• Journal of Microbiology and Biotechnology
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• 제21권5호
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• pp.529-535
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• 2011

Single-chain variable fragment (scFv) is a fusion protein of the variable regions of the heavy (VH) and light (VL) chains of immunoglobulin, connected with a short linker peptide of 10 to about 20 amino acids. In this study, the scFv of a monoclonal antibody against the third domain of human CD4 was cloned from OKT4 hybridoma cells using the phage display technique and produced in E. coli. The expression, production, and purification of anti-CD4 scFv were tested using SDS-PAGE and Western blot, and the specificity of anti-CD4 scFv was examined using ELISA. A 31 kDa recombinant anti-CD4 scFv was expressed and produced in bacteria, which was confirmed by SDS-PAGE and Western blot assays. Sequence analysis proved the ScFv structure of the construct. It was able to bind to CD4 in quality ELISA assay. The canonical structure of anti-CD4 scFv antibody was obtained using the SWISS_MODEL bioinformatics tool for comparing with the scFv general structure. To the best of our knowledge, this is the first report for generating scFv against human CD4 antigen. Engineered anti-CD4 scFv could be used in immunological studies, including fluorochrome conjugation, bispecific antibody production, bifunctional protein synthesis, and other genetic engineering manipulations. Since the binding site of our product is domain 3 (D3) of the CD4 molecule and different from the CD4 immunological main domain, including D1 and D2, further studies are needed to evaluate the anti-CD4 scFv potential for diagnostic and therapeutic applications.

• 한국실천공학교육학회논문지
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• 제2권1호
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• pp.35-41
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• 2010

효소촉매반응은 통상적으로 반응속도가 매우 빠르고 동시에 높은 선택성을 보여 주는 특정이 있다. 단순 효소촉매 반응속도식을 보통 Michaelis-Menten식이라고 부른다. 효소의 활성을 간섭하는 화학물질을 저해제라고 하는데, 효소활성의 저해에는 가역적저해와 비가역적저해의 두 가지 형태로 나타난다. 만일 저해제가 수소결합과 같은 약한 결합으로 효소에 붙게 되면 이런 경우 효소저해는 가역적인 저해로 나타난다. 많은 효소 반응들은 그 반응으로부터 생성되는 생성물 자체에 의해서도 가역적 저해를 받게 된다. 생성물의 생성속도와 더불어 기질의 감소속도식은 비선형 미분방정식으로 나타 낼 수가 있다. 본 연구는 공학 분야의 학부교육을 목적으로 단순 효소반응속도식과 보다 더 복잡한 저해 효소반응속도식에 대한 수치해석의 결과를 보고 하고자 한다.

• BMB Reports
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• 제38권5호
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• pp.584-590
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• 2005

Glucose 6-phosphate dehydrogenase (EC 1.1.1.49) was purified from Aspergillus aculeatus, a filamentous fungus previously isolated from infected tongue of a patient. The enzyme, apparently homogeneous, had a specific activity of $220\;units\;mg^{-1}$/, a molecular weight of $105,000{\pm}5,000$ Dal by gel filtration and subunit size of $52,000{\pm}1,100$ Dal by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The substrate specificity was extremely strict, with glucose 6-phosphate (G6P) being oxidized by nicotinamide adenine dinucleotide phosphate (NADP) only. At assay pH of 7.5, the enzyme had $K_m$ values of $6\;{\mu}m$ and $75\;{\mu}m$ for NADP and G6P respectively. The $k_{cat}$ was $83\;s^{-1}$. Steady-state kinetics at pH 7.5 produced converging linear Lineweaver-Burk plots as expected for ternary-complex mechanism. The patterns of product and dead-end inhibition suggested that the enzyme can bind NADP and G6P separately to form a binary complex, indicating a random-order mechanism. The enzyme was irreversibly inactivated by heat in a linear fashion, with G6P providing a degree of protection. Phosphoenolpyruvate (PEP), adenosinetriphosphate (ATP), and fructose 6-phosphate (F6P), in decreasing order, are effective inhibitors. Zinc and Cobalt ions were effective inhibitors although cobalt ion was more potent; the two divalent metals were competitive inhibitors with respect to G6P, with $K_i$ values of $6.6\;{\mu}m$ and $4.7\;{\mu}m$ respectively. It is proposed that inhibition by divalent metal ions, at low NADPH /NADP ratio, is another means of controlling pentosephosphate pathway.

• Asian Pacific Journal of Cancer Prevention
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• 제16권17호
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• pp.7935-7941
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• 2015

The aim of this study was to implement massive parallel sequencing (MPS) technology in clinical genetics testing. We developed and tested an amplicon-based method for resequencing the BRCA1 and BRCA2 genes on an Illumina MiSeq to identify disease-causing mutations in patients with hereditary breast or ovarian cancer (HBOC). The coding regions of BRCA1 and BRCA2 were resequenced in 96 HBOC patient DNA samples obtained from different sample types: peripheral blood leukocytes, whole blood drops dried on paper, and buccal wash epithelia. A total of 16 random DNA samples were characterized using standard Sanger sequencing and applied to optimize the variant calling process and evaluate the accuracy of the MPS-method. The best bioinformatics workflow included the filtration of variants using GATK with the following cut-offs: variant frequency >14%, coverage ($>25{\times}$) and presence in both the forward and reverse reads. The MPS method had 100% sensitivity and 94.4% specificity. Similar accuracy levels were achieved for DNA obtained from the different sample types. The workflow presented herein requires low amounts of DNA samples (170 ng) and is cost-effective due to the elimination of DNA and PCR product normalization steps.

• Asian Pacific Journal of Cancer Prevention
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• 제16권15호
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• pp.6279-6284
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• 2015

Opisthorchis viverrini (OV)-induced cholangiocarcinoma (CCA) is an important cancer in the Great Mekong region, particularly in Thailand. Limitations of treatment options and the lack of an effective diagnostic tool for early detection of CCA are major concerns for the control of this type of cancer. The aim of the study was to investigate anti-CCA activity of the ethanolic extract of Atractylodes lancea (Thunb.) DC., and the applicability of positron emission tomography-computed tomography (PET-CT) as a tool for detection and monitoring the progression of CCA in Opisthorchis viverrini (OV)/dimethylnitrosamine (DMN)-induced CCA hamsters. Male Syrian hamsters were used for toxicity tests and anti-CCA activity evaluation. Development of CCA was induced by initial feeding of 50 metacercariae of OV, followed by drinking water containing 12.5 ppm of DMN in hamsters. The ethanolic extract of A. lancea (Thunb.) DC. was administered orally for 30 days. PET-CT was performed every 4 weeks after initiation of CCA using 18F-fluorodeoxyglucose ($^{18}F-FDG$). Results from the present study suggest that the ethanolic extract of A. lancea (Thunb.) DC. rhizome exhibited promising anti-CCA activity and safety profile in the OV/DMN-induced hamster model. To successfully apply PET-CT as a tool for early detection of tumor development and progression, modification of radiolabeling approach is required to improve its specificity for CCA cells.

• 한국축산식품학회지
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• 제30권4호
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• pp.590-596
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• 2010

Meat and meat products are a potential source of food-borne pathogens, including Staphylococcus aureus, Salmonella spp., Escherichia coli O157:H7, and Bacillus cereus. A sensitive and specific PCR assay for the detection of these pathogens in meat and meat products was developed in this study, as part of a broader effort to reduce the potential health hazards posed by these pathogens. Initially, PCR conditions were standardized with purified DNA. Under standard conditions, the detection level for PCR was as low as 10 pg of purified bacterial DNA. After overnight growth of bacteria in a broth medium, as few as $10^2$ CFU of bacteria were detected by PCR assay. The primers employed in the PCR assay were found to be highly specific for individual organisms, and evidenced no cross-reactivity with heterologous organisms. Additionally, the multiplex PCR assays also amplified some target genes from the four pathogens, and multiplex amplification was obtained from as little as 10 pg of DNA, thus illustrating the excellent specificity and high sensitivity of the assay. In conclusion, this PCR-based technique provides a sensitive and specific method for the detection of S. aureus, Salmonella spp., E. coli O157:H7, and B. cereus in meat and meat products.

• BMB Reports
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• 제38권4호
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• pp.414-419
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• 2005

The production of recombinant antibodies has been generally recognized as time-consuming and labor-intensive. The aim of our study is to construct mammalian expression vectors containing the cDNA encoding the human constant regions and murine variable regions to massively and cost-effectively produce full-length chimeric antibodies. Unique restriction sites flanking the Ig variable region were designed to allow for the replacement of variable regions generated by PCR. Western blot analysis of the chimeric antibodies revealed that the expressed products were of the predicted size, structure and specificity. The usefulness of the vectors was confirmed by construction of human-mouse chimeric antibody-HCAb which secretes murine antibody against the human colorectal cancer. Selected in medium containing gradually increasing methotrexate (MTX), clones with increased expression of the product gene can be efficiently generated. The secretion of recombinant chimeric antibody-HCAb yielded $30\;pg\;cell^{-1}\;day^{-1}$ at $10^{-6}\;M$ MTX. With this high-level expression from pools, the convenient and rapid production of over 100 milligram amounts per liter of recombinant antibodies may be achieved, which indicates the significant roles of pYR-GCEVH and pYR-GCEVL in the production of chimeric antibodies.

• 한국식품영양과학회지
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• 제41권6호
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• pp.870-874
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• 2012

본 연구에서는 다양한 고추 가공품의 capsaicinoids 함량을 분석하고 분석 방법을 검증하고자 하였다. 본 연구에서는 역상 HPLC를 이용하여 고춧가루 및 고추 가공품의 capsaicin과 dihydrocapsaicin의 함량을 분석하였다. 연구 결과 고춧가루 및 고추 가공품의 capsaicin 함량은 0.21~78.24 mg/100 g이었으며 dihydrocapsaicin 함량은 0.20~38.82 mg/100 g으로 나타났다. 고추장, 초고추장, 쌈장, 소스류에 비해 고춧가루의 capsaicin과 dihydrocapsaicin의 함량이 높게 나타났다. Capsaicinoids의 분석방법을 검증하고자 정밀도, 정확도, 특수성을 분석하였다. 그 결과 회수율은 90% 이상이 었으며 재현성과 반복성의 CV%는 5% 이하로 우수하게 나타났다. 전반적으로 분석 방법의 검증 parameter들은 우수하였다.

• 대한미생물학회지
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• 제14권1호
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• pp.11-15
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• 1979

Identification of group A $\beta$-phemolytic streptococci is very important to provide an appropriate preventive measure of possible rheumatic fever and acute glomerulonephritis. For such purpose bacitracin susceptibility of streptococci because of its simplity has been most widely used despite of its occasional faulty results. Recently, a coagglutination technique was advocated using streptococcal group specific antibodies adsorbed to protein A-containing staphylococci. This study was conducted to evaluate the coagglutination technique using reagents prepared by ourselves. The specificity, reproducibility and stability were ascertained and the following results were obtained. 1. The identification by coagglutination technique using our own reagent gave the same results compared with the Lancefield precipitation technique. The result also agreed with the Phadebact grouping. 2. There were no variation in group A and B identification due to lot difference. However, there were a few discrepant results in group C and G identification which was conducted in different days with different lots of our reagent. 3. The stability of our reagents was less satisfactory compared to the commercial product. An effort to improve the stability was considered necessary. 4. For coagglutination, it was found convenient to use supernatant of Todd-Hewitt broth incubated for 24 hours. Both parafin-ringed slide glass and RPR card gave comparable results and the former could be used when the latter is not available.

• 한국수산과학회지
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• 제30권6호
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• pp.984-991
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• 1997

New tumor suppressor gene, snm23, homologous to human nm23/NDP kinase (human nucleoside diphosphate kinase) gene whose product has a tumor metastasis inhibitory activity, was first cloned from Korean tiger shark (Scyliorhinus forazame) skin cDNA library constructed by using a $\lambda$ ZAP-II cDNA synthesis kit. About $1\times10^5$ plaques were screened and several positive plaques were isolated and confirmed by second screening. The phagemid containing a positive clone from the Uni-Zap XR vector was excised in vivo and the gene containing the tumor metastasis suppressor protein was named as snm23. Cloned gene, snm23, was sequenced with ABI-PRISM 310 Genetic Analyzer. The nucleotide and deduced amino acid sequences of snm23 have shown an open reading frame consisting of 450 base pairs that correspond to a protein of 150 amino acid residues, with a calculated molecular mass of 16.8 kDa. Sequence comparison of snm23 with human nm23/NDP kinase was performed by using Blast protein data base of National Center for Biotechnology Information. In order to determine tissue specificity, reverse transcription-polymerase chain reaction (RT-PCR) was used. Good expression level of snm23/NDP kinase was detected at the tissues from skin, cartilage, and liver of Korean tiger shark.