• 한국생물공학회:학술대회논문집
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• 2001.11a
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• pp.727-730
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• 2001

Various recombinant deletion mutants were constructed from cycloinulo - oligosaccharide fructanotransferase(CFTase) gene of Xanthomonas oryzae #5 . The mutants were expressed in Escherichia coli DH5${\alpha}$. We were able to obtain three recombinant proteins were purified, and examine their CFTase and hydrolyzing activity. N-terminal deletion mutant had both CFTase activity and hydrolyzing activity. however, in C-terminal and N,C-terminal deletion mutant disappeared CFTase activity, but hydrolyzing activity remained. From there results, it seems that the C-terminal region(amino acid $1173{\sim}1333$) is important for cyclization.

• Journal of Microbiology and Biotechnology
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• v.12 no.4
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• pp.563-568
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• 2002

Stably transformed Drosophila melanogaster 52 cells producing recombinant VP7 were obtained, and recombinant VP7 expression was confirmed by Western blot analysis. The molecular weight of recombinant VP7 expressed in 52 cells was approximately 35.5 kDa, and 75% of the total VP7 produced was present in the medium. Recombinant VP7 contained N-linked glycosylated oligosaccharides. Aprotinin, leupeptin, and polyvinylpyrrolidone did not have any noticeable effect on recombinant VP7 production; however, DMSO and sodium butyrate increased its production by 120% and 60%, respectively.

• International Journal of Industrial Entomology and Biomaterials
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• v.4 no.1
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• pp.51-56
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• 2002

A recombinant transfer vector pBacSCF was constructed by inserting huamn stem cell factor (hSCF) cDNA into plasmid pBacPAK8. BmN cells were co-transfected with modified Bombyx mori, nuclear polyhedrosis virus (BmBacPAK) DNA and the recmbinant transfer vector to construct a recombinant baculovirus containing hSCE gene. DNA dot blotting and RNA dot blotting demonstrated that the hSCE gene was contained in the recombinant virus and transcribed. The recombinant baculovirus was infectious to BmN cells and to silkworm. SDS-PAGE analysis showed a specific band of expressed product in the extract of infected cells and in the heamolymph of infected larvae. Bioactivity of the recombinant hSCE was determined with W-1 cell line and MTT colorimetric method in synergy with interlukin-3 (IL-3). These results revealed that the hSCF gene was over-expressed in cultured cells and lavae of silkworm.

• Journal of Microbiology
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• v.38 no.2
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• pp.109-112
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• 2000

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pleiotropic hematopoietic growth factor and an activator of lmature myeloid cells and recombinant GM-CSF is increasingly under clinical studies for the treatment of various diseases including cancer, infectious diseases and hematopoietic diseases. We constructed a reconbinant mouse GM-CSF expression plasmid with pelB leader sequence and His. Tag under T7 promoter control, and showed that the construct produced a 20 kDa recombinant protein in 8M urea. We also showed that the 20 kDa recombinant protein prepared in 8M urea sitmulated colony formation in vitro, indicating that the recombinant mGM-CSF can be renatured to its native form to show the colony stimulating activity.

• Parasites, Hosts and Diseases
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• v.52 no.2
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• pp.137-142
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• 2014

Serologic tests are widely accepted for diagnosing Toxoplasma gondii but purification and standardization of antigen needs to be improved. Recently, surface tachyzoite and bradyzoite antigens have become more attractive for this purpose. In this study, diagnostic usefulness of 3 recombinant antigens (SAG1, SAG2, and SAG3) were evaluated, and their efficacy was compared with the available commercial ELISA. The recombinant plasmids were transformed to JM109 strain of Escherichia coli, and the recombinants were expressed and purified. Recombinant SAG1, SAG2, and SAG3 antigens were evaluated using different groups of sera in an ELISA system, and the results were compared to those of a commercial IgG and IgM ELISA kit. The sensitivity and specificity of recombinant surface antigens for detection of anti-Toxoplasma IgG in comparison with commercially available ELISA were as follows: SAG1 (93.6% and 92.9%), SAG2 (100.0% and 89.4%), and SAG3 (95.4% and 91.2%), respectively. A high degree of agreement (96.9%) was observed between recombinant SAG2 and commercial ELISA in terms of detecting IgG anti-Toxoplasma antibodies. P22 had the best performance in detecting anti-Toxoplasma IgM in comparison with the other 2 recombinant antigens. Recombinant SAG1, SAG2, and SAG3 could all be used for diagnosis of IgG-specific antibodies against T. gondii.

• Korean Journal of Poultry Science
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• v.39 no.4
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• pp.273-278
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• 2012

Staphylococcal enterotoxin B (SEB), which is a bacterial superantigen produced by Staphylococcus aureus, is associated with serious diseases, including food poisoning and atopic dermatitis. This study was performed to produce about 30 kDa of recombinant SEB protein and to immunize in chickens to acquire the specific egg yolk antibody (IgY) against the recombinant SEB. Chickens were immunized with the recombinant SEB intramuscularly in the breast muscle by injection 3 times at intervals of two weeks. Serum- and egg yolk-antibody titers of hens against SEB were highest at 4 weeks after first immunization. In western blot, anti-recombinant SEB IgY was reacted immunospecifically against the recombinant SEB and commercialized SEB. These results suggested that the recombinant SEB antigen could be used as an immunogen to elicit antibody (IgY) against SEB and the anti-recombinant SEB IgY could neutralized staphylococcal enterotoxin B effectively.

• Reproductive and Developmental Biology
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• v.36 no.3
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• pp.193-198
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• 2012

This study was to examine expression of the recombinant full-length adiponectin (recombinant adiponectin) in insect ovarian cell culture system and to characterize structural properties of the recombinant adiponectin secreted in medium. Gene construct encoding the recombinant adiponectin contained N-terminal collagen-like domain (110 Amino Acids, AAs), C-terminal globular domain (137 AAs) and C-terminal peptides for detection with V5 antibody (26 AAs included adaptor peptide) and purification using the 6xHis tag (6 AAs). The approximate molecular weight of the product (monomer) was 35 kDa. Molecular mass species of the expressed recombinant adiponectin were monomer (~35 kDa), dimer (~70 kDa), trimer (~105 kDa) and hexamer (~210 kDa). The major secreted species were the LMW forms, such as monomer, dimer, and trimer. There was MMW of hexamer as minor form. HMW multimers (~300 kDa) were shown as a tracer or not detected on the SDS-PAGE in several experiments (data not shown). The multimer forms in this study were not compatible to those in animal or human serum and adipose tissue by other researcher's study in which the major multimer forms were HMW. By protein denaturing experiments with reducing reagent (${\beta}$-MeOH), anionic detergent (SDS) and heat ($95^{\circ}C$) on the SDS-PAGE, not all adiponectin multimers seemed to have disulfide bond linked structure to form multimers. The recombinant adiponectin which expressed in insect ovarian cell culture system seemed to have the limitation as full physiological regulator for the application to animal and human study.

• Animal cells and systems
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• v.16 no.6
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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.

• International Journal of Industrial Entomology and Biomaterials
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• v.7 no.2
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• pp.197-201
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• 2003

The recombinant plasmids harboring a heterologous gene coding mouse endostatin were transfected and expressed stably in Trichoplusia ni Tn 5B1-4 cells and Bombyx mori BmN cells, respectively. Recombinant endostatin expressed in the stably transformed Tn 5B1-4 and BmN cells was secreted into the medium. BmN cells are relatively lower in maximum cell growth and recombinant endostatin production than Tn 5B 1-4 cells. Recombinant endostatin was also purified to homogeneity using a simple one-step ${Ni^2+}$ affinity fractionation method. Purified recombinant endostatin inhibited endothelial cell proliferation in a dose-dependent manner. The concentration at half-maximum inhibition $({ED_50})$ for recombinant endostatin was approximately 0.35 ${\mu}g$/ml.

• Microbiology and Biotechnology Letters
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• v.13 no.4
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• pp.345-348
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• 1985

Recombinant chimeric plasmid constructed with Xba I digested pUBl10 and -pE194 was transformed by polyethylene glycol induced protoplast transformation system into Bacillus subtilis BR 151 on the mannitol regeneration media, and two genes of antibiotics resistance were expressed simultaneously in the transfromant. Transformation frequency of the recombinant plasmid was 6.5 $\times$ 10$^{-5}$ on the mannitol regeneration agar plate containing neomycin and erythromycin. The replication of recombinant plasmid in the recipient cells was confirmed by the alkaline extraction method and agarose gel electrophoresis.