• Restorative Dentistry and Endodontics
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• 제19권2호
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• pp.485-498
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• 1994

Porphyromonas endodontalis is a black-pigmented anaerobic Gram-negative rod which is associated with endodontal infections and this microorganism possesses a potential for pathogenicity. The purpose of this study was to compare the membrane components of Porphyromonas endodontalis and Porphyromonas gingivalis and to study the immune reaction patterns of Porphyromonas endodontalis with patients with periapical lesion. Porphyromonas endodontalis (ATCC 35406), Porphyromonas gingivals serotypea (381), serotype b(W50), serotype c(A7A1-28) were cultured in anaerobic condition. Rabbit antisera were prepared by intravenous injection of formalized whole cells and human sera were obtained from patients and dental students. Indirect immunofluorescence method was used to study on the cross reaction between Porphyromonas endodontalis and Porphyromonas gingivalis serotype a, b, c antigen. Total membrane protein profiles of Porphyromonas endodontalis antigen were studied by sodium dodecyl sulfate polyacrylamide gel electrophoresis and the reactivity of antigenic components of Porphyromonas endodontalis against sera of patients and rabbit anti-Porphyromonas endodontalis antisera were assessed by Immunoblotting method. The following results were obtained : 1. Antigens of Porphyromonas endodontalis has multiple antigenic components, and both patients with periapical lesion and normal healthy individual showed immune response to this. 2. Patients group and healthy individual group showed a diversity of immune reaction pattern but they showed immune response against 43kd protein. 3. Patients with periapical lesion showed more diverse immune response than healthy individual and in some patients, much more bands appeared to lower molecular weight protein. 4. According to indirect immunofluorescence and Immunoblotting study, Porphyromonas endodontalis did not share common antigen with Porphyromonas gingivalis serotype a, b, c.

• Clinical and Experimental Vaccine Research
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• 제12권4호
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• pp.304-312
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• 2023

Purpose: Due to the many problems with commercially available vaccines, the production of effective vaccines against brucellosis is a necessity. The aim of this study was to evaluate the immune responses caused by the chimeric protein consisting of trigger factor, Bp26, and Omp31 (TBO) along with aluminum hydroxide (AH/TBO) and selenium (Se/TBO) nanoparticles (NPs) as adjuvants in mouse model. Materials and Methods: Recombinant antigen expression was induced in Escherichia coli BL21 (DE3) bacteria using IPTG (isopropyl-d-1-thiogalactopyranoside). Purification and characterization of recombinant protein was conducted through NiFe₃O₄ NPs, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Western blot. NP characteristics, including morphology and particle size, were measured in vitro. The recombinant TBO was loaded on to AH and Se NPs and were administered subcutaneously. After mice immunization, measurement of antibody titter and protection assay was performed. Results: The average sizes of AH and Se NPs were about 60 nm and 150 nm, respectively. The enzyme-linked immunosorbent assay results showed that the serum of mice immunized by subcutaneous injection with both nanovaccines produced significant immunoglobulin G (IgG) responses against the chimeric antigen. The results of TBO-specific IgG isotype (IgG2a/IgG1) analysis showed that both AH and Se NPs induced a type to T-helper immune response. In addition, the results of the challenge with the pathogenic strain of Brucella melitensis 16M showed that vaccinated mice with AH/TBO NPs indicated a higher reduction of bacterial culture than immunized mice with Se/TBO NPs and TBO alone. Conclusion: The results showed that AH NPs carrying chimeric antigen can be a promising vaccine candidate against brucellosis by producing protective immunity.

• Journal of Microbiology and Biotechnology
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• 제10권1호
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• pp.95-98
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• 2000

An immunochemical method has been develooped as the most sensitive tool for studying the expression of quinoproteins containing pyrroloquinoline qinone(PQQ) in E. coli. The PQQ was conjugated to bovine serum albumin (BSA), and the conjugant was purified by using a $KwikSep^{TM}$ dextran desalting column chromatography. The PQQ-BSA conjugant was immunized to rabbits, and the IgG fractions of the antisera were purified. The most sensitive antibody against PQQ-BSA conjugant recognized some nanogram quantity of the antigen on the blot, but had little cross reactivity with BSA. Using this batch of the antibody, all the immunochemical assays of quinoproteins in E. coli were preformed. Some six different PQQ-specfic spots were detected by Western blot analysis of the soluble proteins in E. coli were performed. Some six different PQQ-specific spots were detected by Western blot analysis of the soluble proteins in E. coli after two-dimensional gel electrophoresis. Their molecular weights on the blot were estimated to be about 100-, 90-, 72-, 58-, 52-, and 50kDa. Their pI values fell in the range from 4.8 to 5.5. These results stronly suggest that quinoproteins are present in E. coli, and that the protein moieties were covalently bound to PQQ.

• Journal of Microbiology
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• 제37권1호
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• pp.14-20
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• 1999

Two extracellular proteases, EP I and EP II, from cells of Oligotropha carboxydovorans (formerly Pseudomonas carboxydovorans) DSM 1227 grown in nutrient broth were purified to greater than 95% homogeneity in five steps using azocasein as a substrate. The final specific activities of EPs I and II were 214.9 and 667.4 units per mg of protein. The molecular weights of native EPs I and II were determined to be 23,000. Sodium dodecyl sulfate-gel electrophoresis revealed the two enzymes to be monomers. The enzymes were found to be serine-type proteases. The activity of EP I was stimulated by Ca2+, Mg2+, and Ba2+, but that of EP II was not. The enzymes were completely inhibited by Fe2+, Hg2+, Co2+, Zn2+, and Cd2+. EDTA and EGTA exhibited a strong inhibitory effect on EP I. The optimal pH for the two enzymes was pH 9.0. The optimal temperatures for EP I and II were 60 and 50$^{\circ}C$, respectively. The enzymes were stable under alkaline conditions. The thermal stability of EP I was higher than that of EP II. Cell-free extracts did not inhibit the purified enzymes. The enzymes were active on casein, azocasein, azocoll, and carbon monoxide dehydrogenase, but weakly active with bovine serum albumin.

• Bulletin of the Korean Chemical Society
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• 제4권3호
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• pp.139-143
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• 1983

${\beta}$-N-Acetyl-D-glucosaminidase B was highly purified with the following sequence of steps; DEAE-cellulose, CM-cellulose, and Sephadex G-200 gel filtration chromatograpies. The specific activity of the purified ${\beta}$ -N-acetyl-D-glucosaminidase B was 2.2 units/mg protein with 12.9 % yield and 196.2 fold purity. The purified ${\beta}$-N-acetyl-D-glucosaminidase B showed single band on polyacrylamide gel electrophoresis. The final preparation of ${\beta}$ -N-acetyl-D-glucosaminidase B was completely free friom arylsulfatase and ${\beta}$-glucuronidase. ${\beta}$ -N-Acetyl-D-glucosaminidase B had pH optimum of 4.5 in 0.5 M sodium citrate buffer. The molecular weight of ${\beta}$-N-acetyl-D-glucosaminidase B was 133,000 by Sephadex G-200 gel filtration. The Km value of ${\beta}$-N-acetyl-D-glucosaminidase B using p-nitrophenyl-N-acetyl-${\beta}$-D-glucosaminide as substrate was 1.0 mM and $V_{max}$ was 0.014 ${\mu}$ mole/min. ${\beta}$-N-Acetyl-D-glucosaminidase B was stable at $55^{circ}C$ for 70 minutes. The crude ${\beta}$ -N-acetyl-D-glucosamiinidase in 70 % ammonium sulfate retained 93 % activity after 7 months storage at -$55^{circ}C$. Bovine serum albumin, sodium chloride, and phosphate activated ${\beta}$ -N-Acetyl-D-glucosaminidase B. N-Acetyl-D-glucosamine, ${\alpha}$-methyl-D-mannoside, and acetate inhibited ${\beta}$ -N-acetyl-D-glucosaminidase B.

• BMB Reports
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• 제40권4호
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• pp.459-466
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• 2007

The cDNA sequence of the Japanese flounder (Paralychthys olivaceus) IgD has been previously reported (GenBank accession no. AB052658) and this was followed by the detection of IgD mRNA expression in some flounder organ tissues. However, it has not been determined whether the flounder IgD gene is virtually expressed into IgD protein. To characterize the flounder immunoglobulins utilized in elucidating the mechanism, evolution and diversity of the flounder immune system, antibodies specific to IgD and IgM were necessary. In the present study, partial flounder recombinant IgD (rIgD), IgM (rIgM) and the conserved regions of IgD and IgM (rCIg) were produced by cloning the cDNA sequence using isotype specific primers which were designed to produce unique fragments of IgD and IgM specific amino acid sequences. The production of recombinant Igs was ascertained by SDS-gel electrophoresis and immunoblot analysis using anti-T7$\cdot}$Taq antibody. The produced recombinant Igs were purified using affinity columns, and used as immunogens. Antibodies specific to the isotype of flounder Igs were generated by immunizing rabbits with rfIgs and the antibodies produced were identified by enzyme-linked immunosorbent assay (ELISA) and immunoblotting. Specificities of the generated antibodies were evaluated by testing cross-reactivity between recombinant IgM and IgD. By ELISA, rabbit antibodies against the rfIgD fragment (anti-rfIgD) failed to recognize any kind of flounder serum Igs, whereas respective antibodies against rfCIg (anti-rfCIg) and rfIgM fragments (anti-rfIgM) reacted with serum Igs. Likewise, in immunoblot assays, though anti-rfIgD did not, both anti-rfCIg and anti-rfIgM bound with the ~85 kd flounder IgM heavy chain. By flow cytometry analysis, anti-rfCIg, anti-rfIgD and anti-rfIgM reacted with 6%, 3% and 6.5% of cells, respectively, suggesting that flounder IgD is not secreted in serum but expressed on flounder B-like cell surfaces as in mammals. Antibodies produced against recombinant flounder Igs could be used to develop sandwich assay systems for detecting flounder Igs and for further investigating the flounder immune system.

• 한국임상수의학회지
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• 제28권3호
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• pp.303-306
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• 2011

프로게스테론 제제를 복용해 오던 12세령의 암컷 잡종 견이 복부 종괴의 평가를 위해 내원하였다. 복부 초음파 검사와 방사선 검사를 통해 확장된 자궁과 관련된 종괴를 확인하였다. 혈청 화학 검사에서는 고글로불린혈증이 나타났으며, 전기영동 검사상 급성 염증 패턴으로 판단되었다. 초음파 유도하에 종괴의 세침흡인을 실시하였으며 세포학검사를 통해 선암종이 진단되었다. 탐색적 개복을 실시하였으며 장구체의 종괴를 발견하여 자궁과 함께 종괴를 제거하는 수술을 실시하였다. 조직병리 검사결과 자궁 선암종이 진단되었으며 종양세포의 에스트로겐 수용체와 프로게스테론 수용체 발현 여부 평가를 위해 면역 염색을 실시하였다. 면역염색결과 종양세포는 두 항체에 대해 음성 결과를 보였으며, 정상 간엽세포에서만 양성 결과가 나타났다. 수술 후 1주일에 재검을 위해 내원하였는데 컴퓨터 단층촬영 검사결과 폐 전이가 확인되었다. 환자는 수술 후 40일경에 폐사하였다.

• 한국가축번식학회지
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• 제23권2호
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• pp.119-125
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• 1999

본 연구는 오골계에 있어서 성장에 따른 gamma -globulin, lipoprotein 및 중량변동과, Fabricius 낭과 흉선의 적출시 발생 5일령부터 150일령까지의 혈청 protein 농도와 Ig, 백혈구수 및 림프구수의 변동을 조사하고자 수행하였다. 1. 재래계와 오골계의 성장에 따른 gamma-globulin 농도는 발생 당일로부터 5일령까지는 각각 0.41$\pm$0.01~0.85$\pm$0.05 mg/㎗ 와 0.50$\pm$0.01~0.98$\pm$0.08 mg/㎗ 로 증가하였으나 10일령부터 감소하여 낮은 치를 나타내다가 재래계는 100일령부터 오골계는 발생 50일령 이후부터 증가된 수준을 나타냈다. 2. 오골계의 성장에 따른 혈청 alpha-lipoprotein, beta-liporptein 및 gamma -lipoprotein 농도는 각각 74.1$\pm$6.8~240.2$\pm$9.7 mg/㎗, 89.7$\pm$5.7~225.8$\pm$11.3 mg/㎗ 및 87.6$\pm$4.7~220.3$\pm$10.2 mg/㎗ 수준이었다. 오골계의 성장에 따른 혈청 alpha-lipoprotein 농도는 점차적으로 큰 감소를 나타냈다. 3. 오골계의 Fabricius 낭과 흉선 적출후 성장에 따른 백혈구 및 림프구수는 10일령부터 100일령까지 증가경향을 나타냈으며, 흉선 적출군의 백혈구 및 림프구수는 대조군에 비해 낮게 나타났다. 4. 오골계의 Fabricius 낭과 흉선 적출후 성장에 따른 혈청 Ig 농도는 Fabricius 낭 적출군의 경우 대조군에 비해 큰 차이가 없었으나 50일령 이후에는 증가하기 시작하여 100~150일령에서는 5일령에 비해 높은 치를 나타냈으며, 흉선적출군은 성장에 따른 유의한 증가경향을 나타냈다.

• 한국임상수의학회지
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• 제31권3호
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• pp.212-215
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• 2014

이 연구는 사육황새에 급여하는 먹이(양미리와 전갱이)의 차이에 따른 혈청 단백질 분획의 변화를 평가하기 위한 것이다. 사육황새를 먹이에 따라 두 그룹(그룹 1과 2)으로 나누어 연구를 진행하였다. 그룹 1에는 22마리, 그룹 2에는 29마리의 황새가 포함되었고, 각각 양미리와 전갱이를 급여하였다. 두 그룹에서 전혈구검사, 혈청화학검사, 단백질분획검사(${\alpha}$-, ${\beta}$- 및 ${\gamma}$-글로불린) 및 지단백질 분획검사(HDL 및 LDL)를 진행하고 결과를 비교하였다. 검사 결과 그룹 1에서 ${\alpha}$-글로불린의 감소와 ${\beta}$-글로불린의 증가가 유의적으로 관찰되었다. 그룹 1에서 LDL의 농도는 그룹 2에 비해 유의적으로 증가하였다. 결과적으로 양미리를 급여한 사육황새에서 ${\beta}$-글로불린의 증가가 LDL의 증가에 의한 것임을 확인하였다.

• 한국임상수의학회지
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• 제25권5호
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• pp.340-345
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• 2008

M. pachydermatis는 개에서의 피부 정상 균 종으로서, 과잉 증식되는 가장 흔한 질병 중의 하나가 아토피성 피부염이다. 이 연구의 목적은 개의 아토피성 피부염에서 M. pachydermatis 추출 단백질에 대한 IgG를 이용한 체액성 면역 반응을 알아보는 것이다. M. pachydermatis의 전기 영동과 개의 혈청을 이용한 Western immunoblotting을 이용해 M. pachydermatis의 알러젠을 밝히고 아토피 견과 비 아토피 견에서의 각각의 면역 반응을 비교하였다 결과는 아토피 견의 혈청에서 18, 21, 26, 32, 34, 38, 40, 42, 46, 58, 64, 75, 85, 그리고 120 kDa의 단백질의 반응이 발견된 것에 반해, 비 아토피 견에서는 50 kD을 제외한 다른 단백질의 반응은 발견되지 않았다. 이 연구의 결과로 M. pachydermatis 피부염을 지닌 개의 아토피성 피부염은 IgG의 체액성 면역 반응을 유발하고, 이 면역 반응은 개의 아토피성 피부염의 발병 기전에 중요한 역할을 한다는 것을 알 수 있다. 그러나 개의 아토피성 피부염에서의 M. pachydermatis에 대한 체액성 면역 반응의 역할을 밝히기 위해서는 더 많은 연구가 필요하다.