Application of duck IL7 protein as an adjuvant in the preparation of duck hepatitis A virus type 3 inactivated vaccine

Abstract

This invention discloses the application of duck IL7 protein as an adjuvant in the preparation of duck hepatitis A virus type 3 inactivated vaccine, belonging to the field of biopharmaceuticals. The research of this invention shows that duck IL7 protein as an adjuvant can enhance the immunoprotective efficacy of the DHAV-3 inactivated vaccine and also enhance its heterologous protective ability. This provides a theoretical basis for the development of duck IL7 as a novel duck vaccine adjuvant.

Description

Technical Field

[0001] This invention belongs to the field of immunology, specifically relating to the application of duck IL7 as an adjuvant in duck hepatitis A virus type 3 inactivated vaccine. Background Technology

[0002] Duck hepatitis A virus type 3 (DHAV-3) primarily affects ducklings under 3 weeks old, causing acute, highly lethal infection and leading to enlarged and hemorrhagic livers, resulting in significant economic losses to my country's duck farming industry. Vaccination can stimulate a specific immune response against the pathogen in animals, thereby preventing the spread of the disease. Previously, three different serotypes of DHAV have been reported, with no significant cross-neutralizing effect between the different serotypes. DHAV-3 is currently the predominant genotype circulating in my country.

[0003] Adjuvants can enhance the strength and persistence of vaccine-induced antigen-specific B-cell responses, thereby promoting long-term immune protection. Interleukin-7 (IL7) is a cytokine with broad immune effects, mainly produced by thymic and bone marrow stromal cells. It can promote the differentiation of pluripotent hematopoietic stem cells into lymphoprogenitor cells and stimulate the proliferation and differentiation of lymphocytes, natural killer cells, and dendritic cells. IL7 binds to its receptor (IL7R) and activates the downstream JAK-STAT5 pathway, prolonging B-cell survival and enhancing humoral immune responses by upregulating the expression of anti-apoptotic factors and downregulating the expression of pro-apoptotic factors in the Bcl-2 family. Therefore, IL7 holds promise as an adjuvant in vaccine development to enhance vaccine immunogenicity. Summary of the Invention

[0004] The technical problem to be solved by this invention is: how to improve the immunization effect of DHAV-3 inactivated vaccine.

[0005] The technical solution of the present invention is: the application of duck IL7 protein as an adjuvant in the preparation of duck hepatitis A virus type 3 inactivated vaccine, wherein the amino acid sequence of the duck IL7 protein is shown in SEQ ID No. 3.

[0006] An immunomodulatory agent comprising duck hepatitis A virus type 3 inactivated antigen and duck IL7 protein adjuvant, wherein the amino acid sequence of the duck IL7 protein is shown in SEQ ID No. 3.

[0007] The above-mentioned immunomodulatory agents are used in the preparation of drugs for the prevention and treatment of duck hepatitis A virus type 1 or duck Tembusu virus.

[0008] To investigate the role of duck-derived interleukin 7 (duIL7) in the immunoprotective efficacy of DHAV-3 inactivated vaccine, this invention expresses duck IL7 (duIL7) protein using a recombinant adenovirus Ad5 expression system, and uses vaccines containing or without duIL7 (10... 9 TCID 50 DHAV-3 inactivated vaccine (1×10) for ducks 2 ELD 50 / duck) was immunized with 1-day-old ducks via intramuscular injection. In this study, ducks in each immunized group were challenged with DHAV-3 (10) via intramuscular injection 7 days post-immunization. 3 TCID 50 / duck). Liver samples were collected two days post-infection (2 dpi), and viral titers in the tissues were determined by RT-qPCR. The results showed that the viral copy number in ducks immunized with the DHAV-3 inactivated vaccine containing duIL7 was significantly lower than that in the control group. Furthermore, this study administered intramuscular challenge with duck hepatitis A virus type 1 (DHAV-1, 10...) to each immunized group of ducks seven days post-immunization. 8 copies / duck). Liver samples were collected at 2 dpi, and the viral titer in the tissues was determined by RT-qPCR. The results showed that the copy number of ducks immunized with the DHAV-3 inactivated vaccine containing duIL7 was significantly lower than that of the control group. Furthermore, 7 days post-immunization, ducks in each immunized group were challenged with duck Tembusu virus (DTMUV, 2×10⁻⁶) via intramuscular injection. 5 TCID 50 (Ducks). Brain tissue was collected at 3 dpi, and the viral titer in the tissue was determined by RT-qPCR. The results showed that the viral copy number in ducks immunized with the DHAV-3 inactivated vaccine containing duIL7 was significantly lower than that in the control group. In conclusion, duIL7 can enhance the immunoprotective efficacy of the DHAV-3 inactivated vaccine and also enhance its heterologous protective ability.

[0009] Compared with the prior art, the present invention has the following beneficial effects:

[0010] Duck IL7 protein, when used as an adjuvant, can enhance the immunoprotective efficacy of DHAV-3 inactivated vaccine and also enhance its heterologous protective ability. This provides a theoretical basis for the development of duck IL7 as a novel adjuvant for duck vaccines. Attached Figure Description

[0011] Figure 1 Image of gel electrophoresis results of PCR-amplified duIL7 sequence fragment; M is Marker DL2000; lane 1 is negative control; lane 2 is duIL7 fragment; arrows indicate the size and position of duIL7 fragment.

[0012] Figure 2 Electrophoretic imaging of pShuttle-duIL7 recombinant plasmid identified by PCR; M is Marker DL8000; lane 1 is the size of the fragment obtained from amplified positive colonies; lanes 2-3 are negative bacteria; lane 4 is the negative control; the arrows indicate the location of the target fragment size.

[0013] Figure 3 Gel electrophoresis results of PCR identification of pAd and pAd-duIL7 recombinant plasmids; a: M is Marker DL2000; lane 1 is the negative control; lanes 2-6 and 8 are pAd negative plasmids; lane 7 is the pAd positive plasmid, and the arrow indicates the location of the positive plasmid. b: M is Marker DL2000; lanes 1 and 3-6 are pAd-duIL7 negative plasmids; lane 2 is the pAd-duIL7 positive plasmid; lane 7 is the negative control.

[0014] Figure 4 Image of rAd-duIL7-infected HEK 293A cells.

[0015] Figure 5 Figure 1 shows the results of Western blot analysis for rAd-duIL7 protein expression; M is the pre-stained protein marker; lane 1 is the negative control; lane 2 is the rAd sample; lane 3 is the rAd-duIL7 sample, and the arrows indicate the location of the target protein.

[0016] Figure 6 Figure 1 shows the results of RT-qPCR detection of cytokine mRNA levels in monocytes; a represents the results at 1 day, 2 days, and 1 day post-infection (dpi); b represents the results at 7 days; + indicates inactivated DHAV-3 restimulation; - indicates no inactivated DHAV-3 restimulation.

[0017] Figure 7 Survival rate of ducklings after DHAV-3 infection.

[0018] Figure 8 . Hepatic viral copy number in ducklings after DHAV-3 infection. ns, p>0.05; *, p<0.05; **, p<0.01; ***, p<0.001.

[0019] Figure 9 Images of liver lesions in ducklings caused by DHAV-3 infection; a) Visual lesions of the liver; b) Histopathological findings of the liver tissue.

[0020] Figure 10 Survival rate of ducklings after DHAV-1 infection.

[0021] Figure 11. DHAV-1 liver viral copy number in ducklings after infection; *, p<0.05.

[0022] Figure 12 Histopathological image of duck liver tissue caused by DHAV-1 infection.

[0023] Figure 13 Survival rate of ducklings infected with DTMUV.

[0024] Figure 14 . Copy number of brain virus in ducklings after infection with DTMUV; *, p<0.05.

[0025] Figure 15 Pathological image of brain tissue in ducklings infected with DTMUV. Detailed Implementation

[0026] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were all purchased from commercial sources.

[0027] Example 1: Construction and Identification of duIL7 Recombinant Adenovirus Plasmid

[0028] 1. Construction and identification of duIL7 recombinant adenovirus plasmid

[0029] To ensure successful expression of the duIL7 prokaryotic protein, the nucleotide sequence information of duIL7 (GenBank number: MT663558, CDS sequence as shown in SEQ ID No.1) was first sent to Wuhan Jinkairui Biotechnology Co., Ltd. for duck codon optimization (the optimized CDS sequence is shown in SEQ ID No.2), resulting in the pVAX1-duIL7 plasmid.

[0030] 1) Homologous arm amplification primers were designed to amplify the duck-derived optimized duIL7 fragment containing the pShuttle homologous arm fragment. The duIL7 fragment was ligated into the pShuttle-CMV vector using the homologous arm recombination method, transformed into TOP10 competent cells, and cultured overnight at 37°C for colony identification. The sequence information of the homologous arm amplification primers and identification primers is shown in Table 1.

[0031] Table 1. Sequence information of amplification primers and identification primers for the pShuttle-duIL7 homologous arm.

[0032]

[0033] 2) The duIL7 sequence was amplified using pVAX1-du_IL7 plasmid as a template. The PCR amplification reaction volume was 10 μL, as detailed in Table 2, and the reaction procedure is shown in Table 3.

[0034] Table 2. Reaction system for PCR amplification of duIL7

[0035]

[0036] Table 3. PCR amplification procedure for duIL7

[0037]

[0038] 3) After amplification, electrophoresis was performed on an agarose gel (program: 120V, 20min). Imaging results showed the presence of a specific band consistent with the expected size (643bp) of the duIL7 fragment (see...). Figure 1 ).

[0039] 4) KpnⅠ (Takara) and HindⅢ (Takara) enzymes were selected to digest the pShuttle-CMV plasmid. The digestion system is shown in Table 4, and the digestion procedure is shown in Table 5. MolPure was used to digest the plasmid. ® The PCR Purification Kit (Yisheng Biotechnology Co., Ltd.) recovers the amplified duIL7 fragment and the pShuttle-CMV linearized fragment.

[0040] Table 4. Enzyme digestion reaction system

[0041]

[0042] Table 5. Enzyme digestion reaction procedure

[0043]

[0044] 5) The recovered duIL7 and pShuttle-CMV linearized vector were used for homologous arm recombination using the ClonExpress II One Step Cloning Kit (Nanjing Novizan Biotechnology Co., Ltd.). The recombinant product was transformed into TOP10 competent cells and cultured overnight at 37°C before colony PCR identification. The PCR identification reaction system is detailed in Table 6, and the identification procedure is detailed in Table 7.

[0045] Table 6. Reaction system for PCR identification of pShuttle-duIL7

[0046]

[0047] Table 7. PCR reaction procedure for identifying pShuttle-duIL7

[0048]

[0049] 6) After amplification, electrophoresis analysis was performed on a 1% agarose gel (program: 120V, 20min). The results showed that the specific band was consistent with the expected size (675bp) of the pShuttle-duIL7 fragment (see...). Figure 2 The plasmid was named pShuttle-duIL7.

[0050] 7) TOP10 (pShuttle-duIL7) was expanded and cultured in LB liquid medium containing Kana. The pShuttle-duIL7 plasmid was then extracted. pShuttle-CMV and pShuttle-duIL7 were linearized and recovered using Pme I, and then transformed into BJ5183 competent cells. After overnight incubation at 37°C, colony PCR was performed for identification. The identification system and procedure are shown in Tables 8 and 9. Electrophoresis analysis was performed on a 1 / 2 agarose gel (program: 120V, 20min). Successfully recombined colonies without a band at 500bp were obtained (see Table 8). Figure 3 ).

[0051] Table 8. Reaction system for PCR identification of pAd-duIL7

[0052]

[0053] Table 9. PCR reaction procedure for identifying pAd-duIL7

[0054]

[0055] 8) Single colonies of BJ5183 (pAd) and BJ5183 (pAd-duIL7) were picked and cultured in LB medium containing Kana. The pAd and pAd-duIL7 plasmids were then extracted, linearized using Pac I, and the products were recovered and transfected into HEK 293A cells. Approximately 10 days after transfection, most cells became rounded and detached in a grape-like lesion pattern. The cells were collected and subjected to three freeze-thaw cycles at -80°C, followed by centrifugation at 4°C, 4000 r / min for 5 min. The supernatant was collected, which is the first-generation recombinant adenovirus, designated as F1 generation. With subsequent passages, 80% of the cells developed lesions 2-3 days after inoculation (see...). Figure 4 ).

[0056] 9) Large-scale amplification of F11 generation recombinant adenovirus and Western blotting detection of duIL7 protein expression. A 12% protein gel was prepared, and cell samples were added to wells at 20 μL / well. Protein bands were separated at 80 V for 30 min, then at 120 V for 70 min; the protein gel bands were transferred to a PVDF membrane at 0.22 mA for 60 min; the PVDF membrane was blocked with 5% skim milk for 2 h at room temperature; mouse anti-flag antibody was diluted 5000-fold with 5% skim milk and incubated overnight at 4℃; the PVDF membrane was washed 5 times with TBST for 5 min each time; HRP-labeled goat anti-mouse IgG (H+L) was diluted 10000-fold with 5% skim milk and incubated at room temperature for 2 h; the PVDF membrane was washed 5 times with TBST for 5 min each time; ECL staining showed a specific band at approximately 25 kDa (see...). Figure 5 The size of the amplified rAd-duIL7 protein is consistent with that of the duIL7 protein. This indicates that the duIL7 gene is present in the amplified rAd-duIL7 and can express the protein (its amino acid sequence is shown in SEQ ID No. 3).

[0057] 10) Spread HEK 293A cells evenly into 96-well plates (100 μL / well) and incubate overnight at 37°C in a 5% CO2 incubator. The next day, discard the original liquid in the 96-well plates and dilute the expanded F11 generation rAd-duIL7 and rAd in cell maintenance medium 10-fold (10 μL / well). -3 -10 -10 100 μL / well was added to a 96-well plate, with 6 replicates / gradient, and incubated at 37°C in a 5% CO2 incubator for 10 days. TCID was calculated using the Reed-Muench method after 10 days. 50 1×10 -9.3 TCID 50 / 100 μL and 1×10 -8.6 TCID 50 / 100μL (see Table 10).

[0058] Table 10. TCID 50 Test results

[0059]

[0060] Example 2: Evaluation of duIL7 adjuvant activity

[0061] 2.1 Impact on homologous protection of DHAV-3 inactivated seedlings

[0062] 1) Seventy-six one-day-old ducklings were randomly divided into four groups of 19 each. Specific grouping and immunization information are shown in Table 11. Seven days after immunization, 13 ducklings from each group were randomly selected for a challenge protection test. Each duckling received an intramuscular injection (IM) of DHAV-3 allantoxin (10 mg / L) in its leg. 3 ELD 50 / duck).

[0063] Table 11. Animal Experiment Grouping and Immunization Schedule

[0064]

[0065] 2) Collect jugular venous blood from each group of ducks on days 1, 2, 7, and 1 day post-immunization (dpi), and separate PBMCs. Incubate the PBMCs in 10 cm cell culture dishes for 4 hours to allow them to adhere. Wash twice with PBS, repeating this process twice to obtain monocytes. Collect monocytes from days 1, 2, 7, and 1 day post-immunization. Use RNAisoplus to pipette off adherent monocytes and extract RNA for later use. Obtain monocytes 7 days post-immunization using the same method, and add inactivated 10... 2 ELD 50 DHAV-3 stimulation for 24 hours was followed by RNA extraction using RNAiso plus. Reverse transcription was then performed after concentration determination; the system and procedure are shown in Table 12. RT-qPCR was used to detect the mRNA levels of IL1β, IL6, IFNα, iNOS, and TNFα; primer sequences are shown in Table 13, and the system and procedure are shown in Table 14. The results of day 1 showed that the mRNA levels of IFNα, IL1β, iNOS, and TNFα in group D (DHAV-3 inactivated vaccine + rAd-duIL7) were significantly higher than those in the other three groups (P<0.05), and the IL6 level was also higher in group A (PBS), group C (DHAV-3 inactivated vaccine + rAd) (P<0.05), and group B (DHAV-3 inactivated vaccine) (P>0.05). The results of day 1 epidemiological monitoring (dpi) showed that the mRNA levels of IFNα, IL6, iNOS, and TNFα in group D were significantly higher than those in the other three groups (P<0.05), and the IL1β level was also higher in group D (P>0.05). These results indicate that duIL7 has the ability to promote the response of duck monocytes (see [link to dpi]). Figure 6(a) On day 7, the in vitro detection results of each group without DHAV-3 restimulation showed that the mRNA levels of IFNα, IL6, and TNFα in group D were not significantly different from the other three groups (P>0.05), while IL1β and iNOS were higher in group D than in group C (P<0.05), but not significantly different from groups A and B (P>0.05). The detection results of in vitro DHAV-3 restimulation for 24 h showed that the mRNA levels of IFNα, IL1β, IL6, and TNFα in group D were significantly higher than the other three groups (P < 0.05), while iNOS was not significantly different from the other three groups (P>0.05). This suggests that duIL7 promotes the immune memory response of monocytes (see [reference needed]). Figure 6 (b)

[0066] Table 12 Reverse Transcription System and Procedure

[0067]

[0068] Table 13 RT-qPCR Primers

[0069]

[0070] Table 14 RT-qPCR System and Procedure

[0071]

[0072] 3) Seven days after immunization, each duckling in each group was challenged with 10 doses of the virus. 3 ELD 50 DHAV-3 was administered, and survival rates were statistically analyzed after 10 days. The survival rate was 90% in group D (DHAV-3 inactivated vaccine + rAd-duIL7), 40% in groups B (DHAV-3 inactivated vaccine) and C (DHAV-3 inactivated vaccine + rAd), and 30% in group A (PBS) (see [link to original text]). Figure 7 ).

[0073] 4) Take liver samples from each group for 2 days (dpi) and place them in a grinding tube. Add 1 mL of RNAiso plus and grind at 4°C for 8000 r / min for 5 min. Collect the supernatant and add 200 μL of chloroform. Mix by inversion and incubate on ice for 5 min. Grind at 4°C for 12000 r / min for 15 min. Collect the supernatant and add an equal volume of isopropanol. Mix and incubate on ice for 15 min. Grind at 4°C for 12000 r / min for 15 min. Discard the supernatant and resuspend the precipitate in 1 mL of 75% anhydrous ethanol. Grind at 4°C for 8000 r / min for 5 min. Discard the supernatant and allow to air dry for 15 min. Dissolve the precipitate in 100 μL of DEPC water and determine the concentration. Take 1 μg of RNA for reverse transcription (system and procedure are shown in Table 12) to obtain cDNA. Analyze the cDNA using the DHAV-3 assay established in our laboratory. The VP1 gene was used to determine the viral copy number in liver tissue using an absolute quantitative method. The standard curve equation was: Y = -3.9613X + 37.875. The RT-qPCR primer sequences are shown in Table 15, and the system and procedure are shown in Table 16. The results showed that the viral copy number in duck liver tissue of group D (DHAV-3 inactivated vaccine + rAd-duIL7) was significantly lower than that of the other three groups (P < 0.05) (see Table 16). Figure 8 ).

[0074] Table 15 RT-qPCR System and Procedure

[0075]

[0076] Table 16 RT-qPCR System and Procedure

[0077]

[0078] 5) At 2 dpi, necropsy was performed on the livers of ducklings in each group. Groups A (PBS), B (DHAV-3 inactivated vaccine), and C (DHAV-3 inactivated vaccine + rAd) all showed extensive hemorrhages with obvious hemorrhagic spots in the liver. Group D (DHAV-3 inactivated vaccine + rAd-duIL7) showed no hemorrhagic spots in the liver (see...). Figure 9 (a) Liver tissues were collected from each group, and tissue sections were prepared. The results showed that liver tissues in groups A, B, and C contained a large number of red blood cells (red arrows), disordered hepatic cord structure, and hepatocytes with obvious fatty degeneration, appearing as round swellings with vacuoles in the cytoplasm (black arrows). Group D, however, showed no obvious lesions (see [link]). Figure 9 (b) The above results indicate that DHAV-3 inactivation + rAd-duIL7 can alleviate the pathological damage of DHAV-3 to duck liver tissue.

[0079] 2.2 Impact on the protection of DHAV-3 inactivated seedling sources

[0080] 1) 26 one-day-old ducklings were randomly divided into 2 groups of 13 each. See Table 17 for specific grouping and immunization information.

[0081] Table 17 Animal Immunization Schedule

[0082]

[0083] 2) Twenty-six one-day-old ducklings were randomly divided into two groups of 13 each. Specific grouping and immunization information are shown in Table 17. Seven days after immunization, each duckling in each group received an intramuscular injection of 2×10⁻⁶ ppm in its leg. 8 Copies of DHAV-1. Survival rates were observed for 10 days. Group B (DHAV-3 inactivated vaccine + rAd-duIL7) had a survival rate of 70%, while Group A (DHAV-3 inactivated vaccine + rAd) had a survival rate of 50% (see...). Figure 10 ).

[0084] 3) RNA was extracted from the liver of each group at 2 dpi. After determining the concentration, 1 μg of RNA was reverse transcribed (system and procedure are shown in Table 12) to obtain cDNA. The viral copy number in liver tissue was detected using the absolute quantification method for the DHAV-1 VP0 gene established in our laboratory. The standard curve equation was: Y = -3.371X + 42.679. The RT-qPCR primer sequences are shown in Table 18, and the system and procedure are shown in Table 19. The results showed that the viral copy number in duck liver of group B (DHAV-3 inactivated vaccine + rAd-duIL7) was significantly lower than that of group A (P < 0.05) (see Table 19). Figure 11 ).

[0085] Table 18 RT-qPCR System and Procedure

[0086]

[0087] Table 19 RT-qPCR System and Procedure

[0088]

[0089] 4) Liver tissue samples were collected from two groups at 2 dpi, and sections were prepared. The results showed that the liver tissue of group A (DHAV-3 inactivated vaccine + rAd) contained a large number of red blood cells (red arrows) and hepatocytes showed obvious fatty degeneration (black arrows), while the lesions in group B (DHAV-3 inactivated vaccine + rAd-duIL7) were milder (see...). Figure 12 ).

[0090] 5) Twenty-six one-day-old ducklings were randomly divided into two groups of 13 each. Specific grouping and immunization information are shown in Table 17. Seven days after immunization, each duckling in each group received an intramuscular injection of 2×10⁻⁶ ppm in its leg. 5 TCID 50DTMUV. Survival rates were observed for 10 days. Group B (DHAV-3 inactivated vaccine + rAd-duIL7) had a survival rate of 70%, while Group A (DHAV-3 inactivated vaccine + rAd) had a survival rate of 30% (see...). Figure 13 ).

[0091] 6) RNA was extracted from the brain of each group at 3 dpi. After determining the concentration, 1 μg of RNA was reverse transcribed (system and procedure are shown in Table 12) to obtain cDNA. The viral copy number in brain tissue was detected using the DTMUV NS3 gene absolute quantification method established in our laboratory. The standard curve equation was: Y = -3.566X + 43.431. The RT-qPCR primer sequences are shown in Table 18, and the system and procedure are shown in Table 19. The results showed that the duck liver virus copy number in group B (DHAV-3 inactivated vaccine + rAd-duIL7) was significantly lower than that in group A (P < 0.05) (see Table 19). Figure 14 ).

[0092] Table 18 RT-qPCR System and Procedure

[0093]

[0094] Table 19 RT-qPCR System and Procedure

[0095]

[0096] 7) Brain tissue samples were collected from two groups at 3 dpi, and brain tissue sections were prepared. Results showed that in group A (DHAV-3 inactivated vaccine + rAd), there were numerous small-volume cell clusters in localized areas of the brain tissue, suspected to be glial cell hyperplasia (blue arrows). In contrast, the brain tissue in group B (DHAV-3 inactivated vaccine + rAd-duIL7) had a basically normal structure, normal numbers of neurons and glial cells, and clearly visible neuronal nuclei, with no obvious abnormal lesions (see...). Figure 15 ).

[0097] In summary, we optimized the duck codons of the duck duIL7 (GenBank number: MT663558) nucleotide sequence. Subsequently, we expressed the duIL7 protein using a recombinant adenovirus system and co-immunized ducks with rAd-duIL7 and DHAV-3 inactivated vaccine to investigate the effect of duIL7 as an adjuvant on the immunogenicity of the DHAV-3 inactivated vaccine. The results show that duIL7 as an adjuvant for the DHAV-3 inactivated vaccine can effectively enhance the homologous / heterologous immunoprotective ability of the DHAV-3 inactivated vaccine, providing a theoretical basis and reference for the development and design of novel DHAV-3 inactivated vaccines.

Claims

1. The application of duck IL7 protein as an adjuvant in the preparation of duck hepatitis A virus type 3 inactivated vaccine, wherein the amino acid sequence of the duck IL7 protein is shown in SEQ ID No.

3.

2. An immunomodulatory agent, characterized in that, It contains duck hepatitis A virus type 3 inactivated antigen and duck IL7 protein adjuvant, the amino acid sequence of which is shown in SEQ ID No.

3.

3. The use of the immunomodulatory agent according to claim 2 in the preparation of a drug for preventing and treating duck hepatitis A virus type 1 or duck Tembusu virus.

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