• Nuclear Medicine and Molecular Imaging
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• v.43 no.5
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• pp.487-494
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• 2009

Purpose: Small size of recombinant scFv antibody has many advantages such as rapid blood clearances and improved targeting antibodies to tumor region. On the other hand owing to small size, number of amino group is insufficient in conjugation with chelator and fluorescence labeling. This study is to introduce poly lysine tag to the C-terminal end of scFv lym-1 sequence for fluorescence chelator conjugation. Materials and Methods: Poly lysine scFv lym-1 gene, cloned into pET-22b (+) vector, was expressed in E. coli BL21 (DE3) strain. Antibody purification was performed with Ni-NTA column and then size exclusion column chromatography. Expression and purification levels of poly lysine tagged scFv lym-1 antibody were confirmed by western blot analysis. I-124, I-125, I-131 and Tc-99m were used for radiolabeling of purified poly lysine scFv lym-1. Flow cytometry analysis of FIT( conjugated poly lysine scFv lym-1 was performed for confirmation of immunoreactivity of human Burkitt's lymphoma cells. Results: Poly lysine scFv lym-1 antibody was purified through two steps and identified as molecular weight of 48 KDa. Radiolabeling yields of I-124, I-125, I-131 and Tc-99m into poly lysine scFv lym-1 were >99%, >99%, >95% and >99%, respectively. Flow cytometry analysis of poly lysine scFv and scFv lym-1 was showed similar immunoreactivity to human Burkitt's lymphoma cells. Conclusion: Poly lysine tag was useful for the sufficient number of amino groups to scFv lym-1 antibody for chelator conjugation with minimizing loss of immunoreactivity.

• IMMUNE NETWORK
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• v.3 no.1
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• pp.23-28
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• 2003

Background: Intracellular antibody specific to hepatitis B virus X protein (HBx) might be useful for studying the role of HBx in hepatocellular carcinogenesis and HBV replication. Methods: With variable region genes for H7 monoclonal anti-HBx Ab, we constructed a vector for bacterial expression of single chain Ab (scFv) and a vector for eukaryotic cell expression of it. The expression of H7 scFv and its binding activity against HBx was examined by immunoblotting and immunofluorescence microscopy. Results: H7 scFv expressed in bacterial cells retained reactivity to HBx. We demonstrated its intracytoplasmic expression in CosM6 eukaryotic cells. Conclusion: This is the first study showing the expression of intracellular anti-HBx Ab in eukaryotic cells. H7 scFv may be a good tool to study the function of HBx in HBV infection.

• Journal of Plant Biotechnology
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• v.8 no.1
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• pp.9-14
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• 2006

As an alternative method to produce low cost reagents for immunodiagnosis and protect the plants from viral disease, a gene encoding a single chain variable fragment(scFv) recombinant antibody targeted to the coat protein of cucumber mosaic virus (CMV) was expressed in Nacotiana tabacum. The source of the scFv recombinant antibody gene was from spleen tissue of an immunized mouse. The gene was initially cloned into the pCANTAB5E phagemid and expressed in E. coli. In the following study, the antibody gene was subcloned into the plant expression vector, pCAMBIA-1301 and introduced into tobacco leaf tissue via Agrobacterium tumefacients mediated transformation. After transformation, 56 out of 58 plants were shown to carry the desired anti-CMV scFv gene by PCR analysis. Overall, only 12.5% of the 56 putative transgenic plants were found to express the antibody to a detectable level.

• Journal of Microbiology and Biotechnology
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• v.27 no.4
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• pp.718-724
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• 2017

The combination of rabies immunoglobulin (RIG) with a vaccine is currently effective against rabies infections, but improvements are needed. Genetic engineering antibody technology is an attractive approach for developing novel antibodies to replace RIG. In our previous study, a single-chain variable fragment, scFv57R, against rabies virus glycoprotein was constructed. However, its inherent weak stability and short half-life compared with the parent RIG may limit its diagnostic and therapeutic application. Therefore, an acidic tail of synuclein (ATS) derived from the C-terminal acidic tail of human alpha-synuclein protein was fused to the C-terminus of scFv57R in order to help it resist adverse stress and improve the stability and half-life. The tail showed no apparent effect on the preparation procedure and affinity of the protein, nor did it change the neutralizing potency in vitro. In the ELISA test of molecular stability, the ATS fusion form of the protein, scFv57R-ATS, showed an increase in thermal stability and longer half-life in serum than scFv57R. The protection against fatal rabies virus challenge improved after fusing the tail to the scFv, which may be attributed to the improved stability. Thus, the ATS fusion approach presented here is easily implemented and can be used as a new strategy to improve the stability and half-life of engineered antibody proteins for practical applications.

• Journal of Microbiology and Biotechnology
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• v.9 no.3
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• pp.376-380
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• 1999

Constructs, encoding a single-chain variable fragment of a catalytic antibody 4f4f (scFv-4f4f) with glycosidase activity, were made by combining the coding sequences for the heavy and light chain variable domains with a sequence encoding a linker (GGGGS). Using three different plasmid systems, single-chain antibodies were expressed separately in Escherichia coli, demonstrating significant differences in the expression level and amounts in soluble form of the recombinant protein. The protein expression from pET3a-scFv-4f4f was up to 20% of the total soluble proteins and, more importantly, the proteins were mostly found in a soluble form. An SDS-PAGE analysis of the purified single-chain proteins, yielding higher than 5mg from a 1-1 culture, showed a single band corresponding to its molecular weight of 29,100. A preliminary study shows that the expressed scFv-4f4f is catalytically active. The catalytic parameters for the hydrolysis of p-nitrophenyl-$\beta$-D-glucopyranoside by scFv-4f4f are being investigated.

• The Journal of Korean Institute of Communications and Information Sciences
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• v.34 no.1B
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• pp.67-73
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• 2009

This paper proposes a new fast vertical handover mechanism to optimize and enhance the existing fast vertical handover HMIPv6(FV-HMIP) mechanism in heterogeneous wireless access networks. The proposed mechanism ad opts the IEEE 802.21 Media Independent Handover Function(MIHF), and thus is called the "MFV-HMIP". The Media Independent Handover Service (MIIS) in the IEEE 802.21 MIHF is extended by including new L3 inform ation to provide domain prefixes of heterogeneous neighbouring mobility anchor points(MAPs). This can eliminate the heterogeneous neighbouring MAP discovery phase in the existing FV-HMIP. Thus, the proposed MFV-HMIP can reduce the signalling traffic over wireless link and increase the possibility of predictive mode operation. Therefore, the proposed MFV-HMIP mechanism can be superior to the existing FV-HMIP mechanism for wireless resource efficiency, handover latency, and packet loss. Through analytic performance evaluations and computer si mulations, the proposed mechanism can be shown to optimize and enhance the existing mechanism.

• Macromolecular Research
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• v.17 no.7
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• pp.528-532
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• 2009

The size and shape of free volume (FV) holes available in membrane materials control the rate of gas diffusion and its permeability. Based on this principle, a segmented, thermo-sensitive polyurethane (TSPU) membrane with functional gate, i.e., the ability to sense and respond to external thermo-stimuli, was synthesized. This smart membrane exhibited close-open characteristics to the size of the FV hole and water vapor permeation and thus can be used as smart food packaging materials. Differential scanning calorimetry (DSC), dynamic mechanical analysis (DMA), positron annihilation lifetimes (PAL) and water vapor permeability (WVP) were used to evaluate how the morphological structure of TSPU and the temperature influence the FV holes size. In DSC and DMA studies, TSPU with a crystalline transition reversible phase showed an obvious phase-separated structure and a phase transition temperature at $53^{\circ}C$ (defined as the switch temperature and used as a functional gate). Moreover, the switch temperature ($T_s$) and the thermal-sensitivity of TSPU remained available after two or three thermal cyclic processes. The PAL study indicated that the FV hole size of TSPU is closely related to the $T_s$. When the temperature varied cyclically from $T_s-10{\circ}C$ to $T_s+10^{\circ}C$, the average radius (R) of the FV holes of the TSPU membrane also shifted cyclically from 0.23 to 0.467 nm, exhibiting an "open-close" feature. As a result, the WVP of the TSPU membrane also shifted cyclically from 4.30 to $8.58\;kg/m^2{\cdot}d$, which produced an "increase-decrease" response to the thermo-stimuli. This phase transition accompanying significant changes in the FV hole size and WVP can be used to develop "smart materials" with functional gates and controllable water vapor permeation, which support the possible applications of TSPU for food packaging.

• BMB Reports
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• v.51 no.11
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• pp.572-577
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• 2018

Recent studies showed that the PD-1/PD-L1 checkpoint blockade is a dramatic therapy for melanoma by enhancing antitumor immune activity. Currently, major strategies for the PD-1/PD-L1 blockade have mainly focused on the use of antibodies and compounds. Seeking an alternative approach, others employ endogenous proteins as blocking agents. The extracellular domain of PD-1 (ePD1) includes the binding site with PD-L1. Accordingly, we constructed a PD-1-based recombinantly tailored fusion protein (dFv-ePD1) that consists of bivalent variable fragments (dFv) of an MMP-2/9-targeted antibody and ePD1. The melanoma-binding intensity and antitumor activity were also investigated. We found the intense and selective binding capability of the protein dFv-ePD1 to human melanoma specimens was confirmed by a tissue microarray. In addition, dFv-ePD1 significantly suppressed the migration and invasion of mouse melanoma B16-F1 cells, and displayed cytotoxicity to cancer cells in vitro. Notably, dFv-ePD1 significantly inhibited the growth of mouse melanoma B16-F1 tumor cells in mice and in vivo fluorescence imaging showed that dFv-ePD was gradually accumulated into the B16-F1 tumor. Also the B16-F1 tumor fluorescence intensity at the tumor site was stronger than that of dFv. This study indicates that the recombinant protein dFv-ePD1 has an intensive melanoma-binding capability and exerts potent therapeutic efficacy against melanoma. The novel format of the PD-L1-blocked agent may play an active role in antitumor immunotherapy.

• Journal of Mushroom
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• v.15 no.2
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• pp.78-83
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• 2017

Microsatellite SSR markers were developed and utilized to reveal the genetic diversity of 32 strains of Flammulina velutipes collected in Korea, China, and Japan. From the SSR-enriched library, 490 white colonies were randomly selected and sequenced. Among the 490 sequenced clones, 85 (17.35%) were redundant. Among the remaining 405 unique clones, 201 (49.6%) contained microsatellite sequences. We used 12 primer pairs that produced reproducible polymorphic bands for four diverse strains, and these selected markers were further characterized in 32 Flammulina velutipes strains. A total of 34 alleles were detected using the 12 markers, with an average of 3.42 alleles, and the number of alleles ranged from two to seven per locus. The major allele frequency ranged from 0.42 (GB-FV-127) to 0.98 (GB-FV-166), and values for observed ($H_O$) and expected ($H_E$) heterozygosity ranged from 0.00 to 0.94 (mean = 0.18) and from 0.03 to 0.67 (mean = 0.32), respectively. SSR loci amplified with GB-FV-127 markers gave the highest polymorphism information content (PIC) of 0.61 and mean allele number of five, whereas for loci amplified with GB-FV-166 markers these values were the lowest, namely 0.03 and two. The mean PIC value (0.29) observed in the present study with average number of alleles (3.42). The genetic relationships among the 32 Flammulina velutipes strains on the basis of SSR data were investigated by UPGMA cluster analysis. In conclusion, we succeeded in developing 12 polymorphic SSRs markers from an SSR-enriched library of Flammulina velutipes. These SSRs are presently being used for phylogenetic analysis and evaluation of genetic variations. In future, these SSR markers will be used in clarifying taxonomic relationships among the Flammulina velutipes.

• Journal of Sericultural and Entomological Science
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• v.34 no.2
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• pp.26-31
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• 1992

Flacherie virus (FV) and Densonucleosis virus (DNV) of the silkworm, Bombyx mori, which give the most severest damage to the silk production in korea, were fed on the mulberry wild silkworm, Bombyx mori mandarina, the mulberry pyralid, Gryphodes phyloalis, and the American fall webworm, Hypantria cunea, to investigate cross infectivity by serological and histopathological at observation. By the Ouchterlony's double difusion test the mulberry wild silkworm was infected with both FV and DNV type 1 (DNV-1) and the mulberry pyralid with DNV-1, so those were confirmed the cross infection. But the American fall webworm was not recognized the cross infection by the same method. The infection and multiplication of the FV in the mulberry wild silkworm was observed in the cytoplasm of the goblet cell with the appearance of the virus-specific vesicle. In DNV-1 infection to the mulberry wild silkworm and the mulberry pyralid, the nuclei of columnar cell in the midgut of both insects was hypertrophied and the nuclei of midgut cell of the mulberry pyralid positively stained with the feulgen stain. Multiplication of DNV-1 in the midgut cell of the mulberry wild silkworm was replicated in two different patterens as linear arrays and large masses, while that of DNV-1 in the muberry pyralid was multiplied as virus masses in several portion of the nuclei of the midgut cell.