• 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.

• 대한수의학회지
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• 제38권2호
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• pp.304-313
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• 1998

Sixteen Korean field transmissible gastroenteritis viruses (TGEVs) were isolated using swine testicular cell (STC) and the genomic diversity of them was analyzed. All TGEV isolates produced a typical cytopathic effect in STC and were confirmed as TGEV by immunofluorescence assay using monoclonal antibody against TGEV and PCR using TGEV specific primers. RNAs from TGEV field isolates and vaccine TGEV were extracted and amplified by RT and PCR. The RT-PCR products were digested with selected restriction enzymes and analyzed RFLP patterns. The N-terminal end region of S gene and ORF 3 and 3-1 genes of TGEV amplified by TGEV specific primer pairs seemed to be conserved. Most specific variations were detected in S gene amplified by TGEV 4/6 primer pairs which includes antigenic sites A and D. When the PCR products were treated with Sau3AI and Ssp I, Bvac(vaccine strain), field isolates 133 and 347 were differentiated from Miller and Purdue types. In the case of D5 field isolates, it was classified into Purdue type by Sau 3AI but classified into independent TGEV by Ssp I. Two different TGEV strains from D2 sample were confirmed by plaque purification and RT-PCR-RFLP analysis. To investigate the change occurring in TGEV genome after serial passage, the TGEV P44 strain was passaged through STC. There were specific changes in S gene and a large deletion was observed in ORF 3 and 3-1 genes. These studies showed that a distinct difference in genome exists among TGEV field isolates.

• 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.

• 약학회지
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• 제41권2호
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• pp.212-219
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• 1997

Multiple forms of extracellular phospholipase $A_2$ have been detected in human amniotic fluid (HAF). When HAF was subjected to heparin-Sepharose column chromatography, phospholipase $A_2$ activity was detected in both heparin-non binding and binding fraction. The activity of heparin-non binding fraction was further purified by sequential uses of column chromatographies on butyl-Toy-opearl 650M and DEAE-Sephacel. DEAE-Sephacel fraction contained three different phospholipase $A_2$ activities (Peak I, II, III). The molecular weight of DEAE-Sephacel fraction phospholipase $A_2$ determined by SDS-PAGE were about 52KDa (Peak I). Peak II, III required micromolar $Ca^{2+}$ ion for its maximum activity, but Peak I enzyme showed calcium independent phospholipase $A_2$ activity and showed broad range of pH (6.0~10.0) optimum. All these enzymes were not recognized by a monoclonal antibody raised against phospholipase $A_2$ from human synovial fluid. These results suggest that HAF might contain multiple forms of extracellular phospholipase $A_2$, which may neither belong to the 14KDa group II phospholipase $A_2$ family nor cytosolic phospholipase $A_2$.

• 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.

• 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.

• 한국어병학회지
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• 제21권1호
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• pp.1-11
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• 2008

We report the existence of new type of phosphatidylcholine-hydrolyzing phospholipase D (PLD), which has been characterized and partially purified in the scuticociliate, Uronema marinum. The enzyme from partial purification showed that it was existed in membrane fraction and was a neutral PLD, which catalyzed both transphosphatidylation and hydrolysis reaction. The activity of partially purified membrane-bound PLD was also found to be optimal at pH 7.0-7.5 for 2 hours at 37℃ and depended strictly on the presence of Ca2+ (2.5 mM) and Mg2+ (1.6 mM). Immunoblot analysis indicated that the enzyme was distinct from hPLD1 (human PLD1) and hPLD2 (human PLD2) because it was not recognized by a polyclonal antibody raised to the 12 terminal amino acid of these enzymes. We also found that the membrane-bound PLD is a PIP2-dependent PLD and that GTP-binding proteins are not implicated in the regulation of this enzyme: This enzyme activity is markedly stimulated by phosphatidylinositol 4,5-bisphosphate (PIP2) but not by the small G-protein Arf and GTPrS. In addition, this enzyme was capable of hydrolyzing phosphatidylcholine (PC) but not phosphatidylethanolamine (PE), implying that PC was a preferred substrate.

• 대한수의학회지
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• 제33권2호
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• pp.249-254
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• 1993

설사로 폐사한 자돈의 장 절편을 이용하여 돼지 유행성 설사바이러스(PEDV), 돼지 전염성 위장염 바이러스(TGEV), 돼지 로타 바이러스(PRV)에 대한 병인학적 검사를 형광항체반응을 통하여 조사하였던 바 돼지 유행성 설사 바이러스의 감염에 의한 자돈의 폐사를 확인하였다. 간접형광항체검사시 돼지 유행성 설사 바이러스에 대한 양성반응을 보인 가검재료를 이용 Vero세포에 연속 계대한 후 plaque assay를 통하여 크로닝된 돼지 유행성 설사 바이러스 KPEDV-9주를 작성하였다. 돼지 유행성 설사 바이러스에 대한 면역혈청과 바이러스가 분리된 농장에서 채취된 돼지 혈청을 이용 돼지 유행성 설사 바이러스에 대한 구조단백성분을 분석하였던 바 88K(M.W.), 74K, 70K, 58~54K, 54~46K, 44~40K 및 33~32K에 상당하는 단백성분을 검출할 수 있었다.

• 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.

• 한국임상수의학회지
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• 제25권5호
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• pp.317-323
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• 2008

Canine trypsin-like immunoreactivity (cTLI), which is a mirror of the concentration of trypsin and trypsinogen, is a pancreas-specific enzyme and a suitable marker for canine pancreatitis and especially exocrine pancreatic insufficiency (EPI). To develop the immunochromatographic test kit, monoclonal antibodies that recognize cTLI were prepared. Anionic trypsin, cationic trypsin, and chymotrypsin from canine pancreas were successfully purified to homogeneity, using ammonium sulfate fractionation and benzamidine-affinity chromatography. The purification fold for anionic trypsin was 108 times when compared with that of the homogenation of pancreas. The molecular weights by SDS-PAGE analysis were approximately 23 kDa for chymotrypsin and approximately 20 kDa for cationic trypsin and anionic trypsin, respectively. Using the purified trypsin-like proteins, ten hybridomas which secret canine trypsin-specific monoclonal antibody were prepared. Klotz plot indicated that hybridomas, 5G2H10G4 and 2F4A11, have high affinity constant (Ka) of $4.1\;{\times}\;10^{9}$ and $1.8\;{\times}\;10^{9}$, respectively. Especially, 5F9H3 showed the cationic typsin-specific binding pattern and its Ka was determined to $4.5\;{\times}\;10^{9}$. The development of immunochromatographic test kit using these monoclonal antibodies against cTLI will be very useful in the diagnosis of canine EPI or canine pancreatitis.