DNA vaccine of porcine epidemic diarrhea virus and application of DNA vaccine

Abstract

The present invention relates to a polynucleotide encoding a spine protein of a Porcine Epidemic Diarrhea Virus (PEDV), and to a method for encoding a spine protein of a Porcine Epidemic Diarrhea Virus (PEDV), and to a polynucleotide encoding a spine protein of a Porcine Epidemic Diarrhea Virus (PEDV), an expression vector containing the polynucleotide; the present invention relates to a porcine epidemic dysentery virus expression vector, and a DNA vaccine containing the expression vector, the DNA vaccine being administered in an oral or injection manner, and the DNA vaccine being used for preventing and alleviating symptoms caused by porcine epidemic dysentery viruses.

Description

Technical Field

[0001] The invention relates to a DNA vaccine of porcine epidemic diarrhea virus and application thereof, in particular to a DNA vaccine for preventing and treating diseases caused by porcine epidemic diarrhea virus. Background Art

[0002] Porcine epidemic diarrhea virus (PEDV) is a virus of the genus alphacoronavirus. It mainly infects pigs. Pigs infected with PEDV will show symptoms such as vomiting, dehydration, loss of appetite, acute diarrhea, fever, etc., especially for young pigs, with a very high mortality rate. The virus has caused many major epidemics in Asia and other regions, causing immeasurable serious losses to the pork production industry and the economy.

[0003] DNA vaccine is a new type of vaccine technology that triggers the immune system's response by injecting specific pathogen gene fragments into the human or animal body to prevent and treat diseases. The above gene fragments will be transcribed and translated into proteins by cells, thereby inducing the immune system to produce an immune response to the antigen. The preparation process of DNA vaccine is relatively simple and flexible, and the gene fragments can be adjusted as needed. It does not include live viruses or bacteria, thus reducing the risk of infection and inducing a long-term immune response.

[0004] However, the development of DNA vaccines also faces many challenges. For example, the design of the delivery method is an important consideration. It is necessary to ensure that the gene fragments can enter the cells and trigger an appropriate immune response. In addition, the immune response of DNA vaccines is often insufficient and faces safety issues. The risk of any adverse reactions needs to be carefully evaluated.

[0005] In summary, although there are vaccines for porcine epidemic diarrhea virus on the market, their actual protective effect in clinical practice is not good. In addition, all current PEDV vaccines are intramuscular injection vaccines, which must rely on professionals and trained personnel for injection, making the cost of large-scale vaccination high. Therefore, how to design a vaccine that effectively prevents and treats porcine epidemic diarrhea virus with a low operating threshold and low cost is an urgent problem to be solved in this field. Summary of the invention

[0006] Based on the above content, the present invention provides a polynucleotide encoding the spike protein of porcine epidemic diarrhea virus, wherein the polynucleotide has a nucleotide sequence as shown in SEQ ID NO:1.

[0007] In one aspect, the present invention provides an expression vector comprising the polynucleotide described above.

[0008] In some embodiments, the expression vector is a pTCY expression vector.

[0009] In another aspect, the present invention provides a DNA vaccine comprising the expression vector as described above, and at least one pharmaceutically acceptable carrier, adjuvant, diluent or excipient.

[0010] In some embodiments, the DNA vaccine is administered orally.

[0011] In some embodiments, the DNA vaccine comprises an adjuvant, wherein the adjuvant is a peptide nanotube.

[0012] In some embodiments, the DNA vaccine is administered by injection.

[0013] In certain embodiments, the DNA vaccine comprises an adjuvant, wherein the adjuvant is a CpG moiety.

[0014] In another aspect, the present invention provides a use of the DNA vaccine as described above in the preparation of a pharmaceutical composition for preventing and alleviating symptoms caused by PED virus.

[0015] In some embodiments, it further comprises a booster, which is the DNA vaccine or single-unit vaccine as described above.

[0016] The present invention combines the research and development of DNA vaccines to develop a DNA vaccine that effectively prevents and treats porcine epidemic diarrhea virus. The DNA vaccine contains a polynucleotide sequence that encodes the spike protein antigen epitope of porcine epidemic diarrhea virus. Clinical animal experiments have confirmed that the DNA vaccine can be administered orally and by injection, and can actually enter cells to produce appropriate immune responses, improving the problem that DNA vaccines in the past were difficult to produce immune responses of sufficient intensity. Moreover, the present invention can be administered orally, greatly reducing the operational threshold and cost, and can be simply and effectively administered to pigs on a large scale. In addition, the DNA vaccine described in the present invention will not have adverse effects on pigs and is safe to implement. In summary, the DNA vaccine described in the present invention has great application prospects in the prevention and treatment of diseases caused by porcine epidemic diarrhea virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1This is an analysis of the IgA antibody response of pigs at 0 weeks, 3 weeks, and 6 weeks after the first administration of DNA vaccine. The average values ​​of the graphs a and b are a>b in sequence; the same graph number (e.g., between a and a) indicates no significant difference between the groups; conversely, different graph numbers (e.g., between a and b) indicate significant differences between the groups (p<0.05); and graph numbers ab indicate that this value is not significantly different from both a and b. DETAILED DESCRIPTION

[0018] The existing porcine epidemic diarrhea virus vaccines are insufficient in clinical effectiveness, especially the existing vaccines are particularly ineffective against the PED virus strains in Taiwan, China. This may be due to the variability of porcine epidemic diarrhea virus, which causes mutations in some protein regions and greatly reduces the effectiveness of vaccines developed in the past.

[0019] In view of the above shortcomings of the existing vaccines, the present invention provides a polynucleotide encoding the spike protein of porcine epidemic diarrhea virus, wherein the polynucleotide has a nucleotide sequence as shown in SEQ ID NO: 1. The polynucleotide is used to produce an effective antigen epitope of the spike protein of porcine epidemic diarrhea virus for preparing a DNA vaccine of porcine epidemic diarrhea virus.

[0020] In one aspect, the present invention provides an expression vector comprising the polynucleotide described above, wherein the expression vector may be in any form, including but not limited to plasmid vectors, viral vectors, cosmid vectors, phage plasmid vectors, artificial human chromosomes, etc.

[0021] In another aspect, the present invention provides a DNA vaccine comprising the expression vector as described above, and at least one pharmaceutically acceptable carrier, adjuvant, diluent or excipient.

[0022] In some embodiments, the DNA vaccine is administered orally. Oral administration is simple to operate, has low cost, and reduces the stress of pigs.

[0023] In some embodiments, the DNA vaccine comprises an adjuvant, wherein the adjuvant is a peptide nanotube.

[0024] In some embodiments, the DNA vaccine is administered by injection.

[0025] In certain embodiments, the DNA vaccine comprises an adjuvant, wherein the adjuvant is a CpG moiety.

[0026] In another aspect, the present invention provides a use of the DNA vaccine as described above in the preparation of a pharmaceutical composition for preventing and alleviating symptoms caused by PED virus, wherein the symptoms caused by the PED virus include but are not limited to vomiting, dehydration, anorexia, weight loss, diarrhea, and fever.

[0027] In certain embodiments, the pharmaceutical composition is administered orally to induce the production of gastrointestinal mucosal protective IgA antibodies.

[0028] In some embodiments, it further comprises a booster, which is the DNA vaccine or the single-unit vaccine of the present invention as described above. The booster is used in a common vaccine immunization strategy, which is a Prime-boost immunization strategy.

[0029] It should be understood that the above general description and the following detailed description are exemplary and illustrative, but are not intended to limit the scope of the rights requested by the present invention. Certain details of one or more embodiments of the present invention are described in the following description. From the non-exhaustive list of the following representative embodiments, other features or advantages of the present invention will be apparent from the appended claims.

[0030] Unless otherwise defined below, all scientific and technical terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs. References to the techniques used in the present invention are intended to indicate techniques commonly understood in the art, including variants or equivalents of those techniques or alternative techniques developed later, which are obvious to those skilled in the art.

[0031] It should be noted that, as used herein, the singular terms "a", "an", and "the" include plural referents unless expressly limited to one referent. The term "or" is used interchangeably with the term "and / or" unless the context clearly indicates otherwise.

[0032] The term "about", "approximately" or "approximately" as used herein substantially represents that the numerical value or range is within 20%, preferably within 10%, and more preferably within 5%. The numerical quantities provided in the present invention are approximate values, which means that they can be inferred even if the term "about", "approximately" or "approximately" is not used.

[0033] As used herein, the word "comprising" is open-ended, indicating that such embodiments may include additional elements. Conversely, the word "consisting of" is closed-ended, indicating that such embodiments do not include additional elements (except for trace impurities). The word "consisting essentially of" is partially closed-ended, indicating that such embodiments may also include elements that do not substantially change the basic characteristics of such embodiments.

[0034] Unless otherwise defined in the present invention, the scientific and technical terms used in combination with the present invention should have the meanings commonly understood by those of ordinary skill in the art. In addition, unless the context otherwise requires, singular terms should include the majority, and the majority of terms should include the singular. The methods and techniques of the present invention can generally be carried out according to conventional methods known in the art. In general, the nomenclature described in the present invention for connecting the following technologies, and the techniques of biochemistry, enzyme science, molecular and cell biology, microbiology, genetics, immunology, protein and nucleic acid chemistry and hybrid reactions are all known in the art and frequently used. Unless otherwise indicated, the methods and techniques of the present invention can generally be carried out according to conventional methods known in the art, and are described in various general and more specific references cited and discussed in this specification.

[0035] As used in the present invention, the term "nucleotide sequence" also includes nucleotide sequences having at least 95% identity, including nucleotide sequences having at least 95%, 96%, 97%, 98% or 99% identity.

[0036] As used in the present invention, the term "pharmaceutically acceptable" refers to a composition that is physiologically acceptable for use in living organisms and generally does not cause allergic reactions, such as gastrointestinal problems, fever, etc. As used in the present invention, the term "carrier, excipient and diluent" includes but is not limited to lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, polyvinyl hydropyrrolidone, hydroxybenzoate, talc, magnesium stearate and mineral oil. In addition, fillers, anticoagulants, lubricants, moisturizers, fragrances, emulsifiers, preservatives, etc. are also included.

[0037] As used herein, the term "adjuvant" refers to any substance that can increase the mucosal, humoral or cellular immune response to an antigen, and is generally used to achieve two purposes: slowing the release of the antigen and stimulating the immune response. Non-limiting examples of adjuvants include: aluminum (e.g., aluminum gel and / or aluminum salts, such as aluminum hydroxide, aluminum phosphate, and potassium aluminum sulfate), lipids (e.g., squalene, monophosphoryl lipid A (MPL)), AS03 (e.g., an adjuvant including D,L-α-tocopherol (vitamin E), squalene, and polysorbate 80), AS04 (e.g., an adjuvant including a combination of aluminum hydroxide and MPL), and (For example, an adjuvant comprising squalene).

[0038] As used in the present invention, the term "peptide nanotube" refers to a DNA vaccine adjuvant, wherein the peptide nanotube is a long peptide nanotube and / or a short peptide nanotube, which is used to enhance the mucosal immune response. For a detailed description of the peptide nanotube, please refer to Taiwan Patent No. I756948, which is incorporated herein by reference in its entirety. The term "mucosal immune response" refers to the reaction produced when the mucosal immune system of an animal (including humans) is triggered by foreign substances invading the mucosal immune system, mainly through secretory immunoglobulin A (IgA), which is mainly present in saliva, milk, gastrointestinal fluid, respiratory secretions, etc., and is the first line of defense of the mucosal site against pathogenic microorganisms and harmful substances.

[0039] As used in the present invention, the term "CpG module" refers to a DNA vaccine adjuvant, which contains multiple sets of specific immunostimulatory oligonucleotides or recombinant plasmids containing the same, and does not need to be phosphosulfurized. For a detailed description of the CpG module, please refer to Taiwan Patent No. I425091, which is incorporated into the present invention in its entirety by reference.

[0040] As used in the present invention, the term "spike protein of porcine epidemic diarrhea virus" consists of 1,386 amino acids, and the structural domain of the protein can be divided into the S1 region (amino acids 227-633) and the S2 region (amino acids 640-1,322).

[0041] As used in the present invention, the term "pTCY vector" is derived from the pEGFP-N1 vector (Clontech, Mount View, California, USA), wherein the CMV immediate-early promoter on the pEGFP-N1 vector is replaced with a beta-actin promoter, and the coding region of the enhanced green fluorescent protein is removed, so that other target genes can be inserted and expressed under the control of the beta-actin promoter. For detailed construction of the pTCY vector, please refer to H.-C. Wu et al. (2019) A DNA priming and protein boosting immunization scheme to augment immune responses against parvovirus inducks. J. Appl. Microbiol. 126 (1): 49-57.

[0042] As used in the present invention, the term "Prime-boost immunization strategy" refers to a common vaccine immunization strategy, which stimulates the immune system by using vaccines in two stages to achieve a more effective and lasting immune response. The original immunization (Prime) is for the individual to receive the first dose of vaccine in the first stage to initiate the immune response. This stage is also called the initial immunization, which aims to induce the initial immune memory, but the immune response may be relatively weak. Enhanced immunity (Boost) is a period of time after the original immunization, when the individual receives the second dose of vaccine. This stage is also called booster immunization. The purpose is to stimulate the immune system, strengthen the immune response and prolong the existence of immune memory, thereby providing longer-term protection.

[0043] As used herein, the term "boost" refers to the second dose of vaccine used in the Prime-boost immunization strategy, also known as a booster.

[0044] As used in the present invention, the term "mucosal immune response" refers to the reaction produced when the mucosal immune system of animals (including humans) is triggered when foreign substances invade the mucosal immune system. It mainly reacts through secretory immunoglobulin A (IgA), which is mainly present in saliva, milk, gastrointestinal fluid, respiratory secretions, etc., and is the first line of defense of the mucosal site against pathogenic microorganisms and harmful substances.

[0045] The present invention is further illustrated by the following examples, which should not be interpreted as further limiting in any way. The entire contents of all references cited in the present invention (including references, authorized patents, published patent applications, and patent applications in the same application) are hereby expressly incorporated into the present invention by reference.

[0046] The data statistics in the embodiment of the present invention are statistically analyzed by the least significant difference method (LSD method) after ANOVA analysis. Among them, the average values ​​of the figure numbers a and b are a>b in sequence; the same figure number (for example, between a and a) indicates that there is no significant difference between the groups; on the contrary, different figure numbers (for example, a and b) indicate that there is a significant difference between the groups (p<0.05); as for the figure number ab, it means that this value is not significantly different from both a and b.

[0047] Example 1. Preparation of porcine epidemic diarrhea virus vaccine

[0048] 1.1 Preparation of DNA vaccines

[0049] In this embodiment, the preparation of the DNA vaccine includes: synthesizing the sequence and transferring it into the expression vector; culturing the successfully transferred expression vector in large quantities, extracting the plasmid DNA, and mixing it with an adjuvant after quantification for subsequent clinical animal experiments.

[0050] In this embodiment, the polynucleotide of the present invention has a nucleotide sequence as shown in SEQ ID NO: 1. After the nucleotide sequence is synthesized, it is cloned into the pTCY expression vector to serve as the expression vector of the DNA vaccine of the present invention. The expression vector can drive the successfully cloned nucleotide sequence at the back end to express the PEDV spike protein through the β-actin promoter.

[0051] In this embodiment, after the above-mentioned successfully transfected expression vector is cultured in large quantities, plastid DNA is extracted with a large number of plastid extraction kits, and after quantification, an adjuvant is mixed with 50% by weight to complete the preparation of the DNA vaccine. In some specific embodiments, the adjuvant used in the DNA vaccine administered orally is peptide nanotubes. The exemplary configuration in the embodiment of the present invention is to take 1mL of 260μg / mL plastid DNA and 1mL of 260μg / mL peptide nanotubes to prepare the oral DNA vaccine of the present invention, and the oral DNA vaccine administration dose in the experiment of the present invention is 2mL per pig. In some specific embodiments, the adjuvant used in the DNA vaccine administered by intramuscular injection is a CpG module. The exemplary configuration in the embodiment of the present invention is to take 1mL of 100μg / mL plastid DNA and 1mL of 100μg / mL CpG module to prepare the DNA vaccine for intramuscular injection of the present invention, and the intramuscular injection of the DNA vaccine in the experiment of the present invention is 2mL per pig.

[0052] 1.2 Preparation of unit vaccines

[0053] In this embodiment, the preparation of the conventional subunit vaccine is to emulsify the PEDV spike protein S1 recombinant protein (whose amino acid sequence is shown in SEQ ID NO: 2) at a weight ratio of 50% with ISA 206 adjuvant (SEPPIC, France), and after confirming sterility, administer it to pigs by intramuscular injection. The exemplary configuration in the embodiment of the present invention is to take 1 mL of 50 μg / mL PEDV spike protein S1 recombinant protein and mix it with 1 mL of ISA 206 adjuvant to prepare the subunit vaccine for intramuscular injection of the present invention, and the dosage of the subunit vaccine administered in the experiment of the present invention is 2 mL per pig.

[0054] Example 2. DNA vaccine test of porcine epidemic diarrhea virus

[0055] In this example, in order to test the efficacy and safety of the DNA vaccine of the present invention for preventing and treating porcine epidemic diarrhea virus, 3-week-old specific pathogen free (SPF) piglets were divided into 4 groups, each with 3 piglets, namely, a DNA vaccine oral group, a DNA vaccine injection group, and a blank control group (control), and a Prime-boost immunization strategy was carried out, and then the efficacy was tested by a PEDV challenge test. Among them, the blank control group was administered with 2 mL of saline. In addition, the preparation and administration of the vaccine were as described in Example 1. According to the group design, the piglets were initially immunized with a DNA vaccine at 3 weeks of age, and boosted with the same vaccine type and the same administration method 2 weeks after the initial immunization (i.e., when the piglets were 5 weeks old). For example, the DNA vaccine oral group was administered with a DNA vaccine orally at 3 weeks of age to complete the initial immunization, and the DNA vaccine was administered orally again at 5 weeks of age to complete the booster immunization. After the above groups of experimental pigs completed the Prime-boost immunization strategy, a PEDV challenge test was performed one week after the booster immunization (i.e., when the piglets were 6 weeks old) to verify the efficacy of the vaccine.

[0056] 2.1 Security Testing

[0057] The body temperature of the piglets in each group was measured at 0 hours, 4 hours, 24 hours and 7 days after the initial immunization. The temperature measurement results are shown in Table 1 below. The body temperature of each group was within the normal range of 39.4°C, indicating that the piglets in the DNA vaccine oral group and the DNA vaccine injection group did not develop symptoms of fever.

[0058] Table 1. Average body temperature after primary immunization (°C)

[0059]

[0060] In addition, the weight changes of piglets in each group were measured one week after the first immunization. The weight measurement results are shown in Table 2 below. There was no significant difference in weight gain between the groups 1 week after the first administration of DNA vaccine and the blank control group, indicating that the piglets in the DNA vaccine oral group and the DNA vaccine injection group did not develop symptoms such as anorexia.

[0061] Table 2. Average weight of piglets 1 week after primary vaccination (kg)

[0062] Grouping Before vaccination 1 week after vaccination Weekly weight gain DNA vaccine oral group 7.48±0.58 9.08±1.12 1.6±0.57 DNA vaccine injection group 7.16±0.64 9.21±0.52 2.05±0.60 Blank control group 7.01±0.38 8.53±0.11 1.52±0.27

[0063] The results of this example confirm that the DNA vaccine of the present invention has good safety. The test pigs did not have any adverse reactions after administration of the vaccine and gained weight well.

[0064] 2.2 PEDV challenge test

[0065] One week after the Prime-boost immunization strategy was completed, when the pigs were 6 weeks old, the PEDV G2 virus strain was administered by nasal injection for a challenge test. The virus liquid concentration was 1x 10 6 TCID 50 / mL, each pig was administered 5mL of virus solution. After the virus attack, the clinical symptoms of each group were analyzed and scored. The symptoms and scoring methods are shown in Table 3 below.

[0066] Table 3. Scoring of clinical symptoms after PEDV challenge

[0067]

[0068] The clinical symptom scores of each group were counted for 7 days, and the statistical results are shown in Table 4 below. The total clinical symptom score of the blank control group after the virus challenge was as high as 52 points, indicating that the pigs developed clinical symptoms of PEDV infection after the virus challenge; however, the total clinical symptom scores of the pigs in the DNA vaccine oral group and the DNA vaccine injection group were significantly (p<0.05) reduced, down to 7 and 8 points, respectively, indicating that the DNA vaccine of the present invention can effectively prevent and alleviate the symptoms caused by the PED virus.

[0069] Table 4. Clinical symptom records and statistical scores of each group after PEDV challenge

[0070] Grouping Diarrhea Anorexia Dehydration and weight loss Back hair cover Total score DNA vaccine oral group 7 0 0 0 <![CDATA[7 b ]]> DNA vaccine injection group 6 0 0 2 <![CDATA[8 b ]]> Blank control group 13 15 12 12 <![CDATA[52 a ]]>

[0071] In addition, in this example, the weight gain of the pigs was integrated and statistically analyzed before and after the virus challenge, and the results are shown in Table 5. The pigs in the blank control group had an average weight gain of 0.8 kg 7 days after the virus challenge, which was significantly (p<0.05) lower than the weight gain of the pigs in the DNA vaccine oral group and the DNA vaccine injection group (3.8-3.9 kg), indicating that the DNA vaccine of the present invention has the effect of slowing down the weight loss or anorexia caused by PED virus.

[0072] Table 5. Average body weight of each group before and after challenge (kg)

[0073]

[0074]

[0075] The results of this example demonstrate that the DNA vaccine of the present invention can be effectively used to prevent and alleviate the symptoms caused by PED virus by oral administration or intramuscular injection.

[0076] Example 3. Immunization combinations of different types of PEDV vaccines

[0077] In order to test the efficacy of the Prime-boost immunization strategy of the DNA vaccine combined with the subunit vaccine described in the present invention, 3-week-old SPF piglets were divided into 3 groups, each with 3 piglets, and each group was a DNA vaccine oral group, a DNA vaccine injection group, and a blank control group. The Prime-boost immunization strategy was carried out, and the vaccine efficacy was subsequently detected by a PEDV challenge test. According to the group design, the piglets were first immunized with an oral DNA vaccine or an injection of a DNA vaccine at 3 weeks of age, and the subunit vaccine was administered as a booster immunization 2 weeks after the initial immunization (i.e., when the piglets were 5 weeks old). The preparation and administration of the vaccine are as described in Example 1. For example, the DNA vaccine oral group was administered with a DNA vaccine orally at 3 weeks of age to complete the primary immunization, and the subunit vaccine was administered at 5 weeks of age to complete the booster immunization. In addition, the blank control group was administered with 2 mL of saline at the time of the initial immunization, and 2 mL of saline was administered again at 5 weeks of age.

[0078] 3.1 Analysis of IgA antibodies in serum

[0079] After the initial immunization, serum was collected from each group at 0, 3, and 6 weeks after the first immunization and the IgA antibody reaction in the serum was analyzed. The IgA antibody titer against PEDV in the serum was tested by enzyme immunoassay (ELISA). 25 μg / mL of deactivated PEDV was used as the ELISA bottom antigen. The sample was a 16-fold diluted sample serum. Mouse anti-IgA secondary antibody (mouse anti-IgA-HRP, diluted 5000 times) conjugated with horseradish peroxidase (HRP) was added and the absorbance at a wavelength of 450nm (OD 450nm) was measured. The experimental results are shown in Figure 4. Figure 1 As shown, 3 weeks after the first immunization, the IgA antibody of the pigs in the DNA vaccine oral group was significantly higher (p<0.05) than that in the blank control group, indicating that the DNA vaccine of the present invention can effectively induce mucosal immune response in pigs by oral administration to prevent and alleviate the symptoms caused by PED virus.

[0080] 3.2 PEDV challenge test

[0081] One week after the Prime-boost immunization strategy was completed, the PEDV G2 virus strain was administered to the pigs in each group by nasal injection for PEDV challenge test. The virus liquid concentration was 1x 10 6 TCID 50 / mL, 5mL of virus liquid was administered to each pig, and the temperature changes were measured at 0 hours, 4 hours, 1 day, 2 days, 3 days, 4 days, and 5 days after the virus attack. The results are shown in Table 6 below. The pigs in the blank control group had a significant increase in body temperature 2 days after the virus attack, and exceeded the normal body temperature range, indicating that the pigs in the blank control group had symptoms of fever, and then dropped to the normal range; the body temperatures of the pigs in the DNA vaccine oral group and the DNA vaccine injection group were within the normal body temperature range after the virus attack. The results show that after the Prime-boost immunization strategy of the present invention, pigs can effectively prevent the temperature rise caused by PEDV infection, indicating that the DNA vaccine of the present invention has the ability to prevent and alleviate the fever symptoms caused by PED virus.

[0082] Table 6. Average body temperature after PEDV challenge (°C)

[0083]

[0084] Note: A body temperature exceeding the normal range of 39.4°C indicates clinical symptoms of fever.

[0085] The clinical symptoms of the pigs in each group were observed after PEDV challenge to record and analyze the clinical symptoms. The symptoms included diarrhea, dehydration and weight loss, anorexia, vomiting, depression and hair loss on the back. The characteristics and scoring methods of each symptom are shown in Table 3 above. The clinical symptom scores of each group were counted for 7 days, and the score statistics are shown in Table 7 below. The total score of clinical symptoms of the pigs in the blank control group after PEDV challenge was as high as 44 points, indicating that the pigs had clinical symptoms of PEDV infection after the challenge. However, the total scores of clinical symptoms of the pigs in the DNA vaccine oral group and the DNA vaccine injection group were significantly (p<0.05) reduced, and the total scores dropped to 21 and 20 points, respectively, indicating that the DNA vaccine of the present invention can effectively prevent and alleviate the symptoms caused by PED virus.

[0086] Table 7. Clinical symptom records and statistical scores of each group after PEDV challenge

[0087]

[0088]

[0089] In addition, the weight gain of each group of pigs was integrated and statistically analyzed before and after the challenge, and the results are shown in Table 8. The pigs in the blank control group lost an average of 0.93 kg in weight 7 days after the challenge, while the pigs in the DNA vaccine oral group and the DNA vaccine injection group gained 0.87 and 0.02 kg, respectively, indicating that the DNA vaccine of the present invention has a significant (p<0.05) effect of slowing down the weight loss symptoms caused by PED virus.

[0090] Table 8. Average body weight of each group before and after challenge (kg)

[0091] Grouping Before attack After attack Average total weight gain / 7 days DNA vaccine oral group 20.58±4.24 21.45±5.59 <![CDATA[0.87 a ]]> DNA vaccine injection group 18.95±1.48 18.97±1.77 <![CDATA[0.02 a <!-- 8 -->]]> Blank control group 20.15±1.81 19.22±2.53 <![CDATA[-0.93 b ]]>

[0092] In summary, clinical animal experiments have confirmed that the DNA vaccine of the present invention has good safety, does not cause adverse reactions to pigs after administration, and can effectively prevent and alleviate the symptoms caused by PED virus. In addition, the DNA vaccine of the present invention can be administered to pigs orally and by injection. Compared with traditional subunit vaccines, the DNA vaccine of the present invention administered orally can significantly induce pigs to produce IgA antibodies with gastrointestinal mucosal protection, and has the advantages of simple operation and low cost, and has great application prospects in combating porcine epidemic diarrhea virus.

[0093] The present invention has been specifically demonstrated and described with reference to its preferred specific embodiments, which are only some of the preferred embodiments of the present invention, but are not intended to limit the present invention. Any equivalent changes or modifications made by any person skilled in the art after understanding the above-mentioned technical features and embodiments of the present invention and not departing from the spirit and scope of the present invention are still within the scope of protection covered by the present invention, and the patent protection scope of the present invention shall be subject to the claims.

Claims

1. A polynucleotide encoding the spike protein of porcine epidemic diarrhea virus, characterized in that: The polynucleotide has a nucleotide sequence as shown in SEQ ID NO:

1.

2. A performance carrier, characterized in that: Comprising the polynucleotide of claim 1.

3. The expression carrier according to claim 2, characterized in that: The vector is a pTCY expression vector.

4. A DNA vaccine, characterized in that: The composition comprises the expression carrier according to claim 2 or 3, and at least one pharmaceutically acceptable carrier, adjuvant, diluent or excipient.

5. The DNA vaccine according to claim 4, characterized in that It is administered orally.

6. The DNA vaccine according to claim 5, characterized in that The adjuvant is a peptide nanotube.

7. The DNA vaccine according to claim 4, characterized in that It is administered by injection.

8. The DNA vaccine according to claim 7, characterized in that The adjuvant is a CpG moiety.

9. Use of the DNA vaccine according to claim 4 in the preparation of a pharmaceutical composition for preventing and alleviating symptoms caused by porcine epidemic diarrhea virus.

10. The use according to claim 9, characterized in that It further comprises a booster, which is the DNA vaccine or the single-unit vaccine according to claim 4.

Images