Mucosal immune enhancement type recombinant lactobacillus for expressing PEDV S1 protein as well as construction method and application of mucosal immune enhancement type recombinant lactobacillus

Abstract

The invention discloses mucosal immune-enhanced recombinant lactobacillus for expressing PEDV (porcine epidemic diarrhea virus) S1 protein as well as a construction method and application of the mucosal immune-enhanced recombinant lactobacillus. The mucosal immune-enhanced recombinant lactobacillus contains a recombinant lactobacillus expression vector for expressing PEDV S1 protein, the amino acid of the PEDV S1 protein is fused with M cell targeting peptide (Co1) and dendritic cell targeting peptide (6aa), the carboxyl terminal of the PEDV S1 protein is fused with a mucosal immune adjuvant (LTB), and the connection sequence of all the parts is Co1-6aa-S1-LTB. According to the mucosal immune enhanced recombinant lactobacillus constructed by the invention, through the synergistic effect of the targeting peptide (DC / MC targeting peptide) and the adjuvant (LTB), the mucosal immune response efficiency (including intestinal mucus SIgA and serum IgG levels) of a PEDV S1 antigen is remarkably improved, humoral immunity, cellular immunity and mucosal immune responses of pregnant animals can be remarkably induced, maternal antibodies are generated, and the immune response efficiency of the pregnant animals is remarkably improved. The intestinal SIgA level of newborn animals is improved, and an effective technical means is provided for prevention and treatment of PED.

Description

Technical Field

[0001] The present invention relates to a mucosal immunity-enhancing recombinant lactobacillus expressing PEDV S1 protein, a construction method and application thereof, and belongs to the field of medical technology. Background Art

[0002] Porcine Epidemic Diarrhea (PED) is an acute enteric infectious disease caused by the porcine epidemic diarrhea virus (PEDV). It is widespread worldwide and affects pigs of all ages, with lactating piglets experiencing the highest morbidity and mortality rates. This disease severely restricts the development of the swine industry and causes significant economic losses. PED is characterized by distinct mucosal infection and intestinal mucosal immunity. Lactobacilli are effective antigen delivery vehicles, and immunizing animals can enhance mucosal immunity. Therefore, oral lactobacilli vaccines that mimic natural infection are a novel strategy for the prevention and control of PED.

[0003] M cells are rare, comprising only one ten-millionth of the total epithelial cell population in the intestine and only 10% of follicle-associated epithelial cells in humans and mice. M cells are immune cells that monitor and phagocytose foreign substances such as antigens, microorganisms, and pathogens. They also possess antigen transport capabilities, transporting various foreign substances to the lymphoid tissue within the intestinal mucosa. They are ideal targets for antigen delivery and immune response stimulation, forming a natural epithelial barrier alongside intestinal epithelial cells.

[0004] Dendritic cells (DCs) can recognize, ingest, process and present antigens to T cells, inducing T cell activation and thus activating immune responses. They are the immune cells with the strongest antigen processing and presentation capabilities.

[0005] Wang [Wang X, Wang L, Zheng D, et al. Oral immunization with a Lactobacillus casei-based anti-porcine epidemic diarrhoea virus (PEDV) vaccine expressing microfold cell-targeting peptide Co1 fused with the COEantigen of PEDV [J]. Journal of applied microbiology, 2018, 124 (2): 368-378.] fused the PEDV COE gene with the M cell-targeting peptide Col gene. After oral immunization of mice, the recombinant bacteria induced significant humoral, cellular and mucosal immune responses in mice. However, this technology has the following disadvantages: ① Limitations in antigen selection: The antigen fragment is single, and only the COE domain of PEDV (part of the S1 protein) is used, which does not cover the key neutralizing epitopes of the full-length S1 protein, which may limit the scope of immune protection. The immunogenicity is weak, and the immunogenicity of COE may be lower than that of the complete S1 protein, affecting the broad spectrum and potency of neutralizing antibodies. ② Inadequate targeting design: Single targeting, relying solely on the M cell targeting peptide (Co1) to promote antigen cross-intestinal mucosal barrier, without combining with the dendritic cell (DC) targeting peptide, resulting in limited antigen presentation efficiency and difficulty in fully activating the systemic immune response. ③ Lack of adjuvant synergy: Failure to introduce mucosal immune adjuvants and integration of adjuvant molecules such as LTB resulted in insufficient mucosal immune response. ④ Unverified maternal antibody delivery: The product was not designed for pregnant animal models, and existing technologies have not verified the delivery effect of maternal antibodies (especially intestinal SIgA) in pregnant mouse or pregnant pig models, making it impossible to prove its actual protective value for newborn piglets. Summary of the Invention

[0006] The present invention aims to provide a mucosal immunity-enhancing recombinant lactobacillus expressing PEDV S1 protein and a method for its construction to address the following problems in the prior art: ① Existing PED vaccines have insufficient immune efficacy. Traditional inactivated or subunit vaccines struggle to effectively induce intestinal mucosal immune responses (such as SIgA antibodies), resulting in insufficient local immune protection against PEDV infection. ② Maternal antibody transfer efficiency is low. Existing vaccines induce insufficient levels of maternal antibodies (especially SIgA antibodies in the intestinal mucosa) in pregnant animals, making them unable to effectively protect newborn piglets through colostrum. ③ Existing mucosal vaccines lack targeted design for key intestinal immune cells (such as M cells and dendritic cells), and the synergistic effect of adjuvants (such as LTB) and antigens is not fully optimized.

[0007] In order to achieve the above object, the present invention adopts the following technical means: The present invention provides a mucosal immunity-enhancing recombinant lactobacillus expressing porcine epidemic diarrhea virus S1 protein. The mucosal immunity-enhancing recombinant lactobacillus contains a recombinant lactic acid bacteria expression vector expressing PEDV S1 protein, wherein the amino acids of the PEDV S1 protein are fused with M cell targeting peptide Co1 and dendritic cell targeting peptide 6aa, and the carboxyl terminus is fused with mucosal immune adjuvant LTB (Escherichia coli heat-labile enterotoxin B subunit), and the connection sequence of each part is Co1-6aa-S1-LTB.

[0008] Among them, preferably, the nucleotide sequence encoding the fusion protein is shown as SEQ ID NO.11.

[0009] Among them, preferably, the recombinant lactic acid bacteria expression vector is obtained by inserting the nucleotide sequence encoding the fusion protein into the lactic acid bacteria expression vector pPG-T7g10-PPT.

[0010] Furthermore, the present invention also proposes a method for constructing the mucosal immunity-enhancing recombinant lactobacillus, comprising the following steps: (1) Using primers S1-F1 and S1-R1, PCR amplify the target band with the downstream homology arm of the recombinant plasmid pMD19Ts-S1; using primers LTB-F and LTB-R, PCR amplify the target band with the upstream homology arm of the recombinant plasmid pET28a-LTB, collect and purify, and homologously recombine the two sequences by fusion PCR. Then, use primers S1-F1 / LTB-R to amplify the S1-LTB fragment, whose sequence is shown in SEQ ID NO.10. The S1-LTB fragment is ligated with the pMD19Ts vector to construct a recombinant plasmid. The plasmid is extracted and identified by PCR. The positive recombinant plasmid is named pMD19Ts-S1-LTB. (2) Using primers C6-F and LTB-R, PCR amplification was performed on the recombinant plasmid pMD19Ts-S1-LTB. Two small fragments Co1 and 6aa were introduced upstream of the S1-LTB fragment to obtain the fragment Co1-6aa-S1-LTB, the sequence of which is shown in SEQ ID NO. 11. After gel collection and purification, the fragment Co1-6aa-S1-LTB was ligated with the pMD19Ts vector to construct a recombinant plasmid. The plasmid was extracted and identified by PCR. The positive recombinant plasmid was named pMD19Ts-Co1-6aa-S1-LTB. The primer sequences are as follows: ; (3) The recombinant plasmids pMD19Ts-Co1-6aa-S1-LTB and pPG-T7g10-PPT were double-digested with restriction endonucleases Sac I and Apa I, respectively. After gel recovery, the plasmids were ligated and transformed into TG1. A single colony was picked and expanded for culture, and the plasmids were extracted. PCR identification and double-digestion identification were performed, and the positive recombinant plasmid was named pPG-Co1-6aa-S1-LTB. (4) The recombinant plasmid pPG-Co1-6aa-S1-LTB was electroporated into L. paracasei In the competent cells, a single colony was picked and expanded for culture, and the plasmid was extracted and identified by PCR. The positive recombinant bacteria with correct identification were named pPG-Co1-6aa-S1-LTB / L.paracasei, It is a mucosal immunity-enhancing recombinant lactobacillus that expresses the S1 protein of porcine epidemic diarrhea virus.

[0011] Furthermore, the present invention also proposes the use of the mucosal immunity-enhancing recombinant lactobacillus in the preparation of a drug for resisting porcine epidemic diarrhea virus infection.

[0012] Among them, preferably, the mucosal immunity-enhancing recombinant lactobacillus can significantly improve the mucosal immune response efficiency of PEDV S1 antigen, including intestinal mucus SIgA and serum IgG levels, and can significantly induce the humoral immunity, cellular immunity and mucosal immune response of pregnant animals and produce maternal antibodies, thereby increasing the intestinal SIgA level of newborn animals.

[0013] Furthermore, the present invention also provides an oral lactic acid bacteria vaccine for preventing and treating porcine epidemic diarrhea virus infection, wherein the vaccine contains the mucosal immunity-enhancing recombinant lactobacillus.

[0014] Compared with the prior art, the present invention has the following beneficial effects: ① Significantly improved mucosal immune efficiency: The synergistic effect of the targeting peptide (Co1+6aa) and LTB increased SIgA levels in intestinal mucus (compared to the control group without targeting peptide or adjuvant). Serum IgG neutralizing activity was significantly enhanced.

[0015] ② Efficient transfer of maternal antibodies: The SIgA level in the intestine of newborn mice was higher than that in the control group (immune protection obtained through breast milk).

[0016] ③ Multi-dimensional immune activation: significantly induces Th1 (IFN-γ, IL-2) and Th2 (IL-4, IL-10) cytokines to achieve a balanced immune response.

[0017] ④ Vector stability and safety: The recombinant bacteria have a high survival rate in the gastrointestinal environment, the plasmid is genetically stable, and the growth curve is consistent with that of the wild strain. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The results of PCR and double enzyme digestion identification of the pMD19Ts-DCpep-S1 recombinant plasmid are shown; a) PCR identification results of pMD19Ts-DCpep-S1 plasmid; M: DNA molecular weight marker DL 2000; 1: PCR product of pMD19Ts-DCpep-S1; 2: PCR product without plasmid added to the system; b) Double enzyme digestion identification results of pMD19Ts-DCpep-S1 plasmid; M: DNA molecular weight marker 2K Plus; 1: Double enzyme digestion product; Figure 2 The results of PCR identification and double enzyme digestion of pPG-DCpep-S1 recombinant plasmid are shown; Wherein: a) pPG-DCpep-S1 plasmid PCR identification results; M: DNA molecular weight standard DL 2000; 1: pPG-DCpep-S1 PCR product; 2: PCR product without plasmid added to the system; b) pPG-DCpep-S1 plasmid double enzyme digestion identification results; M: DNA molecular weight standard 2K Plus; 1: double enzyme digestion product; Figure 3 This is the result of PCR identification of the recombinant plasmid pPG-DCpep-S1; Wherein, M: DNA molecular weight standard DL 2000; 1: pPG-DCpep-S1 / L. paracasei ; 2: PCR product without plasmid added to the system; Figure 4 Figure 1 is the immunoblotting result of S1 protein expression; Wherein, M: relative molecular mass standard of pre-stained protein; 1: empty vector pPG-T7g10-PPT / L. paracasei Lysis protein sample; 2: recombinant bacteria pPG-DCpep-S1 / L. paracasei Lyse protein samples; Figure 5 This is the IFA result of S1 protein expression; Note: A: Empty bacteria pPG-T7g10-PPT / L. paracasei ; B: recombinant bacteria pPG-DCpep-S1 / L. paracasei ; Figure 6 This is a growth curve measurement diagram; Figure 7 This is a diagram for analyzing the genetic stability of the recombinant bacteria; Figure 8 The results of PCR and double enzyme digestion identification of the pMD19Ts-Co1-S1 recombinant plasmid are shown; Wherein: a) PCR identification results of pMD19Ts-Co1-S1 plasmid; M: DNA molecular weight standard DL 2000; 1: PCR product of pMD19Ts-Co1-S1; 2: PCR product without plasmid added to the system; b) Double enzyme digestion identification results of pMD19Ts-Co1-S1 plasmid; M: DNA molecular weight standard 2K Plus; 1: Double enzyme digestion product; Figure 9 The results of PCR and double enzyme digestion identification of the pMD19Ts-Co1-6aa-S1 recombinant plasmid are shown; a) PCR identification results of the pMD19Ts-Co1-6aa-S1 plasmid; M: DNA molecular weight marker DL 2000; 1: PCR product of pMD19Ts-Co1-6aa-S1; 2: PCR product without plasmid added to the system; b) Double enzyme digestion identification of the pMD19Ts-Co1-6aa-S1 plasmid; M: DNA molecular weight marker 2K Plus; 1: Double enzyme digestion product; Figure 10 The results of PCR and double enzyme digestion identification of the pMD19Ts-S1-LTB recombinant plasmid are shown; Wherein: a) PCR identification results of pMD19Ts-S1-LTB plasmid; M: DNA molecular weight marker DL 2000; 1: PCR product of pMD19Ts-S1-LTB; 2: PCR product without plasmid added to the system; b) Double enzyme digestion identification results of pMD19Ts-S1-LTB plasmid; M: DNA molecular weight marker 2K Plus; 1: Double enzyme digestion product; Figure 11 The results of PCR and double enzyme digestion identification of the pMD19Ts-Co1-6aa-S1-LTB recombinant plasmid are shown; a) PCR identification results of the pMD19Ts-Co1-6aa-S1-LTB plasmid; M: 2K Plus DNA molecular weight marker; 1: PCR product without plasmid; 2: PCR product of pMD19Ts-Co1-6aa-S1-LTB; b) Double enzyme digestion identification results of the pMD19Ts-Co1-6aa-S1-LTB plasmid; M: 2K Plus DNA molecular weight marker; 1: Double enzyme digestion product; Figure 12 The results of PCR identification and double enzyme digestion of pPG-Co1-S1 recombinant plasmid are shown; Wherein: a) PCR identification results of pPG-Co1-S1 plasmid; M: DNA molecular weight standard DL 2000; 1: PCR product of pPG-Co1-S1; 2: PCR product without plasmid added to the system; b) Double enzyme digestion identification results of pPG-Co1-S1 plasmid; M: DNA molecular weight standard 2K Plus; 1: Double enzyme digestion product; Figure 13 The results of PCR identification and double enzyme digestion of pPG-Co1-6aa-S1 recombinant plasmid are shown; Wherein: a) PCR identification results of pPG-Co1-6aa-S1 plasmid; M: DNA molecular weight marker DL 2000; 1: PCR product of pPG-Co1-6aa-S1; 2: PCR product without plasmid added to the system; b) Double enzyme digestion identification results of pPG-Co1-6aa-S1 plasmid; M: DNA molecular weight marker 2K Plus; 1: Double enzyme digestion product; Figure 14 The results of PCR identification and double enzyme digestion of pPG-S1-LTB recombinant plasmid are shown; a) PCR identification results of pPG-S1-LTB plasmid; M: DNA molecular weight marker DL 2000; 1: PCR product of pPG-S1-LTB; 2: PCR product without plasmid added to the system; b) Double enzyme digestion identification results of pPG-S1-LTB plasmid; M: DNA molecular weight marker 2K Plus; 1: Double enzyme digestion product; Figure 15 This is the result of PCR identification of the recombinant plasmid pPG-Co1-6aa-S1-LTB; Wherein, M: DNA molecular weight standard DL 2000; 1-23: PCR products of pPG-Co1-6aa-S1-LTB; 24: PCR product without plasmid added to the system; Figure 16 This is the result of double enzyme digestion identification of the pPG-Co1-6aa-S1-LTB recombinant plasmid; Wherein, M: DNA molecular weight standard 2K Plus; 1: double enzyme digestion product; Figure 17 This is the result of PCR identification of the pPG-Co1-S1 recombinant plasmid; Wherein, M: DNA molecular weight standard DL 2000; 1-8: pPG-Co1-S1 / L. paracasei; 9: PCR product without plasmid added to the system; Figure 18 Figure 1 is the result of PCR identification of recombinant plasmids pPG-S1-LTB and pPG-Co1-6aa-S1; Wherein: a) PCR identification results of pPG-S1-LTB plasmid; M: DNA molecular weight standard DL 2000; 1: PCR product of pPG-S1-LTB; 2: PCR product without plasmid added to the system; b) PCR identification results of pPG-Co1-6aa-S1 plasmid; M: DNA molecular weight standard DL 2000; 3: PCR product of pPG-Co1-6aa-S1; 4: PCR product without plasmid added to the system; Figure 19 This is the result of PCR identification of the recombinant plasmid pPG-Co1-6aa-S1-LTB; Where, M: DNA molecular weight standard DL 2000; 1: PCR product of pPG-Co1-6aa-S1-LTB; 2: PCR product without plasmid added to the system; Figure 20 Figure 1 is the immunoblotting result of S1 protein expression; Wherein, M: relative molecular mass standard of pre-stained protein; 1: recombinant bacteria pPG-DCpep-S1 / L. paracasei Lysis protein sample; 2: recombinant bacteria pPG-Co1-6aa-S1 / L. paracasei Lysis protein sample; 3: recombinant bacteria pPG-Co1-S1 / L. paracasei Lysis protein sample; 4: empty bacteria pPG-T7g10-PPT / L. paracasei Lysis protein sample; 5: recombinant bacteria pPG-S1-LTB / L. paracasei Lysis protein sample; 6: recombinant bacteria pPG-Co1-6aa-S1-LTB / L. paracasei Lysis protein sample; 7: empty bacteria pPG-T7g10-PPT / L. paracasei Lyse protein samples; Figure 21 This is the IFA result of S1 protein expression; Note: A: Empty bacteria pPG-T7g10-PPT / L. paracasei ; B: recombinant bacteria pPG-Co1-S1 / L. paracasei ; C: recombinant bacteria pPG-Co1-6aa-S1 / L. paracasei ; D: recombinant bacteria pPG-S1-LTB / L. paracasei ; E: recombinant bacteria pPG-Co1-6aa-S1-LTB / L. paracasei ; Figure 22 This is a growth curve measurement diagram; Figure 23 This is a diagram for analyzing the genetic stability of the recombinant bacteria; Figure 24 This is a diagram for analyzing the binding activity of LTB protein expressed by recombinant lactobacilli; Figure 25 This is the result of measuring the anti-PEDV specific IgG level in the serum of immunized mice; Note:* P <0.05,**0.01< P <0.05,*** P <0.01 vs pPG / L. paracasei ;ns, P >0.05; Note: # P <0.05, ## 0.01< P <0.05, ### P <0.01 vs pPG-DCpep-S1 / L. paracasei , * P <0.05, ** 0.01< P <0.05, *** P <0.01 vs PBS; ns, P >0.05; Figure 26 This is the result of measuring the anti-PEDV specific SIgA level in the intestinal mucus of immunized mice; Note: # P <0.05, ## 0.01< P <0.05, ### P <0.01 vs pPG-DCpep-S1 / L. paracasei , * P <0.05, ** 0.01< P <0.05, *** P <0.01 vs PBS; ns, P >0.05; Figure 27 This is the result of measuring the anti-PEDV specific IgG level in the serum of immunized pregnant mice; Note: # P <0.05, ## 0.01< P <0.05, ### P <0.01 vs pPG-Co1-6aa-S1-LTB / L. paracasei ; * P<0.05, ** 0.01< P <0.05, *** P <0.01 vs PBS; ns, P >0.05; Figure 28 This is the result of measuring the level of anti-PEDV specific SIgA in the intestinal mucus of immunized pregnant mice; Note: # P <0.05, ## 0.01< P <0.05, ### P <0.01 vs pPG-Co1-6aa-S1-LTB / L. paracasei ; * P <0.05, ** 0.01< P <0.05, *** P <0.01 vs PBS; ns, P >0.05; Figure 29 This is the result of the test of anti-PEDV specific SIgA level in feces of immunized pregnant mice; Note: # P <0.05, ## 0.01< P <0.05, ### P <0.01 vs pPG-Co1-6aa-S1-LTB / L. paracasei ; * P <0.05, ** 0.01< P <0.05, *** P <0.01 vs PBS; ns, P >0.05; Figure 30 This is the result of the determination of anti-PEDV specific SIgA level in the intestinal mucus of young mice; Note: # P <0.05, ## 0.01< P <0.05, ### P <0.01 vs pPG-Co1-6aa-S1-LTB / L. paracasei ; *P <0.05, ** 0.01< P <0.05, *** P <0.01 vs PBS; ns, P >0.05; Figure 31 This is the test result of cytokine secretion level in the serum of immunized pregnant mice; Among them, a) IFN-γ; b) IL-2; c) IL-4; d) IL-10, A: PBS; B: pPG-Col-S1 / L. paracasei ; C: pPG-6aa-S1 / L. paracasei ;D:pPG-Co1-6aa-S1 / L. paracasei ;E:pPG-Col-6aa-S1-LTB / L. paracasei ;F:pPG-S1-LTB / L.paracasei; Note: # P <0.05, ## 0.01< P <0.05, ### P <0.01 vs pPG-Co1-6aa-S1-LTB / L. paracasei ; * P <0.05, ** 0.01< P <0.05, *** P <0.01 vs PBS; ns, P >0.05. DETAILED DESCRIPTION

[0019] The present invention is further described below with reference to specific examples, which, however, are not intended to limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the art.

[0020] Unless otherwise specified, all reagents and materials used in the following examples were commercially available.

[0021] Example 1 Construction and identification of recombinant lactobacilli expressing PEDV S1 fused to DC targeting peptide 1. Construction and identification of recombinant Lactobacillus expressing PEDV S1 fused to DC targeting peptide (1) Construction and identification of recombinant plasmid pPG-DCpep-S1 The extracted pMD19Ts-S1 plasmid (pMD19Ts-S1 plasmid has been recorded in the following literature: Ma Rumeng. Comparative study on the immune response induced by different Lactobacillus receptor strains expressing porcine epidemic diarrhea virus protective antigen S1 [D]. Northeast Agricultural University, 2023. DOI: 10.27010 / d.cnki.gdbnu.2023.000217.) was used as a template and primers DC-F / DC-R were used for PCR amplification. DCpep-S1 The fragment (nucleotide sequence is shown in SEQ ID NO.1) was recovered from the gel and ligated with pMD-19T Simple Vector using T4 DNA Ligase. The fragment and vector were placed in a 16°C ligator for 18 h. The ligation product was heat-converted to TG1 From the competent E. coli cells, a single colony was picked from the plate for expansion culture and plasmid was extracted. PCR identification was performed using primers 19T-F and 19T-R. The positive clone plasmid was named pMD19Ts-DCpep-S1. The results of PCR and double enzyme digestion identification of the pMD19Ts-DCpep-S1 recombinant plasmid are shown in Figure 1. Figure 1 shown.

[0022] Use restriction enzymes Sac I and Apa I. The recombinant plasmids pMD19Ts-DCpep-S1 and pPG-T7g10-PPT (the plasmid pPG-T7g10-PPT was prepared according to the method disclosed in the patent application with publication number CN107619816A, entitled "A genetically engineered lactic acid bacteria oral vaccine strain for targeted delivery of vaccine antigens and its use in preventing and treating chicken Escherichia coli disease") were double-digested; a gel recovery kit was used to DCpep-S1 Fragments and linearization pPG The vector fragment was purified and ligated overnight at 4°C. The ligation product was heat-converted to TG1 A single colony was picked from the plate for expansion culture and plasmid was extracted. PCR and double enzyme digestion were performed using primers pPG-F and pPG-R. The positive clone plasmid identified correctly was named pPG-DCpep-S1. The results of PCR and double enzyme digestion of pPG-DCpep-S1 recombinant plasmid are shown in Figure 2. Figure 2 The primer sequences are shown in Table 1 below.

[0023] ; Note: Underline The bold part is the LYPPPY polypeptide sequence of porcine CTLA4; bold Underlined part is the Flag tag sequence; Bold italic parts is a rigid Linker sequence; Italic underlined part It is an M cell targeting peptide; Bold, italic, underlined parts Dendritic cell targeting peptide (2) Recombinant Lactobacillus pPG-DCpep-S1 / L. paracasei Construction and identification of The preparation method of lactobacillus competent cells is as follows: the porcine Lactobacillus paracasei stored in the laboratory L. paracasei Take the strain out of the ultra-low temperature freezer, place it on ice and wait for it to thaw naturally. In the clean bench, use an inoculation loop to dip a small amount of bacterial liquid and draw three lines on the anti-MRS solid agar plate. Place the plate in a 37°C constant temperature incubator and incubate for 2 days. Pick several single colonies of similar size and culture them in anti-MRS liquid medium. Add 2 mL of the cultured bacterial liquid to 200 mL of MRS medium containing 1% glycine, cover the bottle, and place it in a 37°C constant temperature incubator and incubate until the OD 600 The pH value was between 0.4 and 0.8. After meeting the requirements, the culture medium was placed on ice for 30 minutes, divided into 50 mL centrifuge tubes in a clean bench, and centrifuged at 5000 rpm at 4°C for 10 minutes. The lactic acid bacteria competent preparation wash solution Solution II (170 g sucrose was added to 90 mL of deionized water, followed by 10 mL of glycerol, stirred to mix evenly, and sterilized by high-pressure steam) was pre-cooled in ice. After centrifugation, the supernatant was discarded in a clean bench, and 30 mL of pre-cooled solution was taken to slowly resuspend the precipitate. The precipitate was centrifuged at 5000 rpm at 4°C for 10 minutes. The previous step was repeated. 1 mL of Solution II was added to each centrifuge tube, and the precipitate was slowly aspirated with a pipette. The precipitate was divided and stored in an ultra-low temperature refrigerator.

[0024] The pPG-DCpep-S1 recombinant plasmid was electroporated into L. paracasei Competent cells. Soak the electroporation cup in alcohol the night before, rinse repeatedly with clean water, and place it in a clean bench overnight under UV light. The next day, pre-cool the electroporation cup in the refrigerator, place the electroporator in the clean bench under UV sterilization for half an hour, start the machine and preheat for 15 minutes. Set the parameters: U = 2200 V, R = 400 Ω, C = 25 F. L. paracaseiRemove the plate from the -80°C freezer and place it in an ice box to thaw; add 1 mg of the pPG-DCpep-S1 recombinant plasmid to the competent cells, mix them slowly with a pipette tip, and place them on ice for 120 s; take out the electroporation cup from the refrigerator, add the mixed competent cells to the cup, and place it in the groove of the electroporator; press the start button and immediately add 1 ml of MRS recovery medium to the cup, mix well, transfer it to a 1.5 mL EP tube, and place it in a 37°C constant temperature incubator for 4 h; centrifuge at 3500 rpm for 5 minutes, discard most of the liquid, resuspend the precipitate with a pipette tip, add the remaining liquid to the MRS solid medium containing 5 µg / mL Cm, use a spreading rod to evenly disperse the liquid, and place the plate in a 37°C constant temperature incubator for 36-48 h.

[0025] After the culture was completed, the bacteria were picked and expanded to culture and the plasmid was extracted. The pPG-T7g10-PPT vector universal primers pPG-F / pPG-R were used to perform PCR identification on the pPG-DCpep-S1 recombinant plasmid. The PCR identification results of the pPG-DCpep-S1 recombinant plasmid were as follows: Figure 3 As shown, the positive recombinant bacteria identified correctly were named pPG-DCpep-S1 / L. paracasei .

[0026] (3) Western blot and indirect immunofluorescence pPG-DCpep-S1 / L. paracasei The recombinant bacteria were inoculated into 10 mL of chloramphenicol-resistant MRS liquid medium and placed in a 37°C constant temperature incubator for overnight culture. The bacterial solution was centrifuged at 12,000 rpm for 1 min. The supernatant in the tube was discarded, and an equal amount of lysozyme solution was added to the EP tube. The tube was mixed by repeated pipetting with a pipette tip. The tube was placed in a 37°C constant temperature incubator for 2 hours and preheated. The tube was centrifuged at 12,000 rpm for 1 min, and PBS was added and mixed by repeated pipetting with a pipette tip. The tube was centrifuged again and repeated three times. The bacterial pellet was resuspended in 500 μL of 1× PBS and transferred to a 2 mL EP tube. The tube was immersed in an ice-water mixture and sonicated. After the sample turned from turbid to transparent, 5× SDS loading buffer was added and boiled. Western blot identification was performed. The immunoblot results of S1 protein expression are shown in Figure 2. Figure 4 shown.

[0027] pPG-DCpep-S1 / L. paracasei and pPG-T7g10-PPT / L. paracaseiEach recombinant bacterium was inoculated with 2 mL of culture medium and cultured overnight in a 37°C static incubator. 500 μL of the bacterial suspension was centrifuged at 3500 rpm for 5 minutes. The EP tube was tilted to allow the entire volume to drain into the waste container. The pellet was resuspended in sterile deionized water and centrifuged again. This process was repeated three times. The supernatant was discarded and each tube was resuspended in 500 μL of PBS. Mouse monoclonal antibody against S1 protein was used as the primary antibody. The cells were incubated at 37°C for 1 hour and washed three times with PBS. A 1:8000 dilution of FITC goat anti-mouse IgG was incubated at 37°C in the dark for 1 hour and washed three times with PBS. Smears were examined under a microscope.

[0028] The results are as follows Figure 4 As shown in the results, the target band was detected at around 101 kDa. L. paracasei Short rod-shaped green fluorescence was observed on the surface of the bacteria, indicating that the S1 protein was successfully expressed in the recombinant bacteria. Figure 5 shown.

[0029] (4) Analysis of biological characteristics Determination of growth curve: streaking and activating wild fungi L. paracasei and recombinant bacteria pPG-DCpep-S1 / L. paracasei , pick bacteria to expand culture; culture to OD 600 ≈1.0, 1:100 After inoculation, the bacterial solution was immediately diluted in a gradient manner and the colony count was performed on the plate; the operation was performed every 2 hours, and the growth of the strain was monitored within 24 hours and a growth curve was drawn. Three groups of repeated experiments were performed for each strain. The results are as follows Figure 6 shown.

[0030] Genetic stability analysis: The recombinant Lactobacillus pPG-DCpep-S1 / L. paracasei The plasmid was extracted every 5 generations and identified by PCR using universal primers of the vector's multiple cloning site. Figure 7 As shown. Samples with expected band sizes were sent to a biological company for sequencing and compared with the original sequence. The comparison results showed that they were consistent with the original sequence.

[0031] Acid and bile resistance test: wild mushrooms L. paracasei and recombinant bacteria pPG-DCpep-S1 / L. paracasei The bacteria were inoculated into the culture medium and cultured at 37℃ for 6 h. The bacterial solution was diluted graded and the colonies were counted on the plates. The experiment was repeated three times for each group. The colony growth at different pH and bile salt concentrations was statistically analyzed. The results are shown in Tables 2 and 3.

[0032] ; Note: a vs a, P >0.05; values represent mean ± standard deviation (n=3) ; Note: a vs a, P >0.05; values represent mean ± standard deviation (n=3) The results showed that the recombinant bacteria had no significant differences in growth characteristics and acid and bile resistance compared with the wild strain, and the plasmid could be stably inherited.

[0033] Example 2 Construction and identification of recombinant lactobacilli expressing PEDV S1 fused with different targeting peptides and LTB adjuvant (1) Construction of pMD19 recombinant vector expressing PEDV S1 fused with different targeting peptides and LTB adjuvant The recombinant plasmid pMD19Ts-S1 was amplified by PCR using primers Co1-F / Co1-R. Co1-S1 The fragment (its sequence is shown in SEQ ID NO.8) was purified by gel collection and ligated with the pMD19Ts vector to construct a recombinant plasmid. The plasmid was extracted and identified by PCR. The positive recombinant plasmid was named pMD19Ts-Co1-S1. The results of PCR and double enzyme digestion identification of the pMD19Ts-Co1-S1 recombinant plasmid are shown in Figure 1. Figure 8 shown.

[0034] The recombinant plasmid pMD19Ts-S1 was amplified by PCR using primers C6-F and Co1-R. Co1-6aa-S1 The fragment (its sequence is shown in SEQ ID NO.9) was purified by gel collection and ligated with the pMD19Ts vector to construct a recombinant plasmid. The plasmid was extracted and identified by PCR. The positive recombinant plasmid was named pMD19Ts-Co1-6aa-S1. The results of PCR and double enzyme digestion identification of the pMD19Ts-Co1-6aa-S1 recombinant plasmid are shown in Figure 1. Figure 9 shown.

[0035] The recombinant plasmid pMD19Ts-S1 was PCR amplified with primers S1-F1 and S1-R1 to obtain the target band with the downstream homology arm; the recombinant plasmid pET28a-LTB (pET28a-LTB has been recorded in the following literature: Ge Junwei. Expression of Escherichia coli heat-labile enterotoxin B subunit in Escherichia coli and its characteristics [J. Journal of Northeast Agricultural University, 2010, 41(6):109-112. Chen Chaoyang. Construction of a recombinant Lactobacillus casei system expressing Escherichia coli LTAK63 and LTB protein and evaluation of its mucosal adjuvant activity [D]. Northeast Agricultural University, 2013.) was PCR amplified with the target band with the upstream homology arm, and the two sequences were homologously recombined by fusion PCR, and then primers S1-F1 / LTB-R were used to amplify the target band. S1-LTBThe fragment (its sequence is shown in SEQ ID NO.10) was connected to the pMD19Ts vector to construct a recombinant plasmid. The plasmid was extracted and identified by PCR. The positive recombinant plasmid was named pMD19Ts-S1-LTB. The results of PCR and double enzyme digestion identification of the pMD19Ts-S1-LTB recombinant plasmid are shown in Figure 1. Figure 10 shown.

[0036] The recombinant plasmid pMD19Ts-S1-LTB was amplified by PCR using primers C6-F and LTB-R, and two small fragments were introduced upstream of the S1-LTB fragment. Co1 、 6aa , get the fragment Co1-6aa-S1-LTB (its sequence is shown in SEQ ID NO.11), gel-collected and purified, it was ligated with the pMD19Ts vector to construct a recombinant plasmid, the plasmid was extracted and PCR identified, and the positive recombinant plasmid was named pMD19Ts-Co1-6aa-S1-LTB. The results of PCR and double enzyme digestion identification of the pMD19Ts-Co1-6aa-S1-LTB recombinant plasmid are shown in Figure 1. Figure 11 shown.

[0037] ; Note: Underline The bold part is the LYPPPY polypeptide sequence of porcine CTLA4; bold Underlined part is the Flag tag sequence; Bold italic parts is a rigid Linker sequence; Italic underlined part It is an M cell targeting peptide; Bold, italic, underlined parts Dendritic cell targeting peptide (2) Construction and identification of recombinant lactobacilli expressing PEDV S1 fused with different targeting peptides and LTB adjuvant Restriction endonucleases Sac I and Apa I. Double enzyme digestion of four recombinant plasmids and pPG-T7g10-PPT; after gel recovery, ligation and transformation TG1 After single colonies were picked and expanded for culture, plasmids were extracted and identified by PCR and double enzyme digestion. The positive recombinant plasmids were named pPG-Co1-S1, pPG-Co1-6aa-S1, pPG-S1-LTB, and pPG-Co1-6aa-S1-LTB, respectively. The results of PCR and double enzyme digestion were shown as follows: Figure 12-16 As shown. The four recombinant plasmids were electroporated into L. paracasei In competent cells, pick a single colony to expand the culture and extract the plasmid, and perform PCR identification. The PCR identification results are as follows Figure 17-19 The positive recombinant bacteria identified correctly were named pPG-Co1-S1 / L. paracasei 、pPG-Co1-6aa-S1 / L. paracasei 、pPG-S1-LTB / L. paracasei 、pPG-Co1-6aa-S1-LTB / L. paracasei Subsequently, Western blot, indirect immunofluorescence and biological characteristics analysis were performed using the same methods as in Example 1.

[0038] The immunoblotting results of S1 protein expression were as follows Figure 20 The IFA results of S1 protein expression are shown in Figure 21 Growth curves were determined as shown in Figure 22 The results of genetic stability analysis of recombinant bacteria are shown in Figure 23 The survival rates of the recombinant bacteria at different pH values and bile salt concentrations are shown in Tables 5 and 6.

[0039] ; Note: a vs a, P >0.05; values represent mean ± standard deviation (n=3) ; Note: a vs a, P >0.05; values represent mean ± standard deviation (n=3) (3) Analysis of binding activity of recombinant Lactobacillus LTB protein Protein samples were processed (100-fold dilution, 10-fold dilution, stock solution, 30-fold concentration, and 10-fold concentration) and used as test samples. ELISA plates were coated with bovine GM1, using anti-LTB MAb as the primary antibody and HRP-labeled goat anti-mouse IgG as the secondary antibody. GM1 binding activity of the target protein was detected by ELISA.

[0040] The results are as follows Figure 24 The results showed that the recombinant Lactobacillus pPG-Co1-6aa-S1-LTB / expressing PEDV S1 fused MC targeting peptide (Co1), DC targeting peptide (6aa) and mucosal immune adjuvant LTB was successfully constructed. L. paracasei Compared with 3 control recombinant lactobacillus pPG-Co1-S1 / L. paracasei 、pPG-Co1-6aa-S1 / L. paracasei and pPG-S1-LTB / L. paracasei Western blot analysis showed that all recombinant strains expressed protein bands of the expected size. Indirect immunofluorescence analysis confirmed protein expression in the recombinant strains. The recombinant strains showed no significant differences in growth characteristics or acid and bile resistance compared to the wild-type strain, and the plasmid was stably inherited. GM1-ELISA assay demonstrated LTB binding activity to GM1 in vitro.

[0041] Example 3 Comparison of the immune effects of recombinant bacteria in the targeting peptide 6aa group and the targeting peptide DCpep group (1) Thirty-five 4-6 week old BALB / c mice were randomly divided into five groups, with seven mice in each group. The grouping and immunization status are shown in Table 7.

[0042] ; (2) Detection of anti-PEDV-specific IgG antibodies in serum Blood was collected from mice in each group on days 0, 7, 14, 21, and 28. The blood was placed in a 37°C incubator for 1 hour and then in a 4°C refrigerator for 2 hours. Serum was collected by centrifugation and stored in a -40°C refrigerator until use. Anti-PEDV-specific IgG antibodies in serum were detected by indirect ELISA.

[0043] The whole PEDV virus was used as an antigen to coat a 96-well microplate, 100 μL per well, and the plate was placed in a 4°C refrigerator for 12 h; the liquid in the plate was discarded, 1×PBST was added, and the plate was placed on a horizontal shaking platform. After 5 min, the liquid was discarded and the operation was repeated three times; 200 μL of blocking solution (5% skim milk) was added to each well and the plate was placed on a 37°C constant temperature shaker for 2 h; the liquid in the plate was discarded, 1×PBST was added, and the plate was placed on a horizontal shaking platform. After 5 min, the liquid was discarded and the operation was repeated three times; the serum was diluted 1:10 and incubated at 37°C for 2 h; the liquid in the plate was discarded, 1×PBST was added, and the plate was placed on a horizontal shaking platform. After 5 min, the liquid was discarded and the operation was repeated three times; the HRP-labeled goat anti-mouse IgG was diluted 1:10000 and incubated at 37°C for 1 h; the liquid in the plate was discarded, 1×PBST was added, and the plate was placed on a horizontal shaking platform. After 5 min, the liquid was discarded and the operation was repeated three times. After 1 minute, pour off the liquid and repeat the operation three times; add TMB colorimetric solution in a dark environment, wrap the well plate with tin foil and incubate it in a 37℃ constant temperature horizontal shaker for 15 minutes; add stop solution and measure OD with a microplate reader 450 Value, the result is Figure 25 shown.

[0044] (3) Detection of PEDV-specific SIgA antibodies in intestinal mucus The jejunal mucus of each group of mice was collected on days 0, 7, 14, 21, and 28 and stored at -40°C. ELISA was used to detect specific SIgA antibodies in the samples. The results were as follows: Figure 26 shown.

[0045] (4) Determination of neutralizing antibody levels in serum and intestinal mucus Serum and intestinal mucus samples collected on day 7 after immunization were centrifuged to obtain the supernatant, filtered with a filter in a clean bench, and then inactivated in a water bath. A 2-fold dilution to 1:256 was performed in a 96-well cell culture plate, with 100 μL per well; 100 μL of 100 TCID 50 The PEDV virus solution was prepared, and negative serum control, virus control, blank control and positive serum control were set up at the same time. The mixture was allowed to stand at 37℃ for 1 hour. The mixture was allowed to act on the confluent monolayer of Vero cells for 2 hours. The supernatant was discarded and the cell culture medium was added. The cells were cultured in a 37℃ incubator with 5% CO2. After the positive control was completely diseased, the neutralization titer was recorded and calculated.

[0046] The results are shown in Table 8, which show that the recombinant lactobacillus pPG-DCpep-S1 / L. paracasei and the recombinant Lactobacillus pPG-6aa-S1 / L. paracasei and pPG-S1 / L. paracasei The results of the immune response induction in mice were compared, and the results of anti-PEDV specific serum IgG antibody and intestinal mucus SIgA antibody detection showed that pPG-6aa-S1 / L. paracasei and pPG-DCpep-S1 / L. paracasei The pPG-S1 / L. paracasei 、Empty vector group pPG / L. paracasei and PBS group ( P <0.05); and pPG-6aa-S1 / L. paracasei The antibody level of the pPG-DCpep-S1 / L. paracasei group, but the difference was not significant ( P >0.05); the results of serum IgG and intestinal mucus SIgA antibody neutralization activity tests showed that the three recombinant bacteria expressing PEDV S1 protein were significantly higher than those in the PBS group and the empty vector group, but the differences among the groups were not significant ( P >0.05), among which pPG-6aa-S1 / L.paracasei The above results show that the immune effect of the recombinant bacteria in the targeting peptide 6aa group is better than that in the targeting peptide DCpep group, so the targeting peptide 6aa was selected for subsequent studies.

[0047] ; Note: a vs b ( P <0.05); a vs a, b vs b ( P >0.05) Example 4: Comparison of the effects of oral administration of recombinant bacteria to induce immune responses in pregnant mice Sixty-eight 8-9 week old BALB / c mice (17 males and 41 females) were randomly divided into six groups: 12 immunization groups and 8 blank control groups. The gestation period of BALB / c mice is 19-21 days. After co-housing males and females, female mice in each immunization group received a primary vaccination at intervals of 2 days, ensuring that female mice gave birth after the second vaccination. The groupings are shown in Table 9.

[0048] ; After the test, the serum anti-PEDV-specific IgG antibodies, intestinal mucus anti-PEDV-specific SIgA antibodies, feces anti-PEDV-specific SIgA antibodies, intestinal mucus anti-PEDV-specific SIgA antibodies of pups were tested, serum and intestinal mucus neutralizing antibody levels, and serum cytokine levels were measured.

[0049] After pregnant mice were orally immunized with the recombinant bacteria, the levels of anti-PEDV specific antibodies in pregnant mice and newborn mice were detected. Figures 27 - 31 As shown in Table 10, the results showed that: oral recombinant bacteria pPG-Co1-6aa-S1-LTB / L.paracasei It can significantly induce pregnant mice to produce anti-PEDV specific serum IgG antibodies and intestinal mucus SIgA antibodies, and both have in vitro neutralizing activity, which is significantly different from other groups ( P <0.05); the levels of IFN-γ, IL-2, IL-4 and IL-10 in the serum of pregnant mice were significantly increased ( P <0.05); recombinant bacteria pPG-Co1-6aa-S1-LTB / L.paracasei The level of anti-PEDV specific SIgA antibodies in the intestinal mucus of newborn mice born from immunized female mice was significantly higher than that in other groups ( P <0.05). This indicates that the recombinant bacteria pPG-Co1-6aa-S1-LTB / L.paracasei It can significantly induce humoral immunity, cellular immunity and mucosal immune responses in pregnant mice and produce maternal antibodies.

[0050]

[0051] Note: Different superscript letters indicate significant differences among groups ( P <0.05) The same superscript letters indicate no significant difference between the groups ( P >0.05).

Claims

1. A mucosal immunity-enhancing recombinant lactobacillus expressing porcine epidemic diarrhea virus (PEDV) S1 protein, characterized in that: The mucosal immunity-enhancing recombinant lactobacillus contains a recombinant lactic acid bacteria expression vector expressing the PEDV S1 fusion protein, wherein the amino acids of the PEDV S1 fusion protein are fused with the M cell targeting peptide Co1 and the dendritic cell targeting peptide 6aa, and the carboxyl end is fused with the mucosal immune adjuvant LTB, and the connection order of the various parts is Co1-6aa-S1-LTB.

2. The mucosal immunity enhancing recombinant lactobacillus according to claim 1, wherein The nucleotide sequence encoding the PEDV S1 fusion protein is shown in SEQ ID NO.

11.

3. The mucosal immunity enhancing recombinant lactobacillus according to claim 1, wherein The recombinant lactic acid bacteria expression vector is obtained by inserting the nucleotide sequence encoding the PEDV S1 fusion protein into the lactic acid bacteria expression vector pPG-T7g10-PPT.

4. A method for constructing the mucosal immunity-enhancing recombinant lactobacillus according to any one of claims 1 to 3, characterized in that: The following steps are involved: (1) Using primers S1-F1 and S1-R1, PCR amplify the target band with the downstream homology arm of the recombinant plasmid pMD19Ts-S1; using primers LTB-F and LTB-R, PCR amplify the target band with the upstream homology arm of the recombinant plasmid pET28a-LTB, collect and purify, and homologously recombine the two sequences by fusion PCR. Then, use primers S1-F1 / LTB-R to amplify the S1-LTB fragment, whose sequence is shown in SEQ ID NO.

10. The S1-LTB fragment is ligated with the pMD19Ts vector to construct a recombinant plasmid. The plasmid is extracted and identified by PCR. The positive recombinant plasmid is named pMD19Ts-S1-LTB. (2) Using primers C6-F and LTB-R, the recombinant plasmid pMD19Ts-S1-LTB was amplified by PCR. Two small fragments Co1 and 6aa were introduced upstream of the S1-LTB fragment to obtain the fragment Co1-6aa-S1-LTB, the sequence of which is shown in SEQ ID NO.

11. After gel collection and purification, the fragment Co1-6aa-S1-LTB was ligated with the pMD19Ts vector to construct a recombinant plasmid. The plasmid was extracted and identified by PCR. The positive recombinant plasmid was named pMD19Ts-Co1-6aa-S1-LTB. The primer sequences are as follows: ； (3) Use restriction endonucleases Sac I and Apa I. Double enzyme digestion of recombinant plasmids pMD19Ts-Co1-6aa-S1-LTB and pPG-T7g10-PPT; after gel recovery, ligation and transformation TG1 After single colonies were picked and expanded for culture, plasmids were extracted and identified by PCR and double enzyme digestion. The positive recombinant plasmid was named pPG-Co1-6aa-S1-LTB. (4) The recombinant plasmid pPG-Co1-6aa-S1-LTB was electroporated into L. paracasei In the competent cells, a single colony was picked and expanded for culture, and the plasmid was extracted and identified by PCR. The positive recombinant bacteria with correct identification were named pPG-Co1-6aa-S1-LTB / L.paracasei, It is a mucosal immunity-enhancing recombinant lactobacillus that expresses the S1 protein of porcine epidemic diarrhea virus.

5. Use of the mucosal immunity-enhancing recombinant lactobacillus according to any one of claims 1 to 3 in the preparation of a medicament for resisting porcine epidemic diarrhea virus infection.

6. The use according to claim 5, characterized in that The mucosal immunity-enhancing recombinant lactobacillus can significantly improve the mucosal immune response efficiency of PEDV S1 antigen, including intestinal mucus SIgA and serum IgG levels, and can significantly induce the humoral immunity, cellular immunity and mucosal immune response of pregnant animals and produce maternal antibodies, thereby increasing the intestinal SIgA level of newborn animals.

7. An oral lactic acid bacteria vaccine for preventing and treating porcine epidemic diarrhea virus infection, characterized in that: The vaccine contains the mucosal immunity-enhancing recombinant lactobacillus according to any one of claims 1 to 3.

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