Recombinant duck plague virus protein combination, subunit vaccine as well as preparation method and application of subunit vaccine

Abstract

The invention discloses a recombinant duck plague virus protein combination, a subunit vaccine as well as a preparation method and application of the subunit vaccine, and belongs to the technical field of vaccine preparation. According to the technical scheme, the recombinant duck plague virus protein combination comprises recombinant gE protein and recombinant gI protein; the amino acid sequence of the recombinant gE protein is as shown in SEQ ID NO: 10, and the amino acid sequence of the recombinant gI protein is as shown in SEQ ID NO: 13. The nucleotide sequence of the nucleic acid molecule for coding the recombinant gE protein is as shown in SEQ ID NO: 12; the nucleotide sequence of the nucleic acid molecule for coding the recombinant gI protein is as shown in SEQ ID NO: 15. The duck plague subunit vaccine provided by the invention adopts gE and gI co-expression, only contains part of antigens of DPV, does not contain genetic materials, does not have toxin expelling and dispersing risks, is good in safety, can enable the spatial conformation of gE protein to be close to that of natural protein by co-expression of gE and gI, can realize 10 / 10 protection of challenge, has the total protein expression amount of about 4000 [mu] g / ml, and can be used for preparing a vaccine for the duck plague. The culture medium can be diluted by 40 times for seedling production, so that the production cost is greatly reduced.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of vaccine preparation, and particularly relates to a recombinant duck plague virus protein combination, a subunit vaccine and a preparation method and application thereof. BACKGROUND

[0002] At present, duck plague, also known as duck viral enteritis, is an acute and septic infectious disease caused by duck plague virus (DPV). It mainly causes blood vessel damage, tissue hemorrhage, digestive tract mucosa erosion, lymphoid organ damage and systemic parenchymal organ degenerative changes in ducks. At present, duck plague has become one of the most serious diseases in waterfowl breeding industry, causing huge economic losses and seriously affecting the development of waterfowl breeding industry.

[0003] DPV belongs to the members of the Mardivirus genus of the Herpesviridae family, and the molecular biology research of the DPV pathogen is relatively backward at present. Similar to other herpes viruses, the DPV genome is a double-stranded linear DNA, and the surface encodes gB, gC, gD, gE, gI and other proteins. Among them, gB is the main fusion protein; gD protein specifically binds to the receptor cell membrane receptor to start the fusion process; gE / gI forms a heterodimer, which is a virulence gene necessary for viral invasion of the central nervous system, and is responsible for viral intercellular transmission. Among them, gD, gE / gI are the first glycoprotein of the subunit vaccine.

[0004] At present, the disease is mainly prevented and controlled by attenuated vaccine and inactivated vaccine. However, the conventional attenuated vaccine has the possibility of recombination with homologous virulent strains or virulence return during large-area use, resulting in immunization failure and even causing new epidemic situation. The inactivated vaccine has the problems of large dose, inability to distinguish between immune population and infected population, and is not conducive to purification. SUMMARY

[0005] In view of the deficiencies in the prior art, the application provides a recombinant duck plague virus protein combination, a subunit vaccine and a preparation method and application thereof, which simultaneously express gE protein and gI protein. The gE gene nucleotide sequence and the gI gene nucleotide sequence are codon optimized to obtain a codon-optimized gE nucleotide sequence SEQ ID NO: 12 and a codon-optimized gI nucleotide sequence SEQ ID NO: 15. The subunit vaccine is prepared by genetic engineering means, only contains part of the antigens of DPV, does not contain genetic material, and has no risk of detoxification and virus dissemination, and has good safety.

[0006] To solve the technical problem, the technical scheme adopted by the application is as follows: In a first aspect of the application, a recombinant duck plague virus protein combination is provided, comprising recombinant gE protein and recombinant gI protein. The amino acid sequence of the recombinant gE protein is shown as SEQ ID NO: 10, and the amino acid sequence of the recombinant gI protein is shown as SEQ ID NO: 13.

[0007] In a second aspect of the present application, a nucleic acid molecule combination encoding the recombinant duck plague virus protein combination is provided, comprising a nucleic acid molecule encoding a recombinant gE protein and a nucleic acid molecule encoding a recombinant gI protein. The nucleotide sequence of the nucleic acid molecule encoding the recombinant gE protein is shown as SEQ ID NO: 12, which is obtained by codon optimization of the nucleotide sequence shown as SEQ ID NO: 11. The nucleotide sequence of the nucleic acid molecule encoding the recombinant gI protein is shown as SEQ ID NO: 15, which is obtained by codon optimization of the nucleotide sequence shown as SEQ ID NO: 14.

[0008] The above technical solution limits the nucleotide sequence of the nucleic acid molecule encoding the recombinant gE protein and the recombinant gI protein, and the two genes are codon optimized to better express in CHO, and the two are expressed together, which improves the protein expression amount of gE and gI, and the expressed protein forms a natural conformation close to the natural conformation, and the immunogenicity is increased.

[0009] In a third aspect of the present application, a biological material is provided, which is selected from any one of the following: A1) an expression cassette comprising the nucleic acid molecule combination; A2) a recombinant vector comprising the nucleic acid molecule combination; A3) a recombinant cell comprising the nucleic acid molecule combination or the recombinant vector.

[0010] In the present application, the expression cassette can further comprise a regulatory element and an enhancer element; the recombinant vector comprises a cloning vector and an expression vector, the cloning vector is used for copying the relevant sequence, and the expression vector is used for expressing the relevant gene, wherein the vector used for constructing the expression vector can be a pEE14.4 plasmid; the recombinant cell can be a CHO cell strain.

[0011] In a fourth aspect of the present application, the recombinant duck plague virus protein combination or the nucleic acid molecule combination encoding the recombinant duck plague virus protein combination is applied to the preparation of a medicament for treating or preventing duck plague.

[0012] As a preferred, the medicament comprises a subunit vaccine.

[0013] In a fifth aspect of the present application, a duck plague subunit vaccine is provided, comprising the recombinant gE protein and the recombinant gI protein.

[0014] The duck plague subunit vaccine provided by the application adopts co-expression of gE and gI, only contains partial antigens of DPV, does not contain genetic material and thus does not exist the risk of detoxification and virus spread, is safe, and the real state of virus protein is difficult to reproduce by separately expressing gE protein, so that the attack is not protected, only the co-expression of gE and gI can make the spatial conformation of gE protein close to the natural protein, so that the attack 10 / 10 protection can be realized, and the total amount of protein expressed by co-expressing gE protein and gI protein is about 4000 mu g / ml, which can be diluted by 40 times to prepare the vaccine, which greatly reduces the production cost.

[0015] Preferably, the duck plague subunit vaccine further comprises an adjuvant.

[0016] In a sixth aspect, the application provides a preparation method of the duck plague subunit vaccine, comprising the following steps: S1, mixing the veterinary white oil and aluminum stearate, heating to 70-90 DEG C, then adding a surfactant, heating to 110-115 DEG C, maintaining for 20-40 min, and cooling to obtain an oil phase; S2, mixing the purified gE protein liquid and gI protein liquid with sterile normal saline to obtain an aqueous phase; S3, emulsifying the aqueous phase into the oil phase to obtain the duck plague subunit vaccine.

[0017] Preferably, the addition amount ratio of the veterinary white oil, aluminum stearate and surfactant is 91:1:5.

[0018] Preferably, the volume ratio of the aqueous phase and the oil phase is 1:1.5-2.

[0019] Preferably, the preparation method of the duck plague subunit vaccine further comprises a preparation step of the purified gE protein liquid and gI protein liquid, and the preparation steps of the purified recombinant gE protein liquid and the purified recombinant gI protein liquid comprise: constructing a recombinant vector containing a nucleic acid molecule encoding a recombinant gE protein and a nucleic acid molecule encoding a recombinant gI protein, and then transforming the recombinant vector into a host cell to obtain a recombinant cell; respectively, the recombinant cell is subjected to flow culture to obtain a culture, and the purified recombinant gE protein liquid and the purified recombinant gI protein liquid are respectively separated and purified from the culture.

[0020] In a seventh aspect, the application provides use of the duck plague subunit vaccine in preparation of a medicine for treating or preventing duck plague.

[0021] Compared with the prior art, the application has the following beneficial effects: This invention provides a duck plague subunit vaccine. The duck plague subunit vaccine provided by this invention uses gE and gI co-expression, contains only a portion of the DPV antigen, does not contain genetic material and therefore does not pose a risk of viral shedding or shedding, and has good safety. Expressing gE protein alone is difficult to reproduce the true state of the viral protein, so it does not provide protection against challenge. Only by co-expressing both gE and gI can the spatial conformation of gE protein be close to that of the natural protein, thus achieving 10 / 10 protection against challenge. Furthermore, this application co-expresses gE and gI proteins, with a total protein expression of approximately 4000 μg / ml, which can be diluted 40 times for vaccine preparation, greatly reducing production costs. Attached Figure Description

[0022] Figure 1 Images of enzyme digestion of plasmids 14.4, pUC57-gDNOPT, gD, gENOPT, and gE provided in the embodiments of the present invention; Figure 2 The PCR diagram for identifying the recombinant plasmids 14.4-gDNOPT, gD, gENOPT, and gE provided in the embodiments of the present invention is shown. Figure 3 Images of enzyme digestion of pEE14.4-gE plasmid, pUC57-gINOPT and gI plasmid provided in the embodiments of the present invention; Figure 4 The PCR diagrams for identifying the pEE14.4-gE+gINOPT and pEE14.4-gE+gI recombinant plasmids provided in the embodiments of the present invention are shown. Figure 5 This is an SDS-PAGE analysis diagram provided in an embodiment of the present invention; Figure 6 The images provided are Western blot images from embodiments of the present invention. Detailed Implementation

[0023] The technical solutions in specific embodiments of the present invention will now be described in detail and completely with reference to the accompanying drawings. Obviously, the described embodiments are merely some specific implementations of the overall technical solution of the present invention, and not all implementations. Based on the overall concept of the present invention, all other embodiments obtained by those skilled in the art fall within the protection scope of the present invention.

[0024] Example 1: Isolation and Identification of Duck Plague Virus In 2024, the applicant isolated a duck plague virus strain from a duck flock at a duck farm in Anhui Province that had already been vaccinated. Diseased samples from infected ducks were added to physiological saline containing antibiotics at a ratio of 1:5 (w / v), ground into a suspension, centrifuged at 1000 rpm for 5 minutes, and the supernatant was inoculated into well-grown CEF cells. The cells were incubated at 37°C with 5% CO2 for 48–72 hours. When more than 70% of the cells showed cytopathic effects, the cells were freeze-thawed three times, centrifuged at 5000 rpm for 10 minutes, and the supernatant was aliquoted, labeled with the harvest date and virus passage number, and stored below -70°C. The harvested virus fluid was purified and analyzed for viral characteristics such as viral content, immunogenicity, specificity, and purity. The results showed that the isolated strain was duck plague virus, free from bacterial, mycoplasma, and exogenous viral contamination. The purified virus strain was named YBDPV2024.

[0025] Sequence analysis was performed on the gD, gE, and gI genes of the screened and identified YBDPV2024 strain. The full-length gD gene is 1269 bp (nucleotide sequence SEQ ID NO:2), encoding 422 amino acids (amino acid sequence SEQ ID NO:1); the full-length gE gene is 1473 bp (nucleotide sequence SEQ ID NO:4), encoding 490 amino acids (amino acid sequence SEQ ID NO:3); and the full-length gI gene is 1089 bp (nucleotide sequence SEQ ID NO:6), encoding 362 amino acids (amino acid sequence SEQ ID NO:5). Comparison of these sequences with the duck plague virus genome sequences in GenBank revealed that the gD, gE, and gI genes of the YBDPV2024 strain showed 99.5% homology with the corresponding genes of the duck plague virus isolated in 2013 (GenBank accession number KF263690). This result indicates that the increased virulence of the duck plague virus YBDPV2024 is not related to these antigen genes.

[0026] Example 2: Optimization of duck plague virus gD, gE, and gI genes The antigenic sites of the gD gene of duck plague virus strain YBDPV2024 were analyzed. Transmembrane and unnecessary regions of the gD gene were removed, and tags and codon optimization were performed. The optimized amino acid sequence of gD is SEQ ID NO:7, the unoptimized nucleotide sequence is SEQ ID NO:8, and the optimized nucleotide sequence is SEQ ID NO:9. The antigenic sites of the gE gene of duck plague virus were analyzed. Transmembrane and unnecessary regions of the gE gene were removed, and tags and codon optimization were performed. The optimized amino acid sequence of gE is SEQ ID NO:10, the unoptimized nucleotide sequence is SEQ ID NO:11, and the optimized nucleotide sequence is SEQ ID NO:12. The antigenic sites of the gI gene of duck plague virus were analyzed. Transmembrane and unnecessary regions of the gI gene were removed, and signal peptides, tags, and codon optimization were performed. The optimized amino acid sequence of gI is SEQ ID NO:13, the unoptimized nucleotide sequence is SEQ ID NO:14, and the optimized nucleotide sequence is SEQ ID NO:15. The optimized gene was synthesized by Shanghai Sangon Biotech Co., Ltd. and ligated into the pUC57 plasmid.

[0027] Example 3: Construction of a recombinant plasmid expressing the E gene 3.1 Construction of 14.4-gDNOPT, gD, gENOPT, and gE recombinant plasmids: gDNOPT refers to the nucleoside sequence of the strain itself (with the transmembrane region removed). gD refers to the optimized codon sequence; gENOPT refers to the nucleoside sequence of the strain itself (with the transmembrane region removed). gE refers to the optimized codon sequence.

[0028] 3.1.1 Enzyme digestion reaction 3.1.1.1 Label the required 1.5mL EP tubes, and add the sample to the 1.5mL EP tubes according to the table below, mixing thoroughly: The reaction system is 50μL, and the sample addition is shown in the table below: Table 1

[0029] 3.1.1.2 Place the 1.5 mL EP tube from step 3.2.1.1 in a 37°C constant temperature water bath and incubate for 2-3 hours.

[0030] 3.1.2 Gel recovery of double enzyme digestion products The above double enzyme digestion system was removed and subjected to agarose gel electrophoresis to recover the DNA fragments. Figure 1 ); Figure 1In the middle, M: from top to bottom, are 100bp, 250bp, 500bp, 750bp, 1000bp, 2000bp, 3000bp, 5000bp, and 8000bp respectively; from left to right, Lane 1: pEE14.4 plasmid digestion; Lane 2: pUC57-gDNOPT recombinant plasmid digestion; Lane 3: pUC57-gD recombinant plasmid digestion; Lane 4: pUC57-gENOPT recombinant plasmid digestion; Lane 5: pUC57-gE recombinant plasmid digestion; (1) Label the sample collection EP tube, adsorption column and collection tube.

[0031] (2) Weigh the marked empty EP tube and record the value.

[0032] (3) Carefully cut the single target DNA band from the agarose gel with a scalpel using a gel cutter and place it into a clean 1.5 mL centrifuge tube.

[0033] (4) Add 600 μL of PC buffer to the 1.5 mL centrifuge tube in step (3) and place it in a 50 °C water bath for about 5 minutes. During this time, gently rotate the centrifuge tube up and down continuously to ensure that the gel is fully dissolved.

[0034] (5) Column equilibration: Add 500 μL of equilibration solution BL to the adsorption column CB2 (the adsorption column is placed in the collection tube beforehand), centrifuge at 12,000 rpm for 1 min, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

[0035] (6) Add the solution obtained in step (5) to the adsorption column CB2, let it stand for 2 min, centrifuge at 10,000 rpm for 30 s, discard the waste liquid in the collection tube, and then put the adsorption column CB2 into the collection tube.

[0036] (7) Add 600 μL of PW buffer to the adsorption column, let stand for 3 min, centrifuge at 10,000 rpm for 30 s, discard the waste liquid in the collection tube, and put the adsorption column CB2 into the collection tube.

[0037] (8) Repeat step (7).

[0038] (9) Centrifuge the empty adsorption column at 12,000 rpm for 2 min to remove as much of the washing solution as possible. Place the adsorption column at room temperature for 10 min to dry completely.

[0039] (10) Place the adsorption column CB2 into the collection tube, add 50 μL of Elutionbuffer (preheated at 65℃) to the middle of the adsorption membrane, let stand for 3 min, and centrifuge at 12,000 rpm for 2 min.

[0040] (11) Remove the centrifuge tube from step (10) from the centrifuge, discard the middle adsorption column CB2, put the cap back on the centrifuge tube, and keep the DNA sample in the centrifuge tube.

[0041] (12) Store the DNA sample from step 11 at 4°C and prepare for agarose gel electrophoresis to identify the DNA fragments recovered from the gel.

[0042] 3.1.3 Connection Reaction (1) Mark the 0.2 mL centrifuge tubes to be used.

[0043] (2) Add the sample to a properly labeled 0.2 mL tube according to the 20 μL reaction system in the table below: Table 2

[0044] (3) After adding the sample, gently blow the liquid several times with a pipette to mix the components.

[0045] (4) Place the 0.2 mL centrifuge tube at 37°C for 30 min. After the reaction is complete, immediately place the reaction tube in an ice-water bath to cool for 5 min.

[0046] (5) The reaction product of step (4) can be directly used for conversion experiments, or it can be stored at -20℃ and thawed for conversion when needed.

[0047] 3.1.4 Transformation Reaction (1) Quickly add 10 μL of ligation reaction solution to 100 μL of competent cells, mix by pipetting, and incubate on ice for 30 min.

[0048] (2) After step (1) is completed, take out the sample tube, place it in a 42℃ water bath for 100s, and then immediately put it in an ice bath for 2 min.

[0049] (3) After step (2) is completed, take out the sample tube, add 600 μL of liquid LB medium to the sample tube in the ultra-clean workbench, and then place the sample tube in a constant temperature shaker at 37℃ and 220 rpm for 1 h.

[0050] (4) Prepare transformation plates. Prepare LB resistance plates for transformation based on plasmid resistance.

[0051] (5) Plate coating: Take out the sample tube from step (3), centrifuge at room temperature at 8,000 rpm for 2 min, remove 600 μL of supernatant, resuspend the bacteria at the bottom of the tube in the remaining supernatant, put the resuspended bacterial solution into the center of the corresponding transformation plate, and spread the bacterial solution in the center of the transformation plate evenly with a plating stick.

[0052] (6) Place the plate from step (5) upright in a biochemical constant temperature incubator and incubate at 37°C for 1 hour. Then, invert the plate and incubate for 15 hours.

[0053] (7) Observe and record the transformation results.

[0054] 3.1.5 Plasmid extraction and PCR identification 3.1.5.1 Plasmid Extraction (1) Use a 10 μL pipette tip to pick up a single colony from the transformation plate and transfer it to 5 ml of LB liquid medium containing ampicillin resistance. Incubate overnight at 37°C and 220 rpm.

[0055] (2) Transfer the bacterial culture into a 1.5 mL EP tube, centrifuge at room temperature, 12,000 rpm, for 2 min, and discard the supernatant.

[0056] (3) Add 250 μL of plasmid extraction reagent P1 buffer to the EP tube in step (2) to completely suspend the bacterial cells.

[0057] (4) Add 250 μL of P2 buffer to the solution in step (3), and immediately gently invert the centrifuge tube 5-10 times to mix. Let stand at room temperature for 2-4 min.

[0058] (5) Add 350 μL of P3 buffer to the solution in step (4), and immediately gently invert the centrifuge tube 5-10 times to mix. Let stand at room temperature for 2-4 min.

[0059] (6) Centrifuge the solution from step (5) at room temperature, 14,000 rpm, for 10 min.

[0060] (7) Transfer the supernatant solution from step (6) to the center of the adsorption column, centrifuge at room temperature, 12,000 rpm for 30 s, and discard the liquid in the collection tube.

[0061] (8) Add 500 μL of Buffer DW1 to the center of the adsorption column, centrifuge at room temperature, 12,000 rpm for 30 s, and discard the liquid in the collection tube.

[0062] (9) Add 500 μL of wash solution to the center of the adsorption column, centrifuge at room temperature, 12,000 rpm for 30 s, and discard the liquid in the collection tube. Repeat once.

[0063] (10) Empty adsorption column, centrifuge at room temperature, 12,000 rpm, 2 min.

[0064] (11) Place the adsorption column into a clean 1.5ml centrifuge tube, add 30μL of Elutionbuffer to the center of the adsorption membrane, let stand at room temperature for 5min, centrifuge at room temperature for 12,000rpm for 2min, and store the DNA solution in the tube at 4℃.

[0065] 3.1.5.2 PCR identification (1) Label the PCR tubes to be used, add samples and mix according to the table below, and the reaction volume is 25 μL: Table 3

[0066] (2) PCR amplification program: Table 4

[0067] (3) Sequencing: Sequencing the plasmids that were positive by PCR ( Figure 2 The samples were sent to a sequencing company for sequencing. Figure 2 In the diagram, M represents 100 bp, 250 bp, 500 bp, 750 bp, 1000 bp, 2000 bp, 3000 bp, 5000 bp, and 8000 bp from top to bottom; from left to right, Lane 1 is digested with pEE14.4-gE recombinant plasmid; Lane 2 is digested with pUC57-gINOPT recombinant plasmid; and Lane 3 is digested with pUC57-gI recombinant plasmid.

[0068] 3.2 Construction of pEE14.4-gE+gINOPT (gE optimized codon sequence; nucleotide sequence of the gI strain itself (transmembrane region removed)) and pEE14.4-gE+gI recombinant plasmid (gE optimized codon sequence; gI optimized codon sequence) 3.2.1 Enzyme digestion reaction 3.2.1.1 Label the required 1.5mL EP tubes, and add the sample to the 1.5mL EP tubes according to the table below, mixing thoroughly: The reaction system is 50μL, and the sample addition is shown in the table below: Table 5

[0069] 3.2.1.2 Place the 1.5 mL EP tube from step 3.2.1.1 in a 37°C constant temperature water bath and incubate for 2-3 hours.

[0070] 3.2.2 The gel recovery procedure for the double enzyme digestion products is the same as in 3.1.2. Figure 3 ); Figure 3In the diagram, M represents 100 bp, 250 bp, 500 bp, 750 bp, 1000 bp, 2000 bp, 3000 bp, 5000 bp, and 8000 bp from top to bottom; from left to right, Lane 1 is digested with pEE14.4-gE recombinant plasmid; Lane 2 is digested with pUC57-gINOPT recombinant plasmid; and Lane 3 is digested with pUC57-gI recombinant plasmid.

[0071] 3.2.3 The connection reaction procedure is the same as in 3.1.3. 3.2.4 The conversion reaction procedure is the same as in 3.1.4. 3.2.5 Plasmid extraction and PCR identification procedures are the same as in 3.1.5 ( Figure 4 ); Figure 4 In the middle, M: from top to bottom, are 100bp, 250bp, 500bp, 750bp, 1000bp, 2000bp, 3000bp, 5000bp, and 8000bp; from left to right, Lane 1: pEE14.4-gE+gINOPT recombinant plasmid PCR; Lane 2: pEE14.4-gE+gI recombinant plasmid PCR; Lane 3: negative control.

[0072] Example 4: Transfection of CHO-K1 cells (1) Preparation: Sterilize the biosafety cabinet with ultraviolet light for 30 minutes; preheat the F-12 culture medium to 37°C in a 37°C water bath.

[0073] (2) Add 2.5 μg of recombinant DNA and P3000 to 125 μl of OPTI-MEM culture medium and mix well. Add 3.75 μl of liposome-convertamine 3000 to 125 μl of OPTI-MEM culture medium and mix well. Mix the liposomes with the recombinant DNA and let stand at room temperature for 10 min.

[0074] (3) Take out the cells from the 6-well plate that were laid flat 24 hours ago from the 37℃ incubator, discard the supernatant culture medium, wash the cells three times with pre-warmed OPTI-MEM culture medium, and discard the OPTI-MEM culture medium.

[0075] (4) Add 2 ml of F-12 culture medium containing 10% and 1% fetal bovine serum with double antibiotics to each cell well.

[0076] (5) Gently add the mixture of recombinant DNA and liposomes to each well of the cell, mix gently, and incubate at 37°C in a 5% CO2 cell culture incubator.

[0077] (6) 72 hours after transfection, the supernatant was collected for protein detection, and the cells were added to G418 for pressure screening.

[0078] (7) After 144 hours, the monoclonal cells are tested, and the positive cells are amplified. This can be used for large-scale protein production.

[0079] Example 5: Construction of cell lines: Cell lines expressing the proteins shown in Table 6 were constructed according to the methods described in the above embodiments. Table 6

[0080] Example 6: Protein Purification and Detection 6.1 Expression and identification of recombinant gDNOPT, gD, gENPOT, gE+gINOPT, and gE+gI proteins in CHO cells: The cells selected in Example 4 were repeatedly passaged and acclimated into suspension cells. The suspension cells were cultured in a fed-batch manner at 37°C for 240 hours, and the supernatant was collected for SDS-PAGE protein analysis. The results showed that the gDNOPT and gENOPT genes were not expressed in CHO cells, while the optimized gD, gE, gE+gINOPT, and gE+gI genes were all expressed in CHO cells.

[0081] 6.2 Protein Content Detection: The supernatant harvested in 6.1 was analyzed for total protein content using a Bradford protein assay kit, and protein purity was determined by SDS-PAGE. The target protein content was then calculated. Figure 5 ). Figure 5 In the middle, M: from bottom to top, are 15KDa, 25KDa, 35KDa, 40KDa, 55KDa, 70KDa, 100KDa, 130KDa, and 180KDa respectively; from left to right, Lane 1: negative control supernatant; Lane 2: gDNOPT supernatant; Lane 3: gD supernatant; Lane 4: gENOPT supernatant; Lane 5: gE supernatant; Lane 6: gE+gINOPT supernatant; Lane 7: gE+gI supernatant.

[0082] Table 7

[0083] 6.3 Chromatographic purification of recombinant gD, gE, and gE+gI proteins The collected supernatants of gD, gE, and gE+gI were subjected to affinity chromatography using GE's Ni Sepharose Excel medium. The collected supernatant was centrifuged at 5000 rpm for 5 min and used as the loading solution.

[0084] 6.3.1 Wash the medium with 5 column volumes of distilled water.

[0085] 6.3.2 Wash the medium with 5 column volumes of equilibration buffer.

[0086] 6.3.3 Sample loading: 4 mg of protein can be loaded per milliliter of column.

[0087] 6.3.4 Wash the medium with 20 column volumes of washing buffer.

[0088] 6.3.5 Elute the protein with 5 column volumes of elution buffer.

[0089] 6.4 Western blot test The protein prepared in 6.3 was subjected to SDS-PAGE using a semi-dry method at 20V for 30 min. The target protein band was transferred to a PVDF membrane, which was blocked overnight with blocking buffer. After washing three times with PBST, the membrane was incubated with 1:500 diluted duck plague virus positive serum at 37℃ for 1.5 h, washed three times with PBST, and then incubated with 1:1000 diluted HRP-labeled goat anti-duck enzyme-labeled antibody at 37℃ for 1.5 h, washed three times with PBST, and incubated with substrate solution for 5 min. The membrane was then developed in a chemiDOC. The results showed that the optimized gD, gE, and gE+gI all specifically bound to the duck plague virus positive serum. Figure 6 This indicates that the expressed gD, gE, and gE+gI proteins have good immunogenicity. Figure 6 In the middle, M: from bottom to top, are 15KDa, 25KDa, 35KDa, 40KDa, 55KDa, 70KDa, 100KDa, 130KDa, and 180KDa respectively; from left to right: Lane 1: negative control supernatant; Lane 2: gD supernatant; Lane 3: gE supernatant; Lane 4: gE+gI supernatant.

[0090] Example 7: Vaccine preparation and efficacy testing 1. Vaccine preparation (1) Preparation of oil phase: Take 95 parts of veterinary white oil and 1 part of aluminum stearate, place them in an oil phase preparation tank and heat to 80°C. Then add 5 parts of Span-80 and maintain the temperature for 30 minutes until it reaches 115°C. After cooling, it is ready for use.

[0091] (2) Preparation of aqueous phase: The protein solution purified in 4.3 was mixed with sterile physiological saline in appropriate proportions to make the protein content in the aqueous phase 40~60 μg / ml. The protein content of the gE+gI group was calculated according to gE.

[0092] (3) Emulsification: Take 1.5 parts of the oil phase and place them in a high-speed shear press. Start the motor and slowly rotate and stir. At the same time, slowly add 1 part of the water phase. Emulsify for 5 minutes at 10000 r / min. After emulsification, take 10 ml and centrifuge at 3000 r / min for 15 minutes. The water precipitated at the bottom of the tube should not exceed 0.5 ml.

[0093] 2. Vaccine efficacy testing Fifty 21-35 day old Muscovy ducks were randomly divided into 5 groups of 10 each. Group 1 received a subcutaneous injection of 0.3 ml of commercially available inactivated duck plague vaccine; Group 2 received a subcutaneous injection of 0.3 ml of gD vaccine; Group 3 received a subcutaneous injection of 0.3 ml of gE vaccine; Group 4 received a subcutaneous injection of 0.3 ml of gE+gI vaccine; and Group 5 served as a control group. Twenty-eight days post-immunization, blood samples were collected from all ducks for neutralizing antibody testing. Simultaneously, 1000 ml of YBDPV2024 virus was injected intramuscularly, and the ducks were observed for 14 days. Results showed that all vaccine groups were positive for neutralizing antibodies (see Table 8). The protection rate of the commercially available inactivated duck plague vaccine was 7 / 10, the gD subunit vaccine group was 1 / 10, the gE subunit vaccine group was 3 / 10, and the gE+gI subunit vaccine group was 10 / 10. All ducks in the control group died (see Table 9).

[0094] Table 8: Results of serum neutralizing antibody detection

[0095] Table 9

[0096] Note: √ represents normal, ★ represents death. In summary, the gD, gE, and gE+gI subunit vaccines of this invention can all produce neutralizing antibodies 28 days after immunization, with neutralizing antibody titers of 1:16, 1:27.2, and 1:86.4, respectively. The challenge experiment results show that the commercially available inactivated vaccine has a protection rate of 7 / 10; the protection rates of the gD and gE subunit vaccine groups alone are lower, at 1 / 10 and 3 / 10, respectively, but the protection rate of our developed gE+gI subunit vaccine group is 10 / 10. This experiment demonstrates that although the gD protein can produce some neutralizing antibodies, it cannot resist challenge with the duck plague virus strain; expressing the gE protein alone is insufficient to reproduce the true state of the viral protein, therefore it does not provide protection against challenge; only by co-expressing both gE and gI can the spatial conformation of the gE protein be made close to that of the natural protein, hence the gE+gI subunit vaccine group provides 10 / 10 protection against challenge. Co-expression of the gE and gI proteins results in a total protein expression of approximately 4000 μg / ml, which can be diluted 40 times for vaccine preparation, significantly reducing production costs.

Claims

1. A recombinant duck plague virus protein combination, characterized in that, Including recombinant gE protein and recombinant gI protein; The amino acid sequence of the recombinant gE protein is shown in SEQ ID NO:10, and the amino acid sequence of the recombinant gI protein is shown in SEQ ID NO:

13.

2. A nucleic acid molecule assembly encoding the recombinant duck plague virus protein assembly of claim 1, characterized in that, This includes nucleic acid molecules encoding recombinant gE protein and nucleic acid molecules encoding recombinant gI protein; The nucleotide sequence of the nucleic acid molecule encoding the recombinant gE protein, as shown in SEQ ID NO:12, is obtained by codon optimization of the nucleotide sequence shown in SEQ ID NO:

11. The nucleotide sequence of the nucleic acid molecule encoding the recombinant gI protein, as shown in SEQ ID NO:15, is obtained by codon optimization of the nucleotide sequence shown in SEQ ID NO:

14.

3. A biomaterial, characterized in that, The biomaterial is selected from any one of the following: A1) An expression cassette comprising the nucleic acid molecule combination of claim 2; A2) A recombinant vector comprising the nucleic acid molecule combination of claim 2; A3) Recombinant cells comprising the nucleic acid molecule combination of claim 2 or the recombinant vector.

4. The use of the recombinant duck plague virus protein combination of claim 1 or the nucleic acid molecule combination encoding the recombinant duck plague virus protein combination of claim 1 in the preparation of a drug for treating or preventing duck plague.

5. The application according to claim 4, characterized in that, The drug includes a subunit vaccine.

6. A duck plague subunit vaccine, characterized in that, Includes the recombinant duck plague virus protein combination as described in claim 1.

7. A method for preparing a duck plague subunit vaccine, characterized in that, Includes the following steps: S1. After mixing veterinary white oil and aluminum stearate, heat to 70-90°C, then add surfactant, heat to 110-115°C, maintain for 20-40 minutes, and cool to obtain oil phase; S2. The purified gE protein solution and gI protein solution were respectively mixed with sterile physiological saline to obtain the aqueous phase; S3. The aqueous phase is added to the oil phase for emulsification to obtain the duck plague subunit vaccine.

8. The preparation method according to claim 7, characterized in that, The ratio of the amount of veterinary white oil, aluminum stearate and surfactant added is 91:1:5; the volume ratio of the aqueous phase and the oil phase is 1:1.5~2.

9. The preparation method according to claim 7, characterized in that, The method for preparing the duck plague subunit vaccine further includes the steps of preparing purified gE protein solution and gI protein solution, wherein the steps of preparing purified recombinant gE protein solution and recombinant gI protein solution include: A recombinant vector containing nucleic acid molecules encoding recombinant gE protein and recombinant gI protein was constructed and then transformed into host cells to obtain recombinant cells; The recombinant cells were fed-batch cultured to obtain cultures, and the recombinant gE protein solution and recombinant gI protein solution were separated and purified from the cultures.

10. The use of the duck plague subunit vaccine according to claim 6 in the preparation of a medicament for treating or preventing duck plague.

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