Gene medicine for treating Chinese sacbrood and preparation and use method thereof
阅读说明：本技术 一种治疗中华囊状幼虫病的基因药物和制备及使用方法 (Gene medicine for treating Chinese sacbrood and preparation and use method thereof ) 是由 王敦 余肖 于 2020-11-11 设计创作，主要内容包括：本发明公开了一种治疗中华囊状幼虫病的基因药物和制备及使用方法,其特征是：该药物是由CSBV-VP1-dsRNA溶液、PBS-S母液和水复配而成。本发明采用特异性的dsRNA和助剂复配,形成的基因药物组合能够有效且特异性地抑制中蜂囊状幼虫病病毒的复制,从而控制病害的发展与传播。本发明的一种治疗中华囊状幼虫病的基因药物属于生物制剂,对环境无污染、无药物残留,是防治中蜂囊状幼虫病的新型高效药剂。(The invention discloses a gene medicine for treating Chinese sacbrood and a preparation and use method thereof, which is characterized in that: the medicine is prepared by compounding CSBV-VP1-dsRNA solution, PBS-S mother solution and water. The invention adopts specific dsRNA and auxiliary agent to compound, and the formed gene drug combination can effectively and specifically inhibit the replication of Chinese bee sacbrood virus, thereby controlling the development and spread of diseases. The gene medicine for treating Chinese sacbrood disease belongs to a biological preparation, has no pollution to the environment and no medicine residue, and is a novel efficient medicine for preventing and treating Chinese sacbrood disease.)
1. A gene medicine for treating Chinese sacbrood disease is characterized in that: the gene medicine at least comprises: CSBV-VP1-dsRNA solution, PBS-S mother solution and water.
2. The gene medicine for treating Chinese sacbrood disease according to claim 1, which is characterized in that: the CSBV-VP1-dsRNA solution, the PBS-S mother solution and the water are prepared by compounding 50mL of the gene medicine with the following raw materials in proportion: the dsRNA content of the CSBV-VP1-dsRNA solution is 100 mu g/mL-1000 mu g/mL, the percentage concentration of the PBS-S mother solution is 10% -50%, and the balance is water.
3. The gene drug for treating Chinese sacbrood according to claim 1 or 2, which is characterized in that: the dsRNA content in the CSBV-VP1-dsRNA solution is 125 mug/mL.
4. The gene drug for treating Chinese sacbrood according to claim 1 or 2, which is characterized in that: the PBS-S mother liquor comprises sucrose, sodium chloride, vitamin C, sodium citrate, disodium hydrogen phosphate, potassium sorbate, potassium dihydrogen phosphate and potassium chloride.
5. The gene drug for treating Chinese sacbrood according to any one of claims 1 to 4, which is characterized in that: the gene medicine is fed in a beehive where the Chinese bee sacbrood disease occurs, and can be fed in each stage of the disease of the Chinese bee sacbrood disease, wherein the gene medicine is fed for 1 time every 1 day and is fed for 5 times in total.
6. A preparation method of a gene medicine for treating Chinese sacbrood disease is characterized in that: and (3) sequentially and uniformly mixing the CSBV-VP1-dsRNA solution and the PBS-S mother solution according to the formula amount, then adding the water according to the formula amount, fully and uniformly mixing, and bottling.
7. The method for preparing the gene medicine for treating Chinese sacbrood disease according to claim 6, which is characterized in that: the gene medicine for treating the Chinese sacbrood disease is frozen and stored at the temperature of-20 ℃, shaken and evenly by force before opening a bottle, and then diluted by 10 times with honey water after opening for feeding.
Technical Field
The invention belongs to the field of economic insect disease control, and relates to a gene medicine for treating Chinese sacbrood and a preparation method and a use method thereof.
Background
Chinese bee sacbrood is an important disease which harms Chinese bee industry in China, the disease mainly occurs in 3-4 months every year, SBV larvae and imagoes can be infected, and particularly 2-3-day-old bee larvae are infected. The imago has no obvious symptoms after infecting CSBV, the bee larva after infection can not pupate normally, the body color gradually turns yellow and deepens from clear white, liquid accumulation can appear under the surface and skin of the larva, the larva can be dried down slowly and finally turns into dark brown and blackened dragon boat shape, so the imago is called as 'rotten son disease' or 'dragon boat disease'. The spread of Chinese sacbrood disease affects the entire Chinese bee colony devastating, and can even lead to collapse of the colony social structure throughout the bee field, loss of the colony, and death of large numbers of individuals. Therefore, there is an urgent need for a drug specifically targeting the virus of Chinese sacbrood disease, which can solve the above problems.
Disclosure of Invention
The invention aims to provide a gene medicine for treating Chinese sacbrood disease and a preparation and use method thereof, and the invention is a gene medicine specially for treating Chinese sacbrood disease. The invention aims at VP1 sequence in CSBV whole genome, and applies specific dsRNA and auxiliary agent to feed the sick bee colony, thereby fundamentally inhibiting the replication of virus and effectively controlling the harm of Chinese bee sacbrood.
The technical scheme of the invention is as follows: a gene medicine for treating Chinese sacbrood disease, which at least comprises: CSBV-VP1-dsRNA solution, PBS-S mother solution and water.
Further, the CSBV-VP1-dsRNA solution, the PBS-S mother solution and the water are prepared by compounding 50mL of the gene medicine with the following raw materials in proportion: the dsRNA content of the CSBV-VP1-dsRNA solution is 100 mu g/mL-1000 mu g/mL, the percentage concentration of the PBS-S mother solution is 10% -50%, and the balance is water.
Furthermore, the dsRNA content of the CSBV-VP1-dsRNA solution is 125 mu g/mL.
Further, the PBS-S mother liquor comprises sucrose, sodium chloride, vitamin C, sodium citrate, disodium hydrogen phosphate, potassium sorbate, monopotassium phosphate and potassium chloride.
Further, the gene medicine is fed in a beehive where the Chinese bee sacbrood is attacked, and can be fed in each stage of the attack of the Chinese bee sacbrood, wherein the gene medicine is fed for 1 time every 1 day and is fed for 5 times in total.
A method for preparing a gene medicine for treating Chinese sacbrood disease comprises the following steps: and (3) sequentially and uniformly mixing the CSBV-VP1-dsRNA solution and the PBS-S mother solution according to the formula amount, then adding the water according to the formula amount, fully and uniformly mixing, and bottling.
Further, the gene medicine for treating the Chinese sacbrood is frozen and stored at the temperature of-20 ℃, shaken and evenly applied before opening the bottle, and then diluted by 10 times with honey water after opening for feeding.
The invention has the beneficial effects that: the invention adopts specific dsRNA and auxiliary agent to compound, and the formed gene drug combination can effectively and specifically inhibit the replication of Chinese bee sacbrood virus, thereby controlling the development and spread of diseases. The invention uses the microorganism product existing in nature as the main component, so the invention has no problems of antibiotic pollution and chemical drug residue, and is particularly suitable for preventing and treating Chinese bee sacbrood. Meanwhile, the invention can be fed conventionally. Therefore, the invention has the characteristics of no pollution, high-efficiency virus inhibition and convenient application.
The present invention will be further illustrated by the following specific examples, which are not intended to limit the scope of the invention.
Detailed Description
The CSBV-VP1-dsRNA solution: constructing an expression bacterium of CSBV-VP1-dsRNA by utilizing a commercial L4440-HT115 dsRNA expression system, inoculating the expression bacterium to an LB liquid culture medium, fermenting for 2h, adding IPTG (isopropyl-beta-thiogalactoside) for inducing for 12h, and then centrifuging at high speed for 3 min at 8000 rpm to collect thalli; after resuspending in STE buffer (0.1M NaCl, 10mM Tris HCl, 1mM EDTA, pH 7.8), ultrasonically disrupting the cells (frequency 20 kHz, treatment time 5 min), centrifuging at high speed at 12000rpm and 4 ℃ to remove the precipitate and obtain a crude extract containing the objective dsRNA; adding absolute ethanol to a final concentration of 12-20%, loading into a CC41 cellulose chromatographic column pre-equilibrated with STE buffer containing the same proportion of ethanol; washing the column with sufficient STE buffer containing 14% -18% ethanol, eluting dsRNA with STE buffer, and collecting eluate; then, an equal amount of pre-cooled isopropanol and 10% NaAc solution was added at 12000rpm, centrifuged at 12000rpm at 4 ℃ for 15 minutes, and the precipitate was washed with 75% ethanol and then dissolved in TE buffer (10 mM Tris HCl, 1mM EDTA, pH 7.0).
The Chinese name of the PBS-S mother liquor is: 25% phosphate-sucrose complex buffer. The 1L PBS-S mother liquor comprises: 50.0g of sucrose, 16.0g of sodium chloride, 10.0g of vitamin C, 6.0g of sodium citrate, 3.0g of disodium hydrogen phosphate, 2.0g of potassium sorbate, 0.5g of monopotassium phosphate and 0.4g of potassium chloride, dissolving in 950mL of sterile water, adjusting the pH value to 6.5-7.4, and fixing the volume to 1000 mL.
Example 1
A gene medicine for treating Chinese sacbrood disease at least comprises: CSBV-VP1-dsRNA solution, PBS-S mother solution and water.
The CSBV-VP1-dsRNA solution, the PBS-S mother solution and the water are prepared from 50mL of gene medicine for treating Chinese sacbrood disease by compounding the following raw materials in proportion: the dsRNA content of the CSBV-VP1-dsRNA solution is 100 mu g/mL-1000 mu g/mL, the percentage concentration of the PBS-S mother solution is 10% -50%, and the balance is water.
The preparation method comprises the following steps: and (3) sequentially and uniformly mixing the CSBV-VP1-dsRNA solution and the PBS-S mother solution according to the formula amount, then adding the water according to the formula amount, fully and uniformly mixing, and bottling.
Preferably, the dsRNA content of the CSBV-VP1-dsRNA solution is 125 mu g/mL.
Example 2
50mL of gene medicine for treating Chinese sacbrood disease is prepared:
taking 1mL of the CSBV-VP1-dsRNA solution of example 1, adding 5mL of PBS-S mother solution, adding water to 50mL, fully mixing uniformly and bottling. (final concentrations are respectively: the target dsRNA content is 2.5mg/L, and the PBS-S mother liquor content is 10%).
Example 3
50mL of gene medicine for treating Chinese sacbrood disease is prepared:
taking 2mL of the CSBV-VP1-dsRNA solution of the example 1, adding 10mL of PBS-S mother solution, supplementing water to 50mL, fully mixing uniformly and bottling. (final concentrations are respectively: the target dsRNA content is 5.0mg/L, and the PBS-S mother liquor content is 20%).
Example 4
50mL of gene medicine for treating Chinese sacbrood disease is prepared:
taking 4mL of the CSBV-VP1-dsRNA solution of the example 1, adding 15mL of PBS-S mother solution, adding water to 50mL, fully mixing uniformly and bottling. (final concentrations are respectively: the target dsRNA content is 10mg/L, and the PBS-S mother liquor content is 30%).
Example 5
50mL of gene medicine for treating Chinese sacbrood disease is prepared:
taking 8mL of the CSBV-VP1-dsRNA solution of the example 1, adding 20mL of PBS-S mother solution, adding water to 50mL, fully mixing uniformly and bottling. (final concentrations are respectively: the target dsRNA content is 20mg/L, and the PBS-S mother liquor content is 40%).
Example 6
50mL of gene medicine for treating Chinese sacbrood disease is prepared:
taking 16mL of the CSBV-VP1-dsRNA solution of the example 1, adding 25mL of PBS-S mother solution, adding water to 50mL, fully mixing uniformly and bottling. (final concentrations are respectively: the target dsRNA content is 40mg/L, and the PBS-S mother liquor content is 50%).
The raw material in the above examples, CSBV-VP1-dsRNA solution, was not only produced by fermentation and extraction in the laboratory, but also other materials were directly available from the market.
Determination of drug efficacy
In examples 1 to 6 of the gene medicine for treating the Chinese sacbrood disease, the Chinese bee sacbrood disease (Chinese bee) is taken as a treatment object, the Chinese bee sacbrood disease is shaken and shaken up forcibly before opening a bottle, diluted by 10 times of honey water after opening the bottle and fed, and the medicine is applied to the middle sac disease onset period, and fed 1 time every 1 day and 5 times in total. And (5) counting prevention and treatment after the last application is completed for 2 weeks. The gene medicine for treating Chinese sacbrood disease is frozen and stored at the temperature of-20 ℃.
(1) The invention relates to a gene medicine working solution for treating Chinese sacbrood disease, which is prepared by the following steps:
reagent to be tested: gene medicine for treating Chinese sacbrood
After addition of a suitable amount of honey, 3 diseased beehives per group were processed in 1 replicate, 3 replicates for each treatment, as follows:
a) control group: equal amount of honey water was fed in the evening.
b) And (3) control group: the gene medicine added with honey for treating Chinese sacbrood is fed in the evening, and each box is fed with 100mL-150 mL. (the control group is 5 groups, and the gene drugs for treating the Chinese sacbrood disease are respectively the gene drugs of the examples 2-6 in sequence).
c) After being fed for 2 weeks, the gene medicine for treating Chinese sacbrood disease counts the incidence of nestic larva and the rate of son spleen capping in beehives.
(2) The gene medicine for treating the Chinese sacbrood disease has the following effects:
control effect on apis cerana cysticercosis
Aiming at the common Chinese sacbrood disease in Chinese bees, after the gene medicine for treating the Chinese sacbrood disease is applied for 2 weeks for the last time, a beehive is opened to check the quantity of the diseased larvae in each honeycomb and the number of the covers of the subspi, and finally the larva susceptibility rate and the subspi cover rate are calculated, and the results are shown in tables 1 and 2: examples 2-6 all produced good control effect to Chinese bee sacbrood, compare with the hydromel contrast, the purpose dsRNA produced very strong prevention and cure effect to Chinese bee sacbrood, the child spleen capping rate after using medicine was all above 90%, the highest 95.9%.
TABLE 1 susceptibility of bee larvae after dosing
TABLE 2 sub-spleen capping rate after dosing
The prevention and treatment effect shows that the gene medicine for treating the Chinese sacbrood disease has obvious effect of preventing and treating the Chinese bee sacbrood disease in production. Because the traditional chemical agent is easy to cause serious drug residue in preventing and treating Chinese bee sacbrood, the traditional chemical agent has great influence on bee products, and easily causes bad stimulation to bee colonies to cause the phenomenon of escaping from the bee colonies. The gene medicine for treating Chinese sacbrood disease can effectively prevent and treat Chinese sacbrood disease, and can remarkably reduce the use of chemical agents and reduce the medicine residue in bee products. Has very important practical significance for improving the quality of bee products and protecting the germplasm resource of Chinese bees.
The gene medicine for treating Chinese sacbrood disease is applied in a beehive where Chinese sacbrood disease occurs, and can be applied in each stage of disease occurrence, wherein the gene medicine is fed for 1 time every 1 day and is fed for 5 times in total.
The gene medicine for treating the Chinese sacbrood is shaken and shaken uniformly by force before opening a bottle, and is diluted by 10 times of honey water after opening for feeding.
The invention has the advantages that: (1) the virus is efficiently inhibited without pollution: the invention utilizes the compounding of specific dsRNA and auxiliary agent, the formed gene drug combination can effectively inhibit the attack of Chinese bee sacbrood, because the invention utilizes the microorganism product existing in nature as the main component, the problems of antibiotic pollution and chemical drug residue (2) are not existed, the application is convenient: the feed is fed conventionally.
The invention utilizes a new technology of specific dsRNA and auxiliary agent, and is a green prevention and control technology of RNAi mechanism existing in organisms. Specific dsRNA and an auxiliary agent are applied to feed the sick bee colony, so that the replication of the virus is fundamentally inhibited, and the harm of Chinese sacbrood is effectively controlled.