Recombinant baculovirus of co-expressed gene type 1 and type 3 duck hepatitis A virus VP1 protein and application of recombinant baculovirus

Abstract

The invention belongs to the technical field of biology, and particularly relates to a recombinant baculovirus capable of simultaneously expressing gene type 1 and gene type 3 duck hepatitis A virus VP1 protein and application of the recombinant baculovirus in preparation of a duck hepatitis A divalent subunit vaccine. The recombinant baculovirus co-expressing the gene type 1 and gene type 3 duck hepatitis A virus VP1 protein is constructed, the virus titer is high, and the two proteins can be expressed. Therefore, the recombinant baculovirus provided by the invention can be used for research and development and epidemic disease prevention and control of novel vaccines for duck viral hepatitis A.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a recombinant baculovirus co-expressing genotype 1 and genotype 3 duck hepatitis A virus VP1 protein and application thereof in preparing a duck hepatitis A virus bivalent subunit vaccine. BACKGROUND

[0002] Duck hepatitis A virus (DHAV) is an acute and highly lethal infectious disease of ducklings caused by duck hepatitis A virus (DHAV). It is the number one killer of the duck industry. For a long time, genotype 1 (DHAV-1) has dominated in duck flocks in China. Since 2013, genotype 3 (DHAV-3) has been introduced into China and has rapidly spread in duck flocks. Currently, DHAV-1 and DHAV-3 are co-prevalent in duck flocks in China, which is a major threat to the duck industry.

[0003] Vaccination is an effective measure to prevent and control duck hepatitis A virus. Currently, live attenuated vaccines and inactivated vaccines against DHAV are mainly used for immunization of duck flocks. Live attenuated vaccines are attenuated viruses of clinically isolated wild strains that are continuously passaged in chicken embryos. This method has the disadvantages of long development cycle, high randomness, and risk of virus virulence returning to strong. Inactivated vaccines are prepared by amplifying wild strains of DHAV in chicken embryos, treating them with chemicals to eliminate virus infectivity, and then adding adjuvants. Inactivated vaccines have the disadvantages of incomplete virus inactivation, high cost, and slow induction of immune response in ducklings. There are live vaccines and inactivated vaccines on the market that target genotype 1 and genotype 3 respectively, as well as bivalent inactivated vaccines that target both genotypes, but bivalent live vaccines have not yet been marketed. It is particularly important to note that the production of live attenuated vaccines and inactivated vaccines relies on chicken embryos, which have the disadvantages of high cost, unstable supply, contamination with endogenous viruses (chicken leukemia virus, etc.), and large amount of biological waste.

[0004] Subunit vaccines based on cell expression systems are one of the effective ways to solve the technical problems of existing DHAV vaccines. The baculovirus expression system is a high-efficiency technology platform for expressing foreign antigen proteins by infecting insect cells with recombinant baculoviruses expressing foreign genes. It has the following advantages: 1) baculovirus only infects arthropods and does not infect vertebrates, so it is relatively safe; 2) insect cells can be cultured on a large scale by fermentation process, which has low production cost and high uniformity; 3) baculovirus has a large genome capacity that can accommodate multiple foreign genes; 4) baculovirus can express foreign proteins using a very late promoter to achieve high antigen yield.

[0005] However, there is currently no new type of DHAV vaccine that covers both genotypes 1 and 3 and does not rely on the production process of chicken embryos. The development of new DHAV vaccines with these characteristics is of great significance for the prevention and control of duck viral hepatitis. Summary of the Invention

[0006] The purpose of this invention is to provide a recombinant baculovirus capable of co-expressing DHAV-1 and DHAV-3 VP1 proteins. This virus exhibits high titers in Sf9 insect cells and correctly expresses the VP1 proteins of both DHAV genotypes.

[0007] The technical solution provided by this invention is as follows:

[0008] A DHAV-1 VP1 consensus protein, the amino acid sequence of which is shown in SEQ ID NO.1.

[0009] A nucleotide molecule encoding the aforementioned DHAV-1 VP1 consensus protein, with its codons optimized for adaptation to insect cell sf9, the sequence of which is shown in SEQ ID NO.2.

[0010] A recombinant plasmid comprising the DHAV-1 VP1 gene, the PH promoter, and the DHAV-3 VP1 gene.

[0011] The present invention also provides a method for constructing the above-mentioned recombinant plasmid, wherein a baculovirus degenerative steroid UDP-glucosyltransferase signal peptide is added to the 5' end of the above-mentioned DHAV-1 VP1 consensus protein and DHAV-3 VP1 protein, respectively, to obtain the sequence shown in SEQ ID NO. 8, and the amino acid sequence of the DHAV-3 VP1 protein is shown in SEQ ID NO. 6; a baculovirus PH promoter is added upstream of the DHAV-3 VP1 protein, and the recombinant plasmid is expressed in tandem in the order of DHAV-1 VP1 consensus protein, PH promoter, and DHAV-3 VP1 protein, and the nucleotide sequence of the recombinant plasmid is shown in SEQ ID NO. 9.

[0012] Furthermore, the gene encoding the DHAV-3 VP1 protein was amplified using primers showing the sequences indicated in SEQ ID NO.3 and SEQ ID NO.4.

[0013] The present invention also provides a shuttle plasmid, the construction method of which includes the following steps: digesting the above-mentioned recombinant plasmid and transfer vector pVL1393 with Xba I and Not I, recovering the target fragment, and ligating it using homologous recombination; transforming the ligation product into T1 competent cells; extracting the plasmid using a plasmid extraction kit, identifying it by EcoRV restriction enzyme digestion, and obtaining the shuttle plasmid.

[0014] The present invention also provides a method for constructing a recombinant baculovirus that co-expresses the VP1 protein of duck hepatitis A virus type 1 and type 3 as described above, comprising the following steps: co-transfecting the above-mentioned shuttle plasmid and linearized baculovirus genomic DNA into host cells to rescue the recombinant baculovirus.

[0015] Furthermore, the host cell is an sf9 cell.

[0016] The present invention also provides a recombinant baculovirus that co-expresses the VP1 protein of duck hepatitis A virus type 1 and type 3, wherein the recombinant baculovirus co-expressing the VP1 protein of duck hepatitis A virus type 1 and type 3 is constructed by the above method.

[0017] The present invention also provides the application of recombinant baculoviruses that co-express the VP1 protein of duck hepatitis A virus type 1 and type 3 as described above in the preparation of DHAV bivalent subunit vaccines.

[0018] A DHAV-1 VP1 consensus protein, wherein the amino acid sequence of the DHAV-1 VP1 consensus protein is a conserved consensus sequence obtained by aligning with published DHAV-1 VP1 sequences in the Genbank database, and the amino acid sequence is shown in SEQ ID NO.1.

[0019] The present invention also provides a nucleotide molecule that encodes the above-mentioned DHAV-1 VP1 consensus protein and optimizes its codons to adapt to insect cell sf9, the sequence of which is shown in SEQ ID NO.2.

[0020] Furthermore, a pair of primers (SEQ ID NO.3 and SEQ ID NO.4) were designed to amplify the VP1 gene of the clinical isolate DHAV-3™ strain using PCR. The nucleotide sequence is shown in SEQ ID NO.5, and the amino acid sequence is shown in SEQ ID NO.6.

[0021] Furthermore, the signal peptide of baculovirus degenerative steroid UDP-glucosyltransferase (EGT) was added to the 5' end of the DHAV-1 VP1 consensus protein and the DHAV-3 VP1 protein, respectively, with the amino acid sequences shown in SEQ ID NO.7 and SEQ ID NO.8.

[0022] Furthermore, a baculovirus PH promoter was added upstream of the DHAV-3 VP1 protein, and the proteins were expressed in tandem in the order of DHAV-1 VP1 consensus protein, PH promoter, and DHAV-3 VP1 protein, as shown in SEQ ID NO. 9. Translation of the DHAV-1 VP1 gene was initiated by the baculovirus PH promoter present in the pVL1393 vector, while translation of the DHAV-3 VP1 gene was initiated by the added PH promoter.

[0023] The present invention also provides a recombinant plasmid comprising the above-mentioned nucleotide molecules, consisting of the DHAV-1VP1 gene, the PH promoter and the DHAV-3VP1 gene.

[0024] Furthermore, the construction of the recombinant plasmid includes the following steps: the plasmid containing the above nucleotide molecules and the transfer vector pVL1393 are digested with Xba I and Not I, the target fragment is recovered, and ligated using homologous recombination; the ligation product is transformed into T1 competent cells; the plasmid is extracted using a plasmid extraction kit, identified by EcoRV enzyme digestion, and the shuttle plasmid is obtained.

[0025] The present invention also provides a method for constructing a recombinant baculovirus co-expressing DHAV VP1 protein of type 1 and type 3, comprising the following steps: co-transfecting the above-mentioned shuttle plasmid and linearized baculovirus genomic DNA into host cells to rescue the recombinant baculovirus.

[0026] Furthermore, the host cell is an sf9 cell.

[0027] The present invention also provides a recombinant baculovirus co-expressing DHAV type 1 and type 3 VP1 proteins, which is constructed by the above method.

[0028] The present invention also provides an antigen prepared by inoculating sf9 cells with a recombinant baculovirus that co-expresses the type 1 and type 3 DHAV VP1 proteins described above.

[0029] The present invention also provides a kit comprising the antigen described above.

[0030] The present invention also provides the application of recombinant baculoviruses co-expressing DHAV type 1 and type 3 VP1 proteins in the preparation of DHAV bivalent subunit vaccines.

[0031] This invention obtains a consensus amino acid sequence (SEQ ID NO.1) of the DHAV-1 VP1 protein through sequence alignment analysis.

[0032] Furthermore, the VP1 genes of DHAV-1 and DHAV-3 were linked, and the PH promoter of baculovirus was added to the 5' end of the DHAV-3 VP1 gene.

[0033] Further, the nucleotide sequence of the target gene was synthesized (SEQ ID NO.9). The target gene was cloned into the transfer vector pVL1393, with cloning sites at XbaI and NotI.

[0034] Furthermore, the recombinant shuttle plasmid was co-transfected with the linearized baculovirus genome into sf9 cells to rescue the recombinant baculovirus. The baculovirus genome was a linearized genome of the alfalfa silver-striped moth polyhedrovirus.

[0035] Another objective of this invention is to express the two VP1 proteins, DHAV-1 and DHAV-3, using the aforementioned recombinant baculovirus.

[0036] Another object of the present invention is to provide the application of the above-mentioned recombinant baculovirus in the preparation of a DHAV-1+DHAV-3 bivalent subunit vaccine.

[0037] Beneficial effects

[0038] On the one hand, there are few vaccine products targeting both DHVA-1 and DHAV-3 genotypes. This invention utilizes bioinformatics methods to obtain a consensus sequence for DHVA-1 VP1, which shows high homology with most published DHAV-1 VP1 sequences. The DHAV-1 VP1 consensus sequence is fused with the VP1 gene of the DHAV-3™ strain to construct a recombinant baculovirus simultaneously expressing the VP1 protein of both genotypes. On the other hand, existing DHAV vaccines heavily rely on chicken embryo systems, exhibiting significant drawbacks. This invention constructs a recombinant baculovirus expressing the VP1 protein, simultaneously expressing protective antigens for both genotypes. Therefore, the vaccine prepared from the recombinant baculovirus co-expressing DHAV-1 and DHAV-3 VP1 proteins provided by this invention can achieve the effect of "one vaccine protecting against two genotype viruses," increasing vaccine coverage and improving disease control effectiveness. It can be used for the research and development of novel vaccines for duck hepatitis A and for disease control. Attached Figure Description

[0039] Figure 1 A phylogenetic tree diagram of the DHAV-1 VP1 consensus sequence provided in an embodiment of the present invention.

[0040] Figure 2 Figure A shows a diagram of the construction of the shuttle plasmid provided in the embodiments of the present invention. Figure B is a schematic diagram of VP1 protein expression and a diagram of the electrophoresis of the shuttle vector enzyme digestion identification.

[0041] Figure 3The images shown are identification diagrams of successfully rescued recombinant baculoviruses provided in the embodiments of the present invention. The left image shows the gp64 protein expression results in transfected cells, and the right image shows the gp64 protein expression results in untransfected cells.

[0042] Figure 4 The images provided in this embodiment of the invention show the identification of exogenous VP1 protein expressed by recombinant baculovirus. The left image shows the expression of DHAV-1 VP1 consensus protein, the middle image shows the expression of DHAV-1 VP1 protein, and the right image shows the control of uninfected cells. Detailed Implementation

[0043] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0044] Example 1

[0045] Obtaining the DHAV-1 VP1 consensus sequence

[0046] 211 complete genome sequences of DHAV-1 VP1 were downloaded from the Genbank database. All sequences were aligned using the ClustalW Multiple Alignment method in BioEdit software, yielding a VP1 consensus amino acid sequence (SEQ ID NO.1). The obtained VP1 consensus protein contains 238 amino acids, and each site in the consensus amino acid sequence represents a conserved amino acid site in most strains.

[0047] Example 2

[0048] Genetic evolutionary analysis of DHAV-1 VP1 consensus protein

[0049] To analyze the homology between the obtained DHAV-1 VP1 consensus protein and the VP1 protein from DHAV-1 isolates, the VP1 consensus sequence was resubmitted to the GenBank database for alignment analysis. It was found that the VP1 consensus protein shared over 95% homology with the VP1 proteins from most DHAV-1 isolates. Phylogenetic analysis showed that the obtained VP1 consensus protein is located at the center of the phylogenetic tree (see attached diagram). Figure 1 This indicates that it is closely related to the VP1 protein of the DHAV-1 isolate and has the potential to cover different strains and variant strains.

[0050] Example 3

[0051] Construction of shuttle plasmids containing DHAV-1 and DHAV-3 VP1 genes

[0052] To simultaneously express VP1 proteins of DHAV-1 and DHAV-3 in insect cells, the corresponding nucleotide coding sequence of the VP1 protein was first deduced based on the amino acid sequence of the obtained DHAV-1 consensus protein. The gene codon was then optimized to adapt to the sf9 insect cell, and the gene sequence corresponding to the VP1 consensus protein was obtained (SEQ ID NO.2). In addition, a pair of primers (SEQ ID NO.3 and SEQ ID NO.4) were designed to amplify the VP1 gene of the DHAV-3™ strain using PCR, and its gene sequence was determined (SEQ ID NO.5). The amino acid sequence was then deduced (SEQ ID NO.6).

[0053] To increase VP1 protein expression, the signal peptide of baculovirus-derived degenerative steroid UDP-glucosyltransferase (EGT) (SEQ ID NO.7 and SEQ ID NO.8) was added to the N-terminus of the VP1 protein in DHAV-1 and DHAV-3, respectively. Furthermore, the baculovirus PH promoter was added upstream of the DHAV-3 VP1 protein gene. The DHAV-1 VP1, PH promoter, and DHAV-3 VP1 were then tandemly linked (see attached image). Figure 2 A) (SEQ ID NO. 9), the gene was synthesized and cloned into the pUC57 vector. The plasmid containing the target gene and the transfer vector pVL1393 were digested with Xba I and Not I, the target fragment was recovered, and ligated using homologous recombinase. The ligation product was transformed into T1 competent cells. The plasmid was extracted using the Axygen plasmid extraction kit and identified by EcoRV digestion (see attached). Figure 2 B), the positive plasmid was sequenced and verified, and the positive plasmid was named pVL-dualVP1.

[0054] Example 4

[0055] Rescue of recombinant baculovirus

[0056] The recombinant shuttle plasmid pVL-dualVP1 and linearized baculovirus genomic DNA were co-transfected into Sf9 cells. The brief steps are as follows:

[0057] (1) Count sf9 cells and seed them into 6-well plates at a density of 1×10⁻⁶. 6 Cells / well, incubated statically at 27°C for 1 h.

[0058] (2) Preparation of the transfection system:

[0059] 1) Transfer 0.1 μg of plasmid and 5 μL of baculovirus genome into a sterile 1.5 mL centrifuge tube and add 50 μL of SF900 II medium to the tube.

[0060] 2) Dilute the transfection reagent: Gently mix 5 μL of Profection transfection reagent with 45 μL of SF900 II medium.

[0061] 3) Add the prepared transfection reagent dropwise to the DNA mixture, vortex mix thoroughly, and incubate at room temperature for 15-20 minutes.

[0062] (3) Add the transfection complex dropwise to Sf9 cells.

[0063] (4) Incubate at 27℃ for 12-24 h, and add 1 mL of 5% FBS Excell 420 medium into the well.

[0064] (5) Continue culturing for 50-60 h, harvest the supernatant and cells respectively, and identify the virus rescue status.

[0065] (6) The baculovirus gp64 protein was detected by indirect immunofluorescence assay to verify the rescue of recombinant virus.

[0066] The results showed that bright GP64 protein fluorescence was visible in transfected cells, while no fluorescence was observed in untransfected cells, indicating that baculovirus rescue was successful. (See appendix) Figure 3 The rescued recombinant baculovirus was named rBac-dualVP1, with generation p1.

[0067] Example 5

[0068] Recombinant Baculovirus Titer Determination and Virus Passage

[0069] The rescued recombinant baculovirus rBac-dualVP1-p1 was inoculated into sf9 cells, with 200 μL of virus solution in each well. After incubation at 27°C for 4 days, the supernatant was harvested, which was the second-generation virus (rBac-dualVP1-p2).

[0070] Determining the viral titer (TCID) of recombinant baculovirus on sf9 cells. 50 The specific process is as follows:

[0071] (1) Seed sf9 cells into 96-well plates, 5 × 10⁶ cells per well. 4 / well, 100 μL / well, incubate at 27℃ for 1 h; during this period, perform 10-fold serial dilution of the virus.

[0072] (2) Take the diluted virus and inoculate the cells, 100 μL / well, and inoculate 6 replicates for each dilution; leave 6 wells and add 100 μL of culture medium as uninfected control, and incubate at 27℃ for 4 days.

[0073] (3) The expression of baculovirus gp64 protein was detected by indirect immunofluorescence assay, the viral infection rate was recorded, and TCID was calculated by Reed-Muehc method. 50 .

[0074] The rescued recombinant baculovirus was inoculated into sf9 suspension cells at a MOI of 0.01 and cultured at 27°C with shaking for 4 days. The supernatant was harvested, which was the second-generation virus. The virus was passaged five times consecutively, and the TCID of the virus was measured in each passage. 50 .

[0075] Table 1. Passage and titer of recombinant baculovirus

[0076] Virus passage Virus titer (TCID 50 / mL) rBac-dualVP1-p2 6 rBac-dualVP1-p3 8.5 rBac-dualVP1-p4 9.7 rBac-dualVP1-p5 8.5

[0077] The results showed that the viral titer of recombinant baculovirus rBac-dualVP1 gradually increased during passage and maintained a high viral titer in the p3-p5 generations, indicating that the yield of recombinant baculovirus was high.

[0078] Example 6

[0079] Identification of DHAV-1 and DHAV-3 VP1 protein expression by recombinant baculovirus

[0080] Using positive sera against DHAV-1 VP1 protein and DHAV-3 VP1 protein as antibodies, the expression of VP1 protein in recombinant baculovirus was identified by IFA. The simplified steps are as follows:

[0081] (1) Seed sf9 cells into 96-well plates, 5 × 10⁶ cells per well. 4 / well, incubate at 27℃ for 1 h.

[0082] (2) Discard the culture supernatant, wash twice with PBS, and inoculate with recombinant baculovirus at a dose of 5 MOI.

[0083] (3) The positive sera of DHAV-1 VP1 protein and DHAV-3 VP1 protein were diluted 200 times as primary antibodies and incubated overnight at 4°C; FITC-labeled goat anti-mouse IgG (1000 times diluted) was used as secondary antibody and incubated at 37°C for 1 h. The results were observed under a fluorescence microscope.

[0084] Experimental results showed that in sf9 cells infected with recombinant baculovirus rBac-dualVP1, the expression of the target protein could be detected using both DHAV-1 positive serum and DHAV-3 VP1 protein-specific serum (see appendix). Figure 4 This indicates that the recombinant baculovirus rBac-dualVP1 can co-express the VP1 protein of DHAV-1 and DHAV-3.

[0085] sequence list

[0086] SEQ ID NO. 1 (Amino acid sequence of DHAV-1 VP1 consensus protein)

[0087] GDSNQLGDDEPVCFLNFETANVPIQGESHTLVKHLFGRQWLVRTVQHASTVQELDLQVPDRGHASLIRFFAYFSGEIILTIVNNGTTPAMVAHSYSMDDLSSEYAVTAMGGVMIPANSA KNISVPFYSVTPLRPTRPPIPGTSEATFGRLFMWTQSGSLSVFMGLKKPALFFPLPAPTSTTLSQKSNDVIPTLNQSGDEVDCHFCEICSKMKRRWKPRGYFRFCLRLKTLAFELNLEIE.

[0088] SEQ ID NO. 2 (nucleotide sequence corresponding to the codon-optimized DHAV-1 VP1 consensus protein)

[0089] GGTGACAGCAACCAGCTGGGTGACGACGAGCCTGTGTGCTTCCTGAACTTCGAAACCGCTAACGTGCCCATCCAGGGCGAAAGCCACACTCTGGTGAAGCACCTGTTCGGCCGCCAGTGGCTGGTCCGCACTGTGCAGCACGCTTCCACTGTGCAGGAGCTGGACCTGCAGGTGCCCGACCGCGGACACGCTTCCCTGATCCGTTTCTTCGCCTACTTCTCCGGTGAAATCATCCTGACCATCGTGAACAACGGCACCACCCCCGCCATGGTGGCTCACTCCTACTCCATGGACGACCTGTCCTCCGAGTACGCTGTGACTGCTATGGGCGGCGTGATGATCCCTGCCAACTCCGCTAAGAACATCTCCGTGCCTTTCTACAGCGTCACCCCTCTGCGTCCTACCCGCCCTATCCCCGGTACCAGCGAAGCCACCTTCGGTCGTCTGTTCATGTGGACCCAGTCCGGTAGCCTGTCCGTGTTCATGGGTCTGAAGAAGCCTGCTCTGTTCTTCCCCCTGCCCGCTCCCACCTCCACCACCTTGAGCCAGAAGAGCAACGACGTGATCCCCACCCTGAACCAGTCCGGCGACGAGGTGGACTGCCACTTCTGCGAAATCTGCTCCAAGATGAAGCGCCGCTGGAAGCCCCGCGGCTACTTCAGGTTCTGCCTGCGCCTGAAGACTCTGGCTTTCGAACTGAACCTGGAAATCGAATAA。

[0090] SEQ ID NO. 3 (Forward primer for amplifying DHAV-3 VP1 gene)

[0091] GGAGACTCCAACCAGCTGGGT。

[0092] SEQ ID NO. 4 (Reverse primer for amplifying DHAV-3 VP1 gene)

[0093] TTATTCGATTTCCAGGTGCAGTTCGAA。

[0094] SEQ ID NO. 5 (Nucleotide sequence corresponding to DHAV-3 VP1 protein)

[0095] GGAGACTCCAACCAGCTGGGTGACGACGAGCCCGTCTGCTTCCTGAACTTCGAAACTGCTAACGTCCCCATCCAGGGTGAAAGCCACACTCTGGTCAAGCACCTGTTCGGCCGCCAGTGGCTGGTGCGTACTGTCCAGCACACTGGCGAAGTCCAGGAGCTGGACCTGCCCGTGCCCGACCAGGGACACGCTAGCCTGCTGCGTTTCTTCGCCTACTTCAGCGGTGAAGTGATCCTGACCATCGTGAACAACGGTACTACTCCCTGCATGGTCGCTCACTCCTACACTATGGACAACCTGACCTCCGAGTACGCCGTCACCGCCATGGGTGGTATCCTGATCCCCGCCAACTCCGCTAAGAACATCAACATCCCCTTCTACTCCGTCACTCCTCTGCGCCCCACTCGTCCCATGCCCGCTTCCCAGGGTGGCGGACTGACTTTCGGCCGTCTGTACATCTGGACCCAGTCCGGTTCCGTCAGCGTGTTCATGGGCCTGCACAAGCCCGCTCTGTTCTTCCCCCTGCCTGCTCCCACCTACACTACCCACACCCGTCTGAACAACATCGAGACTATGAACCTGCACAACCAGAGCGACCAGCCTGACTGCCACCTGTGCAAGATCTGCCGCAAGATGAAGAAGTGGAGCCGCAACCACCGCCCTTTCCGTTTCTGCCTGCGTCTGAAGACTCTGGCCTTCGAACTGCACCTGGAAATCGAATAA。

[0096] SEQ ID NO. 6 (Amino acid sequence of DHAV-3 VP1 protein)

[0097] GDSNQLGDDEPVCFLNFETANVPIQGESHTLVKHLFGRQWLVRTVQHTGEVQELDLPVPDQGHASLLRFFAYFSGEVILTIVNNGTTPCMVAHSYTMDNLTSEYAVTAMGGILIPANSAK NINIPFYSVTPLRPTRPMPASQGGGLTFGRLYIWTQSGSVSVFMGLHKPALFFPLPAPTYTTHTRLNNIETMNLHNQSDQPDCHLCKICRKMKKWSRNHRPFRFCLRLKTLAFELHLEIE.

[0098] SEQ ID NO. 7 (Amino acid sequence of DHAV-1 VP1 expressed in baculovirus; amino acids 1-19 are the EGT signal peptide sequence, amino acids 20-257 are the VP1 consensus sequence)

[0099] MTILCWLALLSTLTAVNAAGDSNQLGDDEPVCFLNFETANVPIQGESHTLVKHLFGRQWLVRTVQHASTVQELDLQVPDRGHASLIRFFAYFSGEIILTIVNNGTTPAMVAHSYSMDDLSSEYAVTAMG GVMIPANSAKNISVPFYSVTPLRPTRPIPPGTSEATFGRLFMWTQSGSLSVFMGLKKPALFFPLPAPTSTTLSQKSNDVIPTLNQSGDEVDCHFCEICSKMKRRWKPRGYFRFCLRLKTLAFELNLEIE.

[0100] SEQ ID NO. 8 (Amino acid sequence of DHAV-3 VP1 protein expressed in baculovirus; amino acids 1-19 are the EGT signal peptide sequence, amino acids 20-259 are the VP1 sequence)

[0101] MTILCWLALLSTLTAVNAAGDSNQLGDDEPVCFLNFETANVPIQGESHTLVKHLFGRQWLVRTVQHTGEVQELDLPVPDQGHASLLRFFAYFSGEVILTIVNNGTTPCMVAHSYTMDNLTSEYAVTAMGG ILIPANSAKNINIPFYSVTPLRPTRPMPASQGGGLTFGRLYIWTQSGSVSVFMGLHKPALFFPLPAPTYTTHTRLNNIETMNLHNQSDQPDCHLCKICRKMKKWSRNHRPFRFCLRLKTLAFELHLEIE.

[0102] SEQ ID NO. 9 (nucleotide sequences corresponding to DHAV-1 and DHAV-3 VP1 proteins expressed in baculovirus; 1-774 nt is the DHAV-1 VP1 consensus sequence; 1019-1110 nt is the PH promoter sequence; 1111-1116 nt is the Kozak sequence; 1117-1896 nt is the DHAV-3 VP1 sequence).

[0103]

Claims

1. A DHAV-1 VP1 consensus protein, characterized in that, The amino acid sequence of the DHAV-1 VP1 consensus protein is shown in SEQ ID NO.

1.

2. A nucleotide molecule, characterized in that, The nucleotide molecule encodes the DHAV-1 VP1 consensus protein of claim 1, and its codons are optimized to adapt to insect cell sf9. The sequence of the nucleotide molecule is shown in SEQ ID NO.

2.

3. A recombinant plasmid, characterized in that, The recombinant plasmid includes the DHAV-1 VP1 gene, the PH promoter, and the DHAV-3VP1 gene.

4. The method for constructing recombinant plasmids according to claim 3, characterized in that, The signal peptide of baculovirus degenerative steroid UDP-glucosyltransferase was added to the 5' end of the DHAV-1 VP1 consensus protein and DHAV-3 VP1 protein as described in claim 1, respectively, to obtain the sequence shown in SEQ ID NO.

8. The amino acid sequence of the DHAV-3 VP1 protein is shown in SEQ ID NO.

6. A baculovirus PH promoter was added upstream of the DHAV-3 VP1 protein, and the two proteins were expressed in tandem in the order of DHAV-1 VP1 consensus protein, PH promoter, and DHAV-3 VP1 protein. The nucleotide sequence of the recombinant plasmid is shown in SEQ ID NO.

9.

5. The method for constructing recombinant plasmids according to claim 4, characterized in that, The gene encoding the DHAV-3 VP1 protein was obtained by primer amplification using the sequences shown in SEQ ID NO.3 and SEQ ID NO.

4.

6. A shuttle plasmid, characterized in that, The method for constructing the shuttle plasmid includes the following steps: digesting the recombinant plasmid and transfer vector pVL1393 as described in claim 3 with Xba I and Not I, recovering the target fragment, and ligating it using homologous recombination; transforming the ligation product into T1 competent cells; extracting the plasmid using a plasmid extraction kit, identifying it by EcoRV enzyme digestion, and obtaining the shuttle plasmid.

7. A method for constructing a recombinant baculovirus co-expressing the VP1 protein of duck hepatitis A virus type 1 and type 3, characterized in that, The procedure includes the following steps: co-transfecting a host cell with the shuttle plasmid as described in claim 6 and linearized baculovirus genomic DNA to rescue the recombinant baculovirus.

8. The method for constructing a recombinant baculovirus co-expressing the VP1 protein of duck hepatitis A virus type 1 and type 3 as described in claim 7, characterized in that, The host cell is an sf9 cell.

9. A recombinant baculovirus co-expressing VP1 protein of duck hepatitis A virus type 1 and type 3, characterized in that, The recombinant baculovirus co-expressing the VP1 protein of duck hepatitis A virus type 1 and type 3 was constructed using the method described in claim 7.

10. The use of the recombinant baculovirus co-expressing duck hepatitis A virus VP1 protein of type 1 and type 3 as described in claim 9 in the preparation of a DHAV bivalent subunit vaccine.

Images