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

Abstract

The invention provides a porcine epidemic diarrhea virus S protein mutant, a nucleic acid molecule, a subunit vaccine and application. Compared with wild type porcine epidemic diarrhea virus S protein, the porcine epidemic diarrhea virus S protein mutant has the advantage that amino acid at the 1076th site, amino acid at the 1077th site, amino acid at the 1016th site and amino acid at the 1017th site are mutated into proline. The natural trimer which is stable and maintains the conformation of the S protein before fusion is constructed, the safe and efficient PEDV subunit vaccine is obtained, and the use effect and immunogenicity of the porcine epidemic diarrhea vaccine can be improved; moreover, compared with a wild type S protein, the porcine epidemic diarrhea virus S protein mutant disclosed by the invention has a higher expression quantity.

Description

Technical Field

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

[0002] Porcine Epidemic Diarrhea (PED) is a highly contagious intestinal disease caused by porcine epidemic diarrhea virus (PEDVirus, PEDV). It is susceptible to pigs of different ages and mainly causes diarrhea, vomiting and dehydration in pigs. Newborn piglets are extremely susceptible.

[0003] PEDV is currently divided into two main genotypes, the classic strain GI type and the variant strain GII type. The GII type is further divided into three genotypes: GII-a, GII-b and GII-c. Effective biosafety prevention and control is an effective means to block the spread of PEDV. In addition, vaccination is one of the most effective measures to prevent and control PEDV. Currently, all commercial PEDV vaccines are inactivated vaccines or attenuated live vaccines. Most commercial vaccines are currently developed for early classic GI type strains, and have poor cross-protection against the currently prevalent variant strain GII type. In addition, the clinical prevention effect of inactivated vaccines is not very ideal, and attenuated vaccines may revert to virulence, posing a major biosafety risk.

[0004] PEDV is a member of the Alphacoronavirus genus of the Coronaviridae family. It is an enveloped, single-stranded, positive-stranded, linear RNA virus. The diameter of the virus particle is about 95-190nm, and the surface of the virus particle contains spikes of about 18nm in size. PEDV S (Spike protein, S) protein is a type I transmembrane protein, embedded in the surface of the virus particle, responsible for the virus and the specific receptor binding on the cell surface to mediate the cell invasion of the virus. Antibodies produced against the S protein can effectively inhibit the invasion of the virus and are one of the ideal antigens for the current development of PEDV subunit vaccines. PEDV is an enveloped RNA virus. During the process of virus invasion of host cells, the S protein on the surface of the virus particle binds to the specific receptor on the cell surface, thereby inducing a change in the conformation of the S protein, from the energy-unstable prefusion (Prefusion) to the energy-stable postfusion (Postfusion) conformation. Therefore, the applicant has studied and found that how to design a stable recombinant S protein trimer that can maintain the S protein in its pre-fusion conformation is the key to the 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, nucleic acid molecule, subunit vaccine and application, so as to obtain a natural trimer that stably maintains the S protein in the pre-fusion conformation, and obtain a safe and efficient PEDV subunit vaccine, thereby solving the technical defects of poor clinical use of porcine epidemic diarrhea vaccine. Summary of the invention

[0006] The main purpose of the present invention is to provide a porcine epidemic diarrhea virus S protein mutant, nucleic acid molecule, subunit vaccine and application to solve the technical problems of how to stabilize and maintain the natural trimer of the S protein in the pre-fusion conformation and how to improve the effectiveness of the porcine epidemic diarrhea vaccine.

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

[0008] The present invention also provides a porcine epidemic diarrhea virus S protein mutant, wherein the S protein mutant has an amino acid sequence as 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, wherein the nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO.4.

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

[0011] The present invention also provides an engineered cell, wherein the engineered cell is capable of expressing any of the porcine epidemic diarrhea virus S protein mutants described above.

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

[0013] The present invention also provides a use of any of the porcine epidemic diarrhea virus S protein mutants described above in immunizing against porcine epidemic diarrhea virus.

[0014] The present invention also provides a subunit vaccine of porcine epidemic diarrhea virus, comprising any porcine epidemic diarrhea virus S protein mutant as described above.

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

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

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

[0018] The present invention constructs a natural trimer that is stable and maintains the pre-fusion conformation of the S protein, obtains a safe and efficient PEDV subunit vaccine, and can improve the efficacy and immunogenicity of the porcine epidemic diarrhea vaccine; and 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, the present invention has found that the isoleucine (I) and leucine (L) at positions 1076-1077 of the PEDV S protein are key amino acid sites for connecting the S2 subunit HR1 (Helix region 1) and CH (Central helix). 1076 IL 1077 Mutation to 2 prolines ( 1076 PP 1077 ), which can destroy the formation of the secondary structure. The present invention adopts the same strategy to mutate the serine (Serine, S) at position 1016 and the valine (Valine, V) at position 1017 connecting the two helical structures into two proline ( 1016 PP 1017 ); Based on the method and strategy for constructing a stable and efficient expressed porcine epidemic diarrhea virus S protein mutant in the present invention, the porcine epidemic diarrhea virus S protein mutant obtained in the present invention can be used to prevent porcine epidemic diarrhea virus infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the structures shown in these drawings without paying creative work.

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

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

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

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

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

[0026] Figure 6 The results of SDS-PAGE and Native-PAGE of PEDV S4P protein in Example 3 of the present invention are shown in the figure.

[0027] (a) is the SDS-PAGE pattern; (b) is the native PAGE pattern; M is the molecular weight of the standard protein; 1 is the PEDV S4P protein; 2 is the BSA protein;

[0028] Figure 7 This is the result of the PEDV indirect ELISA antibody level test in Example 4 of the present invention.

[0029] The realization of the purpose, functional features and advantages of the present invention will be further explained in conjunction with the implementation methods and with reference to the accompanying drawings. DETAILED DESCRIPTION

[0030] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] Furthermore, the technical solutions between the various embodiments of the present invention may be combined with each other, but this must be based on the fact that they can be implemented by ordinary technicians in the field. When the combination of technical solutions is mutually contradictory or cannot be implemented, it should be deemed that such 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 porcine epidemic diarrhea virus S protein mutant, 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 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 may be as shown in SEQ ID NO.1, and the nucleotide sequence encoding the wild-type porcine epidemic diarrhea virus S protein may be as shown in SEQ ID NO.2.

[0034] The S protein in the present invention may refer to the extracellular region of the S protein of porcine epidemic diarrhea virus. The present invention is mainly designed for the extracellular region of the S protein of porcine epidemic diarrhea virus; specifically, the S protein mutant has an amino acid sequence 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 porcine epidemic diarrhea virus S protein mutant 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 a nucleotide sequence as shown in SEQ ID NO. 4. Therefore, the present invention also provides a nucleic acid molecule encoding a porcine epidemic diarrhea virus S protein mutant, the nucleic acid molecule having a 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 porcine epidemic diarrhea virus S protein mutant.

[0037] The recombinant expression vector has a nucleic acid molecule as described in any of the above, which is used to express the porcine epidemic diarrhea virus S protein mutant as described in any of the above; the engineered cell, the engineered cell can express the porcine epidemic diarrhea virus S protein mutant as described in any of the above, and it can specifically express the porcine epidemic diarrhea virus S protein mutant through the nucleic acid molecule as described in any of the above.

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

[0039] Since the porcine epidemic diarrhea virus S protein mutant has excellent immunogenicity, the present invention also provides a use of any of the porcine epidemic diarrhea virus S protein mutants described above in immunizing against porcine epidemic diarrhea virus.

[0040] The present invention also provides a subunit vaccine of porcine epidemic diarrhea virus, comprising any 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 method for preparing a subunit vaccine of porcine epidemic diarrhea virus, comprising: expressing any 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 a use of any of the porcine epidemic diarrhea virus S protein mutants described above in the preparation of porcine epidemic diarrhea virus antibodies.

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

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

[0048] In this example, the PEDV S protein gene was connected to the pcDNA3.4 eukaryotic expression vector through the XbaⅠ and EcoR V restriction sites. The recombinant plasmid map ( Figure 2 ), and the plasmid sequencing results were 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 mutants and construction of recombinant plasmids:

[0055] The three-dimensional structure of PEDV S protein was downloaded from the PDB database (PDB ID: 6u7k). The three-dimensional structure of S protein was analyzed using Pymol software, and the key amino acid sites that maintain the stability of S protein structure were screened out. The multi-site proline (P) mutation strategy was used to design the S protein with stable conformation before fusion. Primers were designed, and the rapid site-directed mutagenesis kit was used to mutate the amino acid sites. The recombinant expression plasmid of PEDV S protein mutant was constructed and named pcDNA3.4-PEDV-S4P.

[0056] In this example, by analyzing the three-dimensional structure of the PEDV S protein, it was found that the isoleucine (I) and leucine (L) at positions 1076-1077 of the PEDV S protein are key amino acid sites for connecting the S2 subunit HR1 (Helix region 1) and CH (Central helix). 1076 IL 1077 Mutation to 2 prolines ( 1076 PP 1077 ), which can destroy the formation of secondary structure, the same strategy was used to mutate the serine (Serine, S) at position 1016 and the valine (Valine, V) at position 1017 connecting the two helical structures into two proline ( 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 site-directed mutagenesis kit. The recombinant plasmid map is shown in ( Figure 4 ), and the plasmid sequencing results were 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 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 recorded as wild-type SWT protein, and the recombinant protein in Example 2 was recorded as mutant S4P protein. The cell density before transfection was 2×10 6 cells / mL, at 37°C, 5% CO 2 , and cultured in a constant temperature shaker at 160 rpm.

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

[0066] (3) Add the transfection mixture dropwise to the cells, gently shake the cells while adding, and place them in a constant temperature shaker for further culture. Turn off the shaker CO 2 .

[0067] (4) 1.0 mL SMS293-SUPI feed 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 supernatant was collected 96 hours after transfection, and the culture product was centrifuged at 3,000g for 30 minutes to collect the culture supernatant, which was then filtered with a 0.45μm filter and replaced with a loading buffer (20mM imidazole, pH=8) using a 50K membrane. The protein was purified by nickel column affinity chromatography. The supernatant was combined with Ni-NTA filler at room temperature for 30 minutes, and the impurities were eluted with Wash buffer (50mM Tris-HCl, 200mM NaCl, 20mMImidazole, pH 8.0) with an imidazole concentration of 20mM, and then the target protein was eluted with Elution buffer (50mM Tris-HCl, 200mM NaCl, 300mM Imidazole, pH 6.0) with an imidazole concentration of 300mM. The purified protein was identified by SDS-PAGE.

[0070] In this example, pcDNA3.4-PEDV-SWT and pcDNA3.4-PEDV-S4P plasmids were simultaneously 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 The results of SDS-PAGE showed that the molecular weight of S4P protein was about 220 kDa, which was consistent with the expected molecular weight of the protein. The S4P protein was analyzed by native PAGE, and the results showed that the molecular weight of S4P protein under native conditions was about 660 kDa, indicating that S4P protein was a natural trimer.

[0072] Example 4

[0073] Piglet Immunization Test:

[0074] 1. Preparation of PEDV subunit vaccine:

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

[0076] 2. Animal experiment grouping:

[0077] Fifteen healthy piglets at 3 days old with negative PEDV maternal antibody test were selected and randomly divided into 3 groups (5 pigs in each group). The piglets in Group 1 (25 μg immunization group) and Group 2 (50 μg immunization group) were vaccinated with 1 mL of subunit vaccine into the neck muscle on day 0. In Group 1 and Group 2, the antigen concentrations of the subunit vaccine were 25 μg / mL and 50 μg / mL, respectively, and the antigen was the mutant S4P protein. Group 3 was the non-immunized control group (blank control group). Blood was collected before and after immunization, and serum was separated for antibody detection.

[0078] 3. PEDV indirect ELISA antibody test:

[0079] (1) PEDV-specific antibodies were detected using the commercial kit of Ingena. All reagents were returned to room temperature before use.

[0080] (2) The serum sample to be tested was diluted at 1:100, and 100 μL of the diluted serum to be tested was added to each well. At the same time, 100 μL of the diluted positive and negative control sera were added and incubated at 37°C for 1 h.

[0081] (3) Wash four times with washing solution, add 100 μL of enzyme-labeled secondary antibody to each well, and incubate at 37°C for 30 min.

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

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

[0084] (6) When the positive control serum OD value minus the negative control serum OD value>0.5 and the negative control OD value<0.15, the result is established, and the S / P value is calculated (S / P value=sample OD value-negative control OD value) / (positive control OD value-negative control OD value). When S / P≥0.35, it is judged as PEDV antibody positive, and when S / P<0.35, it is judged as PEDV antibody negative.

[0085] In this embodiment, see Figure 7 As shown, ELISA antibody detection was performed on the sera before immunization and on days 14 and 21 after immunization; Figure 7 In the figure, the horizontal axes 0, 14, and 21 correspond to 0 days, 14 days, and 21 days respectively; on 0 days, 14 days, and 21 days, the blank control group was closest to the origin of the coordinate, followed by the 25μg immunization group, and the 50μg immunization group was farthest from the origin of the coordinate.

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

[0087] The above technical solutions of the present invention are only preferred embodiments of the present invention, and the patent scope of the present invention is not limited thereto. All equivalent structural changes made by using the contents of the present invention specification and drawings under the technical concept of the present invention, or directly / indirectly applied in other related technical fields are included in the patent protection scope of the present invention.

Claims

1. A porcine epidemic diarrhea virus S protein mutant, characterized in that: Compared with the wild-type porcine epidemic diarrhea virus S protein, the amino acid at position 1076, the amino acid at position 1077, the amino acid at position 1016, and the amino acid at position 1017 are all mutated to proline.

2. A porcine epidemic diarrhea virus S protein mutant, characterized in that: The S protein mutant has an amino acid sequence as shown in SEQ ID NO.

3.

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

4.

4. A recombinant expression vector, characterized in that: The recombinant expression vector has the nucleic acid molecule according to claim 3.

5. An engineered cell, characterized in that: The engineered cells are capable of expressing the porcine epidemic diarrhea virus S protein mutant as described in claim 1 or 2.

6. A method for expressing a porcine epidemic diarrhea virus S protein mutant, characterized in that: The method comprises: constructing a recombinant expression vector and expressing it through engineered cells; the recombinant expression vector has a nucleotide sequence as shown in SEQ ID NO.

4.

7. Use of the porcine epidemic diarrhea virus S protein mutant as claimed in claim 1 or 2 in immunizing against porcine epidemic diarrhea virus.

8. A subunit vaccine for porcine epidemic diarrhea virus, characterized in that: Comprising the porcine epidemic diarrhea virus S protein mutant as described in claim 1 or 2.

9. A method for preparing a subunit vaccine of porcine epidemic diarrhea virus, characterized in that: include: Express the porcine epidemic diarrhea virus S protein as described in claim 1 or 2, and use the porcine epidemic diarrhea virus S protein as an immunogen.

10. Use of the porcine epidemic diarrhea virus S protein mutant according to claim 1 or 2 in the preparation of porcine epidemic diarrhea virus antibodies.

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