Recombinant lactobacillus plantarum as well as construction method and application thereof in resisting porcine epidemic diarrhea

Abstract

The invention belongs to the technical field of bioengineering, and particularly relates to recombinant lactobacillus plantarum as well as a construction method and application thereof in resisting porcine epidemic diarrhea. According to the invention, by constructing a lactobacillus plantarum gene editing plasmid, the S1 protein of the PEDV LJX01 strain is integrated into a lactobacillus plantarum WCFS1 genome, so that stable passage of a virus gene sequence is realized, immunogenicity is expected to be enhanced, an organism is induced to generate stronger immune response, PEDV infection is effectively resisted, and the problem of poor immune effect of a traditional vaccine is solved.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and in particular relates to a recombinant lactobacillus plantarum and a construction method thereof and an application thereof in resisting porcine epidemic diarrhea. Background Art

[0002] Porcine epidemic diarrhea (PED) is a highly contagious, acute infectious disease caused by porcine epidemic diarrhea virus (PEDV), which poses a serious threat to the pig farming industry. PEDV mainly colonizes and replicates in the intestinal epithelial cells of pigs, triggering an inflammatory response, leading to intestinal tissue damage and severe diarrhea. It can infect pigs of all ages, especially newborn piglets, causing high morbidity and mortality, and bringing huge economic losses to the farming industry. PEDV mutates rapidly, and traditional vaccines (such as live vaccines and inactivated vaccines) have many problems. Although live vaccines can simulate natural infection and produce a strong immune response, they have potential pathogenicity and safety risks; inactivated vaccines are relatively safe, but have weak immunogenicity and often require adjuvants or multiple vaccinations. In addition, the existing vaccines are not ideal for the immune effect of PEDV variants, and antibiotic treatment faces problems such as drug-resistant strains and toxic side effects. Summary of the invention

[0003] To solve the above problems, the present invention provides a method for constructing a recombinant Lactobacillus plantarum, comprising the following steps:

[0004] (1) Plasmid transformation: insert the EGFP fragment and the S-layer anchor expression system constructed in the laboratory into position 0537 of the PHSP02 standard plasmid to obtain the transformed plasmid PHSP02-EGFP;

[0005] (2) Plasmid modification: The EGFP portion of the modified plasmid PHSP02-EGFP was replaced with the S1 protein of the PEDVljx strain to obtain the modified plasmid PHSP02-PEDV-S1;

[0006] (3) Competent cell construction: The auxiliary plasmid PLH01 was transferred into the competent cell of Lactobacillus plantarum WCFS1 to obtain the positive strain PW01;

[0007] (4) Screening of positive strains: The modified plasmids PHSP02-EGFP and PHSP02-PEDVSS1 were transferred into PW01 competent cells, the genome was extracted, and positive strains were screened by PCR;

[0008] (5) Verification of positive strains: The obtained positive strains were propagated for 10, 15, and 20 generations, and genomic PCR was performed to prove that they could be stably inherited. A bacterial smear was made and green fluorescence was observed under an electron microscope. It can be considered that the new recombinant lactic acid can autonomously express the target protein.

[0009] The present invention also provides a recombinant Lactobacillus plantarum prepared by the above construction method.

[0010] On the other hand, the present invention also provides the use of recombinant Lactobacillus plantarum in the preparation of a product for preventing or treating porcine epidemic diarrhea.

[0011] The present invention has the following beneficial effects:

[0012] 1. Innovative gene editing strategies

[0013] The RecE / T-assisted CRISPR-Cas9 system is used for genome editing of Lactobacillus plantarum WCFS1, combined with specific plasmids to achieve double-strand breaks, donor introduction and precise insertion of target genes, overcoming traditional defects; constructing an S-anchoring anchoring vector, and after inserting the sequence, efficiently anchoring the expressed protein through synergistic effects, improving efficiency and immunogenicity, and promoting the immune application of recombinant lactic acid bacteria.

[0014] 2. Accurate preparation and integration of target genes

[0015] When preparing the PEDV-S1 target gene, RNA was first extracted from the intestinal lavage fluid of the infected strain and reverse transcribed and amplified, and the fragment was accurately obtained and then the sequence was modified. It was integrated into the plasmid containing the Cas-9 gene and combined with the S layer anchoring system, and transformed into Lactobacillus plantarum by electroporation. After screening and identification, stable integration was achieved, ensuring the stable expression and application of the gene.

[0016] 3. Effective protein expression verification system

[0017] Western blot technology was used to verify the expression of PEDV-S1 protein in the recombinant lactic acid bacteria WCFS1-PEDV-S1. A series of treatments and tests confirmed that the protein was expressed and the size was correct. At the same time, the WCFS1-EGFP strain was used as a control to assist in visualization. Subculture identification showed that the genes of the WCFS1-PEDV-S1 mutant strain were stably inherited, ensuring continuous protein expression.

[0018] 4. Reliable polyclonal antibody preparation method

[0019] The BL21-pET28a-PEDV-S1 prokaryotic expression system was constructed. The recombinant plasmid was prepared and transformed and screened through primer design, PCR amplification, enzyme ligation, etc. The high-purity PEDV-S1 protein was purified by culture and induction and used to immunize New Zealand white rabbits. The reasonable immunization scheme obtained polyclonal antibodies that could recognize the target protein, providing important support for the study of the immune characteristics of recombinant lactic acid bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1 pLH01 plasmid map;

[0022] Figure 2 pHSP02 plasmid map;

[0023] Figure 3 PCR amplification of PEDV-S1

[0024] M: DL10000 DNA Maker; 1: PEDV LJX01 amplified to obtain PEDV-S1 fragment;

[0025] Figure 4 PEDV-S1 sequence;

[0026] Figure 5 pHSP02-PEDV-S1 plasmid map

[0027] Figure 6 pHSP02-EGFP plasmid map;

[0028] Figure 7 Growth of transformed bacteria on plates with and without resistance;

[0029] 1: WCSF1 competent cells do not grow on chloramphenicol resistant plates; 2: WCFS1 competent cells grow well on non-resistant plates; 3: After pLH01 plasmid is transferred into WCFS1 competent cells, a single colony grows on chloramphenicol plates;

[0030] Figure 8 PCR of WCFS1 into pLH01 plasmid

[0031] M: DL10000 DNA Marker; Positive control: pLH01 bacterial solution; Negative control: water; 1-3: PW01;

[0032] Fig. 9 pHSP02-EGFP was transferred into PW02 competent bacterial solution for PCR

[0033] M: DL10000 DNA Maker; 25: WCFS1-EGFP mutant strain; positive control: pHSP02-PEDV-S1 plasmid; negative control: pHSP02 bacterial solution; 1-24, 26-51: WCFS1-EGFP non-mutated strain;

[0034] Fig.10 pHSP02-PEDV-S1 was transferred into PW02 competent bacterial solution for PCR

[0035] M: DL10000 DNA Maker; 20: WCFS1-PEDV-S1 mutant strain; 1-19, 21-27: WCFS1-PEDV-S1 non-mutated strain;

[0036] Fig.11 WCFS1-PEDV-S1 genome PCR

[0037] M: DL2000 DNA Maker; 1: 10 generations of genomic PCR; 2: 15 generations of genomic PCR; 3: 20 generations of genomic PCR;

[0038] Fig.12 The immunofluorescence images of WCFS1-EGFP are white light image, fluorescence image and merged image from left to right;

[0039] Fig.13 PCR PEDV-S1 bases 1143 to 2208;

[0040] M: DL2000 DNA Marker; 1: PEDV-S1 target band;

[0041] Fig.14 pET28a empty vector was digested with enzymes;

[0042] M: DL10000 DNA Maker; 1-4: pET28a double restriction enzyme digestion;

[0043] Fig.15 Enzyme digestion of BL21-pET28a-PEDV-S1 identification diagram;

[0044] M: DL10000 DNA Maker; 1-7: BL21-PEDV-S1 double restriction enzyme digestion;

[0045] Fig.16 SDS-PGAE expression verified PEDV-S1 protein;

[0046] M: Protein Maker; 1: BL21-PEDV-S1 whole bacteria; 2: BL21-PEDV-S1 supernatant; 3: BL21-PEDV-S1 precipitate;

[0047] Fig.17 Lactobacillus WCFS1-PEDV-S1 Western blot;

[0048] M: Protein Maker; 1: WCFS1-PEDV-S1. DETAILED DESCRIPTION

[0049] Now, various exemplary embodiments of the present invention are described in detail. The methods in the examples are conventional methods unless otherwise specified, and the reagents used are conventional commercially available reagents or reagents prepared by conventional methods unless otherwise specified. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, features and embodiments of the present invention.

[0050] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0051] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0052] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0053] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0054] Example

[0055] 1. Transformation of Lactobacillus plantarum editing plasmid

[0056] (1) Preparation of target gene

[0057] RNA was extracted from the intestinal lavage fluid of the PEDV LJX01 strain using the Tiangen virus RNA extraction kit, and then reverse transcribed according to specific steps (70°C water bath for 5 minutes, then ice bath for 10 minutes, adding reverse transcriptase, RNase inhibitor and buffer, 37°C water bath for 2 hours, then stored at -20°C) to obtain cDNA, and the S1 protein primer sequence of the strain was designed based on the NCBI conservative sequence.

[0058] (2) Vector pHSP02 gene synthesis

[0059] The Ori-Cm fragment was amplified using the pNZ5319 lactic acid bacteria gene knockout plasmid as a template and assembled onto the PLP-gba (cut by PshAI) backbone to construct an auxiliary expression vector pLH01. The RecE / T homology arm LVISKB_1732-1730 was amplified and fused with the promoter Porfx, and the homologous fragment of LP_0537 was inserted into the ApaI site of the targeting plasmid pLpCas-p to obtain pLH02. The S1 protein sequence of the PEDV LJXO1 strain was obtained by sequencing. The artificial gene was used to change the restriction site of the PEDV-S1 gene and add the HA tag sequence for later target protein expression detection. The HA tag, EGFP fragment, and S layer anchor expression sequence were sequentially connected to obtain the pHSP02-EGFP sequence to achieve protective antigen fusion expression. Referring to the pHSP02-EGFP sequence, the EGFP part was replaced with PEDV-S1. Plasmids HSP02-EGFP and pHSP02-PEDV-S1 were synthesized at Nanjing GenScript Biotechnology Co., Ltd.

[0060] (3) Preparation of WCFS1 competent cells

[0061] The WCFS1 strain preserved at -80℃ was taken out and thawed, then streaked on an MRS plate, cultured at 37℃ overnight, and a single colony was picked and inoculated into 5ml MRS medium and cultured at 37℃. The next day, it was inoculated into 10ml SGMRS at a ratio of 1%, and cultured when OD600 was 0.3-0.4. The strain was taken out, precooled on ice for 20 minutes, centrifuged at 4℃ and 4200rpm for 10 minutes, and the supernatant was discarded. The strain was resuspended with 1ml precooled SM buffer and centrifuged again at 4℃ and 4200rpm for 10 minutes. The resuspension and centrifugation steps were repeated twice. After the supernatant was aspirated, it was resuspended on ice with 60μl precooled SM buffer, and the strains were divided into 1.5ml centrifuge tubes and stored on ice for later use.

[0062] (4) Transformation of pLH01 plasmid

[0063] Place the homemade WCFS1 competent cell on ice, take 5μL of pLH01 plasmid and add it, transfer the mixture to an electroporation cup and place it in an ice bath for 20 minutes, then perform electroporation according to procedure 4-1-9 under the conditions of 2500V, 200Ω, 25μF to transform it into WCFS1 competent cells, let it stand at room temperature for 5 minutes, add 600μL of SMRS to the electroporation cup and draw it out with a pipette and place it in a 1.5ml centrifuge tube, let it stand in a 37℃ incubator for 3h, take 100μL and spread it on MRS (Cm resistance) culture medium plates, pick positive single colonies every other day, select positive clones to extract plasmid in small amounts, and name the recombinant strain PW01.

[0064] (5) Transformation of plasmid pHSP02 into PW01 competent cells

[0065] The PW01 strain was taken out from -80℃, thawed, and streaked on an MRS plate, cultured at 37℃ overnight, and a single colony was picked and inoculated into 5ml of MRS medium containing Cm for static culture. The next day, it was inoculated into 10ml of SGMRS containing Cm at a ratio of 1%. When the OD was between 0.3-0.4, an inducing peptide with a specific amino acid sequence (MAGNSSNFIHKIKQIIFTHR) was added at a final concentration of 100ng / ml, and the culture was continued until the OD was between 0.4-0.5. The bacterial solution was precooled on ice for 20min, and then centrifuged at 8000rpm for 5min in a precooled 4℃ horizontal centrifuge to collect the bacterial precipitate. The bacterial precipitate was resuspended with 1ml of precooled SM buffer and transferred to a precooled 4℃ horizontal centrifuge for centrifugation at 8000rpm for 5min. This resuspension and centrifugation step was repeated twice. After discarding the supernatant, the suspension was suspended on ice with 60μl of precooled SM buffer, and the suspension was dispensed into 1.5ml centrifuge tubes and stored on ice for later use.

[0066] (6) Transformation of pHSP02-EGFP and pHSP02-PEDV-S1 plasmids

[0067] Take out the PW01 competent cell, take 5 μL of pHSP02-EGFP and pHSP02-PEDV-S1 plasmids and add them, transfer the mixed solution to the electroporation cup and bathe on ice for 20 minutes, and electroporate into the PW01 competent cells under the electroporation conditions of 2500V, 200Ω, and 25μF according to procedure 4-1-9. Let it stand at room temperature for 5 minutes, add 600 μL of SMRS to the electroporation cup and pipette it out and put it into a 1.5ml centrifuge tube, put it in a 30℃ incubator and let it stand for 3 hours, take 100 μL and spread it on the MRS (Cm+Em) medium plate, pick the positive single colony every other day, select the positive clone to prepare the plasmid in small amount, and name the recombinant plasmids WCFS1-EGFP and WCFS1-PEDV-S1 respectively.

[0068] (7) Identification of WCFS1 genome modifications

[0069] To identify the modification of the WCFS1 genome, the cell wall of Lactobacillus plantarum needs to be broken before PCR can be performed: 50 μL of the bacterial solution was centrifuged at 12000 rpm for 30 s, the supernatant was discarded, 50 μL of DDH2O was added to wash twice, and the bacteria were resuspended with an equal volume of DDH2O, in a metal bath at 100°C for 10 min, then in an ice bath for 10 min (oscillated every 5 min during this period), this operation was repeated 3 times, and then centrifuged at 5000 rpm for 1 min, and 1 μL of the treated bacterial solution was taken as a PCR template. The PCR reaction system is as follows: 1 μL of treated bacterial solution, 10 μL of 2×primestarmax, 0.5 μL of F, 0.5 μL of R, 8 μL of DdH20, and a total of 20 μL. The program is 98°C pre-denaturation for 5 min, 95°C melting for 30 s, 65°C annealing for 1 min, 72°C extension for 1 min, and 72°C final extension for 5 min. The PCR product is stored at 4°C, and 10 μL is taken for 1% agarose gel electrophoresis. In addition, the identified single colonies were subcultured in liquid MRS for 10, 15, and 20 generations, and the genome after subculture was extracted using a bacterial genomic DNA rapid extraction kit purchased from Shanghai Shenggong Bioengineering Co., Ltd., and agarose gel was run to verify whether the modified bacteria can be stably inherited.

[0070] 2. Preparation of PEDV-S1 polyclonal antibodies

[0071] (1) Design and amplification of target fragment PEDV-S

[0072] To carry out the relevant operations of the target fragment PEDV-S, primers were designed based on the sequenced PEDV LJXO1 strain, and fragments were cut from the PEDV-S1 and pET28a sequences as upstream and downstream primers. PCR amplification of the target gene PEDV-S1 fragment was performed, and the reaction system was template plasmid PEDV-S12μL, 2×prime starmax 25μL, F-pET28a-PEDV 1μL, R-pET28a-PEDV 1μL, ddH2O 21μL, total volume 50μL, PCR reaction system was 98 degrees pre-denaturation for 20s; 98 degrees denaturation for 10s, 60 degrees annealing for 5s, 72 degrees extension for 2min, denaturation, annealing, extension for 30 cycles; 72 degrees final extension for 1min, PCR amplification vector size is 1233bp, 50μL sample purification specific steps see Omega Gel Extraction Kit user manual.

[0073] (2) pET28a vector digestion and pET28a-PEDV-S1 vector ligation

[0074] The pET28a vector was digested with plasmid as template. The digestion reaction system was 30 μL of PET28a plasmid, 2.15 μL of Buffer, 2 μL of BamHI-F, 2 μL of HindIII-R, 11 μL of ddH2O, and the total volume was 50 μL. After digestion, the target band was purified using a purification kit for later use. Then, pET28a was connected to the PEDV-S1 vector. The connection system was 3 μL of PEDV-S1 fragment, 5 μL of PET28a vector, 1 μL of T4 DNA Ligase, and 1 μL of T4 DNA Ligase Buffer, and the total volume was 10 μL. The reaction was treated in a metal bath at 16°C overnight. Then, DH5α competent cells were transformed. The DH5α competent cells were taken out from -80℃ and placed on ice for 3 minutes to melt until they were slightly viscous. 10 μL of the ligation system was added thereto. After mixing without blowing, it was placed on ice for 20 minutes. The water bath was set to 42℃ in advance. The mixture was heat-shocked for 90 seconds and immediately placed on ice for 5 minutes. 600 μL of LB culture medium was added, and the mixture was placed in a 37℃ shaker at 220 rpm for 1 hour. The mixture was centrifuged at 5000 rpm for 5 minutes, and the excess culture medium on the upper layer was discarded. 200 μL of the bacterial solution was spread on an LB solid plate (100 μg / mL Kan) with a spreading rod, and cultured at 37℃ overnight. A single colony was picked on the next day and inoculated in a 5 mL centrifuge tube for overnight culture, followed by bacterial solution PCR identification.

[0075] 3. Protein Purification

[0076] (1)BL21-PEDV-S1-pET28a SDS-PAGE

[0077] Streak the PEDV-S1-pET28a (BL21) plate, pick a single colony and inoculate it in 5ml of LB (Kan) medium for overnight culture, and then induce the strain in small quantities. When the OD value is detected at 0.6-0.8, add the inducer IPTG, set different concentrations for low-temperature induction at 16°C, collect the bacteria and ultrasonically break them, and take the supernatant and cell precipitate. The supernatant and precipitate are lysed at high temperature and boiled before loading. After running the gel, stain the gel with Coomassie Brilliant Blue staining solution for one and a half hours, then rinse with clean running water, shake on a shaker overnight for decolorization, and observe the protein bands by imaging to determine the specific expression of the PEDV-S1 protein and determine the expression form of the protein, and then perform a large amount of induction purification.

[0078] (2) Denaturation, renaturation and purification of recombinant proteins

[0079] After BL21-PEDV-S1-pET28a was taken out from -80℃ and melted, 5mL of culture medium was inoculated for overnight culture. The next day, 500mL of culture medium was inoculated for continued culture. When the OD was 0.6-0.8, IPTG was used for low-temperature induction overnight. After centrifugation at 4℃ and 10000rpm for 20min, the part with bacterial precipitation was lysed with cell lysis buffer. PAGE showed that the protein content in the precipitation was high and there were few miscellaneous bands. Therefore, the precipitation was used to purify the protein, and the BL21-PEDV-S1-pET28a precipitation was dissolved with 6M guanidine hydrochloride solution. Cut the dialysis bag into appropriate lengths, rinse it, boil it in 200ml 1mM EDTApH8.0 for 10min, and place it in 20% ethanol to cool for later use. After washing the dialysis bag with ddH2O, put the denatured protein solution dissolved in 6M guanidine hydrochloride into the dialysis bag, set up gradient dialysis such as 6M, 4M, 2M guanidine hydrochloride and PBS to renature the protein. At 4M, the dialysis volume and time should be increased because the protein usually begins to renature at 4M-2M guanidine hydrochloride. Pour the renatured protein solution out of the dialysis bag, centrifuge and discard the precipitate, and the supernatant is the purified protein. Determine the protein concentration with BSA and store it in aliquots at -80℃.

[0080] (3) Preparation of rabbit anti-PEDV-S1 polyclonal antibody

[0081] Six-week-old New Zealand white rabbits were purchased for three-week immunization. The immunization scheme was as follows: one immunization was performed on the first day, 0.8 mg protein was mixed with 0.5 mL Freund's complete adjuvant and then injected at multiple points under the back skin; the second immunization was performed on the 8th day, 0.4 mg protein was mixed with 0.25 mL Freund's incomplete adjuvant and then injected at multiple points under the back skin; the third immunization was performed on the 22nd day, 0.4 mg protein was mixed with 0.25 mL Freund's incomplete adjuvant and then injected at multiple points under the back skin. One week after the third immunization, blood was collected from the heart and centrifuged at 4°C and 4000 rpm for 20 minutes to collect serum. This serum is the rabbit-derived PEDV-S1 antibody, which was packaged and stored at -80°C for later use.

[0082] (4) Verification of protein expression of new functional recombinant lactic acid bacteria

[0083] Western blot was used to verify whether the new functional lactic acid bacteria WCFS1 / PLH02-EGFP and WCFS1 / PLH02-PEDV-S1 expressed fusion proteins of the expected size.

[0084] (5) Processing of recombinant lactic acid bacteria protein samples

[0085] The successfully constructed recombinant lactic acid bacteria were plated on MRS plates and placed in a 30°C incubator for 16 hours. The positive clones were picked and inoculated into 5mL MRS liquid medium for activation. After overnight culture, they were transferred to 50mL MRS liquid medium. When the OD value reached 0.3, the inducing peptide was added and anaerobic induction was carried out at 37°C for 6 hours. The induced bacterial solution was centrifuged at 4°C and 5000rcf for 5 minutes to retain the bacterial sediment, and the bacteria were suspended with TES and bathed at 37°C for 30 minutes. Then, the TES was discarded after centrifugation at 4°C and 2500rcf for 10 minutes, and the bacterial sediment was suspended with 1ml PBS, centrifuged at 4°C and 2500rcf for 10 minutes, and then washed again and resuspended with ddH2O. Then it was quickly frozen with liquid nitrogen and dissolved in a 37°C water bath, and the freeze-thaw was repeated three times. Finally, the sample was ultrasonically disrupted.

[0086] (6) Western blot verification of target protein expression

[0087] First, install and clamp the long and short glass plates to prepare the glue, add the separation glue, and seal with isopropanol. After standing, discard the isopropanol, rinse, and dry, add a layer of glue and insert the comb teeth; mix the sample with an equal volume of 2×SDS Loading Buffer, boil it for 10 minutes, centrifuge it at 12000rpm for 10 minutes, take 10μL of the supernatant and add the sample, 10μL per well, 80V 30min, 120V 45min electrophoresis until the front end of the sample is 1cm away from the lower edge of the glass plate; cut the corner mark first when transferring the membrane, discard the upper layer of glue after the electrophoresis, put it into the clamp plate at 600mA constant current for 90min; use 50ml of 5% skim milk powder solution containing 2.5g milk powder to seal the PVDF membrane for 1h; cut the PVDF membrane into appropriate size and use 0.1% TBST The primary antibody was applied with a 1:1000 dilution of PEDV-S1 polyclonal antibody; the membrane was washed with 0.1% TBST for 3 times, 5 min each time; the secondary antibody was incubated with HRP-labeled antibody at room temperature for 1 h; the membrane was washed again according to the above method; finally, the chemiluminescence enhancement reagent was dropped on the PVDF membrane for exposure and development.

[0088] (7) Colony count

[0089] The bacterial solution with OD600 of 1 was diluted to 106, 107, and 108, and 100 μL of each solution was applied to non-anti-MRS solid culture medium and three parallel controls were set up. The optimal feeding amount of recombinant lactic acid bacteria was calculated to be 1×109 CFU / mL.

[0090] Result verification and analysis

[0091] (1) Synthetic plasmids and vectors

[0092] Plasmid maps of pLH01 and pHSP02 are shown in Figure 1 , 2 shown.

[0093] (2) Obtaining PEDV LJX01 fragments and sequences

[0094] The small intestinal lavage fluid of PEDV LJX01 was used to extract viral RNA, and the reverse transcribed cDNA was used as a PCR template to obtain the PCR product of the PEDV-S1 fragment as shown in Figure 3 The obtained PEDV-S1 fragment size is 2213 bp. The PCR product was sequenced to obtain the accurate sequence of PEDV-S1, as shown in FIG. Figure 4 shown.

[0095] (3) Construction of pHSP02-PEDV-S1 plasmid map

[0096] The accurate sequence of PEDV-S1 was obtained in the previous step, and the sequence was integrated into the middle position between 0536 and 0538 of pHSP02 and the HA tag was added to synthesize the pHSP02-PEDV-S1 plasmid as shown in Figure 5 As shown, the EGFP sequence was inserted at the same position as a control. Figure 6 Shown

[0097] (4) Verification of pLH01 transformation into WCFS1 competent cells

[0098] Lactobacillus plantarum WCFS1 was made into competent cells, and the pLH01 plasmid was transferred into the cells. Since the pLH01 plasmid carries chloramphenicol resistance, while Lactobacillus plantarum WCFS1 itself does not carry chloramphenicol resistance, pLH01 positive bacteria can grow on chloramphenicol resistance plates, while negative bacteria cannot grow on chloramphenicol resistance plates, but can grow on non-resistant MRS plates. Figure 7 The results are consistent with expectations, and the positive bacteria are named PW01. The growth is verified again, and the results of bacterial liquid PCR are as follows Figure 8 shown.

[0099] (5) Verify that pHSP02-PEDV-S1 plasmid was transferred into PW01 competent cells

[0100] The PW01 strain obtained above was made competent, and pHSP02-PEDV-S1 and pHSP02-EGFP were transformed into it. The mutant strains were identified by a large number of PCRs and named WCFS1-PEDV-S1 and WCFS1-EGFP. Fig. 9 , 10 Shown

[0101] (6) Subculture

[0102] The obtained WCFS1-PEDV-S1 mutant strain was subcultured, and after extracting genomic DNA, PCR was performed on the bacterial solution to confirm that the recombinant bacteria could be stably inherited. Fig.11As shown, the bands at passages 10, 15, and 20 were all correct.

[0103] (7) Observation of WCFS1-EGFP positive strains under fluorescence microscopy

[0104] like Fig.12 As shown, WCFS1-EGFP is a rod-shaped bacterium under white light, can emit green fluorescence under a fluorescent microscope, and can emit light in the bacterial body after merging. This proves that the new recombinant lactic acid bacteria can autonomously express the target protein.

[0105] (8) Obtaining PEDV-S1 gene

[0106] Since the PEDV-S1 protein has a signal peptide, which causes prokaryotic expression failure, the signal peptide part was cut off and the target band was obtained by PCR amplification, such as Fig.13 The target band was single, so the PCR product was purified and set aside.

[0107] (9) Enzyme digestion of pET28a empty vector

[0108] The empty vector pET28a was double-digested with BamHI and HindⅢ, and the target band size was 5350bp. Fig.14 After the carrier gel is recovered, it is connected with the purified product of PEDV-S1. After being transferred into DH5α competent cells, the plasmid is extracted and then transferred into the prokaryotic expression vector BL21.

[0109] (10) Enzyme digestion verification BL21-PEDV-S1

[0110] The target fragment PEDV-S1 was connected to the vector pET28a and then transferred into the BL21 competent cell. A single colony was picked out for PCR verification. The results were as follows: Fig.15 , bands 2, 3 and 5 are correct, and culture is carried out to the next test.

[0111] (11) BL21-PEDV-S1 protein expression verification

[0112] The BL21-PEDV-S1 positive strain obtained in the previous step was cultured, induced with IPTG, and then ultrasonically broken and paged to verify the expression. The results are as follows Fig.16 The protein expression size was 46KDa and was mainly expressed in the precipitate.

[0113] (12) Rabbit polyclonal antibody protein expression verification

[0114] The prepared rabbit polyclonal antibody was diluted 1:1000 as the primary antibody for incubation, and Western-blot was performed to verify the expression. Fig.17 As shown, the protein size is 46 KDa, which is consistent with the expected result.

[0115] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for constructing a recombinant Lactobacillus plantarum, characterized in that: The following steps are involved: (1) Plasmid transformation: insert the EGFP fragment and the S-layer anchor expression system constructed in the laboratory into position 0537 of the PHSP02 standard plasmid to obtain the transformed plasmid PHSP02-EGFP; (2) Plasmid modification: The EGFP portion of the modified plasmid PHSP02-EGFP was replaced with the S1 protein of the PEDVljx strain to obtain the modified plasmid PHSP02-PEDV-S1; (3) Competent cell construction: The auxiliary plasmid PLH01 was transferred into the competent cell of Lactobacillus plantarum WCFS1 to obtain the positive strain PW01; (4) Screening of positive strains: The modified plasmids PHSP02-EGFP and PHSP02-PEDVSS1 were transferred into PW01 competent cells, the genome was extracted, and positive strains were screened by PCR; (5) Verification of positive strains: The obtained positive strains were propagated for 10, 15, and 20 generations, and genomic PCR was performed to prove that they can be stably inherited. Bacterial liquid smears were made and green fluorescence was observed under an electron microscope. It can be considered that the new recombinant lactic acid can autonomously express the target protein.

2. A recombinant Lactobacillus plantarum, characterized in that Prepared by the construction method described in claim 1.

3. Use of the recombinant Lactobacillus plantarum as claimed in claim 2 in the preparation of a product for preventing or treating porcine epidemic diarrhea.

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