• 대한수의학회지
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• 제43권2호
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• pp.247-253
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• 2003

Actinobacillus pleuropneumoniae causes a highly contagious pleuropneumoniae in swine. The bacterium produces several virulence factors such as exotoxin, LPS, capsular polysaccharide, etc. Among them, the exotoxin, called Apx, has been focused as the major virulence factor, and the toxin consists of 4 gene cluster. apx CABD. apxA is the structural gene of toxin and has four different types, I, II, III, and IV. As the first step of development of a new subunit vaccine, the three different types of apxA gene were amplified from A. pleuropneumoniae isolated from Korea by PCR with primer designed based on the N- and C-terminal of the toxin. The sizes of apxIA, IIA and IIIA were 3,073, 2,971 and 3,159bps, respectively. The comparison of whole DNA sequences of apxIA, IIA and IIIA genes with those of the reference strain demonstrated 98%, 99% and 98% homology, respectively. In addition, the phylogenetic analysis was performed based on the amino acid sequences compared with 12 different RTX toxin family using the neighbor-joining method. ApxA proteins of Korean isolates were identical with reference strains in this study. All ApxA proteins were expressed in E. coli with pQE expression vector and identified using Western blot with polyclonal antibodies against culture supernatants of A. pleuropneumoniae serotype 2 or 5. The sizes of each expressed ApxA protein were about 120, 110, 125 kDa (M.W.), respectively. The results obtained in this study could be used for the future study to develop a new vaccine to porcine pleuropneumoniae.

• Journal of Microbiology and Biotechnology
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• 제16권8호
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• pp.1180-1184
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• 2006

The capsule that surrounds Yersinia pestis cells is composed of a protein-polysacchride complex; the purified protein component is fraction I (F1) antigen. We report the cloning of the cafl gene and its expression in Escherichia coli using the vector pETl02/D-TOPO and the F1-specific monoclonal antibody. The recombinant F1 (rF1) antigen had a molecular size of 17.5 kDa, which was identical to that of the F1 antigen produced by Y. pestis. Recombinant F1 protein was found to react to polyclonal antiserum to Y. pestis Fl. Recombinant F1 was purified by ProBond purification system and induced a protective immune response in BALB/c mice challenged with up to 10$^5$ virulent Y. pestis. Purified rF1 protein was used in an ELISA to evaluate the ability of a method to detect antibodies to Y. pestis in animal sera. These results strongly indicated that the rF1 protein is a suitable species-specific immunodiagnostic antigen and vaccine candidate.

• BMB Reports
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• 제34권2호
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• pp.156-165
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• 2001

Leptin is a circulating non-glycosylated protein that is mainly produced in adipocytes. Leptin acts in the brain to regulate food intake and energy expenditure. Previously we reported our success in the isolation of a partial cDNA of the long form of the leptin receptor, OB-Rb, from rat spleen, and showed that leptin might also play a role in peripheral immune organs. In the present study, for the first time, the complete coding region of OB-Rb cDNA was cloned from rat splenocytes, and its nucleotide sequence was determined. The cDNA was then further expressed in E. coli and mammalian cells, thereby confirming the functional integrity of this receptor. Prokaryotically overexpressed OB-R protein was then used as an immunizing antigen in BALE/c mice to produce leptin receptor-specific antibodies. By using them, we confirmed the cell surface expression of OB-Rb in transfected CHO cells. It is our belief that the reagents, as produced in this study, will be of great use in further studies of the biological role of rat leptin.

• Journal of Microbiology and Biotechnology
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• 제19권2호
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• pp.172-177
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• 2009

A gene(lsd1) encoding dextranase from Lipomyces starkeyi KSM22 has been previously cloned, sequenced, and expressed in Saccharomyces cerevisiae. The gene consisting of 1,824 base pairs and encoding a protein of 608 amino acids was then cloned into and secretively expressed in Pichia pastoris under the control of the AOX1 promoter. The dextranase productivity of the P. pastoris transformant(pPIC9K-LSD1, 134,000 U/I) was approximately 4.2-fold higher than that of the S. cerevisiae transformant(pYLSD1, 32,000 U/I) cultured in an 8-1 fermentor. Over 0.63 g/l of active dextranase was secreted into the medium after methanol induction. The dextranase of the P. pastoris transformant, as analyzed by SDS-PAGE and Western blotting, showed only one homogeneous band. This dextranase of the P. pastoris transformant showed a broad band near 73 kDa. Rabbit monoclonal antibodies against a synthetic LSD1 peptide mix also recognized approximately 73 kDa.

• Parasites, Hosts and Diseases
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• 제41권2호
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• pp.89-96
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• 2003

Among the panel of monoclonal antibodies (mAb) against Toxoplasma gondii, mAb of Tg621 (Tg621) clone blotted 38 kDa protein which localized in the cytoplasm of tachyzoites by immunofluorescence microscopy The protein was not released into the parasitophorous vacuole during or after invasion. The cDNA fragment encoding the protein was obtained by screening a T. gondii cDNA expression library with Tg621. The full length cDNA sequence was completed with 5’-RACE as 1,592 bp, which contained open reading frame of 942 bp. The deduced amino acid sequence of Tg621 consisted of a polypeptide of 313 amino acids, with significant homology to ribosomal P proteins (RPP) of other organisms especially high to those of apicomplexan species. The expressed and purified TgRPP was assayed in western blot with the sera of toxoplasmosis patients and normal sera, which resulted in the 74.0% of positive reactions in toxoplasmosis patients whereas 8.3% in normal group. Therefore, the antibody formation against TgRPP in toxoplasmosis patients was regarded as specific for T. gondii infection and suggested a potential autoantibody.

• Bulletin of the Korean Chemical Society
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• 제34권2호
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• pp.460-464
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• 2013

Single-chain variable fragments of antibodies (scFv) specific to 2,4-dinitrotoluene (DNT) were isolated from a phage library displaying synthetic human scFv fragments with 6 diversified complementary determining regions (CDRs). A DNT derivative that contained an extended amine group was synthesized and conjugated to the NHS-group that was linked to magnetic beads. Phages specific to the immobilized DNT derivatives were isolated from the library after 4 rounds of sequential binding and elution processes. The displayed scFv fragments from the isolated phages showed consensus CDR sequences. One DNT-specific scFv was expressed in E. coli and purified using Ni-affinity chromatography. The purified DNT-specific scFv binds specifically to the immobilized DNT-derivative with $K_D$ value of $6.0{\times}10^{-7}$ M. The scFv and DNT interaction was not disrupted by the addition of 4-nitrotoluene or benzoic acid. These data demonstrate that the screened scFv from the phage displayed library could be used for selective and sensitive detection of explosives such as TNT.

• 생명과학회지
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• 제28권4호
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• pp.478-482
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• 2018

인간 rotavirus는 영아에게 급성 설사를 일으키는 병원체의 하나이다. 본 연구에서는 Escherichia coli의 코돈 선호도를 따라서 인간 rotavirus A (serotype 1 strain WA)의 $VP8^*$ 단백질을 일부분 암호화하도록 인공적인 유전자를 합성하였다. 합성된 $VP8^*$ 유전자는 코돈을 번역틀에 일치시키고 클로닝이 용이하도록 하기 위한 NdeI 및 HindIII 제한효소 절단 부위와 친화적 정제를 위한 6-히스티딘 암호화 서열을 C-말단에 보유하고 있다. 합성된 $VP8^*$ DNA 절편을 pT7-7 발현 벡터에 삽입하여 E. coli BL21 (DE3)로 형질전환한 후에 최종 농도 0.05 mM IPTG로 생산을 유도한 결과 예상했던 대로 19.7-kDa 크기의 $VP8^*$ 단백질이 고농도로 발현되었다. SDS-PAGE에 전개된 단백질들을 대상으로 mouse anti-rotavirus capsid antibody를 사용한 Western blotting의 결과 ~20-kDa $VP8^*$ 단백질 밴드가 관찰되었다. 인공 $Vp8^*$ 단백질이 피하 주사된 토끼의 polyclonal antibody 혈장을 이용한 조사에서도 동일한 크기의 단백질 밴드를 확인할 수 있었다. 이는 합성된 유전자가 바이러스성 질환을 통제할 항원성 백신 후보의 생산 혹은 진단용 항체를 개발하기 위한 쉽고 빠른 방법을 제공할 수 있다는 의미이다.

• 한국식품위생안전성학회지
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• 제33권3호
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• pp.193-199
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• 2018

본 연구에서는 유전자 변형 옥수수(GM 옥수수)에 특이한 단크론성 항체를 개발하고 이에 대한 특성을 확인하는 연구를 수행하고자 하였다. 먼저 형질전환 대장균으로부터 PAT 단백질을 발현시킬 수 있는 시스템을 확립하였고, 재조합 PAT 단백질을 대량 생산하여 항원으로 사용하였다. 준비된 항원을 면역한 결과 재조합 PAT 단백질의 항원성은 매우 높은 것으로 확인되었으며, 세포융합과 클로닝을 통해 12 종의 hybridoma를 확립하였고 western blot 결과 10 종의 hybridoma가 재조합 PAT 단백질과 강한 반응성을 나타내었다. 10종의 hybridoma가 생산하는 항체가 실제 GM 옥수수에 반응하는지를 추가의 western blot으로 분석한 결과 2종의 단크론성 항체(PATmAb-7 and PATmAb-12)가 재조합 PAT 단백질뿐만 아니라 실제 GM 옥수수 중 PAT와 반응하는 것으로 확인되었다. 항체를 대량 생산하고 정제한 후 2종의 항체는 SDS-PAGE 상에서 대표적인 항체의 분리패턴(heavy와 light chain)을 나타내었고, 전형적인 $IgG_1$과 ${\kappa}$ type으로 확인되었다. 정제된 단크론성 항체는 특성을 조사한 결과 다른 GMO에서 발현될 수 있는 재조합 단백질과 non-GM 옥수수 추출물에는 반응성이 없고 PAT 단백질에만 특이적으로 반응하는 것을 확인할 수 있었다. PATmAb-7 를 이용한 간접효소면역분석법의 검출한계는 0.3 ng/mL 수준으로 기준의 유전자변형 콩 면역분석법과 비교했을 때 높은 민감도를 나타내었다. 이상의 결과로 볼 때 개발된 2종의 항체(PATmAb-7 and PATmAb-12)는 GM 옥수수에서 발현되는 PAT 단백질에 특이적으로 반응하는 항체로 확인되었고, 2종의 항체를 이용한 면역분석법과 바이오센서의 개발 가능성을 제시할 수 있었다.

• 한국산학기술학회논문지
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• 제19권3호
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• pp.382-388
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• 2018

본 연구는 치아우식증의 주 원인균으로 작용하는 S. mutans에 특이적인 앱타머 및 그 작용 기전에 관한 것으로 더욱 상세하게는 SELEX방법을 이용하여 S. mutans에 특이적인 앱타머를 선별하고, 앱타머와 결합하는 단백질 분자를 정제 및 동정함으로써 S. mutans의 부착에 관련된 기전을 보다 더 명확하게 규명하고자 하였다. 표적분자에 높은 친화도와 선택성으로 결합할 수 있는 특성을 가지는 앱타머는 3차원적인 특이적 구조에 의해 표적물질에 대해 수 nM ~ 수 pM 수준의 높은 결합력과 선택성을 지니고 있으며, 항체와 유사한 특성을 가지는 핵산으로 여겨지고 있다. 또한, 항체에 비해 안정성이 매우 높아 실온 보관이나 운반이 가능하고, 장시간이나 반복사용이 요구되는 진단용으로의 응용이 매우 용이하다. 또한 생체 내 면역거부반응이 거의 일어나지 않는 점 등을 바탕으로 보다 저렴한 앱타머로 대체하려는 시도가 이루어지고 있으며, 이는 치료용으로의 개발연구에 매우 중요한 장점이 될 수 있다. 이를 위하여 39개의 random sequence를 갖는 DNA library를 제조하고, S. mutans를 앱타머 선별의 대상물질로 하여, SELEX 방법을 통하여 선별하고 pCR2.1 cloning vector에 cloning하고, 그 염기서열을 분석하였다. 그 결과 6종의 서로 다른 염기서열을 갖는 앱타머를 선별하였으며, 선별된 앱타머 중 SM-2를 이용하여 앱타머와 직접 결합하는 단백질을 분리, 동정하였다. 위의 결과로부터 선별된 앱타머들은 S. mutans에 선택적으로 결합함을 확인하였고, 이 앱타머는 세균의 당 대사 및 단백질 합성 관련 단백질에 결합함으로서 세균의 부착을 억제 할 수 있음을 확인하였다.

• Animal cells and systems
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• 제3권2호
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• pp.229-236
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• 1999

The recA gene plays a central role in genetic recombination and SOS DNA repair in Escherichia coli (E. coli). We have previously identified a 42 kDa RecA-like protein inducible by a variety of DNA damages from a fluorescent Pseudomonas strain sp. and characterized its inducible kinetics. In the present study, we cloned and characterized the gene encoding the RecA-like protein by immunological screening of Pseudomonas genomic expression library using polyclonal E. coli anti-RecA antibodies as a probe. From 10$^{5}$ plaques screened, five putative clones were finally isolated. Southern blot analysis indicated that four clones had the same DNA inserts and the recA-like gene was located within the 3.2 kb EcoRI fragment of Pseudomonas chromosomal DNA. In addition, the cloned recA-like gene was transcribed into an RNA transcript approximately 1.1 kb in size, as judged by Northern blot analysis. The cellular level of RNA transcript of the cloned recA-like gene was increased to an average of 5.15- fold upon treatment with DNA damaging agents such as ultraviolet (UV)- light, nalidixic acid (NA), methyl methanesulfonate (MMS), and mitomycin-C (MMC). These results suggest that the cloned gene is inducible by DNA damage similarly to the recA gene in E. coli. However, the cloned gene did not restore the DNA damage sensitivity of the E. coli recA-mutant.