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      • Record: found
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      Production of Polyclonal Antibody against Grapevine fanleaf virus Movement Protein Expressed in Escherichia coli

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          Abstract

          The genomic region of Grapevine fanleaf virus (GFLV) encoding the movement protein (MP) was cloned into pET21a and transformed into Escherichia coli strain BL21 (DE3) to express the protein. Induction was made with a wide range of isopropyl-β-D-thiogalactopyranoside (IPTG) concentrations (1, 1.5, and 2 mM) each for duration of 4, 6, or 16 h. However, the highest expression level was achieved with 1 mM IPTG for 4 h. Identity of the expressed protein was confirmed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blotting. The expressed 41 kDa protein was purified under denaturing condition by affinity chromatography, reconfirmed by Western blotting and plate-trapped antigen enzyme-linked immunosorbent assay (PTA-ELISA) before being used as a recombinant antigen to raise polyclonal antibodies in rabbits. Purified anti-GFLV MP immunoglobulines (IgGs) and conjugated IgGs detected the expressed MP and GFLV virions in infected grapevines when used in PTA-ELISA, double antibody sandwich-ELISA, and Western blotting. This is the first report on the production of anti-GFLV MP polyclonal antibodies and application for the virus detection.

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          One-step preparation of competent Escherichia coli: transformation and storage of bacterial cells in the same solution.

          Michael R. Miller, Tony K H Chung, Perttu S. Niemelä (1989)
          We have developed a simple, one-step procedure for the preparation of competent Escherichia coli that uses a transformation and storage solution [TSS; 1 x TSS is LB broth containing 10% (wt/vol) polyethylene glycol, 5% (vol/vol) dimethyl sulfoxide, and 50 mM Mg2+ at pH 6.5]. Cells are mixed with an equal volume of ice-cold 2 x TSS and are immediately ready for use. Genetic transformation is equally simple: plasmid DNA is added and the cells are incubated for 5-60 min at 4 degrees C. A heat pulse is not necessary and the incubation time at 4 degrees C is not crucial, so there are no critical timing steps in the transformation procedure. Transformed bacteria are grown and selected by standard methods. Thus, this procedure eliminates the centrifugation, washing, and long-term incubation steps of current methods. Although cells taken early in the growth cycle (OD600 0.3-0.4) yield the highest transformation efficiencies (10(7)-10(8) transformants per micrograms of plasmid DNA), cells harvested at other stages in the growth cycle (including stationary phase) are capable of undergoing transformation (10(5)-10(7) transformants per micrograms of DNA). For long-term storage of competent cells, bacteria can be frozen in TSS without addition of other components. Our procedure represents a simple and convenient method for the preparation, transformation, and storage of competent bacterial cells.
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            Movement protein of tobacco mosaic virus modifies plasmodesmatal size exclusion limit.

            C. Deom, Peter Lucas, R Beachy … (1989)
            The function of the 30-kilodalton movement protein (MP) of tobacco mosaic virus is to facilitate cell-to-cell movement of viral progeny in an infected plant. A novel method for delivering non-plasmalemma-permeable fluorescent probes to the cytosol of spongy mesophyll cells of tobacco leaves was used to study plasmodesmatal size exclusion limits in transgenic plants that express the MP gene. Movement of fluorescein isothiocyanate-labeled dextran (F-dextran) with an average molecular mass of 9400 daltons and an approximate Stokes radius of 2.4 nanometers was detected between cells of the transgenic plants, whereas the size exclusion limit for the control plants was 700 to 800 daltons. No evidence of F-dextran metabolism in the leaves of the transgenic plants was found. Thus, the tobacco mosaic virus movement protein has a direct effect on a plasmodesmatal function.
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              Interaction between the tobacco mosaic virus movement protein and host cell pectin methylesterases is required for viral cell-to-cell movement.

              M. Chen, J. Sheng, G Hind … (2000)
              Virus-encoded movement protein (MP) mediates cell-to-cell spread of tobacco mosaic virus (TMV) through plant intercellular connections, the plasmodesmata. The molecular pathway by which TMV MP interacts with the host cell is largely unknown. To understand this process better, a cell wall-associated protein that specifically binds the viral MP was purified from tobacco leaf cell walls and identified as pectin methylesterase (PME). In addition to TMV MP, PME is recognized by MPs of turnip vein clearing virus (TVCV) and cauliflower mosaic virus (CaMV). The use of amino acid deletion mutants of TMV MP showed that its domain was necessary and sufficient for association with PME. Deletion of the PME-binding region resulted in inactivation of TMV cell-to-cell movement.
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                Author and article information

                Journal
                Journal ID (nlm-ta): Plant Pathol J
                Journal ID (iso-abbrev): Plant Pathol. J
                Title: The Plant Pathology Journal
                Publisher: Korean Society of Plant Pathology
                ISSN (Print): 1598-2254
                ISSN (Electronic): 2093-9280
                Publication date (Print): October 2016
                Publication date (Electronic): 01 October 2016
                Volume: 32
                Issue: 5
                Pages: 452-459
                Affiliations
                [1 ]Department of Plant Protection, Faculty of Agriculture, University of Zanjan, Zanjan 45371-38111, Iran
                [2 ]Department of Plant Protection, Faculty of Agriculture, University of Tabriz, Tabriz 51666-16471, Iran
                [3 ]Plant Virology Research Center, College of Agriculture, Shiraz University, Shiraz 71949-84471, Iran
                [4 ]Department of Veterinary Clinical Sciences, Faculty of Veterinary Medicine, University of Tabriz, Tabriz 51666-16471, Iran
                Author notes
                [* ]Corresponding author: Phone) +98-24-3305-2266, FAX) +98-24-3228-3202, E-mail) Koolivand@ 123456znu.ac.ir
                Article
                Publisher ID: ppj-32-452
                DOI: 10.5423/PPJ.OA.01.2016.0031
                PMC ID: 5051564
                PubMed ID: 27721695
                SO-VID: cfac4844-5daa-4573-bada-9fb81daa7ee8
                Copyright © © The Korean Society of Plant Pathology
                License:

                This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                Date received : 23 February 2016
                Date revision received : 19 May 2016
                Date accepted : 30 May 2016
                Categories
                Subject: Research Article

                Keywords: antibody,elisa,expression,grapevine fanleaf virus,recombinant
                Data availability:
                Keywords: antibody, elisa, expression, grapevine fanleaf virus, recombinant

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