CN110856493B - Plant virus attenuated vaccine composition, attenuated vaccine storage method and application thereof - Google Patents
Plant virus attenuated vaccine composition, attenuated vaccine storage method and application thereof Download PDFInfo
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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
Technical Field
The invention relates to the field of agricultural science, in particular to a plant virus attenuated vaccine composition, an attenuated vaccine storage method and application thereof.
Background
The virus disease is one of important diseases in agricultural production, seriously affects the yield and quality of crops and causes huge loss. At present, no specific medicament for preventing and treating crop virus diseases exists in the market, so that the prevention and the treatment of the crop virus diseases are very difficult. Cross protection is an effective means for preventing and treating plant virus diseases. With the progress of the technology, infectious clones of the virus can be constructed at present, a low virulent vaccine which has obviously reduced pathogenicity and can protect crops from being infected by a virulent strain is screened by a reverse genetics technology, and the virus disease can be effectively prevented and treated by inoculating in advance at the seedling stage of the crops.
Attenuated plant virus vaccines require the use of Agrobacterium for production, by means of which plants are inoculated. However, the survival time of the agrobacterium at normal temperature is not long, and the number of live bacteria is greatly reduced after a period of time, so that the agrobacterium carrying the plant virus attenuated vaccine can not be stored for a long time, and the wide application of the plant virus attenuated vaccine is influenced.
The present invention has been made in view of this situation.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a plant virus attenuated vaccine composition, an attenuated vaccine storage method and application thereof. The invention uses light calcium carbonate to dry the agrobacterium tumefaciens carrying the attenuated vaccine, and adds fulvic acid and xanthan gum, thereby prolonging the storage time of the attenuated vaccine, improving the shelf life and being beneficial to the wide application of the attenuated vaccine.
In order to solve the technical problems, the invention adopts the technical scheme that:
the invention provides a plant virus attenuated vaccine composition, which comprises fermented thalli carrying the plant virus attenuated vaccine, light calcium carbonate, fulvic acid and xanthan gum.
In a further scheme, the volume ratio of the fermentation thalli carrying the plant virus attenuated vaccine to the light calcium carbonate is 1: 1.
In a further scheme, the mass ratio of the fulvic acid to the xanthan gum in the composition is 1: 1.
In a further scheme, the relationship between the addition amount of the fulvic acid and the xanthan gum and the mixture of the fermentation thallus and the 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 invention proves that the preservation period can be effectively prolonged by adding the fulvic acid and the xanthan gum into the agrobacterium tumefaciens carrying the plant virus attenuated vaccine, and finally, the formula proportion of 50mg of fulvic acid and 50mg of xanthan gum added into each 10ml of light calcium carbonate powder has the best preservation effect by screening different concentration gradients, and the preservation period of the plant virus attenuated vaccine can be prolonged by more than 2 months.
In a further scheme, the plant virus attenuated vaccine comprises attenuated vaccine for resisting at least one of potato virus X, potato virus Y, cucumber mosaic virus and tobacco mosaic virus.
In a further scheme, the plant virus attenuated vaccine comprises a TVBMV attenuated mutant as a vector, wherein an effective gene fragment capable of inducing cross protection on potexvirus, and/or potyvirus, and/or cucumber mosaic virus, and/or tobacco mosaic virus is embedded in the TVBMV attenuated mutant;
or the plant virus attenuated vaccine comprises a potato virus X and/or potato virus Y and/or cucumber mosaic virus and/or tobacco mosaic virus mutant attenuated strain;
preferably, the plant virus attenuated vaccine comprises a potato Y virus mutant attenuated strain, and K at the 182 th position of HC-Pro is mutated into R.
Further, effective gene fragments inducing cross-protection against potexvirus include the RdRp gene of potexvirus, the nucleotide sequence of which is shown in Seq ID No.13, or Seq ID No.14, or Seq ID No. 15;
alternatively, the effective gene segment inducing cross protection against potyvirus comprises a PVY1 segment and a PVY2 segment, wherein the nucleotide sequence of the PVY1 segment is shown as Seq ID No.20, and the nucleotide sequence of the PVY2 segment is shown as Seq ID No. 21;
or, the effective gene segment which can induce the cross protection to the cucumber mosaic virus comprises the 2b gene of the cucumber mosaic virus, and the nucleotide sequence of the 2b gene is shown as Seq ID No. 24;
alternatively, useful gene segments that induce cross-protection against tobacco mosaic virus include the TMV3 fragment of tobacco mosaic virus, the nucleotide sequence of TMV3 fragment being shown in Seq ID No. 27.
In a further scheme, the fermentation thalli comprise agrobacterium.
The second purpose of the invention is to provide a method for prolonging the preservation time of the plant virus attenuated vaccine, which comprises the following steps:
(1) after the fermentation thalli carrying the attenuated vaccine is centrifugally concentrated, adding light calcium carbonate powder to prepare mixture powder;
(2) adding fulvic acid and xanthan gum into the mixture powder to prepare the plant virus attenuated vaccine composition, so that the shelf life is prolonged;
preferably, the volume ratio of the fermentation thalli carrying the plant virus attenuated vaccine to the light calcium carbonate is 1: 1;
preferably, the mass ratio of the fulvic acid to the xanthan gum is 1: 1;
preferably, the relationship between the addition amount of the fulvic acid and the xanthan gum and the mixture of the fermentation thallus and the 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.
It is a third object of the present invention to provide a composition as described above, or the use of a method as described above, for prolonging the shelf life of a plant virus attenuated vaccine.
The specific technical scheme of the implementation of the invention is as follows:
a. powder conditioning test: the agrobacteria carrying the plant virus attenuated vaccine are divided into two groups, one group is not processed, and the other group is centrifuged to collect thalli and added with light calcium carbonate with the same volume to be mixed evenly into powder. Respectively taking 20 mu L of bacterial liquid and 20 mu L of powder, diluting by 5 times, and coating an LB flat plate; the viable count is 14785/100 muL and 14645/100 muL respectively, which proves that the light calcium carbonate treatment has no influence on the bacterial activity.
b. Optimizing the working concentration of fulvic acid and xanthan gum: and respectively arranging control groups of fulvic acid and xanthan gum with different concentrations, and standing and storing at room temperature.
c. And (4) observing the preservation condition of the vaccine: 100 μ L of powder was taken from each group, the suspension was weighted to resuspend and coat LB plates and the colony growth was observed.
After the technical scheme is adopted, compared with the prior art, the invention has the following beneficial effects.
1. According to the plant virus attenuated vaccine composition, the agrobacterium tumefaciens carrying the attenuated vaccine is dried by using the light calcium carbonate, and the fulvic acid and the xanthan gum are added, so that the storage time of the attenuated vaccine can be prolonged, the shelf life is prolonged, and the wide application of the attenuated vaccine is facilitated.
2. In the method for preserving the plant virus attenuated vaccine, thalli carrying the plant virus attenuated vaccine are centrifugally collected and are uniformly mixed into mixture powder by using light calcium carbonate, the formula proportion of 50mg of fulvic acid and 50mg of xanthan gum is added into every 10ml of mixture powder, the preservation effect is best, and the preservation period of the plant virus attenuated vaccine can be prolonged by more than 2 months.
The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention, are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention without limiting the invention to the right. It is obvious that the drawings in the following description are only some embodiments, and that for a person skilled in the art, other drawings can be derived from them without inventive effort. In the drawings:
FIG. 1 is a schematic diagram of TVBMV genome fragment amplification;
FIG. 2 is a schematic diagram of the pCamTVBMV genome structure;
FIG. 3 is a drawing showing that 20. mu.L of bacterial liquid and 20. mu.L of powder are diluted 5 times and applied to an LB plate;
FIG. 4 shows the effect of the attenuated vaccine compositions of fulvic acid and xanthan gum in different addition amounts after nine weeks of storage by coating LB plate;
FIG. 5 counting colonies on LB plates after nine weeks of storage;
FIG. 6 is a comparison of colony growth after the first coating and the ninth week coating when 50mg of each of fulvic acid and xanthan gum was added to 10ml of the mixture powder.
It should be noted that the drawings and the description are not intended to limit the scope of the inventive concept in any way, but to illustrate it by a person skilled in the art with reference to specific embodiments.
Detailed Description
In order to make the objects, 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 for illustrating the present invention and are not intended to limit the scope of the present invention.
Example one construction of a Multi-site TVBMV attenuated mutant
1. Construction of tobacco vein banding mosaic virus infectious clone
The RNA of tobacco vein banding mosaic virus is taken as a template, and reverse transcription is carried out by using a random primer. According to the restriction map of the whole genome of the existing tobacco vein banding mosaic virus, the amplification can be divided into three parts, and the TVBMV full-length cDNA clone is assembled after enzyme digestion. Firstly, the 35S promoter was fused to the upstream of the fragment from the untranslated region of TVBMV 5' to the cleavage site of HC-pro gene Nru I by Overlap-PCR, which was named p35S-HC by the inventors; PCR-amplifying a fragment from the enzyme cutting site of HC-pro gene Nru I to the enzyme cutting site of 6K2Xho I, and the inventor names the fragment as pHC-6K 2; the fragment from the 6K2Xho I cleavage site to the tail of ploy (A) was PCR amplified and named p6K2-polyA by the inventors (as shown in FIG. 1).
The reverse transcriptase used for cDNA synthesis is Moloney murine leukaemia virus reverse transcriptase (Promega); the total plant RNA is used as a template, and a random primer is used as a reverse transcription primer for reverse transcription.
And carrying out PCR amplification by using the obtained reverse transcription product as a template and corresponding primers. The PCR product was subjected to 1% agarose gel electrophoresis. Cutting and recovering to obtain p35S-HC2110，pHC2111-6K26075And p6K26076-polyA three fragments, which were ligated and double digested with SbfI and Sma I followed by 0.8% agarose gel electrophoresis, recovered and ligated into the agrobacterium-mediated expression vector pCAMBIA0390, the invasive clone constructed by the strategy was named pCamTVBMV (as shown in figure 2).
2. Construction of TVBMV attenuated mutant
After obtaining the tobacco vein banding mosaic virus infectious clone, mutating key sites in an HC-Pro sequence to obtain a TVBMV attenuated mutant, wherein the specific method comprises the following steps:
designing a mutation primer, and carrying out site-directed mutation on a conserved amino acid site of the tobacco vein banding mosaic virus HC-Pro according to the method of Liu et al (2008), wherein the name and the sequence of the mutated primer are shown in Table 1.
TABLE 1TVBMV HC-Pro mutant primer names and sequences
Wherein, the 52-site amino acid of the HC-Pro amino acid sequence is subjected to site-directed mutagenesis by the primer 1 and the primer 2, and arginine is mutated into glutamic acid; the 198 th amino acid of the HC-Pro amino acid sequence is subjected to site-directed mutagenesis by the primer 3 and the primer 4, and aspartic acid is mutated into lysine; the primer 5 and the primer 6 carry out site-directed mutagenesis on the amino acids at the 250 th position and the 251 th position of the HC-Pro amino acid sequence, the isoleucine at the 250 th position is mutated into the aspartic acid, and the glutamine at the 251 th position is mutated into the glutamic acid.
PCR mutagenesis system using pCamTVBMV as template: 5 XPCR Buffer 10. mu.L, dNTP (10mM) 1. mu.L, mutation primer F (10. mu.M) 1. mu.L, mutation primerR (10. mu.M) 1. mu.L, template plasmid 10ng, Phusion DNA polymerase 0.3. mu.L, ddH2The amount of O was made up to 50. mu.L.
PCR mutation procedure: 98 ℃/30 sec; 98 ℃/10sec, Tmno +3 ℃/20sec, 72 ℃/5min, 20 cycles; 98 ℃/10 sec; tmpp/20 sec; 72 ℃/15 min; storing at 4 ℃.
After the mutation PCR is finished, 1 mu L of Dpn I is added into each reaction system, and after the mixture is fully mixed, the mixture is digested for 4 hours at 37 ℃.
After the PCR reaction system is treated by Dpn I, 125 muL of absolute ethyl alcohol (2.5 multiplied by volume) and 5 muL of 3M NaAc pH8.0 are added, and the mixture is evenly mixed and precipitated overnight; 12000r/min, 10min, abandoning the supernatant; washing the precipitate with 1mL of 75% ethanol, and discarding the supernatant; the precipitate was dried and 10. mu.L of ddH2O dissolved the precipitate.
Transforming the mutation precipitation product into escherichia coli, uniformly coating transformed thalli on an LB (lysogeny broth) plate containing Amp antibiotics of X-gal and IPTG (isopropyl-beta-thiogalactoside), selecting a single colony for culturing, extracting plasmids for sequencing, and obtaining the TVBMV attenuated mutant with four site mutations if the sequencing is correct.
3. Research on virulence of TVBMV attenuated mutant
And transferring the obtained mutant plasmid into agrobacterium, and obtaining a recombinant bacterium after colony PCR verification. Then, a single spot was selected and inoculated into liquid LB medium containing kanamycin (50. mu.g/mL), rifamycin (50. mu.g/mL), tetracycline (50. mu.g/mL). Adding 500 μ L of the bacterial liquid into 5mL LB culture medium containing 10 mmol/L2- (N-morpholine) -ethylsulfonic acid (MES), 20 μmol/L Acetosyringone (AS) and the above three antibiotics, and culturing at 28 deg.C under shaking to logarithmic phase.
The cells were collected by centrifugation and resuspended in 10mmol/L MgCl2In 10mmol/L MES, 150. mu. mol/L AS, the concentration was adjusted to OD600About 0.5, and left to stand at room temperature for 3 hours. Taking a 5mL disposable syringe, removing a needle head to absorb the agrobacterium liquid, and infiltrating from the back of common tobacco (5-6 weeks old or 4-6 true leaves) leaves. Each plant was infiltrated with 2 leaves. The infiltrated plants were cultured in a 23 ℃ light incubator (16 hours light/8 hours dark alternating).
A plurality of ordinary tobaccos NC89 with the period of about 6 weeks are selected, TVBMV attenuated mutants are inoculated, and the tobaccos are found to have no symptoms after 15 days of inoculation. The TVBMV attenuated mutant can not cause visible symptoms after being inoculated with plants, can not be spread by aphids and is safe to use.
EXAMPLE two amplification of Gene fragments related to potexvirus and construction of attenuated vaccines
1. Amplification of potato virus X-related gene fragments
Each gene fragment was amplified by RT-PCR using the PVX-1985 genome as a template. The examples provided by the present invention were performed according to conventional experimental conditions, wherein the primer sequences used are as follows:
TABLE 2PVX Gene fragment amplification primer sequences
| Numbering | Sequence (5'-3') | |
| 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 |
Wherein the primers 1 and 2 are used for amplifying the Rd1 region of PVX, the primers 3 and 4 are used for amplifying the Rd2 region of PVX, the primers 5 and 6 are used for amplifying the Rd3 region of PVX, the nucleotide sequence of the Rd1 gene obtained by amplification is shown as Seq ID No.13, the nucleotide sequence of the Rd2 gene is shown as Seq ID No.14, and the nucleotide sequence of the Rd3 gene is shown as Seq ID No. 15.
Using PVX-1985 as template, RT-PCR is used to amplify each gene segment, and the polymerase is Phusion high fidelity polymerase (Finnzymes).
2. Construction of attenuated vaccines
The target fragment of each gene and the TVBMV attenuated mutant are recovered and subjected to double digestion by Xba I and Pac I respectively. Connecting the TVBMV attenuated mutant with the gene fragment, recovering the enzyme digestion product through gel, and connecting according to the ratio of the vector to the fragment molecule number (1: 3-1: 10). And transforming the escherichia coli DH5 alpha, and carrying out sequencing verification on the connected plasmid to obtain the chimeric virus.
Example amplification of the fragment of the Gene associated with the Potato Virus Y and construction of an attenuated vaccine
1. Amplification of potato virus Y-related gene fragments
Each gene fragment was amplified by RT-PCR using the cDNA genome of PVY as a template. The examples provided by the present invention were performed according to conventional experimental conditions, wherein the primer sequences used are as follows:
TABLE 3PVY Gene fragment amplification primer sequences
| Numbering | Sequence (5'-3') | |
| 1 | GCTCTAGAGCCACCACTTGCTTCTCAGACA | Seq ID No.16 |
| 2 | CTTAATTAACAAAGGGATGGTGGCAGGACTTC | Seq ID No.17 |
| 3 | GCTCTAGAGACCTACTTAGAGCCAGAGACTACGG | Seq ID No.18 |
| 4 | CTTAATTAAGTACATCACATTCGCACTACACACTTGG | Seq ID No.19 |
Wherein the primers 1 and 2 are used for amplifying the PVY1 gene segment, and the primers 3 and 4 are used for amplifying the PVY2 gene segment. The nucleotide sequence of the PVY1 gene fragment obtained by amplification is shown as Seq ID No.20, and the nucleotide sequence of the PVY2 gene fragment is shown as Seq ID No. 21.
And (3) carrying out amplification by using PCR (polymerase chain reaction) by using cDNA (complementary deoxyribonucleic acid) of PVY as a template, wherein the polymerase is Phusion high-fidelity polymerase. After the reaction, the PCR product was separated by 1% agarose Gel electrophoresis, the desired Gel strip was cut under an ultraviolet lamp and the PCR product was recovered using the EasyPure Quick Gel Extraction Kit.
2. Vector construction
The vector and the fragment were digested separately using two cleavage sites PacI and XbaI in the multiple cloning site of the tobacco vein banding mosaic virus attenuated mutant pCamTVBMV 1. After the enzyme digestion product is separated by 1 percent agarose gel electrophoresis, the enzyme digestion product is recovered and is connected by T4DNA ligase and is kept stand for 8 hours at the temperature of 4 ℃. E.coli DH5 alpha competent cells were transformed with the ligation products, colonies were verified by sequencing and cells were shake-cultured, plasmid vectors were extracted to obtain 3 single attenuated vaccines, which were named pCamTVBMV1-PVY1 and pCamTVBMV1-PVY2, respectively.
Example four amplification of cucumber mosaic Virus-related Gene fragments and construction of attenuated vaccines
1. Amplification of cucumber mosaic virus-related gene fragment
Each gene fragment was amplified by RT-PCR using CMV-QZ genome as a template. The examples provided by the present invention were performed according to conventional experimental conditions, wherein the primer sequences used are as follows:
TABLE 4CMV Gene fragment amplification primer sequences
Wherein, the primers 1 and 2 are applied to the 2b region of the amplified CMV, and the nucleotide sequence of the amplified 2b gene is shown as Seq ID No. 24. CMV-QZ is taken as a template, and each gene fragment is amplified by RT-PCR, and the polymerase used is Phusion high-fidelity polymerase (Finnzymes). PCR is used for amplification, and Phusion high-fidelity polymerase is used as polymerase. After the reaction, the PCR product was separated by 1% agarose Gel electrophoresis, the desired Gel strip was cut under an ultraviolet lamp and the PCR product was recovered using the EasyPure Quick Gel Extraction Kit.
2. Construction of attenuated vaccines
The target fragment of each gene and the TVBMV attenuated mutant are recovered and subjected to double digestion by Xba I and Pac I respectively. Connecting the TVBMV attenuated mutant with the gene fragment, recovering the enzyme digestion product through gel, and connecting according to the ratio of the vector to the fragment molecule number (1: 3-1: 10). The ligation product is transformed into Escherichia coli DH5 alpha, and the plasmid after ligation is verified by sequencing to obtain the chimeric virus, namely the attenuated vaccine.
Example five amplification of tobacco mosaic virus-related Gene fragment and construction of attenuated vaccine
1. Amplification of tobacco mosaic virus-related Gene fragments
Each gene fragment was amplified by RT-PCR using the cDNA genome of TMV as a template. The examples provided by the present invention were performed according to conventional experimental conditions, wherein the primer sequences used are as follows:
TABLE 5 primer sequences for amplification of TMV Gene fragments
Wherein the primers 1 and 2 are used for amplifying the TMV3 gene segment. The nucleotide sequence of the TMV3 gene fragment obtained by amplification is shown in Seq ID No. 27.
And (3) taking cDNA of TMV as a template, and carrying out amplification by utilizing PCR, wherein the polymerase is Phusion high-fidelity polymerase. After the reaction, the PCR product was separated by 1% agarose Gel electrophoresis, the desired Gel strip was cut under an ultraviolet lamp and the PCR product was recovered using the EasyPure Quick Gel Extraction Kit.
2. Vector construction
The vector and the fragment were digested separately using two cleavage sites PacI and XbaI in the multiple cloning site of the tobacco vein banding mosaic virus attenuated mutant pCamTVBMV 1. After the enzyme digestion product is separated by 1 percent agarose gel electrophoresis, the enzyme digestion product is recovered and is connected by T4DNA ligase and is kept stand for 8 hours at the temperature of 4 ℃. Transforming E.coli DH5 alpha competent cells with the ligation products, sequencing, verifying bacterial colonies, culturing thalli by shaking tables, and extracting plasmid vectors to obtain the single attenuated vaccine named pCamTVBMV1-TMV 3.
Example six Potato Y Virus mutant attenuated strains
Cloning pCamPVY by infecting with Potato Virus YNAs a template, mutagenesis was performed by PCR. The examples provided by the present invention were performed according to conventional experimental conditions, wherein the primer sequences used are as follows:
TABLE 6 site-directed mutagenesis primer sequences
Wherein primers 1 and 2 are used to mutate K to R at position 182 of HC-Pro. Bold letters represent 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.
Cloning pCamPVY by infecting with Potato Virus YNAs template, the mutations were carried out by PCR using Phusion high fidelity polymerase (Finnzymes). After the reaction, 0.5. mu.L of DpnI (20U/. mu.L) was added to the PCR product, treated at 37 ℃ for 2 hours, mixed with 125. mu.L of absolute ethanol and 5. mu.L of 3mol/L sodium acetate (pH 5.2), and precipitated at-20 ℃ overnight. 13000r/min for 10min, abandoning the supernatant, washing the precipitate with 1mL 75% ethanol, then placing the precipitate at room temperature for natural drying, adding 10 μ L ddH2O water for redissolving, transforming Escherichia coli DH5 α, and carrying out sequencing verification on the mutant plasmid to obtain the mutant plasmid named pCamPVY-K182R.
Example seven attenuated vaccines agrobacterium were transformed and attenuated vaccine compositions were prepared.
The various attenuated vaccines obtained in examples two to six were transformed into agrobacterium GV 3101. And obtaining the recombinant bacteria after colony PCR verification. Then, a single spot was selected and inoculated into liquid LB medium containing kanamycin (50. mu.g/mL), rifamycin (50. mu.g/mL), tetracycline (50. mu.g/mL). Adding 500 μ L of the bacterial liquid into 5mL LB culture medium containing 10 mmol/L2- (N-morpholine) -ethylsulfonic acid (MES), 20 μmol/L Acetosyringone (AS) and the above three antibiotics, and culturing at 28 deg.C under shaking to logarithmic phase.
Then the thalli are collected by centrifugation and mixed evenly in a mortar by using light calcium carbonate with the same volume to form mixture powder.
Respectively taking 20 mu L of bacterial liquid and 20 mu L of powder, diluting by 5 times, and coating an LB flat plate; the growth of the colonies is shown in FIG. 3, and the growth of the colonies after the bacterial liquid and the powder are coated is almost the same, which shows that the activity of the cells is not affected by the light calcium carbonate dry cells.
Then adding 50mg of fulvic acid and 50mg of xanthan gum into 10ml of the mixture powder of the fermented thalli and the light calcium carbonate to prepare the plant virus attenuated vaccine composition.
Test examples
Taking a potato X virus attenuated vaccine composition as an example, 10ml of fermented thallus and light calcium carbonate mixture powder are respectively added with the following 11 groups of fulvic acid and xanthan gum with different gradient contents to serve as 11 test groups, and the influence of the addition amount of the fulvic acid and the xanthan gum on the stable period of the composition is examined.
Table 7 additive amounts of fulvic acid and xanthan gum for each test group
| Test group | Fulvic acid/mg | Xanthan gum/ |
| 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 |
Compositions for test groups 1-11 after preparation, 100. mu.L of powder was taken from each group, the suspension was resuspended and the first LB plate was spread, and colony morphology was photographed and counted after 48h of inverted culture at 28 ℃.
Then, the mixture was left at room temperature for two months, 100. mu.L of the powder was taken from each group at the ninth week, the suspension was weighted and resuspended in LB plates, and a colony morphology was photographed after 48 hours of inverted culture at 28 ℃ as shown in FIG. 4, in which the number of colonies in test group 11 was the largest. The colony morphology was introduced into a computer and treated with Photoshop, and each colony was clicked using a brush mode and counted using a keyboard and mouse software, and the results are shown in FIG. 5.
The colony results at the ninth week showed that the number of colonies in test group 11, which were treated with 10ml of the powder mixture of fermented cells and precipitated calcium carbonate and added with 50mg of fulvic acid and 50mg of xanthan gum, was 10228 colonies/100. mu.L at the maximum.
In addition, the colony morphology of the test group 11 coated in the ninth week is compared with the colony morphology after the first coating, and the result is shown in fig. 6, after the test group is placed for two months at normal temperature, the colony number of the test group 11 is basically the same as the colony number of the first coating (as shown in fig. 4), which indicates that the composition and the method of the invention can prolong the storage time of the attenuated vaccine, improve the shelf life, facilitate the wide application of the attenuated vaccine, and greatly reduce the loss.
Although the present invention has been described with reference to a preferred embodiment, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (10)
1. A plant virus attenuated vaccine composition is characterized in that the plant virus attenuated vaccine composition comprises fermentation thalli carrying the plant virus attenuated vaccine, light calcium carbonate, fulvic acid and xanthan gum;
the plant virus attenuated vaccine takes a TVBMV attenuated mutant as a carrier, and the TVBMV attenuated mutant is embedded with an effective gene fragment which can induce the cross protection of potexvirus, and/or potyvirus, and/or cucumber mosaic virus, and/or tobacco mosaic virus;
the TVBMV attenuated mutant is obtained by four site mutations of pCamTVBMV in an HC-Pro sequence, wherein the site mutations are as follows: arginine at position 52 is mutated into glutamic acid, and aspartic acid at position 198 is mutated into lysine; isoleucine at position 250 is mutated into aspartic acid, and glutamine at position 251 is mutated into glutamic acid;
effective gene segments inducing cross-protection against potexvirus include the RdRp gene of potexvirus, the nucleotide sequence of which is shown in Seq ID No.13, or Seq ID No.14, or Seq ID No. 15;
effective gene segments capable of inducing the cross protection of the potyvirus comprise a PVY1 segment and a PVY2 segment, wherein the nucleotide sequence of the PVY1 segment is shown as Seq ID No.20, and the nucleotide sequence of the PVY2 segment is shown as Seq ID No. 21;
the effective gene segment which can induce the generation of cross protection on the cucumber mosaic virus comprises a 2b gene of the cucumber mosaic virus, and the nucleotide sequence of the 2b gene is shown as Seq ID No. 24;
effective gene segments inducing cross protection against tobacco mosaic virus include TMV3 segment of tobacco mosaic virus, and the nucleotide sequence of TMV3 segment is shown in Seq ID No. 27.
2. The composition of claim 1, wherein the volume ratio of the fermentation bacteria carrying the attenuated plant virus vaccine to the light calcium carbonate is 1: 1.
3. The plant virus attenuated vaccine composition of claim 1, wherein the mass ratio of fulvic acid to xanthan gum in said composition is 1: 1.
4. The attenuated plant virus vaccine composition of any one of claims 1 to 3, wherein the amount of fulvic acid and xanthan gum added is related to the mixture of fermented biomass and precipitated calcium carbonate: 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.
5. The attenuated plant virus vaccine composition of claim 1, wherein said fermentive bacteria comprise Agrobacterium.
6. A method for extending the shelf life of a plant virus attenuated vaccine, comprising:
(1) after the fermentation thalli carrying the attenuated vaccine is centrifugally concentrated, adding light calcium carbonate powder to prepare mixture powder;
(2) adding fulvic acid and xanthan gum into the mixture powder to prepare the plant virus attenuated vaccine composition, so that the shelf life is prolonged;
the attenuated vaccine comprises a TVBMV attenuated mutant which is used as a carrier and is embedded with an effective gene fragment which can induce cross protection to potexvirus, and/or potyvirus, and/or cucumber mosaic virus, and/or tobacco mosaic virus;
the TVBMV attenuated mutant is obtained by four site mutations of pCamTVBMV in an HC-Pro sequence, wherein the site mutations are as follows: arginine at position 52 is mutated into glutamic acid, and aspartic acid at position 198 is mutated into lysine; isoleucine at position 250 is mutated into aspartic acid, and glutamine at position 251 is mutated into glutamic acid;
effective gene segments inducing cross-protection against potexvirus include the RdRp gene of potexvirus, the nucleotide sequence of which is shown in Seq ID No.13, or Seq ID No.14, or Seq ID No. 15;
effective gene segments capable of inducing the cross protection of the potyvirus comprise a PVY1 segment and a PVY2 segment, wherein the nucleotide sequence of the PVY1 segment is shown as Seq ID No.20, and the nucleotide sequence of the PVY2 segment is shown as Seq ID No. 21;
the effective gene segment which can induce the generation of cross protection on the cucumber mosaic virus comprises a 2b gene of the cucumber mosaic virus, and the nucleotide sequence of the 2b gene is shown as Seq ID No. 24;
effective gene segments inducing cross protection against tobacco mosaic virus include TMV3 segment of tobacco mosaic virus, and the nucleotide sequence of TMV3 segment is shown in Seq ID No. 27.
7. The method for prolonging the storage life of a plant virus attenuated vaccine as claimed in claim 6, wherein the volume ratio of the fermentation thallus carrying the plant virus attenuated vaccine to the light calcium carbonate is 1: 1.
8. The method for prolonging the shelf life of a plant virus attenuated vaccine of claim 6, wherein the mass ratio of fulvic acid to xanthan gum is 1: 1.
9. The method for prolonging the storage life of a plant virus attenuated vaccine as claimed in claim 6, wherein the relationship between the addition amount of fulvic acid and xanthan gum and the mixture of fermented 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.
10. Use of a composition according to any one of claims 1 to 5 for prolonging the shelf life of a plant virus attenuated vaccine.
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| CN110857438B (en) * | 2018-08-20 | 2022-05-17 | 中国烟草总公司黑龙江省公司牡丹江烟草科学研究所 | Tobacco mosaic virus gene fragment for efficiently generating siRNA, attenuated vaccine, preparation method and application thereof |
| CN110885796B (en) * | 2018-08-20 | 2022-04-08 | 山东农业大学 | Attenuated vaccine for resisting potato virus X, preparation method and application thereof |
| CN112410351A (en) * | 2020-11-12 | 2021-02-26 | 山东农业大学 | Duplex attenuated vaccine for resisting cucumber mosaic virus and potato virus X and application thereof |
| CN116064412B (en) * | 2022-07-06 | 2023-09-22 | 山东农业大学 | Tobacco mosaic virus double-site mutant attenuated vaccine and application thereof |
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