• 한국식품위생안전성학회지
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• 제10권4호
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• pp.225.2-262
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• 1995

Ochratoxin A was produced from Aspergillus ochraceus ATCC 18472 which was then orally administered into the experimental mice to study the toxic levels of ochratoxin A. AELISA (enzyme-linked immunosorbent assay) method which is more rapid and safe than conventional analytical method, was developed by using ochratoxin A antibody. This method was successfully used to measure the levels of ochratoxin Ain blood, liver and kidney of mice. In order to produce a large amount of ochratoxin A to study toxicity in the mice, Aspergillus ochraceus ATCC 18412 was incubated in the rice medium and as a result 0.5 g of ochratoxin A form rice medium (kg) was produced after extraction and purification Feed consumption and gain in body weight of mice with ochratoxin A at a level of $10 \mu\textrm{g}/g$ body weight was significantly (p<0.05) reduced as compared with control during period of 3 weeks. Ochratoxin A-BSA conjugate was made by putting 13 mole of ochratoxin A on I mole of BSA. This conjugate was used to develop ELISA method. The minmum detection level of ochratoxin A by established ELISA method was 0.5 ppb. After oral adminstraton of ochratoxin A dose of $10\;\mu\textrm{g}/g$ every two day for 3 weeks, concentration of ochratoxin A was measured in the blood, liver and kidney by ELISA method. The level of ochratoxin A was $11\;\mu\textrm{g}/dl,\;0.9 \mu\textrm{g}/g\;and\;3.7\;\mu\textrm{g}/g$ in the blood, liver and kidney, respectively.

• 한국미생물·생명공학회지
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• 제20권2호
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• pp.225-232
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• 1992

효소면역측정법에 의한 aflatoxin $B_1(AFB_1)$의 정량법을 개발하기 위하여, 항체를 생산.정제하여 분석법을 확립하고 직접법과 간접법(direct/indirect competitive ELISA)의 특성 및 문제점을 비교. 검토하였다. Bovine serum albumin(BSA)을 carrier protein으로 한 $AFB_1$-1-(O-carboxymethyl)oxime -BSA를 토끼에 면역하여 항$AFB_1$항혈청을 생산하였다. 정량 침강반응에 의하여 항혈청으로부터 항BSA항체를 제거하고 황산암모늄 침전법 및 EDAE-Sephadex A-50 이온교환 크로마토그래피를 통하여 순도 높은 IgG항체를 정제하여 항$AFB_1$항체를 사용하였다.

• Journal of Microbiology and Biotechnology
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• 제7권2호
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• pp.144-150
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• 1997

We developed a simple and efficient method to prepare a Pseudomonas vaccine of outer membrane (OM) proteins free from lipopolysaccharide (LPS). A three step purification process including extraction, ultrafiltration and ultracentrifugation effectively removed LPS from the OM protein fraction. Approximately 2 mg of the OM proteins was obtained from 1 g of wet cell. LPS contaminant in the vaccine preparation was less than 0.003% (w/w) of protein and protease activity was not detectable. To achieve a wide range of protection, OM proteins prepared from four attenuated P. aeruginosa strains were mixed in equal amounts and used as a vaccine, which elicited in rabbits a high titer of antibody reactive to all of the seven Fisher types. The antisera from the immunized rabbit had a strong reactivity to vaccine proteins larger than 25 kDa. In a burned mouse infection model, immunization with the vaccine significantly enhanced bacterial clearance in the Pseudomonas infected skin. The vaccination also provided mice an excellent protection against Pseudomonas infection (11, 16). Data on antigenicity, mutagenicity, acute, subacute toxicity and pharmacological tests confirmed the safety of the vaccine (1, 3, 10, 12, 17). These data demonstrate that this method can be applied to manufacture a bacterial vaccine of OM proteins with safety and prophylactic efficacy at a practical low cost.

• 한국환경성돌연변이발암원학회지
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• 제21권2호
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• pp.149-155
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• 2001

Eighty-one workers including 38 employees directly incinerating industry wastes were recruited from a company located in South Korea. To evaluate the association between urinary 1-hydroxypyrene glucuronide (1-OHPG) levels, as internal dose of polycyclic aromatic hydrocarbon (PAH) exposure, and glycophorin A (GPA) mutation frequency, as an early biologic effect indicator. Urinary 1-OHPG levels were measured by synchronous fluorescence spectroscopy after immunoaffinity purification using monoclonal antibody 8E11. Erythrocyte GPA variant frequency (NN or NO) was assessed in MN heterozygotes with a flow cytometic assay. The GSTM1 and GSTT1 genotypes were assessed by a multiplex PCR method. The GPA NN phenotype frequency was higher in occupationally exposed group (n=14, mean$\pm$S.D. 6.6$\pm$12.0 in 10/SUP 6/ erythrocyte cells) than in non-exposed group (n=22, 2.1$\pm$3.5). Similarly, the GPA(NO or NN) phenotype frequency was higher in exposed group (n=14, 9.7$\pm$17.3) than non-exposed group (n=22, 4.2$\pm$6.3). The above differences failed to reach statistical significance, but a significant increase was seen in GPA variant frequency levels with increase in urinary 1-OHPG levels (Spearman's correlation: p=0.06 (NO), p=0.07 (NO or NN)). When this association was evaluated by GSTM1 genotype status, the association between GPA mutation and urinary 1-OHPG levels was stronger in individuals with GSTM1 present genotype (Spearmans correlation; r=0.50, p=0.02). These results suggest that the association between urinary 1-OHPG and GPA mutation is be modulated by the GSTM1 genotype.

• Journal of Microbiology
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• 제40권4호
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• pp.313-318
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• 2002

To perform the prophylactic study of a vaccine derived from human papillomavirus (HPV) using Balb/c mice, we produced virus like particles consisting of HPV capsid protein L1 which has been reported to induce significant humoral and cellular immunity using various animal model systems. In order to produce HPV16 VLPs, the cDNA of L1 capsid protein in HPV type 16, obtained by polymerase chain reaction, was inserted into yeast expression vector, YEG$\alpha$-HIR525 under the control of GAL10 promoter. The transformation of YEG$\alpha$-HPV16 L1 was performed into the yeast Saccharomyces cerevisiae Y2805 by the lithium acetate method and the yeast clone expressing the highest level of L1 capsid protein of human papillomavirus type 16 was selected by Western blot analysis using anti-HPV16 L1 antibody. The purification of HPV16 VLP has been performed by the ultracentrifugation and gel-filtration methods. To validate the vaccine efficacy of the purified HPV16 VLPs and investigate the properties of HPV16 VLPs to induce humoral immunity, ELISA assay was performed. A significantly increased production of anti-HPV16 VLP antibodies was observed in sera from immunized mice. The neutralization activity of antibodies in the sera from the vaccinated mice was demonstrated by a rapid and simple assay to detect hemagglutihation inhibition activity.

• 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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• 제40권6호
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• pp.1028-1038
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• 2007

Human plasma Hp is classified as 1-1, 2-1, and 2-2. They are inherited from two alleles Hp 1 and Hp 2, but there is only Hp 1 in almost all the animal species. Hp 2-2 molecule is extremely large and heterogeneous associated with the development of inflammatory-related diseases. In this study, we expressed entire bovine Hp in E. coli as a $\alpha\beta$ linear form. Interestingly, the antibodies prepared against this form could recognize the subunit of native Hp. In stead of a complicated column method, the antibody was able to isolate bovine Hp via immunoaffinity and gelfiltration columns. The isolated Hp is polymeric containing two major molecular forms (660 and 730 kDa). Their size and hemoglobin binding complex are significantly larger than that of human Hp 2-2. The amino-acid sequence deducted from the nucleotide sequence is similar to human Hp 2 containing a tandem repeat over the $\alpha$ chain. Thus, the Hp 2 allele is not unique in human. We also found that there is one additional -SH group (Cys-97) in bovine $\alpha$ chain with a total of 8 -SH groups, which may be responsible for the overall polymeric structure that is markedly different from human Hp 2-2. The significance of the finding and its relationship to structural evolution are also discussed.

• Biomolecules & Therapeutics
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• 제8권2호
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• pp.153-159
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• 2000

A novel serine/threonine protein phosphatase with EF-hand motif, which belongs to PPEF family was partially cloned from rat brain cDNA by employing RT-PCR method. The size of the amplified clone was 1.6kbp. The amplified DNA was subcloned into pGEM-T-Easy vector and the resulting plasmid was maned as pGEM-rPPEF2. The nucleuotide sequence is shared by 88% with that of mouse PPEF-2 cDNA, and the deduced amino acid sequence reveal 92% homology with that of mouse PPEF-2 cDNA. The N-terminal region of the cloned rat brain PPEF contains a putative phosphatase catalytic domain (PP domain) and the C-terminal region contains multiple $Ca^{2+}$ binding sites (EF region). The putative catalytic domin (PP) and the EF-hand motif (EF) regions were subcloned into pGEX4T-1 and were overexpressed in E. coli DH5 as glutathione-S-transferase (GST) fusion proteins. Expression of the desired fusion protein was identified by SDS-PAGE and also by immunoblot analysis using monoclonal antibody against GST. The recombinant proteins were purified by glutathione-agarose chromatography. This report is first to demonstrate the cloning of PPEF family from rat brain tissues. The clone reported here would be invaluable for the investigation of the role of this new type-phosphatase in rat brain.

• 한국수산과학회지
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• 제25권5호
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• pp.432-442
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• 1992

동남참게, Eriocheir japonicus의 난황단백전구체 합성부위를 면역조직화학적인 방법으로 구명하기 위하여, 난황단백질을 정제하고, 이 단백질에 대한 항혈청을 제작하여 난황단백질에 대한 면역학적인 성질을 밝힌 후, 난황단백전구체의 항체가 존재하는 항 난황단백혈청을 사용하여 PAP(peroxidase-antiperoxidase)법으로 생식소, 간췌장, 근육조직을 대상으로 면역조직화학 반응을 시켰다. 성숙한 암, 수혈청에 대한 면역전기영동과 Ouch-terlony 면역확산 실험에 의하여 암혈청내에는 암 특이성혈청단백질이 있음이 밝혀졌다. 암, 수혈청에 대한 항혈청과 난황형성중인 암혈청에 대한 조난황단백추출액에 대하여 면역전기영동과 Ouchte-rlony 면역확산 실험을 행한 결과, 암 특이성혈청 단백질은 난황단백전구체임이 밝혀졌다. 조난황단백추출액으로 부터 DEAE 이온교환 chro-matography와 sepharose CL-4B gel 여과법에 의하여 난황단백질을 정제하였으며, 이 난황단백질의 분자량을 sepharose CL-4B gel 여과법에 의하여 추정한 결과 분자량은 약 245,000 dalton이었다. 또한 난황단백질은 면역학적인 분석에 의하여 난황 단백질에 대한 항혈청에는 난황단백전구체에 대한 항체가 존재하고 있음이 증명되었다. 난황단백질에 대한 토끼 항혈청을 이용하여 PAP법에 의한 면역조직화학반응을 시킨 결과, 난황형성중인 난모세포와 성숙한 암참게의 간췌장은 양성반응이 나타는데 반하여 난소내의 여포세포층과 난황형성전 난모세포, 근육층 그리고 성숙한 수참게의 간췌장은 전혀 양성반응이 나타나지 않았다. 따라서 본 종의 난황단백전구체의 합성부위는 간췌장임을 알 수 있다.

• 생명과학회지
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• 제30권5호
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• pp.420-427
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• 2020

Repebody는 비면역 글로블린 인공 항체로 저렴하고 빠르게 생산 가능한 맞춤형 항체이다. 그러나 의료용 repebody의 생산은 저수율 및 복잡한 정제 공정으로 인해 여전히 어려움을 겪고 있다. Pseudomonas fluorescens의 ABC transporter를 사용한다면 생산 공정을 간소화하고 비용을 줄일 수는 있지만 repebody는 양전하를 띠어 분비 효율이 낮다. 따라서 등전점(pI)이 높은 repebody의 등전점을 낮추어 음전하를 띄도록 해야 한다. 이것을 위해 repebody의 N 말단과 C 말단에 연속된 아스파탐산을 붙여 보았지만 분비가 증가하지 않았다. 다른 방법으로 ABC transporter를 통한 repebody 분비 효율을 높이기 위해 repebody의 항원 결합 부위의 반대쪽에 존재하는 열다섯 개의 양전하 아미노산을 아스파탐산으로 변환하여 repebody 표면이 강한 음전하를 띠도록 하였다. 그 결과, 기존 repebody의 발현 단백질 당 분비효율은 21.2%였으나 변형한 과음전하 repebody의 분비효율은 58.5%로 향상되었다. 결론적으로 과음전하를 통해 만들어진 repebody는 P. fluorescens에 의해 세포 바깥에 분비 생산할 수 있었다.