• Journal of Microbiology
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• v.42 no.1
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• pp.25-31
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• 2004

Sf9 cells have obvious advantages for the conventional production technology of vaccine. They are useful tools for high concentration and large-scale cultures. Sf9 cells were grown to maximal concentration, 8${\times}$l0$\^$6/ cells/$m\ell$ in a 500$m\ell$ spinner flask, with a doubling time at the exponentially growing phase of 24.5 hours, using serum-free media. To explore the ability of Sf9 cells to be infected by the Japanese encephalitis (JE) virus Beijing-l strain, Sf9 cells were infected with the virus. By 4-5 days post-infection, 10-15 % of the Sf9 cells showed cytopathic effect (CPE), from granularity to the formation of syncytia and multinucleated giant cells continuously observed over a period of 35 days. Positive fluorescent reactions were detected in 30-40% of cells infected with the JE virus Beijing-l strain, and the uninfected Sf9 cells were completely negative. Virus particles, propagated in Sf9 and Vero cells, were concentrated by sedimentation on 40% trehalose cushions by ultracentrifugation, and showed identical patterns of viral morphogenesis. Complete virus particles, 40 to 50 nm in diameter, were observed, and JE virus envelope (E) proteins, at 53 kDa, were found in the western blot analysis to the anti-JE virus E protein monoclonal antibody and reacted as a magenta band in the same position to the glycoprotein staining. To evaluate whether the infectious virus was produced in Sf9 cells inoculated with the JE virus Beijing-l stain, Sf9 cells were inoculated with the virus, and sample harvested every 5 days. The titers of the JE virus Beijing-l strain rose from 1.0${\times}$l0$\^$5/ to 1.5${\times}$l0$\^$6/ pfu/$m\ell$. The infected Sf9 cells could be subcultured in serum-free medium, with no change in the plaque sizes formed by the JE virus Beijing-l strain in the plaque assay. It is suggested that the ability of the JE virus Beijing-l strain to infect Sf9 cells in serum-free media will provide a useful insect cell system, where the JE virus replication, cytopathogenicity and vaccine immunogen can be studied.

• Microbiology and Biotechnology Letters
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• v.33 no.4
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• pp.274-280
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• 2005

Pasteurella multocida is known to cause widespread infections in husbandry. To induce homologous and heterologous immunity against the infections, outer membrane proteins (OMPs) in the envelope of P. multocida are thought to be attractive vaccine candidates. Outer membrane protein H is considered as the major component of OMPs. In this study, a gene for OmpH was isolated from pathogenic P. multocida serogroup A. The gene was composed of 1,047 nucleotides coding 348 amino acids with signal peptide of 20 amino acids. The amino acid composition showed about 80 to 98 per cent sequence homologies among other 10 strains of P. multocida serogroup A, reported so far. A recombinant ompH, from which signal peptide was truncated, was generated using pRSET A to name 'pRSET A/OmpH-F2'. The pRSET A/OmpH-F2 was well expressed in E. coli BL21(DE3). The truncated OmpH was purified using nickel-nitrilotriacetic acid (Ni-NTA) affinity column chromatography. Its molecular weight was registered to be 40 kDa on SDS-PAGE gel. In order to generate immunesera against the OmpH, 50 ug of the protein was intraperitoneally injected into mice three times. The anti-OmpH immuneserum recognized about $5{\times}10^{-2}$ng quantity of the purified OmpH. It can be used for an effective vaccine production to prevent fowl cholera caused by pathogenic P. multocida (Serogroup A).

• Journal of Microbiology and Biotechnology
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• v.28 no.8
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• pp.1376-1383
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• 2018

The hepatitis B virus (HBV) envelope contains small (S), middle (M), and large (L) proteins. PreS1 of the L protein contains a receptor-binding motif crucial for HBV infection. This motif is highly conserved among 10 HBV genotypes (A-J), making it a potential target for the prevention of HBV infection. In this study, we successfully generated a neutralizing human monoclonal antibody (mAb), 1A8 (IgG1), that recognizes the receptor-binding motif of preS1 using a phage-displayed human synthetic Fab library. Analysis of the antigen-binding activity of 1A8 for different genotypes indicated that it can specifically bind to the preS1 of major HBV genotypes (A-D). Based on Bio-Layer interferometry, the affinity ($K_D$) of 1A8 for the preS1 of genotype C was 3.55 nM. 1A8 immunoprecipitated the hepatitis B virions of genotypes C and D. In an in vitro neutralization assay using HepG2 cells overexpressing the cellular receptor sodium taurocholate cotransporting polypeptide, 1A8 effectively neutralized HBV infection with genotype D. Taken together, the results suggest that 1A8 may neutralize the four HBV genotypes. Considering that genotypes A-D are most prevalent, 1A8 may be a neutralizing human mAb with promising potential in the prevention and treatment of HBV infection.

• Korean Journal of Food Science and Technology
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• v.32 no.1
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• pp.218-224
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• 2000

The antimicrobial activities of chitosan oligosaccharide(chitohexaose) and two types of chitosans M.W.(10,000 and M.W. 100,000) were examined against Escherichia coli O157 : H7(ATCC 43894), Staphylococcus aureus(ATCC 144458) and Candida albicans(KFRI 432). Chitosan with molecular weight of 10,000 showed the strongest antimicrobial activities to E. coil O157 : H7 and S. aureus, whereas chitohexaose acted most strongly against C. albicans. The most effective concentration of chitosan was measured to be 0.1 mg/mL for E. coil O157 : H7 and S. aureus, and that of chitohexaose to be 1 mg/mL for C. albicans. Antimicrobial activities of chitosans and chitohexaose were maintained for 60 min after their treatment. They were found to induce leakage of intracellular proteins and nucleic acids from treated microorganisms. The efflux determined by assaying the ${\beta}-galactosidase$ leaked from the lactose-induced E. coli O157 : H7 cells was observed to reach the highest level within 60 min after treatment with the antimicrobial agents and chitosan with 10,000 molecular weight gave the highest ${\beta}-galactosidase$ activity. Therefore, it is supposed that the antimicrobial activity of chitosan with its unique polycationic nature might be caused by its binding to anionic component(s) of the cell envelope and thereby inhibiting the membrane metabolism and/or leaking intracellular materials.

• Korean Journal of Clinical Laboratory Science
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• v.52 no.4
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• pp.297-309
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• 2020

Coronaviruses were originally discovered as enzootic infections that limited to their natural animal hosts, but some strains have since crossed the animal-human species barrier and progressed to establish zoonotic diseases. Accordingly, cross-species barrier jumps resulted in the appearance of SARS-CoV, MERS-CoV, and SARS-CoV-2 that manifest as virulent human viruses. Coronaviruses contain four main structural proteins: spike, membrane, envelope, and nucleocapsid protein. The replication cycle is as follows: cell entry, genome translation, replication, assembly, and release. They were not considered highly pathogenic to humans until the outbreaks of SARS-CoV in 2002 in Guangdong province, China. The consequent outbreak of SARS in 2002 led to an epidemic with 8,422 cases, and a reported worldwide mortality rate of 11%. MERS-CoVs is highly related to camel CoVs. In 2019, a cluster of patients infected with 2019-nCoV was identified in an outbreak in Wuhan, China, and soon spread worldwide. 2019-nCoV is transmitted through the respiratory tract and then induced pneumonia. Molecular diagnosis based on upper respiratory region swabs is used for confirmation of this virus. This review examines the structure and genomic makeup of the viruses as well as the life cycle, diagnosis, and potential therapy.