Egg drop syndrome virus subunit antigen, its preparation method and application

Abstract

The application belongs to the field of preventive veterinary medicine, and discloses egg drop syndrome virus subunit antigens, and a preparation method and application thereof. The egg drop syndrome virus subunit antigens include egg drop syndrome virus Fiber protein, egg drop syndrome virus Knob-S protein and Acheta domesticus nuclear type polyhedron virus IE0 protein. The egg drop syndrome virus subunit antigen protein prepared by the method provided in the application has a HA of greater than or equal to 22 log2, and the protein is emulsified to prepare a vaccine for immunizing 3-4-week-old SPF chickens. 21 days after emulsification immunization, the serum antibody can reach greater than or equal to 10 log2, which meets the requirements of the regulations and is superior to the effect of an embryo toxin prepared vaccine, and can be used for preparing an egg drop syndrome multi-link subunit vaccine.

Description

Technical Field

[0001] This invention belongs to the field of preventive veterinary medicine, specifically relating to the subunit antigen of egg drop syndrome virus and its preparation method and application. Background Technology

[0002] Egg Drop Syndrome (EDS) is a viral infectious disease caused by Egg Drop Syndrome Virus (EDSV), characterized by thin-shelled or shell-less eggs and a sharp decline in egg production in laying hens. EDSV belongs to avian adenovirus group III, and its viral spike protein (Knob-S or Fiber protein) on the surface of the viral particles is the main immunogenic protein, which can induce the body to produce neutralizing antibodies. Therefore, it can be used as a key protein in the development of subunit vaccines.

[0003] Currently, the vaccines used to prevent and control egg drop syndrome are mainly inactivated vaccines and attenuated vaccines. However, traditional chicken or duck embryo-derived inactivated vaccines have difficulties such as high production costs, long production cycles, potential biosafety risks (such as exogenous virus contamination), and the inability to distinguish between naturally infected and vaccine-immunized animals (DIVA).

[0004] Baculovirus expression systems are eukaryotic expression platforms that utilize insect cells for efficient expression of recombinant proteins. They offer advantages such as strong post-translational modification capabilities, high expression levels, and good biocompatibility, and have been widely used in the production of veterinary and human recombinant protein vaccines. Utilizing baculovirus systems to express the immunogenic protein of EDSV provides a new technological pathway for developing safe and efficient egg drop syndrome subunit vaccines. Summary of the Invention

[0005] The purpose of this invention is to provide a subunit antigen of egg drop syndrome virus, its preparation method, and its application.

[0006] To achieve the objectives of this invention, in a first aspect, this invention provides subunit antigens of egg drop syndrome virus, including egg drop syndrome virus Fiber protein, egg drop syndrome virus Knob-S protein, and silver mosquito nucleopolyhedrovirus IE0 protein; Among them, the Fiber protein is: (a1) A protein consisting of the amino acid sequence shown in SEQ ID NO:1; (b1) A protein derived from (a1) with one or more amino acids substituted, deleted or added to the sequence shown in SEQ ID NO:1 and having the same function; Knob-S protein is: (a2) A protein consisting of the amino acid sequence shown in SEQ ID NO:3; (b2) Proteins derived from (a2) with one or more amino acids substituted, deleted, or added to the sequence shown in SEQ ID NO:3 and having the same function; The IE0 protein is: (a3) A protein consisting of the amino acid sequence shown in SEQ ID NO:5; (b3) Proteins derived from (a3) ​​with the sequence shown in SEQ ID NO:5 substituted, deleted or added with one or more amino acids and having the same function.

[0007] In a second aspect, the present invention provides a nucleic acid molecule encoding the subunit antigen or biological material containing the nucleic acid molecule.

[0008] The biological materials include, but are not limited to, expression cassettes, transposons, plasmid vectors, viral vectors, engineered bacteria, or transgenic cell lines.

[0009] Thirdly, the present invention provides a recombinant baculovirus expression vector comprising an expression cassette for co-expressing the three proteins (Fiber protein, Knob-S protein, and IEO protein).

[0010] Preferably, the nucleotide sequence of the gene encoding the Fiber protein is shown in SEQ ID NO:2, the nucleotide sequence of the gene encoding the Knob-S protein is shown in SEQ ID NO:4, and the nucleotide sequence of the gene encoding the IEO protein is shown in SEQ ID NO:6.

[0011] Fourthly, the present invention provides a recombinant baculovirus whose genome contains an expression cassette of the three proteins (Fiber protein, Knob-S protein and IEO protein).

[0012] Fifthly, this invention provides the silver mosquito noctuid nucleopolyhedrovirus rBac-IE-Fiber-KnobS, classified as Autographa californica multiple nuclecopolyhedrovirus, which is now deposited at the China Center for Type Culture Collection, address: Wuhan University, Wuhan, China, 430072, accession number CCTCC NO: V202633, deposit date: April 22, 2026.

[0013] In a sixth aspect, the present invention provides a transgenic insect cell containing the recombinant baculovirus expression vector, or the cell being infected with the recombinant baculovirus or the silver mosquito nucleopolyhedrovirus (accession number CCTCC NO: V202633).

[0014] In a seventh aspect, the present invention provides a method for preparing the subunit antigen of the egg drop syndrome virus, wherein the transgenic insect cells are cultured under suspension culture conditions to efficiently proliferate the recombinant baculovirus to express the Fiber protein, Knob-S protein and IEO protein, or the nucleopolyhedrovirus of the silver mosquito moth is used to inoculate healthy insect cells at a certain MOI value; and the virus culture medium containing the proteins is harvested.

[0015] Furthermore, when inoculated with recombinant baculovirus, the growth density of the insect cells was 5.0 × 10⁻⁶. 6 -6.0×10 6 Cells / ml were diluted to 2.8 × 10⁶ cells / ml by adding culture medium. 6 -3.0×10 6 Cells / ml, with an inoculation rate of 0.07-0.2.

[0016] Furthermore, the cultivation conditions were as follows: temperature 27±0.5℃, dissolved oxygen 55-65%, stirring speed 20-30 rpm, and pH 6.0-6.4 during the cultivation stage in a 3000L scale reactor.

[0017] Preferably, when the insect cell viability drops below 70% and the density drops to 2×10⁻⁶, 6 When the number of cells / ml drops below a certain threshold, end the culture and harvest the virus culture medium.

[0018] Preferably, the insect cells are SF9 or High5.

[0019] Eighthly, the present invention provides a vaccine for the prevention of egg drop syndrome, comprising a virus culture medium prepared according to the method after inactivation treatment (e.g., inactivation in formaldehyde solution at 37°C for 16 hours) and a vaccine adjuvant.

[0020] In a ninth aspect, the present invention provides the use of the subunit antigen, the nucleic acid molecule encoding the subunit antigen or the biological material containing the nucleic acid molecule, the recombinant baculovirus expression vector, the recombinant baculovirus or the silver mosquito nucleopolyhedrovirus (accession number CCTCC NO: V202633) in the preparation of a vaccine for the prevention of egg drop syndrome.

[0021] In a tenth aspect, the present invention provides a quadrivalent inactivated vaccine for the prevention of chicken diseases, the main components of which are inactivated Newcastle disease virus La Sota strain, H9 subtype avian influenza virus SZ strain, avian infectious bronchitis virus M41 strain, and silver mosquito nucleopolyhedrovirus rBac-IE-Fiber-KnobS culture medium and vaccine adjuvant.

[0022] The preparation method of the quadrivalent inactivated vaccine includes: 1) breeding Newcastle disease virus La Sota strain, H9 subtype avian influenza virus SZ strain, and avian infectious bronchitis virus M41 strain separately using chicken or duck embryos according to conventional methods, and preparing inactivated virus liquid after harvesting the virus liquid through an inactivation process; 2) preparing silver mosquito nucleopolyhedrovirus (CCTCC NO: V202633) culture medium using the aforementioned method for preparing egg drop syndrome virus subunit antigen, and preparing inactivated virus liquid after harvesting the virus liquid through an inactivation process; 3) mixing the four inactivated antigens prepared above and preparing an inactivated adjuvant vaccine according to conventional methods.

[0023] Furthermore, the HA titer of Newcastle disease virus La Sota strain culture medium should be no less than 9 log2, the HA titer of H9 subtype avian influenza virus SZ strain culture medium should be no less than 8 log2, the HA titer of recombinant baculovirus rBac-IE-Fiber-KnobS strain culture medium should be no less than 23 log2, and the HA titer of avian infectious bronchitis virus M41 strain culture medium should be no less than 10 log2. 6.0 EID 50 / 0.1ml; each 0.5ml of vaccine should contain at least 0.025ml of Newcastle disease virus La Sota strain, 0.025ml of H9 subtype avian influenza virus SZ strain, 0.03ml of recombinant baculovirus rBac-IE-Fiber-KnobS strain and 0.03ml of avian infectious bronchitis M41 strain, which are concentrated at 5 times their original concentration.

[0024] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects: (i) The recombinant baculovirus rBac-IE-Fiber-KnobS provided by this invention can rapidly multiply on SF9 and High5 cells and obtain high protein titers in a short time, and can be used to prepare multi-subunit vaccines for avian egg drop syndrome.

[0025] (ii) The subunit antigen protein HA of the egg drop syndrome virus prepared by the method provided in this invention is ≥22log2. The protein is emulsified to prepare a vaccine for immunizing 3-4 week old SPF chickens. 21 days after emulsification immunization, the serum antibody can reach ≥10log2, which meets the requirements of the procedure and is superior to the effect of the vaccine prepared by embryo virus.

[0026] (III) The quadrivalent inactivated vaccine for Newcastle disease, infectious bronchitis, egg drop syndrome and avian influenza (H9 subtype) provided by this invention is of reliable quality and meets the requirements. This quadrivalent vaccine can be used to prevent diseases caused by Newcastle disease, infectious bronchitis, egg drop syndrome and avian influenza (H9 subtype) viruses, achieving the effect of preventing four diseases with one injection, reducing immunization costs and immune stress. Attached Figure Description

[0027] Figure 1 The cell morphology observation results in the preferred embodiment 2 of the present invention are shown in the figures: a) Microscopic morphology of SF9 healthy cells cultured without serum for 3 hours (100×); b) Microscopic morphology of SF9 healthy cells cultured without serum for 48 hours (100×); c) Microscopic morphology of SF9 cells cultured for 48 hours after inoculation with recombinant baculovirus rBac-IE-Fiber-KnobS (100×).

[0028] Figure 2 The image shows the HA detection and identification of the recombinant baculovirus strain rBac-IE-Fiber-KnobS in the preferred embodiment 3 of the present invention, indicating that the HA of the sample is 13 log2.

[0029] Figure 3 The agar amplification test result of the recombinant baculovirus strain rBac-IE-Fiber-KnobS in the preferred embodiment 3 of the present invention shows an agar amplification titer of 1:8.

[0030] Figure 4 The results of SF9 cell morphology observation in the preferred embodiment 4 of the present invention are shown in Figure d, which is the microscopic morphology of fully suspended healthy SF9 cells cultured for 72 hours; and Figure e, which is the microscopic morphology of fully suspended healthy SF9 cells cultured for 72 hours after inoculation with recombinant baculovirus rBac-IE-Fiber-KnobS.

[0031] Figure 5 This is a schematic diagram of the production process of recombinant baculovirus rBac-IE-Fiber-KnobS in High5 suspension cell culture in a 3000 L-scale bioreactor, which is a preferred embodiment of the present invention.

[0032] Figure 6 The results of morphological observation of High5 cells in a 3000 L bioreactor in the preferred embodiment 4 of the present invention are shown in Figure f, which shows the microscopic morphology of healthy High5 cells in a 3000 L bioreactor after 48 h of culture; and Figure g, which shows the microscopic morphology of healthy High5 cells in a 3000 L bioreactor after 72 h of culture following inoculation with recombinant baculovirus rBac-IE-Fiber-KnobS.

[0033] Figure 7 This is a graph showing the HA titer of recombinant baculovirus rBac-IE-Fiber-KnobS in a 3000 L-scale bioreactor in a preferred embodiment of the present invention after harvesting from High5 suspension cell culture.

[0034] Figure 8This is a graph showing the agar amplification titer of recombinant baculovirus rBac-IE-Fiber-KnobS in a 3000 L-scale bioreactor in a preferred embodiment 4 of the present invention after harvesting from High5 suspension cell culture.

[0035] Figure 9 This is a graph showing the healthy cell proliferation curves in three 3000L scale bioreactors in the preferred embodiment 4 of the present invention.

[0036] Figure 10 This is a graph showing the cell number curves after three inoculations of rBac-IE-Fiber-KnobS into a 3000L-scale bioreactor in the preferred embodiment 4 of the present invention.

[0037] Figure 11 In the preferred embodiment 4 of the present invention, the SDS-PAGE image of the rBac-IE-Fiber-KnobS protein solution prepared in a 3000L bioreactor shows that the target protein is expressed at approximately 60KD. Lane 1 is the protein sample suspension of the 3000L scale, lane 2 is the marker, lane 3 is the supernatant of the 3000L scale protein sample centrifugation, lane 4 is the protein sample suspension of the shake flask control experiment, and lane 5 is the supernatant of the shake flask control protein sample centrifugation.

[0038] Figure 12 The image shown is an SDS-PAGE image of the KnobS protein, a subunit of poultry egg drop syndrome, expressed in genetically engineered Escherichia coli in Comparative Example 1 of this invention. It shows that the target protein is expressed at approximately 26 KD. Lane 1 is the marker, lanes 2 and 3 are the supernatant and precipitate after treatment with 0.1 M urea, lanes 4 and 5 are the supernatant and precipitate after treatment with 0.25 M urea, and lanes 6 and 7 are the supernatant and precipitate after treatment with 0.5 M urea. Detailed Implementation

[0039] The present invention aims to provide a protective antigen of avian egg drop syndrome virus with better immunogenicity, its encoding gene and its application.

[0040] The present invention adopts the following technical solution: This invention provides a DNA molecule comprising, in sequence, the IEO gene of the silver mosquito nucleopolyhedrovirus, the Fiber gene of avian egg drop syndrome, and the Knob-S sequence of the Fiber gene of avian egg drop syndrome; The sequence of the Fiber gene for the poultry egg drop syndrome is shown in SEQ ID NO:2; The Knob-S sequence in the Fiber gene of the poultry egg drop syndrome is shown in SEQ ID NO:4; The sequence of the IE0 gene of the silver mosquito noctuid virus is shown in SEQ ID NO:6; This invention optimizes the Fiber gene and Knob-S sequence of avian egg drop syndrome by selecting the optimal sequence and then linking them in a specific order with the IEO gene of the silver mosquito nucleopolyhedrovirus to enhance expression efficiency. This allows for the efficient expression of the target protein using the Bac-to-Bac baculovirus expression system.

[0041] In this invention, it is preferable to transfect cells with recombinant baculovirus containing the above-mentioned DNA molecules, culture them, passage them three times, centrifuge them to collect the supernatant, and obtain a mixture of recombinant baculovirus expressed proteins.

[0042] The present invention also provides a method for preparing a recombinant vector, which uses pFastBac Dual as a vector to insert the Fiber gene of avian egg drop syndrome between the restriction endonuclease sites of Pme I and Hind III of the vector, thereby obtaining a recombinant plasmid vector containing the Fiber gene of avian egg drop syndrome, named pDual-Fiber. The IE0 gene of the silver tussock moth nucleopolyhedrovirus and pDual-Fiber were double-digested with AclⅠ and NcoⅠ and then ligated to obtain a recombinant plasmid vector that simultaneously inserts the Fiber gene of avian egg drop syndrome and the IE0 gene of the silver tussock moth nucleopolyhedrovirus, named pDual-IE-Fiber. The KnobS gene of avian egg drop syndrome was inserted between the Bam HI and XhoI restriction sites of pFastBac Dual to obtain a recombinant plasmid vector containing the KnobS gene of avian egg drop syndrome, named pDual-KnobS. The pDual-IE-Fiber recombinant plasmid vector and the pDual-KnobS recombinant plasmid vector were double-digested with NruI and AvrII, respectively, and then ligated with mixed enzymes to obtain a recombinant plasmid vector containing the IEO gene of the silver mosquito nucleopolyhedrovirus, the Fiber gene of egg drop syndrome, and the KnobS gene of egg drop syndrome, named pDual-IE-Fiber-KnobS; The sequences of the silver mosquito noctuid nucleopolyhedrovirus IE0 gene, the avian egg drop syndrome Fiber gene, and the avian egg drop syndrome KnobS gene are as described above; Preferably, the primer sequences used to amplify the IEO gene of the silver mosquito nucleopolyhedrovirus, the Fiber gene of avian egg drop syndrome, and the KnobS gene of avian egg drop syndrome are shown in SEQ ID NO:7-12.

[0043] In the recombinant vector of this invention, the promoter sequentially initiates the transcription of the IEO gene of the silver mosquito nucleopolyhedrovirus, the Fiber gene of egg drop syndrome, and the KnobS gene of egg drop syndrome. pFastBac Dual is preferably used as the vector.

[0044] The present invention also provides a method for preparing recombinant baculovirus, comprising: 1) The above DNA molecule was cloned into Escherichia coli DH10 Bac competent cells, and after screening and identification, the correct recombinant baculovirus Bacmid was obtained; Preferably, the recombinant vector prepared by the above method is transformed into Escherichia coli DH10 Bac competent cells to clone the above DNA molecule into Escherichia coli DH10 Bac competent cells; 2) Transfect SF9 cells with the recombinant baculovirus Bacmid obtained in step 1), and harvest the recombinant baculovirus after culturing.

[0045] The recombinant baculovirus of the present invention can simultaneously and efficiently express the Fiber protein of avian egg drop syndrome, the KnobS protein of avian egg drop syndrome, and the IEO protein of silver mosquito nucleopolyhedrovirus.

[0046] The present invention also provides a recombinant baculovirus, characterized in that it is prepared by the above method; or, the recombinant baculovirus has the accession number CCTCC NO: V202633.

[0047] The present invention also provides a method for preparing the viral culture medium of the above-mentioned recombinant baculovirus, comprising: (1) Culture of fully suspended cells; preferably cultured in a serum-free fully suspended medium, wherein the fully suspended cells are insect cells, more preferably SF9 cells; (2) Inoculate with the above-mentioned recombinant baculovirus and perform suspension culture; (3) Harvest the virus culture medium; Preferably, in step (2), when inoculating with the above-mentioned recombinant baculovirus, the growth density of the whole suspension cells is 5.0 × 10⁻⁶. 6 -6.0×10 6 Cells / ml, with culture medium added during virus inoculation to dilute the total suspension cell count to 2.8 × 10⁶ cells / ml. 6 -3.0×10 6 cells / ml; and / or, the virus inoculation amount is MOI=0.07-0.2, and the culture conditions are: temperature 27±0.5℃, dissolved oxygen 55-65% (preferably 55-60%), stirring speed 25 rpm, pH 6.0-6.4 (preferably pH 6.0-6.2); Preferably, in step (3), when the viability of the fully suspended cells drops below 70% and the density drops to 2×10⁻⁶, 6 When the number of cells / ml drops below a certain threshold, end the culture, break up the suspended cells, and collect the virus culture medium.

[0048] This invention provides the application of fully suspended cells (such as fully suspended SF9 cells) in the fully suspended culture of baculoviruses. The baculovirus can be AcMNPV (Actovirociraptor nucleopolyhedrovirus), preferably the recombinant baculovirus described above in this invention.

[0049] The method of this invention can be used to prepare Fiber protein and KnobS protein of poultry egg drop syndrome.

[0050] The fully suspended cells of this invention can be SF9 / High5 cell clones.

[0051] The baculovirus strain rBac-IE-Fiber-KnobS prepared using the method described above can rapidly multiply in SF9 and High5 cells, achieving high protein titers in a short time, and can be used to prepare a multi-subunit vaccine for avian egg drop syndrome.

[0052] In the method, the serum-free culture medium used can be any serum-free culture medium suitable for the culture of fully suspended cells (such as fully suspended SF9 cells).

[0053] Prior to inoculation, fully suspended cells are preferably resuscitated and cultured in a stepwise scale-up manner.

[0054] The above-mentioned cultivation can be carried out in 50L, 1000L and / or 3000L bioreactors.

[0055] The method of this invention is applicable to small-scale and 3000L-scale production of baculovirus strain rBac-IE-Fiber-KnobS, providing a technical basis for further efficient production of multi-genetic subunit vaccines for avian egg drop syndrome.

[0056] The present invention also provides a virus culture medium, which is prepared by the above method.

[0057] The present invention also provides the above-mentioned DNA molecules or biological materials, or methods for preparing recombinant vectors, or methods for preparing recombinant baculoviruses, or methods for preparing viral culture media of the above-mentioned recombinant baculoviruses, or the application of the above-mentioned viral culture media in the preparation of vaccines (preferably vaccines for the prevention of avian egg drop syndrome virus).

[0058] The virus culture medium of the present invention can be inactivated (e.g., inactivated in formaldehyde solution at 37°C for 16 hours) and then emulsified to prepare a multivalent vaccine for avian egg drop syndrome.

[0059] This vaccine has been shown to have high immunogenicity and safety through animal testing.

[0060] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0061] The Newcastle disease virus La Sota strain, H9 subtype avian influenza virus SZ strain, and avian infectious bronchitis virus M41 strain used in the following examples were all purchased from the China Institute of Veterinary Drug Control.

[0062] Example 1: Construction of the recombinant vector rBac-IE-Fiber-KnobS 1. In this embodiment, based on the EDS Fiber protein sequence in the NCBI database, the codons were optimized for insect cell preference (SEQ ID NO:1, after codon adaptation optimization and synthesis by a biotechnology company, which can be amplified using primers Fiber-F and Fiber-R), and cloned between the Pme I and Hind III restriction endonuclease sites in the pFastBac Dual vector to obtain a recombinant plasmid vector inserted into the Fiber gene of avian egg drop syndrome, named pDual-Fiber.

[0063] 2. The IE0 gene of the silver mosquito cutworm nucleopolyhedrovirus (SEQ ID NO:3, obtained by amplification with primers IE0-F and IE0-R using a commercially purchased DH10 BAC strain as a template (which contains the ACMNPV genome)) and the plasmid pDual-Fiber were double-digested with AclⅠ and NcoⅠ and then ligated to obtain a recombinant plasmid vector containing both the Fiber gene of avian egg drop syndrome and the IE0 gene of the silver mosquito cutworm nucleopolyhedrovirus, named pDual-IE-Fiber.

[0064] 3. The KnobS gene for egg drop syndrome was codon-adapted (SEQ ID NO:2, which was codon-adapted and synthesized by a biotechnology company, and can be amplified using primers KnobS-F and KnobS-R), and cloned into the pFastBacDual plasmid between the Bam HI and XhoI restriction sites to obtain the plasmid vector containing the KnobS gene for egg drop syndrome, named pDual-KnobS.

[0065] 4. The pDual-IE-Fiber plasmid obtained in step 2 and the pDual-KnobS plasmid obtained in step 3 were double-digested with NruI and AvrII, respectively, and then mixed and ligated to obtain a plasmid vector containing the KnobS gene of avian egg drop syndrome, the IE0 gene of silver mosquito nucleopolyhedrovirus, and the KnobS gene of avian egg drop syndrome, named pDual-IE-Fiber2-KnobS.

[0066] The pFastBac Dual vector (purchased from Beijing Huayueyang Biotechnology Co., Ltd.) is preserved in our laboratory. Primer information for amplifying the optimized Fiber gene and KnobS gene of avian egg drop syndrome, as well as the IE0 gene of silver leafminer nucleopolyhedrovirus, is shown in Table 1.

[0067] Table 1. Amplification primers for each fragment (SEQ ID NO:7-12)

[0068] Example 2: Preparation of recombinant baculovirus pDual-IE-Fiber-KnobS In this embodiment, the recombinant plasmid rBac-IE-Fiber-KnobS obtained in Example 1 was transformed into DH10 Bac competent cells. After recovering resistance for 2 hours in LB liquid medium without antibiotics, the cells were plated on LB solid plates containing tetracycline, gentamicin, kanamycin, IPTG, and X-Gal. The cells were incubated upside down at 37°C for 24-48 hours, and blue-white screening was performed. Single white colonies were picked for PCR testing. Positive clones were inoculated into LB liquid medium containing tetracycline, gentamicin, and kanamycin and cultured overnight with shaking. The recombinant baculovirus Bacmid was then extracted and named rBac-IE-Fiber-KnobS.

[0069] SF9 cells in logarithmic growth phase were divided into groups of 5.0 × 10⁻⁶. 5 -8.0×10 5 After dilution to cells / ml, the cells were evenly distributed in 6-well plates. SF9 cells were transfected using Lipofectamine 3000 transfection reagent and recombinant baculovirus Bacmid rBac-IE-Fiber-KnobS. The cells were cultured at 27°C for 5-7 days until the cell viability was below 40%. The cell supernatant was collected and centrifuged at 5000 rpm for 3 min. The supernatant was then collected, which was the P0 generation strain of recombinant baculovirus rBac-IE-Fiber-KnobS (preservation number CCTCC NO:V202633).

[0070] The P0 generation of recombinant baculovirus rBac-IE-Fiber-KnobS was inoculated into SF9 cells in logarithmic growth phase at a ratio of 1:50 and cultured at 27°C for 5-7 days until cell viability was below 40%. The cell supernatant was then collected, yielding the P1 generation of recombinant baculovirus rBac-IE-Fiber-KnobS. This was passaged using the same method to obtain the P3 generation, which was then subjected to TCID50 assay. 50 The detection showed that the titer of rBac-IE-Fiber-KnobS P3 generation virus was 10. 7.5 TCID 50 / 0.1ml.

[0071] Microscopic morphology (100×) of healthy SF9 cells cultured in serum-free adherence for 3 hours. Figure 1 a. Microscopic morphology of healthy SF9 cells after 48 hours of serum-free adherent culture (100×). Figure 1 b. Microscopic morphology (100×) of serum-free adherent SF9 cells inoculated with recombinant baculovirus rBac-IE-Fiber-KnobS and cultured for 48 h. Figure 1 c.

[0072] Example 3: Expression and identification of recombinant protein in insect cell sf9 It will grow to 5.0 × 10 6 -6.0×10 6 SF9 cells / ml in complete suspension were diluted to 2.8 × 10⁻⁶ cells / ml using serum-free complete suspension medium. 6 -3.0×10 6 Cells / ml were inoculated into the P3 generation rBac-IE-Fiber-KnobS recombinant baculovirus obtained in Example 2, cultured for 5 days until the cell viability decreased to below 70%, and the cells were lysed using a freeze-thaw method. The EDS-Fiber protein and KnobS protein were then detected, and the HA titer reached ≥13 log2. Figure 2 As shown; when tested for agar adenosine titer, the ratio can reach ≥1:8, as... Figure 3 As shown.

[0073] During culture, the morphology of fully suspended SF9 cells was observed daily. Healthy SF9 cells should appear as well-transparent spherical cells, such as... Figure 4 As shown in Figure d; after inoculation with rBac-IE-Fiber-KnobS recombinant baculovirus, the average cell diameter gradually increases by 3-5 μm, reaching a peak average diameter at approximately 72 hours, and the cell number gradually decreases, as shown in Figure d. Figure 4 As shown in e.

[0074] Example 4: Establishment of a fully suspended culture process for recombinant baculovirus pDual-IE-Fiber-KnobS in a 3000L bioreactor This embodiment utilizes fully suspended High5 cells in a 3000L bioreactor for the full suspension culture of recombinant baculovirus rBac-IE-Fiber-KnobS, specifically including the following steps (see flowchart). Figure 5 ): 1. 200 ml of fully suspended High5 cells were cultured in a triangular aerator shake flask. The culture medium used was serum-free fully suspended medium, and the culture conditions were as follows: 27℃, culture time 48 h; 2. The High5 cells obtained in step 1 were subjected to an initial density of 12.0 × 10⁶ cells. 5 The cells / ml were scaled up in shake flasks with a total volume of 1000 ml. The culture medium used was serum-free suspension medium, and the culture conditions were as follows: 27℃, culture time was 48h. 3. Recombinant baculovirus rBac-IE-Fiber-KnobS was passaged in Erlenmeyer SF9 cells in a fully suspended flask to prepare seed virus. The culture medium used was serum-free, fully suspended medium, and the culture conditions were as follows: 27.0℃, culture time 96-120h, and the obtained virus titer was 10. 7.5 TCID 50 / 0.1ml, HA titer reaches ≥13 log2; 4. 50L bioreactor (working volume 30L) High5 healthy fully suspended cell culture: The fully suspended High5 cells obtained in step 2 were cultured at an initial density of 12.0 × 10⁶ cells / year. 5 Cells / ml, with a total shake flask volume of 6000 ml for further scale-up, after 48 h of culture, were inoculated into a 50 L bioreactor for full suspension culture in a serum-free, full suspension medium, with an initial cell density of 100 × 10⁶ cells / ml. 4 -150×10 4 Cells / ml, reactor culture parameters were set as follows: temperature 27.5℃, DO 60%, rotation speed: 70-110 rpm, pH 6.1, cell culture period 48h; 5. High5 Fully Suspension Cell Culture in a 300L Bioreactor (Working Volume 150L): The High5 fully suspended cells obtained in step 4 were seeded into a 300L bioreactor for fully suspended culture. The culture medium was a serum-free fully suspended medium, and the initial cell density was 100-150 × 10⁶ cells / year. 4Cells / ml, reactor culture parameters were set as follows: temperature 27.5℃, DO 60%, rotation speed: 70-90 rpm, pH 6.1, cell culture period 48h; 6. 3000L Bioreactor (Initial Working Volume 750L) High5 Fully Suspension Cell Inoculation and Culture with Recombinant Baculovirus rBac-IE-Fiber-KnobS: The High5 fully suspended cells obtained in step 5 were inoculated into a 3000L bioreactor using serum-free, fully suspended culture medium. The initial cell inoculation density was 100 × 10⁶ cells / year. 4 Cells / ml, cultured for 72 h, when cell density reaches 5.0-6.0 × 10⁻⁶. 6 After reaching a cell / ml concentration, the recombinant baculovirus rBac-IE-Fiber-KnobS prepared in step 3 was inoculated, with an MOI of approximately 0.15. Serum-free suspension medium was added to a final volume of 1600 μL to dilute the cell count to 2.8 × 10⁻⁶ cells / ml. 6 -3.0×10 6 The reactor culture parameters were set as follows: temperature: 27.5℃, DO: 60%, stirring speed: 25 rpm, pH: 6.1; 7. Samples should be taken every few hours for cell density and viability analysis (degree of cytopathic effect). Healthy, fully suspended High5 cells should appear as well-transparent spherical shapes. Figure 6 As shown in Figure f, after inoculation with recombinant baculovirus rBac-IE-Fiber-KnobS, the average cell diameter gradually increases by 4-6 μm, generally reaching its peak diameter within 72 hours, and the cell viability gradually decreases. Figure 6 As shown in g. When cell viability drops below 70%, cell density decreases to 2 × 10⁻⁶. 6 When the cell / ml ratio is below a certain threshold, end the culture and harvest the viral fluid. Perform [testing / analysis] on the viral fluid; the HA titer should reach ≥22 log2. Figure 7 As shown; the ammonia titer can reach ≥1:16, such as Figure 8 As shown.

[0075] The reactor parameters are shown in Table 2. In a 3000L reactor, the cell proliferation pattern is as follows: Figure 9 As shown, the cell number change trend after inoculation with recombinant baculovirus rBac-IE-Fiber-KnobS is as follows. Figure 10 As shown in Table 3, the protein harvesting status of the three batches is as follows.

[0076] SDS-PAGE analysis of a protein solution with good HA titer revealed target protein expression at approximately 60 kDa. Figure 11 As shown.

[0077] Table 2 Reactor Parameters

[0078] Table 3. Protein harvesting status of three batches (protein name: rBAC-Fiber-Knob strain baculovirus vector expression of egg drop syndrome subunit protein)

[0079] Example 5: Preparation of a new type of bronchodilator-reduced bronchitis quadrivalent inactivated vaccine The recombinant baculovirus rBac-IE-Fiber-KnobS strain virus solution obtained from Example 4 through full suspension culture was used to prepare a quadrivalent inactivated vaccine consisting of Newcastle disease virus, H9 subtype avian influenza virus, avian egg drop syndrome virus, and avian infectious bronchitis virus. Three separate batches of the recombinant baculovirus rBac-IE-Fiber-KnobS strain virus solution were used for preparation, as detailed below: 1. Vaccine preparation (1) Newcastle disease virus La Sota strain, H9 subtype avian influenza virus SZ strain, and avian infectious bronchitis virus M41 strain were propagated using chicken or duck embryos according to conventional methods. After harvesting the virus fluid, 10% formaldehyde solution was added to the qualified virus fluid to make the final concentration of formaldehyde solution 0.2%, and the mixture was stirred evenly. The virus fluid was inactivated at 37°C for 16 hours to prepare an inactivated virus fluid. The culture medium of silver mosquito nucleopolyhedrovirus (CCTCC NO: V202633) prepared in Example 4 was harvested and then inactivated to prepare an inactivated virus fluid. The four inactivated antigens prepared above were mixed and an inactivated adjuvant vaccine was prepared according to conventional methods.

[0080] (2) Preparation of oil phase: Take 95 parts (v / v) of white oil for injection, add 5 parts (v / v) of Span-80, heat and stir until completely dissolved, autoclave, and cool for later use. Hold at 115℃ for 30 minutes, and then cool to complete the preparation of oil phase.

[0081] (3) Aqueous phase preparation: Take the inactivated Newcastle disease virus La Sota strain concentrate, H9 subtype avian influenza virus SZ strain concentrate, recombinant baculovirus rBac-IE-Fiber-KnobS strain culture concentrate, and avian infectious bronchitis virus M41 strain concentrate, mix them, and then mix 96 parts of the virus solution mixture with 4 parts of sterile Tween-80 to ensure that the Tween-80 is completely dissolved. Wherein, parts are by weight.

[0082] The HA titer of Newcastle disease virus La Sota strain in culture should be no less than 9 log2; the HA titer of H9 subtype avian influenza virus SZ strain in culture should be no less than 8 log2; the HA titer of recombinant baculovirus rBac-IE-Fiber-KnobS strain in culture should be no less than 22 log2; and the HA titer of avian infectious bronchitis virus M41 strain in culture should be no less than 10 log2. 6.0 EID 50 / 0.1ml; each 0.5ml of vaccine should contain at least 0.025ml of Newcastle disease virus La Sota strain, 0.025ml of H9 subtype avian influenza virus SZ strain, 0.03ml of recombinant baculovirus rBac-IE-Fiber-KnobS strain and 0.03ml of avian infectious bronchitis M41 strain, which are concentrated at 5 times their original concentration.

[0083] (4) Emulsification: Take 3 parts of the oil phase and put them into the emulsification tank. Start the motor and slowly rotate and stir. At the same time, slowly add 1 part of the water phase. Stir at 3500 r / min for 5 minutes and then at 10000 r / min for 5 minutes to complete the emulsification preparation. After emulsification, take 10 ml of the vaccine and add it into a centrifuge tube. Centrifuge at 3000 r / min for 15 minutes. The water separated at the bottom of the tube should not exceed 0.5 ml.

[0084] 2. Characteristics of the vaccine Appearance: Milky white, uniform emulsion.

[0085] Dosage form: water-in-oil emulsion. Take a clean pipette, draw a small amount of vaccine and drop it onto a clean, cold water surface. Except for the first drop, none of the drops spread.

[0086] Stability: Add 10 ml of vaccine to a centrifuge tube and centrifuge at 3000 rpm for 15 minutes. The aqueous phase precipitated at the bottom of the tube should not exceed 0.5 ml.

[0087] 3. Aseptic examination Tests conducted according to the appendix of the current Chinese Veterinary Pharmacopoeia showed that there was no sterile growth.

[0088] Example 6: Safety Testing of a Quadrivalent Inactivated Vaccine Combining Newcastle Disease Virus, Infectious Bronchitis, Egg Drop Syndrome, and H9 Subtype Avian Influenza Virus Safety tests were conducted on the quadrivalent inactivated vaccine prepared in Example 5, which combines Newcastle disease virus, infectious bronchitis virus, egg drop syndrome virus, and H9 subtype avian influenza virus. The specific methods are as follows: Forty 14-day-old SFP chickens were divided into four groups of ten each. Each group received a subcutaneous injection of a quadrivalent inactivated vaccine (batch numbers 2026001, 2026002, and 2026003) containing Newcastle disease virus, infectious bronchitis virus, egg drop syndrome virus, and H9 subtype avian influenza virus (prepared in three batches). The dosage was 2 ml per chicken. The control group of ten SFP chickens received a subcutaneous injection of sterile saline solution in the neck, at a dosage of 2 ml per chicken. All groups were raised and managed under identical conditions and observed for 14 consecutive days. Any deaths were necropsies to examine the internal organs for lesions. Adverse reactions in live chickens were also observed. Fourteen days after vaccination, all surviving chickens were euthanized, and their internal organs were examined for lesions. Local inflammation and tissue lesions were assessed.

[0089] The results of the single-dose repeated vaccination safety experiment are shown in Table 4.

[0090] Table 4. Safety trial results of single-dose repeated vaccination

[0091] The above results indicate that the quadrivalent inactivated vaccine combining Newcastle disease virus, infectious bronchitis, avian egg drop syndrome, and H9 subtype avian influenza virus has high safety.

[0092] Example 7: Immunopotency Test of a Quadrivalent Inactivated Vaccine Combining Newcastle Disease Virus, Infectious Bronchitis, Egg Drop Syndrome, and H9 Subtype Avian Influenza Virus The immunogenicity of the quadrivalent inactivated vaccine prepared in Example 5, which combines Newcastle disease virus, infectious bronchitis, egg drop syndrome, and H9 subtype avian influenza, was tested using the following methods: Sixty 3-week-old SPF chickens were divided into six groups. Groups 1-3 received the three batches of the inactivated Newcastle disease virus + infectious bronchitis + egg drop syndrome + H9 subtype avian influenza quadrivalent vaccine (whole suspension cell culture, batch numbers 2026001, 2026002, and 2026003) from Example 5. Group 4 received the inactivated vaccine prepared from EDS whole virus antigen (available from Qian Yuan Hao Biotechnology Co., Ltd.). Group 5 served as the control group. Each group consisted of 10 SPF chickens. Each chicken received a 0.5 ml intramuscular injection into its pectoral cavity. Blood samples were collected on day 21 to detect HI antibodies. The results are shown in Table 5.

[0093] Table 5. Results of immunogenicity testing of the quadrivalent inactivated vaccine against Newcastle disease virus, infectious bronchitis, avian egg drop syndrome, and H9 subtype avian influenza.

[0094] The above results indicate that when chickens were immunized with a quadrivalent inactivated vaccine consisting of Newcastle disease virus, infectious bronchitis, egg drop syndrome, and H9 subtype avian influenza (0.5 ml / chicken), the HI (infectious immunogenicity) was ≥10 log2 21 days after immunization. This demonstrates that the egg drop syndrome subunit protein expressed by the rBAC-Fiber-Knob strain recombinant baculovirus induced serum antibody titers in SPF chickens that met the prescribed standards, and that the HI titer was superior to that of embryonic toxin antigen immunization. This proves that the protein prepared using the method provided in this invention, after emulsification immunization, has good immunogenicity.

[0095] Example 8: Challenge test of a quadrivalent inactivated vaccine containing Newcastle disease virus, infectious bronchitis, avian egg drop syndrome, and H9 subtype avian influenza. Fifty 140-day-old unvaccinated SPF chickens were divided into 5 groups, with groups 1-4 being the immunization groups and group 5 serving as the control group. A prepared quadrivalent inactivated vaccine combining Newcastle disease virus, infectious bronchitis, egg drop syndrome, and H9 subtype avian influenza was administered. Each immunized chicken in the immunization group received a subcutaneous injection of 0.5 ml in the neck, while the control group received no vaccine. Three weeks after immunization, 10 chickens were... 6.63 TCID 50 A potent strain of egg drop syndrome (AV-127 strain, purchased from the Poultry Research Institute of Shandong Academy of Agricultural Sciences) was administered via intramuscular injection of 1 ml / bird to challenge the chickens. The chickens were observed for 14 days to check for typical symptoms such as soft-shelled eggs, thin-shelled eggs, and decreased egg production. The challenge test group setup is shown in Table 6, and the results are shown in Table 7.

[0096] Table 6. Setup of the challenge test group

[0097] Note: Whole virus antigen vaccine refers to the "Newcastle disease, infectious bronchitis, egg drop syndrome, and avian influenza (H9 subtype) quadrivalent inactivated vaccine (LaSota strain + M41 strain + AV-127 strain + S2 strain)" prepared in accordance with the method in Annex 1 of Announcement No. 1532 of the Ministry of Agriculture and Rural Affairs of the People's Republic of China.

[0098] Table 7 Results of the challenge test

[0099] The results of the challenge test showed that none of the immunized chickens exhibited typical symptoms of the disease, while the control group began to produce thin-shelled and soft-shelled eggs 5 days after challenge. The protection rate against the virulent strain of egg drop syndrome was 100% in all four immunized groups, while the morbidity rate in the control group was 80%, proving the validity of the challenge model. The vaccine prepared from the antigen provided in this invention has a good protection rate against avian egg drop syndrome.

[0100] The subunit protein design method for poultry egg drop syndrome provided by this invention has been verified by the following comparative examples: Comparative Example 1: E. coli expression experiment of egg drop syndrome virus subunit antigen protein To verify the expression system and nucleic acid sequence selected in this invention, this comparative example uses a relatively mature E. coli expression system to express the KnobS protein suitable for the system and to verify its expression level and immunogenicity.

[0101] The optimized sequence provided by this invention, derived from the adaptive codons of *E. coli* (the optimized sequence is shown in SEQ ID NO: 4), was cloned into the NcoⅠ and XhoⅠ restriction sites of the pET-28a vector. After verifying the plasmid's complete correctness, it was transformed into *E. coli* strain BL21(DE3). Upon successful identification, a single colony was picked and named KnobS-BL21(DE3). The positive strain was then transferred to a small tube of culture medium and cultured at 37°C with shaking for 3-4 hours until the OD value reached 0.5%. 600 When the concentration of bacteria reached 0.6-0.8, IPTG was added to a final concentration of 0.5 mM for 6 hours. The E. coli precipitate was then collected by centrifugation. The precipitate was resuspended in PBS solution at a concentration of 10 times the wet weight of the bacteria, the cells were sonicated, and the supernatant was collected by centrifugation for agar immunodiffusion assay. The experiment was validated three times using LB medium and commercial E. coli culture medium (provided by Hongshun Biotechnology Co., Ltd., product code: 005242) (Table 8). Table 8. Agar amplification titer of Escherichia coli KnobS-BL21(DE3) induction test

[0102] A group of bacterial suspensions with relatively good agar amplification titers were taken, resuspended and lysed in urea solutions of different concentrations, and then subjected to SDS-PAGE analysis. The results showed expression of the target protein at 26 kDa, with most of it present in a soluble form. Figure 12 ).

[0103] Two groups of proteins with relatively good agar amplification titers were selected from the two groups of experiments. They were then used to prepare a quadrivalent inactivated vaccine of Newcastle disease virus + infectious bronchitis + avian egg drop syndrome + H9 subtype avian influenza virus according to the preparation method in Example 5 of this invention. The vaccine was then used to immunize 21-day-old SPF chickens. Serum samples were collected at 21 and 35 days after immunization to detect HI. The test results are shown in Table 9.

[0104] Table 9 Vaccine Immunization Results

[0105] Note: Whole virus antigen vaccine refers to the "Newcastle disease, infectious bronchitis, egg drop syndrome, and avian influenza (H9 subtype) quadrivalent inactivated vaccine (La Sota strain + M41 strain + AV-127 strain + S2 strain)" prepared in accordance with the method in Annex 1 of Announcement No. 1532 of the Ministry of Agriculture and Rural Affairs of the People's Republic of China.

[0106] The above results show that the KnobS protein of avian egg drop syndrome virus expressed by genetically engineered Escherichia coli has a certain agar amplification titer, and the agar amplification titer can be improved through process optimization, but it does not reach the serum antibody titer value in Example 6 of this invention. Therefore, the method for high-efficiency expression of the recombinant baculovirus subunit protein of egg drop syndrome provided by this invention has significant advantages.

[0107] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. Egg drop syndrome virus subunit antigen, characterized in that, This includes the egg drop syndrome virus (EDS) fiber protein, the EDS virus Knob-S protein, and the silver mosquito nucleopolyhedrovirus (IE0) protein; Among them, the Fiber protein is: (a1) A protein consisting of the amino acid sequence shown in SEQ ID NO:1; (b1) A protein derived from (a1) with one or more amino acids substituted, deleted or added to the sequence shown in SEQ ID NO:1 and having the same function; Knob-S protein is: (a2) A protein consisting of the amino acid sequence shown in SEQ ID NO:3; (b2) Proteins derived from (a2) with one or more amino acids substituted, deleted, or added to the sequence shown in SEQ ID NO:3 and having the same function; The IE0 protein is: (a3) A protein consisting of the amino acid sequence shown in SEQ ID NO:5; (b3) Proteins derived from (a3) ​​with the sequence shown in SEQ ID NO:5 substituted, deleted or added with one or more amino acids and having the same function.

2. A nucleic acid molecule encoding the subunit antigen of claim 1 or biological material containing said nucleic acid molecule; The biological materials are expression cassettes, transposons, plasmid vectors, viral vectors, engineered bacteria, or transgenic cell lines.

3. A recombinant baculovirus expression vector, characterized in that, It contains an expression cassette for co-expressing the three proteins of claim 1; Preferably, the nucleotide sequence of the gene encoding the Fiber protein is shown in SEQ ID NO:2, the nucleotide sequence of the gene encoding the Knob-S protein is shown in SEQ ID NO:4, and the nucleotide sequence of the gene encoding the IEO protein is shown in SEQ ID NO:

6.

4. A recombinant baculovirus, characterized in that, Its genome contains expression cassettes of the three proteins described in claim 1.

5. Autographa californica multiplenuclecopolyhedrovirus rBac-IE-Fiber-KnobS, accession number CCTCC NO: V202633.

6. A transgenic insect cell, characterized in that, The cells contain the recombinant baculovirus expression vector of claim 3, or the cells are infected with the recombinant baculovirus of claim 4 or the nucleopolyhedrovirus of the silver mosquito moth of claim 5.

7. The method for preparing the subunit antigen of egg drop syndrome virus according to claim 1, characterized in that, The transgenic insect cells of claim 6 are cultured under suspension culture conditions to express the Fiber protein, Knob-S protein and IEO protein, or healthy insect cells are inoculated using the nucleopolyhedrovirus of the silver mosquito moth of claim 5 at a certain MOI value; the virus culture medium containing the proteins is harvested.

8. The method according to claim 7, characterized in that, When inoculated with recombinant baculovirus, the growth density of the insect cells was 5.0 × 10⁻⁶. 6 -6.0×10 6 Cells / ml were diluted to 2.8 × 10⁶ cells / ml by adding culture medium. 6 -3.0×10 6 Cells / ml, virus inoculation rate of MOI = 0.07-0.2; The cultivation conditions were: temperature 27±0.5℃, dissolved oxygen 55-65%, stirring speed 20-30 rpm, and pH 6.0-6.4 during the cultivation stage in a 3000L scale reactor. Preferably, when the insect cell viability drops below 70% and the density drops to 2×10⁻⁶, 6 When the number of cells / ml is below a certain level, end the culture and harvest the virus culture medium. More preferably, the insect cells are SF9 or High5.

9. A vaccine for the prevention of egg drop syndrome, characterized in that, It comprises virus culture medium prepared according to the method of claim 7 or 8 after inactivation treatment and vaccine adjuvant.

10. The use of the subunit antigen of claim 1, the nucleic acid molecule of claim 2 or biological material containing the nucleic acid molecule, the recombinant baculovirus expression vector of claim 3, the recombinant baculovirus of claim 4 or the nucleopolyhedrovirus of the silver mosquito moth of claim 5 in the preparation of a vaccine for the prevention of egg drop syndrome.

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