화학적 특성을 비교분석하였다. 우선, pET28a vector (Novagen)를 이용하여 E. coli BL21(DE3)에서 과발현된 재조합 ML-PLC$\delta$은 $Ni^{2+}$-NTA affinity 크로마토그래피 및 gel filtration 칼럼에 의해서 정제되었다. 미꾸라지 간 조직으로 ML-PLC$\delta$는 open heparin 칼럼 및 분석용 heparin 칼럼등을 통하여 부분 정제하였다. 두개의 재조합 및 wild ML-PLC$\delta$는 phosphatidylinositol 4,5-bis-phosphate ($PIP_2$)에 대한 농도 의존적 PLC 활성을 보여주었고, 그 활성은 포유류 PLC$\delta$ 효소와 유사하게 칼슘 농도에 의존적인 활성을 나타내었다. 재조합 및 wild ML-PLC$\delta$는 각각 pH 7.0 및 7.5에서 가장 큰 PI-가수분해 활성을 나타낸다는 사실을 알 수 있었다. 게다가, 재조합 및 wild ML-PLC$\delta$는 sodium doecylcholate (SDC) 및 phosphatidylethanolamine (PE), phosphatidylcholine (PC)와 같은 지질류에 대하여 농도의존적인 활성을 나타내나, spermine과 같은 polyamine류의 존재하에서는 농도 의존적으로 PLC 활성이 감소됨을 알 수 있었다. 미꾸라지 각 기관들의 ML-PLC$\delta$의 발현양상 및 양등을 측정하여 보았을 때 ML-PLC$\delta$는 포유류 PLC$\delta$와 마찬가지로 다양한 형태의 PLC$\delta$가 존재함을 알 수 있었다. 이와 같은 결과들로 미루어서 미꾸라지로부터 얻은 ML-PLC$\delta$는 포유류의 PLC$\delta$ isozymes과 유사한 형태의 생화학적 특성을 가지나, 포유류 PLC$\delta$1과 PLC$\delta$3 isozyme의 생화학적 특성을 함께 가짐을 알 수 있었다.

• 생명과학회지
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• 제12권6호
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• pp.688-693
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• 2002

Chaperone 분자는 세포 내에서 새로 합성된 polypeptides의 misfolding을 보호하는 역할을 가진다. 이런 chaperone 분자와의 공발현은 활성형 재조합 단백질의 생산을 증가를 기대할 수 있다. 본 연구에서는 E. cozi에서 B. macerans 유래 cyclodextrin glucanotransferase (CGTase)의 활성형 생산에 GroEL/ES chaperone과의 공발현의 효과에 대해 조사하였다. cgt와 groEL/ES 유전자출 발현하는 pTCGT1과 pGro7은 각각 T7 promoter와 araB promoter에 의해 조절되고 이들을 E. coli cell에 co-transformation시켰다. 재조합 E. coli에서 IPTG와 L-arabinose의 최적 농도를 결정하기 위해 행한 결과 1 mM IPTG, 0.3 mg L-arabinose/$m\ell$에서 가장 높은 CGTase 활성을 나타내었다. 그리고 tube에서는 L-arabinose와 IPTG를 각각 0.4~0.5 $OD_{600}$과 0.8~l.0 $OD_{600}$에서 첨가하였을 때 활성형 CGTase의 생산이 증가되었다. GroEL/ES 공발현 조건에서는 가용성 CGTase 활성이 0.7~0.73 unit/$m\ell$로 단독 발현의 0.36~0.56 unit/$m\ell$에 비해 약 1.5 배 정도 증가함을 알 수 있었다. SDS-PAGE 분석에서는 GroEL/ES 공발현 조건에서 총 CGTase의 33.6%정도가 가용성 형태로 생산됨을 알 수 있었다.

• 한국생물정보학회:학술대회논문집
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• 한국생물정보시스템생물학회 2000년도 International Symposium on Bioinformatics
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• pp.13-16
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• 2000

Microorganisms have been widely employed for the production of useful bioproducts including primary metabolites such as ethanol, succinic acid, acetone and butanol, secondary metabolites represented by antibiotics, proteins, polysaccharides, lipids and many others. Since these products can be obtained in small quantities under natural condition, mutation and selection processes have been employed for the improvement of strains. Recently, metabolic engineering strategies have been employed for more efficient production of these bioproducts. Metabolic engineering can be defined as purposeful modification of cellular metabolic pathways by introducing new pathways, deleting or modifying the existing pathways for the enhanced production of a desired product or modified/new product, degradation of xenobiotics, and utilization of inexpensive raw materials. Metabolic flux analysis and metabolic control analysis along with recombinant DNA techniques are three important components in designing optimized metabolic pathways, This powerful technology is being further improved by the genomics, proteomics, metabolomics and bioinformatics. Complete genome sequences are providing us with the possibility of addressing complex biological questions including metabolic control, regulation and flux. In silico analysis of microbial metabolic pathways is possible from the completed genome sequences. Transcriptome analysis by employing ONA chip allows us to examine the global pattern of gene expression at mRNA level. Two dimensional gel electrophoresis of cellular proteins can be used to examine the global proteome content, which provides us with the information on gene expression at protein level. Bioinformatics can help us to understand the results obtained with these new techniques, and further provides us with a wide range of information contained in the genome sequences. The strategies taken in our lab for the production of pharmaceutical proteins, polyhydroxyalkanoate (a family of completely biodegradable polymer), succinic acid and me chemicals by employing metabolic engineering powered by genomics, proteomics, metabolomics and bioinformatics will be presented.

• Biotechnology and Bioprocess Engineering:BBE
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• 제7권1호
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• pp.43-51
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• 2002

The cloning and expression of $\beta-glucosidase$ II, encoded by the gene ${\beta}glu2$, from thermotolerant yeast Pichia etchellsii into Escherichia coli is described. Cloning of the 7.3 kb BamHI/SalI yeast insert containing ${\beta}glu2$ in pUC18, which allowed for reverse orientation of the insert, resulted in better enzyme expression. Transformation of this plasmid into E. coli JM109 resulted in accumulation of the enzyme in periplasmic space. At $50^{\circ}C$, the highest hydrolytic activity of 1686 IU/g protein was obtained on sophorose. Batch and fed-batch techniques were employed for enzyme production in a 14 L bioreactor. Exponential feeding rates were determined from mass balance equations and these were employed to control specific growth rate and in turn maximize cell growth and enzyme production. Media optimization coupled with this strategy resulted in increased enzyme units of 1.2 kU/L at a stabilized growth rate of $0.14\;h^{-l}$. Increased enzyme production in bioreactor was accompanied by formation of inclusion bodies.

• 한방안이비인후피부과학회지
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• 제11권1호
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• pp.1-22
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• 1998

During the last few years, nitric oxide(NO) as a potent macrophage-derived effector molecule against a variety of bacteria, parasites, and tumors has received increasing attention. More recent studies suggest that NO also has antiviral effects in both murine and human cells. The objective of the current study was to determine the effect of Yongdamsagantang(YST) on the production of NO. Stimulation of mouse peritoneal macrophages with YST after the treatment of recombinant $interferon-{\gammer}(rlFN-{\gammer})$ resulted in the increased NO synthesis. YST had no effect on NO synthesis by itself. When YST was used in combination with $rIFN-{\gammer}$, there was a marked cooperative induction of NO synthesis in a dose-dependent manner. The optimal effect of YST on NO synthesis was shown 6 hour after treatment with $rIFN-{\gammer}$. This increase in NO synthesis was reflected as increased amount of inducible NO synthase(iNOS) protein. NO production was inhibited by $N^G-monomethyl-L-arginine$. The increased production of NO from $rIFN-{\gammer}$ plus YST-stimulated cells was decreased by the treatment with staurosporin. In addition, synergy between $rIFN-{\gammer}$ and YST was mainly dependent on YST-induced tumor necrosis $factor-{\alpha}(TNF-{\alpha})$ secretion. These results suggest that the capacity of YST to increase NO production from $rIFN-{\alpha}-primed$ mouse peritoneal macrophages is the result of YST-induced $TNF-{\alpha}$ secretion.

• 생명과학회지
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• 제20권10호
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• pp.1427-1432
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• 2010

본 연구는 WSSV의 재조합단백질 rVP466에 대하여 생산된 항혈청을 사용하여 WSSV에 대한 neutralization (중화) 효과를 확인하고자 수행하였다. 먼저 재조합단백질 rVP466의 생산을 위해 WSSV의 구성단백질 VP466을 암호화하는 유전자인 VP466을 포함하는 재조합 플라스미드 pCold-VP466을 제작한 다음 이것을 발현용 숙주인 E. coli RIPL에서 발현하였다. 발현된 rVP466에 대한 항혈청은 토끼를 사용하여 생산하였으며, 항원 rVP466에 대한 특이면역반응은 Western blot을 통해 확인하였다. WSSV에 대한 항혈청의 중화효과를 확인하기 위해 항혈청과 반응시킨 바이러스액($1{\times}10^4$ 배로 희석된 WSSV)을 이용하여 실험용 새우(Penaeus chinensis)에게 주사 감염을 통해 공격실험(challenge test)을 수행하였다. 실험 결과, WSSV로 공격실험한 감염대조구(positive control)의 새우들은 감염 후 17일째에 100% 누적폐사율을 보였으며, preimmune serum과 WSSV의 혼합액을 challenge한 preimmune control의 새우들은 감염 후 25일째에 83%의 누적폐사율을 보였다. WSSV와 rVP466 항혈청을 1:0.01, 1:0.1, 1:1로 혼합한 액으로 challenge한 새우들은 감염 후 25일째에 각각 73%, 53%, 46%의 누적폐사율을 보였다. 이상의 결과를 통해 WSSV가 rVP466 항혈청에 의해 농도의존적으로 neutralization됨을 확인하였으며, 이는 WSSV 감염과정에 VP466이 관여함을 나타내는 것이다.

• 생명과학회지
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• 제21권6호
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• pp.907-911
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• 2011

인간형 재조합단백질 각질세포 성장인자를 안정적으로 생산하는 누에 배양 세포(Bm5-hKGF cell)을 만들었다. 이 세포에서 분비되어 배지에 포함된 양은 15-20 ng/ml 정도였다. Bm5-hKGF cell에 누에의 PDI를 함께 발현시키면 세포외 분비량이 2배 증가하였다. Wound healing migration assay 결과 누에세포에서 생산된 인간형 재조합단백질 각질세포 성장인자는 세포생장을 촉진하는 활성을 가지고 있었다. 본 실험의 결과는 누에배양세포를 사용하여 저비용으로 양질의 인간형 재조합단백질을 대량생산 할 수 있는 것을 기대한다.