• 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.

• Parasites, Hosts and Diseases
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• v.40 no.1
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• pp.59-64
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• 2002

A Cryptosporidium parvum sporozoite and oocyst λgt11 cDNA library was screened with a hyperimmune rabbit serum that was developed against insoluble fragments of ultrasonicated oocysts. A clone named Cp22.4.1 encoding a protein of 231 amino acids with 4 zinc-finger domains characterized by a Cys-X2-Cys-X4-His-X4-Cys motif was isolated and characterized. There was a complete match between the sequencing data of the coding region of Cp22.4.1 and the corresponding gene at chromosomal level. Cloning in a pBAD-TOPO-TA expression vector permitted to evaluate the antigenicty of the recombinant His-tagged antigen. This antigen was recognized by 2 out of 5 sera from Cruptosporidium immune calves and not by sera from parasite naive animals.

• The Plant Pathology Journal
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• v.16 no.1
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• pp.48-51
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• 2000

Citrus tristeza virus (CTV) was identified form CTV-infected early satsuma mandarin (Citus unshiu) and yuzu (C.junos) by RT-PCR. The total RNAs were isolated from citrus bark and seaf tissues infected with CTV and reverse transcription was followed with primers designed for amplifying CTV coat protein gene. DNA fragments 738 bp were amplified by RT-PCR and these products were colned for sequence analysis. Based on the sequence analysis, this PCR product has 97% sequence homology to CTV (T-385) CP gene isolated from USA. RT-PCR assay for CTV detection was more sensitivity than ELISA assay which was done with anti-CTV CP antibody. This is the frist report about CTV identification in Cheju island Korea.

• IMMUNE NETWORK
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• v.3 no.2
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• pp.126-135
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• 2003

Background: One of the important factors in the prognosis of chronic hepatitis B patient is the degree of replication of hepatitis B virus (HBV). It has been known that HBV DNA polymerase plays the essential role in the replication of HBV. HBV DNA polymerase is composed of four domains, TP (Terminal protein), spacer, RT (Reverse transcriptase) and RNaseH. Among these domains, tyrosine, the $65^{th}$ residue of TP is an important residue in protein-priming reaction that initiates reverse transcription. If monoclonal antibody that recognizes around tyrosine residue were selected, it could be applied to further study of HBV replication. Methods: To produce TP-specific scFv (single-chain Fv) by phage display, mice were immunized using synthetic TP-peptide contains $57{\sim}80^{th}$ amino acid residues of TP domain. After isolation of mRNA of heavy-variable region ($V_H$) and light-chain variable region ($V_L$) from the spleen of the immunized mouse, DNA of $V_H$ and $V_L$ were obtained by RT-PCR and joined by a DNA linker encoding peptide (Gly4Ser)3 as a scFv DNA fragments. ScFv DNA fragments were cloned into a phagemid vector. scFv was expressed in E.coli TG1 as a fusion protein with E tag and phage gIII. To select the scFv that has specific affinity to TP-peptide from the phage-antibody library, we used two cycles of panning and colony lift assay. Results: The TP-peptide-specific scFv was isolated by selection process using TP-peptide as an antigen. Selected scFv had 30 kDa of protein size and its nucleotide sequences were analyzed. Indirect- and competitive-ELISA revealed that the selected scFv specifically recognized both TP-peptide and the HBV DNA polymerase. Conclusion: The scFv that recognizes the TP domain of the HBV DNA polymerase was isolated by phage display.

• Biomedical Science Letters
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• v.14 no.1
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• pp.47-53
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• 2008

Hox genes are known to be transcription factors controlling vertebrate pattern formation along the anteroposterior body axis by regulating many target gene expressions during vertebrate embryogenesis. In order to isolate in vivo Hox responsive target genes, ChIP-cloning technique has been applied using Hoxc8 antibody. Here murine embryo of day 11.5 post coitum (E11.5) highly expressing Hoxc8 gene was used after removing head and tail portions where Hoxc8 is rarely expressing. After fixation with formaldehyde, the chromatin DNAs harboring bound proteins were isolated. After sonication, about 0.5- to 1 Kb chromatin DNAs were immunoprecipitated with anti Hoxc8 antibody. After removing the bound proteins with proteinase K, DNAs were isolated, cloned into the pBluescsript II SK vector, and then sequenced. Total 33 random clones sequenced were anlalyzed to be located at 12 different genomic regions. Among these, 8 turned out to be introns and 4 were intergenic regions localized in random chromosomes. The base composition of total cloned genomic sequences (6608 bp) were AT-rich, i.e., 40% GC. When the Hoxc8 core binding sites, such as TAAT, ATTA, TTAT, and ATAA were analyzed total number of 55, 45, 54, and 55 were found, respectively, which are than twice as many as expected number of 26. Although this in silico analysis does not mean that the ChIP-cloned sequence is real Hoxc8 regulatory element in vivo, these results strongly imply that the DNA fragments cloned through chromatin immunoprecipitation could be very much likely the putative Hoxc8 downstream target genes.

• Journal of Microbiology and Biotechnology
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• v.24 no.5
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• pp.704-713
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• 2014

The highly pathogenic avian influenza A (HPAI) viruses of the H5N1 subtype infect poultry and have also been spreading to humans. Although new antiviral drugs and vaccinations can be effective, rapid detection would be more efficient to control the outbreak of infections. In this study, a phage-display library was applied to select antibody fragments for HPAI strain A/Hubei/1/2010. As a result, three clones were selected and sequenced. A hemagglutinin inhibition assay of the three scFvs revealed that none exhibited hemagglutination inhibition activity towards the H5N1 virus, yet they showed a higher binding affinity for several HPAI H5N1 strains compared with other influenza viruses. An ELISA confirmed that the HA protein was the target of the scFvs, and the results of a protein structure simulation showed that all the selected scFvs bound to the HA2 subunit of the HA protein. In conclusion, the three selected scFVs could be useful for developing a specific detection tool for the surveillance of HPAI epidemic strains.

• Archives of Pharmacal Research
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• v.22 no.5
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• pp.459-463
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• 1999

The effects of the hindered and non-hindered water soluble long-chain disulfide bonds on the stability and cytotoxicity of the ricin A chain (RTA) immunotoxin were examined. The RTA immunotoxins were prepared with the Fab fragments of anti-common acute lymphoblastic leukemia antigen (CALLA) monoclonal antibody (Fab-RTA) using sulfosuccinimidyl-6-[(-methyl-(-2-pyridyldithio)toluamido]toluamido]hexanoate (S-LC-SMPT) and sulfosuccinimidyl-6-[3-(2-pyridyldithio-propionamido]hexanoate (S-LC-SPDP). The prepared Fab-RTA immunotoxins were evaluated for their conjugation yield, immunoreactivity, thermal and disulfide bond stability and cytotoxicity. The conjugation yield of the Fab-RTA immunotoxin from the water soluble long chain crosslinking agents, S-LC-SMPT and S-LC-SPDP, were comparable. Both Fab-RTA immunotoxins exhibited a similar immunoreactivity and thermal stability in aqueous solution. However, S-LC-SMPT -mediated Fab-RTA, sterically hindered, showed an enhanced disulfide bond stability in vitro over S-LC-SPDP mediated one. In the cytotoxicity against antigenic cell Daudi, the S-LC-SMPT -mediated RTA immunotoxin maintained a comparable cytotoxicity, compared with S-LC-SPDP mediated Fab-RTA immunotoxin.

• Journal of Periodontal and Implant Science
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• v.29 no.3
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• pp.663-676
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• 1999

Porphyromonas gingivalis, a Gram negative, anaerobic, asaccharolytic rod, is one of the most frequently implicated pathogens in human periodontal disease and has a requirement for hemin for growth. A 30 kDa (heated 24 kDa) hemin-binding protein whose expression is both hemin and iron regulated has recently been purified and characterized in this oral pathogen. This study has identified a hemin-binding P. gingivalis protein by expression of a P. gingivalis genomic library in Escherichia coli, a bacterium which does not require or transport exogenous hemin. A library of genomic DNA fragments from P. gingivalis was constructed in plasmid pUC18, transformed into Escherichia coli strain $DH5{\alpha}$ , and screened for recombinant clones with hemin-binding activity by plating onto hemin-containing agar. Of approximately 10,000 recombinant E. coli colonies screened on LB-amp-hemin agar, 10 exhibited a clearly pigmented phenotype. Each clone contained various insert DNA. The Hind III fragment transferred to the T7 RNA polymerase/promoter expression vector system produced a sligltly smaller (21 kDa) protein, a precursor form, immunoreactive to the antibody against the 24 kDa protein, suggesting that the cloned DNA fragment probably carried an entire gene for the 24 kDa hemin-binding protein.

• The Plant Pathology Journal
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• v.31 no.1
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• pp.41-49
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• 2015

Sweet cherry is an important fruit crop with increasing economical value in Turkey and the world. A number of viruses cause diseases and economical losses in sweet cherry. Prune dwarf virus (PDV), is one of the most common viruses of stone fruits including sweet cherry in the world. In this study, PDV was detected from 316 of 521 sweet cherry samples collected from 142 orchards in 10 districts of Isparta province of Turkey by double antibody sandwich-enzyme linked immunosorbent assay (DAS-ELISA). The presence of PDV in ELISA positive samples was confirmed in 37 isolates by reverse transcription- polymerase chain reaction (RT-PCR) method. A genomic region of 862 bp containing the coat protein (CP) gene of PDV was re-amplified from 21 selected isolates by RT-PCR. Amplified DNA fragments of these isolates were purified and sequenced for molecular characterization and determining genetic diversity of PDV. Sequence comparisons showed 84-99% to 81-100% sequence identity at nucleotide and amino acid level, respectively, of the CP genes of PDV isolates from Isparta and other parts of the world. Phylogenetic analyses of the CP genes of PDV isolates from different geographical origins and diverse hosts revealed that PDV isolates formed different phylogenetic groups. While isolates were not grouped solely based on their geographical origins or hosts, some association between phylogenetic groups and geographical origins or hosts were observed.

• Parasites, Hosts and Diseases
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• v.57 no.4
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• pp.435-437
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• 2019

Chagas disease is caused by the protozoan parasite Trypanosoma cruzi, and is endemic in many Latin American countries. Diagnosis is based on serologic testing and the WHO recommends two or more serological tests for confirmation. Acidic ribosomal P protein of T. cruzi showed strong reactivity against positive sera of patients, and we cloned the protein after fragmenting it to enhance its antigenicity and solubility. Twelve positive sera of Chagas disease patients were reacted with the fragmented ribosomal P protein using western blot. Detection rate and density for each fragment were determined. Fragments F1R1, F1R2, and F2R1 showed 100% rate of detection, and average density scoring of 2.00, 1.67, and 2.42 from a maximum of 3.0, respectively. Therefore, the F2R1 fragment of the ribosomal P protein of T. cruzi could be a promising antigen to use in the diagnosis of Chagas disease in endemic regions with high specificity and sensitivity.