• International Journal of Industrial Entomology and Biomaterials
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• 제3권1호
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• pp.75-81
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• 2001

We have constructed a novel recombinant Bombyx mori nuclear polyhedrosis virus (BmNPV) producing the green fluorescent polyhedra. For the production of the fluorescent polyhedra, partial polyhedrin gene containing KRKK as nuclear localization site from the BmNPV polyhedrin gene and the green fluorescent protein (gfp) gene were introduced under the control of p10 promoter of BmNPV. The recombinant BmNPV was stably produced fluorescent polyhedra in the infected Bm5 cells and the morphology of the fluorescent polyhedra was similar to that of wild-type BmNPV. The fluorescent polyhedra had 32 kDa native polyhedrin and 41 kDa fusion protein. From these data, we have further developed a novel BmNPV p10-based transfer vector producing recombinant polyhedra with foreign gene Product. The novel BmNPV P10-based transfer vector is composed of partial polyhedrin gene, factor Xa, and multiple cloning sites.

• International Journal of Industrial Entomology and Biomaterials
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• 제26권2호
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• pp.119-123
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• 2013

The insect baculovirus expression vector system (BEVS) is useful for producing biologically active recombinant proteins. However, the overexpression of heterologous proteins using this system often results in misfolded proteins and the formation of protein aggregates. To overcome this limitation, we developed a versatile baculovirus expression and secretion system using Bombyx mori protein disulfide isomerase (bPDI) as a fusion partner. bPDI gene fusion was found to improve the secretions and antibacterial activities of recombinant nuecin and enbocin proteins. Thus, we conclude that bPDI gene fusion is a useful addition to BEVS for the large-scale production of bioactive recombinant proteins.

• Parasites, Hosts and Diseases
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• 제54권3호
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• pp.265-272
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• 2016

Wolbachia is an obligatory intracellular endosymbiotic bacterium, present in over 20% of all insects altering insect reproductive capabilities and in a wide range of filarial worms which is essential for worm survival and reproduction. In Egypt, no available data were found about Wolbachia searching for it in either mosquitoes or filarial worms. Thus, we aimed to identify the possible concurrent presence of Wolbachia within different mosquitoes and filarial parasites, in Assiut Governorate, Egypt using multiplex PCR. Initially, 6 pools were detected positive for Wolbachia by single PCR. The simultaneous detection of Wolbachia and filarial parasites (Wuchereria bancrofti, Dirofilaria immitis, and Dirofilaria repens) by multiplex PCR was spotted in 5 out of 6 pools, with an overall estimated rate of infection (ERI) of 0.24%. Unexpectedly, the highest ERI (0.53%) was for Anopheles pharoensis with related Wolbachia and W. bancrofti, followed by Aedes (0.42%) and Culex (0.26%). We also observed that Wolbachia altered Culex spp. as a primary vector for W. bancrofti to be replaced by Anopheles sp. Wolbachia within filaria-infected mosquitoes in our locality gives a hope to use bacteria as a new control trend simultaneously targeting the vector and filarial parasites.

• 한국잠사곤충학회지
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• 제37권2호
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• pp.181-185
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• 1995

국내 분리주의 BmNPV를 이용한 전이벡터 pBm-KSK1에 외래 유전자로서 E. coli $\beta$-galactosidase 유전자를 클로닝하여 제작한 재조합 바이러스 BmK1-lacZ의 발현 증대를 위해 재조합 바이러스 감염세포주에 누에 체액의 첨가에 따른 발현효율을 조사하였다. 재조합 바이러스의 배양세포주에서 외래 유전자 발현에 대한 누에 체액의 첨가 효과는 FBS가 2% 또는 5% 일때 누에 체액 2%의 소량 첨가만으로도 누에체액이 첨가되지 않았을 때에 비해 약 2배 이상 높은 발현량을 확인하였으며, 그 보다 FBS가 전혀 첨가되지 않고 단지 누에 체액 2% 첨가만으로도 FBS 첨가시와 마찬가지의 높은 발현량을 보여 누에 체액에 의한 FBS의 대체 효과를 나타내었다.

• Parasites, Hosts and Diseases
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• 제46권1호
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• pp.1-15
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• 2008

Introduction of double-stranded RNA (dsRNA) into some cells or organisms results in degradation of its homologous mRNA, a process called RNA interference (RNAi). The dsRNAs are processed into short interfering RNAs (siRNAs) that subsequently bind to the RNA-induced silencing complex (RISC), causing degradation of target mRNAs. Because of this sequence-specific ability to silence target genes, RNAi has been extensively used to study gene functions and has the potential to control disease pathogens or vectors. With this promise of RNAi to control pathogens and vectors, this paper reviews the current status of RNAi in protozoans, animal parasitic helminths and disease-transmitting vectors, such as insects. Many pathogens and vectors cause severe parasitic diseases in tropical regions and it is difficult to control once the host has been invaded. Intracellularly, RNAi can be highly effective in impeding parasitic development and proliferation within the host. To fully realize its potential as a means to control tropical diseases, appropriate delivery methods for RNAi should be developed, and possible off-target effects should be minimized for specific gene suppression. RNAi can also be utilized to reduce vector competence to interfere with disease transmission, as genes critical for pathogenesis of tropical diseases are knockdowned via RNAi.

• Animal cells and systems
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• 제16권6호
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• pp.455-461
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• 2012

Amongst the various antimicrobial peptides, lysozyme plays a central role in initiating and maintaining the antibacterial defense response of insect. Here we propose the biosynthesis and refolding of recombinant lysozyme in Escherichia coli expressed in inclusion body form. The Agrius lysozyme gene was amplified using gene specific primers and then ligated into the pGEX-4T-1 vector, which contained the glutathione S-transferase (GST) gene as a fusion partner. A recombinant lysozyme was expressed in E. coli Rosetta cells using a pGEX-4T-1 expression vector, and the fusion protein was induced by ioporpyl-${\beta}$-D-thiogalactopyranoside (IPTG). The recombinant protein produced as an inclusion body was resolubilized in solubilization buffer, and the resultant solution was dialyzed in refolding buffer. After thrombin cleavage, the recombinant lysozyme was purified by ion exchange chromatography and reverse phase chromatography. The recombinant lysozyme was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and immunoreactivity against the anti-Agrius lysozyme was observed by western blot analysis of this protein. The recombinant lysozyme displayed antibacterial activity against Bacillus megaterium and Micrococcus luteus, which was confirmed by the inhibition zone assay.

• Plant Resources
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• 제6권3호
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• pp.205-210
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• 2003

Ginseng(Panax ginseng C.A. Meyer) is a perennial herbaceous plant which grows very slowly. It takes about 3 to 4 years from seeding to collecting the ripe seeds and the ginseng propagation is very difficult. and so, it is very difficult to breed ginseng plant. Ginseng tissue culture was started from at 1960, and ginseng commercial product by in vitro callus culture was saled, however upto now, regenerants were not planted to soil normally. Recently, plant genetic engineering to produce transgenic plants by introducing useful genes has been advanced greatly. In a present paper, transformation of ginseng plants was achieved by co-cultivation with Agrobacterium harboring the binary vector coding Proteinase-II gene, which confer resistant or tolerant to insect pests, The binary vector for transformation was constructed with disarmed Ti-plasmid and with double 35S promoter. The NPT II gene and introduced genes of the transgenic ginseng plants were successfully identified by the PCR. Especially the transgenic ginseng plants were regenerated using new techniques such as repetitive single somatic embryogenesis.

• 식물병연구
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• 제14권3호
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• pp.226-228
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• 2008

2007년 경북문경의 옥수수 재배지 약 $1000\;m^2$에 RBSDV가 약 $99{\sim}100%$ 발생되었다. 이 포장은 바로 인접지역에 감나무가 재배되고 있었으며 이 나무 아래에 녹비작물로서 보리를 재배하였다. 보리는 RBSDV의 매개충인 애멸구의 겨울철 중간기주로 작용하여 애멸구의 월동에 유리한 환경으로 작용한 것으로 생각된다. 진단은 S9 특이 primer를 사용한 RT-PCR 방법으로 확인하였다. 이 포장에서 매개충인 애멸구를 채집하여 RT-PCR에 의한 보독충율을 조사하였다. 5월 3일, 6월 7일, 8월 4일 3회 애멸구를 채집하여 검정한 결과 각각 2.9%, 4.8%, 4.4%의 보독충율을 나타내었다. 이것은 RBSDV에 이병된 벼 포장에서 나타나는 보독충율과 비슷한 결과를 나타내었다.

• 한국응용곤충학회지
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• 제47권2호
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• pp.127-131
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• 2008

참나무시들음병의 매개충인 광릉긴나무좀, Platypus koryoensis(Murayama)은 한국에서 참나무시들음병원균인 Raffaelea sp.를 매개하는 것으로 알려져 있다. 참나무시들음 발병정도는 광릉긴나무좀의 밀도에 의존적인 것으로 추정되고 있다. 이에 (樹幹)내 천공수와 집중도가 참나무 피해정도에 미치는 영향을 구명하기 위하여 본 연구를 수행하였다. 수락산 피해지에서 고사목 6그루와 피해목 28그루의 신갈나무의 피해정도, 단위면적당 천공수, 천공간 최근거리를 수간의 상부(지표로부터 50cm)와 하부(지표면)에서 조사하였다. 상부와 하부에서의 천공수는 양의 상관을 보였으며 천공간 최근거리 또한 같은 경향을 보였다. 천공수가 증가할수록 수목의 피해도가 심하였으나 수목의 피해도가 심할수록 천공간 최근거리는 감소하였다. 천공의 분포도 수목의 피해도가 증가할수록 집중분포에서 군일분포로 바뀌었다. 광릉긴나무좀이 초기에는 집중적으로 공격을 하나 수간내 밀도가 증가함에 따라 종내경쟁이 일어나고 경쟁의 결과 개체간 간섭현상이 유도되고 천공의 공간적 분포가 균일하게 변환하게 된다는 것을 암시하는 것이다.

• 대한바이러스학회지
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• 제28권2호
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• pp.129-138
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• 1998

Bovine growth hormone (bGH) gene was expressed in an insect Spodoptera frugiperda cell line using a Baculovirus, Hyphantria cunea nuclear polyhedrosis virus (HcNPV). The bGH gene in pbGH plasmid was sequenced and amplified by PCR technique with two primers containing NcoI sites. The bGH gene consisted of 654 bp (217 amino acid residues), the 5'-untranslated region of the cloned bGH cDNA contains 56 bp, and the 3'-untranslated region contains 145 bp and two pallindromic regions. The amplified bGH gene DNA fragment (654 bp) was inserted into the NcoI site of the pHcEVII vector, which was named pHcbGH. The pHcbGH transfer vector DNA and the wild type HcNPV DNA were cotransfected into S. frugiperda cells to construct a recombinant virus. Eight recombinant viruses were selected and named HcbGH. One clone, HcbGH-4-1 showed largest plaque size, therefore the recombinant virus was further studied. The multiplication pattern of the recombinant HcbGH-4-1 was similar to that of the wild type HcNPV. The bGH gene DNA in the HcbGH-4-1 recombinant was confirmed by Southern blot hybridization. The amount of the bGH (217 amino acid residues, 21 kDa) produced in S. frugiperda cells infected with the HcbGH-4-1 recombinant was approximately 5.5 ng per ml ($10^6$ cells) by radioimmunoassay.