Porcine epidemic diarrhea virus S protein mutant, nucleic acid molecule, subunit vaccine and application

Abstract

The present invention provides a porcine epidemic diarrhea virus S protein mutant, a nucleic acid molecule, a subunit vaccine and applications thereof; compared with the wild-type porcine epidemic diarrhea virus S protein, the porcine epidemic diarrhea virus S protein mutant has the 1076th amino acid, the 1077th amino acid, the 1016th amino acid and the 1017th amino acid all mutated into proline. The present invention constructs a stable natural trimer that maintains the prefusion conformation of the S protein, obtains a safe and highly effective PEDV subunit vaccine, and can improve the use effect and immunogenicity of the porcine epidemic diarrhea vaccine; moreover, compared with the wild-type S protein, the porcine epidemic diarrhea virus S protein mutant in the present invention has a higher expression level.

Description

Technical Field

[0001] The present invention belongs to the technical field of the research, development and application of swine coronavirus vaccines, and particularly relates to a mutant S protein of porcine epidemic diarrhea virus, a nucleic acid molecule, a subunit vaccine and applications thereof. Background Art

[0002] Porcine epidemic diarrhea (PED) is a highly contagious intestinal disease caused by porcine epidemic diarrhea virus (PEDV). PEDV is susceptible to pigs of different age groups, mainly causing diarrhea, vomiting and dehydration in pigs, and is extremely susceptible to neonatal piglets.

[0003] PEDV is currently mainly divided into two genotypes, the classical strain GI type and the variant strain GII type. The GII type is further divided into three gene subtypes, namely GII-a, GII-b and GII-c. Effective biosafety prevention and control is an effective means to block the transmission of PEDV. In addition, vaccination is one of the most effective measures for the current prevention and control of PEDV. Currently, commercial PEDV vaccines are all inactivated vaccines or attenuated live vaccines. Most of the current commercial vaccines are developed against the early classical GI type strains, and have poor cross-protection effects against the currently clinically prevalent variant GII type strains. Moreover, the clinical preventive effect of inactivated vaccines is not very ideal, while attenuated vaccines may revert to virulence, posing a great biosafety hazard.

[0004] PEDV is a member of the genus Alphacoronavirus in the family Coronaviridae, and is an enveloped, single-stranded positive-sense, linear RNA virus. The diameter of the virus particles is about 95-190 nm, and the surface of the virus particles contains spikes about 18 nm in size. The PEDV S (Spike protein, S) protein is a type I transmembrane protein embedded on the surface of the virus particles, responsible for the specific receptor binding on the cell surface of the virus and mediating the cell invasion of the virus. Antibodies generated against the S protein can effectively inhibit the invasion of the virus, and it is one of the ideal antigens for the current research and development of PEDV subunit vaccines. PEDV is an enveloped RNA virus. During the process of the virus invading host cells, the S protein on the surface of the virus particles binds to the specific receptor on the cell surface, thereby inducing a conformational change of the S protein, from the prefusion (energy-unstable) conformation to the postfusion (energy-stable) conformation. Therefore, through the research of the applicant, how to design a stable recombinant S protein trimer that can maintain the prefusion conformation of the S protein is the key to the research and development of PEDV subunit vaccines and an effective way to improve the immune effect of porcine epidemic diarrhea vaccines.

[0005] In view of this, it is necessary to provide a porcine epidemic diarrhea virus S protein mutant, a nucleic acid molecule, a subunit vaccine and an application thereof, so as to obtain a native trimer that stably maintains the pre-fusion conformation of the S protein and obtain a safe and highly effective PEDV subunit vaccine, thereby solving the technical defect that the clinical use effect of porcine epidemic diarrhea vaccine is not good. Summary of the Invention

[0006] The main object of the present invention is to provide a porcine epidemic diarrhea virus S protein mutant, a nucleic acid molecule, a subunit vaccine and an application thereof, so as to solve the technical problems of how to stably maintain the native trimer of the S protein in the pre-fusion conformation and how to improve the use effect of porcine epidemic diarrhea vaccine.

[0007] To achieve the above object, the present invention provides a porcine epidemic diarrhea virus S protein mutant, in which the 1076th amino acid, the 1077th amino acid, the 1016th amino acid and the 1017th amino acid are all mutated to proline compared with the wild-type porcine epidemic diarrhea virus S protein.

[0008] The present invention also provides a porcine epidemic diarrhea virus S protein mutant, and the S protein mutant has the amino acid sequence shown in SEQ ID NO.3.

[0009] The present invention also provides a nucleic acid molecule encoding a porcine epidemic diarrhea virus S protein mutant, and the nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO.4.

[0010] The present invention also provides a recombinant expression vector, and the recombinant expression vector has any of the above-mentioned nucleic acid molecules.

[0011] The present invention also provides an engineered cell, and the engineered cell can express any of the above-mentioned porcine epidemic diarrhea virus S protein mutants.

[0012] The present invention also provides a method for expressing a porcine epidemic diarrhea virus S protein mutant, including: constructing a recombinant expression vector and expressing it through an engineered cell; the recombinant expression vector has the nucleotide sequence shown in SEQ ID NO.4.

[0013] The present invention also provides an application of any of the above-mentioned porcine epidemic diarrhea virus S protein mutants in immunizing porcine epidemic diarrhea virus.

[0014] The present invention also provides a subunit vaccine for porcine epidemic diarrhea virus, including any of the above-mentioned porcine epidemic diarrhea virus S protein mutants.

[0015] The present invention also provides a method for preparing a subunit vaccine against porcine epidemic diarrhea virus, comprising: expressing the porcine epidemic diarrhea virus S protein as described above arbitrarily, and using the porcine epidemic diarrhea virus S protein as an immunogen.

[0016] The present invention also provides the use of a porcine epidemic diarrhea virus S protein mutant as described above arbitrarily in the preparation of antibodies against porcine epidemic diarrhea virus.

[0017] The beneficial effects of the present invention at least include:

[0018] The present invention constructs a stable native trimer that maintains the prefusion conformation of the S protein, obtains a safe and highly effective PEDV subunit vaccine, and can improve the use effect and immunogenicity of porcine epidemic diarrhea vaccines; moreover, compared with the wild-type S protein, the porcine epidemic diarrhea virus S protein mutant in the present invention has a higher expression level.

[0019] Specifically, through the research of the present invention, isoleucine (I) and leucine (L) at positions 1076-1077 of the PEDV S protein are key amino acid sites connecting the S2 subunit HR1 (Helix region 1) and CH (Central helix). Mutating 1076 IL 1077 to two prolines ( 1076 PP 1077 ) can disrupt the formation of the secondary structure. The present invention uses the same strategy to mutate serine (S) at position 1016 and valine (V) at position 1017, which connect two helical structures, to two prolines ( 1016 PP 1017 ); based on the strategy of the method for constructing the porcine epidemic diarrhea virus S protein mutant with stable and efficient expression in the present invention, the porcine epidemic diarrhea virus S protein mutant obtained in the present invention can be used to prevent the infection of porcine epidemic diarrhea virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on the structures shown in these drawings without creative efforts.

[0021] Figure 1Analysis results of the transmembrane region of the PEDV S protein in Example 1 of the present invention; in the figure, transmembrane is the transmembrane region; inside is the intracellular region; outside is the extracellular region;

[0022] Figure 2 Plasmid map of pcDNA3.4-PEDV-SWT in Example 1 of the present invention;

[0023] Figure 3 Three-dimensional structure diagram of the PEDV S4P protein in Example 2 of the present invention;

[0024] Figure 4 Plasmid map of pcDNA3.4-PEDV-S4P in Example 2 of the present invention;

[0025] Figure 5 Western blot results of the PEDV S protein in Example 3 of the present invention; in the figure, A is the Western blot map; B is the expression level analysis; 1 is the SWT protein; 2 is the S4P protein;

[0026] Figure 6 SDS-PAGE and Native-PAGE results of the PEDV S4P protein in Example 3 of the present invention; in the figure,

[0027] (a) is the SDS-PAGE map; (b) is the non-denaturing electrophoresis (Native PAGE) map; M is the standard protein molecular weight; 1 is the PEDV S4P protein; 2 is the BSA protein;

[0028] Figure 7 Results of the detection of the PEDV indirect ELISA antibody level in Example 4 of the present invention.

[0029] The realization, functional features and advantages of the object of the present invention will be further described in conjunction with the embodiments with reference to the accompanying drawings. Detailed implementation manners

[0030] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0031] Moreover, the technical solutions between various embodiments of the present invention can be combined with each other, but it must be based on the ability of those of ordinary skill in the art to implement. When the combination of technical solutions results in contradictions or cannot be implemented, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection required by the present invention.

[0032] The present invention provides a mutant S protein of porcine epidemic diarrhea virus. Compared with the wild-type porcine epidemic diarrhea virus S protein, the 1076th amino acid, the 1077th amino acid, the 1016th amino acid, and the 1017th amino acid are all mutated to proline. It should be noted that the positions of the above-mentioned amino acids correspond to the unmodified wild-type porcine epidemic diarrhea virus S protein.

[0033] In the present invention, the amino acid sequence of the wild-type porcine epidemic diarrhea virus S protein can be as shown in SEQ ID NO.1, and the nucleotide sequence encoding the wild-type porcine epidemic diarrhea virus S protein can be as shown in SEQ ID NO.2.

[0034] The S protein in the present invention can refer to the extracellular region of the porcine epidemic diarrhea virus S protein. The present invention is mainly designed for the extracellular region of the porcine epidemic diarrhea virus S protein. Specifically, the amino acid sequence of the S protein mutant is as shown in SEQ ID NO.3 or the amino acid sequence of the S protein mutant is as shown in SEQ ID NO.3. The mutant S protein of porcine epidemic diarrhea virus is the PEDV S4P protein (mutant S4P protein) in the present invention.

[0035] In the present invention, the nucleic acid molecule encoding the S protein mutant has the nucleotide sequence as shown in SEQ ID NO.4. Therefore, the present invention also provides a nucleic acid molecule encoding a mutant S protein of porcine epidemic diarrhea virus, and the nucleic acid molecule has the nucleotide sequence as shown in SEQ ID NO.4 or the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO.4.

[0036] In order to obtain the S protein mutant and highly express the S protein mutant, the present invention also provides a recombinant expression vector, an engineered cell, and an expression method for the mutant S protein of porcine epidemic diarrhea virus.

[0037] The recombinant expression vector has the nucleic acid molecule as described above, and is used to express the mutant S protein of porcine epidemic diarrhea virus as described above; the engineered cell can express the mutant S protein of porcine epidemic diarrhea virus as described above, and specifically, the mutant S protein of porcine epidemic diarrhea virus can be expressed by the nucleic acid molecule as described above.

[0038] The expression method of the porcine epidemic diarrhea virus S protein mutant includes: constructing a recombinant expression vector and expressing it through engineered cells; the recombinant expression vector has the nucleotide sequence shown in SEQ ID NO.4. Specifically, in the present invention, the nucleic acid molecule (S protein mutant gene sequence) is ligated to the pcDNA3.4 vector, and HEK293 cells are used for expression.

[0039] Since the porcine epidemic diarrhea virus S protein mutant has excellent immunogenicity, the present invention also provides an application of the porcine epidemic diarrhea virus S protein mutant as described above in immunizing against porcine epidemic diarrhea virus.

[0040] The present invention also provides a subunit vaccine against porcine epidemic diarrhea virus, including the porcine epidemic diarrhea virus S protein mutant as described above; as a conventional option, the subunit vaccine may also contain a vaccine adjuvant.

[0041] The present invention also provides a preparation method of a subunit vaccine against porcine epidemic diarrhea virus, including: expressing the porcine epidemic diarrhea virus S protein as described above and using the porcine epidemic diarrhea virus S protein as an immunogen.

[0042] The present invention also provides an application of the porcine epidemic diarrhea virus S protein mutant as described above in preparing antibodies against porcine epidemic diarrhea virus.

[0043] The following are specific examples of the present invention:

[0044] Example 1

[0045] Codon sequence optimization of PEDV S protein gene and construction of expression recombinant plasmid:

[0046] The PEDV S protein is a type I transmembrane glycoprotein, containing an extracellular region, a transmembrane region, and an intracellular region. The transmembrane region and intracellular region are predicted using the TMHMM2.0 software, and the extracellular region of the S protein is selected; the signal peptide of the S protein is predicted and analyzed using the SignalP-5.0 software. The extracellular region of the S protein from a clinically isolated strain (PEDV-HuN strain, G2a subtype) is selected, and the IGκ signal peptide sequence, T4 foldon trimerization motif, and His Tag sequence are introduced at the amino terminus and carboxyl terminus. After codon sequence optimization, the S protein gene sequence is ligated to the pcDNA3.4 vector using the XbaⅠ and EcoR V restriction enzyme sites, and named pcDNA3.4-PEDV-SWT.

[0047] The prediction and analysis results of the TMHMM2.0 software show that 1-1327aa of the PEDV S protein is the extracellular region, 1328-1350aa is the transmembrane region, and 1351-1386aa is the intracellular region (Figure 1 ); The prediction result of SignalP-5.0 software shows that 1-25aa is the signal peptide.

[0048] In this example, the PEDV S protein gene was ligated to the pcDNA3.4 eukaryotic expression vector through the XbaⅠ and EcoR V restriction sites, and the recombinant plasmid map ( Figure 2 ), and the plasmid sequencing alignment result was consistent with the target gene sequence.

[0049] In the present invention, the amino acid sequence of the PEDV SWT protein is as follows (SEQ ID NO.1):

[0050]

[0051] In the present invention, the nucleotide sequence encoding the PEDV SWT protein is as follows (SEQ ID NO.2):

[0052]

[0053] Example 2

[0054] Design of PEDV S protein mutant and construction of recombinant plasmid:

[0055] Download the three-dimensional structure of the PEDV S protein (PDB ID: 6u7k) from the PDB database, analyze the three-dimensional structure of the S protein using Pymol software, screen out the key amino acid sites that maintain the stability of the S protein structure, design the S protein with a pre-fusion stable conformation using the multi-site proline (Proline, P) mutation strategy, design primers, and use a rapid site-directed mutagenesis kit to mutate the amino acid sites, and construct a recombinant expression plasmid of the PEDV S protein mutant, named pcDNA3.4-PEDV-S4P.

[0056] In this example, by analyzing the three-dimensional structure of the PEDV S protein, it was found that isoleucine (I) and leucine (L) at positions 1076-1077 of the PEDV S protein are the key amino acid sites connecting the HR1 (Helix region 1) and CH (Central helix) of the S2 subunit. 1076 IL 1077 was mutated to 2 prolines ( 1076 PP 1077 ), which can disrupt the formation of the secondary structure. Using the same strategy, serine (S) at position 1016 and valine (V) at position 1017 connecting the two helical structures were mutated to 2 prolines ( 1016 PP 1017 ); the protein mutant in the present invention is named S4P ( Figure 3 ). The plasmid pcDNA3.4-PEDV-S4P was successfully constructed by the site-directed mutagenesis kit. The recombinant plasmid map is shown in ( Figure 4 ), and the plasmid sequencing result is consistent with the target gene sequence.

[0057] In the present invention, the amino acid sequence of the PEDV S4P protein is as follows (SEQ ID NO.3):

[0058]

[0059] In the present invention, the nucleotide sequence encoding the PEDV S4P protein is shown as follows (SEQ ID NO.4):

[0060]

[0061] Example 3

[0062] Expression and purification of PEDV S protein:

[0063] 1. Expression of PEDV S protein:

[0064] (1) HEK293 cells were used to express the recombinant proteins in Example 1 and Example 2. The recombinant protein in Example 1 was denoted as wild-type SWT protein, and the recombinant protein in Example 2 was denoted as mutant S4P protein; the cell density before transfection was 2×10 6 cells / mL, and they were cultured in a constant temperature shaker at 37°C, 5% CO2, and a rotation speed of 160 rpm.

[0065] (2) On the day of transfection, the cell density was adjusted to 3×10 6 cells / mL. 40 μg of plasmid DNA was diluted with the prepared 150 mM NaCl to a total volume of 1 mL and mixed evenly; 200 μL of PEI transfection reagent was diluted with the prepared 150 mM NaCl to a total volume of 1 mL and mixed evenly; the diluted plasmid DNA and PEI transfection reagent were mixed evenly and placed at room temperature for 10 min.

[0066] (3) The transfection mixture was added dropwise to the cells, and the cells were gently shaken while adding. After shaking, they were placed in a constant temperature shaker for continued culture, and the CO2 of the shaker was turned off.

[0067] (4) 1.0 mL of SMS293-SUPI feeding was added 48 h after transfection, and samples were collected 96 h after transfection.

[0068] 2. Purification of PEDV S protein:

[0069] The protein was purified by nickel column affinity chromatography. The cell culture medium supernatant was collected 96 h after transfection. The culture product was centrifuged at 3,000 g for 30 min to collect the culture medium supernatant, which was then filtered through a 0.45 μm filter, and the buffer was exchanged to the loading buffer (20 mM imidazole, pH = 8) using a 50K membrane package; the protein was purified by nickel column affinity chromatography. The supernatant was combined with Ni-NTA filler at room temperature for 30 min, and the impurity proteins were eluted with Wash buffer (50 mM Tris-HCl, 200 mM NaCl, 20 mM Imidazole, pH 8.0) with an imidazole concentration of 20 mM. Then, the target protein was eluted with Elution buffer (50 mM Tris-HCl, 200 mM NaCl, 300 mM Imidazole, pH 6.0) with an imidazole concentration of 300 mM. The purified protein was identified by SDS-PAGE.

[0070] In this example, pcDNA3.4-PEDV-SWT and pcDNA3.4-PEDV-S4P plasmids were co-transfected into HEK293 cells, and the expression level of the target protein in the cell supernatant was detected by Western blot. The results showed that the expression level of the mutant S4P protein was 4.5 times that of the wild-type SWT( Figure 5 ).

[0071] In this example, the protein was purified by nickel column affinity chromatography; see Figure 6 for understanding. The SDS-PAGE results showed that the molecular weight of the S4P protein was approximately 220 kDa, which was consistent with the expected protein molecular weight. Native PAGE analysis of the S4P protein showed that the molecular weight of the S4P protein was approximately 660 kDa under non-denaturing conditions, indicating that the S4P protein was a native trimer.

[0072] Example 4

[0073] Immunization test for piglets:

[0074] 1. Preparation of PEDV subunit vaccine:

[0075] After determining the protein concentration of the purified PEDV S4P protein (mutant S4P protein) by the BCA method, it was thoroughly shaken and mixed with Seppic Gel 02ST adjuvant at a volume ratio of 1:9 to prepare solutions of 25 μg / mL and 50 μg / mL and stored at 4°C for later use.

[0076] 2. Grouping of animal experiments:

[0077] Fifteen healthy piglets at 3 days old with negative detection of maternally-derived antibodies against PEDV were randomly divided into 3 groups (5 piglets in each group). Piglets in the first group (25 μg immunization group) and the second group (50 μg immunization group) were inoculated with 1 mL of subunit vaccine into the neck muscles on day 0; in the first group and the second group, the antigen concentrations of the subunit vaccine were 25 μg / mL and 50 μg / mL respectively, and the antigen was the mutant S4P protein; the third group was the non-immunized control group (blank control group); blood samples were collected before and after immunization, and the sera were separated for antibody detection.

[0078] 3. Indirect ELISA antibody detection of PEDV:

[0079] (1) Use an Ingnina commercial kit to detect PEDV-specific antibodies. All reagents were restored to room temperature before use in the experiment.

[0080] (2) The serum sample to be tested is diluted at a ratio of 1:100. Add 100 μL of the diluted serum sample to be tested to each well, and at the same time add 100 μL of the diluted positive and negative control sera. Incubate at 37 °C for 1 h.

[0081] (3) Wash 4 times with the washing solution. Add 100 μL of the enzyme-labeled secondary antibody to each well. Incubate at 37 °C for 30 min.

[0082] (4) Wash 4 times with the washing solution. Add 100 μL of the substrate TMB solution to each well. Incubate in the dark at room temperature for 15 min.

[0083] (5) Add 100 μL of the termination solution to each well. Read the OD value at a wavelength of 450 nm.

[0084] (6) When the OD value of the positive control serum - the OD value of the negative control serum > 0.5 and the OD value of the negative control < 0.15, the result is valid. Calculate the S / P value (S / P value = (OD value of the sample - OD value of the negative control) / (OD value of the positive control - OD value of the negative control)). When S / P ≥ 0.35, it is determined to be positive for PEDV antibody; when S / P < 0.35, it is determined to be negative for PEDV antibody.

[0085] In this example, as shown in Figure 7 ELISA antibody detection was performed on the sera before immunization and on the 14th and 21st days after immunization; Figure 7 In [figure], 0, 14, and 21 on the abscissa correspond to day 0, day 14, and day 21 respectively; among day 0, day 14, and day 21, the one closest to the origin of the coordinate is the blank control group, followed by the 25 μg immunization group, and the one farthest from the origin of the coordinate is the 50 μg immunization group.

[0086] The results showed that the antibody level increased significantly on the 14th day after immunization. The average S / P values of the 25 μg and 50 μg immunization groups were 1.24 and 1.23 respectively. The antibody level gradually increased on the 21st day after immunization. The average S / P values of the 25 μg and 50 μg immunization groups were 1.80 and 1.79 respectively. No PEDV antibody level was detected in the blank control group, indicating that this PEDV subunit vaccine has good immunogenicity, and a single immunization with 25 μg and 50 μg can induce the body to produce a high level of antibodies.

[0087] In the above technical solution of the present invention, the above are only the preferred embodiments of the present invention, and do not limit the patent scope of the present invention accordingly. Any equivalent structural transformation made under the technical concept of the present invention by using the content of the specification and drawings of the present invention, or any direct / indirect application in other related technical fields, is included in the patent protection scope of the present invention.

Claims

1. A porcine epidemic diarrhea virus S protein mutant, characterized in that, The amino acid sequence of the S protein mutant is shown in SEQ ID NO.

3.

2. A nucleic acid molecule encoding a mutant S protein of porcine epidemic diarrhea virus, characterized in that, The nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO.

4.

3. A recombinant expression vector, characterized in that, The recombinant expression vector has the nucleic acid molecule as described in claim 2.

4. An engineered cell, characterized in that, The engineered cell is capable of expressing the porcine epidemic diarrhea virus S protein mutant as described in claim 1.

5. A method for expressing a porcine epidemic diarrhea virus S protein mutant, characterized in that, Comprising: constructing a recombinant expression vector and expressing it through an engineered cell; the recombinant expression vector has the nucleotide sequence shown in SEQ ID NO.

4.

6. A subunit vaccine against porcine epidemic diarrhea virus, characterized in that, Comprising the porcine epidemic diarrhea virus S protein mutant as described in claim 1.

7. A method for preparing a subunit vaccine against porcine epidemic diarrhea virus, characterized in that, Comprising: Expressing the porcine epidemic diarrhea virus S protein mutant as described in claim 1 and using the porcine epidemic diarrhea virus S protein mutant as an immunogen.

8. Use of a porcine epidemic diarrhea virus S protein mutant as described in claim 1 in the preparation of porcine epidemic diarrhea virus antibodies.

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