ES2230971B1 - SYSTEM OF SEROLOGICAL DETECTION OF THE VIRUS OF THE YELLOWING VENUES OF CUCUMBER VEIN YELLOWING VIRUS (CVYV). - Google Patents

Abstract

Serological detection system of the cucumber vein yellowing virus, cucumber vein yellowing virus (CVYV). The object of the present invention is a method of serological detection of Cucumber Vein Yellowing Virus (CVYV) using antisera containing specific antibodies against CVYV capsid protein (CP). The method of obtaining the antiserum comprises obtaining the CP protein of CVYV recombinate, by expression in a host cell, (ii) optionally, purification of said CPYV recombinant CP protein obtained in (i), e (iii) Immunization of mammals or other animals or animal cells by using said CVYV recombinant CP protein and subsequent obtaining of the sera of said animals or cell culture supernatants containing said antibodies. The genetic constructs necessary for obtaining the CVYV recombinant CP protein are also part of the present invention.

Description

Procedure for obtaining protein recombinant vein yellowing virus CP capsid of cucumber, cucumber vein yellowing virus (CVYV) and its Applications.

Technical sector

Agricultural Biotechnology Phytopathology Virology vegetable. Detection of pathogenic viruses by techniques serological The invention provides a serological system of detection in plants of the plant pathogen called yellowing of cucumber veins, cucumber vein yellowing virus (CVYV).

State of the art

Plant viruses are pathogens responsible in their entirety for numerous diseases (Hull, 2002). The most numerous taxonomic group of viruses is the one formed by the Potyviridae family, which includes viruses that can affect virtually all crops and cause significant economic losses worldwide. The majority of viruses in this family (more than 100 definitive members and almost as many as possible) belong to the genus Potyvirus . In addition to this genus, at the moment the viruses of this family have been classified into five other genera: Bymovirus, Rymovirus, Tritimovirus, Macluravirus , and Ipomovirus . All members of this family have elongated and flexuous particles, and with the exception of Bymoviruses that are bipartite viruses, the rest of the family viruses contain a single molecular form in their genome, consisting of a single-stranded messenger RNA. This genomic RNA encodes a polyprotein that is proteolytically self-processed to generate the various gene products. An important taxonomic characteristic is the type of vector organism that carries out the transmission of each virus under natural conditions, and which can be insects such as aphids ( Potyvirus and Macluravirus ) or whiteflies ( Ipomovirus ), mites ( Rymovirus and Tritimovirus ) or fungi ( Bymovirus ).

Within the Potyviridae family, the genus Ipomovirus is characterized by including virus transmitted by whiteflies ( Homoptera, Aleyrodidae ) among which are sweet potato mottle virus, sweetpotato mild mottle virus, SPMMV, and brown striatum virus of cassava, cassava brown streak virus, CBSV. A taxonomically related virus with these is the cucumber vein yellowing virus, cucumber vein yellowing virus (CVYV). Initially considered as an unclassified virus (Sela et al., 1980), CVYV has been proposed as a member of the genus Ipomovirus based on biological (whitefly transmission), cytological (presence of cylindrical cytoplasmic inclusions with a typical grinder or " pinwheel "), morphological (elongated and flexuous viral particles) and molecular (partial sequence comparison of NIb and CP proteins) (Lecoq et al. 2000). At this time, CVYV is considered to have a single-stranded and messenger RNA genome like the other ipomoviruses, but its complete sequence is not yet known, and there are only partial sequence data of some viral isolates, all corresponding to region 3 'of the genome (access numbers in Genebank AF233429 and AJ301640). CVYV causes a serious disease in its natural hosts, being found only recently in the Eastern Mediterranean region. However, CVYV has recently been found in our country, seriously threatening the production of important crops such as melon, cucumber, watermelon and zucchini (Cuadrado et al., 2001a).

CVYV is semi-persistent transmissible by the whitefly Bemisia tabaci (Gennadius), and its host range is mainly restricted to cucurbitaceae (Mansour and M usa, 1993), although it has also been detected in plants of other families (Jansen et al . , 2002). Its appearance in Almería during the year 2000 together with its rapid diffusion through the area to different crops of cucurbitaceae have made it a serious threat for the production of crops of great economic importance. Initially CVYV appeared in Spain in the area of the west of Almeria, affecting protected cucumber crops, later finding that it also affects melon and watermelon crops. The problem caused by the virus has led to replace crops with serious consequences for export and with significant losses of image and credibility, which are linked to the direct economic losses caused by the virus. The seriousness of the situation is maintained due to the lack of suitable tolerant varieties in all host species, and the practical difficulties of adequate phytosanitary control of the whitefly that prevents the spread of the virus. As in other virosis, the establishment of effective CVYV control measures depends largely on the availability of virus detection methods that are specific, sensitive and rapid. At the moment, detection can be carried out, thanks to partial sequence knowledge, by molecular hybridization (Aranda and Fernández Marco, 2001) or by RT-PCR techniques (Cuadrado et al., 2001b).

Serological methods of virus detection are among the most widespread for their use in the case of plant virosis (Hampton et al., 1990), including especially those based on ELISA techniques (Clark and Adams, 1977) and western blot (Towbin et al ., 1979). In general, protein detection methods are easier to apply than those based on nucleic acid detection. Apart from the advantages of speed and ease of use, ELISA or western blot serological tests are known and used by most of the sectors involved in the control of virosis. All this makes them a fundamental tool to successfully address phytosanitary measures aimed at controlling a newly introduced virosis. In order to develop the different serological detection methods, it is necessary to have specific antibodies capable of recognizing the pathogen considered.

Antibodies are molecules produced by the mammalian immune system in response to foreign agents. Current technology allows polyclonal or monoclonal antibodies to be obtained. Polyclonal antibodies are derived from the serum of the blood of immunized animals, and contain a population of different antibodies that react against different antigenic determinants. Monoclonal antibodies are obtained by selecting cells that secrete a single type of antibody against a specific epitope of the antigen used for immunization, and can be immortalized with technologies such as hybridoma production (Köhler and Milstein, 1975) or similar ones to allow its production (Shepherd, 2000). It is also possible to apply recombinant DNA technology to antibody production (McCafferty et al ., 1996). In general, to obtain antibodies, it is necessary to previously have the antigen capable of inducing an immune response, that is, the specific immunogenic protein, which can be native or recombinant.

In the case of CVYV, it is especially important to have specific antibodies that serve to detect, identify and properly characterize the virus. To date, no antibodies against CVYV are available, probably due to the difficulties of purification of the viral particles (Mansour and Musa, 1993; Lecoq et al., 2000) to be used as antigens in animal immunization. Therefore, the possibility of expressing the CVYV CP gene in a heterologous system has been considered as a way to obtain an antigen suitable for the immunization of animals, and thus be able to obtain specific antibodies capable of recognizing the virus. The expression of recombinant proteins can be carried out in different systems, using available cloning technology (Sambrook et al., 1989) and different expression systems (Harnes, 1999) that can be used by prokaryotic, eukaryotic organisms (Glorioso and Schmidt, 1999 ) or even in vitro systems (Kudlicki et al ., 1992). Recombinant proteins can be purified with different systems (Roe, 2001) to be subsequently used as antigens. The expression and purification of a CVYV-derived recombinant protein for use in animal immunization and obtaining specific antibodies capable of recognizing the virus has been the objective of the present invention, and its novelty is that it makes available a CVYV serological detection system. , so far nonexistent.

Explanation of the invention.

The present invention provides a serological detection system for cucumber vein yellowing virus (CVYV), using antisera containing specific antibodies against CVYV CP capsid protein.

In one aspect, the invention relates to a antiserum comprising specific antibodies against a part of the CVYV CP protein or against the full CVYV CP.

In another aspect, the invention relates to a procedure for obtaining antisera containing antibodies specific against CVYV CP protein, characterized in that It comprises the following steps: (i) obtaining the CP protein full CVYV recombinant, or a part thereof, by means of its expression in a host cell, (ii) optionally, purification of said CVYV recombinant CP protein obtained in (i), e (iii) Immunization of mammals or other animals or animal cells by using said CVYV recombinant CP protein (complete or a part) and the subsequent obtaining of the sera of said animals or cell culture supernatants containing said antibodies.

In another aspect, the invention relates to a nucleic acid construct comprising: (i) a first nucleotide sequence capable of regulating form expression inducible in host cells with the appropriate characteristics of: (ii) a nucleotide sequence encoding a protein purifiable and (iii) a nucleotide sequence that can comprise a coding sequence for a fragment of the CVYV recombinant CP protein, or CP protein full CVYV recombinant.

In another aspect, the invention relates to a expression vector comprising said acid construct nucleic.

In another aspect, the invention relates to a host cell transformed with said acid construct nucleic or with said vector.

In another aspect, the invention relates to a CVYV recombinant CP protein (complete or a part) obtained after expression in host cells of the construction of described nucleic acid or vector, purified by methods conventional including, among others, affinity chromatography  with an amylose resin column and preferably separated from the fusion protein by proteolytic cutting. The use of this protein to immunize mammals or other animals, or cells of Animals also constitutes an aspect of this invention.

In another aspect, the invention relates to the use of the described antiserum or of the antibodies obtained from said antiserum, in the elaboration of a laboratory reagent for the detection of CVYV in plants infected by the virus, including hosts within the family of Cucurbitaceae or any family of plants, as well as insects or other organisms that can act as vectors of the virus, or in media where virus reservoirs such as water, soil, plant debris or other means could be found.

The invention is novel because it uses the serological detection of a protein component of the particle viral while CVYV detection methods previously Existing (molecular hybridization and RT-PCR) They used nucleic acid detection. In addition the invention provides a system for obtaining the recombinant CP protein whose amino acid sequence is identical to that of the CP protein natural, with the same characteristics as this one and that is capable of induce an immune response after being injected into an animal or animal cell, and so that the antibodies obtained are able to recognize the presence of the virus.

Brief description of the figures

Figure 1A shows an outline of the CVYV genomic organization, showing viral polyprotein as a rectangle indicating the location of the CP protein at its carboxyl end, and the strategy followed to obtain of the construction used by amplification by RT-PCR with oligonucleotides (represented by arrows) and subsequent incorporation of the corresponding sequence (represented by a bar) to a cloning vector, and subsequently to an expression vector.

Figure 1B shows a scheme of the protein of fusion expressed by the corresponding vector, representing the MBP, the cutoff sequence of factor Xa and the position in which Insert the CVYV CP. Under the scheme the sequence of amino acids in the region between the two fused proteins, with indication of the corresponding proteolytic cut-off point (represented by an arrow).

Figure 2 shows the result of an assay western-blot type, using an antiserum obtained in the present invention. Lane 1 shows the position of the intended molecular weight markers (Bio-Rad), lane 2 was loaded with an extract obtained from a plant infected with CVYV virus, and in lane 3 an extract of a non-inoculated plant. The expected size of the CVYV CP is indicated with an arrow

Detailed description of the invention

The present invention provides a method of serological detection of the vein yellowing virus cucumber, cucumber vein yellowing virus (CVYV).

This requires obtaining antisera containing specific antibodies capable of recognizing CP of the isolates of said virus. These antisera and antibodies they contain constitute an object of the present invention.

Antibodies are immunoglobulin molecules produced by an animal's immune system in response to a antigenic stimulus Serum antiserum obtained from the blood of an animal inoculated with an antigen; the antiserum contains a population of antibodies capable of recognizing different antigenic determinants (or epitopes) of the antigen, constituting what is known as polyclonal antibodies. A monoclonal antibody would come from the selection of cells that secrete a single type of antibody against a specific epitope of the antigen used for immunization and therefore it is of homogeneous antibodies in type and specificity by a single epitope There are several techniques that allow obtaining antibodies and antisera many of them described and gathered in the Hampton et al. (1990), and references included in it. Such as used in the present invention, the antiserum encompasses both the serum obtained from immunized animals, and the cell culture supernatants containing the antibodies

To obtain the antisera it is necessary immunize mammals or other animals or animal cells with proteins that are immunogenic, and that can be native or recombinant In the case of CVYV, it is possible to use as antigen a recombinant protein corresponding to the sequence of the CP protein of a CVYV isolate. Immunization with this protein will serve to activate the immune system of animals and generate in response specific antibodies capable of recognizing both the antigen used (recombinant protein) and the native viral protein The protein can be used as an antigen CVYV CP, native or recombinant. The protein used can be complete or part of it, since the distribution of possible epitopes along the protein will depend on the immunogenicity of the various regions; therefore a fragment of antigen can generate immune response against epitopes that contains, and as those same epitopes are found in the protein whole, the antibodies will also recognize the whole protein. It must be considered that a sequence epitope can recognize a discrete number of amino acids from 5 to 7 residues, and a conformational epitope can recognize different number of residues, contiguous or not, but distributed in a certain way in the space: as long as the antibody finds the amino acids or the structure that is able to recognize, will join the epitope and be will produce recognition.

In another aspect, the invention relates to a procedure for obtaining antisera containing antibodies specific against CVYV CP protein, characterized in that It comprises the following steps: (i) obtaining the CP protein CVYV recombinant by gene expression in a cell host, (ii) optionally, purification of said CP protein CVYV recombinant obtained in (i), e (iii) Immunization of mammals or other animals or animal cells by use of said CVYV recombinant CP protein and the subsequent obtaining of the sera of said mammals or others animals or cell culture supernatants containing the mentioned antibodies that constitute the antiserum. The first (i) and the third step (iii) are essential for obtaining the antiserum, while the second step (ii) of purification is not essential but can significantly improve the quality and effectiveness of the antiserum obtained, by avoiding nonspecific responses to other possible components of the preparation.

Recombinant proteins can be obtained through induced expression in a heterologous system that can be based on the use of different types of host cells such as bacteria, fungi or yeasts (of various types, Sambrook et al. 1989), insect cells ( for example by means of baculovirus), cells of mammals or other animals, plants (transgenic or with viral vectors) and whole animals (secretion in milk, for example) (Harnes, 1999; Glorioso and Schmidt, 1999). In addition, there are in vitro protein production systems, transcription followed by translation in cell-free systems (Kudlicki et al ., 1992), some of them already commercial.

In this case the expression in fusion protein host cells that incorporate a sequence corresponding to the CP of CVYV, or part of it, together with any protein with characteristics that facilitate its purification and separation of other products and components, cellular or other type, derived from the chosen expression system. To do this it is It is necessary to obtain adequate genetic constructions. Thus In another aspect, the invention relates to a construction of nucleic acid comprising: (i) a first sequence of nucleotides capable of regulating inducible expression in host cells with the appropriate characteristics of: (ii) a nucleotide sequence encoding a purifiable protein and (iii) a nucleotide sequence that may comprise a coding sequence for a fragment of the CP protein CVYV recombinant, or CVYV recombinant CP protein complete. Obtaining said genetic constructs implies the following steps:

a) Obtaining a genetic construction containing the CVYV full or partial CP

As a preliminary step to the preparation of the genetic constructs provided by the present invention to be able to express the protein that will be used for obtaining the antiserum, it is necessary to clone a DNA fragment containing the coding sequence for the CVYV CP complete or part of it . To do this, a viral CVYV isolate can be used as characterized and described by Aranda and Fernández Marco (2001), named CVYV-A1 LM. The coding sequence for the CP of the selected CVYV isolate can be donated in a cloning vector from an amplified fragment in an RT-PCR reaction. As a template for the reaction, a preparation of RNAs extracted from plants infected with the corresponding isolate can be used, under conditions similar to those described by Aranda and Fernández Marco (2001). The amplification can be carried out using as primers a d T oligonucleotide (therefore complementary to the polyA tail characteristic of all viruses belonging to the Potyviridae family) for the synthesis of the first complementary chain to the viral RNA, and a specific oligonucleotide for the onset of the CP, preceded by a spacer and the target of a suitable restriction enzyme to facilitate subsequent cloning. The length of the oligonucleotide must be sufficient to allow stable hybridization with the template to be amplified, guaranteeing the specificity of the reaction product. Identification of the amino terminal end of the CP of CVYV can be performed by aligning the known partial CVYV sequences with the corresponding products of other viruses of the Potyviridae family in which the onset of their CP has been identified. The process would be the same if we intend to amplify a fragment of the CVYV CP protein, with the difference that the synthesis of the first chain complementary to the viral RNA could be performed with an oligonucleotide complementary to the coding sequence of another internal part of the protein CVYV CP, either the sequence of the second oligonucleotide used in the amplification would correspond to the coding sequence of an internal part of the CVYV CP protein, or both first and second oligonucleotides could amplify an internal fragment of the CVYV CP. In either case, the plasmid containing the insert corresponding to the complete or truncated CVYV CP must be identified, purified and checked by sequencing containing the expected sequence.

b) Obtaining a recombinant genetic construct which merges the CP of CVYV to another protein product used for its purification

Once the coding DNA fragment is cloned for the full CVYV CP or a fragment thereof it is necessary fuse it to the coding sequence of another protein product that allow us its purification; also the DNA fragment that codes for the resulting protein product must be cloned into a vector that allows expression in inducible form in cells host with the appropriate characteristics of said product protein For this, the fragment corresponding to the CP of CVYV can be obtained from a plasmid as described in the previous section, by restricting your DNA with enzymes of suitable restriction, which cut in position 5 'with respect to theoretical start of the CP gene or the fragment considered, and in 3 ' after the CP gene, or the fragment considered, being able to include the 3'UTR region vira) (non-coding) and a fragment of polyA. This fragment must be donated in a targeted manner. expression vector digested with identical or compatible enzymes with the previous ones, so that the genetic construction resulting can be translated using own characteristics of the expression vector used. In particular they must be met the conservation requirements of the reading phase for the case of fusion proteins. In the case of merger of the CP of CVYV at the carboxyl end of the final fusion protein, it keep the termination codon of the CP, or in the case of no be present, a stop codon will be incorporated that prevents Translation of unwanted sequences. If they were done constructions in which the CVYV CP will occupy the amino position, it will be necessary to include a translation initiation sequence to ensure that the product is translated in the system used. The process would be exactly the same if it were to express a part of the CVYV CP protein and is not complete. Plasmid containing the insert must be identified, purified and checked by sequencing that contains the expected sequence, and that is able to be expressed in the heterologous system selected.

The vector or vectors comprising said nucleic acid constructs constitute another aspect of the invention.

In another aspect, the invention relates to a host cell transformed with said acid construct nucleic or with said vector. In another aspect, the invention is relates to a CVYV recombinant CP protein obtained after host cell expression of the nucleic acid construct described or the vector, purified by conventional methods including, among others, affinity chromatography with a amylose resin column and preferably separated from the fusion protein by proteolytic cutting.

Expression in the heterologous system chosen from the protein of merger containing CVYV CP

After obtaining the expression vector it is necessary to transform a host cell prepared for protein expression. As already indicated, there are different types of host cells and even in vitro systems for protein expression. In this case, the expression in bacteria such as Escherichia coli has been chosen. Once the host cell carrying the genetic construct or the vector is obtained, following the specific procedures of the protein expression system used in the previous section, the corresponding protein will be induced and expressed. It will be verified that the protein is expressed, and will proceed to its isolation and purification, separating it from the rest of products or components of the expression system. The properties of the protein fused to the CP of CVYV will be used for this, that is, the corresponding affinity of the fusion product, by means of conventional purification techniques that are applicable, such as, among others, those of affinity chromatography. . The final protein product can be analyzed by electrophoresis in polyacrylamide gels in the presence of sodium dodecyl sulfate (SDS-PAGE) or a similar analysis technique, to assess the degree of purity obtained and quantify the availability of protein to establish adequate conditions of obtaining.

In the event that the fusion protein incorporates some specific protease recognition sequence for allow the digestion and separation of the product corresponding to the CP of CVYV, the appropriate enzyme will be used in the conditions d and use d written for each case to make the separation proteolytic Digestion will be checked by an analysis of the type SDS-PAGE or equivalent, to identify the CP of CVYV as a band with the mobility corresponding to the expected size. Thus the CVYV CP protein that is you get will present the same amino acid sequence as the natural CP protein, incorporating only waste additional necessary for the fusion left by the cut proteolytic once done.

The use of the protein obtained to immunize mammals or other animals or animal cells also constitutes An aspect of the present invention. This will proceed as Indicate below:

a) Preparation of proteins for immunization

The proteins obtained, well the fusion containing the CVYV CP directly obtained after its expression, or CVYV CP after digestion with protease corresponding, and optionally after purification by affinity chromatography, they will be prepared to be used in the immunization of animals, being able to undergo electrophoresis and an elution process in suitable equipment. Yes if necessary, the eluate can be concentrated and dialyzed to get the final product in a buffer suitable for the protein folding, and at a sufficient final concentration for use

b) Immunization of animals or animal cells and obtaining antisera

The purified and prepared protein can be mix with a coadjuvant of choice, and the injections in the chosen animals or animal cells. In in this case rabbits have been chosen, the doses to be used must be suitable for the size of the animal, and the process has to be repeated during an established calendar for each species, up to complete the sufficient number of immunizations to obtain a adequate immune response. Test bleeds may be performed at different times after immunizations, with the purpose to assess the degree of immune response obtained, and the presence in the serum of specific antibodies capable of recognizing antigen, in this case the CVYV CP.

After immunization, the antiserum containing specific antibodies that can be used in the elaboration of a laboratory reagent for the detection of CVYV in plants infected by the virus, including hosts within the Cucurbitaceae family or any family, is obtained from the animal's blood. of plants. Which constitutes an aspect of the invention.

Since CVYV is a virus transmitted by whiteflies, it remains for some time inside the insect. Having a system to detect it could serve to act against the vector in a rational way, avoiding having to constantly perform insecticidal treatments against flies. Likewise, epidemics normally start from an infected host, but they can also start from a reservoir that allows the pathogen to survive in adverse conditions. Thus when there is no cucurbits in the field, the virus can be kept in other infected plants, such as wild plants or weeds, or in residues of previous crops, and even in other possible sources of inoculum, such as soil or cultivation tools , which are contaminated with viruses. Detecting it in these reservoirs can be used to eliminate it, and prevent an epidemic from starting. Thus, the use of the antiserum containing specific antibodies against CVYV CP protein in the preparation of a laboratory reagent for the detection of CVYV in insects or other organisms that can act as vectors of the virus, or in media where reservoirs could be found of the virus such as water, soil, plant debris or other means also constitute an aspect of the present invention.

For all this it is necessary to verify the CVYV detection capacity of said antiserum or of the antibodies obtained from it.

Test of CVYV detection capacity in plants

The antisera obtained from the blood of immunized animals to check their ability to Detection of the viral pathogen called CVYV. For this they will be able to use different serological techniques of the detection of plant virus, starting from host plants infected with the virus, and making relevant comparisons with non-plants infected. Its specificity will be evaluated using in the tests different viruses, and their sensitivity, for which they will be tested known dilutions.

Molecular Biology procedures necessary to cover the successive stages of the process of obtaining and practical use of the specific antiserum can be perform according to the usual techniques described in Laboratory manuals such as Sambrook et al. (1989), and the Serological procedures are based on the techniques described and gathered by Hampton et al. (1990), and references contained in the same.

Detailed description of the content of the figures.

Figure 1A shows an outline of the CVYV genomic organization. Genomic RNA is represented as a bar, indicating at its 5 'end the possible presence of a covalently bound protein (called VPg) and at its 3 'end a polyA tail. Viral polyprotein is shown on RNA as a rectangle, above which the location of the  possible functional domains or gene products, called P1, HC, P3, 6K1, Cl, 6K2, Nla, Nlb and CP (from the amino end to carboxyl). The CP protein is located at the carboxyl end of polyprotein The strategy followed for the obtaining the construction used, which part of the amplification by RT-PCR with the oligonucleotides (represented by arrows) of a sequence (represented by a bar) that includes the entire region of the CP until the start of the queue of polyA. This fragment (corresponding to the sequence SEQ. ID No. 1) is subsequently incorporated into a cloning vector, and subsequently to the expression vector.

Figure 1B shows a scheme of the protein of fusion expressed by the corresponding vector. In amino position terminal represents the MBP, followed by a spacer containing the cutting sequence of factor Xa (corresponding to amino acids of the SEQ. ID. . 2) and the position in which it is inserted CVYV CP. Under the scheme the sequence of amino acids in the region between the two fused proteins, with indication of the proteolytic cut-off point (represented by a arrow) corresponding to digestion with factor Xa.

Figure 2 shows the result of a western-blot test, using an antiserum obtained in the present invention. Lane 1 shows the position of the intended molecular weight markers (Bio-Rad), in lane 2 an extract obtained from a pickle plant ( Cucumis sativus variety SMR-58, Fitó seeds) infected with CVYV virus was loaded, and in lane 3 an extract from a non-inoculated plant. The expected size of the CVYV CP is indicated by an arrow. The samples correspond to 4 µl of total protein extracts at approximately 1: 5 dilution (sheet weight: volume of extraction buffer), and were subjected to SDS-PAGE electrophoresis in 4-10% discontinuous polyacrylamide gels, and subsequently electrotransferred to PVDF membranes to be detected using CVYV anti-CP antiserum at 1: 2000 dilution followed by conjugated anti-species (GAR-PO) at 1: 1000 dilution and colorimetric detection with chloronaphthol.

Example number one

Molecular Biology procedures have been performed in accordance with the usual techniques described in Laboratory manuals such as Sambrook et al. (1989), and the Serological procedures are based on the techniques described gathered by Hampton et al. (1990), and references in it. The materials and the necessary stages for the process of obtaining detailed below.

Viral isolate

The so-called isolate has been used CVYV-A1 LM described by Aranda and Fernández Marco (2001).

Obtaining a genetic construct containing the CP full CVYV

The CP of the CVYV-A1LM isolate is cloned into a pGEM-T Easy II vector (Promega) to from an amplified fragment in a reaction of RT-PCR As a template for the reaction, a Preparation of RNAs extracted from plants infected with the isolate corresponding, under conditions similar to those described by Aranda and Fernández Marco (2001). The amplification was carried out with oligo dT for the synthesis of the first complementary chain to  Viral RNA, and with a specific oligo for the onset of CP, preceded by a spacer and the enzyme target of EcoRI restriction, with the sequence 5 'GAA TTC ACC AAG GCA GAC GAC ATT GAG AAA G 3 '(SEQ. ID. NO. 5).

The plasmid containing the insert was identified, purified and checked by sequencing.

Obtaining a recombinant genetic construct that fuses the MBP to the CP of CVYV

The fragment corresponding to the CP was obtained from the plasmid described in the previous section, by restricting its DNA with EcoRI (enzyme that cuts at 5 'position with respect to the theoretical start of the CP gene) and Spel (target located at 3 'after the CP gene, the 3'UTR viral region and a polyA fragment). This fragment was cloned in an oriented manner into the expression vector pMAL-2c (New England Biolabs) digested with the enzymes EcoRI and Xbal (the latter compatible with Spel). The polylinker of the plasmid pMAL-2c leads following the malE gene MBP one SacI site, a spacer (N_ {10} LG), the target cutting Xa (IEG R / IS factor, where the indicating rod position proteolytic cut), followed by EcoRI-BamHI-XbaI targets (Spel compatible) –SaII-PstI-HindIII. The definitive clone pMAL-CVYV-CP is therefore left with the structure: [MBP] N_ {10} LGIEGR / ISEFTK [CP of CVYV].

This clone was selected, purified and its sequence confirmed by sequencing.

Expression in MBP-CP fusion bacteria of CVYV

E. coli strain BL21 (DE3) transformed with plasmid pMAL-CVYV-CP was used for expression. From pre-cultures grown overnight in agitation at 25 °, the higher volume cultures maintained at the same temperature are started, controlling the growth by optical density readings at 650 nm until reaching a value between 0.5 and 1 units. The induction of the expression is performed by adding IPTG (isopropyl-? -D-thiogalactopyranoside) at a final concentration of 0.3 mM, and at 3 hours the cells are collected by cold centrifugation at 4000 xg for 20 minutes, resuspended in buffer of lysis containing 10 mM Tris-HCl pH 7.8, 200 mM NaCl, 1 mM DTT (dithiothreitol), 0.25% Tween-20 (polyoxyethylene (20) sorbitan monolaurate), 1 mM PMSF (phenylmethylsulfonyl fluoride) and 5 mM EDTA (acid ethylenediaminetetraacetic acid), and frozen in liquid nitrogen to be stored at -80 ° C. The cells were thawed and lysed by adding 2 mg / ml lysozyme to the lysis buffer and incubating 30 minutes with shaking, followed by 5 sonication cycles of 30 seconds duration, and a freeze-thaw cycle. After centrifugation at 9000 xg for 30 minutes, the supernatant was subjected to affinity chromatography on a column of amylose resin (New England Biolabs) equilibrated in 10 mM Tris-HCl, pH 7.8, 200 mM NaCl, 1 mM DTT, 0.25% Tween-20 After washing with the equilibration buffer, elution was carried out using the same buffer containing 10 mM maltose, the successive fractions being collected to be analyzed by electrophoresis in polyacrylamide gels in the presence of sodium dodecyl sulfate (SDS-PAGE). The presence of a majority protein band of the expected size at MBP-CP fusion, approximately 82 kDa, was observed, which was eluted from the column in the successive fractions.

Proteolytic separation of the MBP from the CP of CVYV

The cut was tested with factor Xa (Roche) on CVYV MBP-CP fusion protein, adding to the eluted from the affinity column an amount of protease for reach an approximate ratio between 1:25 and 1:40 (protease: substrate), and incubating for two days at 4 ° C. Digestion is check by analysis by SDS-PAGE, the CP being identified as a band of approximate size 40 kDa, and of mobility greater than the free MBP (42 kDa).

Protein electroelution

The protein expressed after digestion with the factor Xa was subjected to SDS-PAGE electrophoresis and was identified the bands by staining with 0.25% Blue of Coomasie R-250 in distilled water. The band corresponding to the CVYV CP was cut, and faded with several washed in water, then subjecting it to electroelution in the Electro-Eluter Model 422 equipment (Bio-Rad), following the instructions of the maker.

Preparation of proteins for immunization

The electroeluted was concentrated with a Centricon YM-30 (Millipore), gradually replacing the elution buffer with 0.2 M NaHCO 3, 0.02% SDS. Finally the product was dried under vacuum using a Speedvac (Savant) equipment, and resuspended in sterile PBS buffer. The concentration was estimated by SDS-PAGE analysis and staining, compared with a pattern of protein samples of known concentration.

Immunization of rabbits and obtaining antisera

The purified and resuspended protein in PBS is mixed (1: 1) with Freund's complete adjuvant (Sigma). The injections were made in female New Zealand rabbits, neck subcutaneous, using doses ranging from 80 to 350 \ mug of protein, spaced 3 weeks, until completing five immunizations The first test bleeding was performed 8 days after the 4th injection. The animal's sacrifice and sangria Complete was carried out 8 days after the 5th immunization.

The serum was obtained from the samples of blood, subjecting them to incubations for 30 minutes at 37º followed by 16 hours at 4th. The serum released from the clot is clarified by centrifugation at 4000 xg for 10 minutes, and was tested at different dilutions by Western blot and ELISA.

CVYV detection in plants

CVYV was detected in pickle plants ( Cucumis sativus ) of the SMR-58 (Fitó) variety inoculated with the viral isolate CVYV-A1LM. Gherkin plants were infected by mechanical inoculation or experimental transmission by Bemisia tabaci . Once the symptoms appeared (between 6 and 12 days after inoculation), the infection was confirmed by a molecular hybridization assay or by RT-PCR. For serological analyzes, total protein extracts from each plant were obtained, and compared with those obtained from plants of the same variety that had not been inoculated with the virus. They were tested in Western blot, following the usual procedures using the antiserum produced in a rabbit immunized with CVYV CP protein. The antiserum in dilutions 1: 1000 and 1: 2000 recognized in Western blot in the infected plant extracts a corresponding band with the electrophoretic mobility of approximately 40 kDa, which is the theoretical mobility of the CP of the virus. There was no recognition of products of similar size in extracts from uninfected plants.

Example number 2

The immunization of a rabbit with a fusion protein not digested with factor Xa, and which therefore incorporates the CP of CVYV linked to the protein used for purification, in this example the MBP. The materials and necessary stages for the procurement process are detailed at below, and are the same as those described in example number 1 in relation to the viral isolate, to obtain a genetic construction containing the complete CVYV CP, to the obtaining a recombinant genetic construct that fuses the MBP to CVYV CP, and to expression in fusion bacteria CVYV MBP-CP. The fusion protein, about 82 kDa in size, was directly subjected to electrophoresis SDS-PAGE, continuing with your electroelution, protein preparation, rabbit immunization and obtaining corresponding antiserum, acting again in parallel to the described in example 1. The doses of protein used in the immunizations ranged in this example between 60 and 300 µg of protein.

Plants were prepared to perform detection assays similar to those described in example number 1, by Western blotting using antisera produced in a rabbit immunized with the MBP fusion protein with CVYV CP. The antiserum recognized in Western blot in the extracts of infected plants a band corresponding to the electrophoretic mobility of approximately 40 kDa, which is the theoretical mobility of the CP of the virus. There was no recognition of products of the corresponding size in extracts from non-plant
infected.

For preliminary ELISA tests, used an indirect analysis scheme, performing the upholstery of the plates with total plant extracts in buffer pH 9.6 carbonate bicarbonate supplemented with 0.1% of skim milk powder as a blocking agent. After successive incubations with immunoglobulins (purified from antiserum by affinity chromatography with Protein A Sepharose CL-4B from Pharmacia), and Enzyme conjugated anti-species, each step followed by washing with PBST, the incubation with the own colorimetric substrate of the enzyme used and reading automatic at the appropriate wavelength. Readings were obtained of optical density in samples from infected plant that twice exceeded those corresponding to a control plant negative not infected by the virus.

References

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Claims (20)

1. Procedure for obtaining an antiserum comprising specific antibodies against the CPY capsid protein of the CVYV cucumber vein yellowing virus characterized in that it comprises the following steps:

(i)

obtaining the CP protein CVYV recombinant by gene expression in a cell Guest,

(ii)

optionally, purification of said CVYV recombinant CP protein obtained in (i), e

(iii)

immunization of mammals or others animals or animal cells by using said CVYV recombinant CP protein and subsequent obtaining of the sera of said mammals or other animals or supernatants of animal cells containing the aforementioned antibodies that They constitute the antiserum.

2. Method of obtaining an antiserum according to claim 1 characterized in that the obtaining of said CVYV recombinant CP protein is carried out by transformation of a host cell with a nucleic acid construct comprising:

(i)

a first nucleotide sequence inducible by IPTG.

(ii)

a nucleotide sequence encoding a purifiable protein by affinity chromatography.

(iii)

a nucleotide sequence that comprises the coding sequence of the recombinant CP protein CVYV complete or part of it, and optionally.

(iv)

a nucleotide sequence that codes for a cutting site proteolytic as factor Xa that can be used to separate the fusion protein purifiable protein.

3. Method according to claim 2 characterized in that said nucleotide sequence (iii) comprises the sequence identified as SEQ. ID. No. 1 Method according to claim 2, characterized in that the nucleotide sequence (ii) corresponds to the coding sequence for the Escherichia coli malE gene that encodes the protein known as Maltose Binding Protein or MBP. 5. Method according to claim 2 characterized in that the nucleotide sequence (IV) comprises the nucleotide sequence identified as SEQ. ID. No. 3 Method according to claim 2, characterized in that said first nucleotide sequence (i) is constituted by a P lac transcription promoter and the MBP's own translation signals. 7. Method according to claim 2 characterized in that said nucleic acid construction comprises

(i)

a IPTG functional and inducible promoter capable of regulating the expression of a second nucleotide sequence, comprising said second nucleotide sequence:

(ii)

the nucleotide sequence encoding the MBP protein, located preceding

(iii)

the nucleotide sequence identified as SEQ. ID. No. 1,

(iv)

between said sequences of nucleotides (ii) and (iii) the nucleotide sequence identified as SEQ. ID. No. 3

Method according to claim 1, characterized in that the obtaining of said recombinant CP protein (i) is carried out by transformation of a host cell, with a vector comprising a nucleic acid construct defined according to any one of claims 2 to 7. 9. Method according to claim 1 characterized in that the purification of the CVYV recombinant CP protein (ii) is carried out by conventional methods including, among others, affinity chromatography with an amylose resin column. 10. Method according to claim 9 characterized in that after purification the CPYV CP protein is obtained by proteolytic cutting. Method according to claim 1 characterized in that the immunization of mammals or other animals or animal cells (iii) is carried out by the CVYV recombinant CP protein obtained according to any of claims 2 to 10. 12. Nucleic acid construction comprising any of those defined in claims 2 to 7. 13. A gene expression vector characterized in that it comprises a nucleic acid construct according to any of those indicated in claims 2 to 7. 14. Gene expression vector according to claim 1, characterized in that: pMAL-CVYV-CP. 15. A host cell transformed with a nucleic acid construct according to claim 12 or with a gene expression vector according to claims 13 or 14. 16. A host cell according to claim 16 characterized in that it is the cell deposited in the CECT as bacterial strain Escherichia coli with the reference CECT 5757. 17. Use of a construction according to claim 12 or a gene expression vector according to claims 13 or 14, to transform a host cell for induction of expression of CVYV CP protein. 18. CVYV recombinant CP protein obtainable by the procedure described according to any of the claims 15 and 16, and purified according to any of the claims 9 and 10. 19. Antiserum comprising antibodies specific against the CP capsid protein of the virus yellowing of the cucumber veins CVYV obtained by the method described according to any of claims 1 to eleven. 20. Use of the antiserum according claim 19 or the antibodies obtained from said antiserum in the elaboration of a laboratory reagent for the detection of CVYV in:

(i)

virus-infected plants, including hosts within the cucurbitaceae family,

(ii)

in insects or other organisms that can act as vectors of virus, or in means where reservoirs of the Viruses such as water, soil or plant debris.