• 미생물학회지
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• 제29권2호
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• pp.80-84
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• 1991

We cloned and expressed hepatitis B viral core antigen (HBcAg) gene in E. coli using $P_{L}$ promoter system. For optimal expression of the gene, we undertook the studies on the effects of the distance between Shine-Dalgarno (SD) sequence and start codon, copy number of repressor gene, induction temperature, and the stability of the core antigen. The results demonstrated that the induction at 37.deg.C was more efficient than at 42.deg.C, and the 11 base pairs (bp) distance between SD sequence and start codon of HBcAg gene was more efficient than the 15 bp distance in E. coli. The copy number of cI857 repressor gene did not influence on the expression of HBcAg, and the expression level of HBcAg in mutant type (low protease activity) and wild type strains was almost the same. The produced core antigen appeared to be HBcAg not HBeAg judged by two different radioimmunoassat (RIA) kits. This result suggested that the antigen was stable in E. coli.i.

• 미생물학회지
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• 제27권3호
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• pp.169-175
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• 1989

포유동물 세포내에서 간염 바이러스의 내면항원의 발현과 전위내면 항원(precore) 부위의 역할을 규명하기 위하여 고등동물세포 발현용 벡터에 전위내면항원 부위를 갖거나 또는 갖지 않는 내면항원 유전자를 클로닝 하여 COS 세포내에서의 발현을 조사하였다. 전위내면항원 부위를 포함한 내면항원 유전자를 갖는 플라스미드로감염시킨 COS 세포는 항원들이 세포추출물과 배양액에서 검출되었다. 분비된 항원의 증가율은 감염후 2일과 3일 사이가 가장 높았고, 부분결실된 제조합 플라스미드 중 내면항원의 ATG codon에서 180bp 떨어진 것이 가장 발현이 잘 되었다. 전위내면항원을 갖지 않거나 하나의 염기가 첨가되어 변형된 전위내면항원을 갖는 제조합 플라스미드의 경우 항원들이 세포 추출물에서만 검출되었다. 이러한 사실은 전이내면항원 부위가 HBe 항원의 분비에 관여 한다는 경우 항원들이 세포 그러나 대장균이나 효모 세포의 경우는 전위내면항원의 존재와 상관없이 항상 세포추출물에서만 존재하는 것으로 보아 이들 세포의 경우에서는 전위내면항원 부위가 HBe 항원의 분비에 영향을 줄 수 없음을 의미한다.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 1994년도 춘계학술대회 and 제3회 신약개발 연구발표회
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• pp.178-178
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• 1994

Hepatitis B Virus (HBV) is a DNA virus with a 3.2kb partially double-stranded genome. The life cycle of the virus involves a reverse transcription of the greater than genome length 3.5kb mRNA. This pegenomic RNA contains all the genetic information encoded by the virus and functions as an intermediate in viral replication. Tumor suppressor p53 has previously been shown to interact with the X-gene product of the HBV, which led us to hypothesize that p53 may act as a negative regulator of HBV replication and the role of the X-gene product is to overcome the p53-mediated restriction. As a first step to prove the above hypothesis, we tested whether p53 represses the propagation of HBV in in vitro replication system. By transient cotransfection of the plasmid containing a complete copy of the HBV genome and/or the plasmid encoding p53, we found that the replication of HBV is specifically blocked by wild-type p53. The levels of HBV DNA, HBs Ag and HBc/e Ag secreted in cell culture media were dramatically reduced upon coexpresion of wild-type p53 but not by the coexpression of the mutants of p53 (G154V and R273L). Furthermore, levels of RNAs originated from HBV genome were repressed more than 10 fold by the cotransfection of the p53 encoding plasmid. These results clearly states that p53 is a nesative regulator of the HBV replication. Next, to addresss the mechanism by which p53 represses the HBV replication, we performed the transient transfection experiments employing the pregenomic/core promoter-CAT(Chloramphenicol Acetyl Transferase) construct as a reporter. Cotransfection of wild-type p53 but not the mutant p53 expression plasmids repressed the CAT activity more than 8 fold. Integrating the above results, we propose that p53 represses the replication of HBV specifically by the down-regulation of the pregenomic/core promoter, which results in the reduced DNA synthesis of HBV. Currently, the mechanism by which HBV overcomes the observed p53-mediated restriction of replication is tinder investigation.