Body and head lice (Pediculus humanus humanus and Pediculus humanus capitis, respectively) are typical ectoparasites of humans. They differ not only in the ecological habitat but also in the vector competence in spite of their conspecific nature. Only body lice transmit several bacterial pathogens to humans, including Bartonella quintana, Rickettsia prowazekii and Borrelia recurrentis. In this study, the proliferation rates of two model bacteria, a gram positive Staphylococcus aureus and a gram negative Escherichia coli, were determined following bacterial challenge by cuticular injection. Both bacteria proliferated rapidly in body lice at the early stage of bacterial challenge but not in head lice, suggesting that head lice have more sensitive immune responses to these bacteria. In vivo phagocytosis assay revealed that head lice have much higher phagocytic activity against E. coli than body lice whereas only slight differences in phagocytic activity against S. aureus were observed between the two lice species. Taken together, these findings suggest that the reduced phagocytosis activity of body lice contributes, at least in part, to their higher vector competence.
Presently, We have constructed an olig-d(T) primed directional cDNA library from the silkworm Dongchunghacho, an entomopathogenic fungus, of which species is belonging to Paecilomyces tenuipes Jocheon-1. To isolate and screen genes in the fungus, 626 expressed sequence tags(ESTs) were generated by a partial sequencing from the cDNA library. Paecilomyces tenuipes Jocheon-1 cDNA encoding the glyceraldehyde-3-phosphate dehydrogenase(Pt-GAPDH) of Paecilomyces tenuipes Jocheon-1 was cloned from the above cDNA library. The complete cDNA sequence of Pt-GAPDHis comprised of 1,014bp encoding 338 amino acid residues. The deduced protein sequence of Pt-GAPDH showed higher homology with Beauberia bassiana-GAPDH(93% amino acid identity). Hydropathy analysis revealed that Pt-GAPDH protein is hydrophilic. The major three amino acids in its composition of amino acid residues were alanine(11.54%), valine(9.47%) and glycine(8.88%). The cDNA encoding Pt-GAPDH was expressed as a 37 kDa polypeptide in baculovirus-infected insect Sf9 cells. The Pt-GAPDH gene of Paecilomyces tenuipes entomopathogenic fungus consisted of three exons and two introns coding for 338 amino acid residues, and the genomic DNA length of the gene spans 1302bp. The accession number of the gene in GenBank are GU997099 for Pt-GAPDH cDNA and GU997102 for Pt-GAPDH genomic DNA.
In this study, a full-length heat shock protein88 complementary DNA (cDNA) of Paecilomyces tenuipes Jocheon-1 was obtained by screening of P. tenuipesJocheon-1 Uni-Zap cDNA library and 5' RACE polymerase chain reaction. The Paecilomyces tenuipes Jocheon-1 heat shock protein88 cDNA contains an open reading frame of 2,139 bp encoding 713 amino acid residues. The deduced amino acid sequence of the P. tenuipes Jocheon-1 HSP88 cDNA showed 77% identity to N. haematococca HSP88 and 45-76% identity to other fungi HSP88. 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 and P. tenuipes Jocheon-1 HSP88 also contains the conserved ATPase domain at the N-terminal. The cDNA encoding P. tenuipes Jocheon-1 HSP88 was expressed as a 88 kDa polypeptide in baculovirus-infected insect Sf9 cells. Under different stress conditions, mRNA expression of P. tenuipes Jocheon-1 HSP88 were quantified by real-time PCR and the result showed that heat shock stress affected the mRNA expression levels of P. tenuipes Jocheon-1 HSP88.
The genomic structure and phylogenetic relationships of HSP88 genes from P. tenuipes Jocheon-1, P. tenuipes, C. militaris and C. pruinosa are described. The HSP88 genomic DNA from P. tenuipes Jocheon-1, P. tenuipes and C. militaris all contain 5 introns and 6 exons with the length of 13, 62, 32, 1438, 306, 288 bp, encoding 713 amino acid residues. C. pruinosa HSP88 genomic DNA contains 4 introns and 5 exons encoding 713 amino acids. The length of each exon of C. pruinosa HSP88 is 13, 62, 32, 1744, 288 bp and the length of exon 4 is identical to the total length of exon 4 and exon 5 of HSP88 of P. tenuipes Jochoen-1, P. tenuipes, and C. militaris. The deduced amino acid sequence of P. tenuipes Jocheon-1 HSP88 showed 99% identity with the P. tenuipes, 97% identity with the Cordyceps militaris, and 98% identity with the C. pruinosa. Phylogenetic analysis confirmed that the P. tenuipes Jocheon-1, P. tenuipes, C. militaris and C. pruinosa HSP88 are placed together within the ascomycetes group of fungal clade.
본 연구는 경북 의성군 봉양면 소재 복숭아 재배농가에서 개화전인 꽃눈에 이상 증상이 발생하여 피해원인을 구명하기 위하여 실시하였다. 2010년 4. 20일 기온이 급상승하면서 피해가 발생하였는데 6,000㎡의 복숭아 과원에 70% 이상 발생하였 고 주변의 과원에서도 피해가 확인되었다. 피해증상은 개화전인 꽃눈의 중앙 및 상단부가 칼로 절단한 것처럼 보이고 꽃눈을 확대해 보면 수술과 암술대가 일정하 게 잘려져 있다. 일부 꽃눈은 꽃잎을 둘러싸고 있는 인편과 꽃잎이 같이 잘려져 있 는 현상도 관찰되었다. 가해해충은 부산우단풍뎅이(Maladera fusania Murayama) 로 몸 길이는 10㎜ 내외의 난형으로 적갈색 또는 흑색이었다.
Bacillus thuringiensis (Bt) is characterized by its ability to synthesize crystal toxins and also able to produce bacteriocins such as thuricin, tochicin, entomocin and bacthuricin. The present work, for the first time, describes the biological activity of bacteriocins from B. thuringiensis subsp. cameroun (Btc). Supernatant which was produced from a liquid culture of Btc had antimicrobial activity against Bacillus cereus, ending up to making a inhibition zone on an agar medium. A significant reduction in antimicrobial activity was observed when the supernatant was exposed to heat at 75~100°C for 15 min. Proteins were separated from the supernatant by a fast protein liquid chromatography (FPLC) given the thermal instability. A group of FPLC fractions had antimicrobial activity against Bt subsp. palmanyolensis, israelensis, 1-3, morrisoni, toguchini and kurstaki, and B. cereus ACTC21768, ATCC14579 and NRRLB-569. Interestingly, when the supernatant was individually incorporated into the liquid cultures of Bt subsp. israelensis (Bti) and mogi (Btm) with mosquitocidal activity, a vegetative cell growth was observed only in the Btm culture 10 h post-incubation. A possible recovery of vegetative Btm cell growth was observed, compared to a control without the supernatant. These results suggest that Btc produced proteinous antimicrobial substances, one of which may be used as a selection marker to separate Btm after possibly conjugating the two mosquitocidal strains.
To determine differential gene expression profiles in the salivary gland of a water stick-insect, Ranatra chinensis Mayrt, a subtractive cDNA library was constructed by suppression subtractive hybridization. The salivary gland was determined among three salivary gland-like tissues by investigating transcription levels of five trypsin genes isolated from R. chinensis. The major transcripts encoding trypsins (64.4% of the total ESTs) were eliminated from the library and then remaining salivary gland-specific genes were searched. A total of 643 expressed sequence tags (ESTs) were clustered and assembled into 148 contigs (49 multiple sequences and 99 singletons), among which 35 contigs had matched BLASTx hits (E ≤ 1.00E-4). Salivary apyrase occupied 5.6% (36 ESTs) of the library. Apyrase is known to be released by female mosquitoes or blood-sucking assassin bugs to prevent blood clots during blood sucking. Therefore, apyrase in the salivary of R. chinensis might allow R. chinensis to facilitate feeding. Several contigs encoding acid phosphatase, hyaluronidase, prophenoloxidase, and dipeptidylpeptidase IV, commonly found in venoms of Hymenoptera, were also identified from the salivary gland-specific library. Discovery of salivary glandspecific genes should promote further studies on biologically active components in the saliva of R. chinensis.
The silkworm-baculovirus expression system has distinct advantages, such as a high yield and safe usage in vertebrates. Here, we report a novel strategy for the large-scale production of a classical swine fever virus (CSFV) envelope glycoprotein E2 in the larvae of a baculovirus-infected silkworm, Bombyx mori. We constructed a recombinant B. mori nucleopolyhedrovirus (BmNPV) that expressed recombinant polyhedra together with the N-terminal 179 amino acids of CSFV E2 (E2ΔC). BmNPV-E2ΔC-infected silkworm larvae expressed native polyhedrin and approximately 44-kDa fusion protein that was detected using both anti-polyhedrin and anti-CSFV E2 antibodies. Electron and confocal microscopy both demonstrated that the recombinant polyhedra contained both the fusion protein and native polyhedrin were morphologically normal and contained CSFV E2ΔC. The CSFV E2ΔC antigen produced in BmNPV-E2ΔC-infected silkworm larvae reached 0.68 mg per ml of hemolymph and 0.53 mg per larva at 6 days post-infection. Six-week-old female BALB/c mice that were immunized with the E2ΔC protein purified from solubilized recombinant polyhedraelicited CSFV E2 antibodies, which indicated that the CSFV E2ΔC protein from recombinant polyhedra was immunogenic. The virus neutralization test showed that the serum from mice that were treated with E2ΔC protein from recombinant polyhedra contained significant levels of virus neutralization activity. These results demonstrate that the present strategy can be used for the large-scale production of CSFV E2 antigen.
Vespa tropica is a tropical species of Vespa found in Southeast Asia. V. tropica wasps were collected from rural provinces of Cambodia, and their total RNA and venom were extracted on site. To search for novel substances in venom, a subtracted cDNA library specific to the venom gland and sac was constructed and venom protein was analyzed by nano-LC-MS/MS. A total of 1127 expressed sequenced sequence tags (ESTs) were sequenced and assembled into 572 contigs (152 multiple sequences and 420 singletons). The short venom peptides were identified to be encoded from 5 contigs (43 ESTs) by proteomic analysis. In addition, putative antimicrobial peptides together with typical major components of wasp venom (venom allergen 5, mastoparan-like peptide, serine protease, and hyaluronidase) were identified in the EST Library. Additional in-depth annotation would be required for further characterization of many unidentified genes found in the EST library.
Aujeszky's disease (AD), also called pseudorabies, is an infectious viral disease caused by an alpha herpes virus and has domestic and wild pigs, as well as a wide range of domestic and wild animals, as the natural host. Aujeszky's disease virus (ADV) virions contain several envelope glycoproteins. Among them, glycoproteins gB, gC and gD are regarded as the major immunogenicity proteins and the antibodies induced by them can neutralize virus in vitro or in vivo. In this study, we investigated expression of these glycoproteins using the bacterial and baculovirus expressionn system. Successful expression of ADV glycoproteins in E. coli was confirmed by SDS-PAGE and Western blot analysis and their optimal expression condition was determined. However, the recombinant proteins generated in the bacterial expression system which lacks glycosylation process frequently lose their biological activity. We tried to express the ADV glycoproteins using the baculovirus expression vector system. The recombinant gB, gC and gD were detected at approximately 100, 60 and 50 kDa on SDS-PAGE and Western blotting, respectively. The optimal expression conditions were determined for MOI(multiplicity of infection) and post-infection days. One MOI and 4 or 5 days post-infection were the best conditions for the expression of the ADV glycoproteins in Sf21 cells. We are currently investigating the antigenicity of recombinant proteins using experimental animals.
약리반응속도론(Pharmacokinetics) 모델을 적용하여 최근 개발되고 있는 항바 이러스성 생화학 작물보호제의 정량적 약효 검정 및 작용기전 탐색 기술을 개발하 고자 본 연구에서는 이를 위한 약리반응속도론적 파라미터를 아래와 같이 정의하 고 사례 실험을 수행하였다. 시험약제를 식물체 또는 바이러스 매개충에 분무처리 하고 (매개충의 보독화 직후 약제처리 하고 시험 유묘에 충 접종) 경과 시간에 따 라 시험 유묘에서 바이러스의 발병율을 검정하면 약효에 따른 발병율을 무처리와 비교 가능하다. 이 때 시간에 따른 발병율의 회귀 곡선식에서 MVIT50 (바이러스 전달 후 감염율 50%에 도달하는 시간), MAT (Mean Antiviral Time = |MVITtreated- MVITcontrol|), AUG (Area Under Graph), 약효의 정량적 값인 F value (=AUGtreated/ AUGcontrol)를 얻을 수 있다. 애멸구에 의해 영속전염하는 벼줄무늬잎마름병바이러 스(Rice Stripe Virus, RSV)에 대하여 신규 개발된 2종의 항바이러스성 생화학 작 물보호제로 상기와 같이 약효 검정 시험을 수행한 결과, f(x)=a(1-exp(-bx))c의 회 귀곡선식이 얻어졌으며 무처리구에서 MVIT50이 1시간, MAT는 0, F value가 1.00으로 계산되었다. 이에 비해 시험 약제인 KNF2016은 MVIT50 5.75, MAT 4.75, F value 0.71, KNF2022는 MVIT50 5.55, MAT 4.55, F value 0.70으로 확인 되었다. 즉, 본 시험 약제는 무처리와 비교하여 각각 F value로 0.29, 0.30의 항바이 러스 약효가 있었다. 1회 처리에 따른 약효 지속기간도 KNF2016이 13.4일, KNF2022가 14일 이상으로 검정되었다. 애멸구 보독충에는 약제처리를 안하고 시 험 유묘에만 경엽처리 후 보독충을 접종할 때, 보독충에서 바이러스는 검정되면서 항바이러스 효과는 시간에 따라 감소하는 것으로 확인됨에 따라 본 시험 약제들을 처리한 벼를 애멸구가 흡즙하더라도 보독충 체내 바이러스에는 영향을 끼치지 않 는 것으로 추정되었다. 따라서 약리반응속도론 모델을 사용하면 항바이러스 작물 보호제의 정량적 약효 평가가 가능함과 동시에 약제의 작용기전 탐색에도 유용한 기술로 평가되었다.
Porcine Circovirus Type2 (PCV2), a single-stranded DNA virus associated with Postweaning multisystemic wasting syndrome(PMWS) of swine, has two major open reading frames, ORF1 and ORF2. The genomic size and molecular weight of ORF2 is respectively 699bp, 28kDa. ORF2 encodes the capsid protein (structural protein) that has type-specific epitopes and is very immunogenic and associated with the induction of neutralizing antibodies, suggesting its potential use in diagnostic assays as well as vaccine development. For efficient production of the capsid proteins, we expressed the PCV2 ORF2 gene with baculovirus in the insect cells. In this study, PCV2 ORF2 was appropriately ligated into the baculovirus transfer vector, pBacPAK9 and pB9-Acpol19-110-EK. Sf21 cells were transfected with a mixture of the purified recombinant transfer vector and bAcGOZA. We generated and purified recombinant viruses containing PCV2 ORF2, and named rAc-B9-PCV2ORF2 and rAc-B9-19-110-EK-PCV2ORF2, respectively. Expression levels of capsid fusion proteins with a partial polyhedrin region of AcNPV more increased than recombinant proteins from non-fusion expressed. Also, expression efficiency increased over time and differed at MOI. As a results, fusion expression of porcine circovirus type2 ORF2 using baculovirus could be utilized as an alternative expression method to produce recombinant antigen against PCV2 infection and is worthy of further investigation.
Many thousands of recombinant proteins have been successfully produced in baculovirus - infected insect cells and larvae. In this study, to improve its value and the yield of recombinant protein production, we constructed transgenic silkworm using Heat shock genes with regard to protein folding. This time, we adapted GAL4/UAS system to express at necessary time point and to carry genes for foreign protein. First, we generated two transgenic cells and silkworm lines that carried the silkworm heat shock proteins, UAS-HOP and UAS-HSC70 and UAS-HSP70 and UAS-HSP40 construct plus 3xP3-DsRED. Subsequently, to drive the GAL4 gene as activatorvector, we engineered Baculoviruses that contain the GAL4 under the P10 promoter linked to the expression cassette of interest foreign genes under the polyhedron promoter. Also, activator vector linked to the GAL4 was designed expressing 6xHis and 6xHis–GST tag. Infection of silkworm larvae with recombinant virus, His-tagged human C3d gene was more efficiently produced transgenic silkworm than that of wild-type, but not His-GST tagged. We show the possibilityin use of HSPs transgenic silkworm system by GAL4/ UAS BmNPV that can generate the efficient production of foreign protein.