• Journal of Microbiology and Biotechnology
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• 제15권3호
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• pp.544-550
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• 2005

To develop a highly efficient mammalian expression vector, a series of vectors were constructed based on the murine cytomegalovirus (MCMV) immediate-early (IE) promoter and human ${\beta}$-globin second intron. The resulting MCMV promoter was several-fold stronger than the HCMV promoter in various mammalian cell lines, such as the NIH3T3, Neuro-2a, 293T, and HT1080 cell lines, and was only slightly weaker than the HCMV promoter in HeLa and CHO cells. The inclusion of the human ${\beta}$-globin second intron behind the MCMV promoter or HCMV promoter markedly enhanced the promoter activity in various mammalian cell lines, and the resultant MCMV/Glo-I expression system was stronger than the HCMV promoter from 4.7- to 11.2-fold in every cell line tested. Also, the MCMV/Glo-I promoter induced a higher level of the VSV-G protein in a transiently transfected 293T cell line, which is useful for the production of recombinant retrovirus and lentivirus vectors.

• 한국식물병리학회:학술대회논문집
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• 한국식물병리학회 2003년도 정기총회 및 추계학술발표회
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• pp.65.1-65
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• 2003

In addition to the five well-characterized genes of Tobacco mosaic virus (TMV), this virus contains a sixth open reading frame (ORF6) that encodes a 4.8 kDa protein. TMV ORF6 overlaps the ORFs encoding the 30 kDa movement protein and the adjacent 17.5 kDa capsid protein. Although the 4.8 kDa protein could not be detected in vivo, alteration of the AUG codons of this ORF resulted in a mutant virus that attenuated the virulence of the mutated TMV in Nicotiana benthamiana, but not N. tabacum (tobacco). These sequence changes did not affect either the replication or movement of the mutated TMV. Expression of TMV ORF6 from the virus expression vector Potato virus X (PVX) intensified the virulence of this virus in N. benthmiana, but not tobacco, while expression of TMV ORF6 from the virus expression vector Tobacco rattle virus enhanced the pathogenicity observed in both N. benthamima and tobacco. Thus, the TMV ORF6 is a host- and virus-specific. virulence factor. However, two separate assays indicated that the TMV 4.8 kDa protein was not a suppression of RNA silencing. A fusion protein formed between the TMV 4.8 kDa protein and the green fluorescent protein was expressed from the PVX vector and localized to plasmodesmata. Possible roles of the 4.8 kDa protein in pathogenicity will be discussed

• 한국가축번식학회지
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• 제27권1호
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• pp.15-23
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• 2003

본 연구는 VSV-G glycoprotein을 envelope으로 하는 pseudotyped retrovirus vector system을 이용하여 쥐의 유방상피세포인 HC11에서 human Lactadherin 유전자의 발현을 확인하고자 하였다. 실험에 사용한 vector는 개체내에서의 외래 유전자의 지속적인 발현에 의한 생리적인 부작용을 최소화하기 위한 구조로, 조직특이적이며 lactogenic hormone에 의해 유도적인 활성을 가지는 것으로 알려진 WAP promoter의 통제하에 도입하고자 하는 외래 유전자를 위치하도록 하였다. WAP promoter의 대조군으로 지속적인 활성을 나타내는 $\beta$-actin promoter를 사용하였으며, 이 각각의 promoter와 marker gene으로 E. coli LacZ gene을 재조합한 후 retrovirus vector system을 이용하여 HCll에 도입하였다. 세포의 genome 내로의 유전자의 전이는 PCR을 통해 확인하였고, RT-PCR의 수행으로 유전자의 발현을 확인하였다. Lactadherin 유전자를 이용한 실험도 동일한 과정으로 수행하였으며, RT-PCR의 결과에서 HCll 세포에서 Lactadherin 유전자의 발현이 insulin을 단독으로 처리한 군에 비해 insulin, hydrocortisone, prolactin을 동시에 처리한 군에서 우월하게 나타나는 것으로 확인되었다. 그러나 insulin 단독 처리군에서 유전자의 발현이 약하게 나타나는 것으로 관찰되어 WAP promoter의 leakiness에 대한 재고의 필요성이 요구되었다.

• Journal of Animal Science and Technology
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• 제50권2호
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• pp.177-184
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• 2008

Acid-labile subunit(ALS)는 85-kDa 크기의 당단백질로서 7.5-kDa의 insulin-like growth factor(IGF) 및 40~45-kDa IGF-binding protein-3와 결합하여 150-kDa ternary complex를 형성하는 혈장단백질이다. 선행연구에서 본 연구진은 reverse transcription-polymerase chain reaction(RT-PCR) 방법으로 돼지(porcine) ALS(pALS)의 coding sequence를 합성하여 plasmid vector에 삽입시켜 ‘expression construct’를 제작한 바 있다. 그러나 본 expression construct의 pALS coding sequence에는 PCR error로 추정되는 원인으로 말미암아 2개의 bases에서 mis-sense mutation이 일어난 것이 발견되었다. 본 연구에서는 ‘site-directed mutagenesis’ 방법으로 pALS의 올바른 coding sequence를 합성하여 ‘insert DNA’의 마지막 codon 다음에 ‘His-tag’ sequence가 위치한 pET- 28a(+) plasmid expression vector에 삽입하였다. 본 expression construct는 E. coli BL21(DE3) 세포에서 ‘induction’ 시켰고, 발현된 유전자재조합(recombinant) peptide는 Ni-affinity chromato- graphy로 정제하였다. 이렇게 affinity chro- matography로 정제된 peptide는 SDS-PAGE에서 66kDa 위치에 single band를 나타냄으로써 recombinant pALS의 예상된 질량과 일치하였다. 이상의 결과는 본 연구에서 recombinant pALS peptide가 성공적으로 발현정제되었음을 시사한다.

• 한국컴퓨터정보학회논문지
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• 제15권4호
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• pp.47-55
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• 2010

본 논문은 실시간 카메라 영상으로부터 얼굴을 검출하고 얼굴 표정을 인식하여 웃음 치료훈련을 할 수 있는 시스템을 제안한다. 제안된 시스템은 카메라 영상으로부터 Haar-like 특징을 이용하여 얼굴 후보 영역을 검출한 다음, SVM분류기를 이용하여 얼굴 후보 영역이 얼굴 영상인지 아닌지를 검증한다. 그 다음에는 검출된 얼굴 영상에 대해, 조명의 영향을 최소화하기 위한 방법으로 히스토그램 매칭을 이용한 조명 정규화를 수행한다. 표정 인식 단계에서는 PCA를 사용하여 얼굴 특징 벡터를 획득한 후 다층퍼셉트론 인공신경망을 이용해 실시간으로 웃음표정을 인식하였다. 본 논문에서 개발된 시스템은 실시간으로 사용자의 웃음 표정을 인식하여 웃음 양을 화면에 표시해 줌으로써 사용자 스스로 웃음 훈련을 할 수 있게 해 준다. 실험 결과에 따르면, 본 논문에서 제안한 방법은 SVM 분류기를 통한 얼굴 후보 영역 검증과 히스토그램 매칭을 이용한 조명정규화를 이용하여 웃음 표정 인식률을 향상시켰다.

• Journal of Microbiology and Biotechnology
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• 제11권1호
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• pp.85-91
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• 2001

To construct an efficient Bacillus subtilis expression vector, strong promoters were isolated from the chromosomal DNA libraries of Clostridium acetobutylicum ATCC 4259, Thermoactinomyces sp. E79, and Bacillus thermoglucosidasius KCTC 3400. The $P_{C27}$ promoter cloned from the clostridial chromosmal DNA showed a 5-fold higher promoter strength than the $P_{SP02}$ promoter in the expression of the cat gene, and its sequence was estimated as an upstream region of the predicted hypothetical gene (tet-R family bacterial transcription regulator gene) in C. acetobutylicum. As a promoter element, $P_{C27}$ exhibited putative nucleotide sequences that can bind with bacterial RNAP and the 3'end of the 16S rRNA just upstream of the start codon. In addition, the promoter activity of $P_{C27}$ was distinctively repressed in the presence of glucose. Using $P_{C27}$ as the promoter element, a glucose controllable B. subtilis expression vector was constructed and the lipase gene from Staphylococcus haemolyticus KCTC 8957P was expressed in B. subtilis. When compared with the lipase expression by the T7 promoter induced by IPTG in E. coli, the $P_{C27}$ promoter showed about a 1.5-fold higher expression level in B. subtilis than that without induction.

• 미생물학회지
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• 제30권2호
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• pp.141-148
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• 1992

In order to overexpress bacteriophage PRD1 DNA polymerase in E. coli cells, the 2 kb HaeII fragment was isolated from phage genomic DNA. This fragment was then cloned into pEMBL/sup ex/ 3-expression vector. A specific 57bp deletion was performed by using uracil containing ss DNA and oligonucleotide spanning each region to remove an unwanted non-coding region. After this deletion, the PRD1 DNA polymerase gene is totally under the control of the vector promoter and SD sequence. Upon heat induction, a protein with an apparent size of 68 kdal was overexpressed as an active PRD1 DNA polymerase. The expression of PRD1 DNA polymerase was about 1% of total E. coli protein.

• 한국미생물·생명공학회지
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• 제21권3호
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• pp.214-220
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• 1993

The mating type a of yeast, Saccharomyces cerevisiae mutant with hmla2-102 and sir3-8ts was changed to type alpha by changing the growth temperature from 25C to 35C. A temperature-sensitive expression vector system was constructed using mating factor alpha1 (Mfalpha1) gene encoding alpha factor which is expressed in the type alpha cells. Vectors with different copy numbers were constructed by joining the promoter and pre or prepro-secretion single sequence of Mfalpha1 to promoterless PHO5' gene as a reporter gene.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2003년도 생물공학의 동향(XIII)
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• pp.186-189
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• 2003

Helicobacter pylori is the etiologic agent of human gastritis and peptic ulceration and produces urease as the major protein component on its surface. H. pylori urease is known to serve as a potent immunogen as well as major virulence factor. In order to express the recombinant urease in tobacco plants, a DNA fragment containing the minimal H. pylori urease gene cluster was subcloned into a plant expression vector. The recombinant vector was transformed to tobacco plants. The integration of the recombinant plasmids into tobacco chromosomal genome was verified by genomic PCR. Expression to mRNA was confirmed by Northern blot analysis, and expression to recombinant urease protein was observed by Western blot analysis. These results showed that the recombinant urease can be produced in tobacco plants and will be tested for immune response to use as an edible vaccine.

• International Journal of Industrial Entomology and Biomaterials
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• 제2권2호
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• pp.155-160
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• 2001

We have generated a novel baculovirus genome which can be maintained in Escherichia coli that facilitates the rapid and efficient generation of recombinant baculovirus expression vectors. To make $Occ^{+}$ recombinant expression vectors, polyhedrin gene under the control of p10 promoter was inserted to bAcGOZA and this genome was designated bApGOZA. As in bAcGOZA, bApGOZA lacks a portion of the essential ORF1629 gene, but includes a mini-F replicon and selectable kanamycin-resistance marker, This occasion-producing activity of bApGOZA can be used very conveniently for its oral infectivity to insect larvae in mass production of foreign protein and insecticides.