scholarly journals Towards Development of an Edible Vaccine against Bovine Pneumonic Pasteurellosis Using Transgenic White Clover Expressing aMannheimia haemolytica A1 Leukotoxin 50 Fusion Protein

2001 ◽  
Vol 69 (9) ◽  
pp. 5786-5793 ◽  
Author(s):  
Raymond W. H. Lee ◽  
Judith Strommer ◽  
Doug Hodgins ◽  
Patricia E. Shewen ◽  
Yongqing Niu ◽  
...  
Keyword(s):  
Fusion Protein ◽  
White Clover ◽  
Immunoblot Analysis ◽  
Edible Vaccine ◽  
Cauliflower Mosaic Virus ◽  
35S Promoter ◽  
Pneumonic Pasteurellosis ◽  
Western Immunoblot ◽  
Bovine Pneumonic Pasteurellosis ◽  
Western Immunoblot Analysis

ABSTRACT Development of vaccines against bovine pneumonia pasteurellosis, or shipping fever, has focused mainly on Mannheimia haemolytica A1 leukotoxin (Lkt). In this study, the feasibility of expressing Lkt in a forage plant for use as an edible vaccine was investigated. Derivatives of the M. haemolytica Lkt in which the hydrophobic transmembrane domains were removed were made. Lkt66 retained its immunogenicity and was capable of eliciting an antibody response in rabbits that recognized and neutralized authentic Lkt. Genes encoding a shorter Lkt derivative, Lkt50, fused to a modified green fluorescent protein (mGFP5), were constructed for plant transformation. Constructs were screened by Western immunoblot analysis for their ability to express the fusion protein after agroinfiltration in tobacco. The fusion construct pBlkt50-mgfp5, which employs the cauliflower mosaic virus 35S promoter for transcription, was selected and introduced into white clover byAgrobacterium tumefaciens-mediated transformation. Transgenic lines of white clover were recovered, and expression of Lkt50-GFP was monitored and confirmed by laser confocal microscopy and Western immunoblot analysis. Lkt50-GFP was found to be stable in clover tissue after drying of the plant material at room temperature for 4 days. An extract containing Lkt50-GFP from white clover was able to induce an immune response in rabbits (via injection), and rabbit antisera recognized and neutralized authentic Lkt. This is the first demonstration of the expression of anM. haemolytica antigen in plants and paves the way for the development of transgenic plants expressing M. haemolytica antigens as an edible vaccine against bovine pneumonic pasteurellosis.

scholarly journals Phosphorylation of nuclear and flagellar basal apparatus proteins during flagellar regeneration in Chlamydomonas reinhardtii

1993 ◽  
Vol 122 (4) ◽  
pp. 877-886 ◽  
Author(s):  
JD Harper ◽  
MA Sanders ◽  
JL Salisbury
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Transcriptional Activation ◽  
Kinase Inhibitor ◽  
Nuclear Periphery ◽  
Immunoblot Analysis ◽  
Basal Region ◽  
Western Immunoblot ◽  
Flagellar Regeneration ◽  
Basal Apparatus ◽  
Western Immunoblot Analysis

The antiphosphoprotein monoclonal antibody MPM-2 was used to investigate protein phosphorylation during flagellar regeneration in Chlamydomonas reinhardtii. MPM-2 recognizes a phosphorylated epitope and detects several Chlamydomonas proteins by Western immunoblot analysis. Two MPM-2 reactive proteins (34 and 90 kD) increase in Western immunoblot intensity after flagellar excision and decrease in intensity during flagellar regeneration. Immunofluorescence and immunogold labeling revealed MPM-2 staining within the nucleus, especially towards the nuclear periphery, the flagellar basal apparatus, and the nucleus-basal body connector after flagellar excision. Comparison of MPM-2 reactivity in wild-type cells and in the mutant bald-2, which lacks functional basal bodies, demonstrates that the 34-kD protein is localized in the nucleus and the 90-kD protein is localized in the flagellar basal region. MPM-2 reactivity is observed in cells competent for flagellar regeneration. However, when cells were treated with the kinase inhibitor, staurosporine, MPM-2 reactivity did not increase after flagellar excision and flagellar regeneration was impaired. These observations suggest that phosphorylation of the 34- and 90-kD proteins may be important for flagellar regrowth. Possible roles for phosphorylation in flagellar regeneration include transcriptional activation and transport of flagellar precursors to the base of the growing flagella.

Anthocyanin regulatory gene expression in transgenic white clover can result in an altered pattern of pigmentation

2000 ◽  
Vol 27 (7) ◽  
pp. 659
Author(s):  
John de Majnik ◽  
Jeremy J. Weinman ◽  
Michael A. Djordjevic ◽  
Barry G. Rolfe ◽  
Greg J. Tanner ◽  
...  
Keyword(s):  
White Clover ◽  
Regulatory Gene ◽  
Phenylpropanoid Pathway ◽  
Regulatory Genes ◽  
Cauliflower Mosaic Virus ◽  
35S Promoter ◽  
Anthocyanin Accumulation ◽  
Cauliflower Mosaic Virus 35S ◽  
White Clover Plant ◽  
Clover Plant

This study presents the first evidence of heterologous anthocyanin regulatory genes altering anthocyanin expression in stably transformed leguminous plants. Two families of anthocyanin regulatory genes, myc (delila, B-Peru) and myb (myb.Ph2, C1), are involved in the activation of the phenylpropanoid pathway. White clover (Trifolium repens cv. Haifa) plants were transformed with dicotyledonous and monocotyledonous myb or myc genes. Some of these transformed plants exhibited enhanced anthocyanin accumulation in a range of tissues. One plant, transformed with the B-Peru gene driven by the Cauliflower Mosaic Virus 35S promoter, displayed a unique pattern of anthocyanin accumulation in the leaf. The accumulation of anthocyanin in this plant was closely associated with the crescent of leaves, which is normally white. The red pigmentation declined in intensity in the oldest leaf stage. The B-Peru message was detected in all leaf stages of this white clover plant. This anthocyanin pattern was shown to be heritable.

Fructan formation in transgenic white clover expressing a fructosyltransferase from Streptococcus salivarius

2002 ◽  
Vol 29 (11) ◽  
pp. 1287 ◽  
Author(s):  
Colin L. D. Jenkins ◽  
Annette J. Snow ◽  
Richard J. Simpson ◽  
Thomas J. Higgins ◽  
Nicholas A. Jacques ◽  
...  
Keyword(s):  
White Clover ◽  
Carbon Assimilation ◽  
Dry Weight ◽  
Cauliflower Mosaic Virus ◽  
Soluble Carbohydrate ◽  
35S Promoter ◽  
Streptococcus Salivarius ◽  
Clonal Line ◽  
Leaf Extracts ◽  
Storage Carbohydrate

White clover (Trifolium repens L.) is an important pasture legume that does not normally accumulate fructan as a storage carbohydrate. We have generated transgenic white clover plants that accumulate fructan, by expressing the fructosyltransferase (Ftf) enzyme from the bacterium Streptococcus salivarius under the control of the cauliflower mosaic virus (CaMV) 35S promoter. Fructan accumulated in leaves, petioles, stolons, flowers, and roots of transgenic plants. Levels of fructan up to approximately 2% dry weight were measured in leaves. The fructan was of high molecular mass ( > 5000 kDa), typical of bacterial fructans. Ftf enzyme activity up to 120 nmol min–1 g–1 fresh weight was determined in leaf extracts of the transformed plants, and appeared to be stable throughout leaf development. Most transformed lines appeared normal, flowered and produced seed, but the growth rate of some transformed lines decreased. Photosynthetic carbon assimilation and levels of endogenous carbohydrates (hexoses, sucrose and starch) were not substantially changed in a clonal line with relatively low fructan. However, in a clonal line with relatively high fructan accumulation, plant growth was reduced, leaf photosynthesis was decreased by 60%, and carbohydrate contents were reduced. The results are discussed in the context of manipulating soluble carbohydrate composition in pasture species to improve nutritive quality for grazing animals.

scholarly journals The Immunologic Response of Dogs to Bartonella Vinsonii Subspecies Berkhoffii Antigens: as Assessed by Western Immunoblot Analysis

2003 ◽  
Vol 15 (4) ◽  
pp. 349-354 ◽  
Author(s):  
Edward B. Breitschwerdt ◽  
Jiraporn Suksawat ◽  
Bruno Chomel ◽  
Barbara C. Hegarty
Keyword(s):  
Cell Culture ◽  
Immunoblot Analysis ◽  
Progressive Increase ◽  
Protein Recognition ◽  
Molecular Testing ◽  
Serum Samples ◽  
Tissue Samples ◽  
Antigenic Protein ◽  
Western Immunoblot ◽  
Western Immunoblot Analysis

Bartonella vinsonii subspecies berkhoffii is a recently recognized zoonotic pathogen that causes endocarditis, granulomatous rhinitis, and granulomatous lymphadenitis in dogs. Isolation of B. vinsonii ( berkhoffii) from blood or tissue samples is frequently unsuccessful; therefore, diagnosis is primarily dependent on serologic or molecular testing modalities. Because previous canine serologic studies have used an indirect immunofluorescence assay (IFA), without Western immunoblot (WI) confirmation, the overall objective of this study was to examine the diagnostic use of WI for confirmation of B. vinsonii ( berkhoffii) infection in dogs. To confirm that agar-grown and cell culture–grown organisms yielded similar patterns of WI antigenic protein recognition, the 2 preparations were compared using IFA-reactive sera obtained from dogs experimentally infected with B. vinsonii ( berkhoffii). Temporal changes in the pattern of antigenic protein recognition were characterized using sera obtained from dogs at various time points after experimental B. vinsonii ( berkhoffii) infection. The specificity of B. vinsonii ( berkhoffii) WI was examined by testing canine sera that were reactive to B. henselae, B. clarridgeiae, Ehrlichia canis, Rickettsia rickettsii, Babesia canis, Anaplasma phagocytophilum (previously E. equi), or Brucella canis antigens. Clinical accessions including serum samples obtained from B. vinsonii ( berkhoffii) culture–positive dogs and B. vinsonii ( berkhoffii) culture–negative dogs that were IFA seroreactive to B. vinsonii ( berkhoffii) antigens were examined by WI. The results of this study indicate that WI using agar-grown or cell culture–grown B. vinsonii ( berkhoffii) antigens produce identical patterns of antigenic protein recognition. After experimental infection, there is a progressive increase in the number of antigenic proteins that are recognized by WI, with the 33-kD antigen representing the first and the most persistent antigen recognized by B. vinsonii ( berkhoffii)–infected dogs. Regarding specificity, sera from dogs that were reactive to various heterologous antigens did not recognize B. vinsonii ( berkhoffii) antigens by IFA or WI, and sera from dogs experimentally infected with B. henselae did not recognize B. vinsonii ( berkhoffii) antigens by WI. Regarding clinical accessions, there was good agreement between B. vinsonii ( berkhoffii) IFA test results and WI analysis. Western immunoblot analysis can be used to detect or confirm exposure to B. vinsonii ( berkhoffii) in dogs.

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