Recombinant lactobacillus paracasei for fusion expression of swine trefoil factor 3 and epidermal growth factor as well as construction method and application of recombinant lactobacillus paracasei

Abstract

The invention discloses recombinant lactobacillus paracasei for fusion expression of a swine trefoil factor 3 and an epidermal growth factor as well as a construction method and application of the recombinant lactobacillus paracasei, and belongs to the technical field of veterinary medicines. The recombinant lactobacillus paracasei contains recombinant plasmids capable of expressing fusion protein of a swine trefoil factor 3 and an epidermal growth factor. Wherein the porcine trefoil factor 3 is connected with the epidermal growth factor through a rigid Linker, the amino terminal of the fusion protein is further connected with a signal peptide SP sequence, the carboxyl terminal of the fusion protein is connected with a flag tag, and the structure of the obtained fusion protein is SP-pTFF3-rigid Linker-pEGF-flag. According to the invention, TFF3 and EGF are fused into a single protein through genetic engineering, the problem of proportion control of traditional two-factor combined administration is solved, and'repair-regeneration 'dual-channel synergy is realized by utilizing a spatial proximity effect. In-vivo experiments prove that the recombinant bacterium obtained by the invention not only can repair intestinal mucosa, but also can achieve the purpose of treating the inflammatory bowel disease by regulating cell factors such as IL-10 / IL-6 and the like to balance an inflammatory microenvironment.

Description

Technical Field

[0001] The present invention relates to a recombinant Lactobacillus paracasei that fusion-expresses porcine trefoil factor 3 and epidermal growth factor, a construction method thereof, and an application thereof. The present invention belongs to the technical field of veterinary medicine. Background Art

[0002] Inflammatory bowel disease (IBD) is a type of chronic inflammatory disease with recurrent attacks. Currently, the incidence of IBD is tending to be younger, and early-onset IBD increases the risk of colorectal cancer.

[0003] Existing treatments for IBD are mainly divided into two aspects: drug therapy and surgical treatment. Drug therapy is the first choice for treating IBD. Corticosteroids, aminosalicylic acid, antibiotics and immunomodulators are used to treat IBD [FORD AC, ACHKAR JP, KHAN KJ, et al. Efficacy of 5-aminosalicylates in ulcerative colitis: systematic review and meta-analysis [J]. The American journal of gastroenterology, 2011, 106(4): 601-16.]. However, the side effects of drugs are a major problem. With the increasing number of IBD patients, researchers are trying to find new treatments and improve the complications caused by it, such as the use of small molecule drugs and new biological agents. The treatment goal has gradually shifted from relief to mucosal repair and healing.

[0004] Trefoil Factor 3 (TFF3), a key member of the trefoil factor family, has become a research hotspot in the field of gastrointestinal mucosal protection and repair due to its unique structure and extensive biological functions. TFF3 is remarkably stable, exhibiting resistance to acid, proteases, and thermal degradation, which underpins its ability to function in the harsh gastrointestinal environment. In mammals, TFF3 is primarily distributed in intestinal goblet cells, with limited expression in tissues such as the respiratory tract, urogenital tract, and the neural lobe of the porcine pituitary gland. TFF3 participates in immune regulation, reducing inflammation and thereby protecting the gastrointestinal mucosa. NF-κB is considered to be the main regulator of intestinal inflammatory response. Studies have shown that TFF3 inhibits NF-κB activity through TLR4 and ERK signaling pathways, thereby alleviating the inflammatory response [WANG Y, LIANG K, KONG W. Intestinal Trefoil Factor 3Alleviates the Intestinal Barrier Function Through Reducing the Expression ofTLR4 in Rats with Nonalcoholic Steatohepatitis [J]. Arch Med Res, 2019, 50(1): 2-9.]; In addition, related studies have shown that TFF3 is involved in protecting and repairing intestinal mucosal inflammation caused by radiotherapy and chemotherapy [BECK PL, WONG JF, LI Y, et al. Chemotherapy- and radiotherapy-induced intestinal damage is regulated by intestinal trefoil factor [J]. Gastroenterology, 2004, 126(3): 796-808.].

[0005] Epidermal Growth Factor (EGF) is a small molecule polypeptide with a wide range of biological functions. Since its first discovery by Stanley Cohen in 1962, its core role in cell proliferation, tissue repair, and disease treatment has attracted much attention. Porcine EGF (pEGF) is highly conserved in structure and function with human and rodent EGF. It is mainly secreted by Paneth cells, salivary glands, and pancreas, and is widely involved in regulating gastrointestinal homeostasis. EGF can promote cell proliferation. Related studies have shown that EGF promotes the proliferation of human hair follicle mesenchymal stem cells by activating the EGFR / ERK and AKT pathways [CHEN YA, TSAI YC, CHEN YD, et al. Intraventricular Medium B Treatment Benefits an IschemicStroke Rodent Model via Enhancement of Neurogenesis and Anti-apoptosis [J]. Sci Rep, 2020, 10(1): 6596.]. Secondly, EGF can promote cell differentiation. EGF causes endogenous neural stem cells to proliferate and migrate to the infarct focus, and mainly promotes their differentiation into GFAP-positive astrocytes, thereby accelerating the structural recovery of the infarct focus [Lu Shijun, Yang Huike, Ma Wenping, et al. Changes in the expression of vimentin and matrix metalloproteinase-2 during the migration of human umbilical vein endothelial cells induced by epidermal growth factor [J]. Acta Anatomica Sinica, 2013, 44(06): 789-794.]. In addition, EGF can also promote cell migration. Studies have shown that EGF promotes the migration of human umbilical vein endothelial cells (HUVECs). After EGF stimulation, the migration ability of HUVECs increased by 1.8 times, the morphology of migrating cells changed, and the expression of vimentin and MMP-2 was upregulated [WEIS, WANG W, LI L, et al. Recombinant human epidermal growth factor combined with vacuum sealing drainage for wound healing in Bama pigs [J]. Mil Med Res, 2021, 8(1): 18.].EGF has a protective effect on the intestinal mucosal barrier. Studies have shown that oral administration of porcine epidermal growth factor (pEGF) can increase the gene expression of tight junction proteins such as ZO-1, Claudin-1 and Occludin, thereby enhancing the intestinal barrier function of early-weaned piglets [XU S, WANG D, ZHANG P, et al. Oral administration ofLactococcus lactis-expressed recombinant porcine epidermal growth factorstimulates the development and promotes the health of small intestines inearly-weaned piglets [J]. J Appl Microbiol, 2015, 119(1): 225-235.].

[0006] Vandenbroucke Klaas [VANDENBROUCKE K, HANS W, VAN HUYSSE J, et al. Active delivery of trefoil factors by genetically modified Lactococcus lactis prevents and heals acute colitis in mice [J]. Gastroenterology, 2004, 127(2):502-13] et al. constructed a recombinant Lactococcus lactis that secretes TFF. They used in vivo experiments to analyze the effects of the recombinant lactococci on intestinal damage induced by dextran sulfate sodium salt (DSS, MW: 36000-50000 Da) in mice with colitis to determine the protective and therapeutic potential of the recombinant bacteria. The results showed that it not only improved the pathology of acute DSS-induced colitis, but also improved the pathology of confirmed chronic colitis. However, this technology has the following disadvantages: ① MG1363 Disadvantages: Intolerant to gastric acid and bile salts, unable to colonize in the intestines, requiring frequent administration, and poor efficacy. ② Lactic acid bacteria MG1363 The immune regulation function is weak, mainly because it lacks inherent immune regulation ability as a carrier and relies on exogenous gene expression. ③ TFF3 single factor has insufficient efficacy.

[0007] In order to overcome the problems existing in the prior art, the present invention proposes a recombinant Lactobacillus paracasei that fusion-expresses porcine trefoil factor 3 and epidermal growth factor, as well as a construction method and application thereof, providing a new technical means for the treatment of inflammatory bowel disease. Summary of the Invention

[0008] The purpose of the present invention is to provide a recombinant Lactobacillus paracasei that fusion-expresses porcine trefoil factor 3 and epidermal growth factor, as well as a construction method and application thereof.

[0009] In order to achieve the above object, the present invention adopts the following technical means: Firstly, the present invention proposes a recombinant Lactobacillus paracasei that expresses porcine trefoil factor 3 and epidermal growth factor in fusion. The recombinant Lactobacillus paracasei contains a recombinant plasmid that can express the fusion protein of porcine trefoil factor 3 and epidermal growth factor.

[0010] Among them, preferably, the porcine trefoil factor 3 and epidermal growth factor are connected through a rigid linker, the amino terminus of the fusion protein is also connected to a signal peptide SP sequence, and the carboxyl terminus is connected to a flag tag, and the structure of the resulting fusion protein is SP-pTFF3-rigid linker-pEGF-flag.

[0011] Among them, preferably, the nucleotide sequence encoding the fusion protein is shown as SEQ ID NO.31.

[0012] Among them, preferably, the recombinant plasmid is a recombinant lactobacillus expression plasmid containing porcine trefoil factor 3 and epidermal growth factor capable of fusion expression.

[0013] Preferably, the recombinant plasmid is obtained by cloning the nucleotide sequence encoding the fusion protein of porcine trefoil factor 3 and epidermal growth factor shown in SEQ ID NO.31 into the vector plasmid pPG-PPT. SnaB Ⅰ and Apa I restriction endonuclease sites.

[0014] Furthermore, the present invention also proposes a method for constructing the recombinant Lactobacillus paracasei, comprising the following steps: (1) Acquisition of target gene and signal peptide sequence Fresh pig intestinal tissue was taken, total RNA was extracted and reverse transcribed, and the extracted cDNA was used as a template and pTFF3-F / R, pEGF-F / R, and pTE-F / R were used as primer pairs to amplify the pTFF3 、 pEGF and a fusion fragment of the two pTE , pTFF3 、 pEGF and fusion fragments pTE The sequences are shown in SEQ ID NO.10-12 respectively; the signal peptide SP sequence of the S layer protein of Lactobacillus pumilus was obtained from the plasmid pPG-T7g10-SP-pBD2, and the primers SP-F / SP-R1 and SP-F / SP-R2 were used for PCR amplification and recovery respectively. The primer SP-R1 contained the target gene pEGF The homologous sequence at the 5' end, the target gene is contained in the primer SP-R2 pTFF3 Homologous sequence at the 5' end, containing the target gene pEGF The sequence of the SP signal peptide of the homologous sequence at the 5' end is shown in SEQ ID NO.13, containing the target gene pTFF3 The sequence of the SP signal peptide of the homologous sequence at the 5' end is shown in SEQ ID NO.14, and the primer sequence is shown below: ; (2) Construction of pMD19T-SP-pTFF3 and pMD19T-SP-pEGF cloning plasmids The purified and recovered SP Signal peptide genes and acquired pTFF3 and pEGF The genes were subjected to fusion PCR respectively, and the fusion PCR products were used as templates to amplify pPG-pTFF3-F / R and pPG-pEGF-F / R with primers to obtain SP- pTFF3 Fragments and SP-pEGF The gel-collected products were then connected to the pMD19T Simple vector, the system was mixed, and the connection was carried out at 16 ° C for 4 h. The connection products were transformed into Escherichia coli. DH5α Competent cells were used to identify the extracted plasmids by PCR using primer pair 19T-F / 19T-R. The correctly identified plasmids were sequenced and aligned, and named pMD19T-SP-pTFF3 and pMD19T-SP-pEGF. Plasmid pMD19T-SP-pTFF3 was used as a template and primer pair pPG-pTE-F / Linker-pTFF3-R was used for PCR amplification. Plasmid pMD19T-SP-pEGF was used as a template and primer pair Linker-pEGF-F / pPG-pTE-R was used for PCR amplification. The two sequences were recovered by gel excision and homologously recombined by fusion PCR. Primers pPG-pTE-F / R were used to amplify the two sequences. SP-pTFF3-pEGF The sequence was confirmed by agarose gel electrophoresis and then the gel was cut and recovered. It was ligated with the pMD19T Simple vector to construct the positive clone plasmid pMD19T-SP-pTE. The recombinant plasmid was identified by PCR and sequence alignment using 19T-F / 19T-R as primers. The primer sequences are shown below: ; (3) Construction of pPG-SP-pTE expression vector The vector plasmid pPG-PPT and the above-constructed cloning plasmid pMD19T-SP-pTE were used SnaB Ⅰ and Apa I restriction enzyme double digestion at 30℃ for 2 h, and the vector fragment was recovered by gel cutting. pPG With the target fragment SP-pTFF3-rigid linker-pEGF-flag, Abbreviation SP-pTE , whose sequence is shown in SEQ ID NO.31, and then the target fragments were connected to the vector fragments using T4 ligase, and the ligation products were chemically heat-transformed into Escherichia coli TG1 In the competent state, a single colony was picked and cultured overnight. The extracted plasmid was identified by PCR using pPG-F / pPG-R as primers. In addition, the plasmid with the correct size identified by PCR was SnaB Ⅰ and Apa I. Double enzyme digestion identification. After correct sequence alignment, the correct plasmid was named pPG-SP-pTE, which is the recombinant lactobacillus expression plasmid expressing porcine trefoil factor 3 and epidermal growth factor; (4) Preparation of recombinant Lactobacillus paracasei The recombinant plasmid pPG-SP-pTE was transformed into competent cells using electroporation L.paracasei HLJ-27 After culture and PCR identification, a positive strain containing the recombinant plasmid pPG-SP-pTE was obtained and named pPG-SP-pTE / HLJ-27 .

[0015] Furthermore, the present invention also proposes the use of the recombinant Lactobacillus paracasei in the preparation of a drug for treating inflammatory bowel disease.

[0016] Compared with the prior art, the present invention has the following beneficial effects: ① The present invention fuses TFF3 and EGF into a single protein through genetic engineering, overcoming the difficulty of controlling the ratio of traditional dual-factor combined administration, and utilizing the spatial proximity effect to achieve the synergy of the "repair-regeneration" dual pathways, which is superior to a single factor.

[0017] ② Lactobacillus paracasei (with its high safety, acid-bile resistance, and strong colonization ability) as a delivery vehicle offers advantages over common lactic acid bacteria for clinical translation. Lactobacillus paracasei possesses inherent probiotic properties (such as regulating microbial flora and enhancing the barrier barrier), and its synergistic effect with fusion proteins may lead to the development of a combined bacterial-drug therapy.

[0018] ③ Using a specific signal peptide and the expression vector pPG-PPT, we achieved stable secretion of the fusion protein (ELISA showed peak secretion at 18 hours). The TFF3 and EGF fusion protein secreted ≥1 mg / L in vitro. After 20 consecutive passages, the recombinant plasmid remained stably inherited, the recombinant bacterial protein was stably expressed, and the migration rate of intestinal epithelial cells was significantly enhanced.

[0019] ④ In vivo experiments have shown that pTE recombinant bacteria not only repair the intestinal mucosa (goblet cell retention, upregulation of tight junction proteins), but also balance the inflammatory microenvironment by regulating cytokines such as IL-10 / IL-6, thereby achieving the purpose of treating inflammatory bowel disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Figure 1 is the PCR amplification result of pEGF and pTFF3 target genes; Among them, M: DL 2000 DNA Marker; 1~3: pEGF Gene PCR amplification products; 4~6: pTFF3 Gene PCR amplification product; 7: negative control; Figure 2 Figure 1 is the PCR and double enzyme digestion identification results of the pMD19T-SP-pTFF3 plasmid; Wherein: a) pMD19T-SP-pTFF3 PCR identification results; M: DL 2000 DNA Marker; 1-7: SP-pTFF3 Gene PCR amplification product; 8: negative control; b) plasmid pMD19T-SP-pTFF3 SnaB Ⅰ. Apa ⅠDouble enzyme digestion identification results; Figure 3 Figure 1 is the PCR and double enzyme digestion identification results of pMD19T-SP-pEGF plasmid; Wherein: a) pMD19T-SP-pEGF PCR identification results; M: DL 2000 DNA Marker; 1-7: SP-pEGF Gene PCR amplification product; 8: negative control; b) plasmid pMD19T-SP-pEGF SnaB Ⅰ. Apa ⅠDouble enzyme digestion identification results; Figure 4 Figure 1 is the result of PCR and double enzyme digestion identification of pMD19T-SP-pTE plasmid; Among them, a) PCR identification results of plasmid pMD19T-SP-pTE; M: DL 2000 DNA Marker; 1: PCR product of pMD19T-SP-pTE; 2: negative control; b) PCR product of plasmid pMD19T-SP-pTE SnaB Ⅰ. Apa ⅠDouble enzyme digestion identification; Figure 5 The vector map and vector construction strategy diagram of the vector plasmid pPG-PPT; Figure 6 Figure 1 is the PCR identification and enzyme digestion identification results of the pPG-SP-pTFF3 plasmid; Among them, a) PCR identification results of plasmid pPG-SP-pTFF3; M: DL 2000 DNA Marker; 1: negative control; 2: PCR product of pPG-SP-pTFF3; b) SnaB Ⅰ. Apa ⅠDouble enzyme digestion identification; Figure 7 Figure 1 is the result of PCR identification and enzyme digestion identification of pPG-SP-pEGF plasmid; Among them, a) PCR identification results of plasmid pPG-SP-pEGF; M: DL 2000 DNA Marker; 1: PCR product of pPG-SP-pEGF; 2: negative control; b) PCR product of plasmid pPG-SP-pEGF SnaB Ⅰ. Apa ⅠDouble enzyme digestion identification; Figure 8 Figure 1 is the result of PCR identification and enzyme digestion identification of pPG-SP-pTE plasmid; Among them, a) PCR identification results of plasmid pPG-SP-pTE; M: DL 2000 DNA Marker; 1~4: PCR products of pPG-SP-pTE; 5: negative control; b) Plasmid pPG-SP-pTE SnaB Ⅰ. Apa ⅠDouble enzyme digestion identification; Figure 9 Figure 1 is the PCR identification result of the recombinant bacteria; Wherein, M: 2K Plus DNA Marker; 1-3: PCR identification results of plasmid pPG-SP-pEGF; 4-6: PCR identification results of plasmid pPG-SP-pTFF3; 7-9: PCR identification results of plasmid pPG-SP-pTE; 10: negative control; Figure 10 This is the Western blot detection result of the recombinant bacteria expressing pTFF3; Among them, M: small molecular weight protein pre-stained marker; 1: pPG-SP-pTFF3 / HLJ-27 Bacterial supernatant; 2: pPG-SP-pTFF3 / HLJ-27 Bacteria precipitation; 3: pPG / HLJ-27 Bacterial supernatant; 4: pPG / HLJ-27 Bacteria precipitation; Figure 11 The figure shows the Western blot detection results of recombinant bacteria expressing pEGF; Among them, M: small molecular weight protein pre-stained marker; 1: pPG-SP-pEGF / HLJ-27 Bacterial supernatant; 2: pPG-SP-pEGF / HLJ-27 Bacteria precipitation; 3: pPG / HLJ-27 Bacterial supernatant; 4: pPG / HLJ-27 Bacteria precipitation; Figure 12 The figure shows the Western blot detection results of the recombinant bacteria expressing pTE; Among them, M: small molecular weight protein pre-stained marker; 1: pPG-SP-pTE / HLJ-27 Bacterial supernatant; 2: pPG-SP-pTE / HLJ-27 Bacteria precipitation; 3: pPG / HLJ-27 Bacterial supernatant; 4: pPG / HLJ-27 Bacteria precipitation; Figure 13 This is the result of indirect immunofluorescence identification of the protein expressed by the recombinant bacteria; Figure 14 This is the growth curve measurement result diagram; Figure 15 This is the result diagram of genetic stability of recombinant plasmid; Among them, a) pPG-SP-pTFF3 / HLJ-27 ; b) pPG-SP-pEGF / HLJ-27 ; c) pPG-SP-pTE / HLJ-27 ; Figure 16 This is the result diagram of genetic stability of recombinant bacterial protein; Among them, a) pPG-SP-pTFF3 / HLJ-27 ; b) pPG-SP-pEGF / HLJ-27 ; c) pPG-SP-pTE / HLJ-27 ; Figure 17 This is the analysis result of the effects of pTFF3, pEGF and pTE on the proliferation of IPEC-J2 cells; Note:* P <0.05, **0.01< P <0.05,*** P <0.01 vs 0 ng / mL; Figure 18 This is the analysis result of the target protein's effect on promoting IPEC-J2 cell migration; Note: The dotted line indicates the edge of cell migration; Figure 19 This is the result of the analysis of the area of IPEC-J2 cell migration promoted by the target protein; Note:* P <0.05, **0.01< P<0.05,*** P <0.01 vs pPG;# P <0.05,##0.01< P <0.05,### P <0.01 vs pEGF; ns, P >0.05; Figure 20 This is a graph showing the evaluation results of the disease activity index of mice in each treatment group; Note:* P <0.05, **0.01< P <0.05,*** P <0.01 vs DSS;# P <0.05,##0.01< P <0.05,### P <0.01 vs pPG / HLJ-27 ;ns, P >0.05; Figure 21 Figure 2 is the result of measuring the colon length of mice in each treatment group; Note:* P <0.05, **0.01< P <0.05,*** P <0.01 vs DSS;# P <0.05,##0.01< P <0.05,### P <0.01 vs pPG / HLJ-27 ;ns, P >0.05; Figure 22 The graph shows the results of myeloperoxidase activity assay in the colon tissue of mice in each treatment group; Figure 23 Figure 2 is the result of real-time fluorescence quantitative PCR detection of Claudin-2, Occludin and ZO-1 genes in the colon of mice in each treatment group; Among them, a) Claudin-2; b) Occludin; c) ZO-1; Note: * P <0.05, **0.01< P <0.05,*** P <0.01vs DSS;# P <0.05,##0.01< P <0.05,### P <0.01 vs pPG / HLJ-27 ;ns, P >0.05; Figure 24 The figure shows the Western blot detection results of tight junction protein expression in mouse colon; Figure 25 The graph shows the results of measuring the secretion levels of inflammatory factors in mice in each treatment group; Note:* P <0.05, **0.01< P <0.05,*** P <0.01 vs DSS;# P <0.05,##0.01< P <0.05,### P <0.01 vs pPG / HLJ-27 ;ns, P >0.05; Figure 26 The colon H&E staining results of mice in each treatment group. DETAILED DESCRIPTION

[0021] The present invention is further described below with reference to specific examples, which, however, are not intended to limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the art.

[0022] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0023] Example 1 Fusion expression of porcine trefoil factor 3 and epidermal growth factor in recombinant Lactobacillus paracasei 1. Construction and identification of recombinant lactobacilli expressing pTFF3, pEGF, and pTE (1) Acquisition of target gene and signal peptide sequence Fresh pig intestinal tissue was taken, total RNA was extracted and reverse transcribed, and the extracted cDNA was used as a template and pTFF3-F / R, pEGF-F / R, and pTE-F / R were used as primer pairs to amplify the pTFF3 、 pEGF and a fusion fragment of the two pTE , pTFF3 、 pEGF and fusion fragments pTE The sequences are shown in SEQ ID NO.10-12, pEGF and pTFF3 The PCR amplification results of the target gene are as follows Figure 1As shown; the signal peptide SP sequence of the S-layer protein of Lactobacillus pumilus was obtained from the plasmid pPG-T7g10-SP-pBD2 (constructed according to the method disclosed in the patent application with publication number CN118853524A, invention name "Recombinant porcine Lactobacillus casei co-expressing porcine β-defensin 2 and PEDV S1 protein, construction method and application thereof"). PCR amplification and recovery were performed using primer pairs SP-F / SP-R1 and SP-F / SP-R2, respectively. Primer SP-R1 contains the target gene pEGF The homologous sequence at the 5' end, the target gene is contained in the primer SP-R2 pTFF3 Homologous sequence at the 5' end, containing the target gene pEGF The sequence of the SP signal peptide of the homologous sequence at the 5' end is shown in SEQ ID NO.13, containing the target gene pTFF3 The sequence of the SP signal peptide of the homologous sequence at the 5' end is shown in SEQ ID NO. 14. The primer sequences are shown in Table 1: ; (2) Construction of pMD19T-SP-pTFF3 and pMD19T-SP-pEGF cloning plasmids The purified and recovered target gene pTFF3 The SP signal peptide sequence of the 5' end homologous sequence (shown in SEQ ID NO.14) and the target gene pEGF The sequence of the SP signal peptide with homologous sequence at the 5' end (shown in SEQ ID NO.13) SP Signal peptide genes and acquired pTFF3 and pEGF The genes were subjected to fusion PCR respectively. Using the fusion PCR products as templates, primers were used to amplify pPG-pTFF3-F / R and pPG-pEGF-F / R respectively to obtain SP-pTFF3 fragments and SP-pEGF fragments. The amplified products were then connected to the pMD19T Simple vector respectively. After the system was mixed, the connection was carried out in a 16℃ connection instrument for 4 hours, and the connection products were transformed into Escherichia coli. DH5α Competent cells. The extracted plasmids were identified by PCR using primer pair 19T-F / 19T-R. The correctly identified plasmids were sequenced and aligned, and named pMD19T-SP-pTFF3 and pMD19T-SP-pEGF. The PCR and double enzyme digestion identification results of pMD19T-SP-pTFF3 and pMD19T-SP-pEGF plasmids are shown in Figure 1. Figure 2 、 3As shown. Using plasmid pMD19T-SP-pTFF3 as template, primer pair pPG-pTE-F / Linker-pTFF3-R was used for PCR amplification. Using plasmid pMD19T-SP-pEGF as template, primer pair Linker-pEGF-F / pPG-pTE-R was used for PCR amplification. The two sequences were recovered by gel excision. The two sequences were homologously recombined by fusion PCR, and primer pair pPG-pTE-F / R was used to amplify the SP- pTFF3-pEGF The sequence was confirmed by agarose gel electrophoresis and then the gel was cut and recovered. It was connected to the pMD19T Simple vector to construct the positive clone plasmid pMD19T-SP-pTE. The recombinant plasmid was identified by PCR and sequence alignment using 19T-F / 19T-R as primers. The results of PCR and double enzyme digestion identification of pMD19T-SP-pTE plasmid are shown in Figure 2. Figure 4 The primer sequences are shown in Table 2: ; (3) Construction of pPG-SP-pTFF3, pPG-SP-pEGF, and pPG-SP-pTE expression vectors The vector pPG-PPT (pPG-PPT consists of HCE promoter, PgsA anchor, rrnBT1T2 terminator, and the chloramphenicol resistance gene and repA and repC replicons on the original pPG612 vector, see the vector map) was inserted into the pPG-PPT vector. Figure 5 The vector has been described in the following literature: Julong strain. Construction of constitutive lactic acid bacteria expression vector and comparison of expression effects [D]. Northeast Agricultural University, 2014. Deposited and provided by Northeast Agricultural University) and the above-constructed cloning plasmids pMD19T-SP-pTFF3, pMD19T-SP-pEGF and pMD19T-SP-pTE were used respectively. SnaB Ⅰ and Apa I restriction enzyme double digestion at 30℃ for 2 h, and the vector fragment was recovered by gel cutting. pPG With the target fragment SP-pTFF3-flag (Abbreviation SP-pTFF3 , shown in SEQ ID NO.29), SP- pEGF-flag (Abbreviation SP-pEGF , shown in SEQ ID NO.30) and SP-pTFF3-rigid Linker-pEGF-flag (Abbreviation SP-pTE , as shown in SEQ ID NO.31), then the target fragments were ligated into the vector fragments using T4 ligase, and the ligation products were chemically heat-transformed into Escherichia coli. TG1In competent state, single colonies were picked and cultured overnight. The extracted plasmids were identified by PCR using pPG-F / pPG-R as primers. In addition, the plasmids with the correct size identified by PCR were double-enzyme digested ( SnaB Ⅰ and Apa I) Identification: After correct sequence alignment, the correct plasmids were named pPG-SP-pTFF3, pPG-SP-pEGF, and pPG-SP-pTE. PCR identification and enzyme digestion identification of pPG-SP-pTFF3, pPG-SP-pEGF, and pPG-SP-pTE plasmids were performed as follows: Figure 6-8 shown.

[0024] (4) Construction of recombinant bacteria Lactobacillus paracasei L.paracasei HLJ-27 (The Lactobacillus paracasei L.paracasei HLJ-27 The results were recorded in the following literature: Li Fengsai., Mei Zhuyuan., Ju Ning., Sui Ling., Fan Xiaolong., Wang Zi., Li Jiaxuan., Jiang Yanping., Cui Wen., Shan Zhifu., Zhou Han., Wang Li., Qiao Xinyuan., Tang Lijie., Wang Xiaona., Li Yijing.(2022). Evaluationof the immunogenicity of auxotrophic with CRISPR-Cas9D10A system-mediated chromosomal editing to express porcine rotavirus capsid protein VP4. Virulence, 13(1), 1315-1330.) The cells were streaked and activated, and a single colony was picked and placed in liquid MRS medium without antibiotics. The cells were cultured at 37℃ for 14-16 h, and then inoculated into 100 mL expansion medium (MRS liquid medium containing 1% glycine) at a ratio of 1:100. The cells were cultured for about 4 h, and the OD was measured every 1 h. 600 nm value, wait for OD 600When the nm reaches between 0.6 and 0.8, begin preparing competent cells. First, place the bacterial solution in an ice bath for 30 minutes. Pre-cool the 50 mL centrifuge tube, pipette tip, and EP tube. After the ice bath, aliquot the bacterial solution into 50 mL centrifuge tubes in a clean bench. Centrifuge at 4°C at 5000 rpm for 10 minutes. Wash the cells with Solution II stored at 4°C, discarding the supernatant. After two washes, resuspend the centrifuged cells in 2 mL of Solution II, aliquot, and store at -80°C.

[0025] Take 1 μg of recombinant plasmids pPG-SP-pTFF3, pPG-SP-pEGF, pPG-SP-pTE and control plasmid pPG-PPT and add them into competent cells respectively. L.paracasei HLJ-27 In the tube, gently tap the wall of the tube to mix it, place it on ice and let it act for 2 minutes. Disinfect the electroporation cup with 75% alcohol in advance, rinse it clean and irradiate it with ultraviolet light overnight, and pre-cool it before use. Add the mixture to the electroporation cup, place it in the electroporator, and use a voltage of 2200V for electric shock. Immediately add 1mL of MRS recovery medium to the electroporation cup, mix it evenly and transfer it to a 2mL EP tube. Place it in a 37℃ incubator and let it stand for 4 hours. Then take it out and centrifuge it. Resuspend the bacteria in about 200 μL of the remaining supernatant, spread it on an MRS solid agar medium plate containing 5 μg / mL Cm, and let it stand for culturing at 37℃. The recombinant plasmid was identified by PCR using pPG-F / pPG-R as primers, and the correctly identified positive strains were named pPG-SP-pTFF3 / HLJ-27 、pPG-SP-pEGF / HLJ-27 、pPG-SP-pTE / HLJ-27 and pPG / HLJ- 27 The PCR identification results of the recombinant bacteria are as follows Figure 9 shown.

[0026] 2. Identification of pTFF3, pEGF, and pTE protein expression in recombinant bacteria (1) Western blot analysis ①Sample processing: Preparation of bacterial protein precipitate samples: 5 mL of cultured bacterial solution was washed three times with PBS, lysozyme (10 mg / mL) was added to resuspend the precipitate, incubated at 37°C for 1 h, centrifuged at 12,000 rpm for 1 min, washed three times with PBS, resuspended the precipitate in 1 mL of PBS, and ultrasonically disrupted the precipitate (PW 100 W, working time 30 min, ultrasonication 4 s, interval 4 s) and centrifuged at 5,000 rpm for 5 min. The lysed protein sample was collected, 5× SDS loading buffer was added, boiled for 15 min, cooled in an ice bath, and stored at 4°C for later use.

[0027] Preparation of supernatant protein samples: Collect the supernatant of the recombinant bacterial culture medium, add 20% trichloroacetic acid (TCA), mix thoroughly, and allow to settle. Centrifuge at low speed to collect the protein precipitate, treat with acetone, neutralize, and dissolve the protein in an appropriate amount of sterile water to the desired concentration factor. Add 5× SDS loading buffer, boil for 15 minutes, cool on ice, and store at 4°C until ready for use.

[0028] ② The prepared samples were subjected to Western blot analysis. The three recombinant strains were able to express target proteins of approximately 12 kDa, 13 kDa, and 23 kDa. The Western blot results of the recombinant strains expressing pTFF3, pEGF, and pTE are as follows: Figure 10-12 shown.

[0029] (2) Indirect immunofluorescence identification Recombinant Lactobacillus pPG-SP-pTFF3 / HLJ-27 、pPG-SP-pEGF / HLJ-27 and pPG-SP-pTE / HLJ-27 and pPG / HLJ-27 After culturing the control bacteria for 12 hours, centrifuge the bacterial solution at 5000 rpm for 5 minutes, resuspend and wash three times with sterile PBS, take an appropriate amount of bacterial smears, and perform indirect immunofluorescence. The primary antibody is mouse anti-flag tag antibody and the secondary antibody is FITC-goat anti-mouse IgG. After the operation is completed, observe under a fluorescence microscope and you can see obvious green fluorescence, such as Figure 13 shown.

[0030] (3) ELISA quantitative detection Pick the recombinant lactobacillus pPG-SP-pTFF3 / HLJ-27 、pPG-SP-pEGF / HLJ-27 and pPG-SP-pTE / HLJ-27 and pPG / HLJ-27The control strains were cultured statically at 37°C for 16 hours and then inoculated into the corresponding MRS medium at a dilution of 1:100 and continued to be cultured. Samples were taken at 6, 10, 14, 18, 22, and 24 hours. Commercial pTFF3 and pEGF ELISA kits were used to measure the target protein levels in the culture supernatant and bacterial lysate at different time points. The ELISA quantitative test results are shown in Tables 3 and 4. These results show that the expression levels of pTFF3, pEGF, and pTE proteins in the supernatant and bacterial lysate of the three recombinant strains were the highest at 18 hours of culture.

[0031] ; Note: Values are mean ± standard deviation (n=3); bold fonts represent maximum values; different letters indicate significant differences between groups ( P <0.05); the same letters indicate no significant difference between the groups ( P >0.05) ; Note: Values are mean ± standard deviation (n=3); bold fonts represent maximum values; different letters indicate significant differences between groups ( P <0.05); the same letters indicate no significant difference between the groups ( P >0.05) 3. Analysis of biological characteristics of recombinant Lactobacillus The recombinant Lactobacillus pPG-SP-pTFF3 / HLJ-27 、pPG-SP-pEGF / HLJ-27 and pPG-SP-pTE / HLJ-27 Streak activation on Cm-resistant MRS solid plates, strain L.paracasei HLJ-27 Streak and activate on MRS solid plate without antibiotics, incubate at 37℃ for 24 h, pick a single colony in liquid culture medium, and continue to culture until OD 600 nm=0.8; inoculate the bacterial suspension into the corresponding liquid culture medium at a ratio of 1:100, incubate at 37℃ for 24 h, take the bacterial suspension every 2 h, calculate the number of viable bacteria by plate count method, and draw the bacterial growth curve, as shown in Figure 2. Figure 14 Each experiment was repeated three times. The results showed that the growth characteristics of the recombinant Lactobacillus were consistent with those of the wild-type strain.

[0032] The recombinant Lactobacillus pPG-SP-pTFF3 / HLJ-27 、pPG-SP-pEGF / HLJ-27 and pPG-SP-pTE / HLJ-27The cells were subcultured for 20 generations, and plasmids extracted from the 5th, 10th, 15th and 20th generations of each recombinant bacteria were used as templates and pPG-F / pPG-R as primers for plasmid PCR identification. The positive plasmids that were correctly identified were sent to the company for sequencing, and the sequencing results were compared using DNAMAN. Protein samples of the 5th, 10th, 15th and 20th generations of recombinant lactobacilli were processed for protein genetic stability. The results showed that the recombinant plasmids can be stably inherited and the recombinant bacterial protein can be stably expressed. Figure 15 、 16 shown.

[0033] 4. Protein in vitro activity analysis The proliferation effects of pTFF3, pEGF, and pTE on IPEC-J2 cells were detected by CCK-8 assay. The specific steps were as follows: ① Prepare a 96-well plate. After the normally subcultured IPEC-J2 cells filled the cell flask, digest them and add complete medium to adjust the cell density to 2.0×10 5 ② Discard the culture medium and wash three times with sterile PBS. ③ Add 100 μL of protein samples diluted at different ratios to each well, make 6 replicates for each sample, and continue to incubate for 12 hours. ④ Add 10 μL of CCK-8 solution to each well, continue to incubate, and measure the OD value. 450 The nm value is read when it reaches the optimal reading range of CCK-8. Figure 17 As shown in the figure, the results indicate that pTFF3, pEGF and pTE proteins can significantly promote the proliferation of IPEC-J2 cells.

[0034] The effects of pTFF3, pEGF and pTE on the migration ability of IPEC-J2 cells were detected by cell scratch experiments, and their effects on the migration ability of cells were analyzed according to the degree of healing after scratching. The specific operation steps are as follows: After the normally subcultured IPEC-J2 cells fill the cell bottle, they are digested and then added to the complete culture medium. The cell suspension formed by gentle blowing and mixing is plated, and 1 mL of cell suspension is added to each well of the 12-well plate. The plate is placed in a cell culture incubator for culture. When it fills about 80% of the well plate, it is scratched; Use a sterilized 10 μL pipette tip to gently draw a line, discard the original culture medium, add 1 mL of sterile PBS to each well, gently shake the well plate to remove the residual culture medium and the scratched cells, repeat twice, and discard the waste liquid; Add the protein sample diluted with DMEM to each well, place it in a cell culture incubator for continued culture. Then observe the cell changes under an inverted microscope. The results are as follows Figure 18 、 19As shown, the results showed that pTFF3, pEGF and pTE proteins could significantly promote cell migration and heal scratch damage, and the migration area promoted by pTE protein was significantly larger than that of pTFF3 and pEGF, but there was no significant difference when pTE protein was used in combination with pTFF3 and pEGF proteins.

[0035] 5. Study on the repair effect of oral recombinant lactobacillus on intestinal damage in colitis model mice (1) Animal grouping Thirty-two 6-week-old BALB / c mice were selected and housed in a standard environment with a temperature of 22±2°C and a light cycle of 12 h light and 12 h dark. They were allowed to eat and drink freely. The experiment was conducted after one week of adaptation. First, a colitis model was established using 8 mice. The mice were allowed to drink 2% DSS solution freely for 7 days. The DSS solution was replaced every 2 days. After 7 days, the model was completed and the mice were killed as the model group (DSS). Subsequently, the experiment was divided into PBS, pPG-SP-pTE / HLJ-27 、pPG / HLJ-27 There were 3 groups, 8 in each group; the PBS healthy group was not treated, and the pPG-SP-pTE / HLJ-27 and pPG / HLJ-27 Mice in the two groups were fed with 200 μL pPG-SP-pTE for 7 consecutive days. / HLJ-27 、pPG / HLJ-27 After reconstitution of the bacterial solution, mice were fed 2% DSS to establish the model. The specific procedures were the same as those for the model group. The experimental groups and feeding doses are shown in Table 5.

[0036] ;

[0037] (2) Disease activity index The Disease Activity Index (DAI) score was calculated using the following scoring system: DAI = (body mass index + stool form + bleeding) / 3. During DSS modeling, mice were weighed and feces collected daily. Body weight, mental status, and the presence of diarrhea or bloody stools were recorded for each group. Feces were tested for occult blood using a Piramid's hole fecal occult blood kit.

[0038] (3) Colon length measurement On the 8th day after DSS modeling, blood was collected from the eyeballs of mice in each experimental group. After the mice were killed, they were dissected, and the entire colon (from the end of the cecum to the anus) was removed and its length was measured.

[0039] (4) H&E staining of tissue sections The mice were dissected and the colon tissue was measured. Then, a 1 cm length of colon tissue was cut with scissors and fixed in 4% paraformaldehyde for 48 h, followed by H&E staining.

[0040] (5) Myeloperoxidase (MPO) activity assay Myeloperoxidase activity was measured in colon tissue. First, the colon tissue was processed: the tissue was accurately weighed and the homogenate medium prepared according to the instructions was mixed at a weight-to-volume ratio of 1:19 to prepare a 5% tissue homogenate. The tissue homogenate was as uniform as possible without large pieces of tissue. Then, the colon MPO activity of each group of mice was measured according to the instructions of the MPO test kit.

[0041] (6) Real-time fluorescence quantitative PCR detection of gene transcription levels in colon tissue and Western blot detection of expression levels Real-time fluorescence quantitative PCR: First, RNA was extracted from the colon tissue. About 0.1 g of mouse colon tissue was taken from each group and added to 1 mL of sterile PBS. The tissue was homogenized in a tissue homogenizer and centrifuged at 12,000 rpm for 2 min at 4°C. 200 μL of the supernatant was taken for RNA extraction to obtain cDNA of the intestinal tissue for target gene expression. ZO-1 、 Claudin-2 、 Occludin and internal reference genes β-actin The 2-ΔΔCt method was used for qPCR analysis.

[0042] Western blot analysis: Equal amounts of mouse colon tissue were added to 1 mL of Western and IP lysis buffer containing 1% PMSF. Lysis was performed on a shaker at 4°C for 4 h. The cells were centrifuged at 5000 rpm at 4°C for 5 minutes. The supernatant was collected and protein quantified using a BCA protein assay kit. Protein amounts were kept consistent across all tissues. 5× SDS loading buffer was then added to mix thoroughly. The samples were boiled for 15 minutes. After cooling, 20 μL of sample was loaded into each well. Western blot analysis was performed using antibodies selected based on the target protein.

[0043] (7) Serum cytokine determination Serum was collected from mice 14 days after oral administration of the recombinant bacteria and used to measure IL-1β, IL-6, IL-10, and TNF-α cytokine levels using ELISA cytokine assay kits according to the manufacturer's instructions. Standard curves were plotted and corresponding cytokine levels were calculated.

[0044] (8) Results The results are as follows Figure 20-25 As shown, the results show that oral administration of pPG-SP-pTE / HLJ-27 Compared with the DSS model group, the mice's condition was significantly improved, and their appetite and activity were almost unaffected ( Figure 20 ); maintained colon length and significantly reduced colon shortening ( Figure 21 ); Myeloperoxidase (MPO) values were significantly decreased ( Figure 22 ); and significantly upregulated the expression of tight junction proteins Claudin-2, Occludin and ZO-1 and the level of IL-10 in serum ( Figure 23 、 24 , 25); significantly reduced serum IL-6, IL-1β and TNF-α levels ( Figure 25 ). Colon histopathological findings were as follows Figure 26 The results showed that compared with DSS model, oral administration of pPG-SP-pTE / HLJ-27 The lesions in the group were milder, the mucosal structure was relatively intact, a large number of goblet cells were retained, and the crypt structure was relatively intact; the above results showed that oral administration of pPG-SP-pTE / HLJ-27 It can effectively alleviate DSS-induced intestinal damage in mice and has a certain protective effect on the intestine.

Claims

1. A recombinant Lactobacillus paracasei expressing fusion of porcine trefoil factor 3 and epidermal growth factor, characterized in that: The recombinant lactobacillus paracasei contains a recombinant plasmid capable of expressing a fusion protein of porcine trefoil factor 3 and epidermal growth factor.

2. recombinant Lactobacillus paracasei as claimed in claim 1, is characterized in that, The porcine trefoil factor 3 and epidermal growth factor are connected through a rigid linker. The amino terminus of the fusion protein is also connected to a signal peptide SP sequence, and the carboxyl terminus is connected to a flag tag. The structure of the obtained fusion protein is SP-pTFF3-rigid linker-pEGF-flag.

3. recombinant Lactobacillus paracasei as claimed in claim 1, is characterized in that, The nucleotide sequence encoding the fusion protein is shown in SEQ ID NO.

31.

4. recombinant Lactobacillus paracasei as claimed in claim 1, is characterized in that, The recombinant plasmid is a recombinant lactobacillus expression plasmid containing porcine trefoil factor 3 and epidermal growth factor capable of fusion expression.

5. recombinant Lactobacillus paracasei as claimed in claim 4, is characterized in that, The recombinant plasmid is obtained by cloning the nucleotide sequence encoding the fusion protein of porcine trefoil factor 3 and epidermal growth factor shown in SEQ ID NO.31 into the vector plasmid pPG-PPT. SnaB Ⅰ and Apa I restriction endonuclease sites.

6. A method for constructing the recombinant Lactobacillus paracasei according to any one of claims 1 to 5, characterized in that: The following steps are involved: (1) Acquisition of target gene and signal peptide sequence Fresh pig intestinal tissue was taken, total RNA was extracted and reverse transcribed, and the extracted cDNA was used as a template and pTFF3-F / R, pEGF-F / R, and pTE-F / R were used as primer pairs to amplify the pTFF3 、 pEGF and a fusion fragment of the two pTE , pTFF3 、 pEGF and fusion fragments pTE The sequences are shown in SEQ ID NO.10-12 respectively; the signal peptide SP sequence of the S layer protein of Lactobacillus pumilus was obtained from the plasmid pPG-T7g10-SP-pBD2, and the primers SP-F / SP-R1 and SP-F / SP-R2 were used for PCR amplification and recovery respectively. The primer SP-R1 contained the target gene pEGF The homologous sequence at the 5' end, the target gene is contained in the primer SP-R2 pTFF3 Homologous sequence at the 5' end, containing the target gene pEGF The sequence of the SP signal peptide of the homologous sequence at the 5' end is shown in SEQ ID NO.13, containing the target gene pTFF3 The sequence of the SP signal peptide of the homologous sequence at the 5' end is shown in SEQ ID NO.14, and the primer sequence is shown below: ； (2) Construction of pMD19T-SP-pTFF3 and pMD19T-SP-pEGF cloning plasmids The purified and recovered SP Signal peptide genes and acquired pTFF3 and pEGF The genes were subjected to fusion PCR respectively, and the fusion PCR products were used as templates to amplify pPG-pTFF3-F / R and pPG-pEGF-F / R with primers to obtain SP-pTFF3 Fragments and SP-pEGF The gel-collected products were then connected to the pMD19T Simple vector, the system was mixed, and the connection was carried out at 16 ° C for 4 h. The connection products were transformed into Escherichia coli. DH5α Competent cells were used to identify the extracted plasmids by PCR using primer pair 19T-F / 19T-R. The correctly identified plasmids were sequenced and aligned, and named pMD19T-SP-pTFF3 and pMD19T-SP-pEGF. Plasmid pMD19T-SP-pTFF3 was used as a template and primer pair pPG-pTE-F / Linker-pTFF3-R was used for PCR amplification. Plasmid pMD19T-SP-pEGF was used as a template and primer pair Linker-pEGF-F / pPG-pTE-R was used for PCR amplification. The two sequences were recovered by gel excision and homologously recombined by fusion PCR. Primers pPG-pTE-F / R were used to amplify the two sequences. SP-pTFF3-pEGF The sequence was confirmed by agarose gel electrophoresis and then the gel was cut and recovered. It was ligated with the pMD19T Simple vector to construct the positive clone plasmid pMD19T-SP-pTE. The recombinant plasmid was identified by PCR and sequence alignment using 19T-F / 19T-R as primers. The primer sequences are shown below: ； (3) Construction of pPG-SP-pTE expression vector The vector plasmid pPG-PPT and the above-constructed cloning plasmid pMD19T-SP-pTE were used SnaB Ⅰ and Apa I restriction enzyme double digestion at 30℃ for 2 h, and the vector fragment was recovered by gel cutting. pPG With the target fragment SP-pTFF3-rigid Linker-pEGF-flag, Abbreviation SP-pTE , whose sequence is shown in SEQ ID NO.31, and then the target fragments were connected to the vector fragments using T4 ligase, and the ligation products were chemically heat-transformed into Escherichia coli TG1 In the competent state, a single colony was picked and cultured overnight. The extracted plasmid was identified by PCR using pPG-F / pPG-R as primers. In addition, the plasmid with the correct size identified by PCR was SnaB Ⅰ and Apa I. Double enzyme digestion identification. After correct sequence alignment, the correct plasmid was named pPG-SP-pTE, which is the recombinant lactobacillus expression plasmid expressing porcine trefoil factor 3 and epidermal growth factor; (4) Preparation of recombinant Lactobacillus paracasei The recombinant plasmid pPG-SP-pTE was transformed into competent cells using electroporation L.paracasei HLJ-27 After culture and PCR identification, a positive strain containing the recombinant plasmid pPG-SP-pTE was obtained and named pPG-SP-pTE / HLJ-27 .

7. Use of the recombinant Lactobacillus paracasei according to any one of claims 1 to 5 in preparing a medicament for treating inflammatory bowel disease.

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