Construction and application of NGPV and MDPV antigen dual expression vector

Abstract

The invention relates to construction and application of an NGPV and MDPV antigen double expression vector, and belongs to the technical field of veterinary biological products. According to the invention, codon optimization is carried out on novel goose parvovirus VP2 gene and muscovy duck parvovirus VP3 gene sequences, the optimized NGPV VP2 and MDPV VP3 gene sequences are inserted into PH and P10 promoters of pFastBac-Dual plasmids to obtain recombinant baculovirus shuttle plasmids, the recombinant plasmids are transfected to Sf9 cells, and the recombinant baculovirus shuttle plasmids are transfected to obtain recombinant baculovirus. And packaging to obtain the recombinant baculovirus for expressing the novel goose parvovirus VP2 protein and the muscovy duck parvovirus VP3 protein. The subunit vaccine prepared by using insect cells to express the NGPV VP2 protein and the MDPV VP3 protein is good in immune effect and simple and convenient in production process, and provides technical support for prevention and control work of the disease.

Description

Technical field:

[0001] The present invention belongs to the technical field of veterinary biological products and relates to the construction and application of a dual expression vector of NGPV and MDPV antigens. Background technology:

[0002] Both NGPV and MDPV are single-stranded, linear, non-enveloped DNA viruses belonging to the genus Dependovirus of the family Parvoviridae. The two are extremely similar in morphological structure and physicochemical properties. Their genome homology is 81.9% and their amino acid homology is 88.96%, which determines that they have certain homology in antigenicity. However, the inactivated NGPV vaccine (inactivated MDPV vaccine) currently under development cannot produce effective neutralizing antibodies and cross-challenge protection against heterologous MDPV or NGPV after immunization. Both NGPV and MDPV are important epidemic diseases that currently seriously affect the Muscovy duck farming industry in my country. The two are often mixed-infected in clinical practice, causing huge economic losses to the Muscovy duck farming industry.

[0003] NGPV is one of the main pathogens that cause infectious diseases in Muscovy ducks. The mortality rate of ducks infected with NGPV is not high, but the recovery is poor and the growth and development are hindered, which brings serious economic losses to my country's duck farming industry. There is no specific medicine for the new goose parvovirus disease. Due to the continuous mutation of the virus, the cross-protection of existing vaccines and egg yolk antibodies against different strains is insufficient. The development of relevant vaccines for virus variants is the key to preventing this disease. The genome of the new goose parvovirus (NGPV) is a single-stranded linear DNA with a total length of about 5.1kb. The NGPV genome contains two large open reading frames, which encode non-structural proteins NS1 and NS2 and structural proteins VP1, VP2, and VP3, respectively. The structural proteins VP1, VP2, and VP3 are components of the virus capsid, with molecular weights of 87, 70, and 60ku, respectively. Compared with VP1 and VP3, VP2 has stronger immunogenicity and is the preferred protein for the development of NGPV subunit vaccines and detection reagents.

[0004] MDPV mainly causes disease in young Muscovy ducks under 3 weeks old, so the disease is called "three-week disease", with a mortality rate of 50%-80%, posing a serious threat to the development of the waterfowl industry. MDPV is widely prevalent in Fujian, Sichuan, Guizhou, Anhui and other provinces in my country. How to effectively control the spread of MDPV, reduce the harm of the virus to the duck farming industry, and quickly develop an effective vaccine is imminent.

[0005] This study used insect cells to simultaneously express NGPV VP2 protein and MDPV VP3 protein in eukaryotic cells, aiming to develop a subunit vaccine containing protective antigens of both viruses, to provide more effective and comprehensive protection for the immunization and prevention of novel goose parvovirus disease and Muscovy duck parvovirus disease, while providing dual protection with one shot. While providing comprehensive protection, it can also reduce the number of immunizations and save immunization costs. Summary of the invention:

[0006] The purpose of the present invention is to provide a novel goose parvovirus (NGPV) VP2 protein and Muscovy duck parvovirus (MDPV) VP3 protein gene recombinant baculovirus subunit vaccine and its application, in order to solve the problem that the existing technology cannot effectively control the epidemic spread of NGPV and MDPV at the same time.

[0007] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0008] The invention discloses an immunogenic composition, which contains VP2 protein encoded by VP2 gene of novel goose parvovirus and VP3 protein encoded by VP3 gene of Muscovy duck parvovirus.

[0009] In the present invention, the novel goose parvovirus VP2 protein has a Kozak sequence GCCACC and a Flag tag added to its N-terminus, and the Muscovy duck parvovirus VP3 protein has a Kozak sequence GCCACC and a secretion signal peptide Melittin added to its N-terminus, and a 6*His tag added to its C-terminus.

[0010] In the present invention, the genes of the novel goose parvovirus VP2 protein and Muscovy duck parvovirus VP3 protein are codon optimized according to the preference of the insect cell protein expression system. The optimized nucleotide sequences are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively, with a Kozak sequence GCCACC added to the 5' end and a stop codon TAA added to the 3' end.

[0011] In the present invention, the preparation method of the novel goose parvovirus VP2 protein and the Muscovy duck parvovirus VP3 protein is eukaryotic expression, and the eukaryotic expression vector is a recombinant baculovirus vector pFastBac TM Dual.

[0012] In the present invention, the recombinant baculovirus vector is a gene with codon optimization inserted into the downstream of the baculovirus PH and P10 promoters. The recombinant baculovirus was deposited in the China Center for Type Culture Collection on November 28, 2024, with the deposit address: Wuhan University, Wuhan, China, and the deposit number is CCTCCNO: V2024116.

[0013] In the present invention, the host cell of the recombinant baculovirus vector is selected from the Sf9 cell of the insect cell line.

[0014] A subunit vaccine capable of simultaneously preventing and treating NGPV and MDPV, the vaccine comprising an adjuvant and the immunogenic composition of claim 1.

[0015] The subunit vaccine is characterized in that the vaccine is used to prevent and treat new goose parvovirus and Muscovy duck parvovirus.

[0016] The present invention has the following advantages: the present invention simultaneously expresses NGPV VP2 protein and MDPV VP3 protein through insect cells to develop a subunit vaccine containing protective antigens of two viruses. The vaccine has the characteristics of high antigen expression and good immune effect. It can protect against two diseases with one shot. While providing comprehensive protection, it can also reduce the number of immunizations and save immunization costs, laying the foundation for the industrial production of NGPV and MDPV subunit vaccines. Description of the drawings:

[0017] Figure 1 pFastBac TM Dual / NGPV-VP2-MDPV-VP3 expression vector restriction enzyme digestion identification results. Among them, M is DL2000DNAMarker; 1 is pFastBac TM Dual / NGPV-VP2-MDPV-VP3 recombinant plasmid XhoI, KpnI double restriction digestion product, 2 is pFastBac TM Dual / NGPV-VP2-MDPV-VP3 recombinant plasmid EcoRI and SalI double enzyme digestion product.

[0018] Figure 2 pFastBac TM Dual / NGPV-VP2-MDPV-VP3 expression vector PCR identification results. Among them, M is DL15000DNAMarker; 1 is pFastBac TM Dual / NGPV-VP2-MDPV-VP3 recombinant plasmid was identified by PCR using M13 universal primers; 2 is pFastBac TM Dual / NGPV-VP2-MDPV-VP3 recombinant plasmid was run with NGPV detection primers to identify the results; 3 is pFastBac TM The Dual / NGPV-VP2-MDPV-VP3 recombinant plasmid was identified by PCR using MDPV detection primers.

[0019] Figure 3The results of Bacmid / NGPV-VP2-MDPV-VP3 recombinant plasmid transfection into Sf9 insect cells caused cytopathic effects. Figure A is a normal Sf9 insect cell image, and Figure B is a pFastBac TM Cytopathic effect graph 72 hours after Sf9 insect cells were transfected with Dual / NGPV-VP2-MDPV-VP3 recombinant plasmid.

[0020] Figure 4 This is the Western blot detection result of NGPV VP2 protein of recombinant baculovirus rBAC / NGPV-VP2-MDPV-VP3.

[0021] Figure 5 This is the Western blot detection result of MDPV VP3 protein of recombinant baculovirus rBAC / NGPV-VP2-MDPV-VP3.

[0022] Figure 6 This is the IFA identification result of the NGPV VP2 protein of the recombinant baculovirus rBAC / NGPV-VP2-MDPV-VP3.

[0023] Figure 7 This is the IFA identification result of MDPV VP3 protein of recombinant baculovirus rBAC / NGPV-VP2-MDPV-VP3.

[0024] Figure 8 This is the negative control result of IFA test.

[0025] Fig. 9 These are the results of the determination of the production and persistence of NGPV and MDPV antibodies in the serum of egg-laying Muscovy ducks after immunization.

[0026] Fig.10 This is the result of yolk antibody titer determination. Specific implementation method:

[0027] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0029] It should be understood that the experimental methods used in the following examples are conventional methods unless otherwise specified, and the materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.

[0030] As used herein, the terms "comprises," "including," "having," "containing," or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a composition, process, method, or article that comprises the listed elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, process, method, or article.

[0031] Example 1: Construction and identification of recombinant baculovirus co-expression vector

[0032] The vector used for this co-expression is a commercial pFastBac-Dual vector, which contains two promoters, PH and p10. Each promoter has a multiple cloning site downstream, where a target gene can be inserted. The vector is propagated and preserved in the laboratory. The new goose parvovirus (NGPV) GSS02 strain used for this co-expression came from a farm in Sansui County, Guizhou Province, and the Muscovy duck parvovirus (MDPV) GZQ01 strain came from a farm in Zhaoqing City, Guangdong Province. It was propagated and identified by our laboratory, and the NGPV-VP2 and MDPV-VP3 sequences were obtained by sequencing.

[0033] 1.1 Primer design

[0034] 1.1.1 Design of primers for NGPV-VP2 gene amplification

[0035] According to the NGPV-VP2 gene sequence obtained after sequencing and codon optimization, a pair of primers for amplifying the full length of the NGPV-VP2 gene was designed, and EcoRI and SalI restriction sites were added to the 5' ends of the upstream primer and the downstream primer, respectively. The primers were synthesized by General Biotechnology (Anhui) Co., Ltd., and the primer sequences are shown in Table 1 below:

[0036] Table 1 NGPV-VP2 gene primer sequence list

[0037]

[0038] 1.1.2 Design of primers for MDPV-VP3 gene amplification

[0039] According to the MDPV-VP3 gene sequence obtained after sequencing and codon optimization, a pair of primers for amplifying the full length of the MDPV-VP3 gene was designed, and XhoI and KpnI restriction sites were added to the 5' ends of the upstream primer and the downstream primer, respectively. The primers were synthesized by General Biotechnology (Anhui) Co., Ltd., and the primer sequences are shown in Table 2 below:

[0040] Table 2 MDPV-VP3 gene primer sequence list

[0041]

[0042] 1.2 Construction of recombinant transfer plasmid

[0043] The NGPV-VP2 sequence was inserted downstream of the PH promoter of the pFastBac-Dual vector, and the MDPV-VP3 sequence was inserted downstream of the P10 promoter to obtain the recombinant transfer plasmid pFastBac / NGPV-VP2-MDPV-VP3. 1.2.1 NGPV-VP2 sequence amplification

[0044] The virus liquid of NGPV-GSS02 strain isolated in the laboratory was used as a template, and NGPV-VP2-F-ESa and NGPV-VP2-F-ESb were used as primers for PCR amplification. The PCR system was as follows: 2 μL of upstream primer NGPV-VP2-F-ESa (10 μM), 2 μL of downstream primer NGPV-VP2-F-ESb (10 μM), PCR Master Mix 25μL, ddH2O to 50μL. PCR reaction conditions are as follows: 94℃ pre-denaturation for 5min; 94℃ denaturation for 30s, 56℃ annealing for 30s, 72℃ extension for 90s, 35 cycles in total; 72℃ extension for 10min. 10μL of PCR product was identified by 0.8% agarose gel electrophoresis. The detection results were consistent with the expected results, with a size of about 1809bp.

[0045] 1.2.2 Double restriction digestion of NGPV-VP2 sequence and vector

[0046] After confirming that the NGPV-VP2 PCR amplification product is accurate, gel-cut and recovery are performed, and the gel-cut product and pFastBac-Dual vector are double-digested with EcoRI and SalI respectively. The digestion system is: NGPV-VP2 gel-cut DNA / pFastBac-Dual vector plasmid DNA 10μL, EcoRI 1μL, SalI 1μL, ddH2O 6μL, and digestion buffer 2μL. Digestion is carried out at 37℃ for 2h.

[0047] 1.2.3 NGPV-VP2 sequence and vector ligation

[0048] After the digestion is completed, the digestion product is recovered and purified before ligation. Ligation system: NGPV-VP2 PCR product 3μL, pFastBac-Dual vector plasmid DNA 1μL, T4 DNA ligase 1μL, 10×Ligase Buffer 2μL, dd H2O 3μL. After ligation at 2-8℃ for 12h, the ligation product is transformed into E. coli DH5α competent cells. Take 100μL of the transformation product and spread it on LB plate (containing 100μg / mL ampicillin) and invert and culture at 37℃ overnight.

[0049] 1.2.4 Double restriction enzyme digestion verification of recombinant pFastBac / NGPV-VP2 plasmid

[0050] Pick a single white colony and identify it with NGPV-VP2-F-ESa and NGPV-VP2-F-ESb primers to obtain positive bacteria, extract the recombinant transfer plasmid pFastBac / NGPV-VP2, sequence and identify it correctly, and store it for future use.

[0051] 1.2.5 MDP-VP3 sequence amplification

[0052] The virus liquid of MDPV-GZQ01 strain isolated in the laboratory was used as a template, and MDPV-VP3-XK1 and MDPV-VP3-XK2 were used as primers for PCR amplification. The PCR system was as follows: 2 μL of upstream primer MDPV-VP3-XK1 (10 μM), 2 μL of downstream primer MDPV-VP3-XK2 (10 μM), PCR Master Mix 25μL, ddH2O to 50μL. PCR reaction conditions are as follows: 94℃ pre-denaturation for 5min; 94℃ denaturation for 30s, 56℃ annealing for 30s, 72℃ extension for 90s, 35 cycles in total; 72℃ extension for 10min. 10μL of PCR product was identified by 0.8% agarose gel electrophoresis. The detection results were consistent with the expected results, with a size of about 1701bp.

[0053] 1.2.6 Double restriction digestion of MDPV-VP3 sequence and pFastBac / NGPV-VP2 plasmid

[0054] After confirming that the MDPV-VP3 PCR amplification product is accurate, gel-cut and recovered, and the gel-cut product and pFastBac / NGPV-VP2 plasmid are double-digested with two endonucleases, XhoI and KpnI, respectively. The digestion system is: MDPV-VP3 gel-cut DNA / pFastBac / NGPV-VP2 recombinant plasmid DNA 10μL, XhoI 1μL, KpnI 1μL, ddH2O 6μL, and digestion buffer 2μL. Digestion was carried out at 37℃ for 2h.

[0055] 1.2.7 Ligation of MDPV-VP3 sequence and pFastBac / NGPV-VP2 recombinant plasmid

[0056] After the digestion is completed, the digestion product is recovered and purified before ligation. Ligation system: 3μL of MDPV-VP3 PCR product, 1μL of pFastBac / NGPV-VP2 recombinant plasmid DNA, 1μL of T4 DNA ligase, 2μL of 10×Ligase Buffer, 3μL of dd H2O. After overnight ligation at 16℃, the ligation product is transformed into E. coli DH5α competent cells. Take 100μL of the transformation product and spread it on LB plate (containing 100μg / mL ampicillin) and invert and culture at 37℃ overnight.

[0057] 1.2.8 Identification of pFastBac / NGPV-VP2-MDPV-VP3 recombinant plasmid

[0058] Single white colonies were picked and identified by PCR using NGPV-VP2 and MDPV-VP3 gene amplification primers to obtain positive bacteria. The recombinant transfer plasmid pFastBac / NGPV-VP2-MDPV-VP3 was extracted and identified by double digestion with ECoRI and SalI and double digestion with XhoI and KpnI, respectively. The identification results were as follows: Figure 1 shown.

[0059] 1.2.9 Obtaining the recombinant shuttle plasmid Bacmid / NGPV-VP2-MDPV-VP3

[0060] The identified pFastBac / NGPV-VP2-MDPV-VP3 plasmid was transformed into E.coli DH 10Bac competent cells containing the shuttle vector Bacmid. 100 μL of the transformation product was applied to an LB plate containing three antibiotics (kanamycin 100 μg / mL, tetracycline 10 μg / mL, gentamicin 7 μg / mL) and 100 μg / mL X-gal and 20 μg / mL IPTG, and inverted at 37°C for 24 to 48 hours. White colonies were selected and inoculated into LB liquid culture medium containing the above three antibiotics, and cultured at 37°C and 180 rpm for 24 hours. The recombinant baculovirus shuttle plasmid Bacmid / NGPV-VP2-MDPV-VP3 was extracted using a plasmid extraction kit. The recombinant baculovirus shuttle plasmid Bacmid / NGPV-VP2-MDPV-VP3 was identified by PCR using M13 universal primer, NGPV-VP2 detection primer, and MDPV-VP3 detection primer. The identification results were as follows: Figure 2 shown.

[0061] 1.2.10 Bacmid / NGPV-VP2-MDPV-VP3 transfection into Sf9 insect cells

[0062] Using Lipofectamin TM 3000 was used as a transfection reagent to transfect Bacmid / NGPV-VP2-MDPV-VP3 into Sf9 monolayer cells. After transfection, the cells were cultured at 27°C and the cytopathic effects were observed. When 80% to 90% of the cells showed obvious lesions (cells and nuclei became enlarged, stopped growing, fell off, lysed, etc.), the results were as follows: Figure 3 As shown, the cells and supernatant were collected, which were the P1 generation recombinant virus. The P1 generation virus was passaged three times on the Sf9 monolayer cells and plaque purified to obtain a high-titer recombinant baculovirus containing the NGPV-VP2 and MDPV-VP3 genes. This recombinant baculovirus strain was named rBAC / NGPV-VP2-MDPV-VP3 strain.

[0063] 2 Expression and identification of recombinant protein

[0064] 2.1 Western blot detection

[0065] The harvested recombinant protein was subjected to SDS-PAGE electrophoresis to identify the expression of the recombinant protein. After SDS-PAGE separation, the protein was transferred to the NC membrane by wet transfer, blocked with 5% skim milk at room temperature for 2 hours, and then washed with TBST at low speed for 10 minutes, repeated 3 times, and mouse anti-6*His tag antibody (1:1000 dilution) and rabbit anti-Flag tag antibody (1:1000 dilution) were used as primary antibodies, respectively, and incubated at 4°C overnight. After the incubation, it was washed again with TBST at low speed for 10 minutes, repeated 3 times. HRP-labeled goat anti-mouse IgG and goat anti-rabbit (1:1000 dilution) were used as secondary antibodies, incubated at room temperature for 1 hour, and washed with TBST at low speed for 10 minutes, repeated 3 times. After color development with the DAB color development kit, the results were observed.

[0066] The results showed that the product harvested from Sf9 insect cells inoculated with rBAC / NGPV-VP2-MDPV-VP3 recombinant baculovirus produced the expected target band, in which the NGPV VP2 protein was approximately 67KDa ( Figure 4 ), the size of MDPV VP3 protein is about 64KDa ( Figure 5 ).

[0067] 2.2 Amplification of recombinant baculovirus and IFA identification

[0068] Infect Sf9 cells with the P3 virus solution harvested in 1.2.10 and culture them statically for 48 to 72 hours. When the cells show obvious pathological changes, discard the supernatant and fix the cells with pre-cooled acetone: methanol (1:1) for 30 minutes. Block them with 5% skim milk at 4°C overnight. Add rabbit anti-FLag tag antibody and mouse anti-6*His tag antibody and incubate at room temperature for 2 hours. Use Alexa Fluor 555-labeled donkey anti-rabbit IgG antibody and FITC-labeled goat anti-mouse IgG antibody to incubate at room temperature for 30 to 40 minutes, wash three times with PBST each time, and observe the results under a fluorescence microscope. The identification results are as follows: Figure 6 , Figure 7 and Figure 8 shown.

[0069] 3. Immune protection of co-expressed proteins

[0070] 3.1 Preparation of recombinant baculovirus inactivated vaccine

[0071] Culture Sf9 suspension cells until the cell density is about 2×10 6 When the cells were 100 μg / mL, the recombinant rBAC / NGPV-VP2-MDPV-VP3 baculovirus was inoculated at a 1% inoculation ratio, cultured at 27°C and observed continuously, and the cell culture was harvested when the cells showed obvious lesions. The cell culture expressed the recombinant NGPV-VP2-MDPV-VP3 protein antigen, and the AGP titers of the NGPV VP2 and MDPV VP3 proteins were determined respectively. The NGPV VP2 protein with an AGP titer higher than 1:8 and the MDPV VP3 protein with an AGP titer higher than 1:4 were selected, inactivated with formaldehyde solution, and emulsified and prepared in a ratio of 1 part of aqueous phase to 2 parts of oil phase to prepare the recombinant NGPV-VP2-MDPV-VP3 baculovirus inactivated vaccine.

[0072] 3.2 Immunogenicity analysis

[0073] 3.2.1 Determination of antibody production and duration in egg-laying Muscovy ducks

[0074] Forty 173-day-old laying ducks were randomly divided into two groups, 20 in each group. The ducks in the immunized group were injected subcutaneously with 0.5 mL of the inactivated baculovirus vaccine of rBAC / NGPV-VP2-MDPV-VP3 through the nape of the neck; the ducks in the control group were injected subcutaneously with 0.5 mL of saline through the nape of the neck; the two groups were kept in isolation. Blood was collected aseptically at 21 days, 2 months, 3 months, and 4 months after immunization, and the serum was separated and inactivated at 56℃ for 30 minutes to determine the titer of MDPV and NGPV neutralizing antibodies in the serum.

[0075] The virus was diluted to contain 200 ELD per unit dose. 50, mixed with an equal amount of the tested serum diluted twice in series, and placed at 37°C for 1 hour. Three to six 9-11-day-old Muscovy duck embryos were inoculated at each dilution. The duck embryos were placed at 37°C for 144 hours, and the death of each group of duck embryos was recorded. The neutralization titer of the serum was calculated by the Reed-Muench method. The results are as follows: Fig. 9 shown.

[0076] Table 3 Antibody titer determination of egg-laying Muscovy ducks

[0077]

[0078] 3.2.2 Yolk antibody titer determination

[0079] 21 days, 2 months, 3 months, and 4 months after immunization, eggs of laying Muscovy ducks were collected from the vaccine-immunized group and the non-immunized control group. Ten eggs were randomly selected from each group and each time period, and yolk antibodies were extracted for neutralizing antibody titer determination. The results are as follows Fig.10 The neutralizing antibody determination method is the same as 3.2.1.

[0080] Table 4 Yolk antibody titer determination

[0081]

[0082] 3.2.3 Immune challenge test

[0083] Eggs of laying Muscovy ducks were collected and artificially incubated 21 days, 2 months, 3 months, and 4 months after immunization. Twenty male and female Muscovy ducklings were randomly selected from each immunization group and non-immunization group and inoculated intramuscularly with NGPV-GSS02 strain at 3 days of age. The challenge dose was 0.5 mL / bird, 200 ELD 50 / feather, 20 male and female Muscovy ducklings were randomly selected from each group of the immunized group and the non-immunized group and inoculated with MDPV-GZQ01 strain, with a challenge dose of 0.5mL / feather, 100ELD 50 / feather, and the empty control group was injected with the same amount of physiological saline. Each group was raised in isolation.

[0084] After the NGPV-GSS02 strain was challenged, the clinical symptoms of the test ducks (dyskinesia, decreased appetite, mental depression, beak atrophy, tongue eversion, white loose stools, tibia fracture, shortened beak, and growth and development stunting) were recorded every day and observed for 21 days. Death or clinical symptoms were considered to be the cause of the disease in the test ducks, and the standard for the establishment of the experiment was that the incidence rate of the test ducks in the non-immune challenge group was not less than 80%.

[0085] After infection with the MDPV-GZQ01 strain, the death and clinical symptoms (developmental delay, weak feet, panting, diarrhea) of the test ducks were observed and recorded every day for 21 consecutive days. The occurrence of death or clinical symptoms could be considered as the disease in the test ducks. The standard for establishing the experiment was that the disease incidence rate of the test ducks in the non-immune infection group was not less than 80%.

[0086] Table 5 Results of immune protection test of recombinant rBAC / NGPV-VP2-MDPV-VP3 inactivated baculovirus vaccine

[0087]

[0088] The foregoing examples are merely illustrative and are used to explain some features of the method of the present invention. The appended claims are intended to claim the widest possible range that can be conceived, and the embodiments presented herein are only illustrations of selected implementations according to the combination of all possible embodiments. Therefore, it is the applicant's intention that the appended claims are not limited by the selection of examples that illustrate the features of the present invention. Some numerical ranges used in the claims also include sub-ranges within them, and changes in these ranges should also be interpreted as being covered by the appended claims where possible.

Claims

1. An immunogenic composition, characterized in that The immunogenic composition contains VP2 protein encoded by NGPV VP2 gene and VP3 protein encoded by MDPV VP3 gene.

2. The immunogenic composition according to claim 1, characterized in that The NGPV VP2 and MDPV VP3 genes are codon-optimized according to the preference of the insect cell protein expression system. The optimized nucleotide sequence of the NGPV VP2 gene is shown in SEQ ID NO: 1, and the optimized nucleotide sequence of the MDPV VP3 gene is shown in SEQ ID NO:

2.

3. A novel goose parvovirus NGPV and Muscovy duck parvovirus MDPV co-expression vector, characterized in that: The expression vector is a recombinant baculovirus vector pFastBac TM Dual, the MDPV VP3 gene is inserted downstream of the P10 promoter of the vector, and the NGPV VP2 gene is inserted downstream of the PH promoter of the vector.

4. A host cell containing the recombinant baculovirus vector according to claim 3, characterized in that: The host cell is selected from Sf9 cells of the insect cell line.

5. A recombinant baculovirus, characterized in that The recombinant baculovirus contains codon-optimized NGPV VP2 gene and MDPV VP3 gene.

6. The recombinant baculovirus according to claim 5, characterized in that The recombinant baculovirus was deposited in the China Center for Type Culture Collection on November 28, 2024, with the deposit address: Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: V2024116.

7. A subunit vaccine capable of preventing and treating both NGPV and MDPV, characterized in that: The vaccine comprises an adjuvant and the immunogenic composition of claim 1.

8. The subunit vaccine according to claim 7, characterized in that The vaccine is used in preventing and treating novel goose parvovirus and Muscovy duck parvovirus.

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