Expression of Prunus Necrotic Ringspot Virus Coat Protein in E. coli (original) (raw)
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Phytopathologia Mediterranea, 2005
In order to have a more detailed understanding of the various factors influencing a reverse transcriptase polymerase chain reaction (RT-PCR), a number of important parameters such as Mg+2, primer, enzyme concentration and others were optimized for the detection of Prunus necrotic ringspot virus (PNRSV). Using a PNRSV isolate with a pair of primers, complementary DNA of viral genome as template, and an appropriate enzyme together with magnesium chloride, the following optimal conditions were identified: primer concentration between 0.2 and 0.0002 pmol µl-1 and 0.06–2 units µl-1 for Taq DNA polymerase enzyme for a 50 µl reaction volume when other parameters were optimum; magnesium chloride concentration less than 2.5 mM; dNTP concentration between 1 and 10 mM. The optimum cDNA amount should be ~360 ng for a 50 µl reaction mixture. When these optimized concentrations and/or values of the main PCR parameters were brought together for a new RT-PCR, a clear and a reliable PNRSV detection ...
tarimdergisi.yyu.edu.tr
Two methods (DAS-ELISA and RT-PCR) were compared for the detection of Prunus necrotic ringspot virus (PNRSV) in woody host Prunus mahleb isolated from Malatya. Total RNA extractions were made from serially diluted fresh leaf tissue, root, bark and one year old green bark tissue using silica-based method. Purified total RNA extracts were used as template for cDNA synthesis by reverse transcription. PCR products, about 616 base pair in size, was analyzed by gel electrophoresis and visualized by ethidium bromide staining. Comparing ELISA test, RT-PCR was found more sensitive in detecting local PNRSV isolate. Several plant tissues were used for the evaluation of best tissue in the detection of PNRSV by RT-PCR. Leaf tissue of infected P. mahaleb was proved to be more suitable tissue in detection of virus by RT-PCR respect to root, one-year-old green bark tissue and bark tissue.
Iranian Journal of Fisheries Sciences, 2014
Infectious Pancreatic Necrosis Virus (IPNV) is a member of the family Birnaviridae that has been linked to high mortalities in salmonids. Bacterial based systems as live vectors for the delivery of heterologous antigens offer a number of advantages as vaccination strategies. VP2 is a structural viral protein of IPNV with immunogenicity effects. In this study IPNV was isolated from diseased fry of rainbow trout Oncorhynchus mykiss (Walbaum) using CHSE-214. Then an expression vector was constructed for expression of viral protein VP2. The designed vector was constructed based upon pET-26b (+) with T7 promoter. A fragment containing the full length of the VP2 gene of Iranian Sp strain was amplified by PCR using genomic RNA of IPNV as template and cloned inpET-26b(+) plasmid. Recombinant structural viral protein VP2 was expressed as a soluble, N-terminal PelB fusion protein and secreted into the periplasmic space of Escherichia coli BL21(DE3) and Rosetta (DE3). The glucose, Isopropyl-β-D-thiogalactopyranoside (IPTG) was used as a chemical inducer for rVP2 production in 37º C. The rVP2 was extracted from the periplasm by osmotic shock treatment. The presence of gene in bacterial system of E. coli was confirmed by gel electrophoresis technique. The constructed vector could efficiently express the rVP2 into the periplasmic space of E. coli. The successful cloning and expression of the structural viral protein gene into E. coli can be used for developing a useful and safe vaccine to control IPNV infection in Iranian fish industry.
Molecular Variability Among Isolates of Prunus Necrotic Ringspot Virus from Different Prunus spp
Phytopathology®, 1999
Viral sequences amplified by polymerase chain reaction from 25 isolates of Prunus necrotic ringspot virus (PNRSV), varying in the symptomatology they cause in six different Prunus spp., were analyzed for restriction fragment polymorphisms. Most of the isolates could be discriminated by using a combination of three different restriction enzymes. The nucleotide sequences of the RNA 4 of 15 of these isolates were determined. Sequence comparisons and phylogenetic analyses of the RNA 4 and coat proteins (CPs) revealed that all of the isolates clustered into three different groups, represented by three previously sequenced PNRSV isolates: PV32, PE5, and PV96. The PE5-type group was characterized by a 5′ untranslated region that was clearly different from that of the other two groups. The PV32-type group was characterized by an extra hexanucleotide consisting of a duplication of the six immediately preceding nucleotides. Although most of the variability was observed in the first third of t...
acgssr.org
Western blot and dot blot immunoassays (DBIA). The antiserum raised against the expressed CP (BBTV-AS 6xHis ) gave strong BBTV-specific DBIA reactions and very weak background reactions with non-infected tissues, similar to those produced by polyclonal antibodies raised against BBTV purified virion (BBTV-AS). Furthermore, (BBTV-AS 6xHis ) polyclonal antibody reacted specifically with both the denatured recombinant protein and the disrupted BBTV virus particle as well as FBNYV purified particles in DBIA. These results showed that the (BBTV-AS 6xHis ) polyclonal antibody is useful for the detection of BBTV in infected tissues by dot blot tests.
The coat protein (CP) gene of potato leafroll virus (PLRV) was cloned into the pBAD-TOPO expression vector upon induction; the viral protein was expressed as 6XHis-tagged PLRV fusion protein in Escherichia coli (E. coli) BL21 cells. The fusion protein was confirmed by western blot analysis using antibodies specific for the PLRV. The predicted length of the coat protein gene was 627 nucleotides, corresponding to a 209 amino acid coat protein of Mr 23 KDa. The nucleotide sequence of the coat protein gene of PLRV (PLRV-CP) was compared to the sequences of the coat protein genes of other PLRV isolates. The PLRV-CP gene of the Egyptian isolate (PLRV-CP-Eg) was found to be 95% homologous to those of other luteoviruses at the nucleotide level. Antiserum obtained from rabbit after injection with 6XHis-tagged PLRV fusion protein was immuno-reactive towards the PLRV in indirect ELISA.
Serological Characterisation of Mediterranean Prunus Necrotic Ringspot Virus Isolates
Journal of Plant Pathology, 2001
SUMMARY Prunus necrotic ringspot virus (PNRSV), a worldwide pathogen of stone fruits, has many isolates with different biological, serological, and molecular properties. Monoclonal antibodies (MAbs) to a peach isolate of PNRSV were used to investigate the serological variability of PNRSV isolates by DASI-ELISA. Isolates from different stone fruit species and different Mediterranean countries were first identified with polyclonal antisera (PAbs), then 81 isolates were selected from Albania (3), Cyprus (10), Greece (5), Italy (31), Lebanon (6), Malta (5), Tunisia (7), Turkey (7) and Spain (1) and characterised against 10 single MAbs. Six U.S. isolates were included as controls. The virus showed high serological variability as shown by the identification of 34 serogroups, many of which were host-specific (64%) and country-specific (67%).
Plant Pathology, 1998
Three methods were compared for the detection of prunus necrotic ringspot virus in herbaceous and woody plants: DAS-ELISA, nonisotopic dot-blot hybridization and reverse transcriptional polymerase chain reaction (RT-PCR). When purified virus preparations were used, the detection limit of the RT-PCR technique was 1·28 pg mL ¹1 whereas nonisotopic molecular hybridization and DAS-ELISA allowed detection of 0·8 ng mL ¹1 and 4 ng mL ¹1 , respectively. Several sample processing procedures were evaluated for virus detection by the nonisotopic molecular hybridization technique. When a very short and simple sample processing method was used, the detection limit of the nonisotopic molecular hybridization technique was 25 times higher than that of DAS-ELISA and 625 times lower than that of RT-PCR. A comparison of the level of virus accumulation in mature fruits and in leaf tissue showed that, on average, 125 times more virus was found in the fruits.
Fitopatologia Brasileira, 2007
Grapevine leafroll-associated virus 3 (GLRaV-3), the main viral species of the grapevine leafroll complex, causes yield and quality reduction in grapes (Vitis spp.). The coat protein gene was RT-PCR-amplified from total RNA extracted from infected grapevine leaves and the amplified fragment was cloned and completely sequenced. The fragment was subsequently subcloned into the pRSET-C expression vector. The recombinant plasmid was used to transform Escherichia coli BL21:DE3 and express the capsid protein. The coat protein, fused to a 6 His-tag, was purified by affinity chromatography using an Ni-NTA resin. The identity of the purified protein was confirmed by SDS-PAGE and Western blot. The in vitro-expressed protein was quantified and used for rabbit immunizations. The antiserum was shown to be sensitive and specific for the detection of GLRaV-3 in grapevine extracts in Western blot and DAS-ELISA assays, with no unspecific or heterologous reactions against other non-serologically related viruses being observed.