Recombinant Coat Protein Research Articles

Production of polyclonal antiserum against Cowpea mild mottle virus coat protein and its application in virus detection

Cowpea mild mottle virus (CpMMV), the causal agent of stem necrosis disease, has drawn attention of soybean producers in recent years due to yield losses in the main producing regions of Brazil. Serological methods for viral detection require the use of an antiserum of good quality to achieve specificity and sensitivity. The entire coat protein gene of a Brazilian CpMMV isolate was cloned into a bacterial expression vector and transformed into Escherichia coli BL21::DE3 for in vitro expression. The coat protein, fused to a His-tag, was purified under denaturing conditions by affinity chromatography using a Ni-NTA resin. After renaturation, the integrity and identity of the purified recombinant protein was confirmed by SDS-Page and MALDI-ToF/ToF mass spectrometer analyses. A rabbit was immunized with increasing amounts of the recombinant protein. The specificity and sensitivity of the antiserum was demonstrated by Western blot and indirect ELISA assays. The polyclonal antisera raised against recombinant coat protein proved to be a reliable tool for CpMMV detection.

Development of an Indirect ELISA and Immunocapture RT-PCR for Lily Virus Detection

Multiple viruses such as Lily symptomless virus (LSV), Lily mottle virus (LMoV), and cucumber mosaic virus (CMV) are the most prevalent viruses infecting lilies in Korea. Leaf samples and bulbs showing characteristic symptoms of virus infection were collected from Gangwon, Chungnam, and Jeju provinces of Korea in 2008-2011. Coat protein (CP) genes of LSV and LMoV were amplified from collected samples by reverse transcription-polymerase chain reaction (RT-PCR) and cloned into a pET21d(+) expression vector to generate recombinant CPs. The resulting carboxy-terminal His-tagged CPs were expressed in Escherichia coli strain BL21(DE3) by isopropyl-1-thio- beta-D-galactoside induction. The recombinant proteins were purified using Ni-NTA agarose beads, and the purified proteins were used as an immunogen to produce polyclonal antibodies in rabbits. The resulting polyclonal antisera recognized specifically LSV and LMoV from infected plant tissues in Western blotting assays. Indirect enzyme-linked immunosorbent assay and immunocapture RTPCR using these polyclonal antisera were developed for the sensitive, efficient, economic, and rapid detection of Lily viruses. These results suggest that large-scale bulb tests and economic detection of Lily viruses in epidemiological studies can be performed routinely using these polyclonal antisera.

First report of Konjac mosaic virus in elephant foot yam (Amorphophallus paeoniifolius) from India

Natural association of Konjac mosaic virus (KoMV) with mosaic disease of elephant foot yam (Amorphophallus paeoniifolius) in India was identified by enzyme-linked immunosorbent assay using an antiserum raised against KoMV recombinant coat protein (CP) and immunocapture-reverse transcription-polymerase chain reaction using CP gene specific primers. Sequence analysis showed 77.2–98.9 % and 92.1–98.2 % identities at CP nucleotide and amino acid levels, respectively with the reported KoMV isolates.

Fabrication and characterization of gold nano-wires templated on virus-like arrays of tobacco mosaic virus coat proteins

The rod-shaped plant virus tobacco mosaic virus (TMV) is widely used as a nano-fabrication template, and chimeric peptide expression on its major coat protein has extended its potential applications. Here we describe a simple bacterial expression system for production and rapid purification of recombinant chimeric TMV coat protein carrying C-terminal peptide tags. These proteins do not bind TMV RNA or form disks at pH 7. However, they retain the ability to self-assemble into virus-like arrays at acidic pH. C-terminal peptide tags in such arrays are exposed on the protein surface, allowing interaction with target species. We have utilized a C-terminal His-tag to create virus coat protein-templated nano-rods able to bind gold nanoparticles uniformly. These can be transformed into gold nano-wires by deposition of additional gold atoms from solution, followed by thermal annealing. The resistivity of a typical annealed wire created by this approach is significantly less than values reported for other nano-wires made using different bio-templates. This expression construct is therefore a useful additional tool for the creation of chimeric TMV-like nano-rods for bio-templating.

Indian citrus ringspot virus의 ELISA 진단 시스템 구축

Indian citrus ring spot virus (ICRSV) is known to cause a serious disease to citrus, especially to Kinnow mandarin, the popular cultivated citrus species in India. In this study, we developed diagnostic systems based on enzyme-linked immunosorbent assay (ELISA). In order to generate antibodies against ICRSV coat protein, we overexpressed the coat protein in Escherichia coli using the pET15b expression vector containing an optimized ICRSV coat protein gene. The recombinant ICRSV coat protein was overexpressed as soluble form at upon IPTG induction. The protein was purified to 95% in purity by Ni-NTA column chromatography. The purified protein was immunized to rabbit for the generation of polyclonal antibody (PAb). The PAb showed a specific immunoreaction to recombinant ICRSV coat protein in western blot analysis and ELISA. Diluted rabbit antisera (10,000 fold) could detect less than 10 ng and 5 ng of the target protein in western blot and ELISA analysis, respectively.

Diagnostic Potential of Polyclonal Antibodies Against Bacterially Expressed Recombinant Coat Protein of Alfalfa mosaic virus.

Alfalfa mosaic virus (AMV), a pathogen of a wide range of plant species, including Glycine max (soybean), is poorly immunogenic. Polyclonal antibodies were generated against bacterially expressed recombinant coat proteins (rCPs) of two biologically distinct AMV strains in rabbits and compared with those raised against native and glutaraldehyde-treated virions of the same strains. Analyses showed that sera against rCPs had comparable antibody titers in indirect enzyme-linked immunosorbent assay with those raised against virions when soybean sap containing homologous viruses served as antigens. Polyclonal antibodies against rCPs were specific, sensitive, and detected all AMV isolates that originated from soybean fields from geographically different regions of the United States. Comparison of CP genes of these isolates showed 96 to 99 and 96 to 100% nucleotide and amino acid sequence identities, respectively, suggesting that they are all closely related. This was further confirmed by phylogenetic analysis where they were all clustered together along with representative AMV strains belonging to group I. Collectively, our data demonstrate that, despite poor immunogenicity of AMV, polyclonal antibodies against rCP are effective probes for detection and diagnosis of the virus.

In vivo expression and binding activity of scFv-RWAV, which recognizes the coat protein of tomato leaf curl New Delhi virus (family Geminiviridae)

Recombinant antibodies expressed in plants have the potential to interrupt virus infections by blocking essential stages of the infection cycle. Here, we show that the expression of a recombinant single-chain variable fragment (scFv) that recognizes the coat protein of tomato leaf curl New Delhi virus (ToLCNDV) in vitro can also bind to a recombinant coat protein in vivo in the reducing environment of the plant cytosol. The scFv and its target were both expressed as fluorescent protein fusions, one incorporating green fluorescent protein (GFP) and the other DsRed. We found that the incorporation of a nuclear localization signal into the scFv construct resulted in the nuclear import of the antibody-antigen complex, as shown by colocalization of the two fluorescent signals. This demonstrates that recombinant antibodies can be targeted to the nucleus and will bind to geminivirus coat proteins therein, allowing the virus infection cycle to be interrupted during its critical replicative phase.

Production of Polyclonal Antibodies to the Recombinant Potato virus M (PVM) Non‐structural Triple Gene Block Protein 1 and Coat Protein

AbstractThe genes encoding the coat protein (CP) and triple gene block protein 1 (TGBp1) of Potato virus M (PVM) were cloned into expression vector pET‐45b(+) (N‐terminal 6xHis tag) and expressed in E. coli Rosetta gami‐2(DE3). The purified recombinant antigens were used for raising polyclonal antibodies. The antibodies against recombinant CP were successfully used in Western blot analysis, plate‐trapped ELISA and DAS‐ELISA as a coating for PVM detection in infected potato leaf samples. The antibodies against recombinant non‐structural protein detected the TGBp1 only in Western blot analysis. This is the first report of the production of polyclonal antibodies against recombinant coat protein and TGBp1 of PVM and their use for detecting the virus.

Immunodiagnostics of cucumber mosaic virus using antisera developed against recombinant coat protein

Cucumber mosaic virus (CMV), causing mosaic disease in banana cultivars, is the most widespread virus after Banana bunchy top virus. The entire coat protein (CP) gene of CMV was amplified by polymerase chain reaction (PCR) and engineered to be expressed in bacterial expression vector pQE30, under the control of isopropyl-thio-β-d galactopyranoside (IPTG)-inducible T7lac promoter. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) revealed expressed CP as ∼25 kDa protein. Expression conditions for maximum recovery of soluble recombinant protein were standardised. The recombinant CP (r-CP) fused with His-tag was purified from Escherichia coli using nickel-nitrilotriacetic acid (Ni-NTA) resin and used as an antigen for the production of polyclonal antisera. This antiserum was used to develop enzyme-linked immunosorbent assay (ELISA) and immunocapture reverse transcription-polymerase chain reaction (IC-RT-PCR) based assay for the detection of CMV isolates. The antiserum had a titre of 1:8000 when tested with r-CP. The reaction of polyclonal antisera was much better than that of commercial antibody. On comparing IC-RT-PCR with direct antigen coating-enzyme-linked immunosorbent assay (DAC-ELISA) using polyclonal antisera, IC-RT-PCR was found to be more sensitive, hence can be used to detect the presence of virus in tissue culture based banana plant as well as in various host plants.

Complete Nucleotide Sequence of the RNA 3 of Bacopa chlorosis virus and Production of Polyclonal Antibodies to a Recombinant Coat Protein

AbstractThe complete sequence of the RNA 3 of a virus causing chlorosis in Impatiens in Germany was determined and identified as an isolate of Bacopa chlorosis virus (BaCV, genus Ilarvirus). BaCV has previously only been reported from bacopa in the USA, but no coat protein (CP) sequence has been previously available. Both RNA 3 encoded proteins, CP and movement protein, showed highest sequence identity to Parietaria mottle virus, a subgroup 1 ilarvirus. Attempts to purify BaCV failed, so an antiserum was raised against a recombinant CP. The polyclonal antiserum so produced allowed specific detection of BaCV but showed no serological cross‐reaction with other ilarviruses and was unsuitable for immunoelectron microscopy. The host range includes many important flowering plant species, highlighting the potential threat BaCV might pose for the horticultural industry. This is the first report of BaCV occurring in Germany and outside the US.

Functional Characterization of Coat Protein and V2 Involved in Cell to Cell Movement of Cotton Leaf Curl Kokhran Virus-Dabawali

The functional attributes of coat protein (CP) and V2 of the monopartite begomovirus, Cotton leaf curl Kokhran virus- Dabawali were analyzed in vitro and in vivo by their overexpression in E coli, insect cells and transient expression in the plant system. Purified recombinant V2 and CP proteins were shown to interact with each other using ELISA and surface plasmon resonance. Confocal microscopy of Sf21 cells expressing V2 and CP proteins revealed that V2 localized to the cell periphery and CP to the nucleus. Deletion of the N terminal nuclear localization signal of CP restricted its distribution to the cytoplasm. GFP-V2 and YFP-CP transiently expressed in N.benthamiana plants by agroinfiltration substantiated the localization of V2 to the cell periphery and CP predominantly to the nucleus. Interestingly, upon coinfiltration, CP was found both in the nucleus and in the cytoplasm along with V2. These results suggest that the interaction of V2 and CP may have important implications in the cell to cell movement.

A virus capsid component mediates virion retention and transmission by its insect vector

Numerous pathogens of humans, animals, and plants are transmitted by specific arthropod vectors. However, understanding the mechanisms governing these pathogen-vector interactions is hampered, in part, by the lack of easy-to-use analytical tools. We investigated whitefly transmission of Lettuce infectious yellows virus (LIYV) by using a unique immunofluorescent localization approach in which we fed virions or recombinant virus capsid components to whiteflies, followed by feeding them antibodies to the virions or capsid components, respectively. Fluorescent signals, indicating the retention of virions, were localized in the anterior foregut or cibarium of a whitefly vector biotype but not within those of a whitefly nonvector biotype. Retention of virions in these locations strongly corresponded with the whitefly vector transmission of LIYV. When four recombinant LIYV capsid components were individually fed to whitefly vectors, significantly more whiteflies retained the recombinant minor coat protein (CPm). As demonstrated previously and in the present study, whitefly vectors failed to transmit virions preincubated with anti-CPm antibodies but transmitted virions preincubated with antibodies recognizing the major coat protein (CP). Correspondingly, the number of insects that specifically retained virions preincubated with anti-CPm antibodies were significantly reduced compared with those that specifically retained virions preincubated with anti-CP antibodies. Notably, a transmission-defective CPm mutant was deficient in specific virion retention, whereas the CPm-restored virus showed WT levels of specific virion retention and transmission. These data provide strong evidence that transmission of LIYV is determined by a CPm-mediated virion retention mechanism in the anterior foregut or cibarium of whitefly vectors.

An oral nervous necrosis virus vaccine using Vibrio anguillarum as an expression host provides early protection

Oral vaccination is the most desirable immunization method by which to prevent fish disease occurring at the early larval stage. We developed an Escherichia coli-based oral nervous necrosis virus (NNV) subunit vaccine and demonstrated its effectiveness in orange spotted grouper ( Epinephelus coioides) fry. However, the incubation period was determined to be over 2 weeks. To enhance the efficacy and shorten the incubation period, a safe and digestible biological adjuvant should be found. Vibrio anguillarum, a common marine bacterium with immune-stimulatory capability was selected as an expression host. An expression vector containing its heat shock protein 60 gene ( groE) as an inducible promoter was constructed to express recombinant NNV coat protein as an antigen. The recombinant V. anguillarum was inactivated and encapsulated in Artemia nauplii to formulate a V. anguillarum-based oral vaccine. Compared to the E. coli-based oral vaccine, this novel oral vaccine not only elicited a higher expression of Mx (encoding for an anti-viral protein) in the brain and viscera but also a higher specific antibody titer against NNV in the first week after inoculation. Meanwhile, a higher survival rate compared to an equal dosage of an E. coli-based oral vaccine was also noticed in two individual challenge trials, indicating that an antigen expressed in an V. anguillarum-based oral vaccine could enhance efficacy in a shorter incubation period and could reduce the risk of NNV infection.

Recombinant CHIK virus E1 coat protein of 11 KDa with antigenic domains for the detection of Chikungunya

Chikungunya is an acute febrile illness caused by an alpha virus technically called as CHIK virus. A smaller size of CHIK virus E1 coat protein −11 kDa was expressed in prokaryotic expression system. The recombinant protein was purified and confirmed by western blot analysis. The positions of the antigenic domain in the protein were identified and the immunoreactivity of recombinant protein with anti-CHIK IgM antibodies was ascertained. The antigen showed an 88% sensitivity and 100% specificity by Indirect ELISA. No cross reactivity of the antigen was observed with anti-Dengue virus serum samples. The results strongly support that the recombinant CHIK coat protein could be used as a diagnostic antigen for the detection of Chikungunya by Indirect ELISA. The relevance of a smaller size recombinant antigen highlights its large scale application in serodiagnosis of CHIK virus since bacterial expression is more simple and cost effective than eukaryotic system.

Biological, molecular, and serological studies of a novel strain of grapevine leafroll-associated virus 2

In California, a novel closterovirus was detected in "Redglobe" grapevine, associated with graft incompatibility and given a trivial name "Grapevine rootstock stem lesion associated virus (GRSLaV)." The biological properties of the putative virus were ascertained when asymptomatic yet infected Redglobe scion buds were graft-inoculated onto test plants of Cabernet Sauvignon propagated on 18 different rootstocks. It proved lethal on test plants growing on rootstocks 1616C, 5BB, 5C, 3309C, and 1103 P, whereas latent infections occurred on the remaining scion-rootstock combinations. In contrast, GLRaV-2 type (type strain) produced only typical leafroll symptoms. In a different experiment, GLRaV-2 type was successfully sap-transmitted to N. benthamiana, whereas sap transmission of GRSLaV was unsuccessful. Double-stranded RNA was extracted from infected Redglobe grapevines, cloned, sequenced, and determined a genome length of 16,527 nucleotides. Computer-assisted analysis of open-reading frames (ORFs) revealed a genome organization typical of monopartite viruses in the genus Closterovirus with nine ORFs (range 71-79% identity) with GLRaV-2 type, the closest similar virus species within the family Closteroviridae. Also the 3'-UTR of GRSLaV consisted of 223 nucleotides with an extended oligo(A) tract similar to that of GLRaV-2 type, Beet yellow stunt virus, and Beet yellows virus. Recombinant GRSLaV coat protein was expressed in E. coli, purified, and immunized a rabbit to produce polyclonal antiserum. Serological data matched the molecular data, whereby exposed plant tissue extracts of grapevines infected by both viruses (GRSLaV and GLRaV-2) reacted positively with homologous and heterologous viral antisera but not with healthy grapevine extracts in ELISA and Western blot tests. Based on the comparative sequence data and shared antigens, GRSLaV is now considered a strain of GLRaV-2 and redesignated as Grapevine leafroll associated virus-2 Redglobe (GLRaV-2RG). Primers specific for GLRaV-2RG were developed, which did not amplify GLRaV-2 type strain. When both sets of specific primers were used in assays of different grapevine collections, the incidence of the respective viruses varied considerably, e.g., 1.7 and 13.5%, respectively, for GLRaV-2RG and GLRaV-2 type.

English

Sequence alignment of the 897 bp amplicon obtained from a diseased sunn hemp (Crotalaria juncea L.) plant DNA revealed a complete 771 bp coat protein (CP) gene flanked by 3’ regions of the AV2 and AC3 genes. Southern hybridization using (α-32P) dCTP labeled (CP) gene probe of Indian tomato leaf curl virus (IToLCV) demonstrated the association of begomovirus with the leaf curl disease of sunn hemp. Phylogenetic data suggested that, the AV2, CP and AC3 genes have closest genetic relationship with begomovirus isolates from India, China and Bangladesh, respectively. In silico recombination analysis elucidated a 297 nucleotides hot spot (346 to 643 nucleotides) within AV2 overlapping region of CP gene, amenable to genetic rearrangements, with lineage from tomato leaf curl virus Bangalore (ToLCuVB) and Indian cassava mosaic virus-Ind (ICMV) as major and minor parents, respectively. Thus, it is concluded that the recombinant CP genes related to begomoviruses are evolved from the Indian isolates, causing broad host specificity and molecular diversity among the related begomoviruses across the geographical limits of Southeast Asia. Key words: Begomovirus, sunn hemp, coat protein, recombination, phylogenetic analysis, in silico analysis.

Molecular and serological diversity in Apple chlorotic leaf spot virus from sand pear (Pyrus pyrifolia) in China

Apple chlorotic leaf spot virus (ACLSV) isolates from sand pear (Pyrus pyrifolia) were characterized by analyzing the sequences of their coat protein (CP) genes and serological reactivity of recombinant coat proteins (rCPs). The sequences of CP genes from 22 sand pear isolates showed a high divergence, with 87.3–100% identities at the nucleotide (nt) level and 92.7–100% identities at the amino acid (aa) level. Phylogenetic analysis on the aa sequence of CP showed that the analyzed ACLSV isolates fell into different clusters and all isolates from sand pear were grouped into a large cluster (I) which was then divided into two sub-clusters (A and B). Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), western blot and enzyme-linked immunosorbent assay (ELISA) analyses demonstrated that rCPs of eight ACLSV isolates (PP13, PP15-2, PP24, PP43, PE, PP54, PP56 and ACLSV-C) from two sub-clusters had different mobility rates and serological reactivity. The rCPs of five isolates grouped into the sub-cluster A showed stronger reactivity with antibodies against rCPs of a sand pear isolate ACLSV-BD and virions of a Japanese apple isolate P-205 than that with the antibody against a Chinese apple isolate ACLSV-C. Three isolates grouped into the sub-cluster B showed stronger reactivity with the antibody against ACLSV-C. The antigenic determinants of CPs from these eight isolates and isolates ACLSV-BD and P-205 were predicted. These results contribute to a further understanding of molecular diversity of the virus and its implication in serological detection.

Production of antiserum and immunodetection of Cucurbit chlorotic yellows virus, a novel whitefly-transmitted crinivirus

Cucurbit chlorotic yellows virus (CCYV), a whitefly-transmitted virus, is a new member of the Crinivirus genus, that causes major economic losses in cucumber, melon and watermelon crops. To develop immunodiagnostic methods for CCYV, we produced an antiserum by immunizing rabbits with bacterially expressed recombinant coat protein of CCYV and detected CCYV from CCYV-infected plants with western blotting and immunoelectron microscopy. CCYV in extracts from infected plants was also readily detected by double antibody sandwich enzyme-linked immunosorbent assay with high sensitivity and specificity. A tissue blot immunoassay indicated that CCYV localizes in the phloem tissues of the petiole and is distributed in tiny spots within the leaf lamina.

An Induced Hypersensitive-Like Response Limits Expression of Foreign Peptides via a Recombinant TMV-Based Vector in a Susceptible Tobacco

BackgroundBy using tobacco mosaic virus (TMV)-based vectors, foreign epitopes of the VP1 protein from food-and-month disease virus (FMDV) could be fused near to the C-terminus of the TMV coat protein (CP) and expressed at high levels in susceptible tobacco plants. Previously, we have shown that the recombinant TMV vaccines displaying FMDV VP1 epitopes could generate protection in guinea pigs and swine against the FMDV challenge. Recently, some recombinant TMV, such as TMVFN20 that contains an epitope FN20 from the FMDV VP1, were found to induce local necrotic lesions (LNL) on the inoculated leaves of a susceptible tobacco, Nicotiana tabacum Samsun nn. This hypersensitive-like response (HLR) blocked amplification of recombinant TMVFN20 in tobacco and limited the utility of recombinant TMV vaccines against FMDV.Methodology/Principal FindingsHere we investigate the molecular mechanism of the HLR in the susceptible Samsun nn. Histochemical staining analyses show that these LNL are similar to those induced in a resistant tobacco Samsun NN inoculated with wild type (wt) TMV. The recombinant CP subunits are specifically related to the HLR. Interestingly, this HLR in Samsun nn (lacking the N/N′-gene) was able to be induced by the recombinant TMV at both 25°C and 33°C, whereas the hypersensitive response (HR) in the resistant tobacco plants induced by wt TMV through the N/N′-gene pathways only at a permissive temperature (below 30°C). Furthermore, we reported for the first time that some of defense response (DR)-related genes in tobacco were transcriptionally upregulated during HLR.ConclusionsUnlike HR, HLR is induced in the susceptible tobacco through N/N′-gene independent pathways. Induction of the HLR is associated with the expression of the recombinant CP subunits and upregulation of the DR-related genes.

Potent inhibition of the classical pathway of complement by a novel C1q-binding peptide derived from the human astrovirus coat protein

Previous work from our laboratories has demonstrated that purified, recombinant human astrovirus coat protein (HAstV CP) binds C1q and mannose-binding lectin (MBL) inhibiting activation of the classical and lectin pathways of complement, respectively. Analysis of the 787 amino acid CP molecule revealed that residues 79–139 share limited sequence homology with human neutrophil defensin-1 (HNP-1), a molecule previously demonstrated to bind C1q and MBL, inhibiting activation of the classical and lectin pathways of complement, respectively. A 30 amino acid peptide derived from this region of the CP molecule competitively inhibited the binding of wild-type CP to C1q. The parent peptide and various derivatives were subsequently assayed for C1q binding, inhibition of C1 and C4 activation as well as suppression of complement activation in hemolytic assays. The parent peptide and several derivatives inhibited complement activation in these functional assays to varying degrees. One peptide derivative in particular (E23A) displayed superior inhibition of complement activation in multiple assays of classical complement pathway activation. Further analysis revealed homology to a plant defensin allowing development of a proposed structural model for E23A. Based upon these findings, we hypothesize that further rationale optimization of E23A may result in a promising therapeutic inhibitor for the treatment of inflammatory and autoimmune diseases in which dysregulated activation of the classical and lectin pathways of complement contribute to pathogenesis.