PEDV-S1 recombinant protein antigen as well as preparation method and vaccine thereof

Abstract

The invention discloses a PEDV-S1 recombinant protein antigen as well as a preparation method and a vaccine thereof, and belongs to the technical field of recombinant protein vaccines. The CHO eukaryotic expression system is adopted, high expression and complete glycosylation modification of protein can be achieved, and therefore it is guaranteed that the vaccine has strong immunogenicity; through His tag fusion expression design, nickel column affinity chromatography is conveniently adopted for purification, the process is simple and convenient, and the protein recovery rate is also improved; in combination with a serum-free suspension culture technology, the method is suitable for large-scale production, and the stability among batches is good; the prepared vaccine is high in safety and few in side reaction; an immune challenge test further proves that the vaccine can induce a body to generate a high-level antibody and can effectively protect PEDV attack.

Description

PEDV-S1 recombinant protein antigen and its preparation method, vaccine Technical Field

[0001] This invention belongs to the field of recombinant protein vaccine technology, specifically relating to the PEDV-S1 recombinant protein antigen and its preparation method, and the vaccine. Background Technology

[0002] Porcine epidemic diarrhea (PED) is a highly contagious, acute intestinal infectious disease caused by Porcine Epidemic Diarrhea Virus (PEDV). The disease is characterized by severe watery diarrhea, vomiting, dehydration, and high mortality in pigs of all ages, with particularly severe damage to suckling piglets, leading to mortality rates as high as 90%-100%, causing continuous and enormous economic losses to the global pig industry. PEDV belongs to the genus Alphacoronavirus of the family Coronaviridae and is an enveloped, single-stranded, positive-sense RNA virus. Its spike protein (S protein) on the surface of the envelope is the virus's most crucial antigenic protein, responsible for recognizing host cell receptors and mediating viral membrane fusion and invasion. The S protein can be cleaved into two functional subunits, S1 and S2, by viral proteases. The S1 subunit contains a receptor-binding domain (RBD) and a major antigenic epitope, and is the core antigen that stimulates the host to produce high levels of neutralizing antibodies; therefore, it is considered an ideal target for constructing next-generation genetically engineered vaccines.

[0003] Currently, the market primarily relies on traditional inactivated vaccines and live attenuated vaccines for PED control. While inactivated vaccines offer high biosafety, they have inherent limitations such as weak immunogenicity, short-lasting induced immune responses, and the need for multiple immunizations. Live attenuated vaccines can induce relatively comprehensive immune protection, but they still face safety risks such as virulence reversion, genetic recombination with field-circulating strains, and potential threats to immunodeficient pigs. Furthermore, the production of both types of vaccines depends on large-scale cultivation of live viruses, which not only poses a risk of biosafety leaks but also involves complex and costly production processes, making it difficult to meet the high standards of vaccine quality and quantity required for disease eradication.

[0004] With the rapid development of molecular biology and recombinant protein technology, genetically engineered subunit vaccines offer a revolutionary solution to overcome the bottlenecks of traditional vaccines. These vaccines contain only specific protective antigenic components of the pathogen, without viral genetic material, fundamentally eliminating the risks of virulence reversion and viral replication, and possessing unparalleled safety advantages. Furthermore, their production process does not require handling live viruses, significantly reducing biosafety risks, and the product composition is clearly defined, with uniform quality, making it easy to establish a standardized quality control system. However, the efficacy of subunit vaccines largely depends on whether the antigenic protein can maintain its native conformation and immunogenicity in the expression system.

[0005] Therefore, there is an urgent need to develop a safe, efficient, and stable genetically engineered subunit vaccine for porcine epidemic diarrhea. Summary of the Invention

[0006] In view of this, the purpose of this invention is to provide PEDV-S1 recombinant protein antigen and its preparation method, as well as a vaccine. The vaccine of this invention has good safety and few side effects; challenge tests show that it can induce high-titer antibodies and effectively resist PEDV infection.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] In a first aspect, the present invention provides a PEDV-S1 recombinant protein antigen, the amino acid sequence of which is shown in SEQ ID NO:1.

[0009] In a second aspect, the present invention provides a nucleotide sequence of a PEDV-S1 recombinant protein antigen, the nucleotide sequence of which is shown in SEQ ID NO:2.

[0010] Thirdly, the present invention provides a method for preparing the above-mentioned PEDV-S1 recombinant protein antigen, comprising the following steps: synthesizing the nucleotide sequence shown in SEQ ID NO:2 into the pKS001 vector to construct the recombinant plasmid pKS001-PEDV-S1; then introducing the recombinant plasmid pKS001-PEDV-S1 into an engineered CHO-K1Q cell line for serum-free suspension culture, screening out monoclonal cell lines that highly express PEDV-S1 protein; and then collecting and purifying the PEDV-S1 protein.

[0011] The Chinese hamster ovary cell (CHO cell) expression system is currently the most mature and widely used eukaryotic expression platform in the field of recombinant protein biopharmaceuticals. Compared to prokaryotic expression systems (such as E. coli), CHO cells can precisely perform complex post-translational modifications of proteins, such as N-linked glycosylation, which is crucial for maintaining the correct spatial structure, biological activity, and immunogenicity of enveloped viral antigen proteins such as PEDV S1. Furthermore, CHO cells possess significant advantages, including adaptability to serum-free suspension culture, strong proliferation capacity, high protein expression levels, and ease of linear scaling up to industrial production scale, making them an ideal tool for large-scale production of high-quality vaccine antigens.

[0012] Therefore, developing a genetically engineered subunit vaccine against the PEDV S1 antigen based on the CHO cell expression system can not only effectively avoid the technical defects and safety risks of traditional vaccines, providing a safer, more efficient, and quality-controllable immunization tool, but its stable and scalable production process also lays a solid foundation for the commercial supply of the vaccine. This has significant socio-economic value for the scientific prevention and control of porcine epidemic diarrhea and for ensuring the sustainable development of animal husbandry.

[0013] Fourthly, the present invention provides the application of the above-mentioned PEDV-S1 recombinant protein antigen in a porcine epidemic diarrhea vaccine.

[0014] Fifthly, the present invention provides a porcine epidemic diarrhea vaccine comprising the above-mentioned PEDV-S1 recombinant protein antigen.

[0015] Furthermore, it also includes water adjuvants.

[0016] Furthermore, the content of the PEDV-S1 recombinant protein antigen is 40 μg / mL.

[0017] The present invention provides a vaccine preparation method with the following advantages: The CHO eukaryotic expression system enables high protein expression and complete glycosylation modification, thus ensuring strong immunogenicity of the vaccine; the His tag fusion expression design facilitates purification using nickel column affinity chromatography, simplifying the process and improving protein recovery; combined with serum-free suspension culture technology, this method is suitable for large-scale production and exhibits good batch-to-batch stability; the prepared vaccine has high safety and few side effects; and immune challenge experiments further demonstrate that the vaccine can induce high levels of antibodies in the body and provide effective protection against PEDV challenge. Attached Figure Description

[0018] Figure 1 shows the electrophoresis diagram of the recombinant expression vector pKS001-PEDV-S1 after enzyme digestion verification. Lane M: DL2000 bp Marker; primers were PEDV-S1-F and PEDV-S1-R for amplification. Lane 1: Control group, using the original plasmid pKS001 as a template, the target fragment PEDV-S1 was not amplified. Lanes 2-3: Amplified fragments using the genome of the constructed vector pKS001-PEDV-S1 as a template.

[0019] Figure 2 shows the SDS-PAGE analysis of the purified PEDV-S1 protein. Lane M: Protein Marker; Lane 1: CHO-K1Q empty vector sample; Lane 2: Purified protein sample; Lane 3: Unpurified protein sample. Detailed Implementation

[0020] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0021] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0022] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0023] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0024] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0025] Unless otherwise specified, "room temperature" and "normal temperature" in this invention refer to 25±2℃.

[0026] Unless otherwise specified, all raw materials or instruments used in the following embodiments of the present invention are commercially available.

[0027] Example 1. Recombinant Expression Sequence Design and Vector Construction Referring to the porcine epidemic diarrhea PEDV-S1 protein sequence published in NCBI (accession number: ACC95468.1), a recombinant protein sequence suitable for the CHO eukaryotic expression system was designed, consisting of: signal peptide + PEDV-S1 protein + linker + His tag. Using codon optimization technology, the optimized coding sequence was synthesized into the pKS001 prokaryotic vector by Sangon Biotech (Shanghai) Co., Ltd. This gene sequence contains 2701 nucleotides. The recombinant plasmid pKS001-PEDV-S1 was constructed, and the amino acid and nucleotide sequences are shown in SEQ ID NO:1; the codon-optimized nucleotide sequence is shown in SEQ ID NO:2.

[0028] 2. Construction of expression vector (pKS001-PEDV-S1) 2.1 Primers The primers used in this experiment were synthesized by Sangon Biotech (Shanghai) Co., Ltd., and the primers are shown in Table 1.

[0029] Table 1 Primers

[0030] 2.2 Methods: Construction and Validation of Recombinant Protein: Plasmid pKS001-PEDV-S1 was extracted from *E. coli*. Using plasmid pKS001-PEDV-S1 as a template, the PEDV-S1 gene was amplified using primers PEDV-S1-F and PEDV-S1-R. The amplified target fragment was validated by gel electrophoresis. The validation results are shown in Figure 1, which are consistent with the expected results. The constructed recombinant plasmid pKS001-PEDV was preliminarily constructed correctly. It was then sent to a sequencing company for sequencing, and the gene sequence was compared. The results were consistent with the expected target, proving that the recombinant plasmid pKS001-PEDV-S1 was successfully constructed.

[0031] The correct E. coli pKS001-PEDV-S1 was cultured and verified. The recombinant plasmid pKS001-PEDV-S1 was extracted and verified to be correct. Electroporation was used to introduce it into the engineered CHO-K1Q cell line for serum-free suspension culture. A monoclonal cell line (CHO-PEDV-S1) that highly expresses PEDV-S1 protein was screened.

[0032] 3. Expression and Purification of Recombinant Protein: High-expression clones were selected and placed in a bioreactor for serum-free suspension culture to achieve high-density fermentation. The cell supernatant of recombinant porcine epidemic diarrhea (PEDV-S1) protein expressed in the CHO-PEDV-S1 cell line was centrifuged, collected, and filtered through a 0.22 μm filter. Purification was then performed using immobilized metal ion (nickel column) affinity chromatography (IMAC): the column was equilibrated with equilibration buffer at a flow rate of 2 mL / min for approximately 3–5 column volumes. Cell supernatant was loaded into the column at a flow rate of 2 mL / min, and the flow-through was collected. After loading, the column was equilibrated by washing with 5 column volumes of equilibration buffer at a flow rate of 2 mL / min. After baseline equilibration, the target protein was eluted with elution buffer (200mM imidazole, pH=8) to obtain high-purity PEDV-S1 protein. The elution buffer was collected based on the UV absorption peak. This protein is the antigen of the subunit vaccine. The volume of eluted protein was recorded and stored at 4°C.

[0033] 4. Protein Purity and Content Determination: The purity of the collected eluted protein was determined using SDS-PAGE gel electrophoresis and thin-layer chromatography imaging system. The results are shown in Figure 2. The purified protein showed a clear protein expression band at approximately 102.1 kDa, consistent with the expected result. Analysis showed that the purity of the purified protein reached 80%. The total protein content of the purified protein was determined using a BCA protein concentration assay kit, and the result was 3.6 mg / mL. The calculated content of the target protein was 2.88 mg / mL.

[0034] 5. Vaccine Preparation: After sterile filtration through a 0.22 μm filter membrane, the purified recombinant porcine epidemic diarrhea virus (PEDV-S1) protein is diluted to 80 μg / mL with sterile 0.9% physiological saline. It is then mixed with sterile aqueous adjuvant at a certain ratio and stirred at 500 rpm for 30–60 minutes at 30°C to prepare a stable vaccine. The antigen content of the finished vaccine should meet the requirement of 40 μg / mL for recombinant porcine epidemic diarrhea virus (PEDV-S1) protein.

[0035] 6. Testing of Porcine Epidemic Diarrhea Disease Genetically Engineered Subunit Vaccine 6.1 Safety Testing of Porcine Epidemic Diarrhea Disease Genetically Engineered Subunit Vaccine in Pigs 6.1.1 Experimental Animals: 1-month-old piglets and pregnant sows with a gestation age of 20 days before farrowing 6.1.2 Safety Testing of Porcine Epidemic Diarrhea Disease Genetically Engineered Subunit Vaccine in Piglets and Pregnant Sows: Laboratory-prepared trial vaccines were used to conduct single-dose vaccination trials, single-dose repeat trials (14-day interval), and overdose vaccination trials on piglets (1 month old) and pregnant sows (20 days before farrowing) at the recommended use age.

[0036] Table 2 Grouping of experimental animals

[0037] Twenty experimental piglets and twenty experimental pregnant sows were randomly divided into four groups, and the experiment was conducted according to Table 2, with isolated rearing. Observations and records were kept continuously until day 7 post-vaccination, with daily records of the survival, mental state, diet, and local and systemic reactions of all vaccinated experimental pigs. The safety test results for single-dose vaccination of the experimental pigs are detailed in Table 3 below. The safety test results showed that all vaccinated experimental pigs survived during the observation period, with no deaths or significant local or systemic reactions caused by the vaccine. Their mental state was good, and their feed and water intake were normal. There were no visible differences between the immunized and control groups, indicating that the three batches of vaccine are safe for piglets with the youngest age of administration (1 month old) and pregnant sows with a gestation age of 20 days before farrowing.

[0038] Table 3. Safety test results of the experimental vaccine developed from genetically engineered subunits for porcine epidemic diarrhea (PED).

[0039] Note: "R" indicates a red ear tag; "Y" indicates a yellow ear tag; "B" indicates a blue ear tag; "G" indicates a green ear tag.

[0040] 6.2 Efficacy Testing of Porcine Epidemic Diarrhea Genetically Engineered Subunit Vaccine in Piglets and Pregnant Sows To investigate the immunogenicity of the porcine epidemic diarrhea disease (PED) genetically engineered subunit vaccine, efficacy testing was conducted on the laboratory-developed vaccine. The results showed that the laboratory-developed vaccine provided good immunoprotective effects in piglets and pregnant sows.

[0041] 6.2.1 Experimental Grouping and Immunization Animals: One-month-old piglets and pregnant piglets at 20 days gestation were randomly divided into two groups of 10 each (n=5 per group): an immunization group and a control group. The immunization group received 0.5 mL / piglet / administration, with a booster immunization administered 14 days post-immunization using the same method. The control group received the same volume of 0.9% physiological saline. Pregnant piglets were also randomly divided into two groups of 10 each (n=5 per group): an immunization group and a control group. The immunization group received 1.0 mL / piglet / administration, with a booster immunization administered 14 days post-immunization using the same method. The control group received the same volume of 0.9% physiological saline. Animal grouping is shown in Table 4.

[0042] Table 4 Grouping of experimental animals

[0043] 6.2.2 In the neutralization test, each piglet in the vaccine group received a subcutaneous injection of 0.5 mL of vaccine (1 dose) in the neck, while the control group received the same dose of 0.9% saline. A booster immunization was administered 14 days after the initial immunization using the same method. Blood samples were collected and serum separated before the second immunization and 14 days after the second immunization for neutralizing antibody testing.

[0044] Pregnant swine were vaccinated by subcutaneous injection of 1.0 mL of vaccine (1 dose) into the neck of each swine, while the control group received the same dose of 0.9% saline. A booster immunization was administered 14 days after the initial vaccination using the same method. Blood samples were collected and serum separated before the second vaccination and 14 days after the second vaccination for neutralizing antibody testing.

[0045] Cells in both the serum toxicity control wells and the normal cell control wells should be normal. The virus control should contain 200 TCID. 50 / 0.1 mL and 20 TCID 50 / 0.1 mL of cells should all be lesions, 2 TCID 50 / 0.1 mL wells should show 0-2 wells with cytopathic effect and 0.2 TCID. 50 The cells in each 0.1 mL well should be normal. The neutralizing antibody titer in the control group should not exceed 1:4.

[0046] 6.2.3 After blood collection from immunized piglets, each piglet, along with the control group, was orally infected with 2 mL of porcine epidemic diarrhea virus (XJ-DB2 strain) for 7 consecutive days. At least 4 piglets in the control group should develop the disease, and at least 4 piglets in the immunized group should be protected.

[0047] After blood collection from pregnant sows, each sow, along with the control group, was orally infected with 4 mL of 2 mL porcine epidemic diarrhea virus (XJ-DB2 strain) for 7 consecutive days. At least four sows in the control group should develop the disease, and at least four sows in the immunized group should be protected.

[0048] 6.2.4 PEDV Neutralizing Antibody Detection and Challenge Protection Results Based on the PEDV neutralizing antibody detection and challenge protection results, the neutralizing antibody detection results for each group of experimental piglets and pregnant sows are detailed in Table 5 below. Fourteen days after the second immunization, blood was collected from 5 piglets to measure PEDV neutralizing antibodies and challenge them. The results showed that the neutralizing antibody titers of all 5 piglets were high, and 5 / 5 of the immunized piglets were protected after challenge. In contrast, the neutralizing antibody titers of the control group piglets were all below 1:4, and all 5 piglets developed the disease after challenge. Fourteen days after the second immunization, blood was collected from 5 pregnant sows to measure PEDV neutralizing antibodies and challenge them. The results showed that the neutralizing antibody titers of all 5 pregnant sows were high, and 5 / 5 of the immunized pregnant sows were protected after challenge. In contrast, the neutralizing antibody titers of the control group pregnant sows were all below 1:4, and all 5 pregnant sows developed the disease after challenge. Neutralizing antibody titer results showed that the neutralizing antibody titers measured in piglets and pregnant sows were qualified, with all immunized groups showing positive titers and all control groups showing negative titers. This demonstrates that a PEDV neutralizing antibody titer level of 1:32 or higher can completely protect immunized piglets and pregnant sows against challenge with virulent PEDV. Therefore, the porcine epidemic diarrhea virus genetically engineered subunit vaccine prepared in this invention has passed the efficacy test, and immunization with this vaccine can induce antibody production in experimental piglets and pregnant sows.

[0049] Table 5. Results of PEDV neutralizing antibody detection and challenge protection.

[0050] Note: "O" indicates no abnormalities; "E" indicates decreased appetite; "N" indicates lethargy; "F" indicates diarrhea; "V" indicates vomiting.

[0051] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A PEDV-S1 recombinant protein antigen, characterized in that, The amino acid sequence of the PEDV-S1 recombinant protein antigen is shown in SEQ ID NO:

1.

2. A nucleotide of a PEDV-S1 recombinant protein antigen, characterized in that, The nucleotide sequence of the PEDV-S1 recombinant protein antigen is shown in SEQ ID NO:

2.

3. The method for preparing the PEDV-S1 recombinant protein antigen according to claim 1, characterized in that, The procedure includes the following steps: synthesizing the nucleotide sequence shown in SEQ ID NO:2 into the pKS001 vector to construct the recombinant plasmid pKS001-PEDV-S1; then introducing the recombinant plasmid pKS001-PEDV-S1 into an engineered CHO-K1Q cell line for serum-free suspension culture, screening out monoclonal cell lines that highly express PEDV-S1 protein; and then collecting and purifying the PEDV-S1 protein.

4. The use of the PEDV-S1 recombinant protein antigen as described in claim 1 in a porcine epidemic diarrhea vaccine.

5. A porcine epidemic diarrhea vaccine, characterized in that, Includes the PEDV-S1 recombinant protein antigen as described in claim 1.

6. The porcine epidemic diarrhea vaccine according to claim 1, characterized in that, It also includes water-based adjuvants.

7. The porcine epidemic diarrhea vaccine according to claim 6, characterized in that, The content of the PEDV-S1 recombinant protein antigen is 40 μg / mL.

Images