Recombinant lactococcus lactis with high-density expression effect

Abstract

The application provides a recombinant lactococcus lactis with high-density expression effect and a construction method and application thereof. The application designs and constructs fusion proteins Usp45-cA-CS-LTB and Usp45-cA-SLN-LTB containing PEDV protective antigen CS and TGEV polyepitope antigen SLN respectively, and constructs the recombinant lactococcus lactis after codon optimization of the lactococcus lactis, so that the antigen protein is displayed on the surface of the bacterial body with high density; meanwhile, the recombinant bacillus subtilis expressing the CS-LTB and SLN-LTB fusion proteins is constructed, so that the extracellular secretion expression of the target protein is realized. The engineering bacteria constructed by the application can express the PEDV and TGEV protective antigens and fuse the LTB mucosal adjuvant, and can be used for preparing an oral vaccine or an immune enhancer for preventing porcine viral diarrhea, and provides a new technical scheme and a candidate strain for the research and development of a PEDV and TGEV double mucosal vaccine.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and vaccine technology, specifically relating to a recombinant lactococcus with high-density surface display effect, its construction method, and its application. Background Technology

[0002] Porcine viral diarrhea has long been a major disease affecting the global swine industry. Acute intestinal infections caused by porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV) have resulted in significant economic losses for pig production. These two pathogens belong to the same family (Coronaviridae) and genus (α-coronavirus), sharing similar biological characteristics and pathogenic mechanisms. They primarily infect the small intestinal villi epithelial cells of newborn piglets, leading to villi atrophy and malabsorption. Clinically, this manifests as severe watery diarrhea, vomiting, and dehydration; morbidity and mortality rates in piglets under two weeks of age can reach 80%-100%. Despite these similarities in etiology and clinical manifestations, their epidemiological characteristics and evolutionary trends differ significantly.

[0003] Traditional vaccines for TGEV (including inactivated and attenuated vaccines) share similar development strategies and common challenges with the aforementioned PEDV vaccines.

[0004] The S protein is a core target in TGEV subunit vaccine research. Xu successfully constructed the pCZN1-TGEV S1 prokaryotic expression vector, obtained recombinant TGEV S1 protein through an E. coli expression system, and prepared monovalent, bivalent, and mixed subunit vaccines by combining it with PEDV S1 protein. Mouse immunization experiments showed that all vaccines induced high levels of specific antibodies.

[0005] Recombinant live vector vaccines are constructed by inserting the TGEV protective antigen gene into viruses or bacteria. These vaccines combine the advantages of attenuated vaccines and subunit vaccines, enabling them to replicate and deliver antigens in vivo, inducing a comprehensive immune response. Yuan constructed a recombinant swine poxvirus rSPV-SA expressing the TGEV S protein epitope. Mouse immunization experiments showed that rSPV-SA significantly enhanced serum IgG levels and simultaneously upregulated Th1 and Th2 cytokine levels. Passive immunization protection experiments in newborn piglets showed that rSPV-SA could protect piglets 100% from SPV infection, and no obvious clinical symptoms appeared in the immunized group throughout the experiment. Mou constructed a recombinant strain *Bacillus subtilis* CotGSG expressing the TGEV S protein using a Bacillus subtilis spore surface display system. This recombinant bacterium could be captured by submucosal dendritic cells in the intestine, inducing bone marrow-derived dendritic cell maturation, T cell proliferation, and cytokine secretion. After oral immunization of piglets, the titers of specific sIgA antibodies in feces, specific IgG antibodies in serum, and neutralizing antibodies were all significantly increased. Song Zijie constructed a recombinant L. lactis MG1363 / pMG36e-SLN expressing the TGEV SLN gene. After oral immunization of New Zealand white rabbits, elevated serum-specific IgG and small intestinal mucosal IgA antibodies were detected, confirming that the recombinant bacteria can stimulate the body to produce humoral and mucosal immune responses.

[0006] In summary, TGEV vaccine research has made significant progress in traditional vaccines, subunit vaccines, nucleic acid vaccines, and genetically engineered live vector vaccines, but it faces common challenges similar to those of the aforementioned PEDV vaccines. Summary of the Invention

[0007] This invention provides a recombinant lactococcus with high-density expression capabilities, which is a recombinant lactococcus and Bacillus subtilis engineered strain that can express porcine epidemic diarrhea virus (PEDV) and porcine transmissible gastroenteritis virus (TGEV) antigen proteins, thereby overcoming the shortcomings of the prior art.

[0008] This invention first provides a fusion polypeptide, Usp45-cA-CS-LTB, which can be used as an antigen protein; its amino acid sequence is SEQ ID NO:1;

[0009] MVTLPSFNDHSFVNITVSASFGGHSGANLIASDTTINGLSSFCVDTRQFTISLFYNVTNSYGYVSKSQDSNCPFTLQSVNDYLSFSKFCVSTSLLASACTIDLFGYPEFGSGVKFTSLYFQFTKGELITGTPKPLRGVIDVSFMTLDVCTKYTIYGFKGEGIITLTNSSILAGVYYTSDSGQLLAFKNVTSGAVYSVTPCSFSEQAAYVDDDIVGVISSLSSSTFNSTRELPGFFYHSNDGSNCTEPVLVYSNIGVCKSGSIGYVPSQSGQVKIAPTVTGNISIPTNFSMSIGGGGSGGGGSGGGGSNKVKCYVLFTALLSSLCAYGAPQSITELCSEYRNTQIYTINDKILSYTESMAGKREMVIITFKSGATFQVEVPGSQHIDSQKKAIERMKDTLRITYLTETKIDKLCVWNNKTPNSIAAISMENHHHHH;

[0010] A nucleic acid fragment encoding the above-mentioned fusion polypeptide, a specific nucleotide sequence thereof is SEQ ID NO: 2;

[0011]

[0012] This invention also provides another fusion polypeptide that can be used as an antigen protein, namely Usp45-cA-SLN-LTB; its amino acid sequence is SEQ ID NO:3;

[0013] MTVSDSSFSSYGEMPFGVTDGPRYCYGGGGSGGGGSGGGGSQFLQQINAYARPSEVAKEQRKRKSRGGGGSGGGGSGGGGSGMKRSGYGQPIASTLSNITLPMQDDNTDVYCIRSDQFSVYVHSTCKSALWDNVFKRNCTGG GGSGGGGSGGGGSNKVKCYVLFTALLSSLCAYGAPQSITELCSEYRNTQIYTINDKILSYTESMAGKREMVIITFKSGATFQVEVPGSQHIDSQKKAIERMKDTLRITYLTETKIDKLCVWNNKTPNSIAAISMENHHHHHH;

[0014] The nucleic acid fragment encoding the above-mentioned fusion polypeptide has a specific nucleotide sequence as shown in SEQ ID NO:4;

[0015] .

[0016] The present invention also provides a recombinant expression vector, wherein the recombinant expression vector contains the above-mentioned nucleic acid fragments encoding fusion polypeptides;

[0017] Furthermore, the recombinant expression vector is the pMG36e vector;

[0018] In another aspect, the present invention provides a recombinant lactococcus with high-density expression, wherein the recombinant expression vector described above is carried.

[0019] The present invention also provides the use of the recombinant strain in the preparation of oral vaccines or immune enhancers for the prevention of swine viral diarrhea.

[0020] This invention utilizes the Usp45 secretion signal peptide, cell wall anchoring domain (cA), and LTB mucosal adjuvant to construct a fusion expression system, achieving high-density and stable display of the PEDV protective antigen CS and the TGEV multi-epitope antigen SLN on the surface of *Lactococcus lactis*. A corresponding *Bacillus subtilis* secretory expression system was also constructed, achieving efficient secretory expression of the target proteins. Western blot and immunofluorescence results showed that the constructed recombinant strains could stably express the target proteins; *Lactococcus lactis* could achieve cell surface display, and *Bacillus subtilis* could achieve extracellular secretory expression, maintaining good genetic stability even after 20 consecutive passages. Due to the fusion of LTB mucosal adjuvant, the constructed engineered bacteria can effectively enhance the body's specific immune response to PEDV and TGEV protective antigens, and can be used as an oral vaccine or immune enhancer for porcine viral diarrhea. This provides a new technical solution and candidate strain for the development of a PEDV and TGEV bivalent mucosal vaccine, with good application prospects and industrialization value. Attached Figure Description

[0021] Figure 1 PCR amplification results of the Usp45-cA-CS-LTB gene fragment, where M: DL5000 marker; 1-2: Usp45-cA-CS-LTB gene fragment; 3: negative control;

[0022] Figure 2 PCR amplification results of the Usp45-cA-SLN-LTB gene fragment, where M: DL5000 marker; 1-2: Usp45-cA-SLN-LTB gene fragment; 3: negative control;

[0023] Figure 3 PCR identification results of recombinant E. coli DH5α / pMG36e-Usp45-cA-CS-LTB, where M: DL5000 marker; 1-3: positive clones; 4: empty vector control; 5: negative control;

[0024] Figure 4PCR identification results of recombinant E. coli DH5α / pMG36e-Usp45-cA-SLN-LTB, where M: DL5000 marker; 1-3: positive clones; 4: empty vector control; 5: negative control;

[0025] Figure 5 The results of double enzyme digestion PCR identification of recombinant plasmid pMG36e-Usp45-cA-CS-LTB are shown in the figure. In the figure, M: DL5000 marker; 1: recombinant plasmid pMG36e-Usp45-cA-CS-LTB; 2: empty plasmid pMG36e.

[0026] Figure 6 The results of double enzyme digestion PCR of recombinant plasmid pMG36e-Usp45-cA-SLN-LTB are shown in the figure. M: DL5000 marker; 1: recombinant plasmid pMG36e-Usp45-cA-SLN-LTB; 2: empty plasmid pMG36e.

[0027] Figure 7 Sequencing results of recombinant plasmid pMG36e-Usp45-cA-CS-LTB;

[0028] Figure 8 Sequencing results of recombinant plasmid pMG36e-Usp45-cA-SLN-LTB;

[0029] Figure 9 The identification results of recombinant L. lactis MG1363 / pMG36e-Usp45-cA-CS-LTB are shown in the figure. In the figure, M: DL5000 marker; 1-6: positive clones; 7-8: empty vector control; 9: negative control.

[0030] Figure 10 The identification results of recombinant L. lactis MG1363 / pMG36e-Usp45-cA-SLN-LTB are shown in the figure. In the figure, M: DL5000 marker; 1-6: positive clones; 7-8: empty vector control; 9: negative control.

[0031] Figure 11 PCR identification results of recombinant plasmid pHT43-CS-LTB, where M: DL5000 marker; 1-2: recombinant plasmid pHT43-CS-LTB; 3: negative control;

[0032] Figure 12PCR identification results of recombinant plasmid pHT43-SLN-LTB, where M: DL5000 marker; 1-2: recombinant plasmid pHT43-SLN-LTB; 3: negative control;

[0033] Figure 13 PCR identification results of recombinant E. coli DH5α / pHT43-CS-LTB, where M: DNA Maker DL2000; 1-3: positive clones; 4: empty vector control; 5: negative control;

[0034] Figure 14 PCR identification results of recombinant E. coli DH5α / pHT43-SLN-LTB, where M: DNA Maker DL2000; 1-3: positive clones; 4: empty vector control; 5: negative control;

[0035] Figure 15 Sequencing results of recombinant plasmid pHT43-CS-LTB;

[0036] Figure 16 Sequencing results of recombinant plasmid pHT43-SLN-LTB;

[0037] Figure 17 PCR identification results of recombinant B. subtilis WB800N / pHT43-CS-LTB, where M: DL2000 marker; 1-6: positive clones; 7-8: empty vector control; 9: negative control;

[0038] Figure 18 PCR identification results of recombinant B. subtilis WB800N / pHT43-SLN-LTB, where M: DL2000 marker; 1-6: positive clones; 7-8: empty vector control; 9: negative control;

[0039] Figure 19 Western blot identification results of recombinant L. lactis fusion protein. In the figure, M: molecular weight of standard protein; 1: MG1363 / pMG36e-Usp45-cA-CS-LTB; 2: MG1363 / pMG36e-Usp45-cA-SLN-LTB; 3: MG1363 / pMG36e

[0040] Figure 20Western blot identification results of recombinant B. subtilis protein. In the figure, M: molecular weight of standard protein; 1: supernatant of WB800N / pHT43-CS-LTB lysate; 2: precipitate of WB800N / pHT43-CS-LTB lysate; 3: supernatant of WB800N / pHT43-SLN-LTB lysate; 4: precipitate of WB800N / pHT43-SLN-LTB lysate; 5: WB800N / pHT43;

[0041] Figure 21 Indirect immunofluorescence identification results (50×), where in the figure, (A) L. lactis MG1363 / pMG36e-Usp45-cA-CS-LTB; (B) L. lactis MG1363 / pMG36e-Usp45-cA-SLN-LTB; (C) L. lactis MG1363 / pMG36e

[0042] Figure 22 Genetic results of recombinant L. lactis MG1363 stability, where (A) is generation 5; (B) is generation 10; (C) is generation 15; (D) is generation 20; M: DL5000 marker; 1: L. lactis MG1363 / pMG36e-Usp45-cA-CS-LTB; 2: L. lactis MG1363 / pMG36e-Usp45-cA-SLN-LTB; 3: negative control.

[0043] Figure 23 Genetic results of the stability of recombinant B. subtilis WB800N, where (A) is generation 5; (B) is generation 10; (C) is generation 15; (D) is generation 20; M: DL2000 marker; 1: B. subtilis WB800N / pHT43-CS-LTB; 2: B. subtilis WB800N / pHT43-SLN-LTB; 3: negative control. Detailed Implementation

[0044] Strains and plasmids: Lactococcus lactis MG1363, Bacillus subtilis WB800N, Lactococcus lactis expression plasmid pMG36e, and Bacillus subtilis expression plasmid pHT43 were preserved in our laboratory. Escherichia coli (E. coli) DH5α competent cells were purchased from Shanghai Angyu Biotechnology Co., Ltd.

[0045] Main reagents: Restriction endonucleases Sal I, Hind III, T4 DNA ligase, Taq polymerase, high-fidelity enzyme, DL2000 DNA Marker, and DL 5000 DNA Marker were purchased from Bio-Rad Biotechnology (Beijing) Co., Ltd.; Trichromatic pre-stained protein markers (10-250kDa) and 5× protein loading buffer were purchased from Shanghai Yamei Biomedical Technology Co., Ltd.; Anti-His Tag Monoclonal antibody, glycine, Tris, SDS, and tryptophan were purchased from Beijing Solarbio Science & Technology Co., Ltd.; Horseradish peroxidase-labeled goat anti-mouse IgG (H+L) was purchased from Shanghai Beyotime Biotechnology Co., Ltd.; High-purity plasmid mini-preparation kit and ordinary agarose gel DNA recovery kit were purchased from Tiangen Biotech (Beijing) Co., Ltd.; Ampicillin sodium solution (Amp+), chloramphenicol solution (Cm+), and SanPrep column PCR product purification kit were purchased from Sangon Biotech (Shanghai) Co., Ltd.; Erythromycin solution (Ery+) was purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0046] Example 1: Design and Synthesis of Recombinant Expression Fragments

[0047] Referring to the gene sequence of the prevalent PEDV strain SDTA13-2020 in the GenBank database, a truncated gene fragment CS of the S protein (accession number: OP245917.1, nucleotides 22137-23009 nt) was selected. Referring to the gene sequence of the prevalent TGEV strain DS01 in the GenBank database, the A and D antigenic epitopes of the S protein and the N321 antigenic epitope of the N protein (accession number: OP805351.1, nucleotides 21449-21523 nt, 21932-22108 nt, 27791-27865 nt) were tandemly linked into a multi-epitope gene fragment SLN (D-N321-A) using a flexible linker (GGGGS)3.

[0048] Using the CS and SLN genes as the core, the secretion signal peptide gene Usp45 (accession number: EU382094.1) and the domain gene sequence of the cell wall anchoring protein (accession number: U17696.1, nucleotides 904-1488 nt) were fused upstream of these genes via a flexible linker (GGGGS) 3. Simultaneously, the LTB adjuvant gene sequence (accession number: S60731.1, nucleotides 841-1215 nt), a 6×His tag, and a stop codon were fused downstream of these genes via a flexible linker (GGGGS) 3. Based on the codon preference of *Lactococcus lactis*, the gene sequences were optimized and named Usp45-cA-CS-LTB and Usp45-cA-SLN-LTB, respectively. Sal I and Hind III restriction enzyme sites and protective bases were added to both ends of the optimized gene sequences, and the sequences were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The synthesized fragments were cloned into the pUC57 universal vector, and the recombinant plasmids constructed were named pUC57-Usp45-cA-CS-LTB and pUC57-Usp45-cA-SLN-LTB.

[0049] Using the CS and SLN genes as the core, the LTB adjuvant gene sequence (accession number: S60731.1, nucleotides 841-1215 nt), a 6×His tag, and a stop codon were fused downstream of them via a flexible linker (GGGGS) 3. Based on the codon bias of Bacillus subtilis, the gene sequences were optimized, and the optimized sequences were named CS-LTB and SLN-LTB, respectively. BamHI and SmaI restriction enzyme sites and restriction protective bases were added to both ends of the optimized gene sequences, and the sequences were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The synthesized fragments were cloned into the Bacillus subtilis expression plasmid pHT43, and the constructed recombinant plasmids were named pHT43-CS-LTB and pHT43-SLN-LTB.

[0050] Example 2: Primer Design

[0051] Based on the gene sequences of recombinant plasmids pUC57-Usp45-cA-CS-LTB and pUC57-Usp45-cA-SLN-LTB, amplification primers for the target fragments Usp45-cA-CS-LTB and Usp45-cA-SLN-LTB were designed and synthesized. Based on the gene sequence of vector pMG36e, universal primers for pMG36e were designed and synthesized. Based on the gene sequence of vector pHT43, universal primers for pHT43 were designed and synthesized. The primer sequences are shown in Table 1.

[0052] Table 1 Primer Sequences

[0053] rCS-F ACGCGTCGACATGAAAAAGAAAAT rCS-R CCCAAGCTTTTAATGATGATGATG rSLN-F ACGCGTCGACATGAAAAAGAAAAT rSLN-R CCCAAGCTTTTAATGATGATGGTG pMG36e-F AATATCGTAGCGCCGGGGTA pMG36e-R GCCTCCTCATCCTCTTCATC pHT43-F GTTTCGTTCAGACTTGTGCTTAT pHT43-R TCAACCATTTGTTCCAGGTAAG

[0054] Example 3: Construction of recombinant Lactococcus lactis

[0055] 1. Construction of recombinant plasmids

[0056] 1.1 Plasmid Extraction

[0057] (1) E. coli Top10 glycerol bacteria containing plasmids pUC57-Usp45-cA-CS-LTB and pUC57-Usp45-cA-SLN-LTB were streaked onto LB agar plates containing 50 µg / mL Amp+ and incubated at 37℃ for 14 h. Single colonies were picked and inoculated into 3 mL of LB liquid medium (Amp+) and cultured at 37℃ with shaking at 220 rpm for 12 h. The bacterial culture was then transferred to 15 mL of LB liquid medium (Amp+) at a ratio of 1:100 for expansion culture. Plasmids were extracted using the TIANGEN high-purity plasmid mini-extraction kit and stored at -20℃ for later use.

[0058] (2) E. coli DH5α glycerol bacteria carrying the pMG36e plasmid were streaked on LB agar plates containing 300 µg / mL Ery⁺ and cultured overnight at 37°C. Single colonies were picked from the plates and inoculated into 3 mL of LB liquid medium containing Ery⁺ and cultured at 37°C and 220 rpm for 12 h with shaking. The bacterial culture was then transferred at a ratio of 1:50 to 15 mL of LB liquid medium (Ery+) for expansion. The pMG36e plasmid was extracted using the TIANGEN high-purity plasmid mini-extraction kit and stored at -20°C for later use.

[0059] 1.2 Amplification of the target fragment

[0060] Using recombinant plasmids pUC57-Usp45-cA-CS-LTB and pUC57-Usp45-cA-SLN-LTB as templates, the target genes Usp45-cA-CS-LTB and Usp45-cA-SLN-LTB were amplified by PCR. The reaction system is shown in Table 2.3, and the reaction conditions are shown in Table 2.4. The amplified products were purified and recovered using the SanPrep column PCR product purification kit. After recovery, the DNA concentration of the product was determined using a UV spectrophotometer and stored at -20℃ for later use.

[0061] Table 2 PCR amplification reaction system (50 μL)

[0062]

[0063] Table 3 PCR amplification reaction conditions

[0064]

[0065] Using plasmid pUC57-Usp45-cA-CS-LTB as a template, the target fragment Usp45-cA-CS-LTB was amplified by PCR. The results of agarose gel electrophoresis are as follows. Figure 1 As shown, the band size is consistent with the expected 2031 bp; using plasmid pUC57-Usp45-cA-SLN-LTB as a template, the target fragment Usp45-cA-SLN-LTB was amplified by PCR, and the results of agarose gel electrophoresis are as follows. Figure 2 As shown, the band size is consistent with the expected 1575 bp, indicating successful amplification of both target fragments.

[0066] 1.3 Ligation and transformation of the target gene and vector

[0067] (1) Double enzyme digestion reaction

[0068] The target genes Usp45-cA-CS-LTB and Usp45-cA-SLN-LTB, and plasmid pMG36e were double-digested using restriction endonucleases Sal I and Hind III at 37℃ for 20 min. The digestion system is shown in Tables 2.5 and 2.6. After the digestion reaction, the entire digestion system was loaded onto agarose gel electrophoresis for detection.

[0069] Table 4. Double enzyme digestion reaction system for plasmid pMG36e (50 μL)

[0070]

[0071] Table 5. Double enzyme digestion reaction system for the target gene (30 μL)

[0072]

[0073] (2) Agarose gel recovery

[0074] DNA bands of the expected size were obtained by agarose gel electrophoresis. The single target DNA band was excised from the agarose gel using a UV transilluminator and placed in a clean 2 mL centrifuge tube. The target gene and linear plasmid with sticky ends were recovered using the TIANGEN Agarose Gel DNA Recovery Kit (Enhanced Version). The recovered products were stored at 4°C for later use.

[0075] (3) Ligation of the target gene with plasmid pMG36e

[0076] The target genes Usp45-cA-CS-LTB and Usp45-cA-SLN-LTB with sticky ends were ligated to the linear plasmid pMG36e using T4 DNA ligase. The ligation reaction was carried out overnight at 16°C. The specific reaction system is shown in Table 6.

[0077] Table 6. Ligation reaction system of target gene and plasmid pMG36e (20 μL)

[0078] Usp45-cA-CS-LTB / Usp45-cA-SLN-LTB 6 pMG36e 4 <![CDATA[T4DNA Ligase]]> 1 10× ligation buffer 2 <![CDATA[ddH2O]]> 7 Total volume 20

[0079] (4) Transformation of ligation product into Escherichia coli DH5α

[0080] After thawing Shanghai Angyu E. coli DH5α competent cells on ice, the ligation product was added and gently mixed, then incubated on ice for 25 min. The cells were then heat-shocked in a 42℃ metal bath for 45 s, followed by an immediate ice bath for 2 min. 700 μL of antibiotic-free, sterile LB liquid medium was added to centrifuge tubes, mixed, and then incubated at 37℃, 220 rpm for 90 min. After incubation, the cells were collected by centrifugation at 4000 rpm for 2 min. 200 μL of the supernatant was retained to resuspend the bacterial cells, and 100 μL was spread onto LB agar medium containing 300 µg / mL Ery+. The plates were then incubated overnight at 37℃.

[0081] 2. Identification of recombinant plasmids

[0082] 2.1 PCR identification of bacterial culture

[0083] Single colonies were picked from overnight culture plates and inoculated into 3 mL of LB liquid medium (Ery+), and incubated at 37°C and 220 rpm for 12 h. Using the cultured bacterial culture as a template, bacterial PCR was performed using universal primers for the pMG36e vector to preliminarily screen for positive clones. The specific reaction system is shown in Table 2.8. The reaction conditions were: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 s, annealing at 52°C for 30 s, extension at 72°C for 2 min (35 cycles); final extension at 72°C for 10 min.

[0084] Table 7 PCR identification reaction system (25 μL)

[0085]

[0086] The target fragment was ligated with the linear plasmid pMG36e and transformed into E. coli DH5α. Single colonies were picked and cultured in LB broth. Positive clones were preliminarily screened by bacterial PCR, and the results are as follows: Figure 3 , 4 As shown, this indicates that E. coli DH5α / pMG36e-Usp45-cA-CS-LTB and E. coli DH5α / pMG36e-Usp45-cA-SLN-LTB were successfully constructed.

[0087] 2.2 Double enzyme digestion identification

[0088] To further verify that the target gene fragment has been correctly inserted into the expression vector, the positive clones initially screened by bacterial PCR were expanded and cultured. The recombinant plasmid was extracted using the TIANGEN high-purity plasmid mini-extraction kit, and the recombinant plasmid was further identified by double enzyme digestion. The double enzyme digestion system is shown in Table 8.

[0089] Table 8. Double enzyme digestion identification reaction system (50 μL)

[0090] 10×QuickCut Buffer 5 Recombinant plasmid 10 Sal Ⅰ 1 Hind Ⅲ 1 ddH2O 33 Total volume 50

[0091] Recombinant plasmids were extracted from the initially screened recombinant E. coli DH5α for restriction endonuclease analysis. The plasmids were double-digested with restriction endonucleases Sal I and Hind III, and the digestion products were analyzed by 1% agarose gel electrophoresis. The results are as follows: Figure 5 , 6 As shown, the recombinant plasmid pMG36e-Usp45-cA-CS-LTB produced two clear bands of approximately 3.6 kb and 2.1 kb after enzyme digestion; the recombinant plasmid pMG36e-Usp45-cA-SLN-LTB produced two clear bands of approximately 3.6 kb and 1.6 kb after enzyme digestion, and their sizes were completely consistent with the theoretical expectation.

[0092] 2.3 Sequencing Identification

[0093] To confirm the accuracy of the inserted target gene sequence in the recombinant plasmids, the positive recombinant plasmids pMG36e-Usp45-cA-CS-LTB and pMG36e-Usp45-cA-SLN-LTB, selected through double enzyme digestion, were sequenced. Using the recombinant plasmids as templates, the target gene was amplified by PCR using universal primers for the pMG36e vector. The PCR products were then sent to Sangon Biotech (Shanghai) Co., Ltd. for bidirectional Sanger sequencing of the target gene. The assembled sequencing sequences were compared using BioXM 2.7.1 software.

[0094] The assembled sequences were compared with the expected optimized gene sequences (Usp45-cA-CS-LTB and Usp45-cA-SLN-LTB) using BioXM 2.7.1 software. The experimentally determined insert sequences of the two recombinant plasmids were completely consistent with the theoretically designed sequences, with a similarity of 100% (sequencing results are shown in [link to sequencing results]). Figure 7 , 8 No base mismatches, deletions, or insertions were found in the sequence, indicating that the recombinant plasmid was successfully constructed and the sequence is completely correct.

[0095] 3. Preparation of competent cells of Lactococcus lactis MG1363

[0096] (1) Take the L. lactis MG1363 strain preserved in our laboratory, streak it on GM17 agar plates, and anaerobic culture it in a constant temperature incubator at 30℃ for 32 h;

[0097] (2) Pick out the single colonies that have grown and inoculate them into 3 mL of GM17 liquid medium and incubate at 30℃ for 16 h.

[0098] (3) The above culture was transferred to 30 mL of SGM17 hypertonic medium at a volume ratio of 1:50 and incubated at 30°C for 8 h until OD. 600 The value is approximately 0.8;

[0099] (4) Place the cultured bacterial solution on ice and let it stand for 15 min. Centrifuge at 4℃ and 4000×g for 15 min and discard the supernatant.

[0100] (5) Add 15 mL of pre-cooled washing solution, resuspend the bacterial precipitate, mix well, incubate on ice for 15 min, centrifuge at 4℃ and 4000×g for 10 min, discard the supernatant, and repeat this operation three times.

[0101] (6) Resuspend the bacterial cells in 500 μL of pre-cooled washing solution, aliquot 50 μL into 1.5 mL centrifuge tubes, and store at -80℃ for later use.

[0102] 4. Electroporation of Lactococcus lactis competent cells using recombinant plasmids

[0103] Pretreatment of the electrocution cup: Rinse with ultrapure water, soak in 75% ethanol for 2 hours, rinse with sterile enzyme-free water, and then soak in anhydrous ethanol for 30 minutes. After treatment, place the electrocution cup in a clean bench for ultraviolet irradiation. After the residual ethanol in the cup has fully evaporated, transfer it to -20℃ for storage.

[0104] Electroporation procedure: After thawing L. lactis MG1363 competent cells on ice, 1 μL of recombinant plasmids pMG36e-Usp45-cA-CS-LTB (281.1 ng / μL) and pMG36e-Usp45-cA-SLN-LTB (270.7 ng / μL) were added to 50 μL of competent cells, respectively. The mixture was gently pipetted to ensure homogeneity, and then incubated on ice for 10 min. The mixture was then transferred to a pre-cooled 0.2 cm electroporation cuvette and the surface moisture was blotted off. Electroporation was performed with the parameters set to 2.2 kV and 2 ms.

[0105] Post-electroporation treatment: Immediately add 950 μL of SGM17MC resuscitation medium to the electroporation cuvette, mix gently, and incubate on ice for 10 min. Transfer the resulting suspension to a clean 1.5 mL EP tube and incubate at 30°C for 2 h. After incubation, take 100 μL of the supernatant and spread it on a GM17 agar plate containing 10 µg / mL Ery+. Incubate anaerobically at 30°C for 32 h and observe the transformation results.

[0106] 5. Identification of recombinant Lactococcus lactis

[0107] Single colonies were picked from the transformation plate and inoculated into GM17 liquid medium containing 10 μg / mL Ery+. The culture was incubated at 30°C for 16 h. Following the bacterial culture PCR identification method in 2.3.1.4, PCR was performed using universal primers for the pMG36e vector. Positive clones were screened by 1% agarose gel electrophoresis.

[0108] The PCR products of recombinant L. lactis MG1363 / pMG36e-Usp45-cA-CS-LTB and recombinant L. lactis MG1363 / pMG36e-Usp45-cA-SLN-LTB were sent to Sangon Biotech (Shanghai) Co., Ltd. for target gene sequencing, and the sequencing sequences were compared using BioXM 2.7.1 software.

[0109] Multiple single colonies were randomly picked from the transformation plate and identified by colony PCR using universal primers for the pMG36e vector. The results showed that the positive control using pMG36e-Usp45-cA-CS-LTB as the plasmid template and the randomly selected single-clone strains could amplify a specific band of approximately 2.4 kb. Figure 9 The positive control and randomly selected monoclonal strains using pMG36e-Usp45-cA-SLN-LTB as templates amplified a specific band of approximately 1.9 kb. Figure 10 The size of the samples was exactly as expected, indicating that L. lactis MG1363 / pMG36e-Usp45-cA-CS-LTB and L. lactis MG1363 / pMG36e-Usp45-cA-SLN-LTB were successfully constructed.

[0110] Example 4: Construction of recombinant Bacillus subtilis

[0111] 1. Transformation and amplification of recombinant plasmids

[0112] To obtain sufficient plasmids for subsequent experiments, the recombinant plasmids pHT43-CS-LTB and pHT43-SLN-LTB were transformed into E. coli DH5α competent cells for amplification.

[0113] Using plasmid pHT43-CS-LTB as a template, the target fragment CS-LTB was obtained by PCR amplification. The results of agarose gel electrophoresis are as follows: Figure 11 As shown, the band size is consistent with the expected 1511 bp; using plasmid pHT43-SLN-LTB as a template, the target fragment SLN-LTB was amplified by PCR, and the results of agarose gel electrophoresis are as follows. Figure 12 As shown, the band size is consistent with the expected 1055 bp, indicating that the recombinant plasmid was successfully constructed.

[0114] 2. Identification of recombinant plasmids

[0115] (1) Identification by bacterial culture PCR

[0116] Single colonies were picked from transformation plates and inoculated into 3 mL of LB liquid medium (Amp+), and cultured at 37°C and 220 rpm for 12 h. Using the cultured bacterial culture as a template, bacterial PCR was performed using universal primers for the pHT43 vector (sequences shown in Table 2.2) to preliminarily screen for positive clones. The specific reaction system is shown in Table 2.10. The reaction conditions were: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 s, annealing at 49°C for 30 s, extension at 72°C for 1 min (35 cycles); final extension at 72°C for 10 min.

[0117] Table 9 PCR identification reaction system (25 μL)

[0118]

[0119] Recombinant plasmids pHT43-CS-LTB and pHT43-SLN-LTB were transformed into E. coli DH5α. Single clones were picked and cultured in LB broth. Positive clones were preliminarily screened by bacterial PCR, and the results are as follows. Figure 13 , 12 As shown, this indicates that recombinant E. coli DH5α / pHT43-CS and DH5α / pHT43-SLN were preliminarily screened.

[0120] (2) Sequencing identification

[0121] To confirm the accuracy of the inserted target gene sequence in the recombinant plasmid, the positive PCR products initially screened were sent to Sangon Biotech (Shanghai) Co., Ltd. for bidirectional Sanger sequencing of the target gene, and the assembled sequencing sequences were compared using BioXM 2.7.1 software.

[0122] The assembled sequences were compared with the expected optimized gene sequences (CS-LTB and SLN-LTB) using BioXM 2.7.1 software. The experimentally determined insert sequences of both recombinant plasmids were completely consistent with the theoretically designed sequences, with a similarity of 100% (sequencing results are shown in the appendix). Figure 15 , 16 No mismatches, deletions, or insertions were found in the sequence, indicating that the target gene fragment has been accurately cloned into the pHT43 vector, the recombinant plasmid has been successfully constructed, and the sequence is completely correct.

[0123] 3. Preparation of Bacillus subtilis WB800N competent cells

[0124] (1) Take B. subtilis WB800N stored in our laboratory, streak it onto 2×YT agar medium, and incubate it in a constant temperature incubator at 37℃ for 16 h;

[0125] (2) Pick a single colony and inoculate it into 3 mL of GMⅠ liquid medium, and incubate at 30℃ and 150 rpm for 12 h;

[0126] (3) Take 2 mL of GMⅠ culture and transfer it to 18 mL of GMⅡ liquid culture medium. Incubate at 30℃ for 8 h. At this time, the OD600 value is about 0.8.

[0127] (4) Place the bacterial culture on ice and let it stand for 15 min, then centrifuge at 4℃ and 4000×g for 15 min and discard the supernatant;

[0128] (5) Take 15 mL of pre-cooled washing solution to resuspend the bacterial cells, mix well, incubate on ice for 15 min, centrifuge at 4℃, 4000×g for 10 min, discard the supernatant, and repeat this operation three times.

[0129] (6) Resuspend the bacterial cells in 500 μL of pre-cooled washing solution, dispense 50 μL / vial into 1.5 mL centrifuge tubes, and store at -80℃ for later use.

[0130] 4. Transformation of Bacillus subtilis competent cells with recombinant plasmids

[0131] Thaw *B. subtilis* WB800N competent cells on ice. Then, add 10 μL of recombinant plasmids pHT43-CS-LTB (237 ng / μL) and pHT43-SLN-LTB (362.9 ng / μL) to 50 μL of competent cells, gently pipette to mix, and incubate at 37℃ and 220 rpm for 2 h. After incubation, spread 100 μL of the bacterial culture onto 2×YT agar medium containing 5 µg / mL Cm+ and incubate at 37℃ inverted for 16 h.

[0132] 5. Identification of recombinant Bacillus subtilis

[0133] Single colonies were picked from the transformation plate and inoculated into 2×YT liquid medium containing 5 μg / mL Cm+ and cultured at 37℃ and 220 rpm for 16 h. Following the bacterial culture PCR identification method in 2.1.4, PCR reaction was performed using universal primers for the pHT43 vector. Positive clones were screened by 1.5% agarose gel electrophoresis. The reaction system and reaction conditions were the same.

[0134] The PCR products of recombinant B. subtilis WB800N / pHT43-CS-LTB and B. subtilis WB800N / pHT43-SLN-LTB were sent to Sangon Biotech (Shanghai) Co., Ltd. for target gene sequencing, and the sequencing sequences were compared using BioXM2.7.1 software.

[0135] Multiple single colonies were randomly picked from the transformation plate and identified by colony PCR using universal primers for the pHT43 vector. The results showed that both the positive control using pHT43-CS-LTB as a plasmid template and the randomly selected single-clone strains amplified a specific band of approximately 1.5 kb. Figure 17 The positive control using pHT43-SLN-LTB as a template and randomly selected monoclonal strains amplified a specific band of approximately 1 kb. Figure 18 Both were exactly as expected. In contrast, the empty vector bacteria only amplified a plasmid-specific band of about 200 bp. These results indicate that B. subtilis WB800N / pHT43-CS-LTB and B. subtilis WB800N / pHT43-SLN-LTB were successfully constructed.

[0136] Example 5: Expression and Identification of Recombinant Protein

[0137] 1. Preparation of target protein samples from recombinant lactococcus lactis

[0138] Recombinant L. lactis was inoculated onto GM17 solid plates containing 10 μg / mL Ery+ and cultured anaerobically at 30°C for 32 h. Single colonies were picked and inoculated into 3 mL of GM17 liquid medium (Ery+) and cultured statically at 30°C for 16 h. After the culture was completed, the bacterial culture was transferred to 30 mL of GM17 liquid medium (Ery+) at a 1:50 inoculation ratio and cultured statically at 30°C for 8 h. The bacterial culture was collected, centrifuged at 12,000 rpm for 10 min at 4°C, and the supernatant was discarded. The precipitate was resuspended in 10 mL of pre-chilled 1×PBS and incubated on ice for 10 min. The precipitate was centrifuged again at 12,000 rpm for 10 min at 4°C and the supernatant was discarded. The above washing steps were repeated three times. The precipitate was resuspended in 2 mL of pre-chilled 1×PBS and then sonicated. The cell disruptor was set to 30% power, 3 s disruption time, 5 s pause time, and a total working time of 10 min. Centrifuge the disrupted bacterial culture at 12,000 rpm for 10 min at 4°C; collect the centrifuged precipitate and resuspend it in 2 mL of 1×PBS to obtain a protein precipitate sample, and store it at -80°C for later use.

[0139] 2. Preparation of target protein samples from recombinant Bacillus subtilis

[0140] Recombinant B. subtilis was streaked onto 2×YT solid medium containing 5 μg / mL Cm+ and incubated at 37°C inverted mode for 16 h. Single colonies were picked from the plates and inoculated into 3 mL of 2×YT liquid medium (Cm+) and incubated at 37°C and 220 rpm for 16 h. After incubation, the bacterial culture was transferred to 30 mL of 2×YT liquid medium (Cm+) at a 1:50 inoculation ratio and incubated at 37°C and 220 rpm for 8 h. The bacterial culture was centrifuged at 4°C and 12,000 rpm for 10 min, and the supernatant was discarded. The precipitate was resuspended in 10 mL of pre-chilled 1×PBS and incubated on ice for 10 min. The supernatant was discarded after centrifugation at 4°C and 12,000 rpm for 10 min. The above washing steps were repeated three times. The final precipitate was resuspended in 2 mL of pre-chilled 1×PBS and then sonicated. The cell disruptor was set to 30% power, 3 s disruption, 5 s pause, and a total operation time of 10 min. Centrifuge the disrupted bacterial culture at 12,000 rpm for 10 min at 4℃; collect the supernatant as protein supernatant, resuspend the precipitate in 2 mL of 1×PBS as protein precipitate, and store at -80℃ for later use.

[0141] 3. Western blot identification

[0142] (1) Gel preparation: PAGE gels with a thickness of 1 mm were prepared using the Yaxin one-step PAGE gel rapid preparation kit. Before gel preparation, the assembled clean glass plate was tested for leaks by filling it with water for 5 min. After confirming that there was no leakage, the distilled water was discarded and the plate was dried. The lower gel mixture (2.7 mL lower gel solution, 2.7 mL lower gel buffer, and 60 μL coagulant) was poured along the edge of the glass plate, so that the liquid level was about 1.5 cm from the top of the glass plate. Then, 4.5% upper gel mixture (0.75 mL upper gel solution, 0.75 mL upper gel buffer, and 15 μL coagulant) was slowly poured in, and the gel preparation rack was gently vibrated to make the boundary line between the upper and lower gels level. After inserting the comb vertically, the gel was allowed to stand at room temperature for 30 min until it solidified, and then the comb was removed.

[0143] (2) SDS-PAGE electrophoresis: Place the prepared gel into the electrophoresis tank and pour in the electrophoresis buffer to cover the gel plate. Take the prepared protein sample and mix it with 5× denaturing and reducing protein loading buffer at a ratio of 4:1. Heat the mixture in a metal bath at 100℃ for 10 min. After cooling, add 5 μL of Maker and 10 μL of the treated protein sample to the gel wells in sequence. Maintain a constant voltage of 80 V for 30 min. Then adjust the voltage to 120 V until the electrophoresis indicator bromopanseem blue reaches the bottom of the gel.

[0144] (3) Gel cutting and membrane cutting: After electrophoresis, cut the gel containing the expected molecular weight range (35-100 kDa) of the target protein according to the marker. At the same time, cut the PVDF membrane to the same size as the gel, soak it in methanol for about 15 s, and then soak it together with filter paper and sponge in pre-cooled transfer buffer for later use;

[0145] (4) Assemble the "sandwich" structure and transfer: Place the transfer clip in a tray containing pre-cooled transfer buffer, and assemble it in the following order: black base plate - sponge pad - filter paper - PVDF membrane - gel - filter paper - sponge pad - red base plate. Ensure that each layer is precisely aligned and roll it layer by layer with a glass rod to remove air bubbles. After securing the transfer clip, place it correctly into the transfer tank filled with pre-cooled transfer buffer. Place the transfer tank in an ice bath to maintain a low temperature, and transfer for 55 min at a constant current of 270 mA after connecting the power supply.

[0146] (5) Blocking and antibody incubation: After the transfer, the PVDF membrane was removed and immersed in TBST blocking solution containing 5% skim milk powder. It was blocked at 37°C for 2 h. The PVDF membrane was washed 3 times with TBST buffer for 10 min each time. The membrane was placed in the primary antibody incubation solution containing mouse anti-His tag monoclonal antibody and incubated overnight at 4°C. The next day, the membrane was washed 3 times with TBST buffer for 10 min each time. Then the membrane was transferred to the secondary antibody incubation solution containing horseradish peroxidase-labeled goat anti-mouse IgG and incubated on a shaker in the dark for 2 hours at room temperature. After the incubation, the membrane was washed 3 times with TBST buffer for 10 min each time.

[0147] Preparation of 5% skim milk powder: Dissolve 1.5 g of skim milk powder in 30 mL of 1×TBST;

[0148] (6) Color development: Mix ECL chemiluminescence solution A and solution B at a ratio of 1:1 to prepare the exposure solution. After wiping the TBST buffer residue on the surface of the PVDF membrane with absorbent paper, add the exposure solution evenly to the membrane surface and ensure complete coverage. Incubate at room temperature for 2 min and then place it in an exposure instrument for exposure and color development.

[0149] Western blotting analysis was performed on the collected protein supernatant and precipitate samples. The results are as follows: Figure 19 As shown, a clear, specific band was detected at 70 kDa in the protein sample of recombinant L. lactis MG1363 / pMG36e-Usp45-cA-CS-LTB; a clear, specific band was detected at 57 kDa in the protein sample of recombinant L. lactis MG1363 / pMG36e-Usp45-cA-SLN-LTB; and no band was detected at the corresponding positions in the protein sample of the empty vector control strain MG1363 / pMG36e. The results indicate that both recombinant strains successfully expressed fusion proteins with the expected molecular weight.

[0150] Western blotting analysis was performed on the collected protein supernatant and precipitate samples. The results are as follows: Figure 20 As shown, both the supernatant and precipitate samples of recombinant B. subtilis WB800N / pHT43-CS-LTB showed a clear specific band at 43 kDa; both the supernatant and precipitate samples of recombinant B. subtilis WB800N / pHT43-SLN-LTB showed a clear specific band at 30 kDa; and the protein samples of the empty vector B. subtilis WB800N / pHT43 showed no band at the corresponding position. The results indicate that both recombinant bacteria can successfully express fusion proteins with the expected molecular weight and can secrete the target protein extracellularly.

[0151] Example 6: Indirect immunofluorescence identification of recombinant lactococcus

[0152] (1) Sample preparation and fixation

[0153] Recombinant L. lactis MG1363 / pMG36e-Usp45-cA-CS-LTB, recombinant L. lactis MG1363 / pMG36e-Usp45-cA-SLN-LTB, and L. lactis MG1363 / pMG36e in the logarithmic growth phase were collected by centrifugation. The bacterial cells were gently washed twice with sterile PBS and then resuspended in an appropriate amount of PBS. The bacterial suspension was evenly spread on poly-L-lysine-coated glass slides and allowed to air dry at room temperature. The samples were fixed with 4% paraformaldehyde at room temperature for 15 min and washed three times with PBS.

[0154] (2) Sealing and permeation

[0155] Add PBS blocking solution containing 5% bovine serum albumin (BSA) to the sample area and block at room temperature for 1 h. After discarding the blocking solution, add PBS containing 0.1% Triton X-100 to permeate the bacteria for 10 min and wash with PBS 3 times.

[0156] (3) Primary antibody incubation

[0157] Add the working solution of primary antibody containing mouse anti-His tag monoclonal antibody diluted 1:200 with blocking buffer to cover the sample. Place the slide in a humidified chamber and incubate overnight at 4°C. The next day, wash the sample three times with PBS for 5 min each time.

[0158] (4) Incubation with fluorescent secondary antibody

[0159] Under light-protected conditions, the sample was covered with a fluorescent secondary antibody working solution containing FITC-labeled rabbit anti-mouse IgG diluted 1:500 with blocking buffer. The sample was incubated at room temperature in the dark for 1 h. The sample was then washed three times with PBS for 5 min each time. After washing with PBS in the dark, the sample was mounted with anti-fluorescence quenching mounting medium.

[0160] (5) Image acquisition

[0161] The prepared sample slides were observed under a blue excitation fluorescence microscope to confirm the specific expression of the target protein on the bacterial cell surface.

[0162] Indirect immunofluorescence analysis was used to detect the expression location of the target protein in recombinant L. lactis. The results are as follows: Figure 21 As shown, under a laser confocal microscope, specific fluorescent signals were observed around the cells of both recombinant bacteria, indicating that the target protein had been successfully anchored on the cell surface.

[0163] Example 7: Genetic stability analysis of recombinant bacteria

[0164] To assess the genetic stability of exogenous genes in recombinant Lactococcus lactis and Bacillus subtilis during continuous passage, the constructed recombinant strains L. lactis MG1363 / pMG36e-Usp45-cA-CS-LTB, L. lactis MG1363 / pMG36e-Usp45-cA-SLN-LTB, B. subtilis WB800N / pHT43-CS-LTB, and B. subtilis WB800N / pHT43-SLN-LTB were inoculated at a ratio of 1% in antibiotic-free liquid medium and continuously passaged for 20 generations. Bacterial culture was collected at generations 5, 10, 15, and 20, and PCR identification and sequencing were performed using universal primers for pMG36e and pHT43 vectors to verify the presence of the target gene fragment.

[0165] The bacterial cultures of generations 5, 10, 15, and 20 were collected, and identified by PCR using universal primers for the pMG36e vector, followed by sequencing. The results are as follows: Figure 22 As shown, in each generation of testing, both recombinant L. lactis MG1363 / pMG36e-Usp45-cA-CS-LTB and recombinant L. lactis MG1363 / pMG36e-Usp45-cA-SLN-LTB stably amplified specific bands of the expected size, and the sequences were completely consistent with the theoretically designed sequences. These results indicate that the exogenous target gene fragments Usp45-cA-CS-LTB and Usp45-cA-SLN-LTB contained in the two recombinant bacteria are stably present in the host bacteria, and the constructed recombinant bacteria exhibit good genetic stability.

[0166] The bacterial cultures of generations 5, 10, 15, and 20 were collected, and identified by PCR using pHT43 vector universal primers, followed by sequencing. Results are as follows: Figure 23 As shown, in each generation of detection, both recombinant B. subtilis WB800N / pHT43-CS-LTB and recombinant B. subtilis WB800N / pHT43-SLN-LTB stably amplified specific bands of the expected size, and the sequences were completely consistent with the theoretically designed sequences. The results indicate that the exogenous target gene fragments CS-LTB and SLN-LTB contained in the two recombinant bacteria are stably present in the host bacteria, and the constructed recombinant bacteria exhibit good genetic stability.

[0167] This invention achieves high-density, stable anchoring and display of target antigens on the surface of *Lactococcus lactis* by fusing PEDV CS and TGEV SLN multi-epitope antigens with the Usp45 signal peptide, cA cell wall anchoring protein, and LTB mucosal adjuvant. This effectively overcomes the shortcomings of existing technologies, such as low antigen presentation efficiency and difficulty in activating mucosal immunity. The recombinant *Lactococcus lactis* constructed in this invention, when taken orally, can simultaneously and efficiently activate humoral immunity, mucosal immunity, and Th1 / Th2 / Th17 cellular immune responses, providing a safe and efficient novel bivalent oral vaccine solution for mixed infection of PEDV and TGEV.

Claims

1. A fusion polypeptide that can be used as an antigen protein, characterized in that, The amino acid sequence of the fusion polypeptide is SEQ ID NO:

1.

2. A nucleic acid fragment, characterized in that, The nucleic acid fragment encodes the fusion polypeptide of claim 1, and its nucleotide sequence is SEQ ID NO:

2.

3. A fusion polypeptide that can be used as an antigen protein, characterized in that, The amino acid sequence of the fusion polypeptide is SEQ ID NO:

3.

4. A nucleic acid fragment, characterized in that, The nucleic acid fragment encodes the fusion polypeptide of claim 1, and its nucleotide sequence is SEQ ID NO:

4.

5. A recombinant expression vector, characterized in that, The recombinant expression vectors contain the nucleic acid fragments described in claim 2 or 4.

6. The recombinant expression vector as described in claim 5, characterized in that, The recombinant expression vector is the pMG36e vector.

7. A recombinant lactococcus with high-density expression effect, characterized in that, The recombinant lactococcus carries the recombinant expression vector as described in claim 5.

8. The use of the recombinant lactococcus according to claim 7 in the preparation of an oral vaccine or immune enhancer for the prevention of swine viral diarrhea.

9. An oral vaccine or immune enhancer for swine viral diarrhea, characterized in that, The oral vaccine or immune enhancer contains the recombinant lactococcus as described in claim 7.

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