A vaccine for porcine epidemic diarrhea and its preparation method

Abstract

This invention belongs to the field of biomedical technology, specifically relating to a vaccine for porcine epidemic diarrhea (PED) and its preparation method. The PED vaccine is obtained by transforming an expression vector containing a fusion protein into lactic acid bacteria. The fusion protein comprises porcine epidemic diarrhea virus S1 protein and porcine Seneca virus type A VP2 protein, and the amino acid sequence of the fusion protein is shown in SEQ ID NO.1. Experimental results show that administering the PED vaccine prepared according to this invention can induce high levels of specific antibodies, effectively inhibiting porcine epidemic diarrhea and significantly enhancing the immunoprotective effect against it, demonstrating promising application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a vaccine for swine epidemic diarrhea and its preparation method. Background Technology

[0002] Porcine epidemic diarrhea (PED) is a highly contagious acute intestinal disease caused by porcine epidemic diarrhea virus (PEDV). PEDV can infect pigs of all ages, but suckling piglets are most susceptible. Symptoms include severe diarrhea, vomiting, and severe dehydration, with a mortality rate approaching 100%, causing significant economic losses to the pig industry. Senecavirus A (SVA), belonging to the microRNAviridae family, can infect pigs of different ages, with vesicles and erosions at the snout and coronary bands, and acute death in newborn piglets as its main clinical features. In recent years, it has become one of the important pathogens threatening the health of pig herds. Therefore, research on vaccines against porcine epidemic diarrhea virus and SVA is essential for disease prevention.

[0003] Currently, the prevention and control of PED mainly relies on immunization with traditional inactivated vaccines and live attenuated vaccines. However, live attenuated vaccines pose potential biosafety risks such as virulence reversion and recombination with wild-type viruses, and have stringent requirements for transportation and storage conditions. While whole-virus inactivated vaccines have higher safety, they suffer from low viral titers, insufficient antigen content, weak immunogenicity, and the need for multiple booster immunizations. Furthermore, PEDV is difficult to proliferate in in vitro cell culture, resulting in low culture titers and high production costs, limiting the output of inactivated vaccines. Traditional vaccines are mostly administered via injection, which makes it difficult to induce effective secretory IgA antibodies locally in the intestinal mucosa, failing to form a mucosal immune barrier against intestinal pathogens, resulting in unsatisfactory protective effects.

[0004] With the development of genetic engineering technology and mucosal immunity theory, the construction of oral subunit vaccines using lactic acid bacteria as antigen presentation vectors has become a research hotspot. Lactic acid bacteria, as normal probiotics in the intestines of humans and animals, possess unique advantages such as safety and non-toxicity, colonization of the intestinal mucosa, immune adjuvant effect, and low production cost. Therefore, developing an oral vaccine for porcine epidemic diarrhea based on genetically engineered lactic acid bacteria has significant application value for improving the efficiency of swine herd immunity, reducing breeding costs, and ensuring the healthy development of the pig industry. Summary of the Invention

[0005] In order to overcome the shortcomings of the prior art, one of the objectives of the present invention is to provide a vaccine for swine epidemic diarrhea.

[0006] The second objective of this invention is to provide a method for preparing a vaccine for swine epidemic diarrhea.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A vaccine for porcine epidemic diarrhea (PED) is obtained by transforming an expression vector containing a fusion protein into lactic acid bacteria. The fusion protein contains porcine epidemic diarrhea virus S1 protein and porcine Seneca virus type A VP2 protein, and the amino acid sequence of the fusion protein is shown in SEQ ID NO.1.

[0008] Furthermore, the lactic acid bacteria mentioned are Lactobacillus casei.

[0009] According to the above-described method for preparing a vaccine for porcine epidemic diarrhea, the preparation method specifically includes the following steps: (1) Using the amino acid sequence of porcine epidemic diarrhea virus S1 protein (positions 466-718) and the full amino acid sequence of porcine type A Seneca virus SVA (positions 151-434) as N-terminal and C-terminal sequences respectively, a linker with the sequence GGGGSGGGGS was added in the middle to obtain the amino acid sequence of the fusion protein as shown in SEQ ID NO.1. (2) Obtain the nucleotide sequence of the fusion protein as shown in SEQ ID NO.1 based on the amino acid sequence of the fusion protein; design upstream and downstream primers to amplify the corresponding nucleotide sequence of the fusion protein, and connect the nucleotide sequence of the fusion protein amplified by the primers to the pUC57 vector to construct the recombinant plasmid pUC57-S1-VP2. (3) The recombinant plasmid pUC57-S1-VP2 constructed in step (2) and the vector pMG36e were respectively subjected to... Sal I and Kpn After double digestion with enzyme I, the target fragment S1-VP2 and the linearized vector pMG36e were recovered by gel extraction. After ligation with T4 DNA ligase, the recombinant plasmid pMG36e-S1-VP2 was transformed into E. coli MC1061 to obtain the recombinant plasmid pMG36e-S1-VP2. (4) The recombinant plasmid pMG36e-S1-VP2 obtained in step (3) is introduced into competent lactic acid bacteria cells, and recombinant lactic acid bacteria transformants are screened to obtain the vaccine strain for porcine epidemic diarrhea. (5) The porcine epidemic diarrhea vaccine strain obtained in step (4) is inoculated into the culture medium, the bacterial cells are collected, washed, and resuspended in PBS to obtain the porcine epidemic diarrhea vaccine.

[0010] Further, the upstream primer sequence of the corresponding nucleotide sequence of the fusion protein in step (2) is shown in SEQ ID NO.3; the downstream primer sequence is shown in SEQ ID NO.4.

[0011] Further, in step (4), the recombinant plasmid pMG36e-S1-VP2 is introduced into lactic acid bacteria competent cells by electroporation.

[0012] Furthermore, in step (5), the culture medium is MRS medium, and the culture temperature is 28-32℃.

[0013] The present invention has the following advantages over the prior art: (1) This invention expresses the porcine epidemic diarrhea virus S1 protein gene and the porcine type A Seneca virus VP2 protein gene through a flexible linker (GGGGSGGGGS). Among them, the S1 protein acts as a protective immunogen, which can induce the intestine to produce secretory IgA (sIgA), forming the first line of immune barrier of the mucosa and blocking the infection of diarrhea virus through the digestive tract; the VP2 protein acts as an immune enhancer, containing a large number of dominant B cell and T cell epitopes, and as an immune enhancer, it can induce strong humoral immunity and cellular immunity; the two work together to achieve a three-dimensional protective effect of humoral + mucosal + cellular immunity.

[0014] (2) The present invention uses lactic acid bacteria as a live vector system, which has a high survival rate in the gastrointestinal environment and stable plasmid genetics. After immunizing piglets, it can simultaneously induce humoral immunity, cellular immunity and mucosal immunity responses, produce specific antibodies, significantly increase the SIgA level in the piglet intestine, enhance the immune protection of pigs, effectively reduce the challenge effect of swine diarrhea virus, and has broad application prospects in the prevention and control of swine epidemic diarrhea virus infection. Attached Figure Description

[0015] Figure 1 The plasmid map of recombinant plasmid pUC57-S1-VP2; Figure 2 The plasmid map of recombinant plasmid pMG36e-S1-VP2; Figure 3 Western blot analysis results for recombinant pMG36e-S1-VP2; Figure 4 The results show the growth curves of recombinant lactic acid bacteria. Detailed Implementation

[0016] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field. Test methods in the following embodiments that do not specify specific experimental conditions are generally performed according to conventional experimental conditions or experimental conditions recommended by the manufacturer. Unless otherwise specified, the reagents and raw materials used in the present invention are commercially available.

[0017] The Lactobacillus casei used in the embodiments of this invention has the accession number ATCC393.

[0018] Example 1 A vaccine for porcine epidemic diarrhea (PED) is obtained by transforming an expression vector containing a fusion protein into lactic acid bacteria. The fusion protein comprises porcine epidemic diarrhea virus (PEDV) S1 protein and porcine Seneca virus type A (SENECV) VP2 protein. The amino acid sequence of the fusion protein is shown in SEQ ID NO.1. The nucleotide sequence of the fusion protein is shown in SEQ ID NO.2.

[0019] A method for preparing a vaccine for porcine epidemic diarrhea, the method specifically comprising the following steps: (1) Using the amino acid sequence of porcine epidemic diarrhea virus S1 protein (positions 466-718) and the VP2 protein of porcine type A Seneca virus SVA (positions 151-434) as N-terminal and C-terminal sequences respectively, a linker with the sequence GGGGSGGGGS was added in the middle to obtain the amino acid sequence of the fusion protein as shown in SEQ ID NO.1.

[0020] (2) Based on the amino acid sequence of the fusion protein SEQ ID NO.1, the nucleotide sequence of the fusion protein was obtained as shown in SEQ ID NO.2; upstream and downstream primers were designed to amplify the corresponding nucleotide sequence of the fusion protein, and the nucleotide sequence of the fusion protein amplified by the primers was ligated to the pUC57 vector to construct the recombinant plasmid pUC57-S1-VP2. The plasmid map of the recombinant plasmid pUC57-S1-VP2 is shown in the figure below. Figure 1 As shown in SEQ ID NO.3; the downstream primer sequence is shown in SEQ ID NO.4.

[0021] Table 1 Sequence List (3) The recombinant plasmid pUC57-S1-VP2 constructed in step (2) and the vector pMG36e were respectively subjected to... Sal I and Kpn Double digestion with enzymes, the reaction system is shown in Table 2. The target fragment S1-VP2 and the linearized vector pMG36e were recovered by gel extraction, ligated with T4 DNA ligase, and transformed into E. coli MC1061 to obtain the recombinant plasmid pMG36e-S1-VP2. The T4 DNA ligase reaction system is shown in Table 3. Sequencing verification was performed. The plasmid map of recombinant plasmid pMG36e-S1-VP2 is shown below. Figure 2 As shown.

[0022] Table 2. Double enzyme digestion reaction system Table 3. T4 DNA ligase reaction system (4) The recombinant plasmid pMG36e-S1-VP2 obtained in step (3) is introduced into Lactobacillus casei competent cells by electroporation, and recombinant lactic acid bacteria transformants are screened to obtain the vaccine strain for porcine epidemic diarrhea. The preparation method of Lactobacillus casei competent cells includes the following steps: Lactobacillus casei with preservation number ATCC393 is inoculated into MRS medium containing 1% glycine and cultured at 37°C until OD. 600 The bacterial cells were collected by centrifugation after being placed on ice at a concentration of 0.8. They were then washed twice with pre-cooled electroporation buffer (0.5 M sucrose, 10% glycerol), resuspended, and concentrated to obtain electroporated competent cells.

[0023] (5) The porcine epidemic diarrhea vaccine strain obtained in step (4) was inoculated into MRS medium and incubated at 30°C until OD. 600 =0.8, continue culturing until the stationary phase, and take a portion of the bacterial sample for Western blot detection. The results are as follows. Figure 3 As shown, the recombinant lactic acid bacteria pMG36e-S1-VP2 exhibited a specific target band, consistent with the theoretical molecular weight of the S1-VP2 fusion protein, confirming successful expression of the exogenous protein. The remaining bacterial culture was collected by centrifugation at 4 ℃ and 5000 rpm for 10 min, washed twice with PBS, and resuspended in PBS to adjust the bacterial concentration to 1×10⁻⁶. 9 CFU / mL is the concentration of the vaccine for swine epidemic diarrhea.

[0024] Comparative Example 1 The difference between Comparative Example 1 and Example 1 is that the recombinant plasmid pMG36e-S1-VP2 in step (4) of the preparation method of the porcine epidemic diarrhea vaccine is replaced with the recombinant plasmid pMG36e-S1. All other aspects are the same as in Example 1. The preparation method of the recombinant plasmid pMG36e-S1 is the same as in Example 1.

[0025] Experimental Example 1 Growth curve determination of recombinant lactic acid bacteria The recombinant lactic acid bacteria transformants obtained in step (4) of Example 1 and Comparative Example 1, along with empty vector lactic acid bacteria (containing only pMG36e), were transferred to fresh MRS liquid culture medium at an inoculation rate of 2 v / v% and incubated statically at 30°C. Starting from 0 h after inoculation, samples were aseptically taken every 2 h, using MRS culture medium as a blank control, and the absorbance (OD) was measured at a spectrophotometer at a wavelength of 600 nm. 600 Three replicates were set at each time point, and the average value was used to plot the growth curve.

[0026] The results are as follows Figure 4 The image shows the growth curve results of the recombinant lactic acid bacteria. Figure 4 It was found that both the recombinant lactic acid bacteria and the empty vector bacteria reached a plateau at 14 h, and their growth patterns were almost identical, indicating that the recombinant lactic acid bacteria could grow normally. This shows that the introduction and expression of the exogenous antigen gene did not significantly burden the normal proliferation and metabolism of the host lactic acid bacteria, and the recombinant strain has good in vitro growth stability, providing a technological feasibility for subsequent fermentation preparation of vaccine antigens.

[0027] Experimental Example 2 Evaluation of the immunogenicity of recombinant lactic acid bacteria oral vaccine The following are the experimental results of the porcine epidemic diarrhea vaccine obtained in Example 1 and Comparative Example 1: (1) Fifty healthy piglets aged 4 weeks were randomly divided into a blank control group, an empty vector control group, a positive control group, Example 1 group, and a comparative example 1 group, with 10 piglets in each group. The immunization program for each group was as follows: Example 1 group was administered 1×100g of porcine epidemic diarrhea vaccine prepared in Example 1 group via oral gavage. 9 CFU / head (resuspended in 200 μL PBS suspension); Comparative group 1 was administered 1×10⁻⁶ of the porcine epidemic diarrhea vaccine prepared in Comparative group 1 via oral gavage. 9 CFU / head (200 μL PBS suspension); the empty vector control group was administered Lactobacillus casei containing pMG36e empty plasmid 1×10⁻⁶ via oral gavage. 9 CFU / head (200 μL PBS suspension); the positive control group was administered via intramuscular injection in the neck according to the instructions for the commercially available PEDV-SVA bivalent inactivated vaccine; the blank control group was administered an equal volume of PBS orally. All groups received their first immunization on day 0 and a booster immunization with the same dose on day 14.

[0028] (2) Blood samples were collected from the anterior vena cava of piglets in each group before the first vaccination (0 days) and on days 7, 14, 21, 28, and 35 after the first vaccination, and serum was separated. Simultaneously, rectal fecal samples were collected on days 14 and 28. The samples were prepared into a 10% (w / v) suspension with PBS, and the supernatant was collected by centrifugation. The serum PEDV-specific IgG antibody titer and fecal-specific sIgA level were detected using an indirect ELISA method. Serum IgG titer was expressed as the log2 value of the maximum serum dilution factor that could detect a positive reaction; fecal sIgA titer was expressed as the log2 value of the maximum dilution factor with a P / N ≥ 2.1. The results are shown in Tables 4 and 5.

[0029] Table 4 Serum PEDV-specific IgG antibody titer (log 2 , ±SD) The results are shown in Table 4, which presents the serum PEDV-specific IgG antibody titers. As can be seen from Table 4, the PEDV-specific IgG antibody titers in the serum of piglets in all immunization groups showed an increasing trend with prolonged immunization time. Compared with Comparative Example 1, the serum PEDV-specific IgG antibody titers in the group of Example 1 of this invention were significantly increased, indicating that the co-expression of the bivalent antigens S1 and VP2 proteins in the porcine epidemic diarrhea vaccine of Example 1 can synergistically enhance the humoral immune response against PEDV and significantly increase the level of serum-specific IgG antibodies.

[0030] Table 5. Fecal PEDV-specific sIgA levels (log2, ±SD) The results are shown in Table 5, which represents the fecal PEDV-specific sIgA level. As shown in Table 5, the PEDV-specific sIgA level in the feces of piglets in Example 1 of this invention was higher than that in Comparative Example 1, the positive control group, and the blank control group on days 14 and 28 after the first immunization. These results indicate that the porcine epidemic diarrhea vaccine of Example 1 of this invention, which delivers bivalent antigens of S1 and VP2 proteins to the intestinal mucosa via an oral lactic acid bacteria carrier, is significantly superior to Comparative Example 1, which expresses monovalent antigens. It also effectively overcomes the limitation of traditional intramuscularly injected inactivated vaccines in inducing high-titer intestinal sIgA; it can synergistically enhance the intestinal mucosal immune response and form a high-titer sIgA protective barrier on the intestinal mucosal surface.

[0031] (3) The challenge protection test was conducted on day 14 after the second immunization (i.e., when the antibody level reached its peak and stabilized). All groups were challenged with the isolated porcine epidemic diarrhea virus strain HN1301. After challenge, the disease incidence of each group was recorded for 7 days. The results are shown in Table 6.

[0032] Table 6 Results of the challenge protection test The results are shown in Table 6, which presents the results of the challenge protection test. As can be seen from Table 6, the protection rate of the Example 1 group against the porcine epidemic diarrhea virus HN1301 strain was 100%, with no piglets becoming ill or dying. This was superior to the Comparative Example 1 group and the positive control group, indicating that the recombinant lactic acid bacteria oral vaccine can achieve complete protection under the synergistic effect of systemic immunity and mucosal immunity.

Claims

1. A vaccine for porcine epidemic diarrhea, characterized in that, The porcine epidemic diarrhea vaccine is obtained by transferring an expression vector containing a fusion protein into lactic acid bacteria. The fusion protein contains porcine epidemic diarrhea virus S1 protein and porcine type A Seneca virus VP2 protein, and the amino acid sequence of the fusion protein is shown in SEQ ID NO.

1.

2. The vaccine for porcine epidemic diarrhea according to claim 1, characterized in that, The lactic acid bacteria mentioned are Lactobacillus casei.

3. A method for preparing a vaccine for porcine epidemic diarrhea according to any one of claims 1-2, characterized in that, The preparation method specifically includes the following steps: (1) Using the amino acid sequence of porcine epidemic diarrhea virus S1 protein (positions 466-718) and the full amino acid sequence of porcine type A Seneca virus SVA (positions 151-434) as N-terminal and C-terminal sequences respectively, a linker with the sequence GGGGSGGGGS was added in the middle to obtain the amino acid sequence of the fusion protein as shown in SEQ ID NO.

1. (2) Obtain the nucleotide sequence of the fusion protein as shown in SEQ ID NO.1 based on the amino acid sequence of the fusion protein; design upstream and downstream primers to amplify the corresponding nucleotide sequence of the fusion protein, and connect the nucleotide sequence of the fusion protein amplified by the primers to the pUC57 vector to construct the recombinant plasmid pUC57-S1-VP2; (3) The recombinant plasmid pUC57-S1-VP2 constructed in step (2) and the vector pMG36e were respectively subjected to... Sal I and Kpn After double digestion with enzyme I, the target fragment S1-VP2 and the linearized vector pMG36e were recovered by gel extraction. After ligation with T4 DNA ligase, the recombinant plasmid pMG36e-S1-VP2 was transformed into E. coli MC1061 to obtain the recombinant plasmid pMG36e-S1-VP2. (4) The recombinant plasmid pMG36e-S1-VP2 obtained in step (3) is introduced into competent lactic acid bacteria cells, and recombinant lactic acid bacteria transformants are screened to obtain the vaccine strain for porcine epidemic diarrhea. (5) The porcine epidemic diarrhea vaccine strain obtained in step (4) is inoculated into the culture medium, the bacterial cells are collected, washed, and resuspended in PBS to obtain the porcine epidemic diarrhea vaccine.

4. The method for preparing a vaccine for porcine epidemic diarrhea according to claim 3, characterized in that, The upstream primer sequence of the corresponding nucleotide sequence of the fusion protein in step (2) is shown in SEQ ID NO.3; the downstream primer sequence is shown in SEQ ID NO.

4.

5. The method for preparing a vaccine for porcine epidemic diarrhea according to claim 3, characterized in that, In step (4), the recombinant plasmid pMG36e-S1-VP2 is introduced into competent lactic acid bacteria cells by electroporation.

6. The method for preparing a vaccine for porcine epidemic diarrhea according to claim 3, characterized in that, In step (5), the culture medium is MRS medium, and the culture temperature is 28-32℃.

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