• 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.

• Molecules and Cells
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• 제39권3호
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• pp.242-249
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• 2016

A balance between production and degradation of reactive oxygen species (ROS) is critical for maintaining cellular homeostasis. Increased levels of ROS during oxidative stress are associated with disease conditions. Antioxidant enzymes, such as extracellular superoxide dismutase (EC-SOD), in the extracellular matrix (ECM) neutralize the toxicity of superoxide. Recent studies have emphasized the importance of EC-SOD in protecting the brain, lungs, and other tissues from oxidative stress. Therefore, EC-SOD would be an excellent therapeutic drug for treatment of diseases caused by oxidative stress. We cloned both the full length (residues 1-240) and truncated (residues 19-240) forms of human EC-SOD (hEC-SOD) into the donor plasmid pFastBacHTb. After transposition, the bacmid was transfected into the Sf9-baculovirus expression system and the expressed hEC-SOD purified using FLAG-tag. Western blot analysis revealed that hEC-SOD is present both as a monomer (33 kDa) and a dimer (66 kDa), as detected by the FLAG antibody. A water-soluble tetrazolium (WST-1) assay showed that both full length and truncated hEC-SOD proteins were enzymatically active. We showed that a potent superoxide dismutase inhibitor, diethyldithiocarbamate (DDC), inhibits hEC-SOD activity.

• 한국수산과학회지
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• 제30권3호
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• pp.473-479
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• 1997

범가자미, Verasper variegatus의 황단백 질을 증류수로 침전시킨 후 Sepharose CL-6B column chromatography에 의해 분리하였다. 조난황단백추출액에서 분리한 난황단백질이 암컷 특이 혈청단백질과 공통의 항원성을 가짐을 Ouchterony 면역확산 test와 면역전기영동으로 확인하였다. 분리한 난황단백질의 분자량은 약 550kD이었으며, 난황단백질을 구성하고 있는 subunits은 분자량이 각각 108, 85, 31kD인 3종의 major subunits와 103, 80kD인 2종의 minor subunits이었다. 분리한 난황단백질은 vitellin에 대한 항혈청을 이용하여 western blot으로 확인한 바, 면역화학적 반응을 나타냈다.

• 한국환경성돌연변이발암원학회지
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• 제20권1호
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• pp.34-39
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• 2000

Although shipbuilding workers were exposed to a variety of genotoxic compounds including polycyclic aromatic hydrocarbons (PAHs), limited number of studies were conducted to evaluate the biomarkers related to PAH exposure in painting workers in shipbuilding industry. One hundred and thirty three workers including 73 employees using coal tar paints were recruited from a shipbuilding company located in South Korea. Urinary 1-hydroxypyrene glucuronide (1-OHPG), as internal dose of PAH exposure, were measured by synchronous fluorescence spectroscopy after immunoaffinity purification using monoclonal antibody 8E11. Glutathione S-transferase (GST)M1 and GSTT1 genotypes were assessed by multiplex PCR. Information on demographic characteristics, smoking gabit, diet, job title, use of personal protective equipments were collected by self-administered questionnaire. Urinary 1-OHPG were higher in workers using coal tar paints than in workers using general paints, however, the difference was not statistically significant (p=0.20, Mann-Whitney U test). Urinary 1-OHPG levels in smokers were higher than in non-smokers (p<0.05 by Mann-Whitney U test) and there was a significant increase in urinary 1-OHPG levels with the numbers of cigarettes consumed per day (Spearman's correlation coefficient = 0.28, p=0.02). Genetic polymorphisms of GSTM1 and GSTT1 did not influence the level of 1-OHPG in study subjects. Multiple regression analysis show that smoking is the only significant predictor for lon-transformed 1-OHPG (overall model R2=0.1). These results suggest that workers using coal tar paints were exposed to significant amount of PAHs and individual difference in xenobiotic metabolism might affect the levels of internal dose of PAHs.

• 한국임상약학회지
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• 제8권1호
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• pp.29-34
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• 1998

Murine CD38 is a 42 kDa type II glycoprotein expressed on cell surface of both B and T lymphocytes. CD38 is a multifunctional enzyme that catalyzes the formation and hydrolysis of cyclic adenosine diphosphoribose (cADPR): ADP-ribosyl cyclase activity of CD38 catalyzes the formation of cADPR from NAD and cADPR hydrolase activity of CD38 catalyzes the hydrolysis of cADPR to ADP-ribose (ADPR). And also, CD38 has the catalytic activity of NAD glycohydrolase (NADase) which catalyzes the hydrolysis of catalyzes the formation and hydrolysis of cyclic adenosine diphosphoribose (cADPR): ADP-ribosyl cyclase activity of CD38 catalyzes the formation of cADPR from NAD to ADPR. In this study, we attempted to purify CD38 from mouse lymphocytes by using the immobilized anti-CD38 monoclonal antibody. The single step immuno-affinity column chromatography resulted in homogeneous purification, showing a single protein of 42 kDa on a SDS polyacrylamide gel. We have investigated the effects of various inhibitors on the enzyme activities of the purified CD38. Cibacron blue (0.5 mM) inhibited all three enzyme activities of CD38, NADase, ADP-ribosyl cyclase and cADPR hydrolase activities. ADPR (2 mM) showed inhibitory effect on both cADPR hydrolase activity and NADase, but not on ADP-ribosyl cyclase activity. However, ATP (2 mM) inhibited only cADPR hydrolase activity. $Zn^{2+}$ (1 mM) showed similar inhibitory effect as that of ADPR, but activated cyclase activity These results suggest that CD38 has three different catalytic activity domains which might be differentially regulated by their specific inhibitors.

• 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.

• The Plant Pathology Journal
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• 제27권1호
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• pp.37-43
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• 2011

Rice stripe virus (RSV) is one of serious epidemic pathogens for rice species grown in many Asian countries. Therefore, it is necessary to produce a diagnostic detection kit applicable in fields for RSV detection. In this study, RSV proteins that were derived from recombinant proteins and synthetic polypeptides as antigens were generated and were raised in rabbits for antiserum production. Among seven proteins in RSV, genes that code for NCP and NS3 proteins were cloned and subcloned into vector carrying His-tag protein and were expressed in E. coli. Of two recombinant proteins, only anti-NCP displayed stable hybridization signals in western blot analysis. Alternately, synthetic RSV polypeptides for CP, NCP, NS3 and NSvc4 we also generated and only antibodies against CP and NCP were very effective to detect RSV in both RSV infected rice and weed plants. However, antibodies against NS3 and NSvc4 showed weak specific bands as well as strong non-specific background due to the difference of viral proteins produced in the infected leaves. In summary, the antibodies generated against RSV proteins produced in this study will be useful for various assays such as for RSV diagnostic detection, immunoprecipitation, protein purification, and western blot analysis.

• 한국미생물·생명공학회지
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• 제49권3호
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• pp.305-315
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• 2021

The cotton leafworm, Spodoptera littoralis, is a major pest in Egypt and many countries worldwide, and causes heavy economic losses. As a result, management measures to control the spread of the worm are required. S. littoralis nucleopolyhedrovirus (SpliNPV) is one of the most promising bioagents for the efficient control of insect pests. In this study, a chitinase gene (chitA) of a 1.8 kb DNA fragment was cloned and fully characterized from SpliNPV-EG1, an Egyptian isolate. A sequence of 601 amino acids was deduced when the gene was completely sequenced with a predicted molecular mass of 67 kDa for the preprotein. Transcriptional analyses using reverse transcription polymerase chain reaction (RT-PCR) revealed that chitA transcripts were detected first at 12 h post infection (hpi) and remained detectable until 168 hpi, suggesting their transcriptional regulation from a putative late promoter motif. In addition, quantitative analysis using quantitative RT-PCR showed a steady increase of 7.86-fold at 12 hpi in chitA transcription levels, which increased up to 71.4-fold at 120 hpi. An approximately 50 kDa protein fragment with chitinolytic activity was purified from ChitA-induced bacterial culture and detected by western blotting with an anti-recombinant SpliNPV chitinase antibody. Moreover, purification of the expressed ChitA recombinant protein showed in vitro growth inhibition of two different fungi species, Fusarium solani and F. oxysporum, confirming that the enzyme assembly and activity was correct. The results supported the potential role and application of the SpliNPV-ChitA protein as a synergistic agent in agricultural fungal and pest control programs.

• 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.

• Clinical and Experimental Vaccine Research
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• 제11권1호
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• pp.12-29
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• 2022

Purpose: In the present study, whole diphtheria toxin (dt) and fragment B (dtb) genes from Corynebacterium diphtheriae Park William were cloned into Escherichia coli, the purified expressed proteins were evaluated for ultimately using as a candidate vaccine. Materials and Methods: The dt and dtb genes were isolated from bacterial strain ATCC (American Type Culture Collection) no. 13812. Plasmid pET29a+ was extracted by DNA-spin TM plasmid purification kit where genes were inserted using BamHI and HindIII-HF. Cloned pET29a+dt and pET29a+dtb plasmids were transformed into E. coli BL21(DE3)PlysS as expression host. The identity of the sequences was validated by blasting the sequence (BLASTn) against all the reported nucleotide sequences in the NCBI (National Center for Biotechnology Information) GenBank. Production of proteins in high yield by different types and parameters of fermentation to determine optimal conditions. Lastly, the purified concentrated rdtx and rdtb were injected to BALB/c mice and antibody titers were detected. Results: The genetic transformation of E. coli DH5α and E. coli BL21 with the pET-29a(+) carrying the dt and dtb genes was confirmed by colony polymerase chain reaction assay and were positive to grow on Luria-Bertani/kanamycin medium. The open reading frame of dt and dtb sequences consisted of 1,600 bp and 1,000 bp, were found to be 100% identical to dt and dtb sequence of C. diphtheriae (accession number KX702999.1 and KX702993.1) respectively. The optimal condition for high cell density is fed-batch fermentation production to express the rdtx and rdtb at 280 and 240 Lf/mL, dissolved oxygen was about 24% and 22% and the dry cell weight of bacteria was 2.41 g/L and 2.18 g/L, respectively. Conclusion: This study concluded with success in preparing genetically modified two strains for the production of a diphtheria vaccine, and to reach ideal production conditions to achieve the highest productivity.