Mucosal immunity enhanced recombinant lactobacillus expressing pedv s1 protein, and construction method and application thereof

Abstract

The application discloses a mucosal immunity enhanced recombinant lactobacillus expressing PEDV S1 protein and a construction method and application thereof. The mucosal immunity enhanced recombinant lactobacillus contains a recombinant lactic acid bacteria expression vector expressing the PEDV S1 protein, wherein the PEDV S1 protein is fused with an M cell targeting peptide (Col) and a dendritic cell targeting peptide (6aa) at an amino acid end, and is fused with a mucosal immunity adjuvant (LTB) at a carboxyl end; and the connection sequence of each part is Col-6aa-S1-LTB. The mucosal immunity enhanced recombinant lactobacillus constructed by the application significantly improves the mucosal immunity response efficiency (including intestinal mucus SIgA and serum IgG levels) of the PEDV S1 antigen through the synergistic effect of the targeting peptide (DC / MC targeting peptide) and the adjuvant (LTB), and can significantly induce humoral immunity, cellular immunity and mucosal immunity response of pregnant animals and produce maternal antibodies, improves the intestinal SIgA level of newborn animals, and provides an effective technical means for the prevention and treatment of PED.

Description

TECHNICAL FIELD

[0001] The present application relates to a mucosal immunity enhanced recombinant lactobacillus expressing PEDV S1 protein, a construction method and application thereof. BACKGROUND

[0002] Porcine epidemic diarrhea (PED) is an acute enteric infectious disease caused by porcine epidemic diarrhea virus (PEDV), which is prevalent worldwide, and pigs of all ages are susceptible, especially the morbidity and mortality of nursing piglets are the highest, which seriously restricts the development of pig industry and causes huge economic losses to the global pig industry. PED has obvious characteristics of mucosal infection and intestinal mucosal immunity, and lactic acid bacteria is an effective antigen delivery carrier, and immunization of animals can improve mucosal immunity, therefore, lactic acid bacteria oral vaccine simulating the natural infection process is a new strategy for preventing and controlling PED.

[0003] M cells are less in content, accounting for only one ten-thousandth of the number of epithelial cells in the intestine, and only accounting for 10% in the follicle-associated epithelial cells of humans and mice. M cells are a kind of immune cells, which can monitor and phagocytose foreign substances such as antigens, microorganisms and pathogens, have antigen transport function, can transport a variety of foreign substances to the lymphoid tissue in the intestinal mucosa, and are an ideal target for delivering antigens and stimulating immune response, and constitute a natural epithelial barrier with intestinal epithelial cells.

[0004] Dendritic cells (DCs) can recognize, ingest, process and present antigens to T cells, induce T cell activation, and thus activate immune response reaction, and are the immune cells with the strongest antigen processing and presentation ability.

[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 Col fused with the COE antigen 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 for expression, and after oral immunization of mice, the recombinant bacteria could induce significant humoral, cellular and mucosal immune responses in mice. However, this technology has the following shortcomings: ① antigen selection limitation: the antigen fragment is single, only the COE domain of PEDV (part of the S1 protein) is used, which does not cover the key neutralizing epitope of the full-length S1 protein, which may limit the scope of immune protection. The immunogenicity of COE may be lower than that of the complete S1 protein, affecting the broad spectrum and titer of neutralizing antibodies. ② Insufficient targeting design: single targeting, only relying on M cell-targeting peptide (Col) to promote the passage of antigen through the intestinal mucosal barrier, without combining dendritic cell (DC) targeting peptide, resulting in limited antigen presentation efficiency and difficulty in fully activating systemic immune response. ③ Lack of adjuvant synergy: no mucosal immune adjuvant is introduced, and no adjuvant molecules such as LTB are integrated, resulting in insufficient mucosal immune response. ④ Mother's antibody transmission is not verified: no pregnant animal model is designed, and the existing technology has not verified the transmission effect of mother's antibodies (especially intestinal SIgA) in pregnant mouse or pregnant pig models, and the actual protection value for newborn piglets cannot be proved. SUMMARY

[0006] The purpose of the present application is to provide a mucosal immune enhanced recombinant lactobacillus expressing PEDV S1 protein and its construction method, to solve the following problems existing in the prior art: ① The immune efficiency of existing PED vaccine is insufficient, and traditional inactivated vaccine or subunit vaccine is difficult to effectively induce intestinal mucosal immune response (such as SIgA antibody), resulting in insufficient local immune protection against PEDV infection. ② The transmission efficiency of mother's antibodies is low, and the level of mother's antibodies (especially intestinal mucosal SIgA antibodies) induced by existing vaccines in pregnant animals is insufficient, which cannot effectively protect newborn piglets through colostrum. ③ The existing mucosal vaccine lacks targeting 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 application adopts the following technical means:

[0008] The mucosal immunity enhanced recombinant Lactobacillus for expressing PEDV S1 protein of the present application contains a recombinant Lactobacillus expression vector for expressing PEDV S1 protein, wherein the amino acid of the PEDV S1 protein is fused with M cell targeting peptide Col and dendritic cell targeting peptide 6aa, and the carboxy terminal is fused with mucosal immunity adjuvant LTB (E. coli heat-labile enterotoxin B subunit), and the connection sequence of each part is Col-6aa-S1-LTB.

[0009] Preferably, the nucleotide sequence encoding the fusion protein is shown in SEQ ID NO. 11.

[0010] Preferably, the recombinant Lactobacillus expression vector is obtained by inserting the nucleotide sequence encoding the fusion protein into Lactobacillus expression vector pPG-T7g10-PPT.

[0011] Further, the present application also provides a method for constructing the mucosal immunity enhanced recombinant Lactobacillus, comprising the following steps:

[0012] (1) The primer S1-F1 and S1-R1 are used to perform PCR amplification on the recombinant plasmid pMD19Ts-S1 to obtain a target band with a downstream homologous arm; the primer LTB-F and LTB-R are used to perform PCR amplification on the recombinant plasmid pET28a-LTB to obtain a target band with an upstream homologous arm, and the gel is collected and purified; the two sequences are subjected to homologous recombination through fusion PCR; the primer S1-F1 / LTB-R is used to amplify the S1-LTB fragment, and the sequence is shown in SEQ ID NO. 10; the S1-LTB fragment is connected with the pMD19Ts vector to construct a recombinant plasmid, and the plasmid is extracted and subjected to PCR identification; the positive recombinant plasmid is named as pMD19Ts-S1-LTB;

[0013] (2) The primer C6-F and LTB-R are used to perform PCR amplification on the recombinant plasmid pMD19Ts-S1-LTB to introduce two small fragments Col and 6aa at the upstream of the S1-LTB fragment to obtain the fragment Col-6aa-S1-LTB, and the sequence is shown in SEQ ID NO. 11; the gel is collected and purified; the fragment Col-6aa-S1-LTB is connected with the pMD19Ts vector to construct a recombinant plasmid, and the plasmid is extracted and subjected to PCR identification; the positive recombinant plasmid is named as pMD19Ts-Col-6aa-S1-LTB; the primer sequence is as follows:

[0014] ;

[0015] (3) Restriction enzymes Sac I and Apa I were used to double enzyme cut recombinant plasmid pMD19Ts-Co1-6aa-S1-LTB and pPG-T7g10-PPT respectively; after glue recovery, connection and transformation into TG1, single colony picking was carried out to extract plasmid, PCR identification and double enzyme cut identification were carried out, and the positive recombinant plasmid was named as pPG-Co1-6aa-S1-LTB;

[0016] (4) The recombinant plasmid pPG-Co1-6aa-S1-LTB was electroporated into L. paracasei competent cells, single colony picking was carried out to extract plasmid after expansion culture, PCR identification was carried out, and the positive recombinant bacteria correctly identified were named as pPG-Co1-6aa-S1-LTB. L. paracasei, That is, the mucosal immune enhanced recombinant lactobacillus for expressing S1 protein of porcine epidemic diarrhea virus.

[0017] Further, the application also proposes the application of the mucosal immune enhanced recombinant lactobacillus in preparing a medicine for resisting infection of porcine epidemic diarrhea virus.

[0018] Preferably, the mucosal immune enhanced recombinant lactobacillus can significantly improve the mucosal immune response efficiency of PEDV S1 antigen, including intestinal mucus SIgA and serum IgG level, and can significantly induce humoral immunity, cellular immunity and mucosal immune response of pregnant animals and produce maternal antibodies, and improve the intestinal SIgA level of newborn animals.

[0019] Further, the application also proposes an oral lactobacillus vaccine for preventing and treating infection of porcine epidemic diarrhea virus, wherein the vaccine contains the mucosal immune enhanced recombinant lactobacillus.

[0020] Compared with the prior art, the application has the following beneficial effects:

[0021] ①Mucosal immune efficiency is significantly improved: the synergistic effect of the targeting peptide (Co1+6aa) and LTB makes the intestinal mucus SIgA level improved (compared with the control group without targeting peptide or adjuvant). The serum IgG neutralization activity is significantly improved.

[0022] ②Maternal antibody is efficiently transmitted: the intestinal SIgA level of newborn mice is improved compared with the control group (immunoprotection is obtained through breast milk).

[0023] ③Multi-dimensional immune activation: Th1 (IFN-γ, IL-2) and Th2 (IL-4, IL-10) cytokines are significantly induced, and immune response balance is realized.

[0024] (4) Carrier stability and safety: the recombinant bacteria have high survival rate in the gastrointestinal environment, and the plasmid genetic stability is consistent with the growth curve of the wild strain. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 Figure is the PCR and double enzyme digestion identification results of pMD19Ts-DCpep-S1 recombinant plasmid;

[0026] Wherein, a) PCR identification results of pMD19Ts-DCpep-S1 plasmid; M: DNA molecular weight standard DL 2000; 1: PCR product of pMD19Ts-DCpep-S1; 2: PCR product without plasmid in the system; b) Double enzyme digestion identification results of pMD19Ts-DCpep-S1 plasmid; M: DNA molecular weight standard 2K Plus; 1: double enzyme digestion product;

[0027] Figure 2 Figure is the PCR identification and double enzyme digestion identification results of pPG-DCpep-S1 recombinant plasmid;

[0028] Wherein, a) PCR identification results of pPG-DCpep-S1 plasmid; M: DNA molecular weight standard DL 2000; 1: PCR product of pPG-DCpep-S1; 2: PCR product without plasmid in the system; b) Double enzyme digestion identification results of pPG-DCpep-S1 plasmid; M: DNA molecular weight standard 2K Plus; 1: double enzyme digestion product;

[0029] Figure 3 Figure is the PCR identification results of pPG-DCpep-S1 recombinant plasmid;

[0030] Wherein, M: DNA molecular weight standard DL 2000; 1: pPG-DCpep-S1 / L. paracasei ; 2: PCR product without plasmid in the system;

[0031] Figure 4 Figure is the immunoblotting results of S1 protein expression;

[0032] Wherein, M: pre-stained protein relative molecular weight standard; 1: empty carrier pPG-T7g10-PPT / L. paracasei lysis protein sample; 2: recombinant bacteria pPG-DCpep-S1 / L. paracasei lysis protein sample;

[0033] Figure 5 Figure is the IFA results of S1 protein expression;

[0034] Note: A: empty carrier pPG-T7g10-PPT / L. paracaseiB: Recombinant bacteria pPG-DCpep-S1 L. paracasei ;

[0035] Figure 6 Figure 1 is a growth curve determination chart;

[0036] Figure 7 Figure 2 is a genetic stability analysis chart of recombinant bacteria;

[0037] Figure 8 Figure 3 is a PCR and double enzyme digestion identification result chart of pMD19Ts-Co1-S1 recombinant plasmid;

[0038] Figure 3 is a PCR and double enzyme digestion identification result chart of pMD19Ts-Co1-S1 recombinant plasmid; a) PCR identification result of pMD19Ts-Co1-S1 plasmid; M: DNA molecular weight marker DL 2000; 1: PCR product of pMD19Ts-Co1-S1; 2: PCR product without plasmid in the system; b) double enzyme digestion identification of pMD19Ts-Co1-S1 plasmid; M: DNA molecular weight marker 2K Plus; 1: double enzyme digestion product;

[0039] Figure 9 Figure 4 is a PCR and double enzyme digestion identification result chart of pMD19Ts-Co1-6aa-S1 recombinant plasmid;

[0040] Figure 4 is a PCR and double enzyme digestion identification result chart of pMD19Ts-Co1-6aa-S1 recombinant plasmid; a) PCR identification result of 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 in the system; b) double enzyme digestion identification of pMD19Ts-Co1-6aa-S1 plasmid; M: DNA molecular weight marker 2K Plus; 1: double enzyme digestion product;

[0041] Figure 10 Figure 5 is a PCR and double enzyme digestion identification result chart of pMD19Ts-S1-LTB recombinant plasmid;

[0042] Figure 5 is a PCR and double enzyme digestion identification result chart of pMD19Ts-S1-LTB recombinant plasmid; a) PCR identification result of pMD19Ts-S1-LTB plasmid; M: DNA molecular weight marker DL 2000; 1: PCR product of pMD19Ts-S1-LTB; 2: PCR product without plasmid in the system; b) double enzyme digestion identification result of pMD19Ts-S1-LTB plasmid; M: DNA molecular weight marker 2K Plus; 1: double enzyme digestion product;

[0043] Figure 11 Figure 6 is a PCR and double enzyme digestion identification result chart of pMD19Ts-Co1-6aa-S1-LTB recombinant plasmid;

[0044] Wherein, a) PCR identification results of pMD19Ts-Co1-6aa-S1-LTB plasmid; M: DNA marker 2K Plus; 1: PCR product of the system without adding plasmid; 2: PCR product of pMD19Ts-Co1-6aa-S1-LTB; b) double enzyme digestion identification results of pMD19Ts-Co1-6aa-S1-LTB plasmid; M: DNA marker 2K Plus; 1: double enzyme digestion product;

[0045] Figure 12 It is the PCR identification and double enzyme digestion identification results figure of pPG-Co1-S1 recombinant plasmid;

[0046] Wherein, a) PCR identification results of pPG-Co1-S1 plasmid; M: DNA marker DL 2000; 1: PCR product of pPG-Co1-S1; 2: PCR product of the system without adding plasmid; b) double enzyme digestion identification results of pPG-Co1-S1 plasmid; M: DNA marker 2K Plus; 1: double enzyme digestion product;

[0047] Figure 13 It is the PCR identification and double enzyme digestion identification results figure of pPG-Co1-6aa-S1 recombinant plasmid;

[0048] Wherein, a) PCR identification results of pPG-Co1-6aa-S1 plasmid; M: DNA marker DL 2000; 1: PCR product of pPG-Co1-6aa-S1; 2: PCR product of the system without adding plasmid; b) double enzyme digestion identification results of pPG-Co1-6aa-S1 plasmid; M: DNA marker 2K Plus; 1: double enzyme digestion product;

[0049] Figure 14 It is the PCR identification and double enzyme digestion identification results figure of pPG-S1-LTB recombinant plasmid;

[0050] Wherein, a) PCR identification results of pPG-S1-LTB plasmid; M: DNA marker DL 2000; 1: PCR product of pPG-S1-LTB; 2: PCR product of the system without adding plasmid; b) double enzyme digestion identification results of pPG-S1-LTB plasmid; M: DNA marker 2K Plus; 1: double enzyme digestion product;

[0051] Figure 15 It is the PCR identification results figure of pPG-Co1-6aa-S1-LTB recombinant plasmid;

[0052] M: DNA Marker DL 2000; 1-23: PCR products of pPG-Co1-6aa-S1-LTB; 24: PCR products without plasmid in the system;

[0053] Figure 16 Figure for PCR identification results of pPG-Co1-6aa-S1-LTB recombinant plasmid;

[0054] M: DNA Marker 2K Plus; 1: double enzyme digestion product;

[0055] Figure 17 Figure for PCR identification results of pPG-Co1-S1 recombinant plasmid;

[0056] M: DNA Marker DL 2000; 1-8: pPG-Co1-S1 / L. paracasei; 9: PCR products without plasmid in the system;

[0057] Figure 18 Figure for PCR identification results of pPG-S1-LTB and pPG-Co1-6aa-S1 recombinant plasmid;

[0058] M: DNA Marker DL 2000; 1: PCR product of pPG-S1-LTB; 2: PCR products without plasmid in the system; b) PCR identification results of pPG-Co1-6aa-S1 plasmid; M: DNA Marker DL 2000; 3: PCR product of pPG-Co1-6aa-S1; 4: PCR products without plasmid in the system;

[0059] Figure 19 Figure for PCR identification results of pPG-Co1-6aa-S1-LTB recombinant plasmid;

[0060] M: DNA Marker DL 2000; 1: PCR product of pPG-Co1-6aa-S1-LTB; 2: PCR products without plasmid in the system;

[0061] Figure 20 Figure for immunoblotting results of S1 protein expression;

[0062] M: pre-stained protein relative molecular mass marker; 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. paracaseiProtein lysate sample; 4: empty vector pPG-T7g10-PPT L. paracasei Protein lysate sample; 5: recombinant vector pPG-S1-LTB L. paracasei Protein lysate sample; 6: recombinant vector pPG-Co1-6aa-S1-LTB L. paracasei Protein lysate sample; 7: empty vector pPG-T7g10-PPT L. paracasei Protein lysate sample;

[0063] Figure 21 IFA results of S1 protein expression;

[0064] Note: A: empty vector pPG-T7g10-PPT L. paracasei ; B: recombinant vector pPG-Co1-S1 L. paracasei ; C: recombinant vector pPG-Co1-6aa-S1 L. paracasei ; D: recombinant vector pPG-S1-LTB L. paracasei ; E: recombinant vector pPG-Co1-6aa-S1-LTB L. paracasei ;

[0065] Figure 22 Growth curve determination chart;

[0066] Figure 23 Genetic stability analysis chart of recombinant bacteria;

[0067] Figure 24 Binding activity analysis chart of recombinant lactobacillus expressing LTB protein;

[0068] Figure 25 Immunized mouse serum anti-PEDV specific IgG level determination results chart;

[0069] Note: P <0.05, **0.01 P <0.05, *** P <0.01 vs pPG L. paracasei ; ns, P >0.05;

[0070] 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;

[0071] Figure 26 Figure for the results of the determination of the level of SIgA specific to PEDV in the intestinal mucus of immunized mice;

[0072] 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;

[0073] Figure 27 Figure for the results of the determination of the level of IgG specific to PEDV in the serum of immunized pregnant mice;

[0074] 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;

[0075] Figure 28 Figure for the results of the determination of the level of SIgA specific to PEDV in the intestinal mucus of immunized pregnant mice;

[0076] 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;

[0077] Figure 29 Figure for the results of the detection of the level of fecal anti-PEDV specific SIgA in immunized pregnant mice;

[0078] 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;

[0079] Figure 30 Figure for the results of the determination of the level of intestinal mucus anti-PEDV specific SIgA in pups;

[0080] 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;

[0081] Figure 31 Figure for the results of the detection of the level of cytokine secretion in serum of immunized pregnant mice;

[0082] Wherein, 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-Col-6aa-S1 / L. paracasei ; E: pPG-Col-6aa-S1-LTB / L. paracasei ; F: pPG-S1-LTB / L. paracasei;

[0083] Note: # P <0.05, ## 0.01< P <0.05, ### P <0.01 vs pPG-Col-6aa-S1-LTB / L. paracasei ; * P <0.05, ** 0.01< P <0.05, *** P <0.01 vs PBS; ns, P >0.05. DETAILED DESCRIPTION

[0084] The present application will be further described in conjunction with specific examples, but the examples do not limit the present application in any form. Unless otherwise specified, the reagents, methods and equipment used in the present application are conventional reagents, methods and equipment in the technical field.

[0085] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0086] Example 1 Construction and identification of recombinant lactobacillus expressing PEDV S1 fusion DC targeting peptide

[0087] 1. Construction and identification of recombinant lactobacillus expressing PEDV S1 fusion DC targeting peptide

[0088] (1) Construction and identification of recombinant plasmid pPG-DCpep-S1

[0089] Using the extracted pMD19Ts-S1 plasmid (pMD19Ts-S1 plasmid has been described in the following literature: Maju, Comparison of Immune Response Induced by Different Lactobacillus Recipient Bacteria Expressing Porcine Epidemic Diarrhea Virus Protective Antigen S1 [D]. Northeast Agricultural University, 2023. DOI: 10.27010 / d.cnki.gdbnu.2023.000217.) as the template, the primer DC-F / DC-R was used to amplifyDCpep-S1 The fragment (nucleotide sequence as shown in SEQ ID NO. 1) is connected with pMD-19T Simple Vector using T4 DNA Ligase after glue recovery, and the connection is placed in a 16℃ connection instrument for 18 h, and the connection product is heat transformed into TG1 The E. coli competent cells are picked and plated for single colony expansion and plasmid extraction, and primers 19T-F and 19T-R are used for PCR identification, and the positive cloned plasmid is named pMD19Ts-DCpep-S1. The PCR and double enzyme digestion identification results of the pMD19Ts-DCpep-S1 recombinant plasmid are shown in Figure 1 .

[0090] The restriction endonuclease Sac I and Apa I are used for double enzyme digestion of the recombinant plasmid pMD19Ts-DCpep-S1 and pPG-T7g10-PPT (the plasmid pPG-T7g10-PPT is prepared according to the method disclosed in the patent application with publication number CN107619816A and the invention name of “A genetically engineered lactic acid bacteria oral vaccine strain for targeted delivery of vaccine antigens and its use in preventing and treating chicken E. coli disease”); the gel recovery kit is used for purification of DCpep-S1 fragments and linearized pPG vector fragments, 4℃ connection overnight, and the connection product is heat transformed into TG1 , and single colonies are picked and plated for expansion and plasmid extraction, and primers pPG-F and pPG-R are used for PCR and double enzyme digestion identification, and the positive cloned plasmid with correct identification is named pPG-DCpep-S1. The PCR and double enzyme digestion identification results of the pPG-DCpep-S1 recombinant plasmid are shown in Figure 2 . The primer sequences are shown in Table 1.

[0091] ;

[0092] Note: underlined Part of the introduced enzyme cutting site; the bold part is the LYPPPY polypeptide sequence of pig CTLA4; bold underlined portion The underlined part is the Flag tag sequence; bold italicized portion The underlined part is the rigid Linker sequence; italicized underlined portion The underlined part is the M cell targeting peptide; bold italicized underlined portion The underlined part is the dendritic cell targeting peptide

[0093] (2) Construction and identification of recombinant lactobacillus pPG-DCpep-S1 L. paracasei

[0094] ​The preparation method of Lactobacillus competent cells is as follows: Laboratory-preserved porcine Lactobacillus paracasei cells are used... L. paracasei The bacterial culture was removed from the ultra-low temperature freezer and placed on ice to thaw naturally. In a clean bench, a small amount of the bacterial culture was streaked onto a three-zone streak on an antibiotic-free MRS solid agar plate using an inoculation loop. The plate was then placed in a 37°C incubator and incubated for 2 days. Several single colonies of similar size were picked and cultured in antibiotic-free MRS liquid medium. 2 mL of the cultured bacterial culture was added to 200 mL of MRS medium containing 1% glycine. The bottle was capped and placed in a 37°C incubator for static incubation until OD (dose elapsed). 600 The concentration should be between 0.4 and 0.8. After meeting the requirements, place the culture medium on ice for 30 min, then aliquot it into 50 mL centrifuge tubes in a laminar flow hood and centrifuge at 4°C and 5000 rpm for 10 min. Pre-cool the lactic acid bacteria competent cell preparation solution (90 mL of deionized water with 170 g of sucrose and 10 mL of glycerol, stirred until well mixed, and autoclaved) on ice beforehand. After centrifugation, discard the supernatant in a laminar flow hood, slowly resuspend the precipitate in 30 mL of the pre-cooled solution, and centrifuge at 4°C and 5000 rpm for 10 min. Repeat the previous step. Add 1 mL of Solution II solution to each centrifuge tube, slowly pipette the precipitate, aliquot, and store in an ultra-low temperature freezer.

[0095] The pPG-DCpep-S1 recombinant plasmid was electroporated to... L. paracasei In competent cells, the electroporation vessel was soaked in alcohol the night before, rinsed repeatedly with water, and exposed to UV light overnight in a clean bench. The next day, the electroporation vessel was pre-cooled in a refrigerator, and the electroporator was placed in a clean bench for UV sterilization for half an hour. It was then preheated for 15 minutes with the following parameters set: U=2200 V, R=400 Ω, C=25 F. The competent cells were then... L. paracasei Remove from the -80℃ freezer and place in an ice box to thaw; add 1 mg of pPG-DCpep-S1 recombinant plasmid to competent cells, mix slowly with a pipette tip, and place on ice for 120 s; remove the electroporation cuvette from the freezer, add the mixed competent cells to the cuvette, and place it in the groove of the electroporator; press the start button, immediately add 1 ml of MRS recovery medium to the cuvette, mix well, transfer to a 1.5 mL EP tube, and incubate at 37℃ for 4 h; after centrifugation at 3500 rpm for 5 minutes, discard most of the liquid, resuspend the precipitate with a pipette tip, add the remaining liquid to MRS solid medium containing 5 µg / mL Cm, spread the liquid evenly with a spreader, and then incubate at 37℃ for 36–48 h.

[0096] After culturing, bacteria were selected and expanded, and plasmids were extracted. The pPG-DCpep-S1 recombinant plasmid was identified by PCR using the universal primers pPG-F / pPG-R for the pPG-T7g10-PPT vector. The PCR identification results of the pPG-DCpep-S1 recombinant plasmid are as follows: Figure 3 As shown, the correctly identified positive recombinant bacteria were named pPG-DCpep-S1 / L. paracasei .

[0097] (3) Western blot and indirect immunofluorescence

[0098] pPG-DCpep-S1 / L. paracasei The recombinant bacteria were inoculated into 10 mL of chloramphenicol-resistant MRS liquid medium and incubated overnight at 37°C. The bacterial culture was centrifuged at 12000 rpm for 1 min. The supernatant was discarded, and an equal volume of lysozyme solution was added to an EP tube. The mixture was repeatedly pipetted and aspirated, and the tube was incubated at 37°C for 2 h. After centrifugation at 12000 rpm for 1 min, PBS was added and the mixture was repeatedly pipetted and aspirated, followed by centrifugation three times. The bacterial pellet was resuspended in 500 μL of 1×PBS, transferred to a 2 mL EP tube, and sonicated in an ice-water mixture until the sample became clear. Then, 5×SDS loading buffer was added and the sample was boiled. Western blot analysis was performed, and the immunoblotting results for S1 protein expression were as follows: Figure 4 As shown.

[0099] pPG-DCpep-S1 / L. paracasei and pPG-T7g10-PPT / L. paracasei Two mL of each recombinant bacterial culture was incubated overnight at 37°C in a static incubator. 500 μL of the bacterial suspension was centrifuged at 3500 rpm for 5 min. The EP tube was tilted to allow all the liquid to flow into the waste container. The precipitate was resuspended in sterile deionized water and centrifuged again, repeating this process three times. The supernatant was discarded, and each tube was resuspended in 500 μL of PBS. Using mouse anti-S1 protein monoclonal antibody as the primary antibody, the culture was incubated at 37°C for 1 h and washed three times with PBS. FITC goat anti-mouse IgG was diluted 1:8000 and incubated at 37°C in the dark for 1 h and washed three times with PBS. The smears were examined under a microscope.

[0100] The results are as follows Figure 4 As shown, this result indicates that the target band was detected at approximately 101 kDa. Furthermore, in the recombinant strain pPG-DCpep-S1 / L. paracasei Short rod-shaped green fluorescence was observed on the bacterial cell surface, indicating successful expression of the S1 protein in the recombinant bacterial cells, as shown in the results. Figure 5 As shown.

[0101] (4) Biological characteristics analysis

[0102] Determination of growth curves: Streak lines were drawn to activate wild fungi. L. paracasei and recombinant bacteria pPG-DCpep-S1 / L. paracasei Select bacteria for large-scale culture; culture until OD 600 ≈1.0, 1:100. Immediately after inoculation, the bacterial suspension was serially diluted and plate counts were performed. This was repeated every 2 hours to monitor the growth of the strain over 24 hours and plot growth curves. Each strain was tested in triplicate. Results are shown below. Figure 6 As shown.

[0103] Genetic stability analysis: Recombinant Lactobacillus pPG-DCpep-S1 / L. paracasei The culture was continuously passaged for 15 generations, with plasmids extracted every 5 generations. PCR identification was performed using universal primers for the vector's multiple cloning site. The results are as follows: Figure 7 As shown in the figure. Samples with band sizes matching the expectations were sent to a biotechnology company for sequencing, and the results were compared with the original sequence. The comparison results showed that the sequences were consistent with the original sequence.

[0104] Acid and bile salt tolerance tests: wild mushrooms L. paracasei and recombinant bacteria pPG-DCpep-S1 / L. paracasei The bacterial cultures were inoculated into culture media and incubated at 37°C for 6 h. The bacterial cultures were then serially diluted and plate colony counts were performed. Each group was repeated three times. The colony growth at different pH and bile salt concentrations was recorded. The results are shown in Tables 2 and 3.

[0105] ;

[0106] Note: a vs a, P >0.05; numerical values ​​represent mean ± standard deviation (n=3)

[0107] ;

[0108] Note: a vs a, P >0.05; numerical values ​​represent mean ± standard deviation (n=3)

[0109] The results showed that the recombinant bacteria did not differ significantly from the wild-type strain in terms of growth characteristics, acid and bile salt tolerance, and the plasmid could be stably inherited.

[0110] Example 2: Construction and identification of recombinant lactobacilli expressing PEDV S1 fused with different targeting peptides and LTB adjuvant

[0111] (1) Construction of recombinant vectors expressing PEDV S1 fused with different target peptides and LTB adjuvant pMD19

[0112] PCR amplification of recombinant plasmid pMD19Ts-S1 with primers Co1-F / Co1-R Col-S1 The fragment (the sequence of which is shown as SEQ ID NO. 8) was purified by gel recovery, ligated with pMD19Ts vector to construct a recombinant plasmid, the plasmid was extracted for PCR identification, and the positive recombinant plasmid was named as pMD19Ts-Co1-S1. The PCR and double enzyme digestion identification results of pMD19Ts-Co1-S1 recombinant plasmid are shown as follows. Figure 8

[0113] PCR amplification of recombinant plasmid pMD19Ts-S1 with primers C6-F and Co1-R Col-6aa-S1 The fragment (the sequence of which is shown as SEQ ID NO. 9) was purified by gel recovery, ligated with pMD19Ts vector to construct a recombinant plasmid, the plasmid was extracted for PCR identification, and the positive recombinant plasmid was named as pMD19Ts-Co1-6aa-S1. The PCR and double enzyme digestion identification results of pMD19Ts-Co1-6aa-S1 recombinant plasmid are shown as follows. Figure 9

[0114] PCR amplification of recombinant plasmid pMD19Ts-S1 with primers S1-F1 and S1-R1 to obtain a target band with a downstream homologous arm; PCR amplification of 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 research [J. Journal of Northeast Agricultural University, 2010, 41(6): 109-112. Chen Chaoyang. Construction of recombinant lactobacillus casei system expressing Escherichia coli LTAK63 and LTB protein and evaluation of its mucosal adjuvant activity [D]. Northeast Agricultural University, 2013.) with primers LTB-F and LTB-R to obtain a target band with an upstream homologous arm, the two sequences were homologously recombined by fusion PCR, and then a fragment (the sequence of which is shown as SEQ ID NO. 10) was amplified with primers S1-F1 / LTB-R; the fragment was ligated with pMD19Ts vector to construct a recombinant plasmid, the plasmid was extracted for PCR identification, and the positive recombinant plasmid was named as pMD19Ts-S1-LTB. The PCR and double enzyme digestion identification results of pMD19Ts-S1-LTB recombinant plasmid are shown as follows. S1-LTB Figure 10

[0115] PCR amplification of recombinant plasmid pMD19Ts-S1-LTB with primers C6-F and LTB-R to introduce two small fragments Col , 6aa upstream of S1-LTB fragment Col-6aa-S1-LTB ​​​​The sequence of which is shown as SEQ ID NO. 11), purified by gel recovery, ligated with pMD19Ts vector, constructed recombinant plasmid, extracted plasmid for PCR identification, and the positive recombinant plasmid was named as pMD19Ts-Co1-6aa-S1-LTB. The results of PCR and double enzyme digestion identification of pMD19Ts-Co1-6aa-S1-LTB recombinant plasmid are shown as Figure 11 .

[0116] ;

[0117] Note: underlined Part of the introduced enzyme cutting site; the bold part is the LYPPPY polypeptide sequence of pig CTLA4; bold underlined portion Flag tag sequence; bold italicized portion Rigid Linker sequence; italicized underlined portion M cell targeting peptide; bold italicized underlined portion Dendritic cell targeting peptide

[0118] (2) Construction and identification of recombinant lactobacillus expressing PEDV S1 fusion with different targeting peptides and LTB adjuvant

[0119] The four recombinant plasmids and pPG-T7g10-PPT were double digested with restriction enzymes Sac I and Apa I respectively; after gel recovery, ligation and transformation into TG1 , single colonies were picked and expanded for culture, and then the plasmid was extracted, PCR identification and double enzyme digestion identification were performed, and the positive recombinant plasmid was named as pPG-Co1-S1, pPG-Co1-6aa-S1, pPG-S1-LTB, pPG-Co1-6aa-S1-LTB respectively. The results of PCR and double enzyme digestion identification are shown as Figures 12-16 respectively. The four recombinant plasmids were electroporated into L. paracasei competent cells respectively, single colonies were picked and expanded for culture, and then the plasmid was extracted, PCR identification was performed, and the results of PCR identification are shown as Figures 17-19 . The positive recombinant bacteria identified correctly were named as 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 property analysis were performed, and the methods were the same as in Example 1.

[0120] The results of Western blot of S1 protein expression are shown as Figure 20 , and the results of IFA of S1 protein expression are shown as Figure 21The growth curve determination is shown as follows Figure 22 The genetic stability analysis results of the recombinant bacteria are shown as follows Figure 23 The survival rates of the recombinant bacteria under different pH and different concentrations of bile salts are shown in Tables 5 and 6.

[0121] ;

[0122] Note: a vs a, P > 0.05; the numerical values represent mean ± standard deviation (n = 3)

[0123] ;

[0124] Note: a vs a, P > 0.05; the numerical values represent mean ± standard deviation (n = 3)

[0125] (3) Analysis of the binding activity of the recombinant Lactobacillus LTB protein

[0126] After the protein sample is treated (diluted 100 times, diluted 10 times, original solution, concentrated 30 times, and concentrated 10 times), it is used as a detection sample. The enzyme-labeled plate is coated with bovine GM1, the anti-LTB MAb is used as a primary antibody, and the HRP-labeled goat anti-mouse IgG is used as a secondary antibody. The GM1 binding activity of the target protein is detected by ELISA.

[0127] The results are shown as follows Figure 24 The results show that the recombinant Lactobacillus pPG-Co1-6aa-S1-LTB expressing the PEDV S1 fusion MC targeting peptide (Col), the DC targeting peptide (6aa), and the mucosal immune adjuvant LTB is successfully constructed. L. paracasei Compared with three strains of control recombinant Lactobacillus pPG-Co1-S1 L. paracasei , pPG-Co1-6aa-S1 L. paracasei , and pPG-S1-LTB L. paracasei . The Western blot detection results show that the recombinant bacteria can express protein bands of the expected size; indirect immunofluorescence detects that the proteins can be expressed on the recombinant bacteria. The recombinant bacteria do not show obvious differences in growth characteristics and acid and bile salt resistance from the wild strain, and the plasmid can be stably inherited; the LTB has in vitro GM1 binding activity detected by GM1-ELISA.

[0128] Example 3, Comparison of the immune effects of recombinant bacteria in the 6aa targeting peptide group and the DCpep targeting peptide group

[0129] (1) 35 four to six-week-old BALB / c mice were randomly divided into five groups, with seven mice in each group. The grouping and immunization are shown in Table 7.

[0130] ;

[0131] (2) Detection of anti-PEDV specific IgG antibody in serum

[0132] The blood of each group of mice on day 0, 7, 14, 21 and 28 was collected, placed in a 37℃ constant temperature incubator for 1 h, and then placed in a 4℃ refrigerator for 2 h. The serum was collected by centrifugation and stored in a -40℃ refrigerator. The anti-PEDV specific IgG antibody in the serum was detected by indirect ELISA.

[0133] PEDV whole virus was used as an antigen to coat a 96-well microreaction plate, 100 μL per well. The plate was placed in a 4℃ refrigerator for 12 h. The liquid in the plate was discarded, 1×PBST was added, and the plate was placed on a horizontal shaking table for 5 min. The liquid was then 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 incubated in a 37℃ constant temperature shaking incubator for 2 h. The liquid in the plate was discarded, 1×PBST was added, and the plate was placed on a horizontal shaking table for 5 min. The liquid was then discarded and the operation was repeated three times. The serum was diluted 1:10 and incubated at 37℃ for 2 h. The liquid in the plate was discarded, 1×PBST was added, and the plate was placed on a horizontal shaking table for 5 min. The liquid was then discarded and the operation was repeated three times. HRP-labeled goat anti-mouse IgG was diluted 1:10,000 and incubated at 37℃ for 1 h. The liquid in the plate was discarded, 1×PBST was added, and the plate was placed on a horizontal shaking table for 5 min. The liquid was then discarded and the operation was repeated three times. TMB color developing solution was added in a dark environment, the plate was covered with tin foil and incubated in a 37℃ constant temperature horizontal shaking incubator for 15 min. Stop solution was added and the OD value was measured by a microplate reader. 450 Figure 25

[0134] (3) Detection of anti-PEDV specific SIgA antibody in intestinal mucus

[0135] The jejunal mucus of each group of mice on day 0, 7, 14, 21 and 28 was collected and stored at -40℃. The specific SIgA antibody in the sample was detected by ELISA, and the results are shown in Figure 26

[0136] (4) Determination of neutralizing antibody levels in serum and intestinal mucus

[0137] The 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 was performed in a 96-well cell culture plate to 1:256, 100 μL per well. 100 μL of PEDV containing 100 TCID 50 ​​​The PEDV virus liquid was mixed with the prepared serum, and negative serum control, virus control, blank control and positive serum control were set. The mixture was incubated at 37℃ for 1 h, then the mixture was added to the Vero cell monolayer for 2 h, and then the supernatant was discarded and the cell culture solution was added. The mixture was incubated at 37℃ in a 5% CO2 incubator. After the positive control was completely changed, the neutralization titer was recorded and calculated.

[0138] The results are shown in Table 8, which shows that the constructed recombinant Lactobacillus pPG-DCpep-S1 / L. paracasei and the laboratory preserved recombinant Lactobacillus pPG-6aa-S1 / L. paracasei and pPG-S1 / L. paracasei were compared for their effects on inducing immune responses in mice. The results of detection of anti-PEDV specific serum IgG antibodies and intestinal mucus SIgA antibodies showed that the pPG-6aa-S1 / L. paracasei and pPG-DCpep-S1 / L. paracasei groups were significantly higher than the control groups pPG-S1 / L. paracasei , the empty vector group pPG / L. paracasei and the PBS group ( P <0.05); and the antibody level of the pPG-6aa-S1 / L. paracasei group was higher than that of the pPG-DCpep-S1 / L. paracasei group, but the difference was not significant ( P >0.05); the results of detection of serum IgG and intestinal mucus SIgA antibody neutralization activity showed that the three recombinant bacteria expressing PEDV S1 protein were significantly higher than the PBS group and the empty vector group, but the difference between groups was not significant ( P >0.05), and the antibody neutralization activity of the pPG-6aa-S1 / L. paracasei group was the highest. The above results showed that the immune effect of the recombinant bacteria of the target peptide 6aa group was better than that of the target peptide DCpep group, and therefore the target peptide 6aa was selected for subsequent research.

[0139] ;

[0140] Note: a vs b ( P <0.05); a vs a, b vs b ( P >0.05)

[0141] Example 4, Comparison of immune response effects of oral recombinant bacteria on pregnant mice

[0142] Sixty-eight 8-9 week old BALB / c mice were selected, including 17 males and 41 females. They were randomly divided into 6 groups: an immunization group (n=12 per group) and a control group (n=8 per group). The gestation period for BALB / c mice is 19-21 days. After being housed together with males and females, the females in each immunization group were immunized in batches at 2-day intervals to ensure that the females gave birth after the second immunization. The grouping is shown in Table 9.

[0143] ;

[0144] After the procedure, the following tests were performed: detection of serum anti-PEDV specific IgG antibodies, detection of intestinal mucus anti-PEDV specific SIgA antibodies, detection of fecal anti-PEDV specific SIgA antibodies, detection of pup rat intestinal mucus anti-PEDV specific SIgA antibodies, determination of serum and intestinal mucus neutralizing antibody levels, and determination of serum cytokine levels.

[0145] After oral immunization of pregnant mice with the recombinant bacteria, the levels of PEDV-specific antibodies in the pregnant mice and newborn pups were measured. Results are as follows: Figures 27-31 As shown in Table 10, this result indicates that the oral recombinant strain 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, both of which have in vitro neutralizing activity, showing significant differences compared with other groups. P <0.05); induced significant increase in serum IFN-γ, IL-2, IL-4, and IL-10 levels in pregnant mice ( P <0.05); via recombinant strain pPG-Co1-6aa-S1-LTB / L. paracasei The level of anti-PEDV specific SIgA antibody in the intestinal mucus of newborn pups born to 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 L. paracasei It can significantly induce humoral immunity, cellular immunity, and mucosal immune responses in pregnant mice and produce maternal antibodies.

[0146]

[0147] Note: Different superscript letters indicate significant differences between groups. P <0.05) indicates that the difference between groups is not significant (the superscript letters are the same). P >0.05).

Claims

1. A mucosal immune-enhancing recombinant lactobacillus expressing the S1 protein of porcine epidemic diarrhea virus (PEDV), characterized in that, The aforementioned mucosal immune-enhancing recombinant lactobacillus contains a recombinant lactic acid bacteria expression vector expressing the PEDV S1 fusion protein. The PEDV S1 fusion protein is fused with the M cell-targeting peptide Co1 and the dendritic cell-targeting peptide 6aa, and the carboxyl terminus is fused with the mucosal immune adjuvant LTB. The linkage sequence of each part is Co1-Linker-6aa-Linker-S1-LTB. The amino acid sequence of the dendritic cell-targeting peptide 6aa is LYPPPY.

2. The mucosal immune-enhancing recombinant lactobacillus as described in claim 1, characterized in that, 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.

3. The use of the mucosal immune-enhancing recombinant lactobacillus according to claim 1 or 2 in the preparation of a drug for treating porcine epidemic diarrhea virus infection.

4. The application as described in claim 3, characterized in that, The aforementioned mucosal immune-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 humoral immunity, cellular immunity and mucosal immune responses in pregnant animals and produce maternal antibodies, thereby increasing the intestinal SIgA level of newborn offspring.

5. An oral lactic acid bacteria vaccine for the prevention and treatment of porcine epidemic diarrhea virus infection, characterized in that, The vaccine contains the mucosal immune-enhancing recombinant lactobacillus as described in claim 1 or 2.

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