• Animal cells and systems
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• v.11 no.2
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• pp.175-182
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• 2007

The lysozyme II gene of cabbage butterfly Artogeia rapae was cloned from fat body of the larvae injected with E. coli and its nucleotide sequence was determined by the RACE-PCR. It has an open reading frame of 414 bp nucleotides corresponding to 138 amino acids including a signal sequence of 18 amino acids. The estimated molecular weight and the isoelectric point of the lysozyme II without the signal peptide were 13,649.38 Da and 9.11, respectively. The A. rapae lysozyme II (ARL II) showed the highest identity (81%) in the amino acid sequence to Manduca sexta lysozyme among other lepidopteran species. The two catalytic residues ($Glu^{32}$ and $Asp^{50}$) and the eight Cys residue motifs, which are highly conserved among other c-type lysozymes in invertebrates and vertebrates, are also completely conserved. A phylogenetic analysis based on amino acid sequences indicated that the ARL II was more closely related to M. sexta, Hyphantria cunea, Heliothis virescens, and Trichoplusia ni lysozymes. The ARL II gene was expressed in Spodoptera frugiperda 21 insect cells and the recombinant ARL II (rARL II) was purified from cell-conditioned media by cation exchange column chromatography and reverse phase FPLC. The purified rARL II was able to form a clear zone in lysoplate assay against Micrococcus luteus. The lytic activity was estimated to be 511.41 U/mg, 1.53 times higher than that of the chicken lysozyme. The optimum temperature for the lytic activity of the rARL II was $50^{\circ}C$, the temperature dependency of the absolute lytic activity of rARL II was higher than that of the chicken lysozyme at low temperatures under $65^{\circ}C$.

• The Journal of Korean Society of Virology
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• v.28 no.1
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• pp.53-62
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• 1998

Human papillomavirus (HPV) 16, E7 proteins derived from the prototype (Bac73) and natural variant (Bac101) E7 open reading frame were produced in Sf9 insect cells. The variant E7 gene occurred naturally by substitution mutation at the position of 88 nucleotide, resulting serine instead of asparagine. Using E7 specific monoclonal antibody (VD6), both E7 proteins were identified in recombinant baculovirus infected SF9 cells. Radiolabelling and immunoprecipitation analysis revealed that both E7 proteins were phosphoproteins. Immunostaining result showed that E7 proteins were mainly localized in the cytoplasm. Nuclear form of E7 proteins was also detected after a sequential fractionation procedure for removing chromatin structure. Considering that the VD6 recognition site in E7 protein is located within 10 amino acid at the N-terminus, this region appears to be blocked by the nuclear component. Western blot analysis revealed that nuclear form was more abundant than cytoplasmic E7 proteins. Time course immunostaining showed that the primary location of E7 protein was the nucleus and exported to the cytoplasm as proteins were accumulated. These events occurred similarly in both Bac73 and Bac101 infected Sf9 cells, suggesting that these two proteins may have similar biological functions.

• Biomedical Science Letters
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• v.8 no.4
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• pp.211-215
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• 2002

The baculovirus/Sf9 cell expression can be employed as a powerful system for producing large amounts of the human erythrocyte glucose transporter, GLUT1 heterologously In order to exploit the system further, it is necessary to develop a convenient method for demonstrating that the transporter expressed in insect cells is biologically active. To achieve this, we have expressed the human CLUT1 in insect cells and photolabelled the expressed protein with [$^3$H] cytochalasin B, a potent inhibitor of the human erythrocyte glucose transporter. Subsequently, the labelled proteins were analysed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Membranes labelled with [$^3$H] cytochalasln B in the presence of L-Glucose yielded a single sharp peak of labelling of apparent $M_r$ 45,000 on SDS/polyacrylamide gels. The mobility of this peak corresponded exactly to that of the band detected by anti-glucose transporter antibodies on Western blots of membranes prepared from insect cells infected with recombinant virus. In addition, the sharpness of the radioactive peak provides further evidence for the conclusion that the expressed protein is much less heavily and heterogeneously glycosylated than its erythrocyte counterpart. No peak of labelling was seen with the membranes prepared from non-infected Sf9 cells. Furthermore, the incorporation of label into this peak was completely inhibited by the presence of 500 mM-D-Glucose during tile photolabelling procedure, showing the stereoselectivity of the labelling. These evidences clearly show that human glucose transporter expressed in insect cells exhibits native-like biological activity, and that photolabelling with [$^3$H] cytochalasin B can be a convenient means for analysing the biological activity of the transport protein expressed in insect cells.

• The Journal of Korean Society of Virology
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• v.29 no.2
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• pp.137-144
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• 1999

Transformation efficiency, virus multiplication and foreign gene expression were characterized in the insect cells transformed with Autographa californica nuclear polyhedrosis virus (AcNPV) immediate early 1 gene (IE1). Transformation efficiency of insect cells by AcNPV IE1 gene vector horboring foreign gene was approximately 8-fold higher in the Sf9 cells transformed previously with AcNPV IE1 gene than in the normal Sf9 cells. Virus multiplication and foreign gene expression of recombinant baculovirus in the Sf9 cells transformed with AcNPV IE1 gene were similar to those of the normal Sf9 cells. These results suggest that transformed cells displaying foreign gene product by using AcNPV IE1 gene promoter will be useful for the diverse applications of insect cells.

• The Korean Journal of Pesticide Science
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• v.18 no.3
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• pp.181-190
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• 2014

An entomopathogenic bacterium, Bacillus thuringiensis (Bt), can sporulate along with production of insecticidal Cry toxins. Bt Cry toxins exhibit relatively narrow spectrum to target insects due to their specific interactions with midgut receptors. This study designed several strategies to enhance Bt efficacy in target insect spectrum and insecticidal activity. Four Cry toxins were purified from four different Bt strains and showed relatively narrow target insect spectrum. However, the Cry mixtures significantly expanded their target insect spectra. The additional effect of baculovirus to Cry toxin was tested with recombinant baculoviruses expressing Cry1Ac or Cry1Ca. However, the baculovirus was little effective to expand target insect spectrum. Bacterial culture broth of Xenorhabdus nematophila (Xn) significantly suppressed insect cellular immune response and increased Cry toxicity. The addition of Xn culture broth to Cry mixture significantly enhanced Bt efficacy in target insect spectrum and insecticidal activity.

• Journal of Life Science
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• v.24 no.1
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• pp.86-91
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• 2014

Trichoplusia ni cells are used as a host permissive cell line in the baculovirus expression system, which is useful for large-scale production of human sugar transport proteins. However, the activity of endogenous sugar transport systems in insect cells is extremely high. Therefore, the transport activity resulting from the expression of exogenous transporters is difficult to detect. Furthermore, very little is known about the nature of endogenous insect transporters. To exploit the expression system further, the effect of D-fructose on 2-deoxy-D-glucose (2dGlc) transport by T. ni cells was investigated, and T. ni cell-expressed human transporters were photolabeled with [$^3H$] cytochalasin B to develop a convenient method for measuring the biological activity of insect cell-expressed transporters. The uptake of 1 mM 2dGlc by uninfected- and recombinant AcMPV-GTL infected cells was examined in the presence and absence of 300 mM of D-fructose, with and without $20{\mu}M$ of cytochalasin B. The sugar uptake in the uninfected cells was strongly inhibited by fructose but only poorly inhibited by cytochalasin B. Interestingly, the AcMPV-GTL-infected cells showed an essentially identical pattern of transport inhibition, and the rate of 2dGlc uptake was somewhat less than that seen in the non-infected cells. In addition, a sharply labeled peak was produced only in the AcMPV-GTL-infected membranes labeled with [$^3H$] cytochalasin B in the presence of L-glucose. No peak of labeling was seen in the membranes prepared from the uninfected cells. Furthermore, photolabeling of the expressed protein was completely inhibited by the presence of D-glucose, demonstrating the stereoselectivity of labeling.

• Korean Journal of Veterinary Research
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• v.42 no.1
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• pp.89-100
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• 2002

The nonstructural glycoprotein NSP4, encoded by the 10th gene of rotavirus, has been known to play important roles in viral assembly and pathogenesis. The NSP4 genes of human rotavirus Korean isolates, designated as CBNU/HR-1, CBNU/HR-2, CBNU/HR-3, and CBNU/HR-4, were cloned, sequenced and characterized. Also, the NSP4 gene of the CBNU/HR-1 was expressed in a baculovirus-insect cell system. The sequence data indicated that the NSP4 genes of human rotavirus Korean isolates were 750 or 751 bases in length and encoded one open reading frame of 175 amino acids. Two glycosylation sites were recognized in the NSP4 gene of human rotavirus isolates tested. The NSP4 of CBNU/HR-1, CBNU/HR-3, and CBNU/HR-4 exhibited a high degree of amino acid sequence homology with that of NSP4 genotype B viruses, but a low degree of amino acid sequence homology with that of NSP4 genotype A viruses. However, the NSP4 of CBNU/HR-2 exhibited a high degree of amino acid sequence homology with that of NSP4 genotype A viruses, but a low degree of amino acid sequence homology with that of NSP4 genotype B viruses. The Sf9 cells infected with recombinant baculovirus, inserted with NSP4 gene of CBNU/HR-1, produced specific cytopathic effects and the expressed NSP4 was detected by immunofluorescence staining using NSP4-specific monoclonal antibody(MAb). The expressed NSP4 migrated at 16-26 kDa on SDS-PAGE and reacted with NSP4-specific MAb by Western blotting.

• International Journal of Industrial Entomology and Biomaterials
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• v.1 no.2
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• pp.111-114
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• 2000

The female pupae of the silkworms Bombyx mori, were injected with recombinant Autographa californica nuclear polyhedrosis virus (AcNPV) expressing green fluorescent protein (GFP) by percutaneous inoculation. When the 4 day-old female pupae were injected with 1x10$^{7}$ or 2${\times}$10$^{7}$ plaque forming units (pfu) of the recombinant AcNPV, oviposited number and egg weight were significantly decreased. Furthermore, the shape of the eggs was obviously divides into normal and abnormal shapes. The percentage of the eggs with an abnormal shape was 7.8% and 57.1% at 1${\times}$10$^{7}$ and 2${\times}$10$^{7}$ pfu inoculation, respectively. PCR analysis of the genomic DNA extracted from the eggs revealed that gfp and AcNPV ecdysteroid UDP-glucosyltransferase genes were amplified from both types of eggs with normal and abnormal shapes. The results demonstrate that AcNPV DNA, and gfp gene cloned into the AcNPV genome, injected in pupal stage were transmitted to eggs and remained stable through at least next generation.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1995.10a
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• pp.107-113
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• 1995

Basement membrane laminin is a multidomain glycoprotein that interacts with itself, heparin and cells. The distal long arm plays major cell and heparin interactive roles. The long arm consists of three subunits (A, B1, B2) joined in a coiled-coil rod attached to a terminal A chain globule (G). The globule is in turn subdivided into five subdomains (Gl-5). In order to analyze the functions of this region, recombinant G domains (rG, rAiG, rG5, rGΔ2980-3028) were expressed in Sf9 insect cells using a baculovirus expression vector. A hybrid molecule (B-rAiG), consisting of recombinant A chain(rAiG) and the authentic B chains (E8-B)was assembled in vitro. The intercalation of rAiG into E8-B chains suppressed a heparin binding activity identified in subdomain Gl-2. By the peptide napping and ligand blotting, the relative affinity of each subeomain to heparin was assigned as Gl> G2= G4> G5> G3, such that G1 bound strongly and G3 not at all. The active heparin binding site of G domain in intact laminin appears to be located in G4 and proximal G5. Cell binding was examined using fibrosarcoma Cells. Cells adhered to E8, B-rAiG, rAiG and rG, did not bind on denatured substrates, poorly bound to the mixture of E8-B and rG. Anti-${\alpha}$6 and anti-${\beta}$1 integrin subunit separately blocked cell adhesion on E8 and B-rAiG, but not on rAiG. Heparin inhibited cell adhesion on rAiG, partially on B-rAiG, and not on E8. In conclusion, 1) There are active and cryptic cell and heparin binding activities in G domain. 2) Triple-helix assembly inactivates cell and heparin binding activities and restores u6131 dependent cell binding activities.

• Korean Journal of Veterinary Service
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• v.35 no.3
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• pp.175-182
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• 2012

Rotaviruses have been known to be a major etiological agent of gastroenteritis in both infants and young animals. Subsequently new rotaviruses, which were morphologically indistinguishable but antigenically and electrophoretically distinct with each other, were reported from several animals throughout world including Korea. These new rotaviruses were named as non-group A or group B or group C rotaviruses and so on. It has been very difficult to isolate and grow the non-group A rotaviruses in vitro, and this has greatly limited the characterizations of non-group A rotaviruses and serological studies. In this study, monoclonal antibodies (MAbs) against porcine non-group A rotavirus were produced and characterized. The VP6 gene of porcine group C rotavirus Korean isolate(#06-52-1) was cloned and expressed. For expression of VP6 gene, baculovirus expression system was applied. The VP6 gene and expressed protein in the recombinant virus were confirmed by polymerase chain reaction (PCR), indirect fluorescence antibody (IFA) test and Western blot, respectively. The expressed VP6 was used for MAbs production. The MAbs produced in this study would be promising as diagnostic reagents for detection of group C rotavirus infection.