• 생명과학회지
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• 제12권1호
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• pp.96-105
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• 2002

식물 단백질의 영양가 향상을 위한 일환으로 필수아미노산의 조성이 풍부한 인공단백질을 암호화하는 인공유전자를 담배 식물체에서 발현을 시도하기 위하여, 식물에서 외래유전자의 발현에 널리 사용되는 Cauliflower mosaic virus (CaMV)의 35S promoter를 이중으로 중첩되도록 하고, (Lys-Glu-Trp)이 64번 반복되는 인공유전자 및 nopaline synthase (nos) terminator를 갖고있는 binary vector pART4-4를 구성하였다. 이 재조합 플라스미드는 Agrobacterium tumefaciens를 이용한 형질전환에 의해 Nicotiann tabacum (Var. Xanthi)으로 도입되었다. Kanamycin이 포함된 신초 유도 배지 및 뿌리 유도배지를 이용하여 정상적으로 재생된 담배 식물체로부터 도입된 인공유전자의 발현을 분석하였다. 추출한 genomic DNA를 EcoRI으로 자른 다음 Southern blot 분석에 의하면, 효소 절단 시 예상되는 1.1 kb에서 band를 형성하였으며 각각의 형질전환 식물체에 인공유전자가 1 또는 3 개씩 도입되어 있음을 확인하였다. Northern blot 분석에 의하면 약 1.2 kb 전사체가 비교적 안정하게 발현되었으며, 잎, 줄기, 뿌리로부터 RNA를 분리하여 promoter의 조직 특이성 발현을 분석한 결과, 잎에서 생성되는 RNA가 줄기나 뿌리 조직보다 안정하게 발현되었다. 형질전환 식물체에서 Western blot에 의한 단백질 분석 결과, 잎에서 추출한 단백질로부터 원하는 크기인 33 kDa의 인공단백질이 생성됨을 확인하였으며 발현 수준은 전체 세포 단백질의 0.1%로서 낮은 수준이었다.

• 동의생리병리학회지
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• 제23권1호
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• pp.199-205
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• 2009

It is unclear whether Fructus ligustri Lucidi (FLL) extract anti-proliferative effect in human glioma cells. The present study was therefore undertaken to examine the effect of FLL on cell viability and to determine the underlying mechanism in A172 human glioma cells. Cell viability and cell death were estimated by MTT assay and trypan blue exclusion assay, respectively. Apoptosis was measured by Annexin-V binding assay and cell cycle analysis. Activation of kinases and caspase-3 was estimated by Western blot analysis. FLL resulted in apoptotic cell death in a dose- and time-dependent manner. FLL-induced cell death was not associated with reactive oxygen species generation. Western blot analysis showed that FLL treatment caused down-regulation of PI3K/Akt pathway, but not ERK. The PI3K/Akt inhibitor LY984002 sensitized the FLL-induced cell death and overexpression of Akt prevented the cell death. FLL induced caspase-3 activation and the FLL-induced cell death was prevented by caspase inhibitors. These findings indicate that FLL results in a caspase-dependent cell death through a P13K/Akt pathway in human glioma cells. These data suggest that FLL may serve as a potential therapeutic agent for malignant human gliomas.

• Parasites, Hosts and Diseases
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• 제38권4호
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• pp.245-249
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• 2000

A total of 492 cattle sera was screened by IgG-ELISA against Neospora caninum (Nc-1 strain and a Korean isolate, KBA-2) and Toxoplasna gondii. Out of 492, 113 sera (23.0%) reacted positively to either Nc-1 or KBA-2 strains of N. caninum. Among the 113 positive sera, 92 sera (81.4%) reacted with antigens of both strains, but 6 sera (5.3%) with Nc-1 and 15 sera (13.3%) with KBA-2 strain only And with T. gondii antigen, 6 sera (1.2%) were positive but all reacted with N. caninum antigen also. Western blot revealed typical binding pattern according to ELISA values, such that high OD group reacted specifically to the major surface proteins including 43 kDa protein. Seroprevalence of 23.0% indicates that neosporosis seemed to be one of major causes of abortion in cattle. It is suggested here to establish more epidemiological researches nationwide systematically.

• The Plant Pathology Journal
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• 제25권3호
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• pp.225-230
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• 2009

Lily mottle virus (LMoV) causes flower quality reduction in Lilium spp. The coat protein gene was RT-PCR-amplified from total RNA extracted from infected lily leaves and the amplified fragment was cloned into the pRSET expression vector tagged with a His-MBP. The plasmid of recombinant coat protein was used to transform an Escherichia coli strain pLysS and was expressed. The coat protein was purified by affinity chromatography using a Ni-NTA resin. The identity of the purified protein was confirmed by SDS-PAGE. The in vitro-expressed protein was used for immunization of mice. The polyclonal and monoclonal antibodies reacted specifically for the detection of LMoV in lily extracts in Western blot. Moreover the monoclonal antibodies reacted with lily extracts in DAS-ELISA with no unspecific or heterologous reactions against other non-serologically related viruses, but the polyclonal antibodies revealed a weak reaction against both infected lily and healthy control.

• Preventive Nutrition and Food Science
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• 제2권4호
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• pp.328-332
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• 1997

The ripe fruit of Hass avocado contains one of the highest elvels of cytochrome P-450 protein found in the plant kingdom. To determine whether wounded roots of avocado contain P-450 protein, the roots of avocado were wounded by slicing, and then allowed to incubate in sealed plastic bags, in 0.4M mannitol, and in the solution to make protoplast preparation containing cellulysin and macerase during the specified times. The microsomal proteins were extracted from the samples, separated by SDS-PAGE, and then subjected to Western blot analysis using polyclonal antibodies which are generated against the CYP71A1 protein. wounded roots in sealed bags produced CYP71A1 within 6 hours after cutting, and those in 0.4M mannitol did not produce CYP71A1 even after 72 hours, but those in the protoplast preparation by cellulysin and macerase induced and produced CYP71A was induced in only 24 hours. These results indicate that CYP 71A1 plays a role for wound healing for root tissue o avocado, and would-inducible P-450 protein was not detected in the mannitol solution by preventing a synthesis of ethylene in a liquid state, but the softening of tissues by cellulysin and macerase to make protoplast preparation was involved in an activation of CYP 71A1 even in the liquid state.

• Biotechnology and Bioprocess Engineering:BBE
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• 제10권3호
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• pp.275-279
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• 2005

Insect cell transformants, stably expressing human $GFP_{uv}-{\beta}1$, 3-N-acetylglucosaminyltransferase 2 $({\beta}3GnT2)$ as the green fluorescent protein $(GFP_{uv})-fused$ protein, were efficiently isolated on Western blot by the quantification of the densitometric intensity of the fusion protein. From almost 150 transformants containing the fusion gene linked to three different types of signal sequence, two transformants, Tn-pXme4a and -pX28a, were successfully selected, showing 8.3 and 8.6 mU/mL ${\beta}3GnT$ activity, respectively. This method requires a screening time almost one-half that required in the isolation of stably transformed cells with high expression levels, and at the same time allows the handling a large number of transformants.

• BMB Reports
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• 제36권5호
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• pp.456-461
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• 2003

Acetolactate synthase (ALS) catalyzes the first common step in the biosynthesis of valine, leucine, and isoleucine. ALS is the target site for several classes of herbicides, including sulfonylureas, imidazolinones, and triazolopyrimidines. Two forms of ALS (designated ALS I and ALS II) were separated from barley shoots by heparin affinity column chromatography. The molecular masses of native ALS I and ALS II were determined to be 248 kDa and 238 kDa by nondenaturing gel electrophoresis and activity staining. Similar molecular masses of two forms of ALS were confirmed by a Western blot analysis. SDS-PAGE and Western blot analysis showed that the molecular masses of the ALS I and ALS II subunits were identical - 65 kDa. The two ALS forms exhibited different properties with respect to the values of $K_m$, pI and optimum pH, and sensitivity to inhibition by herbicides sulfonylurea and imidazolinone as well as to the feedback regulation by the end-product amino acids Val, Leu, and Ile. These results, therefore, suggest that the two ALS forms are not different polymeric forms of the same enzyme, but isozymes.

• 한국발생생물학회지:발생과생식
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• 제4권2호
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• pp.181-186
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• 2000

생쥐 난소에서 glucosamine-6-phosphate deaminase (GNPDA)의 발현을 조사하였다. Western blot상에서 Mr. 31 kDa의 항원을 검출하였으며 출생 후2주에 급격한 발현의 증가가 확인되었다. 난소절편의 면역염색 결과 협막세포와 간충조직에서는 균질한 발현을 보인 반면 난포내 발현양상은 난포의 발달에 따라 다르게 나타났다. 일부 1차 난포의 난자에서 GNPDA의 발현이 관찰되었으나 강소형성 난포의 난자에서는 관찰되지 않았다. 황체화 과립세포에서의 발현은 황체발달에 따라 증가하였으며 황체퇴화 부위에서 강한 신호가 검출되었다. 난포발달에 따른 GNPDA발현의 차이는 GNPDA가 생쥐 난소 조직 재구성에 관여하고 있음을 암시한다.

• 대한수의학회지
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• 제43권3호
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• pp.449-456
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• 2003

The VP2 gene of aquatic birnavirus, Korean isolate (GC-1) was cloned and expressed using the baculovirus expression system. The VP2 gene and VP2 partial gene, which contained a neutralizing epitope, were constructed for recombinant transfer vectors, for baculovirus expression. The expressed recombinant proteins were confirmed by indirect immuno fluorescence antibody (IFA), SDS-PAGE and Western blot. The level of expression was checked at regular time using IFA and Western blot. To measure the neutralizing activity of recombinant proteins against GC-1 strain, the antisera against recombinant proteins were produced by using guinea pigs. The result showed that the antisera neutralized the GC-1 strain. However, the neutralizing titer was higher in antisera against the VP2 gene expressed recombinant protein than that of VP2 partial gene recombinant protein.

• 대한수의학회지
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• 제48권1호
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• pp.1-8
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• 2008

The testis and epididymis are important organs of the male reproductive system; the functionis to produce, mature, transport, and store sperm. It is important to understand the localization and expressionof specific proteins based for the studies of its physiological processes. In this study, we investigated theexpression and distribution of galectin-3, one of beta-galactoside-binding proteins, in the testis andepididymis of mouse using western blot and imunohistochemistry. Western blot analysis revealed that theexpression of galectin-3, 29 kDa protein, was low in the testis. In the epididymis, high expression wasdetected in the body and tail part, but moderate expression in the head part. By immunohistochemicalanalysis, we found that positive localization of galectin-3 was detected in some myoid cells and Leydigin the epithelium of epididymis, especially in the epithelium of both body and tail of epididymis. Collectively,these results suggest that galectin-3 is constitutively expressed in the testis and epididymis of mouse withvarying intensity, and the role of galectin-3 in the male reproductive organ may be involved in the specificfunction of its structures.