• Biotechnology and Bioprocess Engineering:BBE
• /
• 제10권1호
• /
• pp.29-33
• /
• 2005

A DNA vaccine methodology using eukaryote expression vectors to produce immunizing proteins in the vaccinated hosts is a novel approach to the development of vaccine and immuno-therapeutics, and it has achieved considerable success over several infectious diseases and various cancers. To further enhance its efficiency, attempts were made to develop novel plasmid vectors containing multiple immunostimulatory CpG motifs, for rapid and strong immune response. First, a 2.9 kb compact plasmid vector (pVAC), containing CMV promoter, polycloning site, BGH poly(A) terminator, ampicillin resistance gene and pBR322 origin was constructed. A pVAC-hEPO was also constructed, which contained a human erythropoietin gene, for evaluating the transfection efficiency of naked plasmid DNA both in vitro and in vivo. To examine the adjuvant effect of multi-CpG motifs on naked plasmid DNA, 22 and 44 enriched and unmethylated CpG motifs were introduced into pVAC to generate pVAC-ISS1 and pVAC-ISS2, respectively. $100{\mu}g$ of pSecTagB, pVAC, pVAC-ISS1 or pVAC-ISS2 were each injected intramuscularly into the tibilias anterior muscle of Balb/c mice. The level of interleukin-6 induced in the mice injected with pVAC-ISS1 and pVAC-ISS2 were significantly elevated after 12 hours, which were almost 2 and 2.5 times higher than that in the mice injected with pSecTagB, respectively. These results suggest that DNA vaccine plasmids with enriched CpG motifs can induce rapid secretion of interleukin-6 by lymphocytes. In conclusion, these vectors can contribute to the development of adjuvant-free DNA vaccinations against infectious diseases and various cancers.

• Applied Biological Chemistry
• /
• 제46권4호
• /
• pp.353-360
• /
• 2003

본 실험에서는 acetylcholinesterase(AChE, EC 3.1.1.7)를 이용한 간이 잔류농약 검사법에 필요한, 유기인계 및 카바메이트계 살충제에 대한 감수성이 증가된 AChE(MAChE)를 baculovirus를 이용하여 대량으로 생산하는 시스템을 구축하고 생산된 효소의 특성을 관찰하였다. 한라산에서 채취한 초파리에서 AChE의 cDNA를 합성한 후 PCR을 이용하여 AChE의 lipid anchor부분을 제거하고 site directed mutagenesis에 의해 E107Y, F368L, L408E의 염기서열을 변화시켜 재조합된 MAChE cDNA를 합성하였고 baculovirus vector에 삽입하여 대량생산을 시도하였다. 대량 증식에 필요한 조건으로 감염횟수가 네 번일 때, 그리고 세포수가 $2{\times}10^6$ cell/ml일 때 세포의 증식과 효소의 활성이 극대화됨을 알 수 있었다. His tag을 붙여 Ni-NTA affinity column을 이용하여 MAChE를 정제하였으며, 정제된 효소는 실험조건하에서는 pH(3-10)와 온도$(20-50^{\circ}C)$의 변화에 영향을 받지 않았다. 농약 추출액으로 methanol을 사용했을 때가 ethanol을 사용할 때 보다 효과적임을 알 수 있었다. 대표적인 유기인계와 카바메이트계 농약에 대한 저해율을 조사한 결과 재조합된 MAChE는 대만의 집파리 및 변형되지 않은 AChE에 비하여 전반적으로 농약에 대한 감수성이 높은 것으로 나타났다.

• 한국미생물·생명공학회지
• /
• 제33권2호
• /
• pp.84-89
• /
• 2005

Phosphomannomutase는 진핵 생물과 원핵 생물에 있어서 중요한 효소로, ${\alpha}$-D-mannose 6-phosphate를 ${\alpha}$-D-mannose 1-phosphate로 전환시켜 GDP-mannose를 생산한다. 이 기질은 여러 대사 경로에서 중요하게 작용하는 mannosyl기를 제공하도록 돕는다. 본 논문에서는 Sphingomonas chungbukensis DJ77에서 phosphomannomutase를 암호화하는 유전자를 유전체 library로부터 동정하고 이를 pmmC로 명명하였으며, 이를 발현 vector에 클로닝하고 염기서열을 분석하였다. 유전자 pmmC는 ATG를 개시 코돈으로 사용하고, TAG를 종결 코돈으로 사용하는 750 bp의 open reading frame임을 확인하였고, 이 ORF의 5 bp앞쪽으로 리보좀 결합 부위가 존재한다. 이 ORF로부터 유추되는 아미노산은 249개이며, 단백질 분자량은 약 27.4 kDa이다. 이 유전자를 구성하는 아미노산 서열은 NCBI의 conserved domain search를 통한 분석으로 eukaryotic phosphomannomutase와 약 $86.9\%$ 유사성이 있음을 나타냈고, 기질에 대한 활성을 측정한 결과 pmmC 유전자가 암호화하는 단백질이 phosphomannomutase임을 확인할 수 있었다.

• 한국발생생물학회:학술대회논문집
• /
• 한국발생생물학회 2003년도 제3회 국제심포지움 및 학술대회
• /
• pp.121-121
• /
• 2003

The standard protocol for the production of transgenic mouse from ES-injected embryo has to process via chimera producing and several times breeding steps, In contrast, tetraploid-ES cell complementation method allows the immediate generation of targeted murine mutants from genetically modified ES cell clones. The advantage of this advanced technique is a simple and efficient without chimeric intermediates. Recently, this method has been significantly improved through the discovery that ES cells derived from hybrid strains support the development of viable ES mice more efficiently than inbred ES cells do. Therefore, the objective of this study was to generate transgenic mice overexpressing human resistin gene by using tetrapioid-ES cell complementation method. Human resistin gene was amplified from human fetal liver cDNA library by PCR and cloned into pCR 2.1 TOPO T-vector and constructed in pCMV-Tag4C vector. Human resistin mammalian expression plasmid was transfected into D3-GL ES cells by lipofectamine 2000, and then after 8~10 days of transfection, the human resistin-expressing cells were selected with G418. In order to produce tetraploid embryos, blastomeres of diploid embryos at the two-cell stage were fused with two times of electric pulse using 60 V 30 $\mu$sec. (fusion rate : 93.5%) and cultured upto the blastocyst stage (development rate : 94.6%). The 15~20 previously G418-selected ES cells were injected into tetraploid blastocysts, and then transferred into the uterus of E2.5d pseudopregnant recipient mice. To investigate the gestation progress, two El9.5d fetus were recovered by Casarean section and one fetus was confirmed to contain human resistin gene by genomic DNA-PCR. Therefore, this finding demonstrates that tetraploid-ES mouse technology can be considered as a useful tool to produce transgenic mouse for the rapid analysis of gene function in vivo.

• Journal of Microbiology and Biotechnology
• /
• 제24권2호
• /
• pp.152-159
• /
• 2014

The regulation of protein tyrosine phosphorylation is mediated by protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs) and is essential for cellular homeostasis. Co-expression of PTKs with PTPs in Pichia pastoris was used to facilitate the expression of active PTKs by neutralizing their apparent toxicity to cells. In this study, the gene encoding phosphatase PTP1B with or without a blue fluorescent protein or peroxisomal targeting signal 1 was cloned into the expression vector pAG32 to produce four vectors. These vectors were subsequently transformed into P. pastoris GS115. The tyrosine kinases EGFR-2 and $PDGFR{\beta}$ were expressed from vector pPIC3.5K and were fused with a His-tag and green fluorescent protein at the N-terminus. The two plasmids were transformed into P. pastoris with or without PTP1B, resulting in 10 strains. The EGFR-2 and $PDGFR{\beta}$ fusion proteins were purified by $Ni^{2+}$ affinity chromatography. In the recombinant P. pastoris, the PTKs co-expressed with PTP1B exhibited higher kinase catalytic activity than did those expressing the PTKs alone. The highest activities were achieved by targeting the PTKs and PTP1B into peroxisomes. Therefore, the EGFR-2 and $PDGFR{\beta}$ fusion proteins expressed in P. pastoris may be attractive drug screening targets for anticancer therapeutics.

• 한국통신학회논문지
• /
• 제31권5A호
• /
• pp.517-525
• /
• 2006

본 논문은 다수의 RFID 태그가 사용되고 있는 환경에서 고속 필터링을 수행하기 위한 필터링 엔진을 설계한다. 이를 위하여 우리는 고속 라우터나 방화벽에 적용되었던 고속 패킷 필터링 기법이 RFID 데이터 필터링과 매우 유사함을 보이고 그 중 대표적인 기법인 Bit Parallelism 기반의 Aggregated Bit Vector(ABV)를 고속 RFID 필터링 엔진에 적용한다. 또한, RFID 데이터 필터링의 성향을 관찰한 결과 태그 인식 및 필터 부합의 시간적 중복성을 발견하고 두 가지 캐쉬(태그 캐쉬, 필터 캐쉬)를 적용하여 추가적인 필터링 성능 향상을 꾀하였다. 설계한 RFID 고속 필터링 엔진의 성능 평가를 위해 프로토타입 애플리케이션을 제작하여 시뮬레이션을 수행하였다. 결과로써 기존의 순차적인 RFID 데이터 필터링에 비해 고속의 필터링 성능을 보이며 특히 필터의 수가 증가할수록 필터링의 효율이 높아짐을 보인다.

• Asian-Australasian Journal of Animal Sciences
• /
• 제17권12호
• /
• pp.1641-1646
• /
• 2004

The objective of this study was to generate transgenic mice expressing human resistin gene by using the tetraploidembryonic stem (ES) cell complementation method. Human resistin gene was amplified from human fetal liver cDNA library by PCR, cloned into $pCR^{(R)}$ 2.1 $TOPO^{(R)}$ vector and constructed in pCMV-Tag4C vector. Mammalian expression plasmid containing human resistin was transfected into D3-GL ES cells by Lipofectamine 2,000, and then after 10-12 days of transfection, the human resistin-expressing cells were selected with G418. In order to produce tetraploid embryos, blastomeres of diploid embryos at the two-cell stage were fused with two times of electric pulse using 60 V 30 $\mu$sec (fusion rate: 2,114/2,256, 93.5%) and cultured up to the blastocyst stage (development rate: 1,862/2,114, 94.6%). The selected 15-20 ES cells were injected into tetraploid blastocysts, and then transferred into the uteri of E 2.5 d pseudopregnant recipient mice. To investigate the gestation progress, two E 19.5 mused fetuses were recovered by Cesarean section of which one fetus was confirmed to contain human resistin gene by genomic DNA-PCR. Therefore, our findings demonstrate that tetraploid-ES mouse technology can be considered as a useful tool to produce transgenic mice for the rapid analysis of gene function in vivo.

• 한국미생물학회:학술대회논문집
• /
• 한국미생물학회 2002년도 추계학술대회
• /
• pp.121-124
• /
• 2002

This work was initiated to develope a recombinant oral poliovaccine (OPV), which is highly advanced in safety (minimizing VAPP) by introducing Type 2,3 poliovirus epitopes into our RPS-Vax system. We have introduced several potential vaccine epitopes of poliovirus Type 2, and 3 into RPS-Vax system, resulting in production of recombinant polioviruses. Any of these chimeric viruses, however, were not detected for their foreign gene expression by serotype-specific mouse antiserum. We have designed several folding units to stabilize the introduced vaccine protein and attached short epitope-concatamer or epitope-multimer to them, followed by production of chimeric viruses. Only those who have an HIV-1 Tat-mediated folding unit were nicely detected for the introduced foreign proteins by anti-Tat antiserum and type-specific peptide-induced antisera. Nevertheless, introduced epitopes were not detected in Western blot experiment with each serotype-specific antiserum. None of the mice inoculated with these chimeric viruses showed preventative immunity when challenged with Lansing and Leon wildtype 2 and 3 poliovirus, and the antiserum did not show neutralizing capacity in vitro. Conformational epitope covering B/C loop region of type 2 and 3 were newly designed by computer modeling, and introduced into the RPS-Vax vector system, followed by production of chimeric viruses. Introduced epitope regions were nicely detected by anti-Tag23 mAb or peptide antibody, but still not detected by poliovirus antiserum. Nevertheless, neutralizing antibody was detected in the Tg-PVR mice even when inoculated once with these chimeric viruses. Also, the immunized mice showed perfect preventative immunity against the wild Type poliovirus Lancing or Leon. When boosted appropriately, those chimeric virus-inoculated Tg-PVR mice produced equivalent amounts of neutralizing antibody to those in Sabin 2/3-immunized mice. These data strongly suggest that our recombinant poliovirus (RPS-PV2 and RPS-PV3) can be used as a safe and effective rec-OPV instead of any preexisting poliovaccine.

• 한국통신학회논문지
• /
• 제42권5호
• /
• pp.999-1008
• /
• 2017

본 논문에서는 무선 네트워크 환경에서 효율적인 데이터 전달을 위한 파이프라인 네트워크 코딩(Pipeline Network Coding) 기법과 데이터의 무결성을 검증하기 위한 데이터 인증 기법, 가상 송신자에 대한 노드 인증 기법을 제안한다. 파이프라인 네트워크 코딩 기법은 네트워크 코딩을 수행하는 중계 노드가 송신자 대신 데이터를 전달함으로써 전체적인 네트워크 성능을 향상시키는 기법이다. 그러나 네트워크 코딩은 악의적인 공격자가 데이터를 위 변조하여 네트워크에 주입하는 공격인 오염 공격(pollution attack)에 취약하다. 이를 방어하기 위해 HMAC(Hash-based Message Authentication Code)을 사용한다. 이때 데이터 인증에 사용되는 태그를 생성하기 위해서는 인증을 수행하는 노드들에게 키를 분배해야한다. 키 분배에 따른 오버헤드를 최소화하기 위해 해쉬 체인을 적용하였다. 가상 송신자에 대한 인증 기법으로는 null 벡터를 사용한다. 최종적으로 제안 기법에 대한 안전성과 복잡도를 분석하고, 시뮬레이션을 통해 성능을 분석하였다.

• 생명과학회지
• /
• 제15권6호
• /
• pp.928-934
• /
• 2005

ATF2는 c-Fos와 c-Jun과 같은 전사인자이며 이들은 루신지퍼 단백질이다. 루신지퍼 단백질은 동형이합체 혹은 이형이합체를 형성하며 promoter 영역에 결합하여 전사조절에 중요한 역할을 한다. 세포의 전사인자 ATF2는 ATF/CRE site에 결합하며 특히 선택적인 interfamily 이형이량체를 형성함으로써 전사 조절에 있어서 다양한 메카니즘을 제공할 수 있다. 본 연구에서는 ATF2 cDNA를 6xHis를 가진 expression vector에 subcloning하여 E.cnli BL2l에서 발현시켰다. 6xHis tag은 nickel-chelating chromatography를 가능하게 하였다 발현된 ATF2는 In vitro binding pull-down assay에서 동형이합체를 이를 뿐만 아니라 AP-1 그룹의 인자들과 선택적인 이형이합체를 형성함을 보여 주었다. BATF와는 강하게 결합하였으며 c-Jun과도 안정된 이합체를 형성하였다. 그러나 c-Fos와는 이합체를 형성하지 않으므로서 AP-1그룹 내에서도 이합체 형성이 선택적으로 이루어짐을 알 수 있다.