• Journal of Microbiology and Biotechnology
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• v.12 no.6
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• pp.916-920
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• 2002

This report describes a high-level expression of human alpha-2a interferon ($IFN{\alpha}-2a$) in Escherichia coli and its pilot scale purification by using a monoclonal antibody-independent chromatographic procedure that is based on anion-exchange, cation-exchange, hydrophobic interaction, and gel filtration. The recombinant E. coli produced much more $IFN{\alpha}-2a$ in a soluble form, when cultivated at low temperatures than at high-temperature fermentation. However, if the bacterial growth was taken into consideration, fermentation at $30^{\circ}C$ seemed optimal for the interferon production. By using our new protocol, we recovered approximately 160 mg of $IFN{\alpha}-2a$ with a specific activity of $3.59{\times}10^8$ IU/mg from 201 of the broth. The gel permeation chromatographic and SDS-PAGE indicated that the interferon preparation was purified to homogeneity and was of the correctly folded fast-migrating monomer.

• Journal of environmental and Sanitary engineering
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• v.12 no.2
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• pp.59-64
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• 1997

For purification of a 70kDa toxic protein of mosquitocidal delta-endotoxin from B. thuringiensis strain H9B, immuno-affinity chromatography was performed. After separation of 70kDa toxic proteins from the delta-endotoxin of the strain H9B on SDS-PAGE, the 70kDa toxic protein was subcutaneously injected into rabbit for making a polyclonal antibody. A anti-70kDa toxic protein was purified by a column chromatography packed with protein A-sepharose 4B gels. The 70kDa toxic protein from delta-endotoxin of the strain H9B was also purified by an immuno-affinity chromatography packed with CNBr-activated sepharose 4B gels conjugated anti-70kDa toxic protein after elution with 1/10M citric acid-1/5M Na$_{2}$HPO$_{4}$ buffer(pH3.2) containing 0.5M NaCl. The 70kDa toxic protein was purified through only one step-separation system, was demonstrated by SDS-PAGE and immunoblot.

• KSBB Journal
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• v.13 no.6
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• pp.656-661
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• 1998

Hantaan virus belonging to the genus Hantavirus and family Bunyaviridae causes an acute severe illness of human, Haemorrhagic Fever with Renal Syndrome (HFRS). It is a rodent host-borne pathogen and distributed in Asia and Eastern Europe. Hantaviruses have three major antigens, i.e., G1, G2 glycoproteins and nucleocapsid protein (N). Among them, nucleocapsid protein was reported to be the most invaluable antigen as for diagnosis. We have cloned and expressed Hantaan viral nucleocapsid gene in E. coli BL21(DE3). In this study, we have tried to purify the nucleocapsid protein produced by recombinant E. coli, and could attained a purity of >90% by anti-N monoclonal antibody-coupled immunoaffinity chromatography or phenyl sepharose hydrophobic interaction chromatography.

• Korean Journal Plant Pathology
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• v.14 no.1
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• pp.62-67
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• 1998

Barley yellow mosaic virus (BaYMV-HN) occurring Haenam area was isolated by mechanical inoculation onto barley cultivars, purification and production of antibody. BaYV-HN were purified from infected plants a filamentous virus with 13 nm in diameter and 250∼300 nm and 500∼650 nm in length. Specific antibody made by injecting the purified virus to the muscle of a rabbit. In gel-diffusion tests antibody to BaYMV-HN did not make spur with tow Japanese BaYMV isolates BaYMV-II-1 or BaYMV-III. BaYMV-HN showed the symptom of yellowing and necrosis in host plants. Mechanical inoculation tests with Japanese barley cultivars showed that BaYMV-HN infected New Golden, Akagi Nijo and Tosan Kawa 73, but did not infect Amagi Nijo, Haruna Nijo, Ishukushirazu (ym3), Misato Golden (Ym1), Kashimamugi, Joshushiro Hadaka and Mokusekko 3 (ym1). In Korean barley cultivars, some of the naked barleys which are Hinssalbori, Kinssalbori, Saessalbori and Saechalssalbori were not infected by BaYMV-HN. However, it infected all the covered barley cultivars and the beer barley cultivars. BaYMV-HN had two RNAs, RNA 1 (7.6 Kb) and RNA 2 (3.5 Kb), and one coat protein (33 KDa).

• Korean Journal Plant Pathology
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• v.14 no.1
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• pp.68-73
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• 1998

Barley mild mosaic virus (BaMMV-Kor) was isolated from the southern part of Korea, and by mechanical inoculation onto barley cultivars, purification and production of antibody. BaMMV-Kor purified form infected plants were filamentous particle, with 13 nm in diameter and 250∼300 nm and 500∼650 nm in length. Antibody of BaMMV-Kor was made by injecting the purified virus to the muscle of a rabbit. In gel-diffusion test, antibody to BaMMV-Kor created spur with BaMMV-Kal and BaMMV-M, but did not make spur with BaMMV-Kor infected New Golden, Ishukushirazu, Joshushiro Hadaka and Misato Golden, but did not infect Kashimamugi, Chikurin Ibaraki 1 and Mokusekko 3. In Korean barley cultivars, BaMMV-Kor infected most of the covered barley cultivars, but did not infect Saeolbori. It also infected naked barley cultivars except Chalbori and Hinssalbori. And all the beer barley cultivars were infected by BaMMV-Kor. BaMMV-Kor had two RNAs, RNA 1 (7.5 Kb) and RNA 2 (3.5 Kb), and coat protein (33 KDa).

• BMB Reports
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• v.37 no.5
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• pp.556-564
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• 2004

A recombinant Fab monoclonal antibody (Fab) C37, previously obtained by phage display and biopanning of a random antibody fragment library against Burkholderia pseudomallei protease, was expressed in different strains of Escherichia coli. E. coli strain HB2151 was deemed a more suitable host for Fab expression than other E. coli strains when grown in media supplemented with 0.2% glycerol. The expressed Fab fragment was purified by affinity chromatography on a Protein G-Sepharose column, and the specificity of the recombinant Fab C37 towards B. pseudomallei protease was proven by Western blotting, enzyme-linked immunosorbent assay (ELISA) and by proteolytic activity neutralization. In addition, polyclonal antibodies against B. pseudomallei protease were produced in rabbits immunized with the protease. These were isolated from high titer serum by affinity chromatography on recombinant-Protein A-Sepharose. Purified polyclonal antibody specificity towards B. pseudomallei protease was proven by Western blotting and ELISA.

• Journal of Microbiology and Biotechnology
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• v.6 no.6
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• pp.468-473
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• 1996

The genome of tomato spotted wilt virus (TSWV) is composed of three RNA segments, S, M, and L RNA and the 5.0 kb M RNA encodes two glycoproteins Gl, G2 and NSm protein of unknown function. In an effort to investigate the function of the NSm protein, antibody was raised against NSm fusion protein overexpressed in Escherichia coli. This antibody was used to detect the NSm protein by using western blot analysis and electron microscopic observation after immunogold labelling. For the cloning of the NSm gene, total RNA extracted from a TSWV infected plant was used for cDNA synthesis and polymerase chain reaction (PCR) instead of going through time-consuming virus purification. A protein band specifically reacting to the NSm antibody was detected from TSWV inoculated plants. The NSm protein was detected in the cell wall fraction and in pellet from low speed centrifugation when the infected plant tissue was fractionated into 4 fractions. In the immuno-electron microscopic observation, gold particles were found around the plasmodesmata of infected plant tissue. These results suggest that the NSm protein of TSWV plays some role in cell-to-cell movement of this virus.

• Journal of Microbiology and Biotechnology
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• v.21 no.5
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• pp.529-535
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• 2011

Single-chain variable fragment (scFv) is a fusion protein of the variable regions of the heavy (VH) and light (VL) chains of immunoglobulin, connected with a short linker peptide of 10 to about 20 amino acids. In this study, the scFv of a monoclonal antibody against the third domain of human CD4 was cloned from OKT4 hybridoma cells using the phage display technique and produced in E. coli. The expression, production, and purification of anti-CD4 scFv were tested using SDS-PAGE and Western blot, and the specificity of anti-CD4 scFv was examined using ELISA. A 31 kDa recombinant anti-CD4 scFv was expressed and produced in bacteria, which was confirmed by SDS-PAGE and Western blot assays. Sequence analysis proved the ScFv structure of the construct. It was able to bind to CD4 in quality ELISA assay. The canonical structure of anti-CD4 scFv antibody was obtained using the SWISS_MODEL bioinformatics tool for comparing with the scFv general structure. To the best of our knowledge, this is the first report for generating scFv against human CD4 antigen. Engineered anti-CD4 scFv could be used in immunological studies, including fluorochrome conjugation, bispecific antibody production, bifunctional protein synthesis, and other genetic engineering manipulations. Since the binding site of our product is domain 3 (D3) of the CD4 molecule and different from the CD4 immunological main domain, including D1 and D2, further studies are needed to evaluate the anti-CD4 scFv potential for diagnostic and therapeutic applications.

• Applied Biological Chemistry
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• v.49 no.3
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• pp.196-201
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• 2006

Vitellogenin, which is found in the serum of female and male fishes exposed to environmental endocrine disrupter or estrogen hormone, is used as a biomarker for environmental contamination with an endocrine disrupter. In order to produce antibody against vitellogenin, a synthetic peptide for partial vitellogenin was injected into rabbits. In addition, by using ion exchange chromatography on DE-52, vitellogenin was purified from the serum of carp induced with $17{\beta}$-estradiol. Polyclonal antibody against purified vitellogenin reacted well with vitellogenin in the serum of carp induced with $17{\beta}$-estradiol and the serum of female carp, whereas polyclonal antibody against the vitellogenin peptide did not react with proteins in those samples. This may indicate that vitellogenin proteins, covalently modified largely, could not be detected by Western blotting with the polyclonal antibody against the synthetic vitellogenin peptide.

• Journal of Veterinary Science
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• v.22 no.4
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• pp.45.1-45.13
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• 2021

Background: Peste des petits ruminants (PPR) is an infectious disease caused by the peste des petits ruminants virus (PPRV) that mainly produces respiratory symptoms in affected animals, resulting in great losses in the world's agriculture industry every year. Single-domain variable heavy chain (VHH) antibody fragments, also referred to as nanobodies, have high expression yields and other advantages including ease of purification and high solubility. Objectives: The purpose of this study is to obtain a single-domain antibody with good reactivity and high specificity against PPRV. Methods: A VHH cDNA library was established by immunizing camels with PPRV vaccine, and the capacity and diversity of the library were examined. Four PPRV VHHs were selected, and the biological activity and antigen-binding capacity of the four VHHs were identified by western blot, indirect immunofluorescence, and enzyme-linked immunosorbent assay (ELISA) analyses. ELISA was used to identify whether the four VHHs were specific for PPRV, and VHH neutralization tests were carried out. ELISA and western blot analyses were used to identify which PPRV protein was targeted by VHH2. Results: The PPRV cDNA library was constructed successfully. The library capacity was greater than 2.0 × 10⁶ cfu/mL, and the inserted fragment size was approximately 400 bp to 2000 bp. The average length of the cDNA library fragment was about 1000 bp, and the recombination rate was approximately 100%. Four single-domain antibody sequences were selected, and proteins expressed in the supernatant were obtained. The four VHHs were shown to have biological activity, close affinity to PPRV, and no cross-reaction with common sheep diseases. All four VHHs had neutralization activity, and VHH2 was specific to the PPRV M protein. Conclusions: The results of this preliminary research of PPRV VHHs showed that four screened VHH antibodies could be useful in future applications. This study provided new materials for inclusion in PPRV research.