Preparation method and application of immune response activating protein PPAN for preventing and treating Chinese bee sacbrood virus
By preparing and applying the immune response activation protein PPAN, the treatment problem of Chinese honey bee cystic larvae virus was solved, efficient and safe virus prevention and treatment effects were achieved, and the survival and cure rate of Chinese honey bee larvae were improved.
Patent Information
- Application Number
- CN202510596862.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-09
- Publication Date
- 2025-08-12
AI Technical Summary
The Chinese honey bee cystic larvae virus has serious harm to the Chinese honey bee colony, and the existing drugs are not ideal for treatment, especially for the cure of severely ill bee colony.
The immune response activation protein PPAN was prepared, and the PPAN gene was amplified from the cDNA of Chinese bee larvae by PCR, ligated to the pGEX-4T-1 vector, and expressed and purified to obtain PPAN protein. It was used to prepare an oral preparation against Chinese bee cystic larvae virus and was administered to Chinese bee larvae by spray feeding.
It significantly improves the survival rate of Chinese bee larvae, quickly and effectively inhibits viral infection, has the characteristics of high cure rate, low cost, no drug residues and easy storage, and is especially suitable for emergency prevention and treatment of bee colonies with existing diseases.
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Figure CN120464656A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to a preparation method and application of an immune response activating protein PPAN for preventing and treating cerana apis sac brood virus. Background Art
[0002] The Chinese honey bee, also known as the Chinese honey bee, is a precious local bee species that plays a crucial role in the conservation of my country's livestock and poultry genetic resources. It is listed on the National Livestock and Poultry Genetic Resource Protection List and is one of Liaoning Province's top ten livestock and poultry genetic resource species. Furthermore, it holds irreplaceable ecological value in maintaining the balance and diversity of my country's plant ecosystems. However, the survival and development of the Chinese honey bee is facing severe challenges. One of the most prominent is the rampant spread of sac brood disease in the Chinese honey bee.
[0003] Chinese honeybee sac brood virus (CSBV) is a serious threat to honeybee colonies. It primarily infects young larvae, causing the larvae's cuticle to liquefy, forming sacs that prevent them from pupating. This not only affects the survival rate of honeybees but also severely restricts the development of the honeybee industry. Although bee protection zones have been established in recent years to strengthen the Chinese honeybee industry and attempts have been made to control the spread of the disease through drug treatment, while treatment is available for sac brood, the results are suboptimal, particularly for severely affected colonies.
[0004] The Peter Pan homolog (Ppan) gene is a protein-coding gene with multiple potential functions, and its role in DNA damage repair is currently a hot topic of research. However, the specific functions of PPAN require further research and exploration. Summary of the Invention
[0005] The technical problem to be solved by the present invention is to provide a preparation method and application of an immune response activating protein PPAN for preventing and treating sac brood virus of Chinese honey bees. The preparation method is simple, and the protein can be used to treat sac brood disease of Chinese honey bees with significant efficacy.
[0006] A method for preparing an immune response activating protein PPAN for preventing and treating cerana cerana sac brood virus, comprising the following specific steps:
[0007] (1) Preparation of pGEX-4T-1-PPAN plasmid
[0008] The PPAN gene of the Chinese honey bee larvae was amplified from the cDNA of the Chinese honey bee larvae by PCR. The PCR product was connected to the pGEX-4T-1 vector to obtain the positive recombinant plasmid pGEX-4T-1-PPAN. The primer sequences for amplifying the PPAN gene of the Chinese honey bee larvae are:
[0009] Upstream primer: 5′-GCGGATCCATGGGAGAACATATTGTTGAGCTTA-3′;
[0010] Downstream primer: 5′-GCAAGCTTTTATCATGATTTGCTGAGTTTTTGT-3′;
[0011] (2) Prokaryotic expression of pGEX-4T-1-PPAN plasmid
[0012] Take 2 μL of pGEX-4T-1-PPAN plasmid and transform it into BL21 (DE3) competent cells. The obtained bacterial liquid was inoculated into 10 mL of LB medium containing 100 μg / mL ampicillin and cultured at 37 °C until OD 600 = between 0.6 and 0.8; then transferred to 500 mL of liquid LB medium containing 100 μg / mL ampicillin for large-scale induction, induced at 28°C with 1190 μL of isopropyl-β-D-thiogalactoside for 8 hours, and the induced protein was fully combined with agarose gel 4B and purified to obtain the immune response activating protein PPAN.
[0013] Furthermore, when performing PCR amplification in step (1), the cells were first pre-denatured at 95°C for 5 minutes; then denatured at 95°C for 40 seconds, annealed at 54°C for 40 seconds, and extended at 72°C for 100 seconds, for 35 cycles; and finally, a final extension at 72°C for 10 minutes.
[0014] Furthermore, the nucleotide sequence of the PCR product is shown in SEQ ID NO.1.
[0015] Furthermore, the cDNA of the bee larvae is obtained by reverse transcription of the total RNA of the bee larvae.
[0016] A use of the immune response activating protein PPAN prepared by the above preparation method in the preparation of a medicine for treating sac brood disease.
[0017] Furthermore, the immune response activating protein PPAN was diluted with Chinese honeybee feed to obtain an oral solution against Chinese honeybee sac brood virus.
[0018] Furthermore, the concentration of the immune response activating protein PPAN in the anti-bee sac brood virus oral solution is 0.264 mg / mL.
[0019] Furthermore, the composition of the Chinese bee feed is as follows: when the larvae are 3-4 days old, the Chinese bee feed includes, by volume percentage: 4% glucose, 4% fructose, 0.2% yeast powder, 30% royal jelly, and the balance water; when the larvae are 5 days old - defecation period, the Chinese bee feed includes, by volume percentage: 8% glucose, 4% fructose, 0.2% yeast powder, 24% royal jelly, and the balance water.
[0020] An oral preparation solution of anti-bee sac brood virus containing the immune response activating protein PPAN at a concentration of 0.264 mg / mL diluted with bee feed was administered to the Chinese honey bee larvae by direct spray feeding at a feeding amount of 100 ml / day / frame, twice every other day, for 20 consecutive days.
[0021] The preparation method is simple. The oral preparation for treating sac brood virus of Chinese honey bees produced by this method has the advantages of rapid action, high cure rate, and strong specificity among drugs for treating sac brood disease of Chinese honey bees. Histopathological observations show that the intestinal wall structure of the larvae is protected to a certain extent. Although the symptoms of sac formation are not significantly improved, it can significantly inhibit the blackening of the insect body color and significantly improve the survival rate of larvae after infection. Therefore, it can produce immediate effects after use, especially for emergency prevention when the surrounding disease has already occurred. It is easy to prepare, convenient to use, and safe. The oral preparation for treating sac brood virus of Chinese honey bees also has the characteristics of low production cost, easy use, no drug residue, and convenient storage.
[0022] The oral preparation for resisting Chinese honey bee sac brood virus prepared by this method is used for the treatment of Chinese honey bee sac brood disease. The results of clinical trials show that 30 boxes of bee colonies infected with Chinese honey bee sac brood virus that have been tested and clinically confirmed by RT-PCR were treated. In the bee colonies with milder disease, no larvae were dragged out in 10 days, and new capped larvae began to appear in about 3 weeks. In other bee colonies, no larvae were dragged out in about 15 days, and new capped larvae appeared in about 30 days. In about one month, all 27 boxes of bees were cured, and the pupation rate was 92.625%. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] Figure 1 This is an identification diagram of the PPAN gene amplified by PCR of the present invention; in the figure, M: 2000bp Maker; 1, 2, 3: PCR amplification products of the PPAN gene;
[0024] Figure 2 This is a PCR identification diagram of the recombinant expression plasmid pGEX-4T-1-PPAN of the present invention; in the figure, M: 5000bp Maker; 1: pGEX-4T-1-PPAN plasmid control; 2: double enzyme digestion identification of the recombinant plasmid BamHI and XhoI;
[0025] Figure 3This is an SDS-PAGE analysis of the recombinant protein expression product induced at 25°C for 12 hours. In the figure, M: protein molecular weight standard; 1: uninduced precipitate; 2: 0.25mmol / L IPTG precipitate; 3: 0.5mmol / L IPTG precipitate; 4: uninduced supernatant; 5: 0.25mmol / L IPTG supernatant; 6: 0.5mmol / L IPTG supernatant; 7: empty load;
[0026] Figure 4 This is an SDS-PAGE analysis of the recombinant protein expression product induced at 28°C for 8 hours. In the figure, M: protein molecular weight standard; 1: uninduced precipitate; 2: 0.25mmol / L IPTG precipitate; 3: 0.5mmol / L IPTG precipitate; 4: uninduced supernatant; 5: 0.25mmol / L IPTG supernatant; 6: 0.5mmol / L IPTG supernatant; 7: empty load;
[0027] Figure 5 This is an SDS-PAGE analysis of the recombinant protein expression product induced at 30°C for 5 hours. In the figure, M: protein molecular weight standard; 1: uninduced precipitate; 2: 0.25mmol / L IPTG precipitate; 3: 0.5mmol / L IPTG precipitate; 4: uninduced supernatant; 5: 0.25mmol / L IPTG supernatant; 6: 0.5mmol / L IPTG supernatant; 7: empty load;
[0028] Figure 6 This is a Western-blot identification diagram of the pGEX-4T-1-PPAN purified protein of the present invention;
[0029] Figure 7 This is a Western-blot test result of the hemolymph and epidermis of the honeybee larvae fed with GST-PPAN according to the present invention;
[0030] Figure 8 This is a statistical survival chart of bee larvae fed with different concentrations of the purified pGEX-4T-1-PPAN protein and 4T-1 protein;
[0031] Figure 9 This is a graph showing the expression of Dicer-like, Ago2, Hsp90, DuoX, and NoX in the hemolymph of honeybee larvae detected by qPCR of the present invention;
[0032] Figure 10 is a diagram showing the dosing regimen of the purified pGEX-4T-1-PPAN protein of the present invention and the detection of CSBV by qPCR;
[0033] Figure 11This is a diagram of the present invention collecting uninfected and infected CSBV bee larvae for 48 hours and observing their pathological histological changes; in the figure: PM: peritrophic membrane; IW: intestinal wall outline;
[0034] Figure 12 The present invention is to infect the characteristic symptoms of the bee sac larvae virus and the survival rate statistics. DETAILED DESCRIPTION
[0035] Example 1
[0036] 1. Total RNA Extraction
[0037] Total RNA from honey bee larvae was extracted using the Trizol method: 30 mg of ground honey bee larvae tissue was placed in an RNase-free centrifuge tube. 1 mL of TRI Reagent was added and thoroughly mixed using an electric homogenizer. The tube was centrifuged at 12,000 × g for 10 min at 4°C. The supernatant was aspirated and transferred to a new centrifuge tube and incubated at room temperature for 5 min. 200 μL of chloroform was added, the tube was shaken for 15 s, and the tube was incubated at room temperature for 3 min. The supernatant was aspirated and transferred to a new centrifuge tube. An equal volume of ice-cold isopropanol was added to the supernatant, the tube was inverted several times to mix, and the tube was incubated at room temperature for 10 min. Centrifuge at 12,000 × g for 10 min at 4°C until a precipitate of honey bee larvae RNA was visible at the bottom of the tube. The supernatant was discarded. 1 mL of 75% ethanol was added and the tube was washed by inversion. The tube was centrifuged at 7,500 × g for 5 min at 4°C, and the supernatant was aspirated. After the RNA of the honey bee larvae was dried, 20 μL of DEPC water was added to dissolve it and the RNA was frozen at -80°C for later use.
[0038] 2. Reverse transcription
[0039] Use a nucleic acid analyzer to detect the concentration of total RNA in the previous step and add RNase-free water to adjust the RNA concentration to 1000ng / μL. Use the TransGen reverse transcription kit and perform the experiment according to the system in Table 2: Mix 1μL of RNA diluted to a concentration of 1000ng / μL, 1μL of Anchored Oligo(dT)18 Primer, 6μL of RNase-free water, and 10μL of 2xTS Reaction Mix reagent, then incubate in a 42℃ metal bath for 30min. Then, add 1μL of EasyScript RT / RI Enzyme Mix and 1μL of gDNA Remover, heat at 85℃ for 5s to inactivate, and store the obtained cDNA in a -80℃ refrigerator for later use.
[0040] 3. Amplification and electrophoresis identification of target genes
[0041] Design primers:
[0042] Upstream primer: 5′-GCGGATCCATGGGAGAACATATTGTTGAGCTTA-3′;
[0043] Downstream primer: 5′-GCAAGCTTTTATCATGATTTGCTGAGTTTTTGT-3′;
[0044] 1 μL of the above cDNA template was taken and mixed with 1 μL of upstream primer (10 mM), 1 μL of downstream primer (10 mM), 12.5 μL of 2xTransTaq High Fidelity (HiFi) PCR SuperMix and 9.5 μL of RNase-free water. The mixture was pre-denatured at 95°C for 5 min; 35 cycles of denaturation at 95°C for 40 s, annealing at 54°C for 40 s, and extension at 72°C for 100 s were performed; finally, a final extension at 72°C for 10 min was performed, and PCR amplification was repeated 3 times. The nucleotide sequence of the PCR product is shown in Table 1. The PCR product was detected by electrophoresis. The results are shown in Table 1. Figure 1 As shown, a clear band appeared at 1146 bp, consistent with the theoretical value for the PPAN gene. The target band was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing results showed over 99% homology with the reference sequence (GenBank No. XM_017060463.1), confirming that the PPAN gene was amplified. The PCR product sequence is 1168 bp long, with a PPAN sequence of 1146 bp. The nucleotide sequence of the complete PPAN open reading frame is shown in SEQ ID NO. 1, where bases 3-8 are the BamHI restriction enzyme recognition sequence, bases 9-11 are the start codon, bases 12-1157 are the PPAN gene sequence, bases 1158-1160 are the stop codon, and bases 1161-1166 are the XhoI restriction enzyme recognition sequence.
[0045] Table 1
[0046]
[0047]
[0048] 4. Construction and identification of pGEX-4T-1-PPAN plasmid
[0049] Plasmid pGEX-4T-1 was used as the expression vector, and the plasmid was amplified using E. coli DH5α and purified using the Takara mini-plasmid kit. The PCR product and pGEX-4T-1 plasmid were digested with XhoI and BamHI, and the double enzyme digestion reaction system was as follows: 16 μL of RNase-free water, 2.5 μL of 10× buffer, 5 μL of PCR product or plasmid, 0.75 μL of XhoI, 0.75 μL of BamHI, and digestion at 37°C for 1 hour; after the digestion product was electrophoresed and recovered on gel, the fragment was ligated using T4 DNA ligase, and the reaction system was as follows: 2 μL of 5× buffer, 1 μL of T4 DNA ligase, 6 μL of PCR product, 1 μL of pGEX-4T-1 fragment, and ligation was carried out at 16°C overnight. 2 μL of the ligation product was transformed into BL21 (DE3) competent cells, and the obtained bacterial solution was spread on a plate containing 100 μg / mL ampicillin resistance. The positive recombinant plasmid was named pGEX-4T-1-PPAN.
[0050] The constructed pGEX-4T-1-PPAN recombinant plasmid was double-digested with BamHI and XhoI and detected by agarose gel electrophoresis. A clear band was found at the position of 1146 bp (see Figure 2 ).
[0051] 5. Target protein expression form and optimal induction conditions
[0052] The bacteria identified correctly in the previous step were inoculated into LB medium containing 100 μg / mL ampicillin (Amp) and cultured at 37°C until the OD 600 = 0.6 to 0.8, different final concentrations of inducer IPTG (0, 0.25 mmol / L, 0.5 mmol / L) were added, and the expression was induced at 25°C, 28°C and 30°C, respectively. Samples were taken after 12 h, 8 h and 5 h of induction, and the bacteria were harvested by centrifugation at 12000 rpm for 5 min. The supernatant was discarded, and the precipitate was resuspended in PBS and ultrasonically disrupted. Each ultrasonic disruption was 5 s, and the interval was 10 s until the bacterial solution was clear. The supernatant and precipitate were analyzed by SDS-PAGE, respectively. The results are shown in the figure. Figure 3-Figure 5 As shown in the results, a clear target band appeared at 70.6 kDa (containing the GST tag), which is consistent with the expected target protein size. Therefore, the optimal conditions were set as 28°C, 0.5 mmol / L IPTG induction for 8 hours, and glycerol storage at -80°C.
[0053] 6. Large-scale induction and protein purification
[0054] 100 μL of pGEX-4T-1-PPAN glycerol bacteria stored in glycerol under optimal conditions was taken out from -80°C and inoculated into 10 mL of liquid LB (containing 100 μg / mL Amp) medium for recovery; the fresh bacterial liquid after recovery was inoculated into 500 mL of liquid LB (containing 100 μg / mL Amp) medium for large-scale induction, and the induced protein was fully combined with agarose gel 4B and purified to obtain the purified pGEX-4T-1-PPAN protein (immune response activating protein PPAN). The purified protein was identified by Western-blot, and the results showed (see Figure 6 ), a specific target band appeared at 70.6 kDa.
[0055] 1. Oral delivery of PPAN to honeybee larvae to activate host antiviral immunity and evaluate its safety
[0056] To explore the potential of PPAN protein as an antiviral drug, the purified protein of pGEX-4T-1-PPAN (containing a GST tag), denoted as GST-PPAN, was diluted with sugar water (larval age 5-defecation stage) in Table 2 to three concentrations of bee feed (0.066 mg / mL, 0.132 mg / mL, and 0.264 mg / mL), and the protein concentration was detected by a BCA kit.
[0057] First, a diet containing 0.264 mg / mL of purified pGEX-4T-1-PPAN protein was selected and fed to uninfected bee larvae daily for the next 3 days (0, 24, 48, and 72 hours). The hemolymph and epidermis of the bee larvae were collected at 24, 48, 72, and 96 hours after feeding. The expression of GST-PPAN in the hemolymph was detected 24 hours after feeding and then gradually decreased. After 72 hours, the expression of GST-PPAN was only detected in the epidermis (see Figure 7 ).
[0058] Table 2 Composition of food (sugar water) fed to bee larvae of different ages
[0059]
[0060]
[0061] At the same time, the effects of different protein concentrations on the survival rate of bee larvae were evaluated. 4T-1 protein was used as a control (feed with a protein concentration of 0.264 mg / mL). The results showed that the absorption of different concentrations of pGEX-4T-1-PPAN by bee larvae did not affect their own survival (see Figure 8 ).
[0062] Finally, the same protocol was used to feed the larvae with 0.264 mg / mL pGEX-4T-1-PPAN protein concentration feed and 0.264 mg / mL 4T-1 protein feed. After 72 hours, the hemolymph of the larvae was collected and the expression of key genes in the RNAi signaling pathway and PO response in the hemolymph was detected by RT-qPCR. The results showed that after absorbing pGEX-4T-1-PPAN, the larvae of the bee significantly promoted the expression of Dicer-like, Ago2 and Hsp90. In addition, it also promoted the expression of Nox (see Figure 9 The above experimental results prove that the Chinese honey bee larvae can absorb the in vitro expressed pGEX-4T-1-PPAN through food intake without affecting the normal survival of the organism, and can effectively activate the host's antiviral immune response such as the phenoloxidase system.
[0063] 2. Oral administration of purified pGEX-4T-1-PPAN protein as a potential antiviral drug for the treatment of CSBD
[0064] To verify whether the pGEX-4T-1-PPAN purified protein preparation has antiviral effects, a drug administration and infection scheme was first designed. Three-day-old Chinese honey bee larvae were selected and administered and infected with the virus at the same time 24 hours later (1×10 6 The purified protein pGEX-4T-1-PPAN was diluted into a bee feed with a concentration of 0.264 mg / mL, and the bees were fed with feed containing 0.264 mg / mL of purified protein pGEX-4T-1-PPAN for 4 consecutive days. The samples of the bee larvae infected with the virus were collected at 48 hpi, 72 hpi, and 96 hpi for testing and evaluation of its disease resistance. The purified protein preparation of pGEX-4T-1-PPAN significantly inhibited the replication level of CSBV (see Figure 10 ).
[0065] The histopathological characteristics of the bee larvae at 48 hpi were compared with those of the uninfected bee larvae. In the bee larvae not infected with CSBV (Sugar group), the peritrophic membrane (PM) and the intestinal wall outline (IW) in the intestinal tube can be clearly seen, while in the bee larvae infected with the virus (Sugar+CSBV group), the PM structure disappeared and the IW expanded and ruptured. Compared with the untreated infection group, the bee larvae that took the purified protein of pGEX-4T-1-PPAN (Sugar+PPAN+CSBV group) also had an unclear PM, but had a complete IW and a clear outline (see Figure 11 ).
[0066] Next, the ability of the purified pGEX-4T-1-PPAN protein to alleviate the symptoms of viral infection was evaluated.
[0067] The main symptoms of CSBV infection were assessed and scored: head elevation, cyst formation, and darkening. Each symptom was scored as 1 point, and the total score for each time point and each representative symptom was calculated.
[0068] The results showed that the purified protein of pGEX-4T-1-PPAN did not improve the symptoms of cyst formation caused by CSBV infection, but it could significantly inhibit the blackening of the worm body caused by virus infection (see Figure 12 A). Finally, the effect of oral administration of purified pGEX-4T-1-PPAN protein on the survival rate of Chinese honey bee larvae after infection was evaluated. The results showed that PPAN protein significantly improved the survival rate of larvae after infection with CSBV (see Figure 12 B). These experimental results demonstrate that the purified protein pGEX-4T-1-PPAN can improve the treatment of Chinese honeybee sac brood disease (CSBD). Together, these findings highlight the value of the purified protein pGEX-4T-1-PPAN as a potential therapeutic for CSBD.
[0069] 3. Application of oral anti-CSBV preparations in the prevention and treatment of honeybee sac brood disease
[0070] Ten colonies infected with Chinese honey bee sac brood virus (CSBV) that had been confirmed by conventional PCR and clinical diagnosis were treated with an anti-CSBV oral solution containing 0.264 mg / mL of purified pGEX-4T-1-PPAN protein diluted in sugar water (Table 2). This solution was administered to the larvae of the Chinese honey bee (Apis cerana cerana) by direct spray feeding at a rate of 100 ml / day / frame, twice every other day, for 20 consecutive days. During this application, one colony was treated for queen escape due to poor management, and ultimately no continued treatment was given. In the remaining nine colonies, no larvae were pulled out by the 16th day after treatment, and new capped larvae began to appear on the 25th day. By one month, all nine colonies had been cured, with a pupation rate of 91.75%.
[0071] The above are merely specific embodiments of the present invention and are not intended to limit the present invention. Those skilled in the art will readily appreciate that the present invention is susceptible to various modifications and variations. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.
Claims
1. A method for preparing an immune response activating protein PPAN for preventing and treating Apis cerana cerana sac brood virus, characterized by: Specific steps as follows: (1) Preparation of pGEX-4T-1-PPAN plasmid The PPAN gene of the Chinese honey bee larvae was amplified from the cDNA of the Chinese honey bee larvae by PCR. The PCR product was connected to the pGEX-4T-1 vector to obtain the positive recombinant plasmid pGEX-4T-1-PPAN. The primer sequences for amplifying the PPAN gene of the Chinese honey bee larvae are: Upstream primer: 5′-GCGGATCCATGGGAGAACATATTGTTGAGCTTA-3′; Downstream primer: 5′-GCAAGCTTTTATCATGATTTGCTGAGTTTTTGT-3′; (2) Prokaryotic expression of pGEX-4T-1-PPAN plasmid Take 2 μL of pGEX-4T-1-PPAN plasmid and transform it into BL21 (DE3) competent cells. The obtained bacterial liquid was inoculated into 10 mL of LB medium containing 100 μg / mL ampicillin and cultured at 37 °C until OD 600 = between 0.6 and 0.8; then transferred to 500 mL of liquid LB medium containing 100 μg / mL ampicillin for large-scale induction, induced at 28°C with 1190 μL of isopropyl-β-D-thiogalactoside for 8 hours, and the induced protein was fully combined with agarose gel 4B and purified to obtain the immune response activating protein PPAN.
2. The method for preparing the immune response activating protein PPAN for preventing and treating Apis cerana cerana sac brood virus according to claim 1, wherein: When performing PCR amplification in step (1), the cells were first pre-denatured at 95°C for 5 minutes; then denatured at 95°C for 40 seconds, annealed at 54°C for 40 seconds, and extended at 72°C for 100 seconds, for 35 cycles; and finally, a final extension at 72°C for 10 minutes.
3. The method for preparing the immune response activating protein PPAN for preventing and treating Apis cerana cerana sac brood virus according to claim 1, characterized in that The nucleotide sequence of the PCR product is shown in SEQ ID NO.
1.
4. The method for preparing the immune response activating protein PPAN for preventing and treating Apis cerana cerana sac brood virus according to claim 1, wherein the The cDNA of bee larvae was obtained by reverse transcription of total RNA from bee larvae.
5. Use of the immune response activating protein PPAN prepared by the preparation method according to claim 1 in the preparation of a medicament for treating sac brood disease.
6. Use of the immune response activating protein PPAN according to claim 5 in the preparation of a medicament for treating sac brood disease of bees, characterized in that: The immune response activating protein PPAN is diluted with Chinese honeybee feed to obtain an oral solution against Chinese honeybee sac brood virus.
7. Use of the immune response activating protein PPAN according to claim 6 in the preparation of a medicament for treating sac brood disease of bees, characterized in that: The concentration of the immune response activating protein PPAN in the anti-bee sac brood virus oral solution is 0.264 mg / mL.
8. Use of the immune response activating protein PPAN according to claim 6 in the preparation of a medicament for treating sac brood disease of bees, characterized in that: The composition of the Chinese bee feed is as follows: when the larvae are 3-4 days old, the Chinese bee feed includes, by volume percentage, 4% glucose, 4% fructose, 0.2% yeast powder, 30% royal jelly, and the balance water; when the larvae are 5 days old - defecation period, the Chinese bee feed includes, by volume percentage, 8% glucose, 4% fructose, 0.2% yeast powder, 24% royal jelly, and the balance water.