• Title/Summary/Keyword: i-vector

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Expression and Antibacterial Activity of Recombinant Human Lactoferrin in Methylotrophic Yeast, Pichia pastoris (Methylotrophic Yeast, Pichia pastoris에서 사람 락토페린의 발현 및 항균성 연구)

  • Lee Sang O;Im Eun Mi;Nam Eun Joo;Lee Hyune Hwan
    • Korean Journal of Microbiology
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    • v.40 no.4
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    • pp.348-354
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    • 2004
  • The expression and antibacterial. activity of recombinant human lactoferrin (hLf) was studied from meth­ylotrophic yeast Pichia pastoris. The gene encoding hLf, isolated from human breast cDNA library, was subcloned into the expression vector, pPIC3.5K under the control of AOX1 promoter. The gene was integrated into the host chromosome and was identified by Southern blotting. The expression of the integrated gene was investigated by RT-PCR, Northern blotting, SDS-PAGE and Western blotting. Discrete band corresponding to hLf was detected from the SDS-PAGE, which was confirmed by Western blotting. The expression was also confirmed by RT-PCR and Northern blotting. The antibacterial activity of the recombinant hLf (rhLf) was investigated using Staphy­lococcus aureus ATCC 6538P and Micrococcus flavus ATCC 10240 as test organisms. The rhLf showed strong antibacterial activities against the bacteria. Furthermore, many Gram-negative animal pathogens such as E.coli ATCC8739, 25922, and Salmonella typhimurium 114 and 115, Pseudomonas fluorescens ID 963 I, P. aeruginosa KCCM 11802, and Gram-positive bacteria Bacillus mesentericus were also inhibited in their growth by the rhLf.

Gene Cloning, Nucleotide Sequence and Efficent Expression of Peptidyl proryl cis-trans Isomerase from Bacillus stearothermophilus (Bacillus stearothermophilus의 Peptidyl Prolyl cis-trans Isomerase 유전자 분리 염기배열 및 발현)

  • 김동주
    • The Korean Journal of Food And Nutrition
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    • v.9 no.4
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    • pp.452-458
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    • 1996
  • A PPIase gene of Bacillus stearothermophilus was screened from a genomic library by plaque hybridization using the A-1 primer as a probe. A PPIase positive plaque contained a 3.0kb insert of the chromosomal DNA. A 3.0kb fragment was subcloned into pUC18, resulting pPI1-40. A DNA fragment encoding the N-terminal portion of the PPIase in pPi-40 was amplified by polymerase chain reaction(PCR) method using the A-1 and B-2 primers. The amplified fragment was cloned into the Sma I site of pUC18 and recombinant plasmid was designated as pSN-18. The nucleotide sequence of 167bp fragment was determined. The deduced amino acid sequence of PPIase was completely matched with the determined N-terminal amino acid sequence of PPIase B. stearothermophilus. The translated protein sequence of PPIase B. stearothermophilus was compared with sequence from periplasmic PPIase from Escherichina coil ; homogies of 16 and 58%, respectively, were found. The clond PPIase gene was over-expressed in E. coil cell using pUC19 as an expression vector. The enzyme was partially purified by heat treatment and colum chromatochraphy on DEAE-Sepharose CL-6B. The molecular weight of the enzyme was dermined to be about 18.0 kDal by SDS-PAGE.

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Acquisition of Thermotolerance in the Transgenic Plants with BcHSP17.6 cDNA (BcHSP17.6 cDNA의 도입에 의한 형질전환된 식물의 내열성 획득)

  • Ki Yong Kim;Min Sup Chung;Jin Ki Jo
    • Journal of The Korean Society of Grassland and Forage Science
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    • v.17 no.4
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    • pp.379-386
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    • 1997
  • Recombinant plasmid, pBKH4, containing NPT II and P35S-BcHSP17.6 was constructed by ligation of Bum H I -digested pBKSl-l and BcHSP 17.6 (thermotolerance gene) 6om pBLH4. The tobacco leaf disc was cocultivated with transformed Agmbacterium tumefaciens bearing pBKH4 for 24 hours and transformed shoots were selected on MS-n/B medium containing $100\;{\mu\textrm{g}}/ml$ of kanamycin. Heat-killing temperature of Nicotima tabacum was $50^{\circ}$ for >15min, and transformed tobacco plants with BcHSP17.6 cDNA exhibited thermotolerance at the heat-killing temperature. The transgenic plants were analyzed by Southern blot hybridization with the probe of ${\alpha}^{_32}P$ labelled BcHSP17.6 cDNA. Transcription and expression level of BcHSP17.6 cDNA were also continued by Northern blot analysis and Ouchterlony double immunodiffusion assay. In this study, we suggest that the BcHSP17.6 cDNA introduced to tobacco plant is related to thenuoto-lerance and 17.6-kD LMW HSP acts as a protector from heat damage in plants.

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MAXILLARY ADVANCEMENT USING RIGID EXTERNAL DISTRACTION(RED) IN CLEFT LIP AND PALATE PATIENT : CASE REPORT (견고 구외 골신장술을 이용한 구순구개열 환아의 치험례)

  • Yu, Nan-Young;Kim, Sung-Min;Lee, Ju-Hyun;Seo, Hyun-Woo;Park, Ho-Won
    • Journal of the korean academy of Pediatric Dentistry
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    • v.32 no.4
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    • pp.709-716
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    • 2005
  • Patients with cleft tip and palate present severe maxillary hypoplasia due to scar of lip and palate, often accompanied by compromised mastication, speech abnormalities. Sometimes maxillary hypoplasia persist even though active orthodontic treatment was done. In theses cases, patients born with cleft lip and palate will be potential candidates for maxillary advancement with bone grafting after growth to correct the functional deformities and improve aesthetic facial proportions. But, maxillary advancement using standard surgical approaches has several limitations : increased relapse tendency after maxillary advancement, necessity of additional bone graft and mandibular setback surgery. Distraction osteogenesis is current treatment modality to overcome these limitations, thus has become popular for treatment of maxillary hypoplasia associated cleft lip and palate, craniosyntosis. Especially, rigid external distraction, contrary to internal device, has advantages : better vector control of osteotomized segment, effective traction of the bony segments, the ease of the application and removal the distraction device. This study showed that relatively successful result could be generated by using rigid external distraction osteogenesis(RED) in the case of cleft lip and palate with severe maxillary hypoplasia, 6 years 7 months old.

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Transformation of Plant Cells by Gene Transfer : Construction of a Chimeric Gene Containing Deleted Maize Alcohol Dehydrogenase Intron and ${\beta}-Glucuronidase$ Gene and Its Expression in Potato (유전자 도입에 의한 식물세포의 형질전환 : 옥수수 알코올 탈수소효소 유전자의 절단된 인트론 및 ${\beta}-Glucuronidase$ 유전자를 함유하는 키메라 유전자의 제조와 감자에서의 발현)

  • 이광웅
    • Journal of Plant Biology
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    • v.35 no.3
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    • pp.237-245
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    • 1992
  • To understand the properties of the cauliflower mosaic virus (CaMV) 35S promoter and the effect of the deleted maize alcohol dehydrogenase I-S (Adhl-S) intron 1 on the expression of the CaMV $35S{\beta}-glucuronidase$ (GUS) gene in potato (Solanum tuberosum L. cv. Superior), we constructed a chimeric gene and transferred it into potato with Agrobacterium tumefaciens mediated method. The pLS201, a gene transfer vector of 17.7 kilobase pairs, was composed of the CaMV 35S promoter, the 249 base pairs of deleted maize Adhl-S intron 1, the GUS reporter gene, and the kanamycin resistance gene as a selectable marker for transformation. The GUS activity was examined by histochemical and spectrophotometric assay in transformed potato plants. The GUS activity was found primarily around the vascular tissue cells in stem and root. In the spectorophotometric assay, the level of GUS activity of transgenic potato transformed with CaMV 35S/249 bp of intron 1 fragment-GUS (pLS201) was compared with that of potato transformed with CaMV 35S-GUS (pBI121). The quantitative spectrophotometric assay showed that the level of GUS activity in potato transformed with pLS201 was higher in leaf, stem and root by 30-, 34- and 42-fold, respectively than those in potato transformed with pBI121. This results indicate that the inclusion of the deleted maize Adhl-S intron 1 resulted in increament of the GUS gene expression in transgenic potato.potato.

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Identification and Expression of the cym, cmt, and tod Catabolic Genes from Pseudomonas putida KL47: Expression of the Regulatory todST Genes as a Factor for Catabolic Adaptation

  • Lee Kyoung;Ryu Eun-Kyeong;Choi Kyung-Soon;Cho Min-Chul;Jeong Jae-Jun;Choi Eun-Na;Lee Soo-O;Yoon Do-Young;Hwang In-Gyu;Kim Chi-Kyung
    • Journal of Microbiology
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    • v.44 no.2
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    • pp.192-199
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    • 2006
  • Pseudomonas putida KL47 is a natural isolate that assimilates benzene, 1-alkylbenzene $(C_1-C_4)$, biphenyl, p-cumate, and p-cymene. The genetic background of strain KL47 underlying the broad range of growth substrates was examined. It was found that the cym and cmt operons are constitutively expressed due to a lack of the cymR gene, and the tod operon is still inducible by toluene and biphenyl. The entire array of gene clusters responsible for the catabolism of toluene and p-cymene/p-cumate has been cloned in a cosmid vector, pLAFR3, and were named pEK6 and pEK27, respectively. The two inserts overlap one another and the nucleotide sequence (42,505 bp) comprising the cym, cmt, and tod operons and its flanking genes in KL47 are almost identical (>99 %) to those of P. putida F1. In the cloned DNA fragment, two genes with unknown functions, labeled cymZ and cmtR, were newly identified and show high sequence homology to dienelactone hydrolase and CymR proteins, respectively. The cmtR gene was identified in the place of the cmtI gene of previous annotation. Western blot analysis showed that, in strains F1 and KL47, the todT gene is not expressed during growth on Luria Bertani medium. In minimal basal salt medium, expression of the todT gene is inducible by toluene, but not by biphenyl in strain F1; however, it is constantly expressed in strain KL47, indicating that high levels of expression of the todST genes with one amino acid substitution in TodS might provide strain KL47 with a means of adaptation of the tod catabolic operon to various aromatic hydrocarbons.

Expression of mue Gene on Plasmid pKM101 and pSL4 (플라스미드 pKM101 과 pSL4 의 muc 유전자의 발현에 관한 연구)

  • 전홍기;황유경;이상률;백형석
    • Korean Journal of Microbiology
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    • v.30 no.5
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    • pp.371-376
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    • 1992
  • Plasmid pSL4 of plasmid pKM 101 mutant have high protection effects and mutagenecity for UV and methyl methanesulfonate, The mucA gene and a pan of mucE gene of pKM 101 and pSL4 were sucloned onto lacZ' fusion vector pMC874 and the hybrid plasmids pBH31 and pBH30 were selected. These plsmids were intrduced into $recA^{+}lexA^{-}$, $recA^{-}와lexA^{+}$ strains and determined the activity of $\beta$-galactosidase for UV. In $recA^{+}lexA^{+}$ strain.$\beta$-galactosidase activity of pBH30 included mue region of pSL4 was higher thall pBH31 inclued muc region of pKM 10 I and the tf-galactosidase of two plasmids was not induced in reeA and leeA mutants with or without UV illumination. Without UV illumination. the .$\beta$-galactosidasc of pBH30 was expressed a little higher level than that of pBH3L We suggest that the functional difference of pKM 10l and pSL4 are due to the variety of mue regulatory region. Also. a plasmid pBH 100 earring umuC' -lacZ' gene fusion was constructed in vitro to study the regulation of the umu operon. It was shown that the umu operon is induced by UV and is regulated by the reeA and lexA genes.

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Soluble Expression of a Human MnSOD and Hirudin Fusion Protein in Escherichia coli, and Its Effects on Metastasis and Invasion of 95-D Cells

  • Yi, Shanze;Niu, Dewei;Bai, Fang;Li, Shuaiguang;Huang, Luyuan;He, Wenyan;Prasad, Anand;Czachor, Alexander;Tan, Lee Charles;Kolliputi, Narasaiah;Wang, Feng
    • Journal of Microbiology and Biotechnology
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    • v.26 no.11
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    • pp.1881-1890
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    • 2016
  • Manganese superoxide dismutase (MnSOD) is a vital enzyme that protects cells from free radicals through eliminating superoxide radicals ($O^{2-}$). Hirudin, a kind of small active peptide molecule, is one of the strongest anticoagulants that can effectively cure thrombus diseases. In this study, we fused Hirudin to the C terminus of human MnSOD with the GGGGS linker to generate a novel dual-feature fusion protein, denoted as hMnSOD-Hirudin. The hMnSOD-Hirudin gene fragment was cloned into the pET15b (SmaI, CIAP) vector, forming a recombinant pET15b-hMnSOD-Hirudin plasmid, and then was transferred into Escherichia coli strain Rosetta-gami for expression. SDS-PAGE was used to detect the fusion protein, which was expected to be about 30 kDa upon IPTG induction. Furthermore, the hMnSOD-Hirudin protein was heavily detected as a soluble form in the supernatant. The purification rate observed after Ni NTA affinity chromatography was above 95%. The hMnSOD-Hirudin protein yield reached 67.25 mg per liter of bacterial culture. The identity of the purified protein was confirmed by western blotting. The hMnSOD-Hirudin protein activity assay evinced that the antioxidation activity of the hMnSOD-Hirudin protein obtained was $2,444.0{\pm}96.0U/mg$, and the anticoagulant activity of the hMnSOD-Hirudin protein was $599.0{\pm}35.0ATU/mg$. In addition, in vitro bioactivity assay showed that the hMnSOD-Hirudin protein had no or little cytotoxicity in H9c2, HK-2, and H9 (human $CD_4{^+}$, T cell) cell lines. Transwell migration assay and invasion assay showed that the hMnSOD-Hirudin protein could suppress human lung cancer 95-D cell metastasis and invasion in vitro.

A Feature Re-weighting Approach for the Non-Metric Feature Space (가변적인 길이의 특성 정보를 지원하는 특성 가중치 조정 기법)

  • Lee Robert-Samuel;Kim Sang-Hee;Park Ho-Hyun;Lee Seok-Lyong;Chung Chin-Wan
    • Journal of KIISE:Databases
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    • v.33 no.4
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    • pp.372-383
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    • 2006
  • Among the approaches to image database management, content-based image retrieval (CBIR) is viewed as having the best support for effective searching and browsing of large digital image libraries. Typical CBIR systems allow a user to provide a query image, from which low-level features are extracted and used to find 'similar' images in a database. However, there exists the semantic gap between human visual perception and low-level representations. An effective methodology for overcoming this semantic gap involves relevance feedback to perform feature re-weighting. Current approaches to feature re-weighting require the number of components for a feature representation to be the same for every image in consideration. Following this assumption, they map each component to an axis in the n-dimensional space, which we call the metric space; likewise the feature representation is stored in a fixed-length vector. However, with the emergence of features that do not have a fixed number of components in their representation, existing feature re-weighting approaches are invalidated. In this paper we propose a feature re-weighting technique that supports features regardless of whether or not they can be mapped into a metric space. Our approach analyses the feature distances calculated between the query image and the images in the database. Two-sided confidence intervals are used with the distances to obtain the information for feature re-weighting. There is no restriction on how the distances are calculated for each feature. This provides freedom for how feature representations are structured, i.e. there is no requirement for features to be represented in fixed-length vectors or metric space. Our experimental results show the effectiveness of our approach and in a comparison with other work, we can see how it outperforms previous work.

A Study on the Impact of International Prices on Domestic Prices and Export Prices in Korea (국제물가 변동 충격이 국내물가와 수출물가에 미치는 영향 분석)

  • Kim, Jung Ryol
    • International Commerce and Information Review
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    • v.15 no.4
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    • pp.195-216
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    • 2013
  • In this paper, I investigate how international prices affect domestic prices and export prices in Korea by using vector error correction model(VECM) and estimate its impact on international trade. According to the empirical results, international prices, such as world raw material prices and oil prices, make stronger effects on domestic prices, in order of import, export, producer, and consumer prices. And recent years the effect of international raw material prices on domestic prices becomes larger. It implies importers, exporters and producers are more affected by international prices than consumers are. Therefore, the international trade, import and export, is affected by changes in international prices. Firms, especially importing and exporting companies, should do much efforts on risk managing about raw material prices variation, diversification of raw material suppliers, and oversea resources development. The government is needed to support on firms those efforts while doing its economic policies to cope with economic conditions and the price policy.

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