CN110856493A - Plant virus attenuated vaccine composition, attenuated vaccine storage method and application thereof - Google Patents

Abstract

The invention relates to the field of agricultural science, and discloses a plant virus attenuated vaccine composition, an attenuated vaccine storage method and application thereof. The plant virus attenuated vaccine composition comprises fermented thallus carrying plant virus attenuated vaccine, light calcium carbonate, fulvic acid and xanthan gum. The volume ratio of the fermentation thallus carrying the plant virus attenuated vaccine to the light calcium carbonate is 1: 1. The relationship between the addition amount of fulvic acid and xanthan gum and the mixture of fermentation thallus and light calcium carbonate is as follows: 50mg of fulvic acid and 50mg of xanthan gum are added into 10ml of the mixture of the fermentation thalli and the light calcium carbonate. The composition and the method can prolong the preservation time of the attenuated vaccine, improve the shelf life, facilitate the wide application of the attenuated vaccine and greatly reduce the loss.

Description

A kind of plant virus attenuated vaccine composition, attenuated vaccine preservation method and application thereof

technical field

The present invention relates to the field of agricultural science, and in particular, the present invention relates to a plant virus attenuated vaccine composition, a method for preserving attenuated vaccine and its application.

Background technique

Virus disease is one of the important diseases in agricultural production, which seriously affects the yield and quality of crops and causes huge losses. At present, there is no specific medicament for the prevention and treatment of crop virus diseases on the market, so the prevention and control of crop virus diseases is very difficult. Cross protection is an effective means to control plant virus diseases. With the advancement of technology, it is now possible to construct infective clones of the virus, screen for attenuated vaccines with significantly reduced pathogenicity and protect crops from virulent strains by reverse genetics technology, and inoculate them in advance at the seedling stage of crops , can effectively prevent viral diseases.

Plant virus attenuated vaccines require the use of Agrobacterium to inoculate plants. However, the survival time of Agrobacterium at room temperature is not long, and the number of viable bacteria will drop significantly after a period of time. Therefore, the Agrobacterium carrying the plant virus attenuated vaccine cannot be stored for a long time, which affects the wide application of the plant virus attenuated vaccine.

The present invention has been made in view of this.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is to overcome the deficiencies of the prior art, and to provide a plant virus attenuated vaccine composition, a method for preserving attenuated vaccine and its application. By using light calcium carbonate to dry the Agrobacterium carrying the attenuated vaccine and adding fulvic acid and xanthan gum, the invention can prolong the storage time of the attenuated vaccine, improve the shelf life, and is beneficial to the wide application of the attenuated vaccine.

In order to solve the above-mentioned technical problems, the basic conception of the technical scheme adopted in the present invention is:

The first object of the present invention is to provide a plant virus attenuated vaccine composition, the plant virus attenuated vaccine composition includes fermented thalline carrying the plant virus attenuated vaccine, light calcium carbonate, fulvic acid and xanthan gum.

In a further scheme, the volume ratio of the fermented bacterial cells carrying the plant virus attenuated vaccine to the light calcium carbonate is 1:1.

In a further scheme, the mass ratio of fulvic acid and xanthan gum in the composition is 1:1.

Further scheme, the relationship between the additions of described fulvic acid and xanthan gum and the fermented cell and the light calcium carbonate mixture is: add 50 mg of fulvic acid and 50 mg of fulvic acid to every 10 ml of the fermented cell and the light calcium carbonate mixture. Xanthan gum.

In the present invention, by adding fulvic acid and xanthan gum to the Agrobacterium carrying the plant virus attenuated vaccine, it is proved that the shelf life can be effectively prolonged, and then through the screening of different concentration gradients, it is finally determined that 50 mg of fulvic acid is added per 10 ml of light calcium carbonate powder The formula ratio of acid and 50mg xanthan gum has the best preservation effect, which can prolong the preservation period of plant virus attenuated vaccine by more than 2 months.

In a further scheme, the plant virus attenuated vaccine includes at least one attenuated vaccine against potato X virus, potato Y virus, cucumber mosaic virus, and tobacco mosaic virus.

Further scheme, described plant virus attenuated vaccine comprises taking TVBMV attenuated mutant as carrier, and embedded in TVBMV attenuated mutant can induce to potato X virus, and/or potato Y virus, and/or cucumber mosaic virus, and / or Tobacco mosaic virus to produce effective gene segments for cross-protection;

Or, described plant virus attenuated vaccine comprises potato virus X, and/or potato virus Y, and/or cucumber mosaic virus, and/or the attenuated strain of tobacco mosaic virus mutation;

Preferably, the plant virus attenuated vaccine comprises a mutant attenuated strain of potato Y virus, and K at position 182 of HC-Pro is mutated to R.

In a further scheme, the effective gene segment that can induce cross-protection to Potato X virus includes the RdRp gene of Potato X virus, and its nucleotide sequence is shown in Seq ID No.13, or Seq ID No.14, or Seq ID No. .15 shown;

Alternatively, the effective gene fragments that can induce cross-protection against potato Y virus include PVY1 fragment and PVY2 fragment, the nucleotide sequence of PVY1 fragment is shown in Seq ID No.20, and the nucleotide sequence of PVY2 fragment is shown in Seq ID No.21 shown;

Alternatively, the effective gene fragment that can induce cross protection against cucumber mosaic virus includes the 2b gene of cucumber mosaic virus, and the nucleotide sequence of the 2b gene is shown in Seq ID No.24;

Alternatively, an effective gene fragment that can induce cross-protection against tobacco mosaic virus includes the TMV3 fragment of tobacco mosaic virus, and the nucleotide sequence of the TMV3 fragment is shown in Seq ID No.27.

In a further scheme, the fermented bacterial cells include Agrobacterium.

The second object of the present invention is to provide a kind of method that prolongs the preservation time of plant virus attenuated vaccine, including:

(1) After the fermented thalline carrying the attenuated vaccine is concentrated by centrifugation, light calcium carbonate powder is added to make the mixture powder;

(2) adding fulvic acid and xanthan gum in the mixture powder to make a plant virus attenuated vaccine composition to prolong the shelf life;

Preferably, the volume ratio of the fermented thalline carrying the plant virus attenuated vaccine and light calcium carbonate is 1:1;

Preferably, the mass ratio of fulvic acid and xanthan gum is 1:1;

Preferably, the relationship between the added amount of fulvic acid and xanthan gum and the mixture of fermented cells and light calcium carbonate is: add 50 mg of fulvic acid and 50 mg of yellow humic acid to every 10 ml of the mixture of fermented cells and light calcium carbonate Original gum.

The third object of the present invention is to provide the above-mentioned composition, or the application of the above-mentioned method in prolonging the storage time of the plant virus attenuated vaccine.

The concrete technical scheme that the present invention implements is:

a. Powder treatment test: Divide the Agrobacterium carrying the plant virus attenuated vaccine into two groups, one group is not treated, and the other group is centrifuged to collect the bacteria and add an equal volume of light calcium carbonate and mix to form a powder. Take 20 μL of bacterial liquid and 20 μL of powder respectively, dilute 5 times and spread them on LB plates; the number of viable bacteria are 14785/100 μL and 14645/100 μL respectively, which proves that light calcium carbonate treatment has no effect on bacterial activity.

b. Optimization of working concentration of fulvic acid and xanthan gum: control groups with different concentrations of fulvic acid and xanthan gum were set up and stored at room temperature.

c. Observation of vaccine preservation: 100 μL of powder was taken from each group, the suspension was resuspended and coated on LB plate and the colony growth was observed.

After adopting the above technical solution, the present invention has the following beneficial effects compared with the prior art.

1, the plant virus attenuated vaccine composition of the present invention, by using light calcium carbonate to dry the Agrobacterium carrying the attenuated vaccine, and adding fulvic acid and xanthan gum, the attenuated vaccine storage time can be prolonged, the shelf life is improved, and it is beneficial to Widespread use of attenuated vaccines.

2. In the preservation method of the plant virus attenuated vaccine of the present invention, after centrifugally collecting the thalline carrying the plant virus attenuated vaccine and mixing into the mixture powder using light calcium carbonate, add 50mg fulvic acid and 50mg yellow humic acid in every 10ml mixture powder. The formula ratio of the original gum has the best preservation effect, which can prolong the shelf life of the plant virus attenuated vaccine by more than 2 months.

The specific embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.

Description of drawings

The accompanying drawings, as a part of the present invention, are used to provide further understanding of the present invention, and the exemplary embodiments of the present invention and their descriptions are used to explain the present invention, but do not constitute an improper limitation of the present invention. Obviously, the drawings in the following description are only some embodiments, and for those of ordinary skill in the art, other drawings can also be obtained from these drawings without any creative effort. In the attached image:

Fig. 1 is a schematic diagram of TVBMV genome fragment amplification;

Figure 2 is a schematic diagram of the genome structure of pCamTVBMV;

Figure 3 takes 20 μL of bacterial liquid and 20 μL of powder, respectively, diluted 5 times and then coated on LB plate;

The attenuated vaccine compositions of fulvic acid and xanthan gum with different addition ratios of Fig. 4 observe the preservation effect by coating LB plates after nine weeks of preservation;

Figure 5 counts the number of colonies on the LB plate after nine weeks of preservation;

Figure 6 is the comparison of the colony growth after the first coating and the ninth week coating when 50 mg of fulvic acid and 50 mg of xanthan gum are added to each 10 ml of mixed powder.

It should be noted that these drawings and written descriptions are not intended to limit the scope of the present invention in any way, but to illustrate the concept of the present invention to those skilled in the art by referring to specific embodiments.

Detailed ways

In order to make the purposes, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and the following embodiments are used to illustrate the present invention , but are not intended to limit the scope of the present invention.

Example 1 Construction of multi-site TVBMV attenuated mutants

1. Construction of Infectious Clones of Tobacco Vein Mosaic Virus

Using the RNA of tobacco vein mosaic virus as a template, reverse transcription was performed with random primers. According to the restriction enzyme digestion map of the existing tobacco vein mosaic virus genome, it can be amplified in three parts, and assembled into a TVBMV full-length cDNA clone after enzyme digestion. First, the 35S promoter was fused to the upstream of the fragment from the 5′ untranslated region of TVBMV to the Nru I restriction site of the HC-pro gene by Overlap-PCR, and the inventor named the fragment p35S-HC; PCR amplification of HC- The fragment between the Nru I restriction site of the pro gene and the 6K2Xho I restriction site was named pHC-6K2 by the inventors; the 6K2Xho I restriction site was amplified by PCR to the tail of the ploy(A) fragment, which the inventors named p6K2-polyA (as shown in Figure 1).

The reverse transcriptase used for cDNA synthesis was Moloney murine leukaemia virus reversetranscriptase (Promega); reverse transcription was performed with plant total RNA as a template and random primers as reverse transcription primers.

PCR amplification was performed using the obtained reverse transcription product as a template and corresponding primers. PCR products were subjected to 1% agarose gel electrophoresis. Three fragments of p35S-HC ₂₁₁₀ , pHC _{2111-6K2 6075} and p6K2 ₆₀₇₆ -polyA were obtained after gel cutting and recovery. After ligating the three fragments, they were digested with SbfI and Sma I and then electrophoresed on a 0.8% agarose gel, recovered and ligated. To the Agrobacterium-mediated expression vector pCAMBIA0390, the inventors named the invasive clone constructed by this strategy as pCamTVBMV (as shown in Figure 2).

2. Construction of TVBMV attenuated mutants

After obtaining the invasive clone of Tobacco Vein Mosaic Virus, a TVBMV attenuated mutant was obtained by mutating key sites in the HC-Pro sequence. The specific method is as follows:

Mutation primers were designed. According to the method of Liu et al. (2008), site-directed mutation was performed on the conserved amino acid site of tobacco vein mosaic virus HC-Pro. The names and sequences of the mutated primers are shown in Table 1.

Table 1 Name and sequence of TVBMV HC-Pro mutation primers

Among them, primer 1 and primer 2 site-directed mutation of amino acid 52 of HC-Pro amino acid sequence from arginine to glutamic acid; primer 3 and primer 4 site-directed mutation of amino acid 198 of HC-Pro amino acid sequence, by aspartic acid Acid was mutated to lysine; primers 5 and 6 were site-directed to mutate amino acids 250 and 251 of the HC-Pro amino acid sequence, isoleucine at position 250 was mutated to aspartic acid, and glutamine at position 251 was mutated to Glutamate.

Using pCamTVBMV as template, PCR mutation system: 5×PCR Buffer 10μL, dNTP (10mM) 1μL, mutation primer F (10μM) 1μL, mutation primer R (10μM) 1μL, template plasmid 10ng, Phusion DNA polymerase 0.3μL, ddH ₂ O make up to 50 μL.

PCR mutation program: 98°C/30sec; 98°C/10sec, Tmno+3°C/20sec, 72°C/5min, 20 cycles; 98°C/10sec; Tmpp/20sec; 72°C/15min; 4°C storage.

After mutation PCR, 1 μL of Dpn I was added to each reaction system, mixed well, and then digested at 37°C for 4 hours.

After the PCR reaction system was treated with Dpn I, 125 μL of absolute ethanol (2.5× volume) and 5 μL of 3M NaAc pH 8.0 were added, mixed and precipitated overnight; 12000 r/min, 10 min, discard the supernatant; 1 mL of 75% ethanol washed the precipitate, Discard the supernatant; dry the pellet and dissolve the pellet with 10 μL ddH2O.

The mutant precipitated product was transformed into Escherichia coli, and the transformed bacteria were evenly spread on LB plates containing X-gal and IPTG-Amp antibiotics, and a single colony was selected for cultivation, and the plasmid was extracted for sequencing. If the sequencing was correct, four site mutations were obtained. Attenuated mutants of TVBMV.

3. Study on pathogenicity of TVBMV attenuated mutants

The obtained mutant plasmid was transferred into Agrobacterium, and the recombinant bacteria were obtained after colony PCR verification. Then single spot was inoculated in liquid LB medium containing kanamycin (50 μg/mL), rifamycin (50 μg/mL) and tetracycline (50 μg/mL). Add 500 μL of bacterial liquid to 5 mL of LB medium containing 10 mmol/L 2-(N-morpholine)-ethylsulfonic acid (MES), 20 μmol/L acetosyringone (AS) and the above three antibiotics, at 28°C Shake culture to logarithmic growth phase.

The cells were collected by centrifugation and resuspended in 10 mmol/L MgCl ₂ , 10 mmol/L MES, 150 μmol/L AS, and the concentration was adjusted so that the OD ₆₀₀ was about 0.5, and the cells were allowed to stand at room temperature for 3 hours. Take a 5mL disposable syringe, remove the needle and absorb the Agrobacterium solution, and infiltrate the back of the leaves of common tobacco (5-6 weeks old or 4-6 true leaves). Infiltrate 2 leaves per plant. The infiltrated plants were grown in a light incubator at 23°C (alternating 16 hours light/8 hours dark).

A number of common tobacco NC89 strains about 6 weeks old were selected and inoculated with TVBMV attenuated mutants. After 15 days of inoculation, it was found that the tobacco did not show symptoms. It shows that the TVBMV attenuated mutant will not cause visible symptoms after inoculating plants, will not be transmitted by aphids, and is safe to use.

Example 2 Amplification of Potato X Virus Related Gene Fragments and Construction of Attenuated Vaccine

1. Amplification of potato X virus-related gene fragments

Using the PVX-1985 genome as a template, RT-PCR was used to amplify each gene fragment. The embodiments provided by the present invention are all in accordance with conventional experimental conditions, and the primer sequences used are as follows:

Table 2 PVX gene fragment amplification primer sequences

Numbering Sequence (5'-3') Seq ID Number 1 GCTCTAGAGCCACCACTTGCTTCTCAGACA Seq ID No.7 2 CTTAATTAACAAAGGGATGGTGGCAGGACTTC Seq ID No.8 3 GCTCTAGAGACCTACTTAGAGCCAGAGACTACGG Seq ID No.9 4 CTTAATTAAGTACATCACATTCGCACTACACACTTGG Seq ID No.10 5 GCTCTAGAA ATGGTATTCCTCAAGTCGCAGTGG Seq ID No.11 6 CTTAATTAAAAGAAAGTTTCTGAGGCGGGGA Seq ID No.12

Among them, primers 1 and 2 are used to amplify the Rd1 region of PVX, primers 3 and 4 are used to amplify the Rd2 region of PVX, and primers 5 and 6 are used to amplify the Rd3 region of PVX, and the nucleotides of the Rd1 gene obtained by amplification The sequence is shown in Seq ID No. 13, the nucleotide sequence of the Rd2 gene is shown in Seq ID No. 14, and the nucleotide sequence of the Rd3 gene is shown in Seq ID No. 15.

Using PVX-1985 as a template, RT-PCR was used to amplify its gene fragments, and the polymerase used was Phusion high-fidelity polymerase (Finnzymes).

2. Construction of attenuated vaccine

The recovered target fragments of each gene and TVBMV attenuated mutants were double digested with Xba I and Pac I, respectively. The ligation of the TVBMV attenuated mutant and the gene fragment, after the digestion product is recovered by gel, the ligation is carried out according to the ratio of the number of molecules of the vector and the fragment (1:3-1:10). Escherichia coli DH5α was transformed, and the ligated plasmid was verified by sequencing to obtain a chimeric virus.

Example 3 Amplification of Potato Y Virus Related Gene Fragments and Construction of Attenuated Vaccine

1. Amplification of potato Y virus-related gene fragments

Using the cDNA genome of PVY as a template, each gene fragment was amplified by RT-PCR. The embodiments provided by the present invention are all in accordance with conventional experimental conditions, and the primer sequences used are as follows:

Table 3 PVY gene fragment amplification primer sequences

Numbering Sequence (5'-3') Seq ID Number 1 GCTCTAGAGCCACCACTTGCTTCTCAGACA Seq ID No.16 2 CTTAATTAACAAAGGGATGGTGGCAGGACTTC Seq ID No.17 3 GCTCTAGAGACCTACTTAGAGCCAGAGACTACGG Seq ID No.18 4 CTTAATTAAGTACATCACATTCGCACTACACACTTGG Seq ID No.19

Among them, primers 1 and 2 are used to amplify the PVY1 gene fragment, and primers 3 and 4 are used to amplify the PVY 2 gene fragment. The nucleotide sequence of the amplified PVY1 gene fragment is shown in Seq ID No. 20, and the nucleotide sequence of the PVY 2 gene fragment is shown in Seq ID No. 21.

Using the cDNA of PVY as a template, PCR was used for amplification, and the polymerase used was Phusion high-fidelity polymerase. At the end of the reaction, after the PCR products were separated by 1% agarose gel electrophoresis, the target strip was cut out under UV light and the PCR products were recovered using the EasyPureQuick Gel Extraction Kit.

2. Vector construction

Two enzyme cleavage sites, PacI and XbaI, in the multiple cloning site of the tobacco vein mosaic virus attenuated mutant pCamTVBMV1 were used to digest the vector and the fragment respectively. The digested products were separated by 1% agarose gel electrophoresis, then recovered and ligated with T4 DNA ligase, standing at 4°C for 8 hours. The ligation product was transformed into E.coli DH5α competent cells, the colonies were verified by sequencing and the cells were cultured on a shaker, and the plasmid vector was extracted to obtain three single-linked attenuated vaccines, named pCamTVBMV1-PVY1 and pCamTVBMV1-PVY2 respectively.

Example 4 Amplification of Cucumber Mosaic Virus Related Gene Fragments and Construction of Attenuated Vaccine

1. Amplification of cucumber mosaic virus-related gene fragments

Using the CMV-QZ genome as a template, RT-PCR was used to amplify each gene fragment. The embodiments provided by the present invention are all in accordance with conventional experimental conditions, and the primer sequences used are as follows:

Table 4 CMV gene fragment amplification primer sequences

Among them, primers 1 and 2 are used to amplify the 2b region of CMV, and the nucleotide sequence of the 2b gene obtained by amplification is shown in SeqID No.24. Using CMV-QZ as a template, each gene fragment was amplified by RT-PCR, and the polymerase used was Phusion high-fidelity polymerase (Finnzymes). Amplification was performed by PCR, and the polymerase used was Phusion high-fidelity polymerase. At the end of the reaction, after the PCR products were separated by 1% agarose gel electrophoresis, the target strip was cut out under UV light and the PCR products were recovered using the EasyPure Quick Gel Extraction Kit.

2. Construction of attenuated vaccine

The recovered target fragments of each gene and TVBMV attenuated mutants were double digested with Xba I and Pac I, respectively. The ligation of the TVBMV attenuated mutant and the gene fragment, after the digestion product is recovered by gel, the ligation is carried out according to the ratio of the number of molecules of the vector and the fragment (1:3-1:10). The ligation product is transformed into Escherichia coli DH5α, and the ligated plasmid is verified by sequencing to obtain a chimeric virus, that is, an attenuated vaccine.

Example 5 Amplification of Tobacco Mosaic Virus Related Gene Fragments and Construction of Attenuated Vaccine

1. Amplification of Tobacco Mosaic Virus Related Gene Fragments

Using the cDNA genome of TMV as a template, RT-PCR was used to amplify each gene fragment. The embodiments provided by the present invention are all in accordance with conventional experimental conditions, and the primer sequences used are as follows:

Table 5 TMV gene fragment amplification primer sequences

The primers 1 and 2 were used to amplify the TMV 3 gene fragment. The nucleotide sequence of the amplified TMV3 gene fragment is shown in Seq ID No.27.

The cDNA of TMV was used as a template, and PCR was used for amplification, and the polymerase used was Phusion high-fidelity polymerase. At the end of the reaction, after the PCR products were separated by 1% agarose gel electrophoresis, the target strip was cut out under UV light and the PCR products were recovered using the EasyPureQuick Gel Extraction Kit.

2. Vector construction

Two enzyme cleavage sites, PacI and XbaI, in the multiple cloning site of the tobacco vein mosaic virus attenuated mutant pCamTVBMV1 were used to digest the vector and fragment respectively. The digested products were separated by 1% agarose gel electrophoresis, recovered and ligated with T4 DNA ligase, standing at 4°C for 8 hours. The ligation product was transformed into E.coli DH5α competent cells, the colonies were verified by sequencing and the cells were cultured on a shaker, and the plasmid vector was extracted to obtain a single-linked attenuated vaccine, named pCamTVBMV1-TMV3.

Example 6 Potato Y virus mutant attenuated strain

Mutation was carried out by PCR using the infectious clone pCamPVY ^N of Potato virus Y as a template. The embodiments provided by the present invention are all in accordance with conventional experimental conditions, and the primer sequences used are as follows:

Table 6 Site-directed mutagenesis primer sequences

Among them, primers 1 and 2 were used to mutate K to R at position 182 of HC-Pro. Bold letters indicate mutated nucleotide sites. The nucleotide sequence of HC-Pro before mutation is shown in Seq ID No.30, and the amino acid sequence is shown in Seq ID No.31.

Using the infective clone pCamPVY ^N of potato virus Y as template, mutation was carried out by PCR, and the polymerase used was Phusion high-fidelity polymerase (Finnzymes). After the reaction, 0.5 μL of DpnI (20 U/μL) was added to the PCR product, treated at 37 °C for 2 h, 125 μL of absolute ethanol and 5 μL of 3 mol/L sodium acetate (pH=5.2) were added, and the mixture was mixed, and precipitated at -20 °C overnight. Centrifuge at 13,000 r/min for 10 min, discard the supernatant, wash the precipitate with 1 mL of 75% ethanol, and place it to dry at room temperature. Add 10 μL of ddH2O for redissolving, and transform E. coli DH5α. The mutant plasmid was verified by sequencing, and the obtained mutant plasmid was named pCamPVY-K182R .

Example 7 Attenuated vaccine transforms Agrobacterium and prepares attenuated vaccine composition.

The various attenuated vaccines obtained in Example 2 to Example 6 were transformed into Agrobacterium GV3101. After verification by colony PCR, recombinant bacteria were obtained. Then single spot was inoculated in liquid LB medium containing kanamycin (50 μg/mL), rifamycin (50 μg/mL) and tetracycline (50 μg/mL). Add 500 μL of bacterial liquid to 5 mL of LB medium containing 10 mmol/L 2-(N-morpholine)-ethylsulfonic acid (MES), 20 μmol/L acetosyringone (AS) and the above three antibiotics, at 28°C Shake culture to logarithmic growth phase.

Then the cells were collected by centrifugation and mixed with an equal volume of light calcium carbonate in a mortar to form a mixture powder.

Take 20 μL of bacterial liquid and 20 μL of powder respectively, and then apply LB plate after diluting 5 times; the growth of colonies is shown in Figure 3. After the bacterial liquid and powder are coated, there is basically no difference in the growth of colonies, indicating that the light calcium carbonate drying bacteria The body does not affect the activity of the bacteria.

Then, the amount of 50 mg of fulvic acid and 50 mg of xanthan gum is added to the mixed powder of each 10 ml of fermented bacterial cells and light calcium carbonate mixed powder to prepare a plant virus attenuated vaccine composition.

Test example

Taking the potato X virus attenuated vaccine composition as an example, get 10ml of fermented thalline and light calcium carbonate mixture powder, add the following 11 groups of fulvic acid and xanthan gum with different gradient contents respectively, as 11 test groups, investigate the fulvic Effects of acid and xanthan gum additions on composition stability.

Table 7 The addition amount of fulvic acid and xanthan gum in each test group

test group Fulvic acid/mg Xanthan gum/mg 1 0 0 2 1000 0 3 100 0 4 50 0 5 10 0 6 0 1000 7 0 100 8 0 50 9 0 10 10 100 100 11 50 50

After the compositions of test groups 1-11 were prepared, 100 μL of powder was taken from each group, the suspension was resuspended and coated on LB plates for the first time.

Then placed at room temperature for two months, at the ninth week, 100 μL of powder was taken from each group, the suspension was resuspended and coated on LB plates, and the colony morphology was photographed after inverting at 28°C for 48 hours. The results are shown in Figure 4. The experimental group 11 had the highest number of colonies. The colony morphology map was imported into the computer and processed with Photoshop. Use the brush mode to click on each colony and count it with the keyboard and mouse software. The results are shown in Figure 5.

The results of the colony in the ninth week showed that in the test group 11, the number of colonies treated with 50 mg of fulvic acid and 50 mg of xanthan gum in 10 ml of fermented bacteria and light calcium carbonate mixture powder was the highest, which was 10228/100 μL.

In addition, the colony morphology diagram of the test group 11 coated in the ninth week was compared with the colony morphology diagram after the first coating. There is basically no difference in the number of colonies applied for the first time (as shown in Figure 4 ), indicating that the composition and method of the present invention can prolong the storage time of the attenuated vaccine, improve the shelf life, be beneficial to the wide application of the attenuated vaccine, and can greatly reduce losses.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention in any form. Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Within the scope of the technical solution of the present invention, personnel can make some changes or modifications to equivalent examples of equivalent changes by using the above-mentioned technical content, but any content that does not depart from the technical solution of the present invention is based on the technical solution of the present invention. Substantially any simple modifications, equivalent changes and modifications made to the above embodiments still fall within the scope of the solutions of the present invention.

Claims (10)

1. a plant virus attenuated vaccine composition is characterized in that, the plant virus attenuated vaccine composition comprises the fermented thalline, light calcium carbonate, fulvic acid and xanthan gum that carry the plant virus attenuated vaccine. 2. a kind of plant virus attenuated vaccine composition according to claim 1, is characterized in that, the volume ratio of the fermented thalline carrying the plant virus attenuated vaccine and light calcium carbonate is 1:1. 3. a kind of plant virus attenuated vaccine composition according to claim 1 and 2, is characterized in that, in the described composition, the mass ratio of fulvic acid and xanthan gum is 1:1. 4. according to any described a kind of plant virus attenuated vaccine composition of claim 1-3, it is characterized in that, the addition of described fulvic acid and xanthan gum and fermented thalline and light calcium carbonate mixture The relationship is: 50 mg of fulvic acid and 50 mg of xanthan gum are added to each 10 ml of the mixture of fermented cells and light calcium carbonate. 5. according to any described a kind of plant virus attenuated vaccine composition of claim 1-4, it is characterized in that, described plant virus attenuated vaccine comprises anti-potato virus X, potato virus Y, cucumber mosaic virus, tobacco flower Attenuated vaccine for at least one of the leaf viruses. 6. according to the arbitrary described a kind of plant virus attenuated vaccine composition of claim 1-5, it is characterized in that, described plant virus attenuated vaccine comprises taking TVBMV attenuated mutant as carrier, and embedded in the TVBMV attenuated mutant. an effective gene segment for inducing cross-protection against potato virus X, and/or potato virus Y, and/or cucumber mosaic virus, and/or tobacco mosaic virus; Or, described plant virus attenuated vaccine comprises potato virus X, and/or potato virus Y, and/or cucumber mosaic virus, and/or the attenuated strain of tobacco mosaic virus mutation; Preferably, the plant virus attenuated vaccine comprises a mutant attenuated strain of potato Y virus, and K at position 182 of HC-Pro is mutated to R. 7. according to the arbitrary described a kind of plant virus attenuated vaccine composition of claim 1-7, it is characterized in that, the effective gene fragment that can induce cross-protection to potato X virus comprises the RdRp gene of potato X virus, its nucleoside The acid sequence is shown in Seq ID No.13, or Seq ID No.14, or Seq ID No.15; Alternatively, the effective gene fragments that can induce cross-protection against Potato Y virus include PVY1 fragment and PVY2 fragment, the nucleotide sequence of PVY1 fragment is shown in Seq ID No. 20, and the nucleotide sequence of PVY2 fragment is shown in Seq ID No. 20. 21 shown; Alternatively, the effective gene fragment that can induce cross protection against cucumber mosaic virus includes the 2b gene of cucumber mosaic virus, and the nucleotide sequence of the 2b gene is shown in Seq ID No.24; Alternatively, an effective gene fragment that can induce cross-protection against tobacco mosaic virus includes the TMV3 fragment of tobacco mosaic virus, and the nucleotide sequence of the TMV3 fragment is shown in Seq ID No.27. 8. The plant virus attenuated vaccine composition according to any one of claims 1-7, wherein the fermented thalline comprises Agrobacterium. 9. a method for prolonging the preservation time of plant virus attenuated vaccine, is characterized in that, comprises: (1) After the fermented thalline carrying the attenuated vaccine is concentrated by centrifugation, light calcium carbonate powder is added to make the mixture powder; (2) adding fulvic acid and xanthan gum in the mixture powder to make a plant virus attenuated vaccine composition to prolong the shelf life; Preferably, the volume ratio of the fermented thalline carrying the plant virus attenuated vaccine and light calcium carbonate is 1:1; Preferably, the mass ratio of fulvic acid and xanthan gum is 1:1; Preferably, the relationship between the added amount of fulvic acid and xanthan gum and the mixture of fermented cells and light calcium carbonate is: add 50 mg of fulvic acid and 50 mg of yellow humic acid to every 10 ml of the mixture of fermented cells and light calcium carbonate Original gum. 10. A composition as claimed in any one of claims 1-8, or the application of the method as claimed in claim 9 in prolonging the preservation time of plant virus attenuated vaccines.

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