A Porcine Deltacoronavirus S Protein and Recombinant Adenovirus

Abstract

The present invention discloses a porcine deltacoronavirus S protein and a recombinant adenovirus. The porcine deltacoronavirus S protein expressed by the live vector vaccine has the following characteristics: based on the S protein sequence of the PDCoV SC strain, the MDAMKRGLCCVLLLCGAVFVSN TPA secretion signal peptide sequence is inserted at the N-terminus, the GSGYIPEAPRDGQAYVRKDGEWVLLSTFGSGHHHHHH tag is inserted at the C-terminus, and the S1 and S2 cleavage sites are mutated to GGSGGGGSGGGS, and then the sequence is optimized according to the codon characteristics of Sus Scrofa. The vaccine constructed by the present invention can effectively induce specific immunity in immunized animals and provide a scientific technical means for the prevention and control of PDCoV in clinical applications.

Description

Technical Field

[0001] The present invention relates to the technical field of vaccine preparation, and particularly to a porcine deltacoronavirus S protein and a recombinant adenovirus expressing the protein. Background Art

[0002] Porcine deltacoronavirus (PDCoV) is a highly contagious porcine enteric coronavirus, which is mainly characterized by watery diarrhea, acute diarrhea in infected pig herds and high morbidity in neonatal piglets at low ages, and has been reported in breeding areas across the country. The fecal-oral route is the main transmission route of PDCoV, and feces, vomitus and other pollutants are the main sources of virus transmission. In the diarrhea group, viral RNA persists in feces for 19 - 28 days and persists in oral secretions for up to 42 days.

[0003] After PDCoV infects neonatal piglets or conventional piglets at 5 - 21 days of age by oral administration, clinical symptoms such as diarrhea and vomiting appear 1 - 3 days after infection. PDCoV replication is limited to the epithelial cells of the small intestine and large intestine, and can cause atrophy and acute necrosis of small intestinal villi after infection. In the acute phase of infection, PDCoV antigen mainly exists in the atrophied middle jejunum, and a small amount exists in the duodenum, proximal jejunum and cecum. It occasionally exists in the crypt epithelial cells, lamina propria, Peyer's patches and mesenteric lymph nodes of the jejunum and ileum.

[0004] Serum-negative pigs of all ages are susceptible to PDCoV. In serum-negative breeding farms, the morbidity of piglets can reach up to 100%. It has been reported that the mortality rate in breeding farms in the United States can reach up to 40%, and the outbreak of PDCoV in breeding farms in China and Thailand in Asia can cause 64% - 80% of neonatal piglets to die.

[0005] At present, there are no specific drugs and vaccines available, so developing a safe and effective PDCoV vaccine is of great significance for the prevention and control of this disease clinically. Summary of the Invention

[0006] The purpose of the present invention is to provide a porcine deltacoronavirus S protein and a recombinant adenovirus, which can effectively induce specific immunity in immunized animals, are expected to be applied clinically, and provide a scientific technical means for the prevention and control of PDCoV.

[0007] To solve the above technical problems, the technical solution adopted by the present invention is:

[0008] Design a porcine deltacoronavirus S protein, and the specific method is as follows:

[0009] Based on the S protein sequence of the PDCoV SC strain, the MDAMKRGLCCVLLLCGAVFVSN TPA secretion signal peptide sequence was inserted at the N-terminus, the YIPEAPRDGQAYVRKDGEWVLLSTFGSGHHHHHH tag was inserted at the C-terminus, and the S1 and S2 cleavage sites were mutated to GGSGGGGSGGGS. After sequence optimization according to the codon characteristics of Sus Scrofa, it was synthesized and directionally inserted between the EcoRⅠ and HindⅢ restriction sites of the pDC316-mCMV-EGFP vector. The constructed vector was named pDC316-PDCoV-S.

[0010] Among them, the amino acid sequence is shown in SEQ ID NO.1.

[0011] Among them, the nucleotide sequence is shown in SEQ ID NO.2.

[0012] Furthermore, after sequence optimization according to the codon characteristics of Sus Scrofa, it was synthesized and directionally inserted between the EcoRⅠ and HindⅢ restriction sites of the pDC316-mCMV-EGFP vector.

[0013] Furthermore, the pDC316-PDCoV-S vector and pBHGloxdelE13cre were co-transfected into 293A cells to obtain a recombinant adenovirus expressing the S protein of porcine deltacoronavirus.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] The PDCoV S protein expressed by the present invention has the S protein of the SC strain as the backbone, but the MDAMKRGLCCVLLLCGAVFVSN secretion signal peptide sequence is inserted at its N-terminus, the YIPEAPRDGQAYVRKDGEWVLLSTFGSGHHHHHH tag is inserted at the C-terminus, and the S1 and S2 cleavage sites are mutated to GGSGGGGSGGGS, thereby obtaining a sequence that can secrete and express the S fusion protein. After sequence optimization according to the codon characteristics of Sus Scrofa, it was synthesized and directionally inserted between the EcoRⅠ and HindⅢ restriction sites of the pDC316-mCMV-EGFP vector, and then a recombinant vector expressing the PDCoV S protein was obtained. Furthermore, a recombinant adenovirus expressing the PDCoV S protein was prepared using this vector. Immunizing animals with the recombinant adenovirus constructed by the present invention can effectively induce specific immunity in the immunized animals and is expected to be applied clinically, providing a scientific technical means for the prevention and control of PDCoV. Description of the Drawings

[0016] To more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required for the embodiments. It should be understood that the following drawings only show some embodiments of the present invention, and therefore should not be regarded as a limitation of the scope. For those of ordinary skill in the art, without creative efforts, other related drawings can also be obtained based on these drawings.

[0017] Figure 1 This is the cytopathic effect diagram of the rADV-DS strain of the present invention. Specific embodiments

[0018] In the following text, only some exemplary embodiments are briefly described. As those skilled in the art can recognize, the described embodiments can be modified in various different ways without departing from the spirit or scope of the embodiments of the present invention. Therefore, the drawings and the description are considered to be exemplary in nature rather than restrictive. The embodiments of the present invention will be described in detail below with reference to the drawings.

[0019] Example 1

[0020] This example discloses a recombinant adenovirus expressing the S protein of porcine deltacoronavirus, and the S protein has the following characteristics:

[0021] Using the S protein sequence of the PDCoV SC strain as the backbone, the MDAMKRGLCCVLLLCGAVFVSN TPA secretion signal peptide sequence is inserted at the N-terminus, the YIPEAPRDGQAYVRKDGEWVLLSTFGSGHHHHHH tag is inserted at the C-terminus, and the S1 and S2 cleavage sites in the middle are mutated to GGSGGGGSGGGS. After sequence optimization according to the codon characteristics of Sus Scrofa, it is synthesized and directionally inserted between the EcoRⅠ and HindⅢ restriction enzyme sites of the pDC316-mCMV-EGFP vector. The constructed vector is named pDC316-PDCoV-S.

[0022] Among them, the amino acid sequence expressed by the S protein is shown in SEQ ID NO.1.

[0023] SEQ ID NO.1:

[0024] MDAMKRGLCCVLLLCGAVFVSNQRALLIMTLLCLVRAKFADDLLDLLTFPGAHRFLHKPTSNSTSLHSRANNFDVGVLPGYPTKNVNLFSPLTNSTLPINGLHRSYQPLMLNCLTKITNHTLSMYLLPSEIQTYSCGGAMVKYQTHDAVRIILDLIATDHISVEVVGQHGENYVFVCSEQFNDTTALHKSTFFSLNSALYCFINNTYLGILPPDLTDFTVYRTGQFYANGYLLGTLPITVNYVRLYRGHLPANSAHFALANLTDTLITLTNTTISQITYCDKSVVDSIACQRSSHEVEDGFYSDPKSAVRARQRTIVTLPKLPELEVVQLNISAHMDFGEARLDSVTINGNTSYCVTKPYFRLETNFMCTGCTMNLRTDTCSFDLSAVNNGMSFSQFCLSTESGACEMKIIVTYVWKYLLRQRLYVTAVEGQTHTGTTSVHATDTSSVITDVCTDYTIYGVSGTGIIKPSDLLLHNGIAFTSPTGELYAFKDITTGKTLQVLPCETPSQLIVINNTVVGAITSSNSTENNRFTTTIVTPTFFYSTNATTFNCTKPVLSYGPIS GGSGGGGSGGGSGEVEIPSAFSLSVQTEYLQVQAEQVIVDCPQYVCNGNSRCLQLLAQYTSACSNIEAALHSSAQLDSREIINMFQTSTQSLQLANITNFKGDYNFSSILTTRLGGRSAIEDLLFNKVVTSGLGTVDQDYKACSRDMAIADLVCSQYYNGIMVLPGVVDAEKMAMYTGSLTGAMVFGGLTAAAAIPFATAVQARLNYVALQTNVLQENQKILAESFNQAVGNISLALSSVNYAIQQTSEALNTVAIAIKKIQTVVNQQGEALSHLTAQLSNNFQAISTSIQDIYNRLEEVEANQHVDRLITGRLAALNAYVTQLLNKMSQIRQSRLLAQQKINECVKSQSSRYGFCGNGTHIFSLTQTAPNGIFFMHAVLVPNKFTRVNASAGICVDNTRGYSLQPQLILYQFNNSWRVTPRNMYEPRLPRQADFIQLTDCSVTFYNTTAANLPNIIPDVIDVNQGSG YIPEAPRDGQAYVRKDGEWVLLSTF GSGHHHHHH。

[0025] Furthermore, the nucleotide sequence is as shown in SEQ ID NO.2.

[0026] SEQ ID NO.2:

[0027]

[0028] To facilitate further understanding of the present invention by those skilled in the art, the following will further elaborate on this application in combination with specific cases.

[0029] 1. Construction of recombinant adenovirus vaccine expressing PDCoV S protein:

[0030] 1.1 Construction of recombinant adenovirus vector:

[0031] Based on the S protein sequence of PDCoV SC strain, the MDAMKRGLCCVLLLCGAVFVSN TPA secretion signal peptide sequence was inserted at the N-terminus, and YIPEAPRDGQAYVRKDGEWVLLSTF the GSGHHHHHH tag was inserted at the C-terminus. The S1 and S2 cleavage sites in the middle were mutated to GGSGGGGSGGGS, and then it was synthesized by Nanjing Genscript Biotech Co., Ltd. according to the codon characteristics of Sus Scrofa and directionally inserted between the EcoRⅠ and HindⅢ restriction enzyme sites of the pDC316-mCMV-EGFP vector. The constructed vector was named pDC316-PDCoV-S;

[0032] The amino acid sequence designed in the present invention is shown in SEQ ID NO.1, and the corresponding nucleotide sequence is shown in SEQ ID NO.2.

[0033] 1.2 Small-scale preparation and purification of recombinant adenovirus rADV-DS strain:

[0034] Passage 293A cells to a 12-well plate according to the conventional method. When the cell confluence is about 50%, use a transfection reagent (FuGENE® HD, Promega) to transfect the pDC316-PDCoV-S plasmid and the pBHGloxdelE13cre plasmid. 6 μl of the transfection reagent is used, and 1 μg of each of the pDC316-PDCoV-S plasmid and the pBHGloxdelE13cre plasmid is used. After transfection, the cells are cultured in an incubator at 37°C and 5% carbon dioxide. Observe the cell status daily. Five days after transfection, cytopathic plaques were observed. As Figure 1 shown, continue to culture for 48 hours, and the number of diseased cells increases. The cells are frozen and thawed three times at -80°C, centrifuged at 12000 rpm, and stored at -80°C, designated as the P1 generation virus of the rADVDS strain. Figure 1 In, from left to right are control cells, CPE lesion conditions 5 days and 7 days after transfection.

[0035] Passage 293A cells to a 96-well plate according to the conventional method. Take the P1 generation virus of the rADV DS strain stored at -80°C and perform 10-fold serial dilutions using serum-free DMEM cell culture medium. Take 10 1 ~10 8The inoculum of the dilution was added to the culture wells containing the 293A cell suspension. 100 μl of the virus dilution was added to each well, and there were 24 wells for each dilution gradient. After adding the virus dilution, it was cultured in an incubator at 37 °C with 5% carbon dioxide, and observed daily. When single plaque holes appeared in the lowest dilution, the virus solution in the holes was taken to inoculate 293A for expanded culture. When the cytopathic effect reached 80%, the virus was harvested, frozen and thawed once at -80 °C, centrifuged at 12,000 rpm for 5 min, the supernatant was taken, aliquoted and stored at -80 °C, denoted as the P3 generation virus of the rADV DS strain.

[0036] The P3 generation virus of the rADV DS strain was inoculated into 293A cells according to the conventional method and continuously cultured and propagated to the P5 generation virus of the rADV DS strain. All harvested viruses were stored at -80 °C.

[0037] 293A cells were passaged to a T175 cell flask according to the conventional method. After the cells grew into a confluent monolayer, the cell culture medium was removed, 0.5 ml of the P3 generation virus of the rADV DS strain was inoculated, adsorbed in a cell incubator at 37 °C for 60 minutes, and then 30 ml of DMEM culture medium containing 2% calf serum was added to the inoculated cell flask. It was cultured in an incubator at 37 °C with 5% carbon dioxide. After more than 90% of the cells showed cytopathic effect, the virus solution was harvested, centrifuged at 12,000 rpm for 10 minutes, the supernatant was added to a 100 KD ultrafiltration centrifugal tube (purchased from MERCK), centrifuged at 5,000 rpm until the volume was reduced to 1 / 10 of the original volume, 0.1 M phosphate buffer was added to the original volume, centrifuged at 5,000 rpm until the volume was reduced to 1 / 100 of the original volume, and finally glycerol was added in a ratio of 5:1, aliquoted and stored at -80 °C.

[0038] 293A cells were passaged to a 96-well plate according to the conventional method and cultured in an incubator at 37 °C with 5% carbon dioxide. The P4 and P5 generation viruses of the rADVDS strain and the purified virus were serially diluted 10-fold with 2% serum DMEM to 10 12 , and the diluted viruses were inoculated into the passaged 293A cells, with 8 wells inoculated for each dilution. After inoculation, it was cultured in an incubator at 37 °C with 5% carbon dioxide for 6 days. The cultured 96-well plate was placed under an inverted fluorescence microscope to observe whether cytopathic effect occurred in each dilution well, record the number of cytopathic wells at each dilution, and calculate the TCID of the P4 and P5 generation and purified viruses of the rADV DS strain according to the Reed-Muench method 50 which were 10 7.5 TCID 50 / ml, 10 9 TCID 50 / ml, 10 11 TCID 50 / ml.

[0039] 1.3 Determination of Humoral Immune Effect of rADV-DS Strain in Mice

[0040] Purchase 24 SPF-grade Kunming mice aged 6 - 8 weeks (from Chengdu Dashuo Biotechnology Co., Ltd.), randomly divide them into 4 groups. Take the purified rADV DS strain purified virus and dilute it with serum-free DMEM, and then inject 10 6 、10 7 、10 8 TCID 50 of rADV PS strain virus by intramuscular injection in the leg, and the injection volume is 100 μl for all. Collect sera before immunization, 2 weeks after immunization, and 4 weeks after immunization to measure specific antibodies.

[0041] Use the PDCoV S protein polypeptide FYSDPKSAV, add a cysteine residue at the C-terminus for BSA conjugation. The polypeptide is synthesized by Nanjing Genscript Biotechnology Co., Ltd. and labeled with BSA protein. Take the synthesized and conjugated BSA polypeptide (purity 99%), dissolve it in sterile water for injection to 1 mg / ml, and then dilute it to 4 μg / ml with 0.05 M pH 9.6 carbonate buffer. Take an enzyme-linked immunosorbent assay (ELISA) plate, add 100 μl of the diluted polypeptide solution to each well, and coat it at 2 - 8°C for 12 - 16 hours; remove the coating solution containing the polypeptide, add 0.1 ml of blocking solution (0.15 M PBS, 0.05% Tween 20, 3% BSA, pH 7.4) to each well, and block it at 37°C for 3 h; remove the blocking solution, wash it 5 times with PBST (0.15 M PBS, 0.05% Tween 20, pH 7.4); dilute the collected sera 100-fold, add 100 μl of the diluted sera to each well, set up a dilution control well, and incubate at room temperature for 60 min; remove the sera, wash it 5 times with PBST; add 100 μl of 1:5000 diluted HRP-labeled rabbit anti-mouse secondary antibody (purchased from Sangon Biotech (Shanghai) Co., Ltd.) to each well, incubate at room temperature for 30 min, and wash it 5 times with PBST; add 100 μl of TMB chromogenic solution, incubate at room temperature for 15 min, add 50 μl of 2 M sulfuric acid to terminate the reaction, measure the OD450 light absorption value, and judge as positive when the A450nm of the measurement well is > 2.1 compared with the negative control well. The antibody measurement results after immunization with rADV DS strain are shown in Table 1.

[0042] Table 1 shows the antibody measurement results after immunization with rADV DS strain

[0043]

[0044] As can be seen from Table 1, the antibodies in the immunized groups are all positive, and the 10 8 TCID50 group of rADV DS strain is higher than 10 7 TCID50 group, indicating that the rADV DS strain can induce specific humoral immune responses in mice. The present invention can effectively induce specific immunity in immunized animals and is expected to be applied clinically to provide scientific technical means for the prevention and control of PDCoV.

[0045] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications to these embodiments once they learn the basic creative concept. Therefore, the appended claims are intended to be construed as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0046] The foregoing is only a preferred embodiment of the present invention and is not intended to limit the present invention. It should be noted that any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention should be included within the protection scope of the present invention.

Claims

1. A porcine deltacoronavirus S protein, characterized in that, The porcine deltacoronavirus S protein has the following characteristics: Based on the S protein sequence of the PDCoV SC strain, the MDAMKRGLCCVLLLCGAVFVSN TPA secretion signal peptide sequence was inserted at the N-terminus, the YIPEAPRDGQAYVRKDGEWVLLSTFGSGHHHHHH tag was inserted at the C-terminus, and the S1 and S2 cleavage sites were mutated to GGSGGGGSGGGS. After sequence optimization according to the codon characteristics of Sus Scrofa, it was synthesized and directionally inserted between the EcoRⅠ and HindⅢ restriction sites of the pDC316-mCMV-EGFP vector. The amino acid sequence expressed by the S protein is shown in SEQ ID NO.

1.

2. The porcine deltacoronavirus S protein according to claim 1, characterized in that: The nucleotide sequence of the porcine deltacoronavirus S protein is shown in SEQ ID NO.

2.

3. A recombinant adenovirus expressing the S protein of porcine deltacoronavirus, characterized in that: It contains the nucleotide sequence of the porcine deltacoronavirus S protein described in claim 2.

4. A vaccine, characterized in that: It contains the recombinant adenovirus expressing the porcine deltacoronavirus S protein described in claim 3.

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