CN116769804A - Nucleotide sequence, plasmid vector, preparation method and application thereof - Google Patents

Abstract

The application belongs to the technical fields of plant virology and molecular biology, and particularly relates to a nucleotide sequence, a plasmid vector, a preparation method and application thereof. The inventor of the application deletes 2662-2811 nucleotide sequence of RNA2 based on CMV 2b protein, then sequentially introduces double stop codon and polyclonal enzyme cutting site to obtain artificially modified nucleotide sequence, and constructs mutant vector pCC with the nucleotide sequence _F R2-2bPT IV can accommodate exogenous fragments of at least 450bp and maintain genetic stability, and the mutant vector pCC provided by the application _F R2‑2bPTⅣCan be used for preparing the pentavalent attenuated vaccine, and has wide market application prospect and great economic value.

Description

Nucleotide sequence, plasmid vector, preparation method and application thereof

Technical Field

The application belongs to the technical fields of plant virology and molecular biology, and particularly relates to a nucleotide sequence, a plasmid vector, a preparation method and application thereof.

Background

Cucumber mosaic virus (cucumber mosaic virus, CMV) can infect more than 100 plants of 1200 families, and is one of the most widely distributed and most economically damaging plant viruses with the most hosts currently known. Because plants have no complete immune system and lack of effective control agents and measures, control of plant viral diseases is difficult and, once popular, can cause catastrophic losses. The research on the prevention and control of plant viruses is always a difficult and hot problem of plant diseases and virology research.

The use of the attenuated vaccine created based on the cross protection effect is an effective measure for preventing and treating viral diseases, and has the advantages of no pollution, long period of time, environmental protection and the like. When the multivalent attenuated vaccine is artificially prepared by molecular biology means, the heterologous virus fragment needs to be inserted, so that the inserted heterologous virus fragment with a certain length can trigger gene silencing to have an effect of preventing the heterologous virus, and the attenuated mutant inserted with the heterologous virus fragment is ensured to have genetic stability. Thus, the preparation of multivalent attenuated vaccines requires the study of vaccine construction vectors capable of accommodating longer foreign fragments than attenuated vaccines against the virus alone.

The inventors developed an RNA2 attenuated mutant plasmid vector pCC with partial deletion of CMV 2B protein coding sequence in previous studies (see issued Chinese patent CN 113388637B) _F R2-2bPT III as pCC _F R2-2bPT III is taken as a basic vector, the upper limit of the receivable exogenous fragment is 350bp, 4-valent attenuated vaccines (namely, 4 target viruses can be prepared, however, the limited length of the insertable exogenous fragment can limit the insertion of more target virus fragments from different sources, so that the capability of the basic vector for accommodating more exogenous fragments is very necessary to be further improved in order to prepare multivalent attenuated vaccines.

Disclosure of Invention

In order to solve the problems, the application provides a nucleotide sequence, a plasmid vector, a preparation method and application thereof, and a mutant plasmid vector pCCFR2-2bPT IV containing cucumber mosaic virus CMV isolate RNA2 can have a cross protection effect on CMV; as a vaccine vector, the vector can at least contain 450bp exogenous fragments and maintain genetic stability, and early researches prove that the insertion of the 100bp fragments of the target viruses can play a role in cross protection and prevention and control of the viruses, so that the vector has the capacity of inserting each 100bp fragment of 4 different target viruses based on the mutant vector pCCFR2-2bPT IV, and can be combined with the prevention and control of the basic vector on CMV to create the attenuated vaccine for resisting 5 viruses.

According to one aspect of the present application, there is provided a nucleotide sequence, the method for preparing the nucleotide sequence comprising the step of deleting 2662 to 2811 nucleotide sequences of RNA2 of cucumber mosaic virus CMV.

Optionally, the nucleotide sequence further comprises the step of sequentially introducing a double stop codon and a polyclonal enzyme cutting site after the deletion treatment.

Optionally, the preparation method of the nucleotide sequence comprises the following steps:

(1) Deletion treatment is carried out on nucleotide sequences 2662-2811 of RNA2 of the cucumber mosaic virus CMV;

(2) The double termination codons TAATAG, and the multiple cloning cleavage sites BamH I, spe I and Sma I were introduced sequentially.

Alternatively, the nucleotide sequence has at least 95% identity to SEQ ID No. 1.

According to another aspect of the present application there is provided the use of any one of the nucleotide sequences described above in the preparation of a plasmid vector.

According to a further aspect of the present application there is provided a plasmid vector comprising a nucleotide sequence as any one of the above.

According to a further aspect of the present application there is provided the use of a plasmid vector as described above in the preparation of a multivalent attenuated vaccine.

According to a further aspect of the present application there is provided a multivalent attenuated vaccine prepared from the plasmid vector described above.

According to a further aspect of the present application there is provided the use of a plasmid vector as defined above or a multivalent attenuated vaccine as defined above in the control of cucumber mosaic virus CMV.

According to a final aspect of the application there is provided E.coli, agrobacterium, a kit or a chemical formulation comprising a multivalent attenuated vaccine as described above.

The beneficial effects of the application include, but are not limited to:

(1) The application carries out partial deletion on a 2b protein coding sequence of cucumber mosaic virus, deletes 2662-2811 nucleotide sequence of RNA2, inserts double stop codon TAATAG after 2661G, then sequentially introduces a polyclonal enzyme cutting site GGATCCACTAGTCCCGGG (BamHI, speI, smaI), and obtains a weak virus mutant of cucumber mosaic virus RNA2, and the plasmid is mixed with plasmids containing RNA1 and RNA3 of CMV isolate for inoculation, thereby being capable of preventing infection of CMV virulent strain and being used as a weak virus vaccine for resisting cucumber mosaic virus.

(2) The cucumber mosaic virus attenuated vaccine vector pCCFR2-2bPT IV obtained by the application is used as a basic vector for constructing a multivalent attenuated vaccine, can at least accommodate 450bp exogenous fragments and maintain genetic stability, and provides a choice of more virus types for constructing the multivalent attenuated vaccine.

(3) For the obtained cucumber mosaic virus attenuated mutant, the application can be used as a basic vector for constructing a multivalent attenuated vaccine, and other important plant virus sequences are inserted into the introduced polyclonal enzyme cutting sites to obtain the multivalent attenuated vaccine which can resist CMV and other viruses.

Drawings

The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this specification, illustrate embodiments of the application and together with the description serve to explain the application and do not constitute a limitation on the application. In the drawings:

fig. 1: basic vector pCB301-CMV _Fny -R2, mutant vector pCC _F R2-2bPT IV and exogenous fragment insertion type mutant pCC _F R2-2bPT IV-PDS; in the figure, LB, T-DNA left Loader (LB); RB, T-DNA right sender (RB); 35S, precursor (from gene 2X 35S); RZ, ribozyme (HDV-RZ); NOS, terminator (NOS terminator).

Fig. 2: vector pCC _F An electrophoresis schematic diagram of an enzyme digestion identification result of R2-2bPT IV; wherein Marker is 1kb DNA Ladder molecular weight standard of GenStar company, and the fragment is about 7.5kb after digestion.

Fig. 3: pCC _F The pathogenicity of R2-2bPT IV on the Nicotiana benthamiana and the cross protection effect on CMV are measured; mock, empty agrobacterium solution; r1, CMV _Fny RNA1；R2，CMV _Fny RNA2；R3，CMV _Fny RNA3。

Fig. 4: pCC _F Length limitation determination of R2-2bPT IV accommodating exogenous fragments; r1, CMV _Fny RNA1；R2，CMV _Fny RNA2；R3，CMV _Fny RNA3; p350, P400 and P450 respectively represent PDS sequences with lengths of 350bp, 400bp and 450 bp; dpi, days after inoculation.

Fig. 5: pCC _F Determining the genetic stability of the exogenous fragment insertion type mutant of R2-2bPT IV; marker is GenStar company D2000 molecular weight standard.

Detailed Description

The present application is described in detail below with reference to examples, but the present application is not limited to these examples, and unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs, and unless otherwise indicated, the starting materials and catalysts in the examples of this application are commercially available.

The ability of the base vector to accommodate exogenous fragments is one of the key indicators for the performance of the base vector in the preparation of multivalent attenuated vaccines. CMV is a positive-sense single-stranded trisomy RNA virus encoding a total of 5 proteins, of which the 2b protein is a gene silencing inhibitor that maximally protects the viral genome from degradation by the host plant (Csorba et al 2015). Because the 2b protein of the CMV is a virus-encoded gene silencing inhibitor, the gene silencing effect of host plants on viral genomes is greatly inhibited, and the mutation modification of the encoding gene of the 2b protein is a research hot spot for constructing CMV attenuated mutants.

The inventor develops and designs a cucumber mosaic virus RNA2 attenuated mutant plasmid vector pCC with 2b protein coding sequence deleted in earlier research _F R2-2bPT III can accommodate exogenous fragments with the upper limit of 350bp, and in order to prepare multivalent and attenuated vaccines with stronger functions, the inventor further improves the capacity of accommodating exogenous fragments of a basic vector through research and experimental verification.

Specifically, in order to increase the capacity of the basic vector to accommodate exogenous fragments, the inventor repeatedly explores and experiments prove that the transformation strategy of the cucumber mosaic virus 2b protein coding gene obtains the improvement scheme of the application, and the basic vector provided by the application is obtained by deleting 2662-2811 nucleotide sequence of RNA2, sequentially introducing double termination codons (TAATAG) and polyclonal enzyme cleavage sites GGATCCACTAGTCCCGGG (BamH I, spe I and Sma I) after 2661G to obtain the vector containing mutant CMV _Fny Plasmid vector pCC of RNA2 _F R2-2bPTⅣ。

Plasmid vector pCC _F Mutant CMV in R2-2bPT IV _Fny The nucleotide sequence of RNA2 is shown in SEQ ID NO. 1:

in the above sequences, lowercase letters indicate CMV lacking nucleotides 2662-2811 _Fny RNA2 sequence, capital letters indicate introduced exogenous sequence, wherein italics indicate stop codons, underlined indicates cleavage sites.

The inventor carries out deletion on 2662-2751 in the early stage, constructs an exogenous fragment insertion type mutant vaccine, the maximum limit of the constructed vaccine for accommodating exogenous fragments is only 350bp, and the genetic stability and the prevention effect of the vaccine cannot be guaranteed after exceeding the limit, and the specific content is shown in Chinese patent CN 113388637B.

In a preferred embodiment of the application, a composition comprising a mutant CMV is provided _Fny Plasmid vector pCC of RNA2 _F The construction method of R2-2bPT IV comprises the following steps:

according to CMV published in GenBank _Fny The sequence of RNA2 (accession number: D00355) was designed as primers 2bPT-2812-F and Fny2-del2b-2661-R (see Table 1 for details). With pCB301-CMV _Fny R2 is used as a template, PCR inverse amplification is carried out under the action of Phanta Max Super-Fidelity DNA Polymerase (Vazyme), dpn I (NEB) is utilized to degrade a plasmid template, bamH I digestion is carried out on PCR amplified fragments, then the PCR amplified fragments are self-linked, a ligation product is transformed into escherichia coli DH5 alpha, and the vector pCC is obtained through colony PCR, plasmid digestion identification and DNA sequencing _F R2-2bPT IV. Vector pCC _F R2-2bPT IV is obtained by deleting 2662-2811 sequence of RNA2,and double stop codons (TAATAG) and polyclonal restriction sites GGATCCACTAGTCCCGGG (BamH I, speI, sma I) were introduced sequentially (as shown in FIG. 1).

TABLE 1 primers used in the present application

Table 1 notes: the protecting base is shown in bold, the cleavage site is shown underlined, and the stop codon is shown in italics.

In order to enable those skilled in the art to more clearly understand the technical scheme of the present application, the technical scheme of the present application will be described in detail with reference to specific embodiments.

The test materials used in the examples of the present application, which are not specifically described, are all conventional in the art and are commercially available. Specific experimental conditions and methods are not noted in the examples of the present application, and generally conventional conditions, such as j. Sambrook et al, scientific press, 2002, guidelines for molecular cloning experiments (third edition); D.L. speket et al, scientific press, 2001, guidance of cell experiments; or according to manufacturer recommended conditions.

EXAMPLE 1 vector pCC _F Construction of R2-2bPT IV

With CMV _Fny Infectious clone plasmid pCB301-CMV _Fny R2 is template (CMV) _Fny Construction of the infectious clone plasmid pCB301-Fny2 reference "construction of Agrobacterium-mediated CMV infectious clone and 2b deletion mutant", yao Min et al, china agricultural science, 2011,44 (14): 3060-3068), under the action of Phanta Max Super-Fidelity DNA Polymerase (Vazyme), performing inverse PCR amplification, primer pairs 2bPT-2812-F and Fny2-del2b-2661-R; PCR conditions: pre-denaturation at 95℃for 3min; denaturation at 95℃for 15s, annealing at 50℃for 15s, extension at 72℃for 3min 48s,30 cycles; thoroughly extending at 72 ℃ for 5min; preserving at 4 ℃; amplifying the obtained PCR product, namelyFor linearizing the basic vector pCC _F R2-2bPTⅣ。

Degradation of plasmid template pCB301-CMV in PCR reactions Using Dpn I (NEB) _Fny -R2, the reaction conditions are: 37 ℃ for 1h;80 ℃ for 20min; the product is recovered by an absolute ethanol precipitation method: the product was added to ddH ₂ O to 450. Mu.l, 50. Mu.l of sodium acetate, pH5.2,3.0M, 1ml of absolute ethanol, and precipitation at-80℃for 2h; centrifuging at 12000rpm for 10min, pouring out the liquid, and adding 600 μl 70% ethanol; centrifuging at 12000rpm for 5min, and pouring out the liquid; at normal temperature of 12000rpm, centrifuging for 1min, sucking the liquid in the centrifuge tube, and adding 30 μl ddH ₂ And (5) dissolving back O.

Then, the product was digested with BamH I (Takara), and the digested product was recovered by a DNA recovery kit, followed by digestion with a DNA fragment at T ₄ Under the action of DNA ligase (Takara) at 16 ℃ overnight self-ligation, transforming the ligation product into escherichia coli DH5 alpha, coating LB plate with 100 mug/mL kanamycin, performing colony PCR, plasmid enzyme digestion identification and DNA sequencing, and confirming to obtain vaccine base vector pCC _F R2-2bPTⅣ。

Analysis of results: the PCR products are self-linked after being digested with BamH I, the transformation products are subjected to primary screening by colony PCR, the plasmid is extracted for enzyme digestion identification, bamH I and Sma I are selected for enzyme digestion identification respectively, the enzyme digestion result shows that the plasmid can be linearized by the two enzymes, the length of the linearized plasmid is about 7.5kb (shown in figure 2), the polyclonal sites containing BamH I, spe I and Sma I in the plasmid are basically confirmed, and finally, the plasmid DNA sequencing is confirmed.

The vaccine base vector pCC constructed in this example _F R2-2bPT IV is obtained by deleting the 2662-2811 nucleotide sequence of RNA2 and then sequentially introducing a double-termination codon TAATAG and a polyclonal restriction enzyme site GGATCCACTAGTCCCGGG (BamH I, spe I, smaI).

EXAMPLE 2 plasmid vector pCC _F Biological effect and cross-protection effect measurement of R2-2bPT IV

Agro-infiltration infects Benshi tobacco: first will contain wild type CMV _Fny RNA1, wild type CMV _Fny RNA2, wild type CMV _Fny Plasmid of RNA3 and pCC _F R2-2bPT IV respectively transform Agrobacterium GV3101 are uniformly smeared on LB solid medium (50 mu g/ml kanamycin, 100 mu g/ml rifampicin), after culturing for 48 hours, single spots are picked up for colony PCR verification, and after picking up single spots, the single spots are cultured in 2ml LB culture solution (50 mu g/ml kanamycin, 100 mu g/ml rifampicin) at 28 ℃ for 24 hours under shaking at 200 rpm.

200. Mu.l of the bacterial liquid was cultured in 5ml of LB medium (50. Mu.g/ml kanamycin, 100. Mu.g/ml rifampicin) at 28℃with shaking at 200rpm for 12 hours.

Centrifuging the bacterial liquid in a 10ml centrifuge tube at 6000rpm for 10min at room temperature; the cells were collected and resuspended in 1ml Agrobacterium re-suspension (10 mM MgCl) ₂ 10mM MES,0.15mM AS), adjust the concentration to make the OD ₆₀₀ 1.2, and 3 bacterial solutions OD ₆₀₀ And consistent.

Will adjust the OD ₆₀₀ Bacterial liquids (CMV-containing respectively) _Fny RNA1、CMV _Fny RNA2、CMV _Fny RNA3，CMV _Fny RNA1、pCC _F R2-2bPTⅣ、CMV _Fny RNA 3) and the like, and standing at 28 ℃ for 3h.

Taking 1ml of disposable injector, removing needle head, sucking agrobacterium liquid, selecting 6-8 leaf stage Benshi tobacco, injecting the liquid from the injector between leaf veins on the back of the leaf by pressure, and injecting two leaves for each plant, wherein the injection quantity of each leaf covers at least 1/3 of the leaf.

After 7d of inoculation of the mutants, the plants pre-inoculated with the mutants and the plants not pre-inoculated with the mutants were infected with the target virulent CMV by means of agrobacterium injection, and their cross-protective effect was tested separately.

The inoculated plants were placed at 25℃for cultivation, with 16h light/8 h dark alternation.

Analysis of results: after 14d inoculation, the inoculation symptoms show that mock Ben's tobacco does not attack and has good growth vigor; the wild CMV (wt) inoculated with Benshi tobacco has serious morbidity, dwarf plants and shrunken leaves; the plant symptoms of the Benshiyan inoculated with R2-2bPT IV are basically consistent with those of mock plants, and no obvious virus symptoms exist, which indicates that R2-2bPT IV is CMV _Fny Attenuated mutants of RNA2 (as shown in FIG. 3). Whereas CMV virulent direct inoculation of the early-stage non-inoculated R2-2bPT IV mutant Benshi tobacco, plant leavesThe shrinkage of the sheet is serious and similar to the weight; the CMV virulent inoculation is performed on Benshi tobacco pre-inoculated with the R2-2bPT IV mutant, the plant growth state is good, and the leaves are flat and similar to mock. The above results indicate that R2-2bPT IV is CMV _Fny The attenuated mutant of RNA2 has better prevention effect on CMV, and the relative prevention effect is 90.9% (shown in figure 3).

EXAMPLE 3 plasmid vector pCC _F Determination of the limitation of the accommodation of exogenous fragments by R2-2bPT IV

Plasmid vectors containing PDS sequences with different lengths are constructed, the method is referred to a plasmid construction method in the prior study (patent number: 202011259238.8), total RNA of the Nicotiana benthamiana plant in the 6-leaf stage is extracted, and reverse transcription reaction is carried out by using a reverse primer PDS-BamHI-782-R; then, the cDNA of the reverse transcription product is used as a template, a forward primer PDS-SmaI-333-F and a reverse primer PDS-BamHI-682-R, PDS-BamHI-732-R, PDS-BamHI-782-R are used for respectively carrying out PCR reaction, and partial PDS sequences with the lengths of 350bp, 400bp and 450bp are amplified; vector pCC _F R2-2bPT IV and PDS fragments are subjected to double digestion by BamH I and Sma I and then are recovered by a DNA recovery kit; vector pCC after the same cleavage _F R2-2bPT IV and PDS fragments with different lengths are respectively connected; competent cells of E.coli DH5 alpha are transformed by the ligation product, LB plates with 100 mug/mL kanamycin are coated, and positive clone pCC is obtained by colony PCR screening, enzyme digestion identification and DNA sequencing confirmation _F R2-2bPTⅢ-P350、pCC _F R2-2bPTⅢ-P400、pCC _F R2-2bPTⅢ-P450。

The plasmid vector containing PDS sequence was inoculated as described in example 2, after 14 days of inoculation, the inoculation of the mutant was recorded (as shown in FIG. 4), total RNA was extracted from the upper non-inoculated system leaves, primers CMV-Fny2-2324-F (SEQ ID NO. 8) and CMV-Fny2-R (SEQ ID NO. 9) were designed upstream and downstream of the insertion site of the foreign fragment of RNA2-2bPT IV, RT-PCR detection was performed, and whether the inserted PDS fragment was stably present was judged by the length of the amplified fragment (as shown in FIG. 5), if pCC was _F R2-2bPT III-PDS is stable, and when the length of the inserted PDS is 0bp, 350bp, 400bp and 450bp, the corresponding PCR product fragments are 600bp, 944bp, 994bp and 1044bp.

Analysis of results: inoculation symptoms show that mock Benshi tobacco does not attack, and the growth vigor is good; the wild CMV (wt) inoculated with Benshi tobacco has serious morbidity, dwarf plants and shrunken leaves; the young leaf whitening of the plant is obvious, the leaf is flat, the plant height is similar to that of a mock plant, obvious pathogenic symptoms are avoided, and the exogenous fragment insertion mutants are all attenuated mutants (shown in figure 4).

In fig. 3 and 4, the upper and lower photographs of the same plant at the same position correspond to the observation results of the same plant from different angles.

RT-PCR results showed that the amplified band of the wt inoculated sample was about 726bp, consistent with the expected band size (as shown in FIG. 5, lane 3); the amplified band of the inoculated sample of the mutant R2-2bPT IV is about 600bp, and the amplified band is consistent with the expected band size (as shown in FIG. 5, lane 5), which shows that the mutant R2-2bPT IV without the inserted exogenous fragment can exist in plants stably; the amplified bands of the inoculated samples of the mutants R2-2bPT III-P350, R2-2bPT III-P400 and R2-2bPT III-P450 are about 944bp, 994bp and 1044bp respectively (Lane 7/9/11 respectively as shown in FIG. 5), and the sizes are consistent with the expected band sizes, which indicates that the exogenous fragments inserted into the samples of 350-450bp can exist stably. The results show that the mutants R2-2bPT IV, R2-2bPT IV-P350, R2-2bPT IV-P400 and R2-2bPT IV-P450 have stable inheritance.

Based on the above results, it was demonstrated that the mutant vector pCC _F R2-2bPT IV can at least contain 450bp exogenous fragments and maintain genetic stability, and the mutant vector pCC provided by the application _F R2-2bPT IV can be used for preparing a pentavalent attenuated vaccine, and has wide market application prospect and great economic value.

The above description is only an example of the present application, and the scope of the present application is not limited to the specific examples, but is defined by the claims of the present application. Various modifications and variations of the present application will be apparent to those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the technical idea and principle of the present application should be included in the protection scope of the present application.

Claims (10)

1. A nucleotide sequence is characterized in that the preparation method of the nucleotide sequence comprises the step of deleting 2662-2811 nucleotide sequence of RNA2 of cucumber mosaic virus CMV.

2. The nucleotide sequence according to claim 1, further comprising the step of introducing a double stop codon and a polyclonal enzyme cleavage site in sequence after the deletion treatment.

3. The nucleotide sequence according to claim 2, wherein the method for preparing the nucleotide sequence comprises:

(1) Deletion treatment is carried out on nucleotide sequences 2662-2811 of RNA2 of the cucumber mosaic virus CMV;

(2) The double termination codons TAATAG, and the multiple cloning cleavage sites BamH I, spe I and Sma I were introduced sequentially.

4. A nucleotide sequence according to any one of claims 1 to 3, characterized in that it has at least 95% identity with SEQ ID No. 1.

5. Use of a nucleotide sequence according to any one of claims 1 to 4 for the preparation of a plasmid vector.

6. A plasmid vector comprising the nucleotide sequence according to claim 1 to 4.

7. The use of the plasmid vector of claim 6 for the preparation of multivalent attenuated vaccines.

8. A multivalent attenuated vaccine prepared from the plasmid vector of claim 6.

9. Use of the plasmid vector of claim 6 or the multivalent attenuated vaccine of claim 8 in cucumber mosaic virus CMV control.

10. An escherichia coli, agrobacterium, kit or chemical formulation comprising the multivalent attenuated vaccine of claim 8.