• BMB Reports
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• v.29 no.2
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• pp.175-179
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• 1996

Viral surface proteins are known to play an essential role in attachment of the virus particle to the host cell membrane. In case of the hepatitis B virus (HBV) several reports have described potential receptors on the target cell side, but no definite receptor protein has been isolated yet. As for the viral side, it has been suggested that the preS region of the envelope protein, especially the preS1 region, is involved in binding of HBV to the host cell. In this study, preS1 region was recombinantly expressed in the form of a maltose binding protein (MBP) fusion protein and used to identify and visualize the expression of putative HBV receptor(s) on the host cell. Using laser scanned confocal microscopy and by FACS analysis, MBP-preS1 proteins were shown to bind to the human hepatoma cell line HepG2 in a receptor-ligand specific manner. The binding kinetic of MBP-preS1 to its cellular receptor was shown to be temperature and time dependent. In cells permeabilized with Triton X-100 and treated with the fusion protein, a specific staining of the nuclear membrane could be observed. To determine the precise location of the receptor binding site within the preS1 region, several short overlapping peptides from this region were synthesized and used in a competition assay. In this way the receptor binding epitope in preS1 was revealed to be amino acid residues 27 to 51, which is in agreement with previous reports. These results confirm the significance of the preS1 region in virus attachment in general, and suggest an internalization pathway mediated by direct attachment of the viral particle to the target cell membrane.

• BMB Reports
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• v.40 no.6
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• pp.1002-1008
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• 2007

PreS domain of Hepatitis B virus (HBV) surface antigen is a good candidate for an effective vaccine as it activates both B and T cells besides binding to hepatocytes. This report deals with overexpression and purification of adr subtype of surface antigen that is more prevalent in Pakistan. PreS region, comprising 119 aa preS1 region plus a 55 aa preS2 region plus 11 aa from the N-terminal S region, was inserted in pET21a+ vector, cloned in E. coli $DH5\alpha$ cells and expressed in E. coli BL21 codon+ cells. The conditions for over expression were optimized using different concentrations of IPTG (0.01-5 mM), and incubating the cells at different temperatures (23-$41^{\circ}C$) for different durations (0-6 h). The cells were grown under the given optimized conditions (0.5 mM IPTG concentration at $37^{\circ}C$ for 4 h), lysed by sonication and the protein was purified by ion exchange chromatography. On the average, 24.5 mg of recombinant protein was purified per liter of culture. The purified protein was later lyophilized and stored at $-80^{\circ}C$.

• BMB Reports
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• v.28 no.4
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• pp.353-358
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• 1995

Epitope tagging is the process of fusing a set of amino acid residues that are recognized as an antigenic determinant to a protein of interest. Tagging a protein with an epitope facilitates various immunochemical analyses of the tagged protein with a specific monoclonal antibody. The monoclonal antibody H8 has subtype specificity for an epitope derived from the preS2 region of hepatitis B virus surface antigen. Previous studies on serial deletions of the preS2 region indicated that the preS2 epitope was located in amino acid residues 130~142. To test whether the amino acid sequence in this interval is sufficient to confer on proteins the antigenicity recognizable by the antibody H8, the set of amino acid residues in the interval was tagged to the amino terminal of ${\beta}$-galactosidase and to the carboxyl terminal of the truncated $p56^{lck}$ fragment. The tagged ${\beta}$-galactosidase, expressed in Escherichia coli, maintained the enzymatic activity and was immunoprecipitated efficiently with H8. The tagged $p56^{lck}$ fragment, synthesized in an in vitro translation system, was also immunoprecipitated specifically with H8. These results demonstrate that the amino acid sequence of the preS2 region can be used efficiently for the epitope tagging approach.

• YAKHAK HOEJI
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• v.45 no.6
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• pp.708-714
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• 2001

Many studies have provided evidences that hepatitis B surface antigen (HBsAg) including preS region could be an ideal candidate for a new hepatitis B virus (HBV) vaccine with higher efficacy. We established CHO cell lines, IY-CHO-2 and IY-CHO-11 expressing high levels of HBsAg containing preS2 and S protein by stable transfection method. These cell lines expressed the correct size (about 1 kb in length) of HBsAg mRNA as expected. The purified protein from the culture supernatants of the clones showed the same sizes as those expressed in native hepatitis B virus (24 kDa, 27 kDa, 34 kDa and 36 kDa). Antibody productivity of CHO-derived HBsAg protein at lower dose challenge was higher than the protein containing S region alone expressed in yeast system. These results indicate that CHO-derived HBsAg protein containing preS2 and S region can be effectively used for a better immune response as a HBV vaccine.

• Proceedings of the KSME Conference
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• 2003.04a
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• pp.799-804
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• 2003

Friction phenomenon can be described as two parts which are pre-sliding and sliding regions. In motion of the sliding region, friction forces depend on the velocity of the system and are known as Coulomb, stick-slip, stribeck effect and viscous friction. The pre-sliding region, which is before breakaway, depends on the position of the system. The motion of friction in the sliding region can be described as the LuGre model. But the pre-sliding motion of friction, which has hysteresis characteristics in general, is not known widely. Therefore, an improved friction model, which can describe the motion of friction in the pre-sliding region, is proposed in this paper. And simulation and experimental results show the effectiveness of the proposed friction model for precise tracking control systems.

• Journal of Microbiology
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• v.37 no.2
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• pp.86-89
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• 1999

An OTHBVS cell line from HepG2 was established. This cell line stably expresses the human hepatitis B virus (HBV) middle S protein that includes the preS2 region which is important for HBV particle entry into the hepatocyte. To establish this cell line, the middle S open reading frame (ORF), with a promoter located in the 5' region and enhancer located in the 3' region, was cloned downstream from the metallothionine (MT) promoter of the OT1529 vector. In this vector, expression of the middle S protein was constructed to be regulated by its own promoter and enhancer. Expression of the large S protein which contains the preS1 region in addition to the middle S protein was designed to be regulated by the MT promoter. When extracts of OTHBVS cells were examined with an S protein detection kit (RPHA, Korea Green Cross Co.), an S protein was detected. Total mRNA of OTHBVS cell examined by northern blot analysis with an S ORF probe revealed small/middle S transcripts (2.1 kb). When the MT promoter was induced by Zn, large S transcripts (2.4 kb) were detected. The GP36 and GP33 middle S proteins were presumably detected, but large S proteins were not detected by immunostain analysis using anti-preS2 antibody.

• Journal of Microbiology and Biotechnology
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• v.10 no.6
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• pp.844-850
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• 2000

The specific binding and internalization of viral particles is an essential step for the successful infection of viral pathogens. In the case of the hepatitis B virus (HBV), virions bind to the host cell via the preS domain of the viral surface antigen and are subsequently internalized by endocytosis. HBV-preS specific receptors are primarily expressed on hepatocytes, however, viral DNA and proteins have also been detected in extrahepatic sites, suggsting that celluar recepators for HBV may also exist on extrahepatic cells. Recently, an EBV-transformed B-cell line was identified onto which the preS region binds in a receptor-ligand specific manner. In this study, this specific interaction was further characterized, and the binding region within the preS protein was locaized. Also the internalization after host cell attachment was visualized and analyzed by fluorescence-labeled HBV-preS1 proteins using confocal microscopy. Energy depletion by sodium azide treatment effectively inhibited the internalization of the membrane-bound preS1 ligands, thereby indicating an energy-dependent receptor-mediated endocytotic pathway. Accordingly, the interaction of HBV-pres! with this specific B-cell line may serve as an effective model for an infection pathway in extrahepatic cells.

• Korean Journal of Microbiology
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• v.28 no.1
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• pp.13-18
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• 1990

A DNA sequence encoding the adr subtype preS2 region of hepatitis B virus envelope protein was fused to 5' end of lacZ gene yielding a plasmid pTSZ, in order to produce a preS2-$\beta$-galactosidase fusion protein. Serial deletions from 3' and 5' end of preS2 were constructed in plasmids, which were expressed and their antigenicities were examined with the monoclonal antibody H8. Deletions from amino and carboxy terminal to certain points did not affect the antigenicity, but the longer deletions destroyed the antigenicity. End points of deleted preS2 sequence were determined by DNA sequencing. As a result, each end of preS2 epitope was located in the region of amino acid residue 130-132 and 140-142, respectively. Residue 143 may be supplementary for antigenic epitope since the deletion from carboxy terminal to residue 143 revealed partial defect of antigenicity. In the interval of antigenic epitope the amino acid differences between adr and adw2 subtype occurred ar residue 130, 132, and 141. This result indicated that one or more of the three residues are responsible for the binding specificity of monoclonal antibody H8 to adr subtype preS2 fusion protein.

• Korean Journal of Microbiology
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• v.28 no.1
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• pp.6-12
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• 1990

The preS2 wequence of an adr hepatitis B virus was cloned and expressed in Escherichia coli as a $\beta$-galactosidase fusion polypeptide. Recombinant preS2 product interacted with the preS2-specific monoclonal antibody H8 which was induced by surface antigen particles isolated from a Korean gepatitis patient. The H8 showed only a minor cross-reactivity with recombinant preS2 product of adw2 subtype. Determination of nucleotide sequence of the adr preS2 revealed that twelve amino acid residue substitutions between adr and adw2 subtype sequences. The antigenic determinant to H8 must include some of these differences.

• Journal of The Korean Society of Agricultural Engineers
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• v.58 no.6
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• pp.71-77
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• 2016

This study was to evaluate the feasibility of pre-consolidation pressure distribution characteristic of western and southern coastal region, using correlation of unconfined compressive strength and preceding research equation. Pre-consolidation of western and southern region showed similar trends undrained shear strength and pre-consolidation pressure in proportion to unconfined compressive strength. Predicted results of U.S. NAVY. (1982) equation revealed a small error western 9.7 % and southern 0.4 %. Prediction correlation results of pre-consolidation using unconfined compressive strength revealed an error western 16.8 % and southern 0.7 %. It was reported that less than 20 percent of pre-consolidation pressure prediction result of Casagrande forecasting error. Estimates of pre-consolidation pressure are possible, before the standard consolidation test, because it was reported that less than 20 % of the forecasting errors of Casagrande.