Expression of a part of the Potato virus A non-structural protein P3 in Escherichia coli for the purpose of antibody preparation and P3 immunodetection in plant material (original) (raw)
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Antigenic Analysis of Potato Virus X by Means of Monoclonal Antibodies
Journal of General Virology, 1986
At least three different antigenic determinants were distinguished on the capsid protein of the B strain of potato virus X by their differential reactivity with monoclonal antibodies. One determinant (or group of determinants) was located on the protruding N terminus which, in the assembled virus particles, is readily split off by proteases in crude plant sap or by trypsin. A second determinant (or group of determinants) was located outside the protruding N terminus on the surface of the undisturbed virus particles. In partially denatured preparations containing the protruding N terminus, this determinant became inaccessible. A third determinant (or group of determinants) became exposed only after some denaturation of the virus particles, e.g. when they were applied directly to ELISA plates or nitrocellulose membranes. In contrast to the other two determinants, this determinant was not destroyed by extensive denaturation, such as heating in solution with SDS and 2-mercaptoethanol.
Molecular …, 2000
Antibodies have been expressed in plants to confer novel traits such as virus resistance or altered phenotype. However, not every antibody is suitable for plant expression, and successful intracellular expression of antibody fragments depends primarily on their amino acid sequence in a way that is as yet unpredictable. Therefore it is desirable to assess different constructs before embarking on the production of transgenic plants. We have used a transient expression system based on potato virus X to compare different cDNA constructs for expression and stability of antibody variable gene fragments in plants. Constructs contained an anti-plant enzyme (granule-bound starch synthase I) scFv sequence derived from a naive phage display library together with different combinations of sequences encoding the human IgG κ constant domain, a murine IgG secretory signal sequence, or the endoplasmic reticulum retention signal peptide KDEL. The results obtained with the potato virus X vector correlated with those from Agrobacterium-mediated stable transformation of potato. The best expression levels were obtained by incorporating sequences that target scFv to the lumen of the endoplasmic reticulum and the secretory pathway. The anti-enzyme scFv retained activity during storage of potato tubers for more than five months. The results demonstrate the utility of the potato virus X vector for the analysis and comparison of many scFv with different epitope specificities or sequence modifications. Evaluation of scFv by transient expression from the PVX vector should aid progress in selection of functional scFv for applications in plant biotechnology.
Protein Expression and Purification, 2001
(1) to express the -glucuronidase Some aspects of the expression of a single-chain Fv (GUS) gene. The format now widely used is the inserantibody fragment (scFv) driven by the plant viral tion form in which resident viral genes are unmodified vector Potato virus X (PVX) have been studied by quanand the foreign gene is inserted under the control of a titative ELISA. After inoculation of the infectious tranduplicated coat protein gene subgenomic promoter. script, the vector was stable only for a few passages of After cloning the foreign gene into PVX cDNA, in vitro sap transmission in the inoculated leaves of Nicotiana transcripts are used to infect plants by abrasion (1). benthamiana and the reversal to wild type was more previously described, as a model in order to evaluate 70
Expression of Non-Structural Proteins of Potato Mop-Top Virus in Procaryotic System
Potato mop-top virus (PMTV) is the type member of the genus Pomovirus of soil-borne, fungus-transmitted plant viruses. In order to produce polyclonal antibodies against PMTV proteins we continue in preparing of suitable systems for expression of non-structural recombinant proteins from RNA 1 of PMTV genome. RNA 1 was divided into 6 parts with the length 1 kb aproximately, and these fragments were inserted into pUC-57A suitable for sequencing. The obtained sequences showed high homology (GenBank AY196959). All fragments were inserted into different expression vectors. First of all we used the non-tagged expression vectors, PMPM-Ω-4, 5, 6; pET-21a+ and pTH. To facilitate detection of expressed proteins we used tagged vectors-either with His tag or glutathion S-transferase (GST). GST fusion protein was performed in vectors pGEX-4T-1, 2 and pGEX-KG allowing the removing of tag by thrombin. Proteins with His tag were induced in vectors pQE-40 and pQE-60. The highest yield of expressed protein was obtained from pGEX-4T-2 in E. coli BL 21. Disadvantage of this approach was incomplete cleavage of GST by thrombin. We consider pQE-40 and pQE-60 as more suitable vectors for the purpose of production of polyclonal antibodies against PMTV proteins, because sequence of tag 6xHis has not been found in plant tissue and thus the serum prepared against recombinant proteins should not crossreact with proteins of healthy plant.
Antigenic Characterization of Potato Virus X with Monoclonal Antibodies
Journal of General Virology, 1988
A panel of mouse monoclonal antibodies (MAbs) against potato virus X (PVX) was obtained and three of these which had high affinity to the antigen were characterized in detail. These three antibodies defined two epitopes on PVX and recognized native virus, viral coat protein and denatured viral coat protein in various immunological assays. Two of the MAbs and rabbit anti-PVX polyclonal antibodies bound to the 68 amino acid N-terminal peptide of the PVX coat protein. This implies that the N terminus of the PVX coat protein is exposed at the virus surface and forms a highly immunogenic antigenic determinant. In double antibody sandwich (DAS) ELISA, MAbs and their horseradish peroxidase conjugates reacted with PVX at 10 to 20 ng/ml. Monoclonal antibodies to PVX reacted with virus in potato leaves and tubers and detected the virus in DAS ELISA in various combinations, including in combination with polyclonal antibodies. 0000-8408 © 1988 SGM
Transgenic tobacco plants carrying the non-structural P3 gene of potato virus A
Biologia Plantarum, 2005
Transgenic tobacco (Nicotiana tobacum) plants carrying the gene coding for potato virus A (PVA) non-structural P3 protein were prepared by inoculation with Agrobacterium tumefaciens. Seeds from self-pollinated flowers (T 1 generation) were collected. To estimate the effectiveness of vertical transfer of the introduced gene and usefulness of respective plant lines for further experiments, the T 1 generation was characterized by testing its ability to grow in the presence of kanamycin (Km) and by PCR of both neomycin phosphotransferase (nptII) and PVA P3 genes. Eight and ten of 29 lines showed Mendelian segregation of Km-resistant phenotype 3:1 and ≥15:1, respectively, the T 1 of eleven lines showed low Km resistance. Selected PCR-positive lines were tested for the presence of P3 mRNA. In most cases the transgene transcription was dependent on the presence or absence of Km in the plant growth medium. Prepared transgenic plants were furthermore tested for sensitivity to PVA and potato virus Y (PVY) infection. All of them showed identical symptom development as the non-transgenic control plants.
2013
Polyclonal antibodies to recombinant (r) coat proteins (r CP) of Potato virus X (PVX) and Potato leaf roll virus (PLRV) were developed. The effectiveness of these antisera was determined by indirect ELISA (I-ELISA), immuno blotting, and double antibody sandwich (DAS) enzyme linked immunosorbent assay (ELISA), i.e. DAS-ELISA. The measured titers, with indirect-ELISA, expressed as reciprocal of the dilution end-points were 6000 for PLRV; whereas, PVX had a titer of 5000. The CP genes of PVX, and PLRV were amplified by reverse transcription-polymerase chain reaction (RT-PCR), cloned and expressed into pBAD-C terminal 6xHis Tag TOPO expression vector in Escherichia coli. For immunization, the CP fractions from bacterial lysates of each virus were purified, under native and denatured conditions, by nickel-nitrilotriacetic acid (Ni-NTA) batch chromatography and equally mixed. The fused proteins were 6.6 mg/20 ml for PVX and 9.0 mg/20 ml for PLRV bacterial culture. Antigenicity of the puri...