• Title, Summary, Keyword: apxA

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Mass expression of Apx I and Apx II of Actinobacillus pleuropneumoniae in Escherichia coli (대장균에서 흉막폐렴균 독소 Apx I과 Apx II의 대량발현)

  • Kim, Tae-Jung;Lee, Bong-Joo;Lee, Jae-Il
    • Korean Journal of Veterinary Research
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    • v.45 no.2
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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.

Expression of the Apx Toxins of Actinobacillus pleuropneumoniae in Saccharomyces cerevisiae and Its Induction of Immune Response in Mice

  • Park Seung-Moon;Choi Eun-Jin;Kwon Tae-Ho;Jang Yong-Suk;Yoo Han-Sang;Choi Woo Bong;Park Bong-Kyun;Kim Dae-Hyuk
    • Biotechnology and Bioprocess Engineering:BBE
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    • v.10 no.4
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    • pp.362-366
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    • 2005
  • Actinobacillus pleuropneumoniae is an important pig pathogen, which is responsible for swine pleuropneumonia, a highly contagious respiratory infection. To develop subunit vaccines for A. pleuropneumoniae infection, the Apx toxin genes, apxI and apxII, which are thought to be important for protective immunity, were expressed in Saccharomyces cerevisiae, and the induction of immune responses in mice was examined. The apxI and apxII genes were placed under the control of a yeast hybrid ADH2-GPD promoter (AG), consisting of alcohol dehydrogenase II (ADH2) and the GPD promoter. Western blot analysis confirmed that both toxins were successfully expressed in the yeast. The ApxIA and ApxIIA-specific IgG antibody response assays showed dose dependent increases in the antigen-specific IgG antibody titers. The challenge test revealed that ninety percent of the mice immunized with ApxIIA or a mixture of ApxIA and ApxIIA, and sixty percent of mice immunized with ApxIA survived, while none of those in the control groups survived longer than 36 h. These results suggest that vaccination of the yeast ex­pressing the ApxI and ApxII antigens is effective for the induction of protective immune responses against A. pleuropneumoniae infections in mice.

Responses of Photosynthetic Efficiency and Ascorbate Peroxidase Induced by Salt Stress in Rice (Oryza sativa L.) (벼의 salt stress에 의해 유도된 산화 stress에 대한 ascorbate peroxidase 반응)

  • Koo, Jeung-Suk;Im, Kyoung-Nam;Chun, Hyun-Sik;Lee, Chin-Bum
    • Journal of Life Science
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    • v.20 no.8
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    • pp.1173-1180
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    • 2010
  • We investigated changes in photosynthesis and activity of ascorbate peroxidase (APX) that scavenges ROS as responses to oxidative stress induced by salinity in rice (Oryza sativa L.). Photosynthetic efficiency of rice leaves, monitored in terms of Fv/Fm, declined with the increase of salt concentration (100-300 mM NaCl). Salinity caused an increase of $H_2O_2$ in leaves of rice, with an increase of APX activity. Among total APX isoforms, an isoform of stromal-APX 1 in leaves of rice was completely inactivated by 300 mM NaCl, but was not affected by chilling or drought. The results suggest that salt stress acts in quite a different mechanism in relation to the activity of stromal-APX from that of other stresses such chilling and drought. We carried out RT-PCR for analysis of genes expression of APX isoforms as affected by salt stress. The expression of cytosolic APX/thylakoid-bound APX genes in leaves of rice exposed to salt stress was increased, while stromal APX gene expression rapidly declined.

Regulation of Ascorbate Peroxidase Gene Expression in Response to Stresses and Phytohormone in Rehmannia glutinosa

  • Park Myoung Ryoul;Park Moon Hee;Yoo Nam Hee;Yu Chang Yeon;Yun Song Joong
    • KOREAN JOURNAL OF CROP SCIENCE
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    • v.50 no.4
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    • pp.286-290
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    • 2005
  • Ascorbate peroxidase (APX) plays a crucial role in the detoxification of hydrogen peroxide. APX activity is maintained significantly higher in the paraquat­treated leaves of the paraquat-tolerant Rehmannia glutinos. This study was conducted to understand structural and regulatory characteristics of APX gene in R. glutinosa. A putative APX cDNA clone (RgAPX1) was isolated from a leaf cDNA library using a partially sequenced expressed sequence tag clone. RgAPX1 is consisted of 1148 bp nucleotides and contains an open reading frame encoding a 250 amino acid-long polypeptide. Deduced RgAPX1 amino acid sequence shares higher sequence similarity to cytosolic APXs. RgAPX1. expression was higher in the leaf than in the flower and root. Southern blot result indicates the presence of one or two RgAPX1-related genes in R. glutinosa genome. RgAPX1 transcription was affected differentially by various stresses and phytohormone. The results indicate that RgAPXl is constitutively expressed in most tissues and its expression is modulated for the immediate and efficient detoxification of $H_2O_2$ under normal and stress conditions.

Cloning, Sequencing and Expression of apxIA, IIA, IIIA of Actinobacillus pleuropneumoniae Isolated in Korea (국내 분리 흉막폐렴균의 apxIA, IIA, IIIA 유전자 Cloning, 염기서열 분석 및 단백질 발현)

  • Shin, Sung-jae;Cho, Young-wook;Yoo, Han-sang
    • Korean Journal of Veterinary Research
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    • v.43 no.2
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    • pp.247-253
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    • 2003
  • Actinobacillus pleuropneumoniae causes a highly contagious pleuropneumoniae in swine. The bacterium produces several virulence factors such as exotoxin, LPS, capsular polysaccharide, etc. Among them, the exotoxin, called Apx, has been focused as the major virulence factor, and the toxin consists of 4 gene cluster. apx CABD. apxA is the structural gene of toxin and has four different types, I, II, III, and IV. As the first step of development of a new subunit vaccine, the three different types of apxA gene were amplified from A. pleuropneumoniae isolated from Korea by PCR with primer designed based on the N- and C-terminal of the toxin. The sizes of apxIA, IIA and IIIA were 3,073, 2,971 and 3,159bps, respectively. The comparison of whole DNA sequences of apxIA, IIA and IIIA genes with those of the reference strain demonstrated 98%, 99% and 98% homology, respectively. In addition, the phylogenetic analysis was performed based on the amino acid sequences compared with 12 different RTX toxin family using the neighbor-joining method. ApxA proteins of Korean isolates were identical with reference strains in this study. All ApxA proteins were expressed in E. coli with pQE expression vector and identified using Western blot with polyclonal antibodies against culture supernatants of A. pleuropneumoniae serotype 2 or 5. The sizes of each expressed ApxA protein were about 120, 110, 125 kDa (M.W.), respectively. The results obtained in this study could be used for the future study to develop a new vaccine to porcine pleuropneumoniae.

Manipulation of Antioxidative Mechanism in Chloroplasts

  • Kwon, Suk-Yoon;Lee, Haeng-Soon;Kwak, Sang-Soo
    • Proceedings of the Botanical Society of Korea Conference
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    • pp.79-84
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    • 1999
  • Oxidative stress is one of the major environmental stresses to plants. Reactive oxygen species (ROS) generated during metabolic processes damage cellular functions and consequently lead to cell death. Fortunately plants have in vivo defense system by which the ROS is scavenged by enzymes such as superoxide dismutase (SOD) and ascorbate peroxidase (APX). In attempts to understand the protection mechanism of plant against oxidative stress, we developed transgenic tobacco (Nicotiana tabacum cv. Xanthi) plansts thet expressed both SOD and APX in chloroplast using Agrobacterum-mediated transformation and evaluated their protection capabilities against methyl viologen (MV, paraquat) -mediated oxidative damage. Three double transformants (CAI, CA2, and CA3) expressed the chimeric CuZnSOD and chimeric APX in chloroplast, and one transformant (AM) expressed the chimeric APX and chimeric MnSOD in chloroplast. In addition, we obtained three lines of transformants (C/Al, C/A2, and A/C) that expressed the APX and SOD than control plants, and more resistant to oxidative stress caused by MV. TRansformants (C/A and A/C) overexpressing MnSOD, CuZnSOD and APX at the same time showed the highest resistance to MV-mediated oxidative stress among the transformants.

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Transformation of A Plant by Ascorbate Peroxidase Gene using Agrobacterium tumefaciens (Ascorbate Peroxidase 유전자의 도입에 의한 식물의 형질전환)

  • 이인애;이효신;배은경;김기용;이병현;손대영;조진기
    • Journal of The Korean Society of Grassland and Forage Science
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    • v.22 no.2
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    • pp.101-106
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    • 2002
  • This study was conducted to obtain the transformed tobacco (Nicotiana tubacum) plants with cytosolic ascorbate peroxidase gene(ApxSC7) using Agrobacterium tumefaciens LBA4404. A cDNA encoding the cytosolic ascorbate peroxidase of strawberry, ApxSC7, was introduced into tobacco plants via Agrobacterium-mediated gene transfer system. The expression vector, pIG-AP8, harboring ApxSC7 gene was used for production of transgenic tobacco plants. A large number of transgenic plants were regenerated on a medium containing hygromycin. Integration of ApxSC7 gene was confirmed by PCR and Southern blot analyses with genomic DNA. Northern blot analyses revealed that the pIGap8 gene was constitutively expressed.

Expression Analysis of Oryza sativa Ascorbate Peroxidase 1 (OsAPx1) in Response to Different Phytohormones and Pathogens (벼 ascobate peroxidase 단백질의 병원균 및 식물호르몬에 대한 발현 분석)

  • Wang, Yiming;Wu, Jingni;Choi, Young Whan;Jun, Tae Hwan;Kwon, Soon Wook;Choi, In Soo;Kim, Yong Chul;Gupta, Ravi;Kim, Sun Tae
    • Journal of Life Science
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    • v.25 no.10
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    • pp.1091-1097
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    • 2015
  • We have isolated and characterized an ascorbate peroxidase (APx) gene, OsAPx1 from rice. Northern and Western blot analyses indicated that at young seedling stage, OsAPx1 mRNA was expressed highly in root, shoot apical meristem (SAM) and leaf sheath than leaf. In mature plant, OsAPx1 gene expressed highly in root, stem and flower but weakly in leaf. OsAPx1 gene and protein expression level was induced in leaves inoculated with Magnaporthe oryzae (M. oryzae) and Xanthomonas oryzae pv. oryzae (Xoo). Phytohormones treatment showed that OsAPx1 was up-regulated by jasmonic acid (JA), but was down regulated by ABA and SA co-treatments with JA, resulting that they have antagonistic effect on pathogen responsive OsAPx1 expression. Phylogenetic analysis illustrated that Arabidopsis AtAPx1 has a close relationship with OsAPx1. In AtAPx1 knock out lines, the accumulation of O2- and H2O2 are all highly detected than wild type, revealing that the high concentration of exogenous H2O2 cause the intercellular superoxide anion and hydrogen peroxide accumulation in AtAPx1 knockout plant. These results suggested that OsAPx1 gene may be associated with the pathogen defense cascades as the mediator for balancing redox state by acting ROS scavenger and is associated with response to the pathogen defense via Jasmonic acid signaling pathway.

Enhancement of Apx Toxin Production in Actinobacillus pleuropneumoniae Serotypes 1, 2, and 5 by Optimizing Culture Conditions

  • Dao, Hoai Thu;Do, Van Tan;Truong, Quang Lam;Hahn, Tae-Wook
    • Journal of Microbiology and Biotechnology
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    • v.30 no.7
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    • pp.1037-1043
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    • 2020
  • Actinobacillus pleuropneumoniae (APP) is a causative agent of porcine pleuropneumonia. Therefore, the development of an effective vaccine for APP is necessary. Here, we optimized the culture medium and conditions to enhance the production yields of Apx toxins in APP serotype 1, 2, and 5 cultures. The use of Mycoplasma Broth Base (PPLO) medium improved both the quantity and quality of the harvested Apx toxins compared with Columbia Broth medium. Calcium chloride (CaCl2) was first demonstrated as a stimulation factor for the production of Apx toxins in APP serotype 2 cultures. Cultivation of APP serotype 2 in PPLO medium supplemented with 10 ㎍/ml of nicotinamide adenine dinucleotide (NAD) and 20 mM CaCl2 yielded the highest levels of Apx toxins. These findings suggest that the optimization of the culture medium and conditions increases the concentration of Apx toxins in the supernatants of APP serotype 1, 2, and 5 cultures and may be applied for the development of vaccines against APP infection.

Response of Ascorbate Peroxidase and Dehydroascorbate Reductase in Lettuce (Lactuca sativa L.) Leaves Exposed to Cold Stress (저온 처리한 상추의 잎 내에서 ascorbate peroxidase와 dehydroascorbate reductase의 반응)

  • Kang, Sang-Jae
    • Journal of Life Science
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    • v.18 no.12
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    • pp.1705-1711
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    • 2008
  • To investigate the relationship between cold stress and the activity of ascorbate peroxidase(APX), dehydroascorbate reductase (DHAR), mRNA expression level of two enzymes, hydrogen peroxide content was studied in lettuce leaves under stress condition imposed by cold stress at $4^{\circ}C$ for 24 hr in the dark and following recovery at $20^{\circ}C$ from cold stress. Hydrogen peroxide content increased gradually in lettuce leaves during cold stress, but decreased slightly following recovery from cold stress. Soluble protein content, however, decreased gradually during cold stress, and then rapidly returned to normal levels following recovery. Total chlorophyll content decreased gradually during cold stress, and then keep constant following recovery. The patterns of chlorophyll a and b content similar to that of total chlorophyll content, and carotenoid content didn't change. The ratio of chlorophyll a and total chlorophyll was increased during cold stress, but decreased with rapid during cold stress, and then the ratio returned to normal levels following recovery. During cold stress, the activity of APX and DHAR in the lettuce leaves increased dramatically, and also transcript levels of mRNA of APX and DHAR, as determined by probing 32P-labeled single stranded RNA of APX and DHAR, highly increased and returned to normal levels following recovery, respectively. Relationship between APX and DHAR activity and hydrogen peroxide highly related ($R^2$=0.8715 and 0.8643), whereas between hydrogen peroxide and total chlorophyll content and soluble content related reversely ($R^2$=0.5021 and 0.8915).