• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 1997년도 춘계학술대회
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• pp.82-82
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• 1997

The hepatitis B virus(HBV) is a small, enveloped virus with a circular, double-stranded DNA genome. HBV causes active and chronic hepatitis worldwide, including Korea, and is considered to be a major factor for liver cirrhosis and hepatocellular carcinoma. In contrast to the wealth of knowledge on the gene structure and expressional regulation, immunological and pathological mechanisms for HBV-induced hepatocellular injury are not well known. In the present study, vaccinia virus which has been demonstrated to be a useful eukaryotic expression vector was used to clone the gene for HBV surface antigen, L(S+preS2+preS1). The recombinant vaccinia virus vector, pMJ-L, which contains L surface antigen gene of adr-type HBV was constructed, and subseouently used for making recombinant vaccinia virus VV-$\textrm{HBV}_{L}$. Expression of the HBV antigen was examined by immunofluorescent antibody (IFA) test using mouse monoclonal anti-hepatitis B surface antigen. HBsAg was detected in the recombinant virus indicating that the VV-$\textrm{HBV}_{L}$ expressed S antigen successfully. The HBV-Vaccinia Virus recombinant obtained in this study is currently being used for studying the immunological aspects of HBV infection.

• BMB Reports
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• 제33권6호
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• pp.440-447
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• 2000

In this study HepG2 cells were used to express and purify HBV pol proteins. In order to facilitate purification of HBV pol proteins, HBV pol and its deletion mutants were fused to MBP (Maltose Binding Protein). As a result we successfully expressed and partially purified both wild type and mutant recombinant HBV pol proteins by using an amylose resin and anti-MBP antibody. In the case of wild type, the anti-MBP antibody detected three bands. One was full-length and the others were generated by proteolysis of the terminal domain region. The expressed MBP/POL proteins were localized both in the cytoplasm and in the perinuclear region. The purified proteins had polymerase activity toward an exogenous homo-polymer template. The MBP/POL protein also had DNA synthesis activity in vivo, since the MBP/POL expression construct was able to complement a HBV polymerase mutant in trans.

• Pediatric Infection and Vaccine
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• 제7권1호
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• pp.136-142
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• 2000

목 적 : 혈장백신의 항체의 지속성에 관한 보고는 다양하지만, 유전자재조합 백신의 추적관찰에 대한 연구는 많지 않아 저자들은 신생아시기에 유전자재조합 백신을 기본접종 후 접종방법에 따른 5년 후 항체의 지속성과 기본접종 후 각각의 항체역가에 따른 5년 후 항체음전율에 대해 알아보고자 본 연구를 시도하였다. 방 법 : 1993년 4월부터 12월까지 부산 문화병원에서 태어난 신생아 420례중 유전자재조합 백신을 기본접종 후 5년까지 추적관찰이 가능했던 114례를 대상으로 하였다. 기본접종 후와 5년 후 항체역가의 검사는 RIA로 시행하였다. 결 과 : 1) 2개월 접종을 하였을 경우 5년째 항체음성률은 5%이고, 6개월 접종을 하였을 경우 5년째 항체 음성률은 25.5%이었다(P value <0.05). 2) 2개월접종시 항체역가가 199.9 이하시 5%, 200~499.9시 0%, 500~999.9시 0%, 1,000 이상시 0%가 5년 추적검사시 항체음전이 되었다. 3) 6개월 접종시 항체역가가 199.9 이하시 66.7%, 200~499.9시 40%, 500~999.9시 23.9%, 1,000 이상시 22.5%가 5년 추적검사시 항체음전이 되었다. 결 론 : 이 연구에서는 유전자재조합 백신의 5년후 항체음전율은 5~25.5%였고, 항체가의 지속성은 접종방법과는 통계학저인 유의성을 보이지 않았고, 기본접종 후의 항체역가에 따른 5년 후 항체음전율과도 통계학적인 유의성은 보이지 않았다.

• BMB Reports
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• 제37권5호
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• pp.546-551
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• 2004

The increasing pace of development in molecular biology during the last decade has had a direct effect on mass testing and diagnostic applications, including blood screening. We report the model Microarray that has been developed for Hepatitis B virus (HBV) and Hepatitis D virus (HDV) detection. The specific primer pairs of PCR were designed using the Primer Premier 5.00 program according to the conserved regions of HBV and HDV. PCR fragments were purified and cloned into pMD18-T vectors. The recombinant plasmids were extracted from positive clones and the target gene fragments were sequenced. The DNA microarray was prepared by robotically spotting PCR products onto the surface of glass slides. Sequences were aligned, and the results obtained showed that the products of PCR amplification were the required specific gene fragments of HBV, and HDV. Samples were labeled by Restriction Display PCR (RD-PCR). Gene chip hybridizing signals showed that the specificity and sensitivity required for HBV and HDV detection were satisfied. Using PCR amplified products to construct gene chips for the simultaneous clinical diagnosis of HBV and HDV resulted in a quick, simple, and effective method. We conclude that the DNA microarray assay system might be useful as a diagnostic technique in the clinical laboratory. Further applications of RD-PCR for the sample labeling could speed up microarray multi-virus detection.

• Archives of Pharmacal Research
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• 제19권6호
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• pp.469-474
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• 1996

A rapid and sensitive method has been developed to detect hepatitis B virus DNA (HBV) by nested polymerase chain reaction (PCR) technique with primers specific for the surface and core regions in capillary thermal cycler within 80 min. The lower limit for detection by present PCR method is $10^{-5}$ pg of recombinant HBV DNA which is equivalent to that determined by one round of PCR amplification and Southern blot hybridization analysis. When boiled HBV positive serum was serially diluted 10-fold, HBV DNA was successfully determined in $1{\mu}l-10^{-3}$ of serum. HBV DNA was detected by present method in 69 clinical samples including HBsAg positives and negatives by enzyme-linked immunosorbent assay (ELISA). When serum samples were amplified by nested PCR using surface and core region primers, HBV DNAs were detected in 37 of 69 samples (53.6%) and 18 of 69 samples (26.1%), respectively. These results can inform the infectious state of HBsAg positive pateints. A simple and rapid nested PCR protocol by using boiled serum as DNA template has been described for the clinical utility to determine HBV DNA in human serum.

• 한국미생물·생명공학회지
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• 제14권6호
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• pp.455-460
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• 1986

간염 보균자의 혈액으로부터 Dane 입자를 분리하였다. Dane 입자의 핵으로부터 분리해낸 DNA는 $\alpha$-($^{32}$P) dNTP 존재하의 DNA 폴리머레이즈 반응 후 액체 씬틸레이션 카운터와 한천 전기영동 및 가이거 뮐러 카운터에 의하여 간염의 DNA임이 확인되었다. 간염 바이러스에 의한 감염을 막기 위한 백신으로서의 B형 간염 바이러스 표면항원을 생산하기 위하여 산성포스파테이즈 프로모터를 갖는 재조합 프라스미드를 함유하는 효모균주를 사용하였다. 재조합 프라스미드는 pHBV 130 및 pAM 82로부터 제작되었으며 대장균에 변환되어진 후 효모균주에 전달되었다. 간염 표면항원은 조절된 무기 인산 농도하에서 버크홀더 최소배지에서의 저해 해제로 생산되었다. 간염 표면항원의 생산 속도도 조사하였다. 전체 간염 표면항원 활성은 인산이 없는 배지에 옮겨진 뒤 3시간 내지 6시간에서 급격히 증가하였으며 9시간째에 최대에 도달하였다. 인산이 없는 배지에 옮기는 것은 고농도 인산 배지에서의 세포 배양을 6시간동안 수행한 뒤에 하는 것이 최적의 결과를 나타내었다.

• BMB Reports
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• 제38권6호
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• pp.683-689
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• 2005

Antibody to hepatitis B surface antigen (HBsAb) is the important serological marker of the hepatitis B virus (HBV) infection. Conventionally, the hepatitis B surface antigen (HBsAg) obtained from the plasma of HBV carriers is used as the diagnostic antigen for detection of HBsAb. This blood-origin antigen has some disadvantages involved in high cost, over-elaborate preparation, risk of infection, et al. In an attempt to explore the suitable recombinant HBsAg for the diagnostic purpose, the HBV S gene was expressed in Pichia pastoris and the product was applied for detection of HBsAb. Hepatitis B virus S gene was inserted into the yeast vector and the expressed product was analyzed by sodium dodecyl sulphate polyacrolamide gel electrophoresis (SDS-PAGE), immunoblot, electronic microscope and enzyme linked immunosorbent assay (ELISA). The preparations of synthesized S protein were applied to detect HBsAb by sandwich ELISA. The S gene encoding the 226 amino acid of HBsAg carrying ahexa-histidine tag at C terminus was successfully expressed in Pichia pastoris. The His-Tagged S protein in this strain was expressed at a level of about 14.5% of total cell protein. Immunoblot showed the recombinant HBsAg recognized by monoclonal HBsAb and there was no cross reaction between all proteins from the host and normal sera. HBsAb detection indicated that the sensitivity reached 10 mIu (micro international unit)/ml and the specificity was 100% with HBsAb standard of National Center for Clinical Laboratories. A total of 293 random sera were assayed using recombinant S protein and a commercial HBsAb ELISA kit (produced by blood-origin HBsAg), 35 HBsAb positive sera and 258 HBsAb negative sera were examined. The same results were obtained with two different reagents and there was no significant difference in the value of S/CO between the two reagents. The recombinant HBV S protein with good immunoreactivity and specificity was successfully expressed in Pichia pastoris. The reagent for HBsAb detection prepared by Pichia pastoris-derived S protein showed high sensitivity and specificity for detection of HBsAb standard. And a good correlation was obtained between the reagent produced by recombinant S protein and commercial kit produced by blood-origin HBsAg in random samples.

• 한국동물학회지
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• 제28권3호
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• pp.166-178
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• 1985

한국형 B형 간염바이러스(HBV) DNA의 표면항원 유전자를 포유동물 세포에서 발현시켜 항원의 검출과 유전자의 분자유전학적인 연구를 하기 위하여 pre-surface antigen 지역과 표면항원 유전자 그리고 poly(A) addition site가 포함된 DNA 조각을 simian virus 40(SV 40)의 DNA 복제 원점과 promoter가 포함된 유전자 운반체에 재조함 시켰다. 우선, HBV DNA가 들어있는 pHBV 107을 Bam HI으로 부분절단한뒤 self-ligation시켜 두 HBV DNA가 같은 방향으로 들어간 pHBVD 107을 만들었다. 이 plasmid를 Bgl II로 절단하였을때 pre-surface지역과 표면항원 유전자 그리고 poly(A) addition site가 함께 포함된 2.7 kb의 insert DNA 조각을 얻었다. 유전자 운반체로는 포유동물세포에서 복제할 수 있도록 하기 위하여 SV40의 DNA 복제 원점부위와 72 bp repeats(enhancer)가 포함된 pSVOE를 만든다음 이 vector의 Pvu II 절단자리에 Bam HI linker를 붙여 insert DNA가 vector의 SV40 late promoter지역 가까이에 들어갈 수 있도록 변형시킨 pSVOB를 만들었다. 이상과 같이 만들어진 pre-surface 지역-표면항원유전자-poly(A)-addition site가 포함된 2.7 kb DNA 절편을 pSVOB promoter 뒤의 Bam HI site에 삽입하여 재조합된 plasmid pSVBS를 얻었다. 예비실험으로 pSVBS를 T-antigen이 생산되는 COS cell에 이주시켰더니 $HB_sAg$가 발현됨을 보았다.

• Journal of Microbiology and Biotechnology
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• 제27권7호
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• pp.1336-1344
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• 2017

Hepatitis B virus (HBV) is a major cause of liver cirrhosis and hepatocellular carcinoma. With recent identification of HBV receptor, inhibition of virus entry has become a promising concept in the development of new antiviral drugs. To date, 10 HBV genotypes (A-J) have been defined. We previously generated two murine anti-preS1 monoclonal antibodies (mAbs), KR359 and KR127, that recognize amino acids (aa) 19-26 and 37-45, respectively, in the receptor binding site (aa 13-58, genotype C). Each mAb exhibited virus neutralizing activity in vitro, and a humanized version of KR127 effectively neutralized HBV infection in chimpanzees. In the present study, we constructed a humanized version (HzKR359-1) of KR359 whose antigen binding activity is 4.4-fold higher than that of KR359, as assessed by competitive ELISA, and produced recombinant preS1 antigens (aa 1-60) of different genotypes to investigate the binding capacities of HzKR359-1 and a humanized version (HzKR127-3.2) of KR127 to the 10 HBV genotypes. The results indicate that HzKR359-1 can bind to five genotypes (A, B, C, H, and J), and HzKR127-3.2 can also bind to five genotypes (A, C, D, G, and I). The combination of these two antibodies can bind to eight genotypes (A-D, G-J), and to genotype C additively. Considering that genotypes A-D are common, whereas genotypes E and F are occasionally represented in small patient population, the combination of these two antibodies might block the entry of most virus genotypes and thus broadly neutralize HBV infection.

• IMMUNE NETWORK
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• 제10권4호
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• pp.126-134
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• 2010

Background: $CD8^+$ T cells contribute to the clearance of Hepatitis B virus (HBV) infection and an insufficient $CD8^+$ T cell response may be one of the major factors leading to chronic HBV infection. Since the HBx antigen of HBV can up-regulate cellular expression of several immunomodulatory molecules, we hypothesized that HBx expression in hepatocytes might affect $CD8^+$ T cell activity. Methods: We analyzed the activation and apoptosis of $CD8^+$ T cells co-cultured with primary hepatocytes rendered capable of expressing HBx by recombinant baculovirus infection. Results: Expression of HBx in hepatocytes induced low production of $interferon-{\gamma}$ and apoptosis of CD8+ T cells, with no effect on CD8 T cell proliferation. However, transcriptional levels of H-2K, ICAM-1 and PD-1 ligand did not correlate with HBx expression in hepatocytes. Conclusion: Our results suggest that HBx may inhibit $CD8^+$ T cell response by regulation of $interferon-{\gamma}$ production and apoptosis.