• 한국잠사곤충학회지
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• 제38권2호
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• pp.144-149
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• 1996

곤충 바이러스인 Baculoviridae의 subgroup A에 속하는 핵다각체병 바이러스는 미생물 살충제로서, 또는 유용물질의 생산을 위한 발현벡터로서 현재 널리 연구·이용되고 있다. 본 연구에서는 이러한 NPV중 국내에서 분리된 SeNPV의 다각체 단백질 유전자의 구조적 특성을 구명함으로써 새로운 발현벡터를 제작하고자 하였다. 이를 위해 SeNPV의 다각체 모양을 전자현미경으로 관찰하고, 다각체 단백질의 분자량과 그 유전자의 구조를 각각 SDS-PAGE 및 염기서열 결정법에 의해 결정하였다. 그 결과, SeNPV의 다각체는 분자량 30 kDa의 단일 단백질로 이루어진 부정형의 구조였으며, 다각체 단백질 유전자의 염기서열을 포함한 876 염기의 서열을 결정하였다. 또한, SeNPV 전체 DNA상에서 다각체 단백질 유전자는 Xho I 3.0 Kb와 Nco I 6.0 Kb에 각각 존재함을 확인하고, 각각 cloning하여 제한효소 지도를 작성하였다.

• International Journal of Industrial Entomology and Biomaterials
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• 제19권2호
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• pp.229-235
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• 2009

We have cloned and characterized a lipocalin from the bumblebee Bombus ignitus (Bi-lipocalin). The Bi-lipocalin gene spans 2284 bp and consists of four exons coding for 270 amino acid residues. Sequence analysis revealed that Bi-lipocalin possesses three structurally conserved regions (SCTs) that characterize lipocalins. Recombinant Bi-lipocalin, expressed as a 37 kDa protein in baculovirus-infected insect cells, was N-glycosylated, indicating that the carbohydrate moieties are necessary for secretion. Tissue distribution analysis revealed ubiquitous expression of Bi-lipocalin in all tissues examined. Bi-lipocalin transcripts were upregulated by stress, such as wounding, $H_2O_2$ exposure, and external temperature shock. These results indicate that Bi-lipocalin is a stress-inducible protein that acts on wounding, $H_2O_2$ overexposure and temperature stimulation.

• 대한화학회지
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• 제59권5호
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• pp.466-470
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• 2015

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2002년도 생물공학의 동향 (X)
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• pp.62-65
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• 2002

We have previously shown that the addition of silkworm hemolymph to a culture medium increases the longevity of insect and mammalian cells by inhibiting apoptosis. This indicates that the component which inhibits apoptosis is contained in the silkworm hemolymph, The apoptosis-inhibiting component was isolated from silkwonn hemolymph and characterized in our previous study. A database search using the N-terminal amino acid sequence of this component as a template resulted in a 95% homology with a low molecular weight lipoprotein, the so called ’30K protein' of unknown function. In this study, the 30K protein gene was expressed in mammalian and insect cells to confirm the apoptosis-inhibiting effect. The overexpression of 30K protein in mammalian cell inhibited the staurosporin-induced apoptosis by the prevention of the activation of caspase 3. Using an Autographa californicanuclear polyhedrosis virus (AcNPV) system, the 30K protein was overexpressed also in insect cells. The expression of the 30K protein increased the longevity of baculovirus-infected insect cells by inhibiting apoptosis. These results suggest that the 30K protein is a novel anti-apoptotic protein.

• 한국응용곤충학회지
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• 제36권4호
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• pp.331-340
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• 1997

곤충 유전자를 이용한 항세균성 펩타이드 생산 및 응용에 관한 기초 연구로서 비병원성 세균(Escherichia coli K12)으로 면역 반응을 유도한 누에로부터 발현량이 증가하는 유전자 중 Hyalophora cecropia의 attacin과 cDNA 상동성을 나타내는 BmInc6 클론을 분리하고 그 특성을 조사하였다. BmInc6 cDNA의 전체염기서열을 분석한 결과 그 크기는 852 bp이고, 35번째 염기에서 변역이 개시되어 679 bp 위치에서 종결되는 open reading frame을 가지며 812번째 위치에 잠정 전사 종결 신호의 존재가 확인되었다(GenBank, AF005384). BmInc6에 의해 coding되는 아미노산은 214개이며, hydropathy 분석 결과 친수성을 나타내는 단백질이었다. 그리고 BmInc6 유전자에 의해 연역되는 펩타이드를 nuecin으로 명명하였다. Nuecin 유전자를 baculovirus 발현 백터계를 이용하여 곤충세포주에서 발현시킨 결과 전사체의 크기는 약 950 bp였고, 세포내 벌현 펩타이드의 분자량은 약 23 kDa이었다. 세포내에서 발현된 nuecin 전구체로 추정되는 23 kDa 펩타이드는 세포외로 분비되는 과정에서 약 3 kDa의 signal 펩타이드가 제거됨으로서 약 20 kDa의 성숙 nuecin으로 된다는 사실을 단백질 전기영동상으로 확인하였다. Nuecin 단백질의 항세균 활성을 수종의 그람 음성 및 양성 세균에 대해 검정한 결과, 특히 E. coli와 Bacillus subtilis에 높은 활성을 나타내었으며, attacin이 한정된 그람 음성 세균에만 항세균 활성을 나타내는데 비해 nuecin은 그람 음성은 물론 그람 양성에도 항세균 활성을 나타내어, 보다 넓은 항세균 스펙트럼을 가지는 것을 알 수 있었다.

• International Journal of Industrial Entomology and Biomaterials
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• 제28권2호
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• pp.71-84
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• 2014

The full-length heat shock protein 88 (HSP88) complementary DNA (cDNA) of Paecilomyces tenuipes Jocheon-1 was obtained by screening the Paecilomyces tenuipes (P. tenuipes) Jocheon-1 Uni-Zap cDNA library and performing 5' RACE polymerase chain reaction (PCR). The P. tenuipes Jocheon-1 HSP88 cDNA contained an open reading frame (ORF) of 2,139-basepair encoding 713 amino acid residues. The deduced amino acid sequence of the P. tenuipe s Jocheon-1 HSP88 cDNA showed 77% identity to Nectria haematococca HSP88 and 45-76% identity to other fungal homologous HSP88s. Phylogenetic analysis and BLAST program analysis confirmed that the deduced amino acid sequences of the P. tenuipes Jocheon-1 HSP88 gene belonged to the ascomycetes group within the fungal clade. The P. tenuipes Jocheon-1 HSP88 also contained the conserved ATPase domain at the N-terminal region. The cDNA encoding P. tenuipes Jocheon-1 HSP88 was expressed as an 88 kilodalton (kDa) polypeptide in baculovirus-infected insect Sf9 cells. Under higher temperature conditions for the growth of the entomopathogenic fungus, mRNA expression of P. tenuipes Jocheon-1 HSP88 was quantified by real time PCR (qPCR). The results showed that heat shock stress induced a higher level of mRNA expression compared to normal growth conditions.

• 한국동물위생학회지
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• 제37권3호
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• pp.143-150
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• 2014

Porcine reproductive and respiratory syndrome virus (PRRSV) is the etiological agent of PRRS characterized by reproductive losses in sows and respiratory disorders in piglets. The PRRSV is a small enveloped virus containing a positive-sense, single-stranded RNA genome and divided into two genotype, type 1 (European) and type 2 (North American), respectively, by nucleotide identity. In this study, ORF7 gene of the type 1 and type 2 PRRSV was cloned and expressed in Baculovirus expression system. Also, monoclonal antibodies (MAbs) against ORF7 were produced and characterized. The expressed ORF7 proteins in the recombinant virus were confirmed by indirect fluorescence antibody (IFA) test using His6 and PRRSV-specific antiserum. A total of eight MAbs were produced and characterized. One (3G12) MAb was type 1 PRRSV ORF7-specific and two (6B10 and 16H8) were type 2 PRRSV ORF7-specific. Other five (1A1, 2A4, 4B4, 12C4 and 13F11) MAbs reacted with both type 1 and type 2 PRRSV. Some PRRSV ORF7-specific MAbs recognized the porcine tissues infected with PRRSV by IFA or immunohistochemistry (IHC) assay. From this experiment, it was confirmed that MAbs produced in this study were PRRSV ORF7-specific and could be used as reliable reagents for type 1/type 2 PRRSV detection.

• 한국미생물·생명공학회지
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• 제29권1호
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• pp.18-24
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• 2001

누에 핵다각체병 바이러스의 gp64 프로모터를 이용한 transient 발현 벡터를 제작하기 위해서 gp64 유전자의 구조를 분석하였다. Southern blotting 분석을 통해 genome DNA에서 gp64 유전자를 탐색하기 gp64 구조유전자를 포함하는 2,277 nucleotide의 염기를 분석하였으며 gp64의 early, late 프로모터 발현을 조절하는 인자들을 확인하였다. gp64프로모터를 이용한 transient 발현 벡터를 제작하고 외래유전자로써 lacZ 유전자를 Bm5 세포주에서 transient 발현시켰다. 세포주 내에 도입된 플라스미드 DNA의 안정성을 확인하였으며, gp64 프로모터의 외래유전자 발현성 여부를 조사하기 위하여 gp64 프로모터 하에 laxZ 유전자를 가지는 재조합 바이러스를 제작하고 $\beta$-galactosidase in 냐셔 staining을 수행한 결과 전체적인 발현량은 매우 약한 것으로 판단되었다. BmNPV-K1의 gp64 프로모터를 이용한 벡터는 더욱 민감한 표지 유전자를 발현시켜 재조합 바이러스의 분리에 이용하거나 숙주세포에 독성을 보이는 유전자 산물의 소량 발현에 더욱 유용할 것으로 판단된다.

• International Journal of Industrial Entomology and Biomaterials
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• 제3권1호
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• pp.43-49
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• 2001

We have cloned and characterized an immediate early-1 gene, iel, which is activated immediately upon entrance of the viral genome into the cell nucleus, from Bombyx mori nuclear polyhedrosis virus (BmNPV) K1 strain. This gene encodes a protein 584 amino acids with a predicted molecular weight of 67 kDa. The promoter and coding regions of BmNPV-K1 ie1 showed high homology with Autographa californica nuclear polyhedrosis virus and BmNPV T3 strain. The BmNPV-K1 ie1 was different from amino acid sequence at 4 positions in BmNPV T3. The location of ie1 gene in the BmNPV-K1 genome was confirmed by Southern blot analysis and its expression patterns at the transcriptional level in the infected cells were confirmed by Nerthern hybridization analysis.

• International Journal of Industrial Entomology and Biomaterials
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• 제3권1호
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• pp.25-29
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• 2001

We have cloned and characterized an antiapoptotic gene, p35, which blocks apoptosis, from Bombyx mori nuclear polyhedrosis virus (BmNPV) K1 strain. The 897 bp p35 has an open reading frame of 299 amino acids. The BmNPV-K1 p35 showed a high identity to Autographa californica nuclear polyhedrosis virus and BmNPV T3 strain. The BmNPV-K1 p35 was different from the amino acid sequences of BmNPV T3 at 6 positions. The p35 gene of BmNPV-K1 was 99.2% identical at the nucleotide level and 98% identical at the amino acid level to BmNPV T3. The location of p35 gene in the BmNPV-K1 genome was confirmed by Southern blot analysis and its expression patterns at the transcriptional level in the infected cells were con- firmed by Northern hybridization analysis.