The Riptortus (stinkbug) has a specialized symbiotic organ, M4 midgut, to harboring symbiont Burkholderia. M4 midgut is located in abdomen and surrounded with insect hemolymph. Recently our group demonstrated that symbiotic Burkholderia showed different physiology after adapting in M4 gut compare with in vitro cultured Burkholderia. And population of symbiotic Burkholderia in the M4 midgut is regulated by special organ. However, the molecular mechanism to prevent spreading and migrating symbiont bacteria to other host tissues from symbiotic organ is not clear. Therefore, we assumed that symbiont Burkholderia are susceptible to host humoral immunity after established infection in M4 midgut to prevent spreading and migrating into the other host tissues through Riptortus hemolymph. To prove this assuming, we tested the susceptibility and survival rate of symbiont Burkholderia in hemolymph of Riptortus in vitro and in vivo. We also examined the susceptibility of symbiont Burkholderia using purified antimicrobial peptides (AMP), pyrrhocoricin-like, thanatin-like and defensin-like AMPs. Finally, we tested inducing ability for AMPs by systemic infection of symbiotic Burkholderia. Gene expression of purified AMPs was not different after systemic infection of both symbiont and in vitro cultured Burkholderia. Surprisingly, in vitro cultured Burkholderia resisted on bacteria injected hemolymph and purified AMPs but symbiont Burkholderia were highly susceptible in bacteria injected hemolymph and purified AMP. These results suggest that symbiont Burkholderia can't survive in the hemolymph after escaping symbiotic organ. Moreover, humoral immunity of host Riptortus is important to prevent spreading and migrating symbiont Burkholderia into the other host tissue or organ from symbiotic organ.

Insect constitute the largest and most diverse group of animals on world and also serve as the hosts or nutrient sources. In addition, several insects have a strong influence on people's emotion. To utilize the preference and interest of insects in the field of mental healthcare, a survey study was conducted with individual living in Korea. As results, the most people had a high preference and interest of insect, but some were disagreeable to the insect itself. The preference and interest of insect were high on male, adult and practician experienced insect-related events than female, student and non-practician, respectively. The most favored insects were familiar or pet insects such as Papilio xuthus, Lucanus maculifemoratus, Allomyrina dichotoma and Lampyridae. These results may be useful to develop a healing program for mental healthcare using insects. Further research is needed to determine the effects of these insect in the mental therapy for this purpose.

Polyhedrin is the major component of the nuclear viral occlusions produced during replication of the baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV). To enhance the production efficiency of foreign protein in baculovirus expression system, the effects of various polyhedrin fragments were investigated by fusion expressing them with the enhanced green fluorescent protein (EGFP). Recombinant viruses were generated to express EGFP fused with polyhedrin fragments based on the previously reported minimal region for self-assembly and the KRKK nuclear localization signal (NLS). The marked increase of EGFP by these fusion expressions was confirmed through protein and fluorescence intensity analyses. Among the fusion-expressed protein in nucleus and cytoplasm, the most hyper-expression was observed in the fusion of amino acids 19 to 110 and 32 to 59 of polyhedrin. Also these fragments, some degradation of only the fused polyhedrin was observed in the fusion of amino acids 19 to 85 and 32 to 85. The production of E2 protein, which is a major antigen of classical swine fever virus, was dramatically increased by fusion expression with polyhedrin amino acids 19 to 110, and its preliminary immunogenicity was verified using experimental guinea pigs. The production of luciferase was approximately two folds increased by fusion expression with polyhedrin amino acids 32 to 59, and its activity was measured using Luminometer. This study suggests a new option for higher expression of useful foreign recombinant protein using the partial polyhedrin fusion expression in baculovirus.

To enhance the production efficiency of foreign protein in baculovirus expression system, the effects of polyhedrin fragments were investigated by fusion expression them with the enhanced green fluorescence protein (EGFP). Recombinant viruses were generated to express EGFP fused with polyhedrin fragments based on the minimal region for self-assembly and the KRKK nuclear localization signal (NLS). The increase of EGFP production by fusion expressions was confirmed through protein and fluorescence intensity analyses. The importance of nuclear localization for enhanced production of EGFP was shown by the mutation of the NLS within the fused polyhedrin fragment. Among the fusion expressed protein in cytoplasm, the most hyper-expression was observed in the fusion of amino acids 32 to 59 of polyhedrin. Polyhedrin fragment fusion expression with classical swine fever virus E2 protein also resulted hyper-enhanced expression of E2 protein. However, the fusion expression of porcine circovirus ORF2 with polyhedrin fragment did not show significant enhance of ORF2 production. These results suggested that the enhancement of foreign protein production when fused with polyhedrin is caused by the enhanced stability of expressed protein.

Hyphantria cunea is a fall webworm is considered an agricultural pest. It is a major pest of many board-leaved trees. H. cunea nucleopolyhedrovirus (HcNPV) and H. cunea granulovirus (HcGV) were isolated from the fall webworm cadavers in Korea. To better understand HcNPV and HcGV, their genomic sequences were determined, analyzed and compared to two viruses together. The entire nucleotide sequence of the HcNPV genome was fully sequenced using 454 pyrosequencing. The genome of the HcNPV was 131,302 bp with a 45 % G+C content. Computer assisted analysis predicted 146 open reading frames (ORFs) of 50 or more amino acids that showed minimal overlap. Further more, when the phylogenetic relationship was analyzed, HcNPV was closely related to Orgyia pseudotsugata MNPV (OpMNPV) which belong to Group I NPV. The HcGV genome was 114,557 bp with a 39% G+C content and contained 130 putative ORFs of 50 or more amino acids. When phylogenetic relationships were analyzed, HcGV was closely related to Xestia c-nigrum granulovirus, which belong to the Type-II GV. HcNPV shares 48 ORFs with HcGV. The most significant difference between HcNPV and HcGV is fgf gene. HcNPV contains one fgf gene, whereas HcGV contains three fgf genes. The presence of fgf reduces the time and efficient systemic infection it takes the virus to kill its host. The difference of fgf number from HcNPV and HcGV suggested that different affect for the speed of systemic infection.

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.

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.

The four genetically distinct isolates have been identified previously from Bombyx mori nucleopolyhedroviruses (BmNPVs) isolated in Korea. To further understand the complex of viruses infecting Bombyx mori, the genome of BmNPV-K1 and K4 strains was completely sequenced and analyzed in comparison with the genome of other sequenced baculoviruses including previously reported BmNPV. BmNPV-K1 consisted of 127,542 bp and 133 open reading frames (ORFs) of 150 nucleotides or longer with minimal overlap have been identified. In contrast, BmNPV-K4 consisted of 128,615 bp and 134 open reading frames (ORFs). Although gene arrangement is virtually identical, the genome of BmNPV-K4 is 1,073 bp longer than BmNPV-K1. This was related to the more existence of bro genes in BmNPV-K4. To investigate the relationship between BmNPV-K1 and K4, phylogenetic analysis with each member of the paired ORFs was performed. The sequence data suggest that BmNPVK1 and BmNPV-K4 are closely related but have diverged and evolved into two separate strains. This was study to identify highly related but separately evolving viruses in the same insect host and geographic location. We are currently comparing the differences of these BmNPV genomes to elucidate characteristics of each virus.

Polyhedrin is the major component of the nuclear viral occlusions produced during replication of the baculovirus Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV). To enhance the expression level of baculovirus vector system, we constructed several fusion vectors using various fragments of the polyhedrin. The polyhedrin fragments were genetically fused to the enhanced green fluorescent protein (eGFP) under the control of polyhedrin promoter, and their expressions were analyzed in Sf21 insect cells. Expression of the fusion protein was identified by SDS-PAGE and Western blot analysis using anti-GFP and anti-Polyhedrin. The expression level of eGFP was markedly increased by the fusion of partial polyhedrin. Also, the fluorescence intensity of fusion proteins was higher than that of non-fusion protein. Confocal laser scanning microscopy demonstrated that fusion proteins were localized to the cytosol or nucleus of insect cells. In additional, the glycoprotein E2 (gE2) of classical swine fever virus (CSFV) expressed by the these vectors was dramatically increased and its immunogenicity was proofed using experimental animal guinea pigs that were immunized with the partial polyhedrin containing gE2. This study provides a new option for the higher expression of useful foreign recombinant protein by using the partial polyhedrin in BEVS.

Mamestra brassicae nucleopolyhedrovirus-K1 (MabrNPV-K1) was isolated from naturally infected Mamestra brassicae (Lepidoptera: Noctuidae) larvae in Korea. Restriction endonuclease fragment analysis using EcoRI, PstI, and BamHI estimated that the total genome size of MabrNPV-K1 is about 150 Kb. The full genome sequences of MabrNPV-K1 were determined, analyzed and compared to those of other baculoviruses. The MabrNPV-K1 genome consisted of 152,471 bp and had an overall G + C contents of 39.90 %. Computer-assisted analysis predicted 159 open reading frames (ORFs) of 150 nucleotides or greater that showed minimal overlap. The gene content and arrangement in MabrNPV-K1 were most similar to those of Mamestra configurata nucleopolyhedrovirus-B (MacoNPV-B), including three polh, p10 and lef-8 gene homologues. The MabrNPV-K1 genome contains four homologous repeat regions (hr1,hr2,hr3,hr4) that account for 3.1% of the genome. The genomic positions of MabrNPV-K1 regions hr1– hr4 are conserved with the genomic positions of MacoNPV-B hr1–hr4. This indicates that the position of MabrNPV–K1 hrs is conserved with regard to both the upstream and downstream genes. Given that hrs share higher similarity within a virus strain than any hrs between species, this evidence further indicates that hrs play a fundamental role in viral life cycle and replication process appears to be tightly linked to functional conservation. The dot plot analysis, percent identity of the gene homologues and a phylogenetic analysis suggested that MabrNPV-K1 is a Group II NPV that is closely related to MacoNPV but with a distinct genomic organization.

The Classical Swine Fever Virus (CSFV) is a member of the Pestivirus genus of the Flaviviridae. The polyprotein composed of eight nonstructural and four structural proteins (nucleocapsid protein C and three envelope glycoprotein E0, E1 and E2). E2, the most immunogenic of the CSFV glycoproteins, induces a protective immune response in swine. The objective of this study was to enhance production of E2 protein by fusion with partial polyhedrin of nucleopolyhedrovirus in insect cells. We generated various E2 form by fusion with different combinations of the partial polyhedrin and deletion of the C-terminal transmembrane region (TMR). Expression of the E2 protein was identified by SDS-PAGE and Western blot analysis using anti-CSFV E2 monoclonal antibodies. The fusion expression of an E2 protein with the partial polyhedrin markedly increased expression levels. Also, expression of E2 proteinlacking TMR region was higher than that of intact E2 protein. As a result, the fusion expression of E2 protein lacking the C-terminal TMR with partial polyhedrin was significantly increased in insect cells. These suggest that the fusion of target foreign protein with partial polyhedrin could enhance significantly the production of target protein.

In agricultural fields, the entomopathogenic fungal species have been investigated for their potential as the biological control agents due to their role of natural enemies for insects. To address the requirements of a potential South Korea based biocontrol effort using entomopathogenic fungi, we investigated the occurrence of various entomopathogenic fungi in 1080 soil samples representing from various area and locations in South Korea. Entomopathogenic fungi were isolated from soils using semiselective medium SDA-D50 contained saboraund dextrose agar, 50 ug/ml dodine, 100 ug/ml chloramphenicol and 50 ug/ml streptomycin. The isolated putative fungi were identified by the determination of internal transcribed spacer (ITS) region sequences of the nuclear ribosomal analysis. As a result, entomopathogenic fungi were found to occur in 30.8% of the soil samples studied. The most abundant species were Beauveria bassiana (Bals.) Vuill. and Metarhizium anisopliae (Metschn.) Sorok. Isolates of B. brongniartii, Cordyceps sp., Lecanicillium sp., Isaria sp. and Tolypocladium cylindrosporum were also found. The occurrence of entomopathogenic fungi was analyzed by the area and soil types. These positive entomopathogenic fungi may have potential against variety pests in agriculture and forest

The porcine reproductive and respiratory syndrome virus (PRRSV) has three major structural proteins which designated as GP4, GP5, and M. They have been considered very important to arouse the humoral and cellular immune responses against PRRSV infection and proposed to be the excellent candidate proteins in the design of PRRS bioengineering vaccine. However, the PRRSV structural proteins are produced in low levels in the infected cells because it forms insoluble protein and possesses several transmembrane regions. To overcome this problem, we fused the GP4, GP5, and M with SUMO (Small ubiquitin-related modifier), and expressed the fused gene in Bm5 cells and silkworm larvae. Expression of the proteins were analyzed by 12% SDS-PAGE and western blotting using 6xHis tag and porcine anti-PRRSV antibodies. In results, SUMO fused proteins were expressed at a high level in Bm5 cells. The levels of protein using the silkworm larvae is higher than that using Bm5 cells. The fused protein was purified by Ni-NTA affinity chromatography. This study demonstrated that SUMO, when fused with PRRSV structural proteins, was able to promote its soluble expression. This may be a better method to produce PRRSV structural proteins for vaccine development.

In agricultural fields, the entomopathogenic fungal species have been investigated for their potential as the biological control agents due to their role of natural enemies for insects. To address the requirements of a potential South Korea based biocontrol effort using entomopathogenic fungi, we investigated the occurrence of various entomopathogenic fungi in 1080 soil samples representing from various area and locations in South Korea. Entomopathogenic fungi were isolated from soils using semiselective medium SDA-D50 contained saboraund dextrose agar, 50 ug/ml dodine, 100 ug/ml chloramphenicol and 50 ug/ml streptomycin. The isolated putative fungi were identified by the determination of internal transcribed spacer (ITS) region sequences of the nuclear ribosomal analysis. As a result, entomopathogenic fungi were found to occur in 30.8% of the soil samples studied. The most abundant species were Beauveria bassiana (Bals.) Vuill. and Metarhizium anisopliae (Metschn.) Sorok. Isolates of B. brongniartii, Cordyceps sp., Lecanicillium sp., Isaria sp. and Tolypocladium cylindrosporum were also found. The occurrence of entomopathogenic fungi was analyzed by the area and soil types. These positive entomopathogenic fungi may have potential against variety pests in agriculture and forest

The Classical Swine Fever Virus (CSFV) is a member of the Pestivirus genus of the Flaviviridae. The genome of CSFV is a positive single-stranded RNA molecule 12.3 kb and contains a single large open reading frame (ORF). The polyprotein composed of eight nonstructural and four structural proteins (nucleocapsid protein C and three envelope glycoprotein E0, E1 and E2). E2, the most immunogenic of the CSFV glycoproteins, induces a protective immune response in swine. To determine the characteristics of the CSFV, LOM strain, we investigated the nucleotide sequence of the glycoprotein E0, E1 and E2. Comparison of the LOM with the other strains revealed nucleotide sequence identity ranging from 97 to 98%. Expression of the glycoprotein E2 was identified by SDS-PAGE and Western blot analysis using anti-CSFV E2 monoclonal antibodies in Sf21 cells. The expression levels of glycoprotein E2 were observed from day 3 and 5 days maximum. In addition, its expression efficiency by media and cell line was investigated. The result showed that High-Five cells and Grace’s insect media for Sf21 were the best conditions for the expression of the glycoprotein E2.

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. AD affects many countries and regions in the world, causing important economic losses, mainly due to international trade restrictions. In this study, to determine the characteristics of the Aujeszky’s disease virus (ADV), NYJ strain, which was isolated from the serum of an infected pig in 1987, we investigated the nucleotide sequence and expression of the glycoproteins gB, gC, and gD using the bBpGOZA system. We found that the glycoproteins gB, gC, and gD of NYJ consisted of 2751 bp, 1443 bp, and 1203 bp, respectively. Comparison of the NYJ with the other strains revealed nucleotide sequence identity ranging from 91.tito 99.0%. To better understand the genetic relationships between other strains, phylogenetic analyses were performed. The NYJ strain was formed a distinct branch with high bootstrap support. The expression of glycoprotein gD in insect cells was characterized by SDS-PAGE and Western blotting with an anti-ADV polyclonal antibody. Glycoprotein gD of approximately 45 kDa was detected. The results of this study have implications for both the taxonomy of ADV and vaccine development.

To determine the characteristics of the Korean porcine reproductive and respiratory syndrome virus (PRRSV), CA, which was isolated from the serum of an infected pig in 2006, we investigated the nucleotide sequence and expression of the structural ORFs (ORFs 2 to 7) using the bApGOZA system. We found that the structural ORFs 2 to 7 of CA consisted of 3188 nucleotides that were the same as those formed from VR-2332. Comparison of the CA with the other strains revealed nucleotide sequence identity ranging from 89.8 to 99.5%. To better understand the genetic relationships between other strains, phylogenetic analyses were performed. The CA strain was closely related to the other North American genotype strains but formed a distinct branch with high bootstrap support. Additionally, expression levels of the PRRSV proteins in Sf21 cells were strong or partially weak. The results of this study have implications for both the taxonomy of PRRSV and vaccine development.

The porcine reproductive and respiratory syndrome virus (PRRSV) has six structural proteins which encoded by ORFs 2 to 7 are designated as GP2, 3, 4, 5, M and N, repectively. In this study, we determined the expression of each protein using novel transfer vector, pBmKSK4 which has the polyhedrin promoter of BmNPV and 6xHis tag. The recombinant transfer vector was co-transfected into Bm5 cells along with bBpGOZA DNA. Recombinant virus was purified by plaque assay and amplified in Bm5 cells. Expression of each protein was identified by SDS-PAGE and Western blot analysis using anti-6xHis monoclonal antibody. The expression levels of the structural proteins in Bm5 cells were stronger than the expression system using pBacPAK9 transfer vector in Sf21 cells. As expected, GP5 was expressed at low levels from its structural properties and its toxicity for cells. In addition, each recombinant protein was purified using Ni-NTA spin columns. The ability to produce each protein in the baculovirus system indicates that these could be major candidates for the development of a vaccine against PRRSV.

Background : The study about ginseng cultured roots have been reported mainly ginsenosides in saponins family. Other phytochemical such as non-saponins of fatty acid has been revealed its bioactive activity including anti-oxidation, whitening, anti-cancer. Supercritical extraction (SE) process mainly refer to the extraction with CO2, is usually from a solid matrix, is a sample preparation step for analytical purposes. SE produce no residual solvent and possess high stability of the extract component, which is advantageous for fatty acid analysis. Methods and Results : Fermented ginseng cultured roots used in the experiment were used for fermentation using Pediococcus pentosaceus. SE performed at different temperature, pressure and extraction time using non-fermented and fermented ginseng roots. Further we fractionated from fermented ginseng using Methanol, Hexane, Ethanol, Ethyl acetate and Butanol. We compared fatty acids contents ginseng extractions by GC analysis. Methyl linoleate contents was 44% of fatty acids supercritical extraction contained. The contents of Methyl linoleate was the most dominant component among 37 types of fatty acids by SE and other extractions solvent. Total fatty acids contents obtained by SE process from fermented ginseng (1325.61ppm) was twice than from non-fermented ginseng (618.47ppm). Conclusion : Fatty acids contents by SE was increased at high pressure. The best condition for fatty acids contents extraction was 60℃, 350bar and 3h.