• Journal of Microbiology and Biotechnology
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• 제10권6호
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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.

• 생명과학회지
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• 제15권6호
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• pp.1034-1036
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• 2005

배추 종자 및 유묘에 대하여 GUS발현 또는 hepatitis B surface antigen (HBsAg)발현 벡터를 지니는 Agrobacterium tumefaciens LBA4404 세포를 이용하여 진공침윤(agroinfiltration)에 의한 형질전환을 시도하였다. 특히 ELISA를 이용한 HBsAg발현의 정량적 분석에서 agroinfiltration방법은 형질전환효율이 매우 저조하게 나타났다. 그러나 차아염소산나트륨 용액을 발아 전 또는 발아 중인 배추종자에 처리한 후 agroinfiltration을 실시한 경우 형질전환 효율이 $2\~5$배 증가하였다. 따라서 차아염소산나트륨 등의 화학연마제에 의한 종자의 상처발생이 Agrobacterium의 감염을 용이하게 함으로써 배추유묘에서의 일시유전자발현을 증대시키는 것으로 제안되고있다.

• 미생물학회지
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• 제27권1호
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• pp.16-20
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• 1989

The effect of E. coli plasmid DNA sequences contained by chimeric vectors on plasmid replication was investigated. We constructed YRp7- or 2.$\mu$m circle-based plasmids containing E. coli plasmid DNA sequences and those not containing it. By examining their maintenance in yeast, we showed that plasmid without E. coli plasmid DNA sdquences was nore stable and presented higher copy number, and espressed higher level of hepatitis B viral surface antigen as a foreign gene. This result suggested that E. coli plasmid DNA sequences within chimeric plasmid somehow inhibited plasmid replication in yeast.

• 농업생명과학연구
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• 제45권6호
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• pp.193-199
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• 2011

An efficient transient expression system has been developed and characterized for the production of foreign genes in seedlings. The seedlings can be easily produced from commercial seeds used for vegetable sprouts. In principal, a chemical abrasive was employed to generate wounds in seedlings prior to vacuum-infiltration with Agrobacterium tumefaciens bearing the target gene. This optimized chemical wounding-assisted agro-infiltration process resulted in up to 15-fold increase in $\beta$-glucuronidase (GUS) enzyme activity. This procedure has been used efficiently to express hepatitis B surface antigen (HBsAg) protein in a transient mode. Therefore, seedlings with proper wounds can be suggested as a convenient tool for the production of useful recombinant proteins.

• 한국균학회지
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• 제39권3호
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• pp.235-238
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• 2011

팽이균사체의 형질전환을 위하여 Agrobacterium 세포를 사용하였다. 특히, Agrobacterium 세포의 감염단계 전에 약한 NaOH용액을 처리하였으며 이로써 균사체 세포들의 표면 상해 발생을 기대하였다. 그 결과, hygromycin 저항성 ($hyg^r$) 균사체는 NaOH 처리를 거친 경우에서만 출현하였다. 형질전환 균사체의 $hyg^r$ 유전자 도입은 PCR로 확인되었으며 또한 Southern blot hybridization과 western blotting 분석에 의하여 단일 유전자 copy의 삽입과 외래유전자의 발현을 확인할 수 있었다. 본 연구는 팽이균사체에 대한 효율적인 Agrobacterium 이용 형질전환수단을 보여주고 있다.

• 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 Plant Biotechnology
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• 제7권4호
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• pp.219-223
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• 2005

We investigated chemical-wounding effect on Agrobacterium-mediated Chinese cabbage transformation via vacuum infiltration. Pre-germinated or germinating Chinese cabbage seeds were infiltrated with Agrobacterium tumefaciens LBA4404 cells carrying either GUS gene (pBI121) or hepatitis B virus surface antigen DNA (pBIHBsAg). Prior to agroinfiltration process, the seeds were soaked in sodium hydrosulfite (SHS) solution or just in sterile water as a control. Comparative transformation efficiency was determined by both of histochemistry and ELISA. We could demonstrate that SHS solution treatment especially to 1-day or 2-days old germinating seeds efficiently improved transformation process, and therefore, transient expression level. This strongly indicated that Agrobacterium infection could be facilitated indeed by SHS-causing wounds on Chinese cabbage seeds.

• 식물조직배양학회지
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• 제21권6호
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• pp.353-356
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• 1994

사람 B형 간염 바이러스의 pre-S2 표면항원에 결합하는 키메라 항체 유전자(카파 및 감마사슬의 cDNA클론)를 식물체에서 발현시키기 위해 식물체 발현벡터인 pBKS-1에 XbaI 자리를 이용하여 클로닝하였다. 이들 유전자를 포함하는 대장균의 플라스미드 핵산을 추출하여 아그로박테리움에 형질전환 시켰다. 다음 담배의 조직절편과 아그로박테리움을 공동배양함으로써 담배의 형질 전환을 시도하였다. 카나마이신이 포함된 신초유기배지에서 나온 신초를 시료로 하여 Western blot을 실시함으로써 이들 유전자가 형질전환 담배에서 안정하게 발현됨을 확인하였다.

• BMB Reports
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• 제31권2호
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• pp.123-129
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• 1998

Foreign proteins, $endo-{\beta}-1,4-glucanase$ of Bacillus subtilis, preS1+S2 region of hepatitis B virus large surface antigen, human ${\beta}_2-adrenergic$ receptor ($h{\beta}_{2}AR$), and bovine growth hormone (bGH) were expressed in Saccharomyces cerevisiae and secreted into the medium. These proteins were expressed using the alcohol dehydrogenase I (ADH1) promoter of Saccharomyces cerevisiae and secreted by signal sequence of the 97 K killer toxin gene of doublestranded linear DNA plasmid (pGKL1) of S. cerevisiae. All these proteins underwent severe modifications; in particular, N-glycosylation in the case of $endo-{\beta}-1,4-glucanase$, $h{\beta}_2AR$, and preS1+S2. Seventy four percent of the expressed $endo-{\beta}-1,4-glucanase$ was secreted into the culture medium. Highly modified proteins were detected in the culture medium and in the cell. Expressed $h{\beta}_2AR$, which has seven transmembrane domains, remained in the cell. The degrees of secretion and modification and the states of proteins in the culture medium and in the cell were quite different. These results indicated that the nature of the protein has a critical role in its secretion and modifications.

• IMMUNE NETWORK
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• 제2권1호
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• pp.41-48
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• 2002

Background: Viral antigens presented on the cell surface in association with MHC class I molecules are recognized by CD8+ T cells. MHC restricted peptides are important in eliciting cellular immune responses. As peptide antigens have a weak immunigenicity, pH-sensitive liposomes were used for peptide delivery to induce effective cytotoxic T lymphocyte (CTL) responses. In the previous study, as the HBx peptides could induce specific CTLs in vitro, we tested whether the HLA-A2/$K^b$ transgenic mice that were immunized by HBx-derived peptides could be protected from a viral challenge. Methods: HBx-peptides encapsulated by pH-sensitive liposomes were prepared. $A2K^b$ transgenic mice were immunized i.m. on days one and seven with the indicated concentrations of liposome-encapsulated peptides. Three weeks later, mice were infected with $1{\times}10^7pfu$/head of recombinant vaccinia virus (rVV)-HBx via i.p. administration. The ovaries were extracted from the mice, and the presence of rVV-HBx in the ovaries was analyzed using human TK-143B cells. IFN-${\gamma}$ secretion by these cells was directly assessed using a peptide-pulsed target cell stimulation assay with either peptide-pulsed antigen presenting cells (APCs), concanavalin A ($2{\mu}g/ml$), or a vehicle. To generate peptide-specific CTLs, splenocytes obtained from the immunized mice were stimulated with $20{\mu}g/ml$ of each peptide and restimulated with peptide-pulsed APC four times. The cytotoxic activity of the CTLs was assessed by standard $^{51}Cr$-release assay and intracellular IFN-${\gamma}$ assay. Results: Immunization of these peptides as a mixture in pH-sensitive liposomes to transgenic mice induced a good protective effect from a viral challenge by inducing the peptide-specific CD8+ T cells. Mice immunized with $50{\mu}g/head$ were much better protected against viral challenge compared to those immunized with $5{\mu}g$/head, whereas the mice immunized with empty liposomes were not protected at all. After in vitro CTL culture by peptide stimulation, however, specific cytotoxicity was much higher in the CTLs from mice immunized with $5{\mu}g/head$ than $50{\mu}g/head$ group. Increase of the number of cells that intracellular IFN-${\gamma}$ secreting cell among CD8+ T cells showed similar result. Conclusion: Mice immunized with XEPs within pH-sensitive liposome were protected against viral challenge. The protective effect depended on the amount of antigen used during immunization. XEP-3-specific CTLs could be induced by peptide stimulation in vitro from splenocytes obtained from immunized mice. The cytotoxic effect of CTLs was measured by $^{51}Cr$-release assay and the percentage of accumulated intracellular IFN-${\gamma}$ secreting cells after in vitro restimulation was measured by flow cytometric analysis. The result of $^{51}Cr$-release cytotoxicity test was well correlated with that of the flow cytometric analysis. Viral protection was effective in immunized group of $50{\mu}g/head$, while in the in vitro restimulation, it showed more spectific response in $5{\mu}g$/head group.