CN117625672A - Recombinant vector for expressing pepper vein mottle virus and pepper infection method, preparation method and application thereof - Google Patents
Recombinant vector for expressing pepper vein mottle virus and pepper infection method, preparation method and application thereof Download PDFInfo
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention relates to the technical field of genetic engineering, and discloses a recombinant vector for expressing pepper vein mottle virus, a preparation method and application thereof, wherein the method is used for obtaining a ChiVMV whole genome sequence from pepper, constructing a ChiVMV whole genome infectious clone pCB301-ChiVMV, and then inserting a Myc tag into the C end of pCB301-ChiVMV infectious clone NIb protein. The constructed recombinant vector pCB301-ChiVMV NIb-Myc has the following characteristics and advantages: (1) The host can be systematically infected, and the pathogenicity is consistent with that of ChiVMV infectious clone; (2) After infecting a host, the antigen can be efficiently detected by Myc antibodies, and is suitable for large-scale detection of disease resistance of the host; (3) Can express stable NIb-Myc fusion protein, and is suitable for efficiently screening NIb interaction host proteins by utilizing the aspect of immunoprecipitation-mass spectrometry.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to a recombinant vector for expressing pepper vein mottle virus, a pepper infection method, a preparation method and application thereof.
Background
The pepper vein mottle virus (Chilli veinal mottle virus, chiVMV) is an important worldwide virus belonging to Potyvirus and Potyvirus (Potyvirus), and can infect various crops such as pepper, tobacco, tomato, eggplant and the like of the solanaceae plants under natural conditions, so that the yield and quality are reduced. Currently ChiVMV has become one of the main viruses in pepper production, causing important yield and quality loss and even failure. The application of the disease-resistant variety is the most important means for preventing virus hazard, but at present, no effective ChiVMV antiviral variety exists in China, and large-scale disease resistance screening is required to be carried out on materials so as to promote the breeding of the disease-resistant variety. For the detection of virus resistance, the detection needs to be determined by symptom evaluation combined with nucleic acid or protein research means, and in the large-scale detection, the detection of protein is the first choice due to the complicated operation and high cost of the nucleic acid means. In the aspect of virus detection antibody selection, commercial antibodies are preferred because of the advantages of mature technology, low price, easy availability and the like. Therefore, the ChiVMV invasive clone which can stably express the tag protein is of great significance for screening disease-resistant hosts.
ChiVMV is a forward single stranded RNA virus whose genome is about 9.7kb in length, which upon transcription produces a polyprotein, which by itself cleaves or frameshifts to produce 11 mature proteins, including P1, HC-Pro, P3, PIPO, 6K1, CI, 6K2, VPg, NIa, NIb and CP. Of these, the NIb protein is an RNA-dependent RNA polymerase (RNA-dependent RNA polymerase, rdRp), whose function is closely related to replication of the viral genome. In addition to acting primarily as RNA replicase enzymes, NIb has also been shown to play a key role in a variety of viral and host interactions, including mediating allergic reactions, involving in protein post-translational modifications, autophagy, nuclear transport and cell motility, and promoting viral infection. The mechanism of its pathogenesis is currently still unclear as the most central protein for viral replication, NIb.
Therefore, large-scale screening of host factors of the NIb protein becomes an effective means for rapidly researching the functions of the NIb protein. Candidate proteins can be screened from cDNA libraries of plants by constructing bait protein vectors and utilizing yeast two-hybrid technology, etc., but the method is laborious and high in cost. The immunoprecipitation-mass spectrometry combined analysis method enables large-scale screening of the NIb interaction host proteins to be possible, but no existing NIb protein antibody exists at present, and the newly manufactured antibody is influenced by factors such as high price, complicated operation, unstable quality, difficulty in meeting test requirements and the like, so that the construction of a protein fusion carrier is a direction of researchers. The expression modes of different viral proteins are different, 11 viral proteins of ChiVMV have unique expression strategies, and slight sequence variation, insertion or frame shift can cause the viruses to lose pathogenicity or weaken pathogenicity. At present, the construction of the ChiVMV and other virus NIb fusion proteins does not exist, and the insertion position and the insertion type of the exogenous sequence are all blank in research.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a recombinant vector for expressing pepper vein mottle virus, a pepper infection method, a preparation method and application thereof; through various construction strategy experiments, a recombinant infectious clone of the ChiVMV-NIb protein fusion commercialized Myc tag is obtained, and pathogenicity analysis shows that the recombinant vector has systematic pathogenicity to a host, can stably express the NIb-Myc fusion protein, can be used for high-efficiency detection of viruses, analyzes the interaction host factor of important protein NIb, and provides an effective tool for in-depth research on biological functions of the ChiVMV and the coded NIb protein thereof.
The aim of the invention is realized by the following technical scheme:
the invention provides a recombinant vector for expressing pepper vein mottle virus, which comprises pCB301-ChiVMV recombinant infectious clone, wherein the C end of NIb protein is connected with Myc tag;
and the recombinant vector has the sequence shown in SEQ ID: no. 15.
The invention also provides a method for infecting the capsicum by the recombinant vector, which comprises the following steps:
firstly, introducing a recombinant vector into agrobacterium, inoculating the agrobacterium-mediated recombinant vector into Benshi tobacco, observing symptoms, and performing RT-PCR detection to determine that the recombinant vector has pathogenicity;
step two, the inoculated benthamiana attack leaves in the step one are in friction transfer to a first pair of true leaves of the peppers, and symptom observation is carried out on the pepper leaves to determine pathogenicity of the recombinant vector on the peppers;
and thirdly, taking new system leaves of Nib and capsicum annuum, extracting total protein, and determining the existence of recombinant protein NIb-Myc through Western blot analysis.
In the invention, the inventor finds that the inoculation of the capsicum by using the agrobacterium-mediated inoculation method can cause necrosis and shedding of the inoculated leaves no matter the cotyledons and the true leaves of the capsicum are inoculated, so that the capsicum cannot be infected by the recombinant infectious clone; the infection of the cigarette sample is used as an intermediate, and the infection is transferred to cotyledons and true leaves of the capsicum through friction, so that pathogenicity of the vector obtained by the invention on the capsicum can be identified.
The preparation method of the recombinant vector comprises the steps of obtaining a ChiVMV whole genome sequence from capsicum, constructing a ChiVMV whole genome infectious clone pCB301-ChiVMV, and then inserting a Myc tag into the C end of a pCB301-ChiVMV infectious clone NIb protein to obtain the recombinant vector pCB301-ChiVMV: NIb-Myc.
In a preferred embodiment of the present scheme, the preparation method of the recombinant vector specifically includes the following steps:
s1: obtaining ChiVMV whole genome sequence of infected capsicum
Chi-1F/1R, chi-2F/2R, chi-3F/3R is used as a primer, cDNA of a ChiVMV infected pepper sample is used as a template, and a high-fidelity DNA polymerase is utilized to amplify a band containing the ChiVMV whole genome sequence in three sections;
wherein, the nucleotide sequence of the Chi-1F/1R, chi-2F/2R, chi-3F/3R is shown as SEQ ID: no. 1-6;
s2: construction of ChiVMV Whole genome infectious clone pCB301-ChiVM1V
Selecting StuI and SmaI double-enzyme tangential pCB301 vector, carrying out gel recovery and purification on the product and the strip in the step S1, carrying out homologous recombination reaction, then converting the product into escherichia coli, and carrying out positive clone screening and sequencing verification to obtain clone pCB301-ChiVMV with the correct ChiVMV genome nucleotide complete sequence;
s3, constructing a recombinant infectious clone pCB301-ChiVMV of the NIb fusion tag: NIb-Myc
Taking pCB301-ChiVMV plasmid as a vector, and tangentially transforming the pCB301-ChiVMV vector by BamHI and HpaI double enzymes;
synthesizing a plasmid containing (NIb partial sequence+Myc sequence+sequence B+CP partial sequence) DNA Fragment III in a biological company, taking the plasmid as a template, and adopting a CHi-Myc-insert-F/R primer pair to amplify the plasmid to obtain a PCR product;
then purifying the PCR product, recombining and connecting the PCR product with a pCB301-ChiVMV vector subjected to BamHI and HpaI double enzyme digestion, then converting the product into escherichia coli, and obtaining a recombinant vector pCB301-ChiVMV with correct recombination through positive clone screening and sequencing verification: NIb-Myc;
wherein the Fragment III has the sequence ID: a gene sequence shown in No. 15;
the CHi-Myc-insert-F/R primer pair has the sequence of SEQ ID: the gene sequences shown in No.16 and No. 17.
In a preferred embodiment of the present solution, step S3 is followed by a step of infecting peppers;
the step of infecting the peppers is the step of claim 2.
The recombinant vector is applied to large-scale detection of antiviral agents of hosts such as capsicum, tobacco and the like.
Furthermore, the scheme can prove that the NIb-Myc fusion protein in the Nicotiana benthamiana and capsicum can be effectively enriched through immunoprecipitation experiments.
The recombinant vector is applied to efficient screening of host factors interacted with ChiVMV-NIb.
The improvement of the infectious clone with the commercial label Myc can be used as an effective tool for infecting host plants and researching plant disease resistance, and the advantages of easy acquisition, stability, high efficiency, low price and the like of the commercial label can be utilized to greatly promote the detection of viruses, so that the large-scale screening of plant material virus resistance can be performed. The NIb protein carries Myc tag, and can efficiently screen the interaction host protein of NIb by utilizing the high expression of virus, thus being an effective tool for researching NIb function.
The invention has the beneficial effects that:
according to the invention, through construction of a series of recombinant vectors, through exploration of exogenous sequence introduction positions and sequence composition testing, a ChiVMV invasive clone with a Myc tag carried by a NIb protein is successfully constructed, and a recombinant vector pCB301-ChiVMV with pathogenicity to both Nicotiana benthamiana and capsicum is obtained: the NIb-Myc can ensure that the NIb protein can be stably expressed after carrying a Myc label, and does not influence the pathogenicity of viruses and the functions of other viral proteins;
the recombinant vector pCB301-ChiVMV obtained by the invention: the NIb-Myc has the following features and advantages:
(1) The host can be systematically infected, and the pathogenicity is consistent with that of ChiVMV infectious clone;
(2) After infecting a host, the antigen can be efficiently detected by Myc antibodies, and is suitable for large-scale detection of disease resistance of the host;
(3) Can express stable NIb-Myc fusion protein, and is suitable for efficiently screening NIb interactive host proteins by using an immunoprecipitation-mass spectrometry analysis method.
Drawings
FIG. 1 is a graph showing the results of the symptoms and virus detection of recombinant vector inoculation into Nicotiana benthamiana; in the figure, (A) symptoms of plants after 10 days of inoculation of ChiVMV-NIb-Myc-3 (pCB 301-ChiVMV: NIb-Myc-3 vector), chiVMV-NIb-Myc-2 (pCB 301-ChiVMV: NIb-Myc-2 vector), chiVMV-NIb-Myc-1 (pCB 301-ChiVMV: NIb-Myc-1 vector), chiVMV-Myc-NIb (pCB 301-ChiVMV: myc-NIb vector) and Mock (pCB 301 empty vector control), respectively, into Ben's tobacco; (B) For the RT-PCR result of each Benshi smoke sample, the virus amplification primer Chi-F/R is shown as SEQ ID: no. 18-19;
FIG. 2 shows the wild-type infectious clone pCB301-ChiVMV (abbreviated as ChiVMV), and the recombinant infectious clone pCB301-ChiVMV: constructing a schematic diagram of an NIb-Myc-3 (ChiVMV: NIb-Myc) vector;
FIG. 3 is a graph showing symptoms and virus detection results of vector inoculation into capsicum; in the figure, (A') is the infectious clone ChiVMV and the recombinant infectious clone ChiVMV: NIb-Myc after 7 days on Benshisha, the symptoms of virus infection of plants, and the inoculation of pCB301 empty vector is used as a control (Mock); (B') the RT-PCR results of each Bentonia sample; (C ') carrying out Western blot detection (A') and processing the expression condition of NIb-Myc protein of each Bentonite sample; western blot analysis was performed using Myc antibodies;
FIG. 4 (A ") is ChiVMV and ChiVMV. NIb-Myc infected Benshisha tobacco was rubbed onto capsicum for 11 days, and the symptoms of plant infection virus were compared with PBS rubbed sample (Mock); (B ") is the RT-PCR result of each pepper sample; (C ") carrying out Western blot detection (A") to treat the expression condition of the NIb-Myc protein of each pepper sample; western blot analysis was performed using Myc antibodies;
FIG. 5 shows immunoprecipitation results of ChiVMV NIb-Myc vector infesting samples of Nib-Nicotiana benthamiana and Capsicum annuum; input is a sample which is initially loaded after each sample is processed by an IP buffer; myc is the sample after each sample is processed by Myc beads; "1" is Benshi smoke sample and "2" is capsicum sample.
Detailed Description
The technical scheme of the present invention is described in further detail below with reference to specific embodiments, but the scope of the present invention is not limited to the following description.
Examples
Example 1
The embodiment provides a specific method for inserting a label into a NIb protein of a pepper vein mottle virus and a recombinant vector thereof;
the method firstly constructs the ChiVMV whole genome infectious clone, and then inserts Myc into pCB301-ChiVMV infectious clone NIb protein, and specifically comprises the following steps:
(1) Chi-1F/1R, chi-2F/2R, chi-3F/3R (shown as SEQ ID: no. 1-6) is used as a primer, cDNA of a ChiVMV infected pepper sample is used as a template, and a high-fidelity DNA polymerase is utilized to amplify a band containing the ChiVMV whole genome sequence in three sections;
(2) Selecting StuI and SmaI double-enzyme tangential pCB301 vector, carrying out gel recovery and purification on the product and the fragment in the step (1), carrying out homologous recombination reaction, then converting the product into escherichia coli, and carrying out positive clone screening and sequencing verification to obtain clone pCB301-ChiVMV with a correct ChiVMV genome nucleotide complete sequence;
(3) Recombinant vector construction is carried out by adopting a mode that the N end and the C end of the NIb protein are respectively inserted into Myc labels:
for introducing an N-terminal Myc exogenous sequence, using a pCB301-ChiVMV plasmid as a template and Myc-NIb-1F/1R, myc-NIb-2F/2R, myc-NIb-3F/3R as a primer (shown as SEQ ID: no. 7-12), amplifying the sequence, and introducing a recombinant vector pCB301-ChiVMV of the Myc sequence at the N-terminal of the NIb protein by homologous recombination: myc-NIb;
for introducing a C-terminal Myc exogenous sequence, constructing a vector by adopting BamHI and HpaI double-enzyme tangential pCB301-ChiVMV vectors, respectively synthesizing Fragment I (NIb sequence+Myc sequence+CP partial sequence shown as SEQ ID: no. 13), fragment II (NIb sequence+Myc sequence+sequence A+CP partial sequence shown as SEQ ID: no. 14), fragment III (NIb sequence+Myc sequence+sequence B+CP partial sequence shown as SEQ ID: no. 15), amplifying by adopting CHi-Myc-insert-F/R primer pairs (shown as SEQ ID: no.16 and No. 17) to obtain PCR products, performing enzyme digestion connection on the products with the pCB301-ChiVMV vectors subjected to BamHI and HpaI double-enzyme digestion, converting the products into escherichia coli, and obtaining recombinant correct pCB301-ChiVMV through positive cloning and sequencing verification: NIb-Myc-1, pCB301-ChiVMV: NIb-Myc-2 and pCB301-ChiVMV: a NIb-Myc-3 vector;
(4) Recombinant pCB301-ChiVMV: myc-NIb, pCB301-ChiVMV: NIb-Myc-1, pCB301-ChiVMV: NIb-Myc-2 and pCB301-ChiVMV: the NIb-Myc-3 vector plasmids are respectively introduced into agrobacterium, inoculated to Benshi smoke through agrobacterium mediation, and symptoms are observed and RT-PCR detection is carried out to identify pathogenicity of the recombinant vector to a host. As shown in FIG. 1A, only pCB301-ChiVMV: the NIb-Myc-3 vector is capable of eliciting a leaf curl phenotype in the Nicotiana benthamiana system, and RT-PCR results indicate that only pCB301-ChiVMV: nib-Myc-3 vector inoculated Nib-Myc-3 Nicotiana benthamiana detected the presence of ChiVMV (FIG. 1B), indicating that this construction (FIG. 2) was able to achieve systemic infection of the host;
(5) pCB301-ChiVMV and recombinant pCB301-ChiVMV: the NIb-Myc-3 vector is inoculated to Benshi smoke through agrobacterium mediation respectively, the pathogenesis condition is observed (figure 3A '), the RNA of a sample is extracted, a ChiVMV virus amplification primer Chi-F/R (shown as SEQ ID: no. 10-11) is adopted for PCR detection (figure 3B') after reverse transcription, and as can be seen from figures 3A 'and 3B', the phenotype and the virus accumulation amount detection of the two vectors infecting Benshi smoke are approximately the same, which shows that pCB301-ChiVMV and recombinant pCB301-ChiVMV: the NIb-Myc vectors are pathogenic and have no obvious difference in pathogenicity.
Taking the new system leaves of Nicotiana benthamiana and extracting total protein, and determining pCB301-ChiVMV by Western blot analysis as shown in FIG. 3 (C'): recombinant protein NIb-Myc was present in Nib-Myc vector treated Nib-Myc tobacco System leaves.
Example 2
pCB301-ChiVMV and the recombinant pCB301-ChiVMV obtained in step (3) of example 1 were subjected to: the NIb-Myc-3 vector is inoculated to the capsicum through agrobacterium mediation, the capsicum is inoculated by injection in a mode of inoculating cotyledons and true leaves of the capsicum, and symptoms of the capsicum are observed and RT-PCR detection is carried out after inoculation. As shown in Table 1, the injected leaves of capsicum were necrotic and shed 3-8d after inoculation, and the presence of ChiVMV was undetectable by RT-PCR, regardless of the cotyledon or pepper true leaves.
Meanwhile, in example 1, pCB301-ChiVMV and recombinant pCB301-ChiVMV were taken: the NIb-Myc-3 clone inoculated with 0.5g of positive sample of Benshi tobacco, added with 2mL PBS buffer solution, ground into juice, rubbed and inoculated to capsicum, and performed by two methods of rubbed capsicum cotyledon and true leaf, and performed with symptom observation and RT-PCR detection, it can be seen that only part (11/15, 73.3%) of capsicum rubbed cotyledon is infected with virus and ChiVMV virus is detected, and 100% infection of tested capsicum plants after rubbed true leaf (Table 1). The infection phenotype and RT-PCR detection results of the capsicum are shown in fig. 4 (A ") and 4 (B"), respectively, and the infection phenotype and the virus accumulation amount of the capsicum infected by the two vectors are approximately the same, which shows that pCB301-ChiVMV and recombinant pCB301-ChiVMV: the NIb-Myc vector can cause systemic infection of the capsicum by rubbing and transferring the capsicum true leaves, and the pathogenicity is not obviously different.
Taking fresh system leaves of capsicum and extracting total protein, and performing Western blot analysis, as shown in fig. 4 (C'), the total protein can be obtained in pCB301-ChiVMV: the detection of recombinant proteins NIb-Myc of stable expected size in the NIb-Myc vector-infected capsicum suggests that commercial antibodies Myc can be used to perform batch detection of samples for large-scale detection of host virus resistance.
Table 1 cases where the inoculation methods infest capsicum
Example 3
Because of the lack of the NIb protein antibody, the existing method for detecting the replication of ChiVMV-NIb can only be carried out by a nucleic acid detection method, and is limited in the aspect of large-scale detection of samples. The invention obtains pCB301-ChiVMV: after the NIb-Myc recombinant vector, the sample can be detected on a large scale by a protein detection method. Taking 100 samples for detection as an example, compared with the cost and time consumption of the two methods, the protein detection method adopted on the basis of the invention can save 77.4% of cost, reduce 63.6% of time consumption and has obvious effect in large-scale detection of host virus as shown in table 2.
TABLE 2 comparison of nucleic acid detection and protein detection costs and time consumption
Example 4
Recombinant vector and application of the insertion tag of the NIb protein of pepper vein mottle virus, using the recombinant correct pCB301-ChiVMV in embodiment 1: the NIb-Myc-3 vector was enriched for host NIb interacting proteins by immunoprecipitation. The method specifically comprises the following steps:
(1) 0.2g of Nib-Myc treated Bentonite and Capsicum annuum samples were taken from Mock and ChiVMV, 1mL of IP buffer (containing 40mM Tris-HCl,150mM NaCl,5mM MgCl) 2 2mM EDTA,5mM DDT,0.1% Triton-X100,2% glycerol; protease inhibitor cocktail), gently sucking and beating, mixing, and incubating at 4deg.C for 10min;
(2) 16000g, centrifuging at 4deg.C for 15min;
(3) Pouring the supernatant into a new 1.5mL centrifuge tube, and centrifuging at 4 ℃ for 15min with 16000 g;
(4) Taking the supernatant into a new 1.5mL centrifuge tube, and sucking 100 mu L as Input loading;
(5) Adding 20 mu L Myc beads into the rest sample, and incubating at 4 ℃ for 2 hours;
(6) Centrifuging at 4deg.C for 5min at 2500g, and discarding supernatant;
(7) Washing with 1mL IP buffer for 4 times, and incubating for 5min at 4 ℃ each time;
(8) Discarding the supernatant, adding 80 mu L of IP buffer, adding 2 xLoading with equal volume to the Input sample in the step (4), and denaturing for 5min at 95 ℃;
(9) Western blot detection is carried out on the sample, electrophoresis separation is carried out by using 12% PAGE gel, semi-drying is carried out, nylon membranes are transferred, 5% skimmed milk is used for sealing for 30min, myc antibodies are added after washing, shaking and incubation are carried out at normal temperature for 1.5h, murine antibodies are added after washing, and color development is carried out after washing.
As shown in FIG. 5, via pCB301-ChiVMV: the Input loading of the Nib-Myc vector treated Nicotiana benthamiana and capsicum can detect the CHiVMV-NIb-Myc fusion protein of about 60kDa which is consistent with the expectations, and the fusion protein is obviously enriched after Myc beads treatment, so that the method can be used for efficiently screening host proteins interacted with the ChiVMV-NIb by an immunoprecipitation mass spectrometry combination method, and has wide application prospect.
The foregoing is merely a preferred embodiment of the invention, and it is to be understood that the invention is not limited to the form disclosed herein but is not to be construed as excluding other embodiments, but is capable of numerous other combinations, modifications and environments and is capable of modifications within the scope of the inventive concept, either as taught or as a matter of routine skill or knowledge in the relevant art. And that modifications and variations which do not depart from the spirit and scope of the invention are intended to be within the scope of the appended claims.
Claims (7)
1. A recombinant vector for expressing a pepper vein mottle virus, characterized in that:
the method comprises pCB301-ChiVMV recombinant infectious clone, wherein the C end of the NIb protein is connected with Myc tag;
and the recombinant vector has the sequence shown in SEQ ID: no. 15.
2. A method of infecting capsicum with the recombinant vector of claim 1, comprising the steps of:
firstly, introducing a recombinant vector into agrobacterium, inoculating the agrobacterium-mediated recombinant vector into Benshi tobacco, observing symptoms, and performing RT-PCR detection to determine that the recombinant vector has pathogenicity;
step two, the inoculated benthamiana attack leaves in the step one are in friction transfer to a first pair of true leaves of the peppers, and symptom observation is carried out on the pepper leaves to determine pathogenicity of the recombinant vector on the peppers;
and thirdly, taking new system leaves of Nib and capsicum annuum, extracting total protein, and determining the existence of recombinant protein NIb-Myc through Western blot analysis.
3. The method for preparing the recombinant vector according to claim 1, wherein: the method comprises the steps of obtaining a ChiVMV whole genome sequence from capsicum, constructing a ChiVMV whole genome infectious clone pCB301-ChiVMV, and then inserting a Myc tag into the C end of pCB301-ChiVMV infectious clone NIb protein to obtain a recombinant vector pCB301-ChiVMV: NIb-Myc.
4. A method for preparing a recombinant vector according to claim 3, comprising the steps of:
s1: obtaining ChiVMV whole genome sequence of infected capsicum
Chi-1F/1R, chi-2F/2R, chi-3F/3R is used as a primer, cDNA of a ChiVMV infected pepper sample is used as a template, and a high-fidelity DNA polymerase is utilized to amplify a band containing the ChiVMV whole genome sequence in three sections;
wherein, the nucleotide sequence of the Chi-1F/1R, chi-2F/2R, chi-3F/3R is shown as SEQ ID: no. 1-6;
s2: construction of ChiVMV Whole genome infectious clone pCB301-ChiVM1V
Selecting StuI and SmaI double-enzyme tangential pCB301 vector, carrying out gel recovery and purification on the product and the strip in the step S1, carrying out homologous recombination reaction, then converting the product into escherichia coli, and carrying out positive clone screening and sequencing verification to obtain clone pCB301-ChiVMV with the correct ChiVMV genome nucleotide complete sequence;
s3, constructing a recombinant infectious clone pCB301-ChiVMV of the NIb fusion tag: NIb-Myc
Taking pCB301-ChiVMV plasmid as a vector, and tangentially transforming the pCB301-ChiVMV vector by BamHI and HpaI double enzymes;
synthesizing a plasmid containing a DNA Fragment III in a biological company, and using the plasmid as a template to obtain a PCR product by adopting a CHi-Myc-insert-F/R primer pair for amplification;
then purifying the PCR product, recombining and connecting the PCR product with a pCB301-ChiVMV vector subjected to BamHI and HpaI double enzyme digestion, then converting the product into escherichia coli, and obtaining a recombinant vector pCB301-ChiVMV with correct recombination through positive clone screening and sequencing verification: NIb-Myc;
wherein the Fragment III has the sequence ID: a gene sequence shown in No. 15;
the CHi-Myc-insert-F/R primer pair has the sequence of SEQ ID: the gene sequences shown in No.16 and No. 17.
5. The method for preparing the recombinant vector according to claim 4, further comprising a step of infecting capsicum after the step S3;
the step of infecting the peppers is the step of claim 2.
6. Use of the recombinant vector according to any one of claims 1 to 5 for the large-scale detection of antiviral activity in pepper, tobacco and other hosts.
7. Use of the recombinant vector of any one of claims 1-5 for efficient screening of host factors interacting with ChiVMV-NIb.
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