• Proceedings of the Korean Society of Applied Pharmacology
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• 1994.04a
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• pp.261-261
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• 1994

Bovine milk xanthine oxidase (E.C.1.1.3.22, XO) purchased from Sigma Chemical Co. had the three protein fragments below 150 kDa on 7.5％ SDS-PAGE, which did not show enzyme activity. To remove these fragments, the enzyme preparation was further purified through Sephadex G-200 column chromatography. Two peaks exhibiting enzymatic activity were separated very closely to the void volume, which were revealed as two different enzyme forms, dimeric and monomeric, confirmed by activity staining on native PAGE. Anti sera-against each of the two enzyme forms were raised by subcutaneous injection at multiple sites on the back of rabbits during 4 weeks. On the immunodiffusion test, it was found that both of the antisera of the two forms could react with each other, which implied that their epitopes were identical In the Western blot analysis of mouse liver cytosol fraction, it was found that rabbit anti-XO antibody bound well with the protein band of monomeric mouse liver XO of about 150kDa. Based on this result, mouse liver cDNA 1 ibrary was screened by in situ hybridizat ion wi th rabbi t anti -XO antibody as probe. Through the immunological screening, recombinant phages giving positive signal by the production of XO were selected and further purified. To validate these clones, purified phages were lysogenized in E. coli Y1089 and their lysates were analysed for enzyme activity and immunoreactivity, It was verified that lysates of the purified recombinant phage lysogens exhibited the enzymatic activity as well as bound wi th XO antibody, when induced by IPTG. The above results assert that selected recombinant phage carries mouse liver XO gene.

• Journal of Microbiology and Biotechnology
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• v.23 no.8
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• pp.1047-1054
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• 2013

Witches' broom of lime is a disease caused by Candidatus Phytoplasma aurantifolia, which represents the most significant global threat to the production of lime trees (Citrus aurantifolia). Conventional disease management strategies have shown little success, and new approaches based on genetic engineering need to be considered. The expression of recombinant antibodies and fragments thereof in plant cells is a powerful approach that can be used to suppress plant pathogens. We have developed a single-chain variable fragment antibody (scFvIMP6) against the immunodominant membrane protein (IMP) of witches' broom phytoplasma and expressed it in different plant cell compartments. We isolated scFvIMP6 from a naïve scFv phage display library and expressed it in bacteria to demonstrate its binding activity against both recombinant IMP and intact phytoplasma cells. The expression of scFvIMP6 in plants was evaluated by transferring the scFvIMP6 cDNA to plant expression vectors featuring constitutive or phloem specific promoters in cassettes with or without secretion signals, therefore causing the protein to accumulate either in the cytosol or apoplast. All constructs were transiently expressed in Nicotiana benthamiana by agroinfiltration, and antibodies of the anticipated size were detected by immunoblotting. Plant-derived scFvIMP6 was purified by affinity chromatography, and specific binding to recombinant IMP was demonstrated by enzyme-linked immunosorbent assay. Our results indicate that scFvIMP6 binds with high activity and can be used for the detection of Ca. Phytoplasma aurantifolia and is also a suitable candidate for stable expression in lime trees to suppress witches' broom of lime.

• Biomedical Science Letters
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• v.4 no.2
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• pp.137-142
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• 1998

We examined mRNAs, isolated from the rat brain, to ascertain if there is any polymorphism for S-100 beta protein gene. As templates for polymerase chain reaction (PCR) the reverse-transcribed cDNA from the rat brain or phage DNAs isolated from the rat brain cDNA libraries were used. Although PCR products turned out to be exactly same as the expected size based on the previously reported mRNA sequence a single base substitution (CAT to CAC) was identified at nucleotide level. This change was considered as polymorphism since it did not cause any change of the primary structure for S-100 beta protein. This result should facilitate the understanding of the overall structure of the gene for S-100 beta protein.

• Journal of Microbiology and Biotechnology
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• v.28 no.12
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• pp.2113-2120
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• 2018

Cross-reactive material 197 ($CRM_{197}$) is a non-toxic mutant of diphtheria toxin containing a single amino acid substitution of glycine 52 with glutamic acid. $CRM_{197}$ has been used as a carrier protein for poorly immunogenic polysaccharide antigens to improve immune responses. In this study, to develop a sandwich ELISA that can detect $CRM_{197}$ and $CRM_{197}$ conjugate vaccines, we generated a human anti-$CRM_{197}$ monoclonal antibody (mAb) 3F9 using a phage-displayed human synthetic Fab library and produced mouse anti-$CRM_{197}$ polyclonal antibody. The affinity ($K_D$) of 3F9 for $CRM_{197}$ was 3.55 nM, based on Bio-Layer interferometry, and it bound specifically to the B fragment of $CRM_{197}$. The sandwich ELISA was carried out using 3F9 as a capture antibody and the mouse polyclonal antibody as a detection antibody. The detection limit of the sandwich ELISA was <1 ng/ml $CRM_{197}$. In addition, the 3F9 antibody bound to the $CRM_{197}$-polysaccharide conjugates tested in a dose-dependent manner. This ELISA system will be useful for the quantification and characterization of $CRM_{197}$ and $CRM_{197}$ conjugate vaccines. To our knowledge, this study is the first to generate a human monoclonal antibody against $CRM_{197}$ and to develop a sandwich ELISA for $CRM_{197}$ conjugate vaccines.

• Korean Journal of Microbiology
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• v.31 no.2
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• pp.123-128
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• 1993

Genes for dinitrogenase reductase (nifH) and dinitogenase a subunit (nifD) were found to be located on 7.9 kb of EcoRI, 6.5 kb of Sail, 7.3 kb of HindlII and 4.4 kb of Pstl fragments of the genomic blot of Rhizobium sp. SNU003. a symbiotic strain from root nodule of Canavalia lineata. Nine recombinant phage nif-clones were selected from the genomic library constructed by using EMBL-3 BamHI arms of bacteriophage lambda. Among them. Rnif-6 had insert DNA of 15.3 kb. in which 7.6 kb of BamHI!SacI fragment contained nifHD region. Therefore, the 7.6 kb fragment was subcloned into pUC19 and partial restriction map was constructed. As the results, nifH and nifD were found to be located continuously on 4.5 kb of BamHI/BglIl in the genome of Rhizobium sp. SNU003 strain.

• Korean Journal of Microbiology
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• v.30 no.1
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• pp.30-36
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• 1992

nif (nitrogen fixation)-H.D, K genes of Frankia sp. SNU 014201. a symbiotic strain isolated from root nodule of Alnus hirsura, were found to be located in the genome on 13.5 kb of EcoRI, 18.0 kb of BamHI, 10.5 kb of BglII and 4.5 kb of KpnI fragments. Using EMBL-3 BamHI arms of bacteriophage lambda. the genomic library was constructed. from which fourteen recombinant phage nif-clones were selected. Among them, Ahnif-I2 had insert DNA of 18 kb, in which 7.9 kb of BamHl fragment contained nif-H, D, K and 3.6 kb of HindlIl/KpnI had nif-H and partial -D. Therefore, the 7.9 kb and 3.6 kb fragments were subcloned and partial restriction maps were constructed. As the results, nif-F1, D.K genes were found to be located continuously on the 6.5 kb of HindII/BamHI and 5.2 kb of SalIIBamHI fragment in the genome of Frankia sp. SNU 014201.

• IMMUNE NETWORK
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• v.3 no.3
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• pp.219-226
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• 2003

Background: Phage display is the most widely used technique among display methods to produce monoclonal antibody fragment with a specific binding activity. Having a large library for efficient antibody display/selection is quite laborious process to have more than $10^9$ members of transformants. To overcome these limitations, several in vitro selection approaches have been reported. Ribosome display that links phenotypes, proteins, directly to genotype, mRNA, is one of the in vitro display methods. Ribosome display can reach the size of scFv library up to $10^{14}$ molecules and it can be further diversified during PCR steps. To select the high affinity scFv from one pot library, we established ribosome display technique by modifying the previously reported eukaryotic translation system. Methods: To establish the antibody selection system by ribosome display, we used 3D8, anti-DNA antibody. A 3D8 scFv was synthesized in vitro by an in vitro transcription-translation system. The translated 3D8 scFv and the encoding 3D8 mRNA are connected to the ribosome. These scFv-ribosome-mRNA complexes were selected by binding to their specific antigens. The eluted mRNAs from the complexes are reverse transcribed and re-amplified by PCR. To apply this system, antibody library from immunized mouse with terminal protein (TP)-peptide of hepatitis B virus DNA polymerase TP domain was also used. This TP-peptide encompasses the 57~80 amino acid residues of TP. These mRNA/ribosome/scFv complexes by our system were panned three times against TP-peptide. The enrichment of antibody from library was determined by radioimmunoassay. Results: We specifically selected 3D8, anti-DNA antibody, against ssDNA as a model system. The selected 3D8 RNAs sequences from translation complexes were recovered by RT-PCR. By applying this model system, we enriched TP-peptide-specific scFv pools through three cycles of panning from immunized library. Conclusion: We show that our translating ribosome complexes are well maintained and we can enrich the TP-specific scFv pools. This system can be applied to select specific antibody from an antibody library.

• IMMUNE NETWORK
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• v.1 no.1
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• pp.7-13
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• 2001

Currently 18 monoclonal antibodies were approved by FDA for inj ection into humans for therapeutic or diagnostic purpose. And 146 clinical trials are under way to evaluate the efficacy of monoclonal antibodies as anti-cancer agents, which comprise 9 % of clinical trials in cancer therapy field. When considering a lot of disappointment and worries existed in this field during the past 15 years, this boom could be called as resurrection. Antibodies have several merits over small molecule drug. First of all it is easier and faster in development, as proper immunization of the target proteins usually raises good antibody response. The side effects of antibodies are more likely to be checked out in immunohistomchemical staining of whole human tissues. Antibody has better pharmacokinetics, which means a longer half-life. And it is non-toxic as it is purely a "natural drug. Vast array of methods was developed to get the recombinant antibodies to be used as drug. The mice with human immunoglobulin genes were generated. Fully human antibodies can be developed in fast and easy way from these mice through immunization. These mice could make even human monoclonal antibodies against any human antigen like albumin. The concept of combinatorial library was also actively adopted for this purpose. Specific antibodies can be screened out from phage, mRNA, ribosomal library displaying recombinant antibodies like single chain Fvs or Fabs. Then the coding genes of these specific antibodies are obtained from the selected protein-gene units, and used for industrial scale production. Both $na\ddot{i}ve$ and immunized libraries are proved to be effective for this purpose. In post-map arena, antibodies are receiving another spotlight as molecular probes against numerous targets screened out from functional genomics or proteomics. Actually many of these antibodies used for this purpose are already human ones. Through alliance of these two actively growing research areas, antibody would play a central role in target discovery and drug development.

• YAKHAK HOEJI
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• v.50 no.1
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• pp.33-39
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• 2006

We have previously reported the minimum criteria that can be applied to evaluate efficacy and safety of a DNA vaccine with use of Streptococcus pneumoniae DNA vaccine (SPDNA). The SPDNA was formulated by inserting the DNA sequences that are codons specific for the carbohydrate epitope in the capsule of S. penumoniae by phage display peptide library. Administration of the SPDNA into mice induced both humoral and cell-mediated immunities. The induction was protective even in the absence of CD4+ T lymphocyte in mice. Profiles of cytokine and isotyping of antibody displayed tendency of the Th1. In continuation of these studies, we examined if the efficacy of the SPNDA was provoked by the peptide recognized by codons specific for the capsule. Results showed that the peptide vaccine formulae (SPP) induced protective antibody in mice as did the SPDNA. Involvement of the cell-mediated immunity was also determined. Possible side effects of autoimmune diseases such as myositis and C3a production and tumor-formation were undetectable in mice given 7 times of SPDNA vaccination during entire of 92 days. Even after the frequent immunization, immunogenicity of the SPDNA was observed as determined for antibody production, suggesting that there was no immunotolerance provoked. All together, these examining factors would be applied to measurement of a DNA vaccine safety regarding the immunopathological aspect.

• BMB Reports
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• v.37 no.3
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• pp.356-361
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• 2004

To develop a simplified method that can rapidly prepare DNA microarray probes in a massive scale, a lambda phage genomic DNA-fragments library was constructed for the microarray-probes collection. Four methods of DNA band recovery from the first PCR products were tested and compared. The DNA microarray probes were collected by a novel method of nested PCR that was mediated by gel isolation of the first PCR products. This method was named GIN-PCR. The probes that were prepared by this GIN-PCR technique were used as subjects to fabricate a DNA microarray. The results showed that a wooden toothpick was superior to the other 3 methods, since this technique can steadily transfer the DNA bands as the template of the second PCR after the first PCR. A group of probes were successfully collected and DNA microarrays were constructed using these probes. Hybridization results demonstrated that this technique of DNA recovery and probe preparation was rapid, efficient, and effective. We developed a cost-effective and less labor-intensive method for DNA microarray probe preparation by nested PCR that is mediated by wooden toothpick transfer of the DNA bands in the gel after electrophoresis.