TGEV-S1 recombinant protein antigen as well as preparation method and vaccine thereof
By constructing the TGEV-S1 protein using the CHO cell expression system, a safe and highly effective genetically engineered subunit vaccine against porcine transmissible gastroenteritis was prepared. This solved the safety and production cost issues of existing vaccines, and achieved efficient and stable immunization effects and large-scale production.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-31
- Publication Date
- 2026-04-10
AI Technical Summary
Existing TGEV vaccines have problems such as insufficient safety, poor immunization effect, high production cost and high biosafety risk. Traditional vaccines have many defects in the production process.
A high-efficiency TGEV-S1 protein expression system was constructed using the CHO cell expression system. A safe, efficient, and stable genetically engineered subunit vaccine against porcine transmissible gastroenteritis was prepared through protein purification and adjuvant emulsification processes.
It achieves a highly safe and low-side-effect immunization effect, is suitable for immunizing pigs of all ages, has strong immunogenicity, is suitable for large-scale production, has good vaccine stability, can induce high levels of antibodies, and effectively resist TGEV attack.
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Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of recombinant protein vaccine, and particularly relates to a TGEV-S1 recombinant protein antigen and a preparation method and vaccine thereof. BACKGROUND
[0002] Transmissible gastroenteritis (TGE) is an acute enteric infectious disease caused by porcine transmissible gastroenteritis virus (TGEV), with the main clinical features of vomiting, severe watery diarrhea, dehydration and extremely high mortality (especially the mortality of piglets under 2 weeks old can reach 100%). The disease spreads rapidly and has a high incidence, causing huge economic losses to the global pig industry. TGEV belongs to the alpha coronavirus genus of the coronavirus family and is a single-stranded RNA virus with a capsule. The surface of the virus particle contains various structural proteins, among which the spike protein (S protein) is the main antigenic protein of the virus, responsible for binding to host cell receptors and mediating viral invasion. The S protein can be further hydrolyzed into S1 and S2 functional subunits, and the S1 subunit contains the main antigenic determinant of the virus and is a key antigen for inducing the host to produce neutralizing antibodies, thus becoming an ideal target for developing subunit vaccines.
[0003] At present, commercial vaccines against TGEV mainly include traditional inactivated vaccines and attenuated live vaccines. Inactivated vaccines are safer, but have the disadvantages of weak immunogenicity, multiple immunization times, short protection period, etc. Attenuated live vaccines have better immunization effect, but still have potential risks such as virulence return, recombination with field strains, and possible harm to immunosuppressed pigs. In addition, both of these two traditional vaccines involve large-scale culture of viruses in the production process, which has the problems of biosafety risk and high production cost.
[0004] With the development of molecular biology and genetic engineering technology, genetic engineering subunit vaccines provide a new direction to solve the above problems. Such vaccines only contain specific protective antigen proteins of pathogens, and do not contain the genetic material of viruses, so they have extremely high safety. At the same time, it avoids the biosafety risk brought by the operation of live viruses, and is easy to realize the standardized control of product quality. However, the success of such vaccines is highly dependent on the efficient expression of antigen proteins, correct post-translational modification (such as glycosylation), and strong immunogenicity.
[0005] Therefore, a safer and more effective prevention and control tool is urgently needed, and its stable and controllable production process lays a solid foundation for the large-scale production and commercial application of vaccines, which has great significance for effectively controlling the prevalence of porcine transmissible gastroenteritis and ensuring the healthy development of the pig industry. SUMMARY
[0006] Therefore, the application aims to provide a TGEV-S1 recombinant protein antigen and a preparation method and a vaccine thereof, which utilizes a CHO cell expression system to construct an engineering cell strain for efficiently expressing TGEV-S1 protein, and through protein purification and adjuvant emulsification processes, a safe, efficient and stable porcine transmissible gastroenteritis disease genetic engineering subunit vaccine is prepared.
[0007] In order to achieve the above-mentioned purpose, the application provides the following technical solutions. In a first aspect, the application provides a TGEV-S1 recombinant protein antigen, the amino acid sequence of which is shown in SEQ ID NO: 1.
[0008] In a second aspect, the application provides a nucleotide of a TGEV-S1 recombinant protein antigen, the nucleotide sequence of which is shown in SEQ ID NO: 2.
[0009] In a third aspect, the application provides a preparation method of the TGEV-S1 recombinant protein antigen, comprising the following steps: synthesizing the nucleotide sequence shown in SEQ ID NO: 2 into a pKS001 vector to construct a recombinant plasmid pKS001-TGEV-S1; then introducing the recombinant plasmid pKS001-TGEV-S1 into an engineered CHO-K1Q cell strain for serum-free suspension culture, and screening a monoclonal cell strain with high expression of TGEV-S1 protein through a monoclonal antibody screening technology; and then collecting TGEV-S1 protein for purification.
[0010] The Chinese hamster ovary cell (CHO cell) expression system is the most commonly used and mature eukaryotic expression system in the production of recombinant protein drugs and the most advanced vaccines. Compared with a prokaryotic expression system (such as Escherichia coli), the CHO cell can complete complex and accurate post-translational modifications of eukaryotic proteins, such as glycosylation and phosphorylation, which are crucial for maintaining the correct conformation, stability and immunogenicity of the proteins. In addition, the CHO cell has the advantages of being capable of suspension culture, fast proliferation, high expression, easy scaling up to a bioreactor for industrial large-scale production, etc., making it an ideal platform for producing high-quality viral antigen proteins.
[0011] The application realizes the large-scale production of TGEV-S1 protein through high-density fermentation of the CHO cell strain in a bioreactor by serum-free suspension culture technology. Compared with traditional vaccines (inactivated vaccines, attenuated live vaccines, etc.), the application has high safety, few side reactions, strong immunogenicity, better immune effect, good stability and more controllable production process, and is suitable for large-scale production.
[0012] In a fourth aspect, the application provides the use of the TGEV-S1 recombinant protein antigen in the preparation of a porcine transmissible gastroenteritis disease vaccine.
[0013] In a fifth aspect, the present application provides a vaccine for porcine transmissible gastroenteritis, comprising the TGEV-S1 recombinant protein antigen as described above.
[0014] Further, the water adjuvant is also included.
[0015] Further, the content of the TGEV-S1 recombinant protein antigen is 40 μg / ml.
[0016] At least the following beneficial technical effects are included: The present application utilizes the CHO eukaryotic expression system, and has high protein expression amount, complete glycosylation modification, and strong immunogenicity; the Fc tag fusion expression is adopted, which is convenient for Protein A affinity purification, and has simple process and high recovery rate; the serum-free suspension culture technology is suitable for large-scale production, and has good batch stability; the vaccine has high safety and few side reactions, and is suitable for immunization of pigs of all ages; the immune challenge test shows that the vaccine can induce high level of antibody and effectively resist TGEV attack. BRIEF DESCRIPTION OF DRAWINGS
[0017] Figure 1 : Restriction enzyme digestion verification electrophoretogram of recombinant expression vector pKS001-TGEV-S1. Lane M1: DL2000 bp Marker; Lane 1: control group, using the original plasmid pKS001 as a template, no target fragment TGEV-S1 is amplified; Lanes 2-4: amplified fragments using the genome of the constructed vector pKS001-TGEV-S1 as a template; Lane 5: control group, using water as a template, no target fragment TGEV-S1 is amplified; Lane M2: DL10000 bp Marker.
[0018] Figure 2 : SDS-PAGE analysis of TGEV-S1 protein after purification. Lane M: Protein Marker; Lane 1: unpurified protein sample; Lane 2: flow-through liquid; Lane 3: 0.1M eluent. DETAILED DESCRIPTION
[0019] The various illustrative embodiments of the present application will now be described in detail below, which should not be considered as limiting the present application, but should be understood as a more detailed description of certain aspects, characteristics and embodiments of the present application.
[0020] 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.
[0021] 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.
[0022] 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.
[0023] 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.
[0024] Unless otherwise specified, "room temperature" and "normal temperature" in this invention refer to 25±2℃.
[0025] Unless otherwise specified, all raw materials or instruments used in the following embodiments of the present invention are commercially available.
[0026] Example 1. Recombinant expression sequence design and vector construction Based on the TGEV-S1 protein sequence of porcine transmissible gastroenteritis published in NCBI (accession number: ACC95468.1), a recombinant protein sequence suitable for the CHO eukaryotic expression system was designed, consisting of: signal peptide + TGEV-S1 protein + linker + Fc tag. Using codon optimization technology, the optimized coding sequence was synthesized into the pKS001 vector by Sangon Biotech (Shanghai) Co., Ltd. This gene sequence contains 2785 nucleotides. The recombinant plasmid pKS001-TGEV-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.
[0027] 2. Expression vector construction (pKS001-TGEV-S1) 2.1 Primer The primers in this experiment were synthesized by Shenguo Bioengineering (Shanghai) Co., Ltd., and the primers are shown in Table 1.
[0028] Table 1 Primer
[0029] 2.2 Method Construction and verification of recombinant protein: The plasmid of E. coli pKS001-TGEV-S1 was extracted, and the TGEV-S1 gene was amplified using primers TGEV-S1-F and TGEV-S1-R with the plasmid pKS001-TGEV-S1 as the template, and the amplified target fragment was verified by gel electrophoresis. The electrophoresis map of the verification result is shown in Figure 1 The results are consistent with the expected results, and the constructed vector recombinant plasmid pKS001-TGEV is preliminarily constructed correctly. Then it was sent to a sequencing company for sequencing. The gene sequence was compared, and the results were consistent with the expected target, proving that the recombinant plasmid pKS001-TGEV-S1 was successfully constructed.
[0030] The correct E. coli pKS001-TGEV-S1 was cultured, the recombinant plasmid pKS001-TGEV-S1 was extracted, and after verification, the electroporation technology was used to introduce the engineered CHO-K1Q cell strain for serum-free suspension culture, and through the monoclonal antibody screening technology, a monoclonal cell strain (CHO-TGEV-S1) with high expression of TGEV-S1 protein was screened out.
[0031] 3. Expression and purification of recombinant protein The high-expression clone strain selected was placed in a bioreactor for serum-free suspension culture to achieve high-density fermentation. The cell supernatant of the recombinant porcine transmissible gastroenteritis TGEV-S1 protein expressed by the CHO-TGEV-S1 cell strain was centrifuged, the supernatant was collected and filtered through a 0.22 µm filter membrane, and then purified using a Protein A affinity chromatography column: equilibrate the chromatography column with 2 mL / min flow rate of equilibration buffer, equilibrate for about 3~5 times the column volume, then load the cell supernatant into the chromatography column at a flow rate of 2 mL / min, and collect the flow-through. After loading is completed, load 5 times the column volume of equilibration buffer into the chromatography column at a flow rate of 2 mL / min for miscellaneous washing equilibration. After baseline equilibration, elute the target protein with elution buffer to obtain high-purity TGEV-S1 protein. The eluate was collected according to the ultraviolet absorption peak, and the collected sample was adjusted to pH 7.0 with 2M Tris-Base. This protein is the antigen of a subunit vaccine. The volume of the eluted protein was recorded, and the protein was stored at 4°C.
[0032] 4. Determination of protein purity and content The purity of the collected eluted proteins was determined using SDS-PAGE gel electrophoresis and thin-layer chromatography imaging system. The results are shown in [Figure number missing]. Figure 2 The purified protein showed a clear protein expression band at approximately 99 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 3.75 mg / ml. The calculated content of the target protein was 3.0 mg / ml.
[0033] 5. Vaccine preparation The purified recombinant porcine transmissible gastroenteritis TGEV-S1 protein was aseptically filtered through a 0.22 μm filter membrane, diluted with sterile 0.9% physiological saline, and mixed with sterilized 803 water adjuvant at a certain ratio. The mixture was 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 transmissible gastroenteritis TGEV-S1 protein.
[0034] 6. Testing of genetically engineered subunit vaccines against porcine transmissible gastroenteritis (TGE) 6.1 Safety testing of porcine transmissible gastroenteritis virus (TGEV) genetically engineered subunit vaccines in piglets and pregnant sows 6.1.1 Experimental Animals 1-month-old piglets and gestation pigs with a gestation age of 20 days before farrowing 6.1.2 Safety testing of porcine transmissible gastroenteritis virus (TGE) genetically engineered subunit vaccines in piglets and pregnant sows The laboratory-prepared trial vaccine was used to conduct single-dose vaccination trials, single-dose repeated (14-day interval) and overdose vaccination trials on recommended-use piglets (1 month old) and pregnant pigs (20 days before farrowing).
[0035] Table 2 Grouping of experimental animals
[0036] 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.
[0037] Table 3. Safety test results of the experimental vaccine developed from the genetically engineered subunit of porcine transmissible gastroenteritis virus.
[0038] 6.2 Efficacy testing of porcine transmissible gastroenteritis virus (TGE) genetically engineered subunit vaccines in piglets and pregnant sows To investigate the immunogenicity of a genetically engineered subunit vaccine against porcine transmissible gastroenteritis (TGEV), efficacy testing was conducted on a laboratory-developed vaccine. Results showed that the laboratory-developed vaccine provided good immunoprotective effects against piglets and pregnant sows.
[0039] 6.2.1 Experimental grouping and immunization Experimental animals: 1-month-old piglets and pregnant sows 20 days before farrowing Experimental piglets: Ten experimental piglets were randomly divided into two groups of 5 each: an immunization group and a control group. The immunization group received 0.5 ml / pig / dose, and a booster immunization was administered 14 days post-immunization using the same method. The control group received the same volume of 0.9% physiological saline. Experimental pregnant sows: Ten experimental pregnant sows were randomly divided into two groups of 5 each: an immunization group and a control group. The immunization group received 1.0 ml / pig / dose, and a booster immunization was administered 14 days post-immunization using the same method. The control group received the same volume of 0.9% physiological saline. The grouping of experimental animals is shown in Table 4.
[0040] Table 4 Grouping of experimental animals
[0041] 6.2.2 Neutralization Test Each piglet in the vaccine group received an intramuscular 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 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.
[0042] Pregnant pigs in the vaccine group received an intramuscular injection of 1.0 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 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.
[0043] Cells in both the serum toxicity control wells and the normal cell control wells should be normal, and the virus control should contain 200 TCID. 50 / 0.1ml and 20TCID 50 / 0.1ml of cells should all be lesions, 2TCID 50 / 0.1ml wells should have 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.
[0044] 6.2.3 Immune challenge After blood collection from piglets, each piglet, along with the control group, was orally infected with 2 ml of a 2 MLD (2 ml / day) virulent strain of porcine transmissible gastroenteritis virus (TGEV), and observed 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.
[0045] After blood collection from pregnant sows, each sow, along with the control group, was orally infected with 4 ml of a 2 MLD (2 ml / day) virulent strain of porcine transmissible gastroenteritis (TGE), and observed for 7 consecutive days. At least 4 sows in the control group should develop the disease, and at least 4 sows in the immunized group should be protected.
[0046] 6.2.4 Detection of TGEV neutralizing antibodies and results of challenge protection Based on the results of TGEV neutralizing antibody detection and challenge protection, the results of neutralizing antibody detection in each group of experimental piglets and pregnant sows are detailed in Table 5 below. Fourteen days after the second immunization, blood was collected from five piglets to measure TGEV neutralizing antibodies and challenge them. The results showed that the neutralizing antibody titers of all five 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 five piglets developed the disease after challenge. Similarly, fourteen days after the second immunization, blood was collected from five pregnant sows to measure TGEV neutralizing antibodies and challenge them. The results showed that the neutralizing antibody titers of all five 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 piglets were all below 1:4, and all five pregnant sows developed the disease after challenge. The neutralizing antibody titer results showed that the neutralizing antibody titers measured in both piglets and pregnant sows were within acceptable limits; all were positive in the immunized group, and all were negative in the control group. This study demonstrates that a neutralizing antibody titer of 1:32 or higher against TGEV can completely protect immunized piglets and pregnant sows from attack by virulent TGEV. Therefore, the porcine transmissible gastroenteritis (TGEV) genetically engineered subunit vaccine prepared in this invention has passed efficacy testing, and immunization with this vaccine can induce antibody production in experimental piglets and pregnant sows.
[0047] Table 5. Detection results of TGEV neutralizing antibodies and protection against viral challenge.
[0048] Note: "O" indicates no abnormalities; "E" indicates decreased appetite; "N" indicates lethargy; "F" indicates diarrhea; "V" indicates vomiting.
[0049] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made 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 TGEV-S1 recombinant protein antigen, characterized in that, The amino acid sequence of the TGEV-S1 recombinant protein antigen is shown in SEQ ID NO:
1.
2. A nucleotide of a TGEV-S1 recombinant protein antigen, characterized in that, The nucleotide sequence of the TGEV-S1 recombinant protein antigen is shown in SEQ ID NO:
2.
3. The method for preparing the TGEV-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-TGEV-S1; then introducing the recombinant plasmid pKS001-TGEV-S1 into an engineered CHO-K1Q cell line for serum-free suspension culture, and screening for monoclonal cell lines that highly express TGEV-S1 protein using monoclonal antibody screening technology; and then collecting and purifying the TGEV-S1 protein.
4. The use of the TGEV-S1 recombinant protein antigen of claim 1 in the preparation of a porcine transmissible gastroenteritis vaccine.
5. A vaccine for porcine transmissible gastroenteritis, characterized in that, Includes the TGEV-S1 recombinant protein antigen as described in claim 1.
6. The porcine transmissible gastroenteritis vaccine according to claim 1, characterized in that, It also includes water-based adjuvants.
7. The porcine transmissible gastroenteritis vaccine according to claim 6, characterized in that, The content of the TGEV-S1 recombinant protein antigen is 40 μg / ml.