• 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 $-80^{\circ}C$.

• Journal of Yeungnam Medical Science
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• v.13 no.2
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• pp.272-278
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• 1996

Four subtypes of hepatitis B surface antigen are useful in the epidemiologic studies of the route of virus transmission and clinical significance of simultaneous occurance of hepatitis B surface antigen and antibody to hepatitis B surface antigen in the same serum as well as useful marker for population migration. The sera were obtained from 214 HBs Ag positive patients who are diagnosed as chronic liver disease and following up in the Yeungnam university hospital. The subtypes were determined by solid-phase sandwich EIA using monoclonal antibodies. Among 214 specimens, the subtype adr was 93.9%, adw was 2.8%, ayr was 0.9%, ar was 0.9%, adwr was 1.4% and ayw was not detected. There were no correlation between subtype pattern and disease. In summary, the subtype adr was prominent in our study and the difference of subtype pattern by severity of disease was not significant. However, to determine the prognostic value of HBs Ag subtype and relationship between subtype and disease progression, long-term follow up will be needed.

• Journal of Microbiology and Biotechnology
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• v.10 no.6
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• pp.823-828
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• 2000

The complete nucleotide sequence of hepatitis B virus DNA isolated from Korean patient serum was determined and characterized, and its phylogenetic relation was then investigated. The viral genome was 3,215 base pairs long and included four well known open reading frames (i.e. surface antigens, core antigens, X protein and DNA polymerase). The sequence of the surface antigen showed that the HBV genome under investigation, designated HBV 315, was characteristic of subtype adr. A phylogenetic analysis using the total genome sequence revealed that HBV315 was grouped into genomic group C together with isolates from Japan, China, Thailand, Polynesia, and New Caledonia. The mean percent similarity between HBV315 and other HBV isolates in genomic group C was 97.25%, and that with other genomic groups ranged from 86.16% to 91.25%. The predicted amino acid sequences of HBV315 were compared with two closely related subtype adr isolates, M38636 and D12980. The results showed that the X gene product was identical in the three strains, while there were significant amino acid sequence differences between HBV315 and M38636 in the Pre-S1 and Pre-S2 regions.

• 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.

• 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.

• The Korean Journal of Nuclear Medicine Technology
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• v.15 no.1
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• pp.121-125
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• 2011

Purpose: The DNA-type virus HBV, discovered by D. Dane and others in 1976, is approximately 42nm big and known as the main cause of liver-related diseases around the world. HBsAg has 4 kinds of subtypes including adw, adr, ayw and ayr and besides common antigen factor a, there are d, y, r, w. From the methods of serologically testing HBV, IRMA, EIA and CLIa were developed for testing HBsAg and are being used in examining the surface antigen of HBV. In this study, among the methods for testing HBV, the recently developed RIAKEY Ultrasensitive HBsAg IRMA kit's sensitivity level and performance in detection of mutant forms were measured and compared with CLIA. Materials and methods: Two certified reference materials, which are WHO 1st International Standard 1985(80/549) and WHO 2nd International Standard 2003(00/588. subtype adw2, genotypeA), were used in the examination and the sensitivity level was measured by diluting these materials from 0.08 IU/ml to 0.005 IU/ml. The materials for examining the detection of mutant forms included 9 kinds of subtype 'ad' and one kind of subtype 'ay' purchased from DSI company. Also, with the use of positive and negative samples, they was compared with CLIA. Result: Ultrasensitive HBsAg kit based on IRMA method showed the detection of up to 0.01 IU/ml not only for WHO 1st International Standard 1985(80/549) but also for WHO 2nd International Standard 2003(00/588. subtype adw2, genotypeA) and the sensitivity level was measured as 0.01 IU/ml by WHO standard. In testing the performance for detection of mutant forms, the 9 kinds of subtype 'ad' and one kind of subtype 'ay' mutant materials were detected, demonstrating the capacity of detecting various types of mutant forms. Conclusions: With the clinical importance of sensitivity level and performance in detection of mutant forms increasing in the field of HBsAg diagnosis, the examination of IRMA's effectiveness using RIA method in the aspects of the sensitivity level and performance in detection of mutant forms was carried out and its result is as follows. The sensitivity level was measured as 0.01 IU/ml by WHO standard and it was possible to measure various types of mutant forms with high sensitivity. Thus it is suggested that more speedy and accurate reports could be produced from a nuclear medicine laboratory for clinical practitioners requiring results of various situations.