• 생명과학회지
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• 제9권3호
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• pp.253-261
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• 1999

Leptin gene, an obesity gene, has been known to involve in the regulation of food intake and body weight. It is also thought to be related to the glucose metabolism, insulin secretion and type II diabetes mellitus. Recently, the production of recombinant leptin protein has been attempted for the application in the treatment of obesity and the correction of hereditary obesity and type II diabetes. In the present study, leptin cDNA was cloned from mouse fat cells by RT-PCR and prokaryotic expression of leptin was attempted in order ot prepare a leptin-specific antigen. Immunization of a rabbit with the leptin-specific antigen into a rabbit resulted in the generation of leptin-specific antiserum that could be useful in the detection of leption expressed in various tissues. The sequence of leptin cDNA prepared in the present study wa identical to the previously reported one. Transformation of E. coli(DH5a) cells with the leptin cDNA-inserted translation vector, pGEX-4T-3-leptin followed by treatment with IPTG (0.1mM) resulted in the expression of a large amount of GST-leptin fusion protein with a molecular weight of 44 KDa as an inclusion body. Denaturation of the insoluble fusion protein by 8M urea, 6M guanidium-HCI or 0.1% 2-mercaptoethanol followed by a slow oxidation could not solubilize the inclusion body. The cell extract was subjected to SDS-PAGE and GST-leptin protein electroeluted from the gel was then injected into a rabbit subcutaneously for the immunization. Anti-GST-leptin rabbit antiserum which had a cross reactivity to the GST-leptin protein was generated. Leptin protein expressed in mouse brain and fat tissues was detected by Western blot immunodetection system using the antiserum generated in the present study.

• The Journal of Advanced Prosthodontics
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• 제7권6호
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• pp.468-474
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• 2015

PURPOSE. The aim of this study was to characterize and compare bacterial diversity on the removable partial denture (RPD) framework over time. MATERIALS AND METHODS. This descriptive pilot study included five women who were rehabilitated with free-end mandibular RPD. The biofilm on T-bar clasps were collected 1 week ($t_1$) and 4 months ($t_2$) after the RPD was inserted ($t_0$). Bacterial 16S rDNA was extracted and PCR amplified. Amplicons were cloned; clones were submitted to cycle sequencing, and sequences were compared with GenBank (98% similarity). RESULTS. A total of 180 sequences with more than 499 bp were obtained. Two phylogenetic trees with 84 ($t_1$) and 96 ($t_2$) clones represented the bacteria biofilm at the RPD. About 93% of the obtained phylotypes fell into 25 known species for $t_1$ and 17 for $t_2$, which were grouped in 5 phyla: Firmicutes ($t_1=82%$; $t_2=60%$), Actinobacteria ($t_1=5%$; $t_2=10%$), Bacteroidetes ($t_1=2%$; $t_2=6%$), Proteobacteria ($t_1=10%$; $t_2=15%$) and Fusobacteria ($t_1=1%$; $t_2=8%$). The libraries also include 3 novel phylotypes for $t_1$ and 11 for $t_2$. Library $t_2$ differs from $t_1$ (P=.004); $t_1$ is a subset of the $t_2$ (P=.052). Periodontal pathogens, such as F. nucleatum, were more prevalent in $t_2$. CONCLUSION. The biofilm composition of the RPD metal clasps changed along time after RPD wearing. The RPD framework may act as a reservoir for potentially pathogenic bacteria and the RPD wearers may benefit from regular follow-up visits and strategies on prosthesis-related oral health instructions.

• 한국작물학회:학술대회논문집
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• 한국작물학회 2006년도 한국약용작물학회 공동춘계학술발표회
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• pp.150-151
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• 2006

• Journal of Microbiology and Biotechnology
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• 제18권5호
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• pp.926-932
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• 2008

Human heavy chain (H-) and light chain (L-) ferritins were amplified from a human cDNA library. Each ferritin gene was inserted downstream of the T7 promoter of bacterial expression vectors, and two types of coexpression vectors were constructed. The expression levels of recombinant ferritins ranged about 26-36% of whole-cell protein. H-ferritin exhibited a lower expression ratio compared with L-ferritin, by a coexpression system. However, the coexpression of HL-ferritins was significantly increased above the expression ratio of H-ferritin by cultivation without IPTG induction overnight. Purified recombinant H-, L-, HL-, and LH-ferritins were shown to be homo- and heteropolymeric high molecular complexes and it was indicated that their assembled subunits would be able to work functionally in the cell. Thus, these results indicate an improvement in the expression strategy of H-ferritin for heteropolymeric production and studies of ferritin assembly in Escherichia coli.

• 대한의생명과학회지
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• 제16권4호
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• pp.299-305
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• 2010

KCNE1 is the causal gene of long QT syndrome. KCNE1 gene is located in chromosome 21. In compliance with this KCNE1 gene the proteins come out. KCNE1 is responsible for $K^+$ channel which maintains the normal function of the heart muscle for contraction. Affected individuals manifest prolongation of the QT interval on electrocardiongrams, a sign of abnormal cardiac repolarization. The clinical features of LQT result from episodic cardiac arrhythmias, such as torsade de pointes and ventricular fibrllation. Blood DNA was isolated and kept in $4^{\circ}C$ refrigerator. The KCNE1 gene was amplified by PCR method and about 414 bp band was identified by agarose gel electrophoresis. PCR products were inserted into pGEX-4T-1 vector in order to express KCNE1 protein after treatment with IPTG SDS-PAGE was carried out and the protein band which was about 47 kDa was clearly odserved. Results of this study would contribute to the detailed understanding of KCNE1 protein function and to designing better treatment of Long QT symdrome.

• 식물조직배양학회지
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• 제22권3호
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• pp.149-155
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• 1995

국내에서 분리 동정한 오이모자이크 바이러스(CMV)로부터 외피단백질(CP) 유전자를 분리하였고 이의 염기서열을 결정한 결과 129번째 아미노산이 proline으로서 mosaic symptom을 보이고 염기 및 아미노산 서열의 유사성을 비교한 결과 subgroup I (type I)에 속한다는 것을 확인하였다. 분리한 CP 유전자를 식물형질전환용 운반체에 삽입하여 담배 재배종인 NC82와 바이러스 증식용인 Samsun에 잎절편을 통하여 형질전환시켰다. 형질전환된 담배의 DNA를 분리하여 Southern blotting과 PCR을 수행한 결과 대부분의 형질전환체에 CP 유전자가 삽입되였음을 확인하였고 T$_1$ 종자를 kanamycin이 첨가된 배지에서 후대 항생제 저항성 검정을 실시한 결과 1개의 CP 유전자가 삽입된 경우가 50%에 달했고 2개와 3개의 유전자가 삽입된 경우도 각각 39%와 11%에 달했다.

• 미생물학회지
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• 제27권1호
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• pp.63-69
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• 1989

흙으로부터 에탄올을 이용하여 성장하는 분홍색 통성 에단올 자화세율을 분리하여 Methylobacteriu~η sp. strain SY 1이라 명명하였다. 이 세균은 그람음성세균으호 약간 굽은 간균이고 한쪽 끝에 달련 한개의 편모로 운동하였다. 세균의 군략은 매끈 하고 밝은 분홍맺을 띄우며 정성이 높았다. 이 세균이 지니는 DNA의 G+C 함량은 약 66%로 나타났다. 이 제균은 칠대호가 성으호 catalase와 oxidase 활성을 나타내었고, carotenoid 색소와 poly-$\beta$-hydroxy butyrate를 가지고 있었다. 이 세균은 또 한 세가지 종류의 큰 plasmid DNA (45,000, 38,500, 23,000)을 가졌으며, $30^{\circ}C$와 pH7.0에서 0.5%(v/v)의 에탄올을 이용 하여 빼른 성장을 하였다($t_{d}$=6.5시간), 이 세균은 메탄올은 울온 여러까지 종듀의 당, 유기산, 아미노산, 아민 및 알코올을 이용하여 성장할 수 있었고, 메탄올은 serine pathway를 통하여 이 세균의 세포 구성물질로 전환됨을 알 수 있었다.

• Journal of Plant Biotechnology
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• 제34권1호
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• pp.11-17
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• 2007

수박작물의 대목용으로 사용하는 수박공대에 CGiMMV-CP 유전자를 도입하여 개발된 LM 수박공대의 CGMMV 내성 정도를 격리하우스와 노지 포장내에서 조사하였다. 격리온실에서의 $T_{3}$ 형질전환 수박대목의 CGMMV 내성은 접종 후 70일까지 유지되는 반면 대조구는 접종 후 20일에 전부 이병되었다. 인위적 토양전염 포장에서 형질전환체는 접종후71일까지 약 40%의 내성률을 보였으며, 대조구는 접종 후 37일에 모두 이병되었다. 인위적 접촉전염 포장에서 형질전환체는 대조구에 비해 약 10일 정도 지연효과를 보였다. 따라서 CGMMV-CP 형질전환체는 CGMMV에 저항성을 가진 것이 아니라 감염시기를 지연시키는 부분 내성으로 나타났다. CGMMV-CP homozygous T 세대를 진전시켜서 형질전환 수박공대 계통을 $BC_{1}T_{5}$ 세대에서 선발하였다. 또한 LM 수박공대에 형질전환 되지 않은 접수 (슈퍼금천수박)를 접목하여 non-LM 수박을 생산하고 CGMMV-CP 유전자에 관련된 물질의 이동 여부를 조사하였다. PCR, northern, western 분석한 결과 수박공대 대목에서 형성되는 DNA, RNA, protein 물질이 접수로 이동되지 않음을 확인하였다.

• 미생물학회지
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• 제31권4호
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• pp.279-285
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• 1993

p53 유전자의 변화는 인간의 여러 암에서 가장 흔하게 발견되며 종양세포내에서는 이러한 변형된 p53 단백질의 양의 증가가 초래된다. 세포내에 축적된 p53 단백질의 발견은 인간의 암증세를 판단할 유용한 기중이 되기도 한다. 본 연구에서는 이러한 면역조직화학 검사에 쓰일 수 있는 폴리클로날 항체를 만들기 위햐여 사람의 p53 유전자를 glutathione S-transferase 와의 융합 단백질의 형태로서 대장균내에서 발현시켰다. p53 의 아미노산 1-158번을 코딩하고 있는 NeoI fragment 와 아미노산 159-393 번을 코딩하는 NocI-BamHI fragment 를 BamHI linker 를 이용하여 in frame 으로 pGEX-2T 의 BamHI 자리에 삽입하여 재조합 플라스미드 pGTNS 와 pGTNL 을 각각 만들었다. 또 PCR 에 의한 증폭에 의햐여 아미노산 38-145번을 코딩하는 유전자 부위를 증폭하였으며 BamHI 과 PvuII 로 절단하여 pGEX-2T의 BamHI 과 SmaI 자리에 삽입함으로써 pGTBP 를 제조하였다. 이들 재조합 균주들을 IPTG 로 4시간 induction 한 후 세포 추출물로부터 glutathione Sepharose bead 를 이용하여 융합단백질을 분리하였다. Bead 에 결합된 단백질은 10% SDS-polyacrylamide gel 에서 전기영동하였으며, 각각의 분자량은 54 kDa, 53 kDa 와 40 kDa 였다. 이러한 방법으로 1리터 배양으로부터 약 1mg 의 단백질을 정제하였다.

• 한국발생생물학회지:발생과생식
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• 제23권4호
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• pp.385-390
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• 2019

Upon gene inactivation in animal models, the zebrafish (Danio rerio) has become a useful model organism for many reasons, including the fact that it is amenable to various forms of genetic manipulation. Genome editing is a type of genetic engineering in which DNA is inserted, deleted, modified, or replaced in the genome of a living organism. Mainly, CRISPR (clustered regularly interspaced short palindromic repeats) Cas9 (CRISPR-associated protein 9) is a technology that enables geneticists to edit parts of the genome. In this study, we utilized this technology to generate an mmp15b mutant by using zebrafish as an animal model. MMP15 is the membrane-type MMP (MT-MMP) which is a recently identified matrix metalloproteinase (MMP) capable of degrading all kinds of extracellular matrix proteins as well as numerous bioactive molecules. Although the newly-established mmp15b zebrafish mutant didn't exhibit morphological phenotypes in the developing embryos, it might be further utilized to understand the role of MMP15 in liver-related diseases, such as liver fibrosis, and associated pathogeneses in humans.