• Korean Journal of Food Science and Technology
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• v.43 no.2
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• pp.176-181
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• 2011

Cronobacter sakazakii and Salmonella enterica Typhimurium are hazardous pathogens, especially for ready-toeat foods. For control of pathogens, the virulent bacteriophages were isolated, identified, and applied to infant formula milk and vegetable juice. The phages were isolated from swine feces and identified by morphology and molecular characteristics. ES2 phage for C. sakazakii and ST2 phage for S enterica Typhimurium were identified as Myoviridae and Siphoviridae, respectively. Their burst sizes were $52{\pm}5PFU/cell$ for ES2 phage and $21{\pm}3PFU/cell$ for ST2 phage after latent period of 30-40 minutes. ST2 phage showed higher heat stability at $60^{\circ}C$ than ES2 phage. ES2 phage held the growth of C. sakazakii untill 6 hr afterwhich the number decreased when applied to the infant formula milk and vegetable juice. ST2 phage also showed growth inhibition so that the number of S. enterica Typhimurium decreased. Therefore, virulent bacteriophages might be an agent for the growth inhibition of C. sakazakii and S. enterica Typhimurium in such the ready-to-eat foods.

• Journal of Life Science
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• v.13 no.4
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• pp.541-550
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• 2003

E2 glycoprotein of hepatitis C virus (HCV) comprises a surface of viral particle together with E1 glycoprotein, and is thought to be involved in the attachment of HCV viral particle to receptor (s) on the permissible cells including hepatocytes, B cells, T cells, and monocytes. We constructed a phage library expressing cellular proteins of hepatocytes on the phage surface, which turned out to be 8.8${\times}$$10^5$ cfu of diversity and carried inserts in 95% of library. We screened both cDNA phage library and 12-mer peptide library to identify the cellular proteins binding to E2 protein. Some intracellular proteins including tensin and membrane band 4.1 which are involved in signal transduction of survival and cytoskeleton organization, were selected from cDNA phage library through several rounds of panning and screening. On the contrary, membrane proteins such as CCR7, CKR-L2, and insulin-like growth factor-1 receptor were identified through screening of peptide library. Phages expressing peptides corresponding to those membrane proteins were bound to E2 protein specifically as determined by neutralization of binding assay. Since it is well known that HCV can infect T cells as well as hepatocytes, we examined to see if E2 protein can bind to CCR7, a member of C-protein coupled receptor family expressed on T cells, using CCR7 transfected tells. Human CCR7 cDNA was cloned into pcDNA3.1(-) vector and transfected into human embryonic kidney cell, 293T, and expressed on the surface of the cell as shown by flow cytometer. Binding assay of E2 protein using CCR7 transfected cells indicated that E2 protein bound to CCR7 by dose-dependent mode, giving rise to the possibility that CCR7 might be a putative cellular receptor for HCV.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.28 no.2
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• pp.209-218
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• 1995

Bacteriophage T7 gene 2.5 protein, a single-stranded DNA binding protein, has been implicated in T7 DNA replication, recombination, and repair. Purified gene 2.5 protein has been shown to interact with the phage encoded gene 5 protein (DNA polymerase) and gene 4 proteins (helicase and primase) and stimulates their activities. Genetic analysis of T7 phage defective in gene 2.5 shows that the gene 2.5 protein is essential for T7 DNA replication and growth. T7 phage that contain null mutants of gene 2.5 were constructed by homologous recombination. These mutant phage $(T7\Delta2.5)$ cannot grow in Escherichia coli. After infection of E. coli with $T7\Delta2.5$, host DNA synthesis is shut off, and $T7\Delta2.5$ DNA synthesis is reduced to less than $1\%$ of wild-type phage DNA synthesis (Kim and Richardson, 1993, Proc. Natl. Aca. Sci. USA, 90, 10173-10177). A truncated gene 2.5 protein $(GP2.5-\Delta21C)$ deleted the 21 carboxyl terminal amino acids was constructed by in vitro mutagenesis. $GP2.5-\Delta21C$ cannot substitute for wild-type gene 2.5 protein in vivo; the phage are not viable and exhibit less than $1\%$ of the DNA synthesis observed in wild-type phage-infected cells. $GP2.5-\Delta21C$ has been purified to apparent homogeneity from cells overexpressing its cloned gene. Purified $GP2.5-\Delta21C$ does not physically into「act with T1 gene 4 protein as measured by affinity chromatography and immunoblot analysis. The mutant protein cannot stimulate T7 gene 4 protein activity on RNA-primed DNA synthesis and primer synthesis. These results suggest that C-terminal domain of gene 2.5 protein is essential for protein-protein interactions.

• 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.

• Journal of Microbiology
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• v.44 no.5
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• pp.530-536
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• 2006

Bacteriophage P4, a satellite phage of coliphage P2, is a very useful experimental tool for the study of viral capsid assembly and cos-cleavage. For an in vitro cos-cleavage reaction study of the P2-P4 system, new shortened and selectable markers containing P4 derivative plasm ids were designed as a substrate molecules. They were constructed by swapping the non-essential segment of P4 DNA for either the kanamycin resistance (kmr) gene or the ampicillin resistance (apr) gene. The size of the genomes of the resulting markers were 82% (P4 ash8 delRI:: kmr) and 79% (P4 ash8 delRI:: apr) of the wild type P4 genome. To determine the lower limit of genome size that could be packaged into the small P4-size bead, these shortened P4 plasmids were converted to phage particles with infection of the helper phage P2. The conversion of plasmid P4 derivatives to bacteriophage particles was verified by the heat stability test and the burst size determination experiment. CsCl buoyant equilibrium density gradient experiments confirmed not only the genome size of the viable phage form of shortened P4 derivatives, but also their packaging into the small P4-size head. P4 ash8 delRI:: apr turned out to be the smallest P4 genome that can be packaged into P4-sized head.

• Journal of Microbiology
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• v.45 no.6
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• pp.528-533
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• 2007

In a previous study we generated an anti-Hepatitis B Virus (HBV) preS1 humanized antibody (HzKR127) that showed in vivo HBV-neutralizing activity in chimpanzees. However, the antigen-binding affinity of the humanized antibody may not be sufficient for clinical use and thus affinity maturation is required for better therapeutic efficacy. In this study, phage display technique was employed to increase the affinity of HzKR127. All six amino acid residues (Glu95-Tyr96-Asp97-Glu98-Ala99-Tyr100) in the heavy (H) chain complementary-determining region 3 (HCDR3) of HzKR127 were randomized and phage-displayed single chain Fv (scFv) library was constructed. After three rounds of panning, 12 different clones exhibiting higher antigen-binding activity than the wild type ScFv were selected and their antigen-binding specificity for the preS1 confirmed. Subsequently, five ScFv clones were converted to whole IgG and subjected to affinity determination. The results showed that two clones (B3 and A19) exhibited an approximately 6 fold higher affinities than that of HzKR127. The affinity-matured humanized antibodies may be useful in anti-HBV immunotherapy.

• Biomedical Science Letters
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• v.10 no.3
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• pp.211-217
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• 2004

For discrimination of ductal and ascinar cells, we isolated a single-chain variable domain fragment (scFv) antibody against ductal cells of salivary gland using phage display technique. From the spleen of a mouse immunized with ductal cell lysate, total RNA was prepared and used as a template for cDNA synthesis of antibody genes. The scFv genes were constructed with variable domain genes of heavy and light chain and were introduced into pCANTAB5E to construct phage scFv library. The phage particles specific for acinar cells were screened by subtraction using immunotubes coated with acinar and ductal cell lysate and enzyme-linked immunoabsorbance assay (ELISA). The characteristics of the scFv were determined by immunohistochemistry (IHC) and the result indicated that the isolated scFv has the specificity against ductal cells of salivary glands and tubules of kidney. And the scFv has an unique binding activity specific for Hashimoto's thyroiditis. The nucleotide sequence of isolated scFv gene was determined and revealed that V/sub H/ belongs to the mouse H-chain family subgroup IB and V/sub L/ to the mouse L-chain family subgroup III.

• Journal of Microbiology and Biotechnology
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• v.30 no.10
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• pp.1552-1558
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• 2020

With an increase in the consumption of non-heated fresh food, foodborne shiga toxin-producing Escherichia coli (STEC) has emerged as one of the most problematic pathogens worldwide. Endolysin, a bacteriophage-derived lysis protein, is able to lyse the target bacteria without any special resistance, and thus has been garnering interest as a powerful antimicrobial agent. In this study, rV5-like phage endolysin targeting E. coli O157:H7, named as LysECP26, was identified and purified. This endolysin had a lysozyme-like catalytic domain, but differed markedly from the sequence of lambda phage endolysin. LysECP26 exhibited strong activity with a broad lytic spectrum against various gram-negative strains (29/29) and was relatively stable at a broad temperature range (4℃-55℃). The optimum temperature and pH ranges of LysECP26 were identified at 37℃-42℃ and pH 7-8, respectively. NaCl supplementation did not affect the lytic activity. Although LysECP26 was limited in that it could not pass the outer membrane, E. coli O157: H7 could be effectively controlled by adding ethylenediaminetetraacetic acid (EDTA) and citric acid (1.44 and 1.14 log CFU/ml) within 30 min. Therefore, LysECP26 may serve as an effective biocontrol agent for gram-negative pathogens, including E. coli O157:H7.

• Biomedical Science Letters
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• v.9 no.3
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• pp.111-121
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• 2003

Recombinant single chain Fv (scFv) antibodies offer many advantages over mouse monoclonal antibodies such as faster clearance from blood, improved tumor localization, reduced human anti-mouse antibody (HAMA) response, and the availability to manipulate the scFv through genetic approaches. The recombinant phage display was constructed using lym-l hybridoma cells as a source of genetic starting material. mRNA was isolated from the corresponding antibodies hybridoma cells. VH and VL cDNA were amplified with RT-PCR and linked with ScFv by linker DNA to form ScFv DNA, which then were inserted into phagemid pCANTAB5E. The phage of positive clones selected with tube containing raji lymphoma cell and infected by competent E. coli HB2151 to express soluble scFv. The scFv lym-l was secreted into the cytosol and culture supernatant and shown to be of expected size (approximately 32 kDa) by western blot. An active scFv lym-l could be produced in E. coli with soluble form and high yield from hybridoma cell line, using phage display system. Immunoreactivity indicated that scFv lym1 showed a potential biding affinity against the raji lymphoma cell as its parental antibody (intact lym-l Ab).

• Journal of Microbiology
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• v.40 no.3
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• pp.205-210
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• 2002

To study interactions between $C_{4}$-dicarboxylic acid transport protein D and E$\sigma$$^{54}$ in the dctA promoter regulatory region, we used the challenge phage system. An ant＇-｀lac fusion was recombined onto the challenge phage, and this ant＇-｀lac fusion along with Pant and the R. meliloti dctA promoter regulatory region were cloned onto a plasmid. The plasmid bearing the ant＇-｀lac fusion was used as a reporter plasmid in a coupled transcription-translation system. Addition of purified $\sigma$$^{54}$ to the coupled system specifically repressed transcription of the plasmid-borne ant＇-｀lac fusion. When DCTD was added along with $\sigma$$^{54}$ to the coupled system, transcription of the ant＇-｀lac fusion was even further repressed, suggesting that DCTD may stabilize closed complexes between E$\sigma$$^{54}$ and the dctA promoter.