Shrna for inhibiting gene expression of duck circovirus

Abstract

The application belongs to the field of genetic engineering and relates to an shRNA for inhibiting duck circovirus gene expression, wherein the shRNA is artificially synthesized, can inhibit duck circovirus Rep and Cap gene expression, and thus plays a role in inhibiting virus replication in a duck body. Experiments prove that injection of recombinant plasmids containing the shRNA interference sequence can significantly inhibit virus replication in a duck body, and compared with a control group, the virus copy number is reduced by 100 times at most, which has important significance in clinical prevention and control of duck circovirus.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of genetic engineering and its application, and relates to a shRNA for inhibiting gene expression of duck circovirus. BACKGROUND

[0002] Duck circovirus (DuCV) is a member of the Circoviridae family and the Circovirus genus, which is non-enveloped and has a single-stranded circular DNA structure. The virus was first reported in Germany by Hanemann in 2003. In China, Jiang Shijin et al. (2007) first detected DuCV from duck samples collected in Fujian Province in 2006. Liu Shaoning et al. (2008-2009) detected 343 tissue samples from 36 duck flocks in five provinces, including Shandong, Jiangsu, Sichuan, Fujian and Guangdong, and found that the detection rate of DuCV was as high as 81.63%, and the positive rate of duck flocks was 94.44%. Among them, the positive rate of 18 broiler duck farms in Shandong was 88.89%, and the positive rate of 12 breeding duck farms was 75%. These data show that duck circovirus has been widely infected in duck flocks in major duck breeding provinces and cities in China for more than a decade. According to the detection data of duck flocks from all over the country from 2018 to 2021, the infection rate of DuCV is still very high, and it is the virus with the highest infection rate in duck flocks. It is often mixed with Escherichia coli, Riemerella anatipestifer, adenovirus, duck parvovirus, Tembusu virus, reovirus, influenza virus and other pathogens. For the treatment of mixed infection duck flocks, it is difficult to achieve good treatment effect as long as DuCV is mixed. DuCV infection is slow and usually does not cause obvious clinical symptoms, so it is very easy to be ignored. Since it mainly damages the immune system of ducks, it can easily cause secondary infection of other pathogens, seriously affecting the treatment effect and causing huge economic losses.

[0003] Since DuCV is difficult to proliferate and culture in animal cells and avian embryos at present, there is no commercial vaccine at present. Egg yolk antibody is a commonly used biological preparation against viruses, which is an antibody against specific antigens extracted from immune eggs. Since only specific IgY antibodies are present in egg yolk, it is called egg yolk immunoglobulin IgY. At present, laboratory studies have shown that anti-duck circovirus egg yolk antibody is effective in the clinical treatment of duck circovirus infection. The inventors' team also used liver tissue of ducklings infected with duck circovirus to prepare tissue inactivated vaccine, and immunized laying hens to prepare anti-duck circovirus egg yolk antibody and conducted clinical effect test. The results show that the anti-duck circovirus egg yolk antibody alone can indeed significantly reduce the infection rate and morbidity, but the inhibitory effect is limited, and further improvement of the treatment effect is still needed. Therefore, developing new anti-duck circovirus infection drugs is an urgent task. SUMMARY

[0004] In view of the above problems in the prior art, the inventors of the present application provide an shRNA for inhibiting duck circovirus gene expression, which is artificially synthesized and can inhibit the expression of Rep and Cap genes of duck circovirus, thereby playing a role in inhibiting the replication of the virus in the duck body. Experiments have confirmed that injection of the recombinant plasmid containing the shRNA interference sequence can significantly inhibit the replication of the virus in the duck body, and the viral copy number is reduced by 100 times at most compared with the control group, which has important significance in the clinical prevention and control of duck circovirus.

[0005] The specific principle of the present application is as follows:

[0006] RNA interference (RNAi) exists in various eukaryotic cells and is an evolutionarily conserved gene silencing mechanism that can degrade corresponding mRNA in a sequence-specific manner, thereby causing silencing of the target gene. The antiviral effect of RNAi was first discovered in plants and is an important antiviral way in plants. This mechanism provides a new strategy for antiviral treatment in animals. At present, viral mRNA degradation mediated by small interfering RNA and short hairpin RNA (siRNA and shRNA) has been achieved in animals, thereby inhibiting the proliferation of viruses in the body. Short hairpin RNA is a double-stranded DNA molecule that can be cloned into an expression vector and express siRNA. Compared with the use of synthetic siRNA, shRNA has many advantages in silencing target genes, such as high stability, simple delivery method, low cost, etc., and can directly inhibit viral replication in the animal body by injection, thereby having broad prospects in the treatment of viral diseases in animals. Therefore, the inventors designed an shRNA interference sequence for inhibiting DuCV gene expression, and direct injection of the recombinant plasmid containing the interference sequence can significantly inhibit the replication of the virus in the duck body. If further used in combination with anti-duck circovirus yolk antibody, a more excellent effect of resisting duck circovirus infection can be achieved, which is worthy of clinical popularization and application.

[0007] The specific technical solutions of the present application are as follows:

[0008] An shRNA for inhibiting duck circovirus gene expression, specifically any one of Rep-1, Rep-2, Cap-1, and Cap-2, each shRNA interference sequence comprising a positive strand and a negative strand, wherein:

[0009] Rep-1 positive strand: 5'-GATCC CTGTGAAGCCACAGATG GG TTTTTTA-3' GCAAGAGGTGGGTCTTTACCATTAAT 5'-GATCC CTGTGAAGCCACAGATG GG TTTTTTA-3' 5'-GATCC CTGTGAAGCCACAGATG GG TTTTTTA-3' ATTAATGGTAAAGACCCACCTCTTGC 5'-GATCC CTGTGAAGCCACAGATG GG TTTTTTA-3' as shown in SEQ ID NO. 1;

[0010] Rep-1 negative strand: 5'-GATCC CTGTGAAGCCACAGATG GG TTTTTTA-3' GCAAGAGGTGGGTCTTTACCATTAAT 5'-GATCC CTGTGAAGCCACAGATG GG TTTTTTA-3' 5'-GATCC CTGTGAAGCCACAGATG GG TTTTTTA-3' 5'-GATCC CTGTGAAGCCACAGATG GG TTTTTTA-3' ATTAATGGTAAAGACCCACCTCTTGC G-3’ as set forth in SEQ ID NO. 2;

[0011] Rep-2 positive strand: 5'-GATCC CTGTGAAGCCACAGATG GG TTTTTTA-3' GCCTAATCGTCGAGACGCAACGTGAT 5'-GATCC CTGTGAAGCCACAGATG GG TTTTTTA-3' 5'-GATCC CTGTGAAGCCACAGATG GG TTTTTTA-3' ATCACGTTGCGTCTCGACGATTAGGC 5'-GATCC CTGTGAAGCCACAGATG GG TTTTTTA-3' as set forth in SEQ ID NO. 3;

[0012] Rep-2 negative strand: 5'-GATCC CTGTGAAGCCACAGATG GG TTTTTTA-3' GCCTAATCGTCGAGACGCAACGTGAT 5'-GATCC CTGTGAAGCCACAGATG GG TTTTTTA-3' 5'-GATCC CTGTGAAGCCACAGATG GG TTTTTTA-3' 5'-GATCC CTGTGAAGCCACAGATG GG TTTTTTA-3' ATCACGTTGCGTCTCGACGATTAGGC G-3’ as set forth in SEQ ID NO. 4;

[0013] Cap-1 positive strand: 5'-GATCC CTGTGAAGCCACAGATG GG TTTTTTA-3' GCGATTCGTAGCCTTCGTCTTCTGAA 5'-GATCC CTGTGAAGCCACAGATG GG TTTTTTA-3' 5'-GATCC CTGTGAAGCCACAGATG GG TTTTTTA-3' TTCAGAAGACGAAGGCTACGAATCGC 5'-GATCC CTGTGAAGCCACAGATG GG TTTTTTA-3' as set forth in SEQ ID NO. 5;

[0014] Cap-1 negative strand: 5'-GATCC CTGTGAAGCCACAGATG GG TTTTTTA-3' GCGATTCGTAGCCTTCGTCTTCTGAA 5'-GATCC CTGTGAAGCCACAGATG GG TTTTTTA-3' 5'-GATCC CTGTGAAGCCACAGATG GG TTTTTTA-3' 5'-GATCC CTGTGAAGCCACAGATG GG TTTTTTA-3' TTCAGAAGACGAAGGCTACGAATCGC G-3’ as set forth in SEQ ID NO. 6;

[0015] Cap-2 positive strand: 5'-GATCC CTGTGAAGCCACAGATG GG TTTTTTA-3' GCCACTCCTGTTGTGTTGTCTGGTTT 5'-GATCC CTGTGAAGCCACAGATG GG TTTTTTA-3' 5'-GATCC CTGTGAAGCCACAGATG GG TTTTTTA-3' AAACCAGACAACACAACAGGAGTGGC 5'-GATCC CTGTGAAGCCACAGATG GG TTTTTTA-3' as set forth in SEQ ID NO. 7;

[0016] Cap-2 negative strand: 5'-GATCC CTGTGAAGCCACAGATG GG TTTTTTA-3'GCCACTCCTGTTGTGTTGTCTGGTTT 5'-GATCC CTGTGAAGCCACAGATG GG TTTTTTA-3' 5'-GATCC CTGTGAAGCCACAGATG GG TTTTTTA-3' 5'-GATCC CTGTGAAGCCACAGATG GG TTTTTTA-3' AAACCAGACAACACAACAGGAGTGGC G-3’ As shown in SEQ ID NO.8;

[0017] In addition, the inventors also provided the following negative control sequence:

[0018] shRNA control positive strand: 5'-GATCC CTGTGAAGCCACAGATG GG TTTTTTA-3' TCCCGTTAATCTGAGGGTAAAGTTAG 5'-GATCC CTGTGAAGCCACAGATG GG TTTTTTA-3' 5'-GATCC CTGTGAAGCCACAGATG GG TTTTTTA-3' G CTAACTTTACCCTCAGATTAACGGGA ​ As shown in SEQ ID NO.9;

[0019] shRNA control negative strand: ​ TCCCGTTAATCTGAGGGTAAAGTTAG ​ ​ ​ CTAACTTTACCCTCAGATTAACGGGA G-3’ As shown in SEQ ID NO.10;

[0020] The loop sequence in the above sequence is CTGTGAAGCCACAGATGGG, as shown in SEQ ID NO.11;

[0021] The shRNA control does not have antiviral activity. The inventor set up this control to prove that the designed sequence is effective rather than the vector backbone. The inventor hereby clarifies this.

[0022] The inventors further provided a method for constructing the expression vector of the above-mentioned shRNA, the specific steps of which are as follows:

[0023] (1) Design and synthesize effective siRNA targets for the DuCV Rep and Cap genes (the location of the target in the DuCV genome is as follows: ​ As shown in SEQ ID NO.12, the effective target sequence Rep-1 targeting the DuCV Rep and Cap genes was obtained: 5'-CAAGAGGTGGGTCTTTACCATTAAT-3'.

[0024] Rep-2: 5'-CCTAATCGTCGAGACGCAACGTGAT-3', as shown in SEQ ID NO.13;

[0025] Cap-1: 5'-CGATTCGTAGCCTTCGTCTTCTGAA-3', as shown in SEQ ID NO.14;

[0026] Cap-2: 5'-CCACTCCTGTTGTGTTGTCTGGTTT-3', as shown in SEQ ID NO.15;

[0027] (2) Add restriction enzyme sites and loop sequences to the siRNA target site. The shRNA interference sequence structure is as follows: ​ As shown, shRNA is processed to generate siRNA as follows: ​ As shown;

[0028] (3) Synthesize the positive and negative strands of shRNA interference sequences that inhibit the expression of DuCV Rep and Cap genes, respectively;

[0029] (4) After mixing the positive and negative strands, annealing is performed to form complementary double-stranded DNA fragments with sticky ends;

[0030] (5) The empty shRNA expression vector was double-digested and recovered to form a linear shRNA expression vector with sticky ends;

[0031] (6) The linearized vector and double-stranded DNA were ligated using T4-DNA ligase and transformed into competent E. coli cells. Based on the type of antibiotic resistance gene on the plasmid, initial screening was performed using culture dishes containing the corresponding antibiotic, and positive clones were obtained after PCR and sequencing identification.

[0032] The above method for constructing the shRNA expression vector, wherein the empty expression vector is pBAsi-hU6;

[0033] The application of the shRNA expression vector obtained above in suppressing the expression of DuCV Rep and Cap genes.

[0034] In summary, this invention designs shRNAs that inhibit the expression of DuCV Rep and Cap genes and constructs corresponding shRNA expression vectors. These shRNA expression vectors are introduced into ducks via injection after being mixed with egg yolk antibodies, thereby effectively inhibiting DuCV replication in vivo. This method reduces the amount of antibody used while improving the therapeutic effect. It is simple to operate, has a stable process, and good applicability. Attached Figure Description

[0035] ​ A schematic diagram of shRNA silencing sites in the DuCV genome.

[0036] The figure shows the size of the duck circular genome at 1993bp. The arrows indicate the transcription direction of the corresponding genes. The black squares for shRNA-Cap-1 / shRNA-Cap-2 / shRNA-Rep-1 / shRNA-Rep-2 represent the positions of the shRNA sequences in the duck circular genome.

[0037] ​ This is a schematic diagram of the shRNA interference sequence structure.

[0038] BamHI and HindIII are restriction enzyme sites. The 5' and 3' ends at these sites are sticky ends generated after the double strand synthesis of the target sequence. After ligation by T4-DNA ligase, they bind complementary to the sticky ends on the vector. The N in the black box containing the target sequence can represent any one of A / T / C / G, and its specific meaning is related to the target sequence. The Terminator sequence is the RNA polymerase transcription termination signal. shRNA containing the Loop sequence can be cleaved by the intracellular Dicer enzyme, and then form the corresponding siRNA.

[0039] ​ This is a schematic diagram illustrating the processing and generation of siRNA sequences.

[0040] Intracellular RNA polymerase recognizes the U6 promoter (a promoter that initiates transcription), which then initiates transcription, converting the DNA sequence into hairpin RNA containing the target sequence and the loop sequence. The hairpin RNA is then cleaved by the intracellular Dicer enzyme to form siRNA that does not contain the loop sequence. NN can be any of A / T / C / G (depending on the target sequence), and U is uracil, whose role in RNA is similar to that of T in DNA.

[0041] ​ This is a schematic diagram of agarose gel electrophoresis detection of the purity of five shRNA recombinant expression vectors.

[0042] The band on the left is the marker, which indicates the size of the DNA sequence being detected; the five wells on the right are the electrophoresis images of the corresponding shRNA recombinant expression vector plasmids. Because plasmids have multiple conformations, their electrophoresis rates are different from the linear DNA used for the markers; there are some differences between their electrophoresis rates and the sizes indicated by the markers, which is normal.

[0043] ​ This is a schematic diagram of the pBAsi-hU6 shRNA expression vector.

[0044] Ori is the origin of replication of the vector in E. coli; Amp is the resistance gene carried by the plasmid, which is essential for the long-term existence of the plasmid in E. coli; U6 promoter is the transcription promoter; BamHI and HindIII are restriction enzyme sites.

[0045] ​ The bar chart shows the inhibitory effect of shRNA on DuCV in PBMCs.

[0046] Relative virus content is the relative viral concentration. An asterisk (*) above the bar indicates a significant difference in viral copy number between the shRNA treatment group and the PBS group (P<0.05), a ** indicates an extremely significant difference (P<0.01), and ns indicates no significant difference (P>0.05).

[0047] ​ Bar chart showing the inhibitory effect of shRNA on DuCV in ducks.

[0048] Relative virus content represents the relative viral concentration. An asterisk (*) above the bar indicates a significant difference in viral copy number between the shRNA treatment group and the PBS group (P<0.05), a ** indicates an extremely significant difference (P<0.01), and ns indicates no significant difference (P>0.05). 48, 72, and 96 represent the time (h) after treatment. Detailed Implementation

[0049] The invention will be further described in detail below with reference to the accompanying drawings. It should be understood that these embodiments are for illustrative purposes only and not for limiting the scope of protection of the invention. The specific techniques used in the embodiments are all conventional techniques in the art, and the biological materials used are all known biological materials obtained by the inventors through legitimate channels during the research process. The inventors list the relevant techniques as follows: However, other specific techniques not listed are also known techniques, and the inventors will not elaborate further.

[0050] Example 1

[0051] Construction and validation of shRNA vectors

[0052] (1) Interference sequence design

[0053] Interference sequences were designed and synthesized based on the DuCV genome information in GenBank (GenBank: ON227548.1). The information of the interference sequences is shown in Table 1.

[0054] Table 1 shRNA Insertion Sequence

[0055]

[0056]

[0057] (2) Restriction enzyme digestion of shRNA expression vector

[0058] Map of shRNA expression vector pBAsi-hU6 as follows ​ As shown

[0059] Prepare 60 μL enzyme digestion reaction systems according to the proportions shown in Table 2. After mixing by pipetting, briefly incubate in a PCR instrument at 37°C for 40 min. Separate the enzyme digestion products by 1% agarose gel electrophoresis, and recover the target bands from the gel.

[0060] Table 2. shRNA expression plasmid digestion system

[0061] ​ ​ ddH2O 42 ​ 6 ​ 6 ​ 3 ​ 3 ​ 60

[0062] (3) Double-stranded DNA synthesis

[0063] The synthesized single-stranded DNA was dissolved in Oligo diluent to a final concentration of 20 μM, placed in a PCR instrument and heated at 90 °C for 15 min. After heating, it was cooled to room temperature to form double-stranded DNA with sticky ends. The double-stranded DNA had the same restriction enzyme sites at both ends as the linearized vector.

[0064] (4) Ligation of double-stranded DNA to vector

[0065] The recovered linearized vector was ligated with the five synthetic double-stranded DNAs mentioned above at 25°C for 1 h using T4 DNA Ligase V2. The ligation reaction system is shown in Table 3.

[0066] Table 3 T4 Connection Reaction System

[0067] ​ ​ ​ 1 ​ 1 ​ 2 ​ 0.2 ddH2O 15.8 ​ 20

[0068] (5) Conversion of linker products

[0069] Add 10 μL of the ligation product to 100 μL of commercial JM109 E. coli competent cells, gently pipette to mix, incubate on ice for 5 min, heat shock at 42℃ for 45 s, incubate on ice for 3 min, then spread evenly on Amp antibiotic LB plates and incubate upside down at 37℃ for 12 h.

[0070] (5) Identification of positive colonies

[0071] Single colonies were picked from the plate using a sterile pipette tip and amplified using LB medium containing Amp. The amplified bacterial culture was sequenced and verified after 12 hours.

[0072] (6) Plasmid extraction

[0073] The correctly sequenced bacterial culture was inoculated into 15 mL of LB medium containing Amp and amplified at 37°C and 220 rpm for 12 h. Plasmids were then extracted using an endotoxin-free plasmid mini-prep kit. The steps are as follows:

[0074] 1. Collect the amplified bacterial culture into a 5 mL centrifuge tube and centrifuge at 13000 rpm for 2 min;

[0075] 2. Pour off the supernatant, add 250 μL of bacterial resuspension and vortex to completely suspend the bacterial precipitate;

[0076] 3. Add 250 μL of lysis buffer, invert and mix 4-6 times, and let stand at room temperature for 3 minutes to allow the cells to fully lyse.

[0077] 4. Add 250 μL of stop solution, invert and mix 8-10 times, let stand at room temperature for 5 min to allow the bacterial genome to be fully encapsulated by the precipitate, and centrifuge at 13000 rpm for 5 min.

[0078] 5. Transfer the supernatant to a new centrifuge tube, add 225 μL of isopropanol, and mix by inverting the tube.

[0079] 6. Add 200 μL of equilibration solution to the adsorption column that has been loaded into the collection tube, centrifuge at 13000 rpm for 1 min, and discard the liquid in the collection tube;

[0080] 7. Add the mixed liquid from step 5 into the adsorption column, centrifuge at 13000 rpm for 1 min, and discard the waste liquid in the collection tube;

[0081] 8. Add 750 μL of washing solution to the adsorption column and centrifuge at 13000 rpm for 1 min; discard the waste liquid in the collection tube.

[0082] 9. Centrifuge the adsorption column and collection tube at 13000 rpm for 1 min;

[0083] 10. Place the adsorption column in a new sterile centrifuge tube, add 50 μL of elution buffer, incubate at room temperature for 5 min, centrifuge at 13000 rpm for 2 min, collect the liquid and determine the plasmid purity. The results are as follows: ​ As shown.

[0084] Example 2: Antiviral effect of shRNA in in vitro cell culture

[0085] The inhibitory effect of shRNA on DuCV replication in duck peripheral blood lymphocytes was detected by qRT-PCR.

[0086] 1. Isolation of peripheral blood lymphocytes from ducks

[0087] Blood was collected from the hearts of 10-day-old ducklings (qRT-PCR test for duck porcine circovirus was negative and no other pathogens were present), and peripheral blood lymphocytes were isolated. The procedure was as follows:

[0088] (1) Aseptically collect blood from the heart and collect it in an anticoagulant tube. Dilute the anticoagulant blood with PBS at a 1:1 ratio.

[0089] (2) Use a Pasteur pipette to spread the diluted anticoagulated blood evenly on top of the PBMC separation solution, and centrifuge at 3000 rpm for 10 min in a horizontal centrifuge;

[0090] (3) After centrifugation, aspirate the lymphocyte layer fluid and gently mix it with the cell washing solution, then centrifuge at 1500 rpm for 10 min.

[0091] (4) Discard the supernatant, add RPMI 1640 containing 10% FBS to resuspend the cells, and adjust the cell density to 2 × 10⁻⁶. 6 Cells / mL, evenly seeded in 6-well plates for culture.

[0092] 2. DuCV infection and shRNA electroporation of duck peripheral blood lymphocytes in vitro

[0093] (1) The prepared duck circovirus solution (obtained using strain CGMCC No. 19296 and its described culture method) was diluted to 2×10⁻⁶. 6 copies / mL. The diluted duck circovirus solution was inoculated into 6-well plates, 200 μL per well. The control group was treated with 200 μL of PBS. After gentle vortexing, the plates were incubated at 37°C (5% CO2) for 12 h. PBMCs were grouped as follows:

[0094] Group A consisted of PBMCs infected alone, plus a PBS control group.

[0095] Group B consisted of PBMC infection alone plus pBAsi-hU6-shRNA-Rep-1 treatment.

[0096] Group C consisted of PBMC infection alone plus pBAsi-hU6-shRNA-Rep-2 treatment.

[0097] Group D consisted of PBMC infection alone plus pBAsi-hU6-shRNA-Cap-1 treatment.

[0098] Group E consisted of PBMC infection alone plus pBAsi-hU6-shRNA-Cap-2 treatment.

[0099] Group F was the PBMC-only infection plus pBAsi-hU6-shRNA control treatment group.

[0100] (2) Collect the above cells, centrifuge at 1000g for 8 min, discard the supernatant, wash the cells twice with Opti-MEM and adjust the cell density to 2×10⁻⁶. 7 / mL.

[0101] (3) Electroporation: The parameters were set to 200V, 5ms. 2μg of the corresponding plasmid was added to each group. The mixture was gently mixed to avoid generating air bubbles. Immediately after the electroporation was completed, 500μL of complete culture medium was added to the electroporation cup. The mixture was gently pipetted and then transferred to a 12-well cell culture plate and cultured at 37℃ in a cell culture incubator containing 5% CO2 for 48h.

[0102] (4) Collect cells and extract total DNA using a DNA extraction kit. Use qRT-PCR to detect the viral copy number in each group of cells.

[0103] Results: The inhibitory effect of shRNA on DuCV proliferation in PBMCs by each group 48 h after transfection was determined by qRT-PCR. ​ As shown, all four shRNAs in the treatment group inhibited DuCV replication in PBMCs, with differences among the groups. shRNA-Rep-1 showed the most significant inhibitory effect (P<0.01), reducing viral load by more than 30-fold compared to the PBS control group; shRNA-Cap-2 showed the second most significant effect. These results indicate that shRNA-Rep-1 and shRNA-Cap-2 can significantly inhibit DuCV replication in PBMCs.

[0104] Example 3: Inhibitory effect of shRNA recombinant plasmid on DuCV in laboratory-raised ducks.

[0105] The inhibitory effect of shRNA on DuCV replication in ducks was detected by qRT-PCR.

[0106] Experimental Groups:

[0107] Sixty one-day-old Cherry Valley ducks hatched in the laboratory (tested negative for duck circovirus by qRT-PCR and showed no other pathogen infection) were injected intramuscularly with porcine circovirus (culture preservation number CGMCC No. 19296, specific source as in Example 2) to infect the one-day-old ducklings, with an infection dose of not less than 1×10⁻⁶. 7 Copies. 60 infected ducklings were divided into groups of 10 each, as follows:

[0108] Group A consisted of DuCV infection alone plus a PBS control group.

[0109] Group B consisted of DuCV infection alone plus pBAsi-hU6-shRNA-Rep-1 treatment.

[0110] Group C consisted of DuCV infection alone plus pBAsi-hU6-shRNA-Rep-2 treatment.

[0111] Group D consisted of DuCV infection alone plus pBAsi-hU6-shRNA-Cap-1 treatment.

[0112] Group E consisted of DuCV infection alone plus pBAsi-hU6-shRNA-Cap-2 treatment.

[0113] Group F was the DuCV-only infection plus pBAsi-hU6-shRNA control treatment group.

[0114] There were a total of 6 groups, all with the same age and feeding method. At 14 days old, the infants were treated with intramuscular injection. Except for group A, all other groups were injected with PBS containing 20 μg (dissolved in 500 μL PBS) of the corresponding recombinant plasmid.

[0115] DNA extraction and viral load qRT-PCR detection:

[0116] Three ducks were randomly selected from each group 48h, 72h and 96h after injection of shRNA recombinant plasmid and euthanized. The livers were harvested and total DNA was extracted using a DNA extraction kit. The viral copy number in the liver tissue of each group was detected using qRT-PCR.

[0117] Results: Changes in liver viral load in each group at 48h, 72h, and 96h after shRNA treatment were determined by qRT-PCR. ​ As shown, after treatment with shRNA, all four treatment groups exhibited significant inhibitory effects on DuCV replication within 48 hours, and maintained good inhibitory effects even after 96 hours of injection. The shRNA-Rep-1 treatment group showed the most significant inhibitory effect at all time points (P<0.01), with an inhibition fold of approximately 100-fold; the inhibitory effect of shRNA-Cap-2 was slightly lower. These results indicate that shRNA-Rep-1 and shRNA-Cap-2 have significant inhibitory effects on viral replication in the clinical treatment of DuCV infection.

[0118] Example 4: The effect of shRNA in clinical treatment

[0119] Sixty healthy one-day-old ducklings hatched at a hatchery were isolated and raised in a small-scale farm. Simultaneously, they were artificially infected with duck circovirus (culture preservation number CGMCC No. 19296, specific source the same as in Example 2) via intramuscular injection, with an infection dose of not less than 1×10⁻⁶. 7Copies were raised in the same manner as the main flock. At 5 days of age, blood samples were collected and PCR was used to confirm infection. The ducks were randomly divided into three groups (A, B, and C), with 20 ducks in each group, and kept in isolation. At 10 days of age, group A was injected with 1.0 mL of physiological saline + 20 μg of pBAsi-hU6-shRNA-Rep-1, group B was injected with 1.0 mL of physiological saline, and group C was injected with physiological saline + 20 μg of pBAsi-hU6-shRNA-control. The ducks were observed for 14 days, and the disease severity and mortality were recorded for each group. They were then euthanized, and the positivity rate of the virus in the liver was detected using PCR.

[0120] Results: In Group A, 2 days after injection, the feed intake of sick ducklings began to increase, and their mental state improved significantly. No ducklings died within 14 days. After 14 days, the ducks were culled and dissected. Most ducks showed mild or no symptoms upon necropsy, and the positive rate of duck circovirus in the liver using PCR was 6 / 20. In Group B, after injection, the feed intake and mental state of sick ducks did not improve significantly. Dying ducks began to die from the second day after injection, and the mortality rate within 14 days was 30%. At the end of the 14-day observation period, the surviving ducks were culled. Most ducks showed symptoms such as yellowing of the liver, localized swelling, and hemorrhages. The positive rate of duck circovirus in the liver (including the frozen livers of dead ducks) using PCR was 20 / 20. In Group C, after injection, the feed intake and mental state of sick ducks did not improve significantly. Dying ducks began to die from the second day after injection, and the mortality rate within 14 days was 35%. After 14 days of observation, surviving ducks were culled. Most sick ducks exhibited symptoms such as yellowing of the liver, localized swelling, and petechiae. PCR testing revealed a positivity rate of duck porcine circovirus (PCV) in the livers (including frozen livers from dead ducks) of 20 / 20. The results indicate that injection of pBAsi-hU6-shRNA-Rep-1 significantly alleviated clinical symptoms and reduced the PCV positivity rate.

[0121] ​ ​ ​ A 30％ 0％ B 100％ 30％ C 100％ 35％

[0122] Example 5: Preparation of Anti-Duck Circovirus Egg Yolk Antibody

[0123] (1) Duck circovirus tissue inactivated vaccine (culture preservation number CGMCCNo.19296, specific source is the same as in Example 2) was used to immunize laying hens one month before the start of laying in 4 times. The inoculation dose was 1.5 mL / bird, and the interval between each immunization was 2 weeks.

[0124] The preparation method for duck circovirus tissue-inactivated vaccine is as follows: Duck circovirus antigen is obtained by intraperitoneal injection of the virus into 1-day-old ducklings, followed by booster injections every 7 days via intravenous injection, with each infection containing at least 1×10⁻⁶ viruses. 7The entire liver tissue of infected ducks was collected at 25 days of age, and the viral load in the liver was detected by quantitative real-time PCR with a viral load ≥1×10⁻⁶ copies. 8 The original antigen tissue can be prepared by adding 5 times the amount of water to the antigen tissue and homogenizing it. The antigen solution is then inactivated by adding 3‰ formaldehyde at 37℃ for 48 hours, and then 10% glycerol is added and mixed well to prepare the tissue-inactivated vaccine.

[0125] (2) Two weeks after the fourth immunization, start collecting immunized eggs and continue collecting eggs, maintaining immunization once every 1-2 months. Use an egg yolk separator to separate the yolk from the egg white. Dilute the yolk with purified water preheated to 35°C at a volume ratio of 3.5:1. Stir well to obtain the diluted yolk solution and preheat at 35°C for 1 hour.

[0126] (3) Add PEG6000 with a final concentration of 3.5% to the egg yolk dilution, mix it with the egg yolk and stir it thoroughly. Let it stand for 4 hours to allow it to react fully.

[0127] (4) Extract the supernatant after the reaction and filter it first with a 100-mesh filter and then with a 200-mesh filter.

[0128] (5) The filtered supernatant is placed into a sterile container for secondary sedimentation, and the sedimentation time is 48 hours.

[0129] (6) Extract the supernatant from the secondary precipitation, first filter it through a 200-mesh filter, and then filter it through a 0.22μm filter membrane for sterilization.

[0130] (7) Add a certain amount of preservative or formaldehyde at a concentration of 0.1% to the supernatant after sterilization to obtain refined egg yolk antibodies.

[0131] (8) The titer of the agar diffusion test for detecting antibodies against duck circovirus and adenovirus is ≥1:64.

[0132] (9) Dispensing and storage. Under aseptic conditions, dispense the filtrate into sterile vaccine vials, seal with rubber stoppers, press aluminum caps, affix labels, and store for later use at a temperature of 4-8℃.

[0133] (10) Quality testing of egg yolk antibodies

[0134] ① Safety inspection

[0135] Twenty healthy 1-day-old Cherry Valley ducklings were injected intramuscularly with 2.0 mL of the egg yolk antibody prepared in Example 4 of this invention at multiple sites. After 14 days of observation, all susceptible ducklings survived healthily, indicating that the egg yolk antibody of this invention has good safety.

[0136] ② Sterility test

[0137] The egg yolk antibody of the present invention was tested in accordance with the Veterinary Pharmacopoeia of the People's Republic of China (2015 edition) and found to be free of bacterial, mycoplasma and exogenous viral contamination.

[0138] ③ Virus neutralization efficacy test

[0139] Forty healthy 1-day-old Cherry Valley ducklings (negative for duck circovirus antigen and antibody in serum tests) were randomly divided into two groups, A and B, with 20 ducklings in each group. Group A received 0.5 mL of egg yolk antibody prepared in Example 4 and 0.5 mL of liver homogenate supernatant containing duck circovirus (>1×10⁻⁶). 7 Mix (copies) and incubate at 4°C for 4 hours; Group B is the control group, receiving 0.5 mL of physiological saline and 0.5 mL of liver homogenate supernatant containing duck circovirus (>1×10⁻⁶ copies). 7 The two treatment solutions were mixed and incubated at 4°C for 4 hours. Twenty ducklings were injected intramuscularly with each solution. Three days after challenge, ten ducklings were euthanized, and their livers were collected for PCR testing to detect viral positivity. The remaining ten ducklings were observed at 14 days of age, and their clinical symptoms were recorded. They were then euthanized and tested for viral positivity.

[0140] The results showed that all 20 ducks in the egg yolk antibody group (Group A) were negative for duck circovirus in their livers, while all 20 ducks in the control group (Group B) were positive for duck circovirus. Furthermore, Group A showed no clinical symptoms during the observation period, while some ducks in Group B exhibited symptoms such as lethargy, poor appetite, and reluctance to move. These results indicate that the egg yolk antibody is effective and can neutralize viral infection.

[0141] Example 6: The effect of combined use of shRNA and egg yolk antibody in clinical treatment

[0142] Sixty healthy 1-day-old ducklings were artificially infected with duck circovirus. At 5 days of age, blood samples were collected and PCR was used to confirm infection. The ducklings were randomly divided into three groups (A, B, and C), with 20 ducklings in each group, and kept in isolation. At 10 days of age, group A was injected with 2.0 mL of egg yolk antibody prepared in Example 5 + 20 μg of pBAsi-hU6-shRNA-Rep-1, group B was injected with 2.0 mL of egg yolk antibody, and group C was injected with 2.0 mL of physiological saline. The ducks were observed for 14 days, and the disease condition and mortality of each group were recorded. They were then euthanized, and the positivity rate of the virus in the liver was detected by PCR.

[0143] Results: In Group A, 2 days after injection, the feed intake of sick ducklings began to increase, and their mental state improved significantly. No ducklings died within 14 days of treatment. After 14 days of treatment, the ducks were culled and necropsed. Most ducks showed mild or no symptoms upon necropsy, and the positive rate of duck circovirus in the liver using PCR was 2 / 20. In Group B, 2 days after injection, the feed intake of sick ducklings also began to increase, and their mental state improved significantly. One duckling died within 14 days of treatment. After 14 days of treatment, the ducks were culled and necropsed. Most ducks showed mild or no symptoms upon necropsed, and the positive rate of duck circovirus in the liver using PCR was 5 / 20. In Group C, after injection, the feed intake and mental state of sick ducks did not improve significantly. Ducklings began to die from the second day after injection, and the mortality rate of sick ducks within 14 days of treatment was 25%. During the observation period, surviving ducks were culled after 14 days of treatment. Most ducks were found to have symptoms such as yellowing of the liver, local swelling, and bleeding points. The positive rate of duck porcine circovirus in the liver (including the frozen livers of dead ducks) was 20 / 20 when detected by PCR technology.

[0144] The above experimental results show that the combination of shRNA-Rep-1 and egg yolk antibody of the present invention has good safety, good preventive effect and high cure rate. The combined effect of 20μg shRNA and egg yolk antibody is significantly better than egg yolk antibody alone. It can be used for the prevention and treatment of duck circovirus infection, and has significant economic and social benefits.

Claims

1. A shRNA that inhibits the expression of duck circovirus genes, characterized in that: Specifically, it refers to any one of Rep-1, Rep-2, Cap-1, and Cap-2, with each shRNA interference sequence including a positive and a negative strand; among which: Rep-1 positive strand: 5'-GATCCGCAAGAGGTGGGTCTTTACCATTAATCTGTGAAGCCACAGATGGGATTAATGGTAAAGACCCACCTCTTGCTTTTTTA-3', as shown in SEQ ID NO.1; Rep-1 negative chain: 5'-AGCTTAAAAAAGCAAGAGGTGGGTCTTTACCATTAATCCCATCTGTGGCTTCACAGATTAATGGTAAAGACCCACCTCTTGCG-3', as shown in SEQ ID NO.2; Rep-2 positive strand: 5'-GATCCGCCTAATCGTCGAGACGCAACGTGATCTGTGAAGCCACAGATGGGATCACGTTGCGTCTCGACGATTAGGCTTTTTTA-3', as shown in SEQ ID NO.3; Rep-2 negative chain: 5'-AGCTTAAAAAAGCCTAATCGTCGAGACGCAACGTGATCCCATCTGTGGCTTCACAGATCACGTTGCGTCTCGACGATTAGGCG-3', as shown in SEQ ID NO.4; Cap-1 positive chain: 5'-GATCCGCGATTCGTAGCCTTCGTCTTCTGAACTGTGAAGCCACAGATGGGTTCAGAAGACGAAGGCTACGAATCGCTTTTTTA-3', as shown in SEQ ID NO.5; Cap-1 negative chain: 5'-AGCTTAAAAAAGCGATTCGTAGCCTTCGTCTTCTGAACCCATCTGTGGCTTCACAGTTCAGAAGACGAAGGCTACGAATCGCG-3', as shown in SEQ ID NO.6; Cap-2 positive chain: 5'-GATCCGCCACTCCTGTTGTGTTGTCTGGTTTCTGTGAAGCCACAGATGGG AAACCAGACAACACAACAGGAGTGGCTTTTTTA-3', as shown in SEQ ID NO.7; Cap-2 negative chain: 5'-AGCTTAAAAAAGCCACTCCTGTTGTGTTGTCTGGTTTCCCATCTGTGGCTTCACAGAAACCAGACAACACAACAGGAGTGGCG-3', as shown in SEQ ID NO.8.

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