• The Plant Pathology Journal
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• 제26권1호
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• pp.25-31
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• 2010

Papaya ringspot virus (PRSV) causes the most widespread and devastating disease in papaya. Isolates of PRSV originating from different geographical regions in south India were collected and maintained on natural host papaya. The entire coat protein (CP) gene of Papaya ringspot virus-P biotype (PRSV-P) was amplified by RTPCR. The amplicon was inserted into pGEM-T vector, sequenced and sub cloned into a bacterial expression vector pRSET-A using a directional cloning strategy. The PRSV coat protein was over-expressed as a fusion protein in Escherichia coli. SDS-PAGE gel revealed that CP expressed as a ~40 kDa protein. The recombinant coat protein (rCP) fused with 6x His-tag was purified from E.coli using Ni-NTA resin. The antigenicity of the fusion protein was determined by western blot analysis using antibodies raised against purified PRSV. The purified rCP was used as an antigen to produce high titer PRSV specific polyclonal antiserum. The resulting antiserum was used to develop an immunocapture reverse transcription-polymerase chain reaction (IC-RT-PCR) assay and compared its sensitivity levels with ELISA based assays for detection of PRSV isolates. IC-RT-PCR was shown to be the most sensitive test followed by dot-blot immunobinding assay (DBIA) and plate trapped ELISA.

• BMB Reports
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• 제39권1호
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• pp.16-21
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• 2006

The coat protein (CP) of Papaya ringspot virus (PRSV) was analyzed for presentation of the antigenic peptide of animal virus, Canine parvovirus (CPV), in Escherichia coli (E. coli). The 45 nucleotides fragment coding for the 15-aa peptide epitope of the CPV-VP2 protein was either inserted into the PRSV-cp gene at the 5', 3' ends, both 5' and 3' ends or substituted into the 3' end of the PRSV cp gene. Each of the chimeric PRSV cp genes was cloned into the pRSET B vector under the control of the T7 promoter and transformed into E. coli. The recombinant coat proteins expressed from different chimeric PRSV-cp genes were purified and intraperitoneally injected into mice. All of the recombinant coat proteins showed strong immunogenicity and stimulate mice immune response. The recombinant coat proteins containing the CPV epitope insertion at the C terminus and at both N and C termini elicited ten times higher specific antisera in immunized mice compared with the other two recombinant coat proteins which contain the CPV epitope insertion at the N terminus and substitution at the C terminus.

• Journal of Microbiology and Biotechnology
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• 제25권6호
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• pp.887-892
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• 2015

Uricase is an important microbial enzyme that can be used in the clinical treatment of gout, hyperuricemia, and tumor lysis syndrome. A total of 127 clinical isolates of Pseudomonas aeruginosa were tested for uricase production. A Pseudomonas strain named Ps43 showed the highest level of native uricase enzyme expression. The open reading frame of the uricase enzyme was amplified from Ps43 and cloned into the expression vector pRSET-B. Uricase was expressed using E. coli BL21 (DE3). The ORF was sequenced and assigned GenBank Accession No. KJ718888. The nucleotide sequence analysis was identical to the coding sequence of uricase gene puuDof P. aeruginosa PAO1. We report the successful expression of P. aeruginosa uricase in Escherichia coli. E. coli showed an induced protein with a molecular mass of about 58 kDa that was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. We also established efficient protein purification using the Ni-Sepharose column with activity of the purified enzyme of 2.16 IU and a 2-fold increase in the specific activity of the pure enzyme compared with the crude enzyme.

• 한국화재소방학회논문지
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• 제15권1호
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• pp.93-99
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• 2001

본 연구는 현재 급속히 확산되고 있는 대형 할인점의 지하 매장에 대한 피난성능 개선방안을 제시하기 위한 것으로 주요 내용은 다음과 같다. 할인매장의 건축적 공간 특성 및 피난 상 문제점 분석 지하매장의 거주밀도 분포조사 피난용량 적정성 검토를 위한 피난 시뮬레이션 연구결과, 매장의 실질적인 수용인원을 기준으로 적정피난용량을 결정해야 하며, 피난동선상에 있는임시 판매대, 쇼핑 카트, 그리고 피난층의 계산대가 피난에 중요한 장애요소로서 이에 대한 개선과 제도적 정비가 필요함을 알 수 있었다. 피난시간을 기준 한 성능기준 피난설계 분석결과, 사례분석 대상 지하매장은 적정피난용량을 확보하지 못하고 있음을 보여주었다.

• Journal of Microbiology and Biotechnology
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• 제14권2호
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• pp.344-349
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• 2004

A gene encoding inulin-degrading enzyme of Bacillus sp. snu-7 with ORF of 1536 nucleotides was cloned. And it was overexpressed as His-tagged protein in E. coli BL21(DE3) pLysS using pRSET B vector containing mature enzyme sequence. Maximum enzyme production was achieved by IPTG (0.1 mM) induction at $OD_{600}$ 1.2 and $30^{\circ}C$ followed by 6 h incubation. The expressed protein purified through immobilized metal affinity chromatography showed molecular mass of 60 kDa on SDS-PAGE. Results of thin-layer chromatography using inulin as a substrate showed the enzyme to be an exotype inulinase capable of producing only monomeric fructose as a product. $K_m$ and $k_{cat}$, for the hydrolyses of inulin and sucrose were $2.28\pm0.08$ mM and 358.05$\pm$20.38 $min^{-l}$, and 22.02$\pm$0.41 mM and 4619.11$\pm$215.12 $$min^{-1}, respectively. Optimal activity of the exoinulinase occurred at pH 7.0 and $50^{\circ}C$.

• 대한수의학회지
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• 제45권2호
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• pp.185-189
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• 2005

Actinobacillus pleuropneumoniae is the causative agent of a porcine contagious pleuropneumonia. Among several virulence factors including exotoxin (Apx toxins), LPS, transferrin-binding proteins, OMPs, and some proteases, Apx toxins have been major targets for the protection study. In this study, cloning and expression of A. pleuropneumoniae Apx I and Apx II toxin, which are produced by all highly virulent strains, were performed by Escherichia coli expression system. Genes coding Apx I and II toxin were amplified from the A. pleuropneumoniae serotype 5 genomic DNA using polymerase chain reaction and cloned to a prokaryotic expression vector, pRSET. Expression of the Apx I and Apx II coding sequences in E. coli resulted in the formation of insoluble inclusion bodies purified according to a denaturing purification protocol, which employs the use of guanidium. Recombinant proteins were purified using $Ni^{2+}$-charged resin affinity purification. This expression and purification system made it possible to produce Apx I and Apx II in large amounts for further immunologic studies.

• 한국화재소방학회논문지
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• 제30권5호
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• pp.18-25
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• 2016

국내 성능위주설계 방법 및 기준에서는 다양한 입력 데이터들에 대한 입력 규정이 따로 마련되어있지 않으며, 설계자별로 인용하는 근거 데이터가 상이하여 피난허용시간과 피난완료시간에 많은 차이를 보이고 있다. 이는 성능위주 화재와 피난 시뮬레이션에 대한 신뢰도 문제와도 직결되고 있다. 시뮬레이션에 입력되는 다양한 데이터들을 표준화함으로서 설계자의 경험이나 기술능력에 무관하게 동일한 위험도의 건물에서는 동일한 결과가 도출되어야 한다. 또한 그 위험도 합당한 소방 방재설비가 설치되고 유사한 성능위주 대상 건물에서는 개연성 있는 초기투자비용이 소요됨으로서 효율적이고 효과적인 안전 확보가 이루어져야 할 것으로 판단된다.

• 대한안전경영과학회지
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• 제23권3호
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• pp.97-102
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• 2021

This study developed a scenario to understand the reaction rate and operational time according to RTI value of rate of rise detector in each type in case of fire mattress. In the results of analyzing the reaction rate and operational time of detector in each scenario, in case when installing a single detector, the elevated temperature per minute was raised to 8℃/min ~ 9℃/min. In case when installing two detectors, it was raised to 9℃/min ~ 10℃/min. In case when installing three detectors, it was raised to 10℃/min. The horizontal distance between detector and mattress was 1.8m~2.5m. Whenever the number of detectors was increased, the horizontal distance was decreased. The operational time of detector was within maximum 540 seconds and minimum 420 seconds. As the research tasks in the future, there should be the researches on the effects of reaction rate of detector on the evacuation in case of fire through the result value of RSET by setting up the latency until the detector operates, and the researches on the safety by understanding if the operational time of detector is suitable for the evaluation standard of performance-centered design.

• 대한안전경영과학회지
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• 제25권4호
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• pp.67-72
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• 2023

Human and material damage can be reduced if the risk is evaluated by engineering analysis of fire combustion products, smoke concentration, and smoke movement speed in the event of a fire in apartment houses and officetels. In this study, a lot of research on related safety evaluation in the basement needs to be studied and reflected in design, so experimental research was conducted to analyze the flow of smoke through computer simulation and provide analysis data through evacuation safety evaluation. First of all, the five-story underground parking lot subject to simulation has a large floor area, which is advantageous for improving evacuation safety performance, but it uses temperature detectors to increase detection time and fire spread speed. Second, it was analyzed that the evacuation time at all evacuation ports did not exceed the evacuation time, and as the time from the start of evacuation to the evacuation time was 216.9% compared to the travel time, it was evaluated that the safety performance of the evacuation was secured. Third, the above simulation results are a comprehensive safety evaluation based on the non-operation of fire extinguishing facilities in the fire room to increase safety, which means that smoother evacuation safety performance can be secured by linking fire extinguishing facilities.

• Food Science and Biotechnology
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• 제17권5호
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• pp.990-995
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• 2008

A thermostable $\alpha$-L-arabinofuranosidases ($\alpha$-L-AFase) is an industrially important enzyme for recovery of L-arabinose from hemicellulose. The recombinant $\alpha$-L-AFase from Thermotoga maritima was expressed in Escherichia coli by using a constitutive pHCE or an inducible pRSET vectors. In batch fermentation, the constitutive expression system resulted in slightly faster growth rate (0.78 vs. 0.74/hr) but lower enzyme activity (2,553 vs. 3,723 units/L) than those of the induction system. When fed-batch fermentation was performed, biomass and enzyme activity reached the highest levels of 36 g/L and 9,152 units/L, respectively. The fed batch cultures performed superior results than batch culture in terms of biomass yield (4.62-5.42 folds) and enzyme synthesis (3.39-4.00 folds). In addition, the fed-batch induction strategy at high cell density resulted in the best productivity in cell growth as well as enzyme activity rather than the induction method at low cell density or the constitutive expression.