WO2021190634A1 - 高表达抗菌肽cathelicidin基因的乳酸菌 - Google Patents

高表达抗菌肽cathelicidin基因的乳酸菌 Download PDF

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WO2021190634A1
WO2021190634A1 PCT/CN2021/083282 CN2021083282W WO2021190634A1 WO 2021190634 A1 WO2021190634 A1 WO 2021190634A1 CN 2021083282 W CN2021083282 W CN 2021083282W WO 2021190634 A1 WO2021190634 A1 WO 2021190634A1
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cramp
usp45
group
lactic acid
acid bacteria
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PCT/CN2021/083282
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English (en)
French (fr)
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孙嘉
潘礼龙
迪安那朱利安
张明
陈卫
张灏
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江南大学
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Priority claimed from CN202010230656.8A external-priority patent/CN111333713B/zh
Priority claimed from CN202010229298.9A external-priority patent/CN111411054B/zh
Application filed by 江南大学 filed Critical 江南大学
Publication of WO2021190634A1 publication Critical patent/WO2021190634A1/zh
Priority to US17/529,396 priority Critical patent/US11479588B2/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0008—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4723—Cationic antimicrobial peptides, e.g. defensins
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/11—Lactobacillus
    • A23V2400/157—Lactis
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/11—Lactobacillus
    • A23V2400/169—Plantarum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K2035/11—Medicinal preparations comprising living procariotic cells
    • A61K2035/115—Probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/523—Bacterial cells; Fungal cells; Protozoal cells expressing foreign proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A61K2039/542—Mucosal route oral/gastrointestinal
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/58—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence

Definitions

  • the invention relates to a lactic acid bacterium that highly expresses an antibacterial peptide cathelicidin gene, and belongs to the technical field of genetic engineering.
  • Antibacterial peptides are the main components of innate immunity and defense of many hosts such as plants, invertebrates and vertebrates (including humans).
  • Cathelicidins are a major class of antimicrobial peptides, which are characterized by a conservative anionic N-terminal precursor sequence called cathelin. The conservation of cathelin sequence indicates that various members of this family evolved from the duplication and modification of common ancestor genes.
  • CRAMP Cathelicidin-Related AntiMicrobial Peptide
  • contains 34 amino acids GLLRKGGEKIGEKLKKIGQKIKNFFQKLVPQPEQ
  • CRAMP 1mM CRAMP can directly cause the immediate permeabilization of the inner membrane of E. coli.
  • Anti-CRAMP antiserum showed abundant expression in bone marrow precursors and neutrophils. Similar to Cecropin A, 50mM CRAMP did not show any hemolytic activity on human red blood cells.
  • studies have found that CRAMP has strong antibiotic activity against some pathogenic fungi (Candia alicans and Aspergillus fumigatus) and tumor cells. In vitro studies have confirmed that CRAMP can significantly inhibit the proliferation of Helicobacter pylori; the absence of CRAMP can cause aggravation of gastrointestinal conditions in mice. Treatment of CRAMP knockout mice with gastrointestinal diseases by CRAMP-expressing lactobacillus has a significant alleviation effect.
  • CRAMP expressed in the intestinal tract has a significant decrease in CRAMP levels due to the destruction of the intestinal barrier, resulting in the inability to exert its immune effect and regulate the balance of intestinal flora. Therefore, select a safe and non-toxic vector that can survive in the intestine and express CRAMP.
  • the system enables CRAMP to play a role in the intestinal tract, which is of great significance for regulating the balance of intestinal flora.
  • Lactococcus lactis and Lactobacillus plantarum have high adhesion characteristics on their surface molecules, which can make them successfully colonize the intestinal tract of animals and become the dominant flora in the intestinal tract to improve immunity and promote nutrient absorption. And maintain the balance of the intestinal flora and other functions.
  • the formed biologically stable barrier is an important guarantee for maintaining the balance of intestinal microbes.
  • the Lactococcus lactis expression system and the Lactobacillus plantarum expression system as prokaryotic expression systems have the following advantages: (1) As a food-grade bacteria, it is safer as a live vector vaccine; (2) Exogenous Genes can be expressed in the cell, displayed on the cell surface or secreted outside the cell; (3) Safe, no endotoxin, no need to purify the expressed foreign protein, and take it directly with the bacteria; (4) Can It is planted on the surface of the body's mucosa (belonging to the common mucosal immune system). Vaccination at a certain site of the mucosa can induce a systemic mucosal immune response; last and most important, this immune method can help the body acquire longer-term immune memory, so that To resist the invasion of pathogens for a long time.
  • the technical problem to be solved by the present invention is to overcome the defects in the prior art that oral CRAMP is easily reduced by digestive tract enzymes, cannot achieve intestinal targeted delivery of CRAMP, and maximizes its local immune regulation effect, and provides a recombinant expression of CRAMP protein. Lactic acid bacteria and its applications.
  • the present invention provides a gene encoding CRAMP protein, which contains the nucleotide sequence shown in SEQ ID NO:1.
  • the gene is also fused with a nucleotide sequence encoding Usp45 signal peptide.
  • the nucleotide sequence encoding the Usp45 signal peptide is shown in SEQ ID NO: 3, and the gene sequence of the signal peptide is connected. As shown in SEQ ID NO: 4.
  • nucleotide sequence encoding the Usp45 signal peptide there is a linker sequence between the nucleotide sequence encoding the Usp45 signal peptide and the gene sequence of the CRAMP protein, and the nucleotide sequence is shown in SEQ ID NO: 10.
  • the present invention also provides a vector carrying the gene.
  • the vector is pMG36e or pNZ8148.
  • the vector is pMG36e-CRAMP, pNZ8148-CRAMP, pNZ8148-Usp45-CRAMP, pMG36e-Usp45-CRAMP, pMG36e-Usp45-Linker-CRAMP or pNZ8148-Usp45-Linker-CRAMP.
  • the third object of the present invention is to provide lactic acid bacteria expressing CRAMP protein, which express the gene shown in SEQ ID NO.1, or express CRAMP protein; the CRAMP protein is (a) or (b):
  • the lactic acid bacteria are Lactobacillus plantarum or Lactococcus lactis.
  • the Lactobacillus plantarum FCQHC24L1 is used as a host.
  • the Lactobacillus plantarum FCQHC24L1 was published in a 2019 paper entitled "Study on the Differences in the Genome and Main Physiological Characteristics of Lactobacillus Plantarum in Different Niches", and the applicant promises to report to you within 20 years from the filing date The strain is distributed to the public who implement the present invention in a legal way.
  • the Lactobacillus plantarum uses pMG36e or pNZ8148 as a vector to express the CRAMP protein-encoding gene.
  • the Lactobacillus plantarum also introduces Usp45 signal peptide to promote CRAMP protein expression.
  • the Usp45 is connected to the CRAMP gene through a linker, and the linker includes 2 or more amino acid residues selected from Gly and Ser.
  • the amino acid sequence of the linker is GGGGS; the nucleotide sequence encoding the linker is shown in SEQ ID NO.9.
  • the Lactococcus lactis NZ9000 is a host.
  • the Lactococcus lactis uses pMG36e or pNZ8148 as a carrier.
  • the Lactococcus lactis also introduces Usp45 signal peptide to promote CRAMP protein expression.
  • the present invention also provides a method for constructing the Lactobacillus plantarum, which is to link the gene encoding CRAMP protein shown in SEQ ID NO. 1 with a vector, and then transform it into Lactobacillus plantarum cells; the vector is pMG36e or pNZ8148 .
  • the usp45 signal peptide is connected to the pMG36e or pNZ8148.
  • the Usp45 is connected to the CRAMP gene through a linker, and the nucleotide sequence of the linker is GGCGGTGGCGGCAGC.
  • the method includes the following steps:
  • step (2) Connect the synthesized gene in step (1) to pMG36e to obtain the recombinant plasmid pMG36e-CRAMP; use the electrotransformation method to introduce the pMG36e-CRAMP recombinant plasmid into Lactobacillus plantarum FCQHC24L1 to obtain recombinant Lactobacillus plantarum L. plantarum FCQHC24L1/pMG36e-CRAMP.
  • the method includes the following steps:
  • step (2) Connect the synthesized gene in step (1) to pMG36e to obtain the recombinant plasmid pMG36e-Usp45-Linker-CRAMP; use the electrotransformation method to introduce the pMG36e-Usp45-Linker-CRAMP recombinant plasmid into Lactobacillus plantarum FCQHC24L1 , To obtain recombinant Lactobacillus plantarum FCQHC24L1/pMG36e-Usp45-Linker-CRAMP.
  • the specific operation of the electrotransformation method is: take the competent cells of L. plantarum FCQHC24L1, add the recombinant plasmid, mix and transfer to the electrotransformation cup, add the recovery medium MRS medium after the electric shock, After ice bathing, they were allowed to stand and cultivate, and high-copy transformants were screened on the plate.
  • the present invention also provides a method for constructing any one of the above-mentioned recombinant Lactococcus lactis, which is to link the gene encoding CRAMP protein shown in SEQ ID NO. 1 with a vector, and then transform it into a Lactococcus lactis cell; the vector It is pMG36e or pNZ8148.
  • the usp45 signal peptide is connected to the pMG36e or pNZ8148.
  • the method includes the following steps:
  • step (2) Connect the synthesized gene in step (1) to pMG36e to obtain the recombinant plasmid pMG36e-Usp45-CRAMP; use the electrotransformation method to introduce the pMG36e-Usp45-CRAMP recombinant plasmid into L. lactis NZ9000 to obtain recombinant lactic acid Lactococcus L. lactis NZ9000/pMG36e-Usp45-CRAMP.
  • the method includes the following steps:
  • step (2) Connect the synthesized gene in step (1) to pNZ8148 to obtain the recombinant plasmid pNZ8148-Usp45-CRAMP; use the electrotransformation method to introduce the pNZ8148-Usp45-CRAMP recombinant plasmid into L. lactis NZ9000 to obtain recombinant lactic acid Lactococcus L. lactis NZ9000/pNZ8148-Usp45-CRAMP.
  • the electrotransformation method is: take the competent cells of L. lactis NZ9000, add the recombinant plasmid, mix and transfer to the electrotransformation cup, add the recovery medium after the electric shock, and then let stand culture after an ice bath , Plate screening of high-copy transformants.
  • the present invention also provides an edible or medicinal composition, which contains the lactococcus lactis and/or the lactobacillus plantarum.
  • the content of the Lactococcus lactis and/or Lactobacillus plantarum in the composition is ⁇ 1 ⁇ 10 5 CFU/mL or 1 ⁇ 10 5 CFU/g.
  • the composition is a medicine and contains a pharmaceutically acceptable carrier.
  • the fourth objective of the present invention is to provide the application of the said Lactococcus lactis and/or Lactobacillus plantarum in preparing vaccines.
  • the vaccine is an oral vaccine to prevent acute colitis.
  • the application is to cultivate the Lactococcus lactis for cultivation, and then use the whole culture of Lactococcus lactis as an oral vaccine.
  • the application includes the following steps: inoculating Lactococcus/pMG36e-Usp45-CRAMP in GM17 liquid medium and standing for overnight culture, transferring to GM17 liquid medium at a certain ratio, and continuing to cultivate until the bacteria In the logarithmic growth phase, the whole culture is directly used as an oral vaccine.
  • the temperature of the static culture is 28-30°C.
  • the transfer is to inoculate L. lactisNZ9000/pMG36e-Usp45-CRAMP in a GM17 medium at a volume ratio of 1:100.
  • the OD value of the bacterial culture solution in the logarithmic growth phase is 0.4 to 0.6.
  • the application is to cultivate the Lactobacillus plantarum for cultivation, and then use the whole culture of Lactobacillus plantarum as an oral vaccine.
  • the application is to cultivate the recombinant Lactobacillus plantarum, and then use the whole culture of Lactobacillus plantarum as the oral vaccine or the main component of the oral vaccine.
  • the application includes the following steps: inoculating the recombinant Lactobacillus plantarum/pMG36e-Usp45-Linker-CRAMP in MRS liquid medium and standing for overnight culture, and transferring to the MRS liquid medium at a certain ratio, Continue the culture until the bacteria enter the logarithmic growth phase, and the whole culture is directly used as an oral vaccine.
  • the temperature of the static culture is 28-30°C.
  • the transfer is to inoculate L. plantarum FCQHC24L1/pMG36e-Usp45-Linker-CRAMP in the MRS medium at a volume ratio (1-10):100.
  • the OD value of the bacterial culture solution in the logarithmic growth phase is 0.4 to 0.6.
  • the present invention also claims to protect the application of the Lactobacillus plantarum in the preparation of medicines for preventing or treating acute colitis.
  • the drug is used in combination with nisin.
  • nisin or a nisin-containing drug is then taken to achieve the effect of directional release of CRAMP and increasing the amount of CRAMP secreted in the intestinal tract.
  • the present invention also claims to protect the application of the Lactococcus lactis and/or Lactobacillus plantarum in the preparation of a product that can be introduced into the intestinal tract; the product has at least one of the following functions:
  • the present invention also claims to protect the application of the Lactococcus lactis and/or Lactobacillus plantarum in the preparation of medicines for preventing or treating inflammatory bowel disease, diarrhea or intestinal homeostasis or related diseases.
  • Diseases caused by or related to state imbalance include, but are not limited to, liver disease, metabolic endocrine disease, circulatory system disease, etc., such as diabetes, pancreatitis, or metabolic syndrome.
  • the present invention also claims to protect the use of the lactococcus lactis and/or lactobacillus plantarum in the preparation of medicines for the prevention or treatment of acute colitis.
  • the present invention provides an optimized gene encoding CRAMP protein, and adding Usp45 signal peptide to promote the secretion and expression of CRAMP gene, so that the expression level of CRAMP protein in Lactococcus lactis can reach 40ng/ ⁇ L, which can be used in plants.
  • the expression level in Lactobacillus can reach 20ng/ ⁇ L or even higher. Coupled with the probiotic properties of Lactococcus lactis and Lactobacillus plantarum as probiotics, this lactic acid bacteria expression system becomes a food-grade expression system that can be taken together with the bacteria.
  • the present invention uses the constructed Lactococcus lactis and Lactobacillus plantarum expressing CRAMP protein to prepare vaccines against intestinal flora disorder regulation, which has advantages in the regulation of intestinal flora and intestinal immune response and maintenance.
  • the whole culture can be directly used as an oral vaccine to stimulate mice and cause a strong cellular immune response.
  • the recombinant Lactococcus lactis can be used as a new oral vaccine product with good industrial prospects, which has a positive effect on reducing intestinal inflammation. It has important practical significance for promoting the healthy development of intestines.
  • the vaccine containing recombinant Lactobacillus plantarum and/or Lactococcus lactis prepared by the present invention can specifically adjust the intestinal flora disorder, help the regulation of the intestinal flora and the intestinal immune response and maintenance.
  • the oral vaccine prepared by the present invention can stimulate mice and cause a strong cellular immune response. It can be used as a new oral vaccine product with good industrial prospects. It has a positive effect on reducing intestinal inflammation and promoting intestinal inflammation. The healthy development of Taoism has important practical significance.
  • Figure 1 shows the PCR amplification results of CRAMP and Usp45-CRAMP gene fragments
  • 1 is DL2000 DNA Marker
  • 2-3 is the PCR amplification of Usp45-CRAMP gene fragments
  • Figure 2 shows the PCR identification results of recombinant E. coli MC1061/pMG36e-Usp45-CRAMP group
  • 1 is DL2000 DNA Marker
  • 2 is PCR identification of recombinant E.coli MC1061/pMG36e-Usp45-CRAMP group
  • 3 is PCR identification of recombinant E.coli MC1061/pNZ8148-Usp45-CRAMP group
  • Figure 3 shows the PCR identification results of recombinant L. lactis NZ9000/pMG36e-Usp45-CRAMP group and L.lactis NZ9000/pNZ8148-Usp45-CRAMP group; 1 is DL2000 DNA Marker; 2 is L.lactis NZ9000/pMG36e- PCR identification of CRAMP in Usp45-CRAMP group; 3 is PCR identification of L.lactisNZ9000/pNZ8148-Usp45-CRAMP group;
  • Figure 4 shows the results of immunoblotting of CRAMP in recombinant Lactococcus lactis; 1 is protein Marker; 2 is the expression of CRAMP in the supernatant of L.lactisNZ9000/pMG36e-Usp45-CRAMP group; 3 is L.lactisNZ9000/pMG36e-Usp45-CRAMP The expression level of CRAMP in the cell of the group; 4 is the expression level of CRAMP in the supernatant of the L.lactisNZ9000/pNZ8148-Usp45-CRAMP group; 5 is the expression of CRAMP in the cell of the L.lactisNZ9000/pNZ8148-Usp45-CRAMP group;
  • Figure 5 shows the ELISA results of recombinant Lactococcus lactis CRAMP
  • Figure 6 shows the expression of CRAMP in E. coli by previous studies; 1 is E. coli lysate; 2 is E. coli lysate supernatant; 3 is E. coli lysate precipitation; 4 is the elution of GST-CRAMP on the elution column Buffer; 5 is protein Marker;
  • Figure 7 shows the changes in body weight of mice in each group during the establishment of the colitis model
  • Figure 8 is a comparison of the colon length of mice in each group (A) and (B) a statistical diagram of each group;
  • Figure 9 shows the scores of clinical indicators of colitis
  • Figure 10 is the histology of colon (A) pathological morphology observation and (B) score;
  • Figure 11 shows the changes of intestinal tight junction proteins (A) ZO-1, (B) ZO-2 and (C) occludin measured by qPCR;
  • Figure 12 is a qPCR measurement of the expression of inflammatory cytokines (A) IL-6, (B) IL-1 ⁇ , (C) TNF- ⁇ and (D) IL-10;
  • Figure 13 shows the changes in phosphorylation levels of key transcription factors in the inflammation signaling pathway determined by Western blot:
  • A Western blot experiment results p-ERK, ERK, p-p38, p38, p-NF-kB and NF-kB band diagram ,
  • B p-ERK/ERK gray analysis statistics,
  • C p-p38/p38 gray analysis statistics,
  • D p-NF-kB/NF-kB gray analysis statistics;
  • Figure 14 shows the PCR amplification results of CRAMP and Usp45-Linker-CRAMP gene fragments; 1 is DL2000 DNA Marker; 2-5 is the PCR amplification of CRAMP gene fragments, and 6-9 is the PCR amplification of Usp45-Linker-CRAMP gene fragments increase;
  • Figure 15 is the PCR identification result of recombinant E.coli MC1061/pMG36e-Usp45-Linker-CRAMP, 1 is DL2000 DNA Marker, 2-3 is the PCR identification of recombinant E.coli MC1061/pMG36e-CRAMP, 4- 5 is the PCR identification of recombinant Escherichia coli MC1061/pMG36e-Usp45-Linker-CRAMP;
  • Figure 16 shows recombinant Lactobacillus plantarum FCQHC24L1/pMG36e-CRAMP, L.plantarum FCQHC24L1/pNZ81848-CRAMP, L.plantarum FCQHC24L1/pMG36e-Usp45-Linker-CRAMP, L.plantarum FCQ45HC24L1 L1/pNZ8148-Usp45 PCR identification results of CRAMP; 1 is DL2000 DNA Marker; 2 is PCR identification of CRAMP in L.plantarum FCQHC24L1/pMG36e-CRAMP; 3 is PCR identification of CRAMP in L.plantarum FCQHC24L1/pNZ8148-CRAMP; 4 is L.plantarum FCQHC24L1 /pMG36e-Usp45-Linker-CRAMP in the PCR identification of Usp45-Linker-CRAMP; 5 is the PCR identification of L.plantarum FCQHC24L1/pNZ8148-Usp45-
  • Figure 17 shows the results of immunoblotting of CRAMP in recombinant Lactobacillus plantarum; 1 is protein Marker; 2 is the expression level of CRAMP in L.plantarum FCQHC24L1/pMG36e-CRAMP; 3 is L.plantarum FCQHC24L1/pMG36e-CRAMP supernatant The expression level of CRAMP; 4 is the expression level of CRAMP in L.plantarum FCQHC24L1/pNZ81848-CRAMP; 5 is the expression level of CRAMP in the supernatant of L.plantarum FCQHC24L1/pNZ81848-CRAMP; 6 is the expression level of CRAMP in the supernatant of L.plantarum FCQHC24L1/pMG36e- The expression level of CRAMP in Usp45-Linker-CRAMP bacteria; 7 is the expression level of CRAMP in the supernatant of L.plantarum FCQHC24L1/pMG36e-Usp45-Linker-
  • Figure 18 shows the ELISA results of recombinant Lactobacillus plantarum CRAMP
  • Figure 19 shows the expression of CRAMP in E. coli in the prior art
  • 1 is the E. coli lysate
  • 2 is the supernatant of the E. coli lysate
  • 3 is the precipitation of the E. coli lysate
  • 4 is the GST-CRAMP wash on the elution column Unbuffered solution
  • 5 is protein Marker
  • Figure 20 shows the changes in body weight of mice in each group during the establishment of the colitis model
  • Figure 21 is a comparison of the length of the colon of each group of mice (A) and a statistical diagram of the length of each group (B);
  • Figure 22 shows the scores of clinical indicators of colitis
  • Figure 23 shows the histopathological morphological observation of colon (A) and histopathological score (B);
  • Figure 24 shows the changes of intestinal tight junction proteins (A) ZO-1, (B) ZO-2 and (C) occludin measured by qPCR;
  • Figure 25 shows the qPCR measurement of the expression of inflammatory cytokines (A) IL-6, (B) IL-1 ⁇ , (C) TNF- ⁇ and (D) IL-10;
  • Figure 26 shows the changes in phosphorylation levels of key transcription factors in the inflammation signaling pathway determined by Western blot:
  • A Western blot experiment results p-ERK, ERK, p-p38, p38, p-NF-kB and NF-kB band diagram ,
  • B p-ERK/ERK grayscale analysis statistics,
  • C p-p38/p38 grayscale analysis statistics,
  • D p-NF-kB/NF-kB grayscale analysis statistics,
  • E CRAMP/ ⁇ -actin gray scale analysis statistics chart.
  • p-ERK, ERK, pp38, p38, p-NF-kB, NF-kB, and CRAMP were determined by Western Blot.
  • the method refers to the document "Dietary squalene supplementation improvement DSS-induced acute colitis by downregulating p38 MAPK and NFkB signaling pathways”.
  • Codon preference optimization and synthesis of gene sequence According to the sequence of the target gene CRAMP gene and the characteristics of the expression vector pMG36e, as well as the signal peptide sequence Usp45 added for the purpose of high-efficiency secretion and expression, artificially synthesized The 228bp codon optimized sequence of Usp45-CRAMP gene was sent to the company for synthesis.
  • Xbal-Usp45-CRAMP-F is the upstream primer containing the restriction site Xbal fused with pMG36e and the first sequence of the 5'end of the signal peptide Usp45-CRAMP
  • Usp45-CRAMP-Sph1-R is the signal peptide Usp45-CRAMP gene Reverse primer.
  • primers pNZ1 and pNZ2 for PCR detection and sequencing of recombinant plasmids were also designed, which were designed based on the 70-90bp region of MCS upstream and downstream of empty plasmid pMG36e.
  • the optimized and synthesized Usp45-CRAMP sequence is shown in SEQ ID NO: 4; the optimized and synthesized Xbal-Usp45-CRAMP-F and Usp45-CRAMP-Sph1-R primer sequences are shown in SEQ ID NO: 5-6, respectively.
  • step (3) Construction of the recombinant plasmid pMG36e-Usp45-CRAMP: The PCR product recovered in step (2) was subjected to double enzyme digestion with Sph1 and Xbal, and a band with a size of about 228 bp was recovered by the gel; the pMG36e empty plasmid was treated in the same way After double enzyme digestion, a band with a size of about 3600 bp was recovered by the gel.
  • PCR identification use the colony to be tested as a template, add 1 ⁇ L of high-fidelity DNA polymerase KOD-Plus-(1.0U/ul), 0.3 ⁇ M primers Xbal-Usp45-CRAMP-F, Usp45-CRAMP-Sph1-R 1.5 ⁇ L each, Template 1.5 ⁇ L, 25mM MgSO4 2 ⁇ L, 2mM dNTPs 5 ⁇ L, 10x Buffer for KOD-Plus-5 ⁇ L, make up to 50 ⁇ L with ddH20, PCR reaction program: 94°C pre-denaturation 5min; 94°C denaturation 30s, 55°C annealing 30s, 72°C Extend for 1 min, 35 cycles; after 72°C, extend for 10 min.
  • Codon preference optimization and synthesis of gene sequence According to the method of step 1 (1) of Example 1, the sequence of Usp45-CRAMP gene as shown in SEQ ID NO: 4 is optimized and synthesized; Sph1-Usp45-CRAMP-F is The upstream primer containing the restriction site Xbal expressed in fusion with pNZ8148 and the first sequence of the 5'end of the signal peptide Usp45-CRAMP. Usp45-CRAMP-Xbal-R is the reverse primer of the signal peptide Usp45-CRAMP gene. The optimized and synthesized Sph1-Usp45-CRAMP-F and Usp45-CRAMP-Xbal-R primer sequences are shown in SEQ ID NO: 7-8, respectively.
  • the positive bacterial solution is subjected to plasmid extraction with a plasmid DNA extraction kit. And carry out double enzyme digestion identification and sequencing determination, that is, to obtain the recombinant plasmid pMG36e-Usp45-CRAMP.
  • step 1 (5) of Example 1 The specific operation steps are the same as step 1 (5) of Example 1, except that the recombinant plasmid pNZ8148-Usp45-CRAMP is added to L.lactis NZ9000 competent cells Recombinant plasmid. Pick a single colony after transformation and culture for PCR identification.
  • the specific operation process is as described in step (2) of this example.
  • the PCR product is detected by 1% agarose gel electrophoresis, and an amplified band of about 228 bp can be seen ( Figure 3)
  • the positive recombinant expression strain was named L. lactis NZ9000/pNZ8148-Usp45-CRAMP.
  • the recombinant strain L.lactis NZ9000/pMG36e-Usp45-CRAMP constructed in Example 1 was inoculated into GM17 liquid medium containing 5ug/mL erythromycin at a ratio of 1:100, and the recombinant strain constructed in Example 2
  • L.lactis NZ9000/pNZ8148-Usp45-CRAMP and L.lactis NZ9000/pMG36e-Usp45-CRAMP recombinant Lactococcus lactis oral vaccine the recombinant bacteria L.lactis NZ9000/pMG36e-Usp45-CRAMP constructed in Example 1 Inoculate 5ug/mL erythromycin GM17 liquid medium at a volume ratio of 100 to 5ug/mL, and inoculate the recombinant bacteria L.lactis NZ9000/pNZ8148-Usp45-CRAMP constructed in Example 2 at a volume ratio of 1:100 to 5ug/mL.
  • Chloramphenicol-based GM17 liquid medium incubated overnight at 30°C, inoculate the overnight culture in 10 mL of GM17 liquid medium containing the corresponding antibiotics at a ratio of 1:100, and continue to culture for about 2.5 hours until the bacteria enter the logarithmic growth phase (Using gradient dilution coating to determine the concentration of recombinant bacteria up to the order of 10 12 CFU/mL), optionally, L. lactis NZ9000/pNZ8148-Usp45-CRAMP is cultured to the logarithmic growth phase by adding nisin to induce Cultivate for 2-6h. At this time, the whole culture is used directly as an oral vaccine, or the bacterial cells are collected by centrifugation, and the bacterial cells are used as the main component of the oral vaccine.
  • the oral vaccine containing the recombinant L. lactis NZ9000/pNZ8148-Usp45-CRAMP and the whole culture of L. lactis NZ9000/pMG36e-Usp45-CRAMP prepared in Example 4 was used to prevent acute colitis.
  • 84 male Balb/c mice aged 6 to 8 weeks old were randomly divided into 6 groups, 5 in each group, the first group was a normal saline control, the second group was an acute colitis model, and the third group was L.
  • lactis NZ9000/pMG36e group 4th group L.lactis NZ9000/pNZ8148 group, 5th group L.lactis NZ9000/pMG36e-Usp45-CRAMP group, 6th group L.lactis NZ9000/pNZ8148-Usp45-CRAMP group (i.e. oral vaccine ).
  • oral immunization was performed after 7 days of drinking water with 3% DSS by gavage for 4 consecutive days at a dose of 160 ⁇ L/head. Then the mice were sacrificed for 10 consecutive days, and the intestinal barrier and inflammation-related factors were determined. The results show (Figure 6- Figure 13):
  • the changes of colonic tight junction proteins in each group are: the second group compared with the first group: ZO-1 (p ⁇ 0.01), ZO-2 (p ⁇ 0.0001) and occludin (p ⁇ 0.0001) expression levels are significant Decrease; group 5 compared with group 2: ZO-1 (p ⁇ 0.05), ZO-2 (p ⁇ 0.05) and occludin (p ⁇ 0.05) expression increased significantly; group 6 compared with group 2 :ZO-1 (p ⁇ 0.05), ZO-2 (p ⁇ 0.05) and occludin (p ⁇ 0.05) expression increased significantly; it can be seen that oral vaccine can make the expression of ZO-1, ZO-2 and occludin relative to The colitis group recovered about 50%;
  • the changes in the protein levels of key colonic transcription factors in each group were: the second group compared with the first group: p-ERK/ERK (p ⁇ 0.01), p-p38/p38 (p ⁇ 0.01) and p-NF- kB/NF-kB (p ⁇ 0.01) significantly increased; group 5 compared with group 2: p-p38/p38 (p ⁇ 0.05) and p-NF-kB/NF-kB (p ⁇ 0.05) significantly decreased , P-ERK/ERK (p>0.05) has no significant difference; the 6th group is compared with the second group: p-ERK/ERK (p ⁇ 0.05), p-p38/p38 (p ⁇ 0.05) and p- NF-kB/NF-kB (p ⁇ 0.05) decreased significantly.
  • L.lactis NZ9000/pNZ8148-Usp45-CRAMP was administered to mice by gavage for a period of time.
  • the L. lactis NZ9000/pMG36e-Usp45-CRAMP is colonized in the intestine, and then the treated object is allowed to ingest nisin, or a product with nisin content, to achieve the effect of timing and directional release of CRAMP in the intestine.
  • the CRAMP gene (GGACTTCTCCGCAAAGGTGGGGAGAAGATTGGTGAAAAGCTTAAGAAAATTGGCCAGAAAATTAAGAATTTTTTTCAGAAACTTGTACCTCAGCCAGAG) expressed by it was not codon-optimized and could not promote the intracellular self-cleavage of Usp45 signal peptide and CRAMP gene.
  • CRAMP is secreted extracellularly, and the content of CRAMP protein secreted in the supernatant is low, and the expression product is about 1.5ng/ ⁇ L.
  • the amount of protein is about 40ng/ ⁇ L, which is 27 times higher than the expression amount of the comparative example (1.5ng/ ⁇ L).
  • the expression of CRAMP protein secreted by recombinant bacteria L.lactis NZ9000/pNZ8148-Usp45-CRAMP to the outside of the cell is about 60ng/ ⁇ L , 40 times higher than Comparative Example 1 (1.5ng/ ⁇ L).
  • Codon preference optimization and synthesis of gene sequence According to the sequence of the target gene CRAMP gene and the characteristics of the expression vector pMG36e, the 108bp codon optimized sequence of CRAMP gene is sent to the company for synthesis by artificial synthesis.
  • Xbal-CRAMP-F is an upstream primer containing the restriction site Xbal (TCTAGA) fused with pMG36e and the first sequence of the 5'end of the signal peptide CRAMP.
  • CRAMP-Sph1-R is an upstream primer with restriction site Sph1(GCATGC ) CRAMP gene reverse primer.
  • the optimized and synthesized CRAMP sequence is shown in SEQ ID NO:1; the optimized and synthesized Xbal-CRAMP-F and CRAMP-Sph1-R primer sequences are shown in SEQ ID NO: 11 and SEQ ID NO: 6, respectively.
  • Codon preference optimization and synthesis of gene sequence The nucleotide sequence of CRAMP gene was synthesized according to the method of step (1) in Example 6.
  • Sph1-CRAMP-F is an upstream primer containing the restriction site Sph1 (GCATGC) fused with pMG36e and the first sequence of the 5'end of the signal peptide CRAMP
  • CRAMP-Xbal-R is an upstream primer with restriction site Xbal (TCTAGA )
  • CRAMP gene reverse primers optimized synthesized Sph1-CRAMP-F
  • CRAMP-Xbal-R primer sequences are shown in SEQ ID NO: 12 and SEQ ID NO: 8, respectively.
  • PCR amplification of CRAMP gene fragments use the optimized synthetic CRAMP gene as a template and primers Sph1-CRAMP-F and CRAMP-Xbal-R for PCR amplification.
  • the PCR system and reaction conditions are the same as those in step 1 of Example 1. (2). After the PCR reaction was completed, the product was observed and recovered on a 1.0% agarose gel, and an amplified band of about 102 bp in size was seen, which was consistent with the expected result ( Figure 14). The recovered product will be used as a ligation template to obtain the addition of Sph1- Complete fragment of CRAMP-Xbal sequence.
  • the positive bacterial liquid was subjected to plasmid extraction with a plasmid DNA extraction kit, and double-enzyme digestion identification and sequencing determination were carried out, that is, the recombinant plasmid pNZ8148-CRAMP was obtained.
  • Codon preference optimization and synthesis of gene sequence According to the sequence of the target gene CRAMP gene and the characteristics of the expression vector pMG36e, as well as the signal peptide sequence Usp45 added for the purpose of high-efficiency secretion and expression, artificially synthesized The 243bp codon optimized sequence of Usp45-Linker-CRAMP gene was sent to the company for synthesis.
  • Xbal-Usp45-Linker-CRAMP-F is an upstream primer containing the restriction site Xbal fused with pMG36e and the first sequence of the 5'end of the signal peptide Usp45-Linker-CRAMP, Usp45-Linker-CRAMP-Sph1-R is Signal peptide Usp45-Linker-CRAMP gene reverse primer.
  • primers pNZ1 and pNZ2 for PCR detection and sequencing of recombinant plasmids were also designed, which were designed based on the 70-90bp region of MCS upstream and downstream of empty plasmid pMG36e.
  • the optimized synthesized Usp45-Linker-CRAMP sequence is shown in SEQ ID NO: 10; the optimized synthesized Xbal-Usp45-Linker-CRAMP-F, Usp45-Linker-CRAMP-Sph1-R primer sequence is shown in SEQ ID NO: 5 ⁇ 6 shown.
  • step (3) Construction of the recombinant plasmid pMG36e-Usp45-Linker-CRAMP:
  • the PCR product recovered in step (2) of this example was subjected to double enzyme digestion treatment with Sph1 and Xbal, and a band with a size of about 243 bp was recovered by the gel.
  • the plasmid was subjected to double enzyme digestion according to the method of step 1 (3) in Example 1, and the digested and recovered plasmid fragments were ligated with the Usp45-Linker-CRAMP recovered by the gel.
  • the ligation product was transformed into E. coli MC1061 competent cells, and the transformant was cultured.
  • the recombinant plasmid pNZ8148-CRAMP, the recombinant plasmid pMG36e-Usp45-Linker-CRAMP constructed in Example 8 and the recombinant plasmid pNZ8148-Usp45-Linker-CRAMP constructed in Example 9 were mixed gently; A cold 2mm electric shock cup, quickly give a single pulse, the parameters are set to 2kV, 25F, 200Q, immediately after the electric shock, gently add 1mL ice pre-cooled recovery medium MRS medium, and then suck all the bacterial liquid into a sterile centrifuge.
  • erythromycin MRS plate respectively divide the bacterial solution containing plasmid pNZ8148-CRAMP or plasmid pNZ8148-Usp45-Linker-CRAMP into 10 ⁇ L, 100 ⁇ L, and 900 ⁇ L and evenly spread it on the M17 plate containing 5 ug/mL chloramphenicol. Incubate at 30°C for 1-2 days. Pick a single colony and take the colony for PCR identification. The PCR product was detected by 1% agarose gel electrophoresis. An amplified band of about 243 bp was seen ( Figure 16). The positive recombinant expression bacteria were named L.plantarum FCQHC24L1/pMG36e.
  • Example 11 Induced expression of secretory recombinant Lactobacillus plantarum containing CRAMP gene in vitro
  • Recombinant bacteria L.plantarum FCQHC24L1/pMG36e-CRAMP and recombinant bacteria L.plantarum FCQHC24L1/pMG36e-Usp45-Linker-CRAMP were respectively inoculated into MRS liquid medium containing 5ug/mL erythromycin at a ratio of 1:100.
  • the bacteria L.plantarum FCQHC24L1/pNZ8148-CRAMP and the recombinant bacteria L.plantarum FCQHC24L1/pNZ8148-Usp45-Linker-CRAMP were respectively inoculated in the MRS liquid medium containing 5ug/mL chloramphenicol at a ratio of 1:100.
  • L.plantarum FCQHC24L1/pNZ8148-CRAMP and recombinant bacteria L.plantarum FCQHC24L1/pNZ8148-Usp45-Linker-CRAMP were added to the culture system by adding 40ng/mL nisin to induce 4h, centrifuge at 10000rpm for 5min at 4°C, and collect The culture supernatant was subjected to SDS-PAGE electrophoresis and Western Blot analysis.
  • the recombinant bacteria L.plantarum FCQHC24L1/pMG36e-CRAMP and the recombinant bacteria L.plantarum FCQHC24L1/pMG36e-Usp45-Linker-CRAMP were respectively inoculated into MRS liquid medium at a volume ratio of 1:100, and the recombinant bacteria L.plantarum FCQHC24L1 /pNZ8148-CRAMP and recombinant bacteria L.plantarum FCQHC24L1/pNZ8148-Usp45-Linker-CRAMP were
  • mice 84 male Balb/c mice aged 6-8 weeks were randomly divided into 10 groups, each with 10 mice.
  • the first group is the normal saline control group
  • the second group is the acute colitis model group
  • the third group is the normal saline control group.
  • Group is the L.plantarum FCQHC24L1/pMG36e group
  • the fourth group is the L.plantarum FCQHC24L1/pNZ8148 group
  • the fifth group is the L.plantarum FCQHC24L1/pMG36e-CRAMP group
  • the sixth group is the L.plantarum FCQHC24L1/pNZ8148-CRAMP group.
  • the seventh group is L.plantarum FCQHC24L1/pMG36e-Usp45-Linker-CRAMP group
  • the eighth group is L.plantarum FCQHC24L1/pNZ8148-Usp45-Linker-CRAMP group.
  • the weight of the mice in the first group was 1.084g
  • the second group was 2.85688g
  • the third group was 1.89644g
  • the fourth group was 1.73336g.
  • the fifth group decreased by 0.61004g
  • the sixth group decreased by 0.36816g
  • the seventh group increased by 0.52568g
  • the 8th group increased by 1.23516g;
  • the changes of colonic tight junction proteins in each group are: the second group compared with the first group: ZO-1 (p ⁇ 0.01), ZO-2 (p ⁇ 0.0001) and occludin (p ⁇ 0.0001) expression levels are significant Decrease; Groups 5 to 6 compared with group 2: ZO-1 (p ⁇ 0.05) and occludin (p ⁇ 0.05) expression increased significantly, ZO-2 has no significant difference; Groups 7 to 8 and group 2 Compared: ZO-1 (p ⁇ 0.01), ZO-2 (p ⁇ 0.01) and occludin (p ⁇ 0.05) expression increased significantly;
  • IL-6 p ⁇ 0.0001
  • IL-1 ⁇ IL-1 ⁇
  • TNF- ⁇ p ⁇ 0.0001
  • IL-10 p ⁇ 0.0001
  • the 5th to 8th group compared with the second group: IL-6 (p ⁇ 0.05), IL-1 ⁇ (p ⁇ 0.05), TNF- ⁇ (p ⁇ 0.05)
  • IL-10 p ⁇ 0.05
  • plantarum FCQHC24L1/pMG36e-Usp45-Linker-CRAMP full culture all have a good recovery of the body weight of colitis mice, Restore colon length, reduce colon inflammation, inhibit the secretion of inflammatory cytokines, inhibit the activation of inflammatory signaling pathways, restore the intestinal barrier, thereby treating colitis, and the seventh group L.plantarum FCQHC24L1/pNZ8148-Usp45-Linker-CRAMP group, No. 7
  • the 8 groups of L.plantarum FCQHC24L1/pMG36e-Usp45-Linker-CRAMP group have better treatment effect.
  • Lactobacillus plantarum and nisin were administered to mice by gavage for a period of time.
  • Lactobacillus plantarum FCQHC24L1/pNZ8148-CRAMP or L.plantarum FCQHC24L1/pNZ8148-Usp45-Linker-CRAMP are colonized in the intestines, and then let the treated subjects ingest nisin or products with nisin content to achieve CRAMP in the intestines The effect of timing and directional release in the channel.
  • the CRAMP gene expressed by it has not been codon optimized (GGACTTCTCCGCAAAGGTGGGGAGAAGATTGGTGAAAAGCTTAAGAAAATTGGCCAGAAAATTAAGAATTTTTTTTTCAGAAACTTGTACCTCAGCCAGAG), and there is no Usp45 signal peptide to promote CRAMP extracellular secretion and cannot promote the secretion of CRAMP outside the cell and cannot promote Usp45 signal peptide.
  • CRAMP gene is self-shearing in the cell, and the content of CRAMP protein secreted in the supernatant is low, and the expression product is about 1.5ng/ ⁇ L.
  • Example 11 The recombinant bacteria constructed in Example 11 were compared with the recombinant bacteria of Comparative Example 1 in their ability to express CRAMP, and it was found by ELISA ( Figure 18) that there was no recombinant Lactobacillus plantarum L.plantarum FCQHC24L1/pMG36e-CRAMP and L induced by Usp45.
  • .plantarum FCQHC24L1/pNZ8148-CRAMP The expression level of extracellular CRAMP protein is about 10ng/ ⁇ L, which is 6-8 times higher than that of the recombinant bacterial extracellular protein secretion in Comparative Example 1.; Recombinant Lactobacillus plantarum FCQHC24L1/pNZ8148-Usp45- The CRAMP protein expression level of Linker-CRAMP is about 20ng/ ⁇ L, which is 13 times higher than that of Comparative Example 1 (1.5ng/ ⁇ L); the CRAMP protein of recombinant Lactobacillus plantarum FCQHC24L1/pMG36e-Usp45-Linker-CRAMP The amount of extracellular secretion is higher than 50ng/ ⁇ L, which is 30 times higher than the expression amount of Comparative Example 1 (1.5ng/ ⁇ L) under the same conditions.

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Abstract

提供了一种编码CRAMP蛋白的基因、表达载体、表达乳酸菌及其用途、以及含有该乳酸菌的组合物和疫苗。

Description

高表达抗菌肽cathelicidin基因的乳酸菌 技术领域
本发明涉及高表达抗菌肽cathelicidin基因的乳酸菌，属于基因工程技术领域。
背景技术
抗菌肽是植物、无脊椎动物和脊椎动物(包括人类)等多种宿主的先天免疫和防御的主要成分。Cathelicidins是一类主要的抗菌肽，其特征在于保守的阴离子N-末端前体序列，称为cathelin。cathelin序列的保守性表明该家族的各种成员是从共同祖先基因的复制和修饰进化而来的。CRAMP(Cathelicidin-Related AntiMicrobial Peptide)含有34个氨基酸(GLLRKGGEKIGEKLKKIGQKIKNFFQKLVPQPEQ)，对革兰氏阳性菌和革兰氏阴性菌具有强大的抗菌活性，但对人红细胞没有溶血活性。1mM的CRAMP能够直接导致大肠杆菌内膜的立即透化。抗CRAMP的抗血清在骨髓前体和中性粒细胞中显示出丰富的表达。与天蚕素A类似，50mM CRAMP未显示出对人红细胞的任何溶血活性。此外，研究发现CRAMP对一些致病真菌(Candia alicans和Aspergillus fumigatus)和肿瘤细胞具有强大的抗生素活性。体外研究证实CRAMP能显著抑制幽螺旋杆菌的增殖；而CRAMP缺失会导致小鼠肠胃病情加重，通过表达CRAMP的乳酸杆菌治疗患有肠胃病的CRAMP敲除小鼠则显著缓解效果。
表达于肠道的CRAMP由于肠道屏障的破坏导致CRAMP水平显著下降，导致不能发挥其免疫效果，调节肠道菌群平衡，因此，选择安全无毒，能够在肠道中存活并能表达CRAMP的载体系统，使得CRAMP能够在肠道发挥作用，对调节肠道菌群平衡具有重要意义。
乳酸乳球菌和植物乳杆菌因其表面分子有高黏附性的特征，能使其成功地定植于动物机体的肠道内并成为在肠道内的优势菌群而发挥提高机体免疫力、促进营养物质吸收及维持肠道内菌群平衡等多种功能。所形成的生物学稳固屏障是维持肠道微生物平衡的重要保障。在表达外源基因方面上，乳酸乳球菌表达系统和植物乳杆菌表达系统作为原核表达系统，有以下优势：(1)作为食品级细菌，作为活载体疫苗安全性更高；(2)外源基因能被表达在细胞内，也能被表达展示在细胞表面或被分泌到细胞外；(3)安全、无内毒素，无需纯化表达的外源蛋白，直接同菌体服用；(4)能定植在机体黏膜表面(属于共同黏膜免疫系统)，接种黏膜某一位点便可诱导全身的黏膜免疫反应；最后的也是最重要的，这种免疫方式能帮机体获得的更长久免疫记忆力，以便于长期抵御病原体的侵犯。
发明内容
本发明所要解决的技术问题是克服现有技术中口服CRAMP易被消化道酶降低、无法实现肠道靶向递送CRAMP、最大化实现其局部免疫调节效果的缺陷，提供一种分泌表达CRAMP蛋白重组乳酸菌及其应用。
本发明的提供了一种编码CRAMP蛋白的基因，含有SEQ ID NO：1所示的核苷酸序列。
在一种实施方式中，所述基因还融合有编码Usp45信号肽的核苷酸序列，编码所述Usp45信号肽的核苷酸序列如SEQ ID NO：3所示，连接了信号肽的基因序列如SEQ ID NO:4所示。
在一种实施方式中，编码所述Usp45信号肽的核苷酸序列与CRAMP蛋白的基因序列之间还具有linker序列，其核苷酸序列如SEQ ID NO:10所示。
本发明还提供携带所述基因的载体。
在一种实施方式中，所述载体为pMG36e或pNZ8148。
在一种实施方式中，所述载体为pMG36e-CRAMP、pNZ8148-CRAMP、pNZ8148-Usp45-CRAMP、pMG36e-Usp45-CRAMP pMG36e-Usp45-Linker-CRAMP或pNZ8148-Usp45-Linker-CRAMP。
本发明的第三个目的是提供表达CRAMP蛋白的乳酸菌，其表达SEQ ID NO.1所示的基因，或表达CRAMP蛋白；所述CRAMP蛋白为(a)或(b)：
(a)如SEQ ID NO.2所示的CRAMP蛋白；
(b)在(a)的基础上对一个或几个氨基酸进行缺失、取代或增减且具有抑菌特性的由(a)衍生的蛋白质。
在一种实施方式中，所述乳酸菌为植物乳杆菌或乳酸乳球菌。
在一种实施方式中，所述植物乳杆菌以植物乳杆菌FCQHC24L1为宿主。
在一种实施方式中，所述植物乳杆菌FCQHC24L1公开于2019年题为《不同生态位的植物乳杆菌的基因组及主要生理特性差异研究》的论文中，申请人承诺自申请日起20年内向合法途径下实施本发明的公众发放该菌株。
在一种实施方式中，所述植物乳杆菌以pMG36e或pNZ8148为载体表达所述编码CRAMP蛋白的基因。
在一种实施方式中，所述植物乳杆菌还引入了Usp45信号肽促进CRAMP蛋白的表达。
在一种实施方式中，所述Usp45通过linker与CRAMP基因连接，所述linker包含2个或更多个选自Gly和Ser的氨基酸残基。
在一种实施方式中，所述linker的氨基酸序列为GGGGS；编码所述linker的核苷酸序列如SEQ ID NO.9所示。
在一种实施方式中，所述乳酸乳球菌以乳酸乳球菌NZ9000为宿主。
在一种实施方式中，所述乳酸乳球菌以pMG36e或pNZ8148为载体。
在一种实施方式中，所述乳酸乳球菌还引入了Usp45信号肽促进CRAMP蛋白的表达。
本发明还提供一种构建所述植物乳杆菌的方法，是将SEQ ID NO.1所示的编码CRAMP蛋白的基因与载体连接，再转化至植物乳杆菌细胞中；所述载体为pMG36e或pNZ8148。
在一种实施方式中，所述pMG36e或pNZ8148上连接有usp45信号肽。
在一种实施方式中，所述Usp45通过linker与CRAMP基因连接，所述linker的核苷酸序列为GGCGGTGGCGGCAGC。
在一种实施方式中，所述方法包括如下步骤：
(1)合成SEQ ID NO.1所示的编码CRAMP蛋白的基因；
(2)将步骤(1)合成的基因连接至pMG36e中，获得重组质粒pMG36e-CRAMP；采用电转化法将pMG36e-CRAMP重组质粒导入植物乳杆菌L.plantarum FCQHC24L1中，获得重组植物乳杆菌L.plantarum FCQHC24L1/pMG36e-CRAMP。
在一种实施方式中，所述方法包括如下步骤：
(1)合成SEQ ID NO.10所示的编码CRAMP蛋白的基因；
(2)将步骤(1)合成的基因连接至pMG36e中，获得重组质粒pMG36e-Usp45-Linker-CRAMP；采用电转化法将pMG36e-Usp45-Linker-CRAMP重组质粒导入植物乳杆菌L.plantarum FCQHC24L1中，获得重组植物乳杆菌L.plantarum FCQHC24L1/pMG36e-Usp45-Linker-CRAMP。
在一种实施方式中，所述电转化法的具体操作为：取L.plantarum FCQHC24L1的感受态细胞，加入重组质粒，混匀并转入电转化杯，电击后加入恢复培养基MRS培养基，冰浴后静置培养，平板筛选高拷贝转化子。
本发明还提供一种构建上述任一所述重组乳酸乳球菌的方法，是将SEQ ID NO.1所示的编码CRAMP蛋白的基因与载体连接，再转化至乳酸乳球菌细胞中；所述载体为pMG36e或pNZ8148。
在一种实施方式中，所述pMG36e或pNZ8148上连接有usp45信号肽。
在一种实施方式中，所述方法包括如下步骤：
(1)合成SEQ ID NO.1所示的编码CRAMP蛋白的基因；
(2)将步骤(1)合成的基因连接至pMG36e中，获得重组质粒pMG36e-Usp45-CRAMP；采用电转化法将pMG36e-Usp45-CRAMP重组质粒导入乳酸乳球菌L.lactis NZ9000中，获得重组乳酸乳球菌L.lactis NZ9000/pMG36e-Usp45-CRAMP。
在一种实施方式中，所述方法包括如下步骤：
(1)合成SEQ ID NO.4所示的编码CRAMP蛋白的基因；
(2)将步骤(1)合成的基因连接至pNZ8148中，获得重组质粒pNZ8148-Usp45-CRAMP；采用电转化法将pNZ8148-Usp45-CRAMP重组质粒导入乳酸乳球菌L.lactis NZ9000中，获得重组乳酸乳球菌L.lactis NZ9000/pNZ8148-Usp45-CRAMP。
在一种实施方式中，所述电转化法为：取L.lactis NZ9000的感受态细胞，加入重组质粒，混匀并转入电转化杯，电击后加入恢复培养基，冰浴后静置培养，平板筛选高拷贝转化子。
本发明还提供一种食用或药用组合物，该组合物中含有所述乳酸乳球菌和/或所述植物乳杆菌。
在一种实施方式中，所述乳酸乳球菌和/或植物乳杆菌在组合物中的含量≥1×10 5CFU/mL或1×10 5CFU/g。
在一种实施方式中，所述组合物为药物，含有药学上可接受的载体。
本发明的第四个目的是提供所述乳酸乳球菌和/或植物乳杆菌在制备疫苗中的应用。
在一种实施方式中，所述疫苗为预防急性结肠炎的口服疫苗。
在一种实施方式中，所述应用是培养所述乳酸乳球菌进行培养，再以乳酸乳球菌的全培养物作为口服疫苗。
在一种实施方式中，所述应用包括如下步骤：将乳球菌/pMG36e-Usp45-CRAMP接种于GM17液体培养基静置培养过夜，以一定比例转接于含GM17液体培养基，继续培养至细菌进入对数生长期，全培养物直接作为口服疫苗。
在一种实施方式中，所述静置培养的温度为28～30℃。
在一种实施方式中，所述转接是将L.lactisNZ9000/pMG36e-Usp45-CRAMP以体积比1:100 的比例接种于GM17培养基。
在一种实施方式中，所述对数生长期的细菌培养液的OD值为0.4～0.6。
在一种实施方式中，所述应用包括如下步骤：将L.lactis NZ9000/pMG36e-Usp45-CRAMP重组表达菌以1:100的比例接种于含有GM17培养基，继续培养2～3h至细菌进入对数生长期(OD 600＝0.4～0.6)；培养至重组菌的浓度达10 12CFU/mL数量级，收集诱导后的全培养物作为口服疫苗。
在一种实施方式中，所述应用是培养所述植物乳杆菌进行培养，再以植物乳杆菌的全培养物作为口服疫苗。
在一种实施方式中，所述应用是培养所述重组植物乳杆菌，再以植物乳杆菌的全培养物作为口服疫苗或口服疫苗的主要成分。
在一种实施方式中，所述应用包括如下步骤：将重组植物乳杆菌/pMG36e-Usp45-Linker-CRAMP接种于MRS液体培养基静置培养过夜，以一定比例转接于含MRS液体培养基，继续培养至细菌进入对数生长期，全培养物直接作为口服疫苗。
在一种实施方式中，所述静置培养的温度为28～30℃。
在一种实施方式中，所述转接是将L.plantarum FCQHC24L1/pMG36e-Usp45-Linker-CRAMP以体积比(1～10):100的比例接种于MRS培养基。
在一种实施方式中，所述对数生长期的细菌培养液的OD值为0.4～0.6。
在一种实施方式中，所述应用包括如下步骤：将L.plantarum FCQHC24L1/pMG36e-Usp45-Linker-CRAMP重组菌以(1～10):100的体积比接种于含有MRS的培养基，继续培养2～3h至细菌进入对数生长期(OD 600＝0.4～0.6)；培养至重组菌的浓度达10 12CFU/mL数量级，收集诱导后的全培养物作为口服疫苗。
本发明还要求保护所述植物乳杆菌在制备预防或治疗急性结肠炎的药物中的应用。
在一种实施方式中，所述药物与nisin联合使用。
在一种实施方式中，所述药物摄入肠道后，再摄入nisin或含nisin的药物，达到定向释放CRAMP、提高CRAMP在肠道内分泌量的效果。
本发明还要求保护所述乳酸乳球菌和/或植物乳杆菌在制备可引入肠道的产品中的应用；所述产品具有如下至少一种功能：
(a)抑制肠道炎症；
(b)重塑肠黏膜屏障；
(c)改善肠黏膜通透性；
(d)预防和治疗肠道炎症以及肠道炎症引起的疾病。
本发明还要求保护所述乳酸乳球菌和/或植物乳杆菌在制备预防或治疗炎症性肠病、腹泻或肠道稳态失衡引起的或相关的疾病的药物中的应用，所述肠道稳态失衡引起的或相关的疾病包括但不限于肝脏疾病、代谢内分泌疾病、循环系统疾病等，例如糖尿病、胰腺炎或代谢综合症。
本发明还要求保护所述乳酸乳球菌和/或植物乳杆菌在制备预防或治疗急性结肠炎的药 物中的应用。
有益效果：(1)本发明提供了一段优化的编码CRAMP蛋白的基因，并添加Usp45信号肽促进CRAMP基因的分泌表达，使CRAMP蛋白在乳酸乳球菌中的表达量可达40ng/μL，在植物乳杆菌中的表达量可达20ng/μL甚至更高。加上乳酸乳球菌、植物乳杆菌作为益生菌的益生特性，使得此乳酸菌表达系统成为一个食品级表达系统，可以连同菌体一起服用。
(3)本发明将构建的表达CRAMP蛋白的乳酸乳球菌、植物乳杆菌用于制备针对肠道菌群紊乱调节的疫苗，对肠道菌群的调节以及肠道免疫应答与维持上有优势，全培养物可直接作为口服疫苗刺激小鼠并引起较强的细胞免疫应答，该重组乳酸乳球菌可以作为一种具有良好产业前景的新型口服疫苗产品，对减轻肠道炎症起到积极的作用，对促进肠道健康发展具有重要的实践意义。
(4)本发明制备得到的含重组植物乳杆菌和/或乳酸乳球菌的疫苗可针对性调节肠道菌群紊乱，有助于肠道菌群的调节以及肠道免疫应答与维持，经动物实验证实，本发明制备的口服疫苗可刺激小鼠并引起较强的细胞免疫应答，可以作为一种具有良好产业前景的新型口服疫苗产品，对减轻肠道炎症起到积极的作用，对促进肠道健康发展具有重要的实践意义。
附图说明
图1为CRAMP和Usp45-CRAMP基因片段的PCR扩增结果；1为DL2000 DNA Marker；2-3为Usp45-CRAMP基因片段的PCR扩增；
图2为重组大肠杆菌E.coli MC1061/pMG36e-Usp45-CRAMP组的PCR鉴定结果，1为DL2000 DNA Marker，2为重组大肠杆菌E.coli MC1061/pMG36e-Usp45-CRAMP组的PCR鉴定，3为重组大肠杆菌E.coli MC1061/pNZ8148-Usp45-CRAMP组的PCR鉴定；
图3为重组乳酸乳球菌L.lactis NZ9000/pMG36e-Usp45-CRAMP组、L.lactis NZ9000/pNZ8148-Usp45-CRAMP组的PCR鉴定结果；1为DL2000 DNA Marker；2为L.lactis NZ9000/pMG36e-Usp45-CRAMP组中CRAMP的PCR鉴定；3为L.lactisNZ9000/pNZ8148-Usp45-CRAMP组的PCR鉴定；
图4为重组乳酸乳球菌中CRAMP的免疫印迹结果；1为蛋白Marker；2为L.lactisNZ9000/pMG36e-Usp45-CRAMP组上清中CRAMP的表达量；3为L.lactisNZ9000/pMG36e-Usp45-CRAMP组菌体中CRAMP的表达量；4为L.lactisNZ9000/pNZ8148-Usp45-CRAMP组上清中CRAMP的表达量；5为L.lactisNZ9000/pNZ8148-Usp45-CRAMP组菌体中CRAMP的表达量；
图5为重组乳酸乳球菌CRAMP的ELISA结果；
图6为前人研究大肠杆菌中CRAMP的表达情况；1为大肠杆菌裂解液；2为大肠杆菌裂解液上清；3为大肠杆菌裂解液沉淀；4为洗脱柱上的GST-CRAMP洗脱缓冲液；5为蛋白Marker；
图7为结肠炎模型建立期间各组小鼠体重的变化情况；
图8为各组(A)小鼠结肠长度比较及(B)各组统计图；
图9为结肠炎临床指标评分；
图10为结肠组织学(A)病理学形态观察及(B)评分；
图11为qPCR测定肠道紧密连接蛋白(A)ZO-1、(B)ZO-2和(C)occludin的变化情况；
图12为qPCR测定炎症细胞因子(A)IL-6、(B)IL-1β、(C)TNF-α和(D)IL-10的表达情况；
图13为Western blot测定炎信号通路关键转录因子的磷酸化水平变化情况：(A)Western blot实验结果p-ERK、ERK、p-p38、p38、p-NF-kB和NF-kB条带图,(B)p-ERK/ERK灰度分析统计图，(C)p-p38/p38灰度分析统计图,(D)p-NF-kB/NF-kB灰度分析统计图；
图14为CRAMP和Usp45-Linker-CRAMP基因片段的PCR扩增结果；1为DL2000 DNA Marker；2-5为CRAMP基因片段的PCR扩增，6-9为Usp45-Linker-CRAMP基因片段的PCR扩增；
图15为重组大肠杆菌E.coli MC1061/pMG36e-Usp45-Linker-CRAMP的PCR鉴定结果，1为DL2000 DNA Marker，2-3为重组大肠杆菌E.coli MC1061/pMG36e-CRAMP的PCR鉴定，4-5为重组大肠杆菌E.coli MC1061/pMG36e-Usp45-Linker-CRAMP的PCR鉴定；
图16为重组植物乳杆菌L.plantarum FCQHC24L1/pMG36e-CRAMP、L.plantarum FCQHC24L1/pNZ81848-CRAMP、L.plantarum FCQHC24L1/pMG36e-Usp45-Linker-CRAMP、L.plantarum FCQHC24L1 L1/pNZ8148-Usp45-Linker-CRAMP的PCR鉴定结果；1为DL2000 DNA Marker；2为L.plantarum FCQHC24L1/pMG36e-CRAMP中CRAMP的PCR鉴定；3为L.plantarum FCQHC24L1/pNZ8148-CRAMP中CRAMP的PCR鉴定；4为L.plantarum FCQHC24L1/pMG36e-Usp45-Linker-CRAMP中Usp45-Linker-CRAMP的PCR鉴定；5为L.plantarum FCQHC24L1/pNZ8148-Usp45-Linker-CRAMP的PCR鉴定；
图17为重组植物乳杆菌中CRAMP的免疫印迹结果；1为蛋白Marker；2为L.plantarum FCQHC24L1/pMG36e-CRAMP菌体中CRAMP的表达量；3为L.plantarum FCQHC24L1/pMG36e-CRAMP上清中CRAMP的表达量；4为L.plantarum FCQHC24L1/pNZ81848-CRAMP菌体中CRAMP的表达量；5为L.plantarum FCQHC24L1/pNZ81848-CRAMP上清中CRAMP的表达量；6为L.plantarum FCQHC24L1/pMG36e-Usp45-Linker-CRAMP菌体中CRAMP的表达量；7为L.plantarum FCQHC24L1/pMG36e-Usp45-Linker-CRAMP上清中CRAMP的表达量；8为L.plantarum FCQHC24L1/pNZ8148-Usp45-Linker-CRAMP菌体中CRAMP的表达量；9为L.plantarum FCQHC24L1/pNZ8148-Usp45-Linker-CRAMP上清中CRAMP的表达量；
图18为重组植物乳杆菌CRAMP的ELISA结果；
图19为现有技术用大肠杆菌表达CRAMP的表达情况；1为大肠杆菌裂解液；2为大肠杆菌裂解液上清；3为大肠杆菌裂解液沉淀；4为洗脱柱上的GST-CRAMP洗脱缓冲液；5为蛋白Marker；
图20为结肠炎模型建立期间各组小鼠体重的变化情况；
图21为各组小鼠结肠长度比较(A)及各组长度统计图(B)；
图22为结肠炎临床指标评分；
图23为结肠组织学病理学形态观察(A)及组织病理学评分(B)；
图24为qPCR测定肠道紧密连接蛋白(A)ZO-1、(B)ZO-2和(C)occludin的变化情况；
图25为qPCR测定炎症细胞因子(A)IL-6、(B)IL-1β、(C)TNF-α和(D)IL-10的表达情况；
图26为Western blot测定炎信号通路关键转录因子的磷酸化水平变化情况：(A)Western blot实验结果p-ERK、ERK、p-p38、p38、p-NF-kB和NF-kB条带图，(B)p-ERK/ERK灰度分析统计图，(C)p-p38/p38灰度分析统计图，(D)p-NF-kB/NF-kB灰度分析统计图，(E)CRAMP/β-actin灰度分析统计图。
具体实施方式
下面结合说明书附图和具体实施例，进一步阐述本发明。这些实施例仅用于说明本发明而不用于限制本发明的范围。下例实施例中未注明具体条件的实验方法，通常按照本领域常规条件或按照制造厂商建议的条件。除非另行定义，文中所使用的所有专业与科学用语与本领域技术人员熟悉的意义相同。
小鼠体重、结肠长度、DAI评分参照文献《Curcumin Prevents the Development of Dextran Sulfate Sodium(DSS)-Induced Experimental Colitis》；
ZO-1、ZO-2、occludin、IL-10、IL-1β、TNF-α和IL-6的qPCR测定方法参照文献《Neutralization of IL-6 and TNF-αameliorates intestinal permeability in DSS-induced colitis》的方法进行；
p-ERK、ERK、pp38、p38、p-NF-kB、NF-kB、CRAMP用Western Blot进行测定，方法参考文献《Dietary squalene supplementation improves DSS-induced acute colitis by downregulating p38 MAPK and NFkB signaling pathways》。
实施例1 重组菌L.lactis NZ9000/pMG36e-Usp45-CRAMP的构建
1、重组质粒pMG36e-Usp45-CRAMP的构建
(1)基因序列的密码子偏好性优化与合成：根据目的基因CRAMP基因的序列和表达载体pMG36e的特点，以及为达到高效分泌表达的目的而增加的信号肽序列Usp45，采用人工合成的方法将Usp45-CRAMP基因的228bp的密码子优化序列送公司进行合成。Xbal-Usp45-CRAMP-F为含有与pMG36e融合表达的酶切位点Xbal和信号肽Usp45-CRAMP的5'端最初一段序列的上游引物，Usp45-CRAMP-Sph1-R为信号肽Usp45-CRAMP基因反向引物。同时还设计了用于重组质粒的PCR检测和测序的引物pNZ1及pNZ2，是以pMG36e空质粒的MCS上游和下游70～90bp左右的区域为依据设计的。优化合成的Usp45-CRAMP序列如SEQ ID NO：4所示；优化合成的Xbal-Usp45-CRAMP-F，Usp45-CRAMP-Sph1-R引物序列分别如SEQ ID NO：5～6所示。
(2)Usp45-CRAMP基因片段的PCR扩增：以含优化合成的Usp45-CRAMP基因为模板，加入高保真DNA聚合酶KOD-Plus-(1.0U/μL)1μL,0.3μM的引物Xbal-Usp45-CRAMP-F，Usp45-CRAMP-Sph1-R各1.5μL，模板1.5μL，25mM MgSO 4 2μL，2mM dNTPs 5μL，10×Buffer for KOD-Plus-5μL，用ddH 2O补至50μL，PCR反应程序为：94℃预变性5min；94℃变性30s，55℃退火30s，72℃延伸1min，35个循环；72℃后延伸10min。PCR反应完成，将产物进 行1.0％琼脂糖凝胶观察与回收，可见大小约228bp的扩增条带，与预期结果一致(如图1)，回收的产物将作为连接模板用于获得添加Usp45-CRAMP序列的完整片段。
(3)重组质粒pMG36e-Usp45-CRAMP的构建：将步骤(2)中回收的PCR产物用Sph1、Xbal进行双酶切处理，胶回收大小约228bp的条带；以同样的方法对pMG36e空质粒进行双酶切，胶回收大小约3600bp的条带。分别取4μL双酶切后胶回收的Usp45-CRAMP基因片段和1μL双酶切后胶回收的pMG36e空质粒，将Usp45-CRAMP和pMG36e按照摩尔比6：1添加，并加入10×ligation buffer 2μL，T4 DNA Ligase(350U/μL)1μL，用ddH 2O补至20μL，混匀后置于4℃条件下连接过夜，将连接产物转化E.coli MC1061感受态细胞，在含有5μg/mL红霉素(Erythromycin,Er)的LB琼脂培养板中，37℃培养两天，然后挑取单个菌落进行PCR鉴定。PCR鉴定以待检菌落为模板，加入高保真DNA聚合酶KOD-Plus-(1.0U/ul)1μL,0.3μM的引物Xbal-Usp45-CRAMP-F，Usp45-CRAMP-Sph1-R各1.5μL，模板1.5μL，25mM MgSO4 2μL，2mM dNTPs 5μL，10x Buffer for KOD-Plus-5μL，用ddH20补至50μL，PCR反应程序为：94℃预变性5min；94℃变性30s，55℃退火30s，72℃延伸1min，35个循环；72℃后延伸10min。PCR反应完成，将产物进行1.0％琼脂糖凝胶观察与回收，可见大小约228bp的扩增条带，与预期结果一致(如图2)，对检测阳性的菌液用质粒DNA抽提试剂盒进行质粒抽提，并进行双酶切鉴定和测序测定，即为获得了重组质粒pMG36e-Usp45-CRAMP。
(4)乳酸乳球菌电转化感受态细胞的制备：将冻存的L.lactis NZ9000乳酸乳球菌划GM17平板复苏，挑取单个菌落在GM17液体培养辈中30℃培养过夜，以1：100的比例接入50mL新的GM17液体培养基30℃培养，监测OD 500至0.3-0.4，迅速置冰上冷却，4℃6000×g离心20min，弃上清；用50mL预冷的0.5M蔗糖、10％甘油溶液重悬菌体，4℃6000×g离心20min，弃上清；用25mL预冷的0.5M蔗糖、10％甘油、50mM EDTA溶液重悬菌体，4℃6000×g离心15min，弃上清；再用15mL预冷的0.5M蔗糖、10％甘油溶液重悬菌体，4℃6000×g离心15min，弃上清；最后用500μL预冷的0.5M蔗糖、10％甘油溶液重悬菌体，即为乳酸乳球菌感受态细胞，50μL每管分装，-80℃保存备用。
(5)乳酸乳球菌的电击转化及转化子的PCR鉴定：各取50μL L.lactis NZ9000感受态细胞，冰浴上融解，加入1μL步骤1构建的重组质粒pMG36e-Usp45-CRAMP，轻轻混匀；分别将以上混合物转入冰预冷的2mm电激杯，迅速给予一个单脉冲，参数设置为2kV，25F，200Q，电击后立即轻柔加入1mL冰预冷的恢复培养基GM17培养基，再分别将菌液全部吸入一灭菌离心管，盖紧管盖，冰浴5min后30℃静置培养2h；将含质粒pMG36e-Usp45-CRAMP的菌液分为10μL、l00μL、900μL均匀涂布于含有5ug/mL红霉素的GM17平板，30℃静置培养1-2天。挑取单个菌落，取菌落进行PCR鉴定，具体操作过程如前述步骤(2)所述，区别在于将模板换成待检重组乳酸乳球菌菌液，PCR产物用1％琼脂糖凝胶电泳进行检测，可见约228bp的扩增条带(图3)，阳性重组表达菌命名为L.lactis NZ9000/pMG36e-Usp45-CRAMP。
实施例2 重组菌L.lactis NZ9000/pNZ8148-Usp45-CRAMP的构建
(1)基因序列的密码子偏好性优化与合成：按照实施例1步骤1(1)的方法优化合成如SEQ ID NO：4所示Usp45-CRAMP基因的序列；Sph1-Usp45-CRAMP-F为含有与pNZ8148 融合表达的酶切位点Xbal和信号肽Usp45-CRAMP的5'端最初一段序列的上游引物，Usp45-CRAMP-Xbal-R为信号肽Usp45-CRAMP基因反向引物。优化合成的Sph1-Usp45-CRAMP-F，Usp45-CRAMP-Xbal-R引物序列分别如SEQ ID NO：7～8所示。
(2)Usp45-CRAMP基因片段的PCR扩增：具体实施方式同实施例1步骤1(2)，区别在于，引物替换为Sph1-Usp45-CRAMP-F和Usp45-CRAMP-Xbal-R，扩增、回收获得大小约228bp的扩增条带(如图1)。
(3)重组质粒pNZ8148-Usp45-CRAMP的构建：具体实施方式同实施例1步骤1(3)，区别在于，将质粒pMG36e替换为pNZ8148，即采用Sph1、Xbal进行双酶切处理pNZ8148空质粒，并将双酶切、胶回收后的质粒片段与双酶切后胶回收的Usp45-CRAMP基因连接；采用引物Sph1-Usp45-CRAMP-F，Usp45-CRAMP-Xbal-R进行菌落PCR。PCR反应完成，将产物进行1.0％琼脂糖凝胶观察与回收，可见大小约228bp的扩增条带(如图2)，对检测阳性的菌液用质粒DNA抽提试剂盒进行质粒抽提，并进行双酶切鉴定和测序测定，即为获得了重组质粒pMG36e-Usp45-CRAMP。
(4)乳酸乳球菌电转化感受态细胞的制备：具体操作步骤同实施例1的步骤1(4)。
(5)乳酸乳球菌的电击转化及转化子的PCR鉴定：具体操作步骤同实施例1的步骤1(5)，区别在于，向L.lactis NZ9000感受态细胞中加入重组质粒pNZ8148-Usp45-CRAMP重组质粒。挑取转化、培养后的单个菌落进行PCR鉴定，具体操作过程如本实施例步骤(2)所述，PCR产物用1％琼脂糖凝胶电泳进行检测，可见约228bp的扩增条带(图3)，阳性重组表达菌命名为L.lactis NZ9000/pNZ8148-Usp45-CRAMP。
实施例3 含CRAMP基因的分泌型重组乳酸乳球菌体外诱导表达
将实施例1构建的重组菌L.lactis NZ9000/pMG36e-Usp45-CRAMP重组表达菌以1:100的比例分别接种于含有5ug/mL红霉素的GM17液体培养基，将实施例2构建的重组菌L.lactis NZ9000/pNZ8148-Usp45-CRAMP以1:100的体积比接种于含有5ug/mL氯霉素的GM17液体培养基，30℃静置培养过夜；将过夜培养物以1:50的比例接种于10mL含有对应抗生素的液体培养基，继续培养约2.5h至细菌进入对数生长期(OD 500＝0.4～0.6)，向L.lactis NZ9000/pNZ8148-Usp45-CRAMP培养体系中加入40ng/mL的乳酸链球菌素(nisin)诱导4h，4℃10000rpm离心5min，收集培养上清，经SDS-PAGE电泳并进行Western Blot分析，结果显示，L.lactis NZ9000/pNZ8148-Usp45-CRAMP和L.lactis NZ9000/pMG36e-Usp45-CRAMP培养上清中检测到17KDa的目的条带(如图4)，表明目的基因已经得到分泌表达。
实施例4 乳酸乳球菌制备疫苗中的应用
L.lactis NZ9000/pNZ8148-Usp45-CRAMP和L.lactis NZ9000/pMG36e-Usp45-CRAMP重组乳酸乳球菌口服疫苗的制备：将实施例1构建的重组菌L.lactis NZ9000/pMG36e-Usp45-CRAMP以1：100的体积比分别接种于5ug/mL红霉素GM17液体培养基，将实施例2构建的重组菌L.lactis NZ9000/pNZ8148-Usp45-CRAMP以1：100的体积比分别接种于5ug/mL氯霉素的GM17液体培养基，30℃静置培养过夜，将过夜培养物以1:100的比例接种于10mL含有相应抗生素的GM17液体培养基，继续培养约2.5h至细菌进入对数生长期(采用梯度稀释涂板测定重组菌的浓度达10 12CFU/mL数量级)，可选地，将乳酸乳球 菌L.lactis NZ9000/pNZ8148-Usp45-CRAMP，在培养至对数生长期后加入nisin诱导培养2～6h。此时的全培养物直接作为口服疫苗使用，或离心收集菌体，将菌体作为口服疫苗的主要成分。
实施例5 乳酸乳球菌在预防急性结肠炎中的应用
将实施例4制备的含重组乳酸乳球菌L.lactis NZ9000/pNZ8148-Usp45-CRAMP和L.lactis NZ9000/pMG36e-Usp45-CRAMP全培养物的口服疫苗用于预防急性结肠炎。将84只6～8周龄周龄的雄性Balb/c小鼠随机分为6组饲养，每组5只，第1组为生理盐水对照，第2组急性结肠炎模型，第3组L.lactis NZ9000/pMG36e组，第4组L.lactis NZ9000/pNZ8148组，第5组L.lactis NZ9000/pMG36e-Usp45-CRAMP组，第6组L.lactis NZ9000/pNZ8148-Usp45-CRAMP组(即口服疫苗)。预饲一周后，采用灌胃的方式在3％DSS饮水7天后进行口服免疫，连续免疫4天，剂量为160μL/只。然后连续10天处死小鼠，对肠道屏障及炎症相关因子测定。结果显示(图6-图13)：
(1)各组小鼠第10天的小鼠比第7天平均体重相比：第1组增重1.084g，第2组下降2.19688g，第3组下降1.984g，第4组下降1.658g，第5组增重0.948g，第6组增重0.732g；
(2)各组第10天的平均结肠长度为：第1组为9.66厘米，第2组为5.32厘米，第3组为6.43厘米，第4组为6.41厘米，第5组为6.88厘米，第6组为6.86厘米；
(3)各组在第10天时DAI评分结果为：第1组0.2，第2组为7.2，第3组为6.6，第4组为6.4，第5组为4.2，第6组4.0；
(4)各组结肠形态学评分结果为：第1组0.2，第2组为3.8，第3组为3.0，第4组为3.4，第5组为2.4，第6组2.4；
(5)各组结肠紧密连接蛋白变化情况为：第2组与第1组相比：ZO-1(p<0.01)、ZO-2(p<0.0001)和occludin(p<0.0001)表达量显著下降；第5组与第2组相比：ZO-1(p<0.05)、ZO-2(p<0.05)和occludin(p<0.05)表达量显著增加；第6组与第2组相比：ZO-1(p<0.05)、ZO-2(p<0.05)和occludin(p<0.05)表达量显著增加；可见，口服疫苗可使ZO-1、ZO-2和occludin的表达量相对于结肠炎组恢复约50％；
(6)各组结肠炎症因子变化情况为：第2组与第1组相比：IL-6(p<0.0001)、IL-1β(p<0.0001)、TNF-α(p<0.0001)显著上升，IL-10(p<0.0001)显著下降；第5组与第2组相比：IL-6(p<0.05)、IL-1β(p<0.05)、TNF-α(p<0.05)显著下降，IL-10(p<0.05)显著上升；第6组与第2组相比：IL-6(p<0.05)、IL-1β(p<0.05)、TNF-α(p<0.01)显著下降，IL-10(p<0.05)显著上升；可见，口服疫苗可使炎症因子IL-6、IL-1β、TNF-α水平相对于结肠炎模型组降低30～50％，并使IL-10升高至少一倍；
(7)各组结肠关键转录因子蛋白水平变化为：第2组与第1组相比：p-ERK/ERK(p<0.01)、p-p38/p38(p<0.01)和p-NF-kB/NF-kB(p<0.01)显著增加；第5组和第2组相比：p-p38/p38(p＜0.05)和p-NF-kB/NF-kB(p＜0.05)显著降低，p-ERK/ERK(p＞0.05)无显著差异性；第6组和第2组相比：p-ERK/ERK(p<0.05)、p-p38/p38(p<0.05)和p-NF-kB/NF-kB(p<0.05)显著降低。
以上结果表明，应用含L.lactis NZ9000/pMG36e-Usp45-CRAMP或L.lactis NZ9000/pNZ8148-Usp45-CRAMP的口服疫苗能够很好的恢复肠道屏障，减轻炎症细胞浸润， 抑制炎症细胞因子分泌，具有很好的恢复效果。
发明人还尝试将L.lactis NZ9000/pNZ8148-Usp45-CRAMP与nisin联合使用，例如将未经nisin诱导的乳酸乳球菌L.lactis NZ9000/pMG36e-Usp45-CRAMP对小鼠灌胃处理一段时间后，使乳酸乳球菌L.lactis NZ9000/pMG36e-Usp45-CRAMP在肠道内定植，再让处理对象摄入nisin，或内容物为nisin的产品，可达到CRAMP在肠道内定时、定向释放的效果。
对比例1 含CRAMP基因的分泌型重组乳酸乳球菌
以现有技术中含CRAMP基因的分泌型重组乳酸乳球菌作为对照，其表达的CRAMP基因(GGACTTCTCCGCAAAGGTGGGGAGAAGATTGGTGAAAAGCTTAAGAAAATTGGCCAGAAAATTAAGAATTTTTTTCAGAAACTTGTACCTCAGCCAGAG)未经过密码子优化，不能促进Usp45信号肽和CRAMP基因在胞内自剪切作用，不能促进CRAMP在细胞外分泌，上清中分泌的CRAMP蛋白含量低，表达产物约为1.5ng/μL。
经ELISA检测比较对比例1与实施例1～2构建的重组菌表达CRAMP的能力，结果显示(图5)，重组菌L.lactis NZ9000/pMG36e-Usp45-CRAMP和重组菌L.lactis NZ9000/pNZ8148-Usp45-CRAMP菌体的CRAMP蛋白表达量约为20ng/μL，比现有技术相比(1.5ng/μL)高13倍，重组菌L.lactis NZ9000/pMG36e-Usp45-CRAMP分泌至胞外的蛋白量约为40ng/μL，比对比例的表达量(1.5ng/μL)高27倍，重组菌L.lactis NZ9000/pNZ8148-Usp45-CRAMP分泌至胞外的CRAMP蛋白表达量约为60ng/μL，比对比例1(1.5ng/μL)高40倍。
实施例6 重组质粒pMG36e-CRAMP的构建
(1)基因序列的密码子偏好性优化与合成：根据目的基因CRAMP基因的序列和表达载体pMG36e的特点，采用人工合成的方法将CRAMP基因的108bp的密码子优化序列送公司进行合成。Xbal-CRAMP-F为含有与pMG36e融合表达的酶切位点Xbal(TCTAGA)和信号肽CRAMP的5'端最初一段序列的上游引物，CRAMP-Sph1-R为带有酶切位点Sph1(GCATGC)的CRAMP基因反向引物。优化合成的CRAMP序列如SEQ ID NO：1所示；优化合成的Xbal-CRAMP-F，CRAMP-Sph1-R引物序列分别如SEQ ID NO：11和SEQ ID NO:6所示。
(2)CRAMP基因片段的PCR扩增：具体操作同实施例1的步骤1(2)；
(3)重组质粒pMG36e-CRAMP的构建：具体操作同实施例1的步骤1(3)，区别在于，双酶切处理后，胶回收大小约102bp的条带；采用引物Xbal-CRAMP-F，CRAMP-Sph1-R进行菌落PCR。PCR反应完成，将产物进行1.0％琼脂糖凝胶观察与回收，可见大小约102bp的扩增条带(图15)，与预期结果一致，对检测阳性的菌液用质粒DNA抽提试剂盒进行质粒抽提，并进行双酶切鉴定和测序测定，即为获得了重组质粒pMG36e-CRAMP。
实施例7 重组质粒pNZ8148-CRAMP的构建
(1)基因序列的密码子偏好性优化与合成：按照实施例6步骤(1)的方法合成CRAMP基因的核苷酸序列。Sph1-CRAMP-F为含有与pMG36e融合表达的酶切位点Sph1(GCATGC)和信号肽CRAMP的5'端最初一段序列的上游引物，CRAMP-Xbal-R为带有酶切位点Xbal (TCTAGA)的CRAMP基因反向引物，优化合成的Sph1-CRAMP-F，CRAMP-Xbal-R引物序列分别如SEQ ID NO：12和SEQ ID NO：8所示。
(2)CRAMP基因片段的PCR扩增：以含优化合成的CRAMP基因为模板，以引物Sph1-CRAMP-F、CRAMP-Xbal-R进行PCR扩增，PCR体系和反应条件同实施例1步骤1(2)。PCR反应完成，将产物进行1.0％琼脂糖凝胶观察与回收，可见大小约102bp的扩增条带，与预期结果一致(如图14)，回收的产物将作为连接模板用于获得添加Sph1-CRAMP-Xbal序列的完整片段。
(3)重组质粒pNZ8148-CRAMP的构建：具体操作同实施例6的步骤(3)，用Xbal、Sph1进行双酶切处理pNZ8148，胶回收大小约3100bp的条带。将双酶切后胶回收的CRAMP基因片段和双酶切后胶回收的pMG36e空质粒连接、回收。转化至E.coli MC1061感受态细胞后，进行菌落PCR验证，PCR反应完成，将产物进行1.0％琼脂糖凝胶观察与回收，可见大小约102bp的扩增条带(图15)，与预期结果一致，对检测阳性的菌液用质粒DNA抽提试剂盒进行质粒抽提，并进行双酶切鉴定和测序测定，即为获得了重组质粒pNZ8148-CRAMP。
实施例8 重组质粒pMG36e-Usp45-Linker-CRAMP的构建
(1)基因序列的密码子偏好性优化与合成：根据目的基因CRAMP基因的序列和表达载体pMG36e的特点，以及为达到高效分泌表达的目的而增加的信号肽序列Usp45，采用人工合成的方法将Usp45-Linker-CRAMP基因的243bp的密码子优化序列送公司进行合成。Xbal-Usp45-Linker-CRAMP-F为含有与pMG36e融合表达的酶切位点Xbal和信号肽Usp45-Linker-CRAMP的5'端最初一段序列的上游引物，Usp45-Linker-CRAMP-Sph1-R为信号肽Usp45-Linker-CRAMP基因反向引物。同时还设计了用于重组质粒的PCR检测和测序的引物pNZ1及pNZ2，是以pMG36e空质粒的MCS上游和下游70～90bp左右的区域为依据设计的。优化合成的Usp45-Linker-CRAMP序列如SEQ ID NO：10所示；优化合成的Xbal-Usp45-Linker-CRAMP-F，Usp45-Linker-CRAMP-Sph1-R引物序列分别如SEQ ID NO：5～6所示。
(2)Usp45-Linker-CRAMP基因片段的PCR扩增：具体操作步骤同实施例1步骤1(2)，区别在于，以含优化合成的Usp45-Linker-CRAMP基因为模板，用引物Xbal-Usp45-Linker-CRAMP-F、Usp45-Linker-CRAMP-Sph1-R扩增Usp45-Linker-CRAMP基因片段。PCR反应完成，将产物进行1.0％琼脂糖凝胶观察与回收，可见大小约243bp的扩增条带，与预期结果一致(如图14)，回收的产物将作为连接模板用于获得添加Usp45-Linker-CRAMP序列的完整片段。
(3)重组质粒pMG36e-Usp45-Linker-CRAMP的构建：将本实施例步骤(2)中回收的PCR产物用Sph1、Xbal进行双酶切处理，胶回收大小约243bp的条带。按照实施例1中步骤1(3)的方法对质粒进行双酶切处理，将酶切、回收后的质粒片段与胶回收的Usp45-Linker-CRAMP连接。将连接产物转化E.coli MC1061感受态细胞，并培养转化子。用引物Xbal-Usp45-Linker-CRAMP-F，Usp45-Linker-CRAMP-Sph1-R进行菌落PCR验证。PCR反应完成，将产物进行1.0％琼脂糖凝胶观察与回收，可见大小约243bp的扩增条带(图15)，与预期结果一致，对检测阳性的菌液用质粒DNA抽提试剂盒进行质粒抽提，并进行双酶切 鉴定和测序测定，即为获得了重组质粒pMG36e-Usp45-Linker-CRAMP。
实施例9 重组质粒pNZ8148-Usp45-Linker-CRAMP的构建
(1)基因序列的密码子偏好性优化与合成：按照实施例8的方法设计并合成Usp45-Linker-CRAMP基因。Sph1-Usp45-Linker-CRAMP-F为含有与pNZ8148融合表达的酶切位点Xbal和信号肽Usp45-Linker-CRAMP的5'端最初一段序列的上游引物，Usp45-Linker-CRAMP-Xbal-R为信号肽Usp45-Linker-CRAMP基因反向引物。优化合成的Sph1-Usp45-Linker-CRAMP-F，Usp45-Linker-CRAMP-Xbal-R引物序列分别如SEQ ID NO：7～8所示。
(2)Usp45-Linker-CRAMP基因片段的PCR扩增：按照实施例8的方法扩增Usp45-Linker-CRAMP基因片段，区别在于，引物为Sph1-Usp45-Linker-CRAMP-F，Usp45-Linker-CRAMP-Xbal-R。PCR反应结果与预期结果一致(如图14)，回收的产物将作为连接模板用于获得添加Usp45-Linker-CRAMP序列的完整片段。
(3)重组质粒pNZ8148-Usp45-Linker-CRAMP的构建：具体操作步骤同实施例8，区别在于，将质粒替换为pNZ8148。菌落PCR所用的引物为Sph1-Usp45-Linker-CRAMP-F，Usp45-Linker-CRAMP-Xbal-R。PCR反应完成，将产物进行1.0％琼脂糖凝胶观察与回收，可见大小约243bp的扩增条带(图15)，与预期结果一致，对检测阳性的菌液用质粒DNA抽提试剂盒进行质粒抽提，并进行双酶切鉴定和测序测定，即为获得了重组质粒pMG36e-Usp45-Linker-CRAMP。
实施例10 含CRAMP基因的分泌型重组植物乳杆菌的构建
(1)植物乳杆菌电转化感受态细胞的制备：将冻存的L.plantarum FCQHC24L1植物乳杆菌划MRS平板复苏，挑取单个菌落在MRS液体培养辈中30℃培养过夜，以1：100的比例接入50mL新的MRS液体培养基30℃培养，监测OD 500至0.3-0.4，迅速置冰上冷却，4℃6000×g离心20min，弃上清；用50mL预冷的0.5M蔗糖、10％甘油溶液重悬菌体，4℃6000×g离心20min，弃上清；用25mL预冷的0.5M蔗糖、10％甘油、50mM EDTA溶液重悬菌体，4℃6000×g离心15min，弃上清；再用15mL预冷的0.5M蔗糖、10％甘油溶液重悬菌体，4℃6000X g离心15min，弃上清；最后用500μL预冷的0.5M蔗糖、10％甘油溶液重悬菌体，即为植物乳杆菌感受态细胞，50μL每管分装，-80℃保存备用。
(2)植物乳杆菌的电击转化及转化子的PCR鉴定：各取50μL L.plantarum FCQHC24L1感受态细胞，冰浴上融解，分别加入1μL实施例6构建的重组质粒pMG36e-CRAMP、实施例7构建的重组质粒pNZ8148-CRAMP、实施例8构建的重组质粒pMG36e-Usp45-Linker-CRAMP和实施例9构建的重组质粒pNZ8148-Usp45-Linker-CRAMP，轻轻混匀；分别将以上混合物转入冰预冷的2mm电激杯，迅速给予一个单脉冲，参数设置为2kV，25F，200Q，电击后立即轻柔加入1mL冰预冷的恢复培养基MRS培养基，再分别将菌液全部吸入一灭菌离心管，盖紧管盖，冰浴5min后30℃静置培养2h；分别将含质粒pMG36e-CRAMP或质粒pMG36e-Usp45-Linker-CRAMP的菌液分为10μL、l00μL、900μL均匀涂布于含有5ug/mL红霉素的MRS平板，分别将含质粒pNZ8148-CRAMP或质粒pNZ8148-Usp45-Linker-CRAMP的菌液分为10μL、l00μL、900μL均匀涂布于含有5ug/mL 氯霉素的M17平板，30℃静置培养1-2天。挑取单个菌落，取菌落进行PCR鉴定，PCR产物用1％琼脂糖凝胶电泳进行检测，可见约243bp的扩增条带(图16)，阳性重组表达菌分别命名为L.plantarum FCQHC24L1/pMG36e-CRAMP、L.plantarum FCQHC24L1/pNZ8148-CRAMP、L.plantarum FCQHC24L1/pNZ8148-Usp45-Linker-CRAMP和L.plantarum FCQHC24L1/pMG36e-Usp45-Linker-CRAMP。
实施例11 含CRAMP基因的分泌型重组植物乳杆菌体外诱导表达
将重组菌L.plantarum FCQHC24L1/pMG36e-CRAMP和重组菌L.plantarum FCQHC24L1/pMG36e-Usp45-Linker-CRAMP分别以1:100的比例接种于含有5ug/mL红霉素的MRS液体培养基，将重组菌L.plantarum FCQHC24L1/pNZ8148-CRAMP和重组菌L.plantarum FCQHC24L1/pNZ8148-Usp45-Linker-CRAMP分别以1:100的比例分别接种于含有5ug/mL氯霉素的MRS液体培养基，30℃静置培养过夜；分别将过夜培养物以1:50的比例接种于10mL含有对应抗生素的液体培养基，继续培养约2.5h至细菌进入对数生长期(OD 500＝0.4～0.6)，分别向重组菌L.plantarum FCQHC24L1/pNZ8148-CRAMP和重组菌L.plantarum FCQHC24L1/pNZ8148-Usp45-Linker-CRAMP的培养体系中加入40ng/mL的乳酸链球菌素(nisin)诱导4h，4℃10000rpm离心5min，收集培养上清，经SDS-PAGE电泳并进行Western Blot分析，结果显示，L.plantarum FCQHC24L1/pMG36e-CRAMP、L.plantarum FCQHC24L1/pNZ8148-CRAMP、L.plantarum FCQHC24L1/pNZ8148-Usp45-Linker-CRAMP和L.plantarum FCQHC24L1/pMG36e-Usp45-Linker-CRAMP培养上清中检测到17KDa的目的条带(如图17)，表明目的基因已经得到分泌表达。
实施例12 植物乳杆菌制备疫苗中的应用
L.plantarum FCQHC24L1/pMG36e-CRAMP、L.plantarum FCQHC24L1/pNZ8148-CRAMP、L.plantarum FCQHC24L1/pNZ8148-Usp45-Linker-CRAMP和L.plantarum FCQHC24L1/pMG36e-Usp45-Linker-CRAMP重组植物乳杆菌口服疫苗的制备：将重组菌L.plantarum FCQHC24L1/pMG36e-CRAMP和重组菌L.plantarum FCQHC24L1/pMG36e-Usp45-Linker-CRAMP以1：100的体积比分别接种于MRS液体培养基，将重组菌L.plantarum FCQHC24L1/pNZ8148-CRAMP和重组菌L.plantarum FCQHC24L1/pNZ8148-Usp45-Linker-CRAMP以1：100的体积比分别接种于的MRS液体培养基，30℃静置培养过夜，将过夜培养物以1:100的比例接种于10mL含有相应抗生素的MRS液体培养基，继续培养约2.5h至细菌进入对数生长期(采用梯度稀释涂板测定重组菌的浓度达10 12CFU/mL数量级)，可选地，将植物乳杆菌L.plantarum FCQHC24L1/pNZ8148–CRAMP、L.plantarum FCQHC24L1/pNZ8148-Usp45-Linker-CRAMP在培养至对数生长期后加入nisin诱导培养2～6h。此时的全培养物直接作为口服疫苗使用，或离心收集菌体，将菌体作为口服疫苗的主要成分。
实施例13 植物乳杆菌在预防急性结肠炎中的应用
将实施例11制备的分别含重组植物乳杆菌L.plantarum FCQHC24L1/pMG36e-CRAMP、L.plantarum FCQHC24L1/pNZ8148-CRAMP、L.plantarum FCQHC24L1/pNZ8148-Usp45-Linker-CRAMP和L.plantarum  FCQHC24L1/pMG36e-Usp45-Linker-CRAMP全培养物的口服疫苗用于预防急性结肠炎。
将84只6～8周龄周龄的雄性Balb/c小鼠随机分为10组饲养，每组10只，第1组为生理盐水对照组，第2组为急性结肠炎模型组，第3组为L.plantarum FCQHC24L1/pMG36e组，第4组为L.plantarum FCQHC24L1/pNZ8148组，第5组为L.plantarum FCQHC24L1/pMG36e-CRAMP组，第6组为L.plantarum FCQHC24L1/pNZ8148-CRAMP组，第7组为L.plantarum FCQHC24L1/pMG36e-Usp45-Linker-CRAMP组，第8组为L.plantarum FCQHC24L1/pNZ8148-Usp45-Linker-CRAMP组。预饲一周后，采用灌胃的方式在3％DSS饮水7天后用含不同重组菌全培养物的口服疫苗进行口服免疫，连续免疫4天，剂量为160μL/只。然后连续10天处死小鼠，对肠道屏障及菌群变化进场测定，结果显示(图20～26)：
(1)各组小鼠第10天的小鼠比第7天平均体重相比：第1组增重1.084g，第2组下降2.85688g，第3组下降1.89644g，第4组下降1.73336g，第5组下降0.61004g，第6组下降0.36816g，第7组增重0.52568g，第8组增重1.23516g；
(2)各组第10天的平均结肠长度为：第1组为9.66厘米，第2组为5.32厘米，第3组为6.32厘米，第4组为6.36厘米，第5组为7.18厘米，第6组为7.44厘米，第7组为8.2厘米，第8组为8.32厘米；
(3)各组在第10天时DAI评分结果为：第1组0.2，第2组为7.2，第3组为6.6，第4组为6.0，第5组为4.6，第6组4.8，第7组为3.6，第8组为3.6；
(4)各组结肠形态学评分结果为：第1组0.2，第2组为3.8，第3组为3.2，第4组为3.2，第5组为2.6，第6组2.6，第7组为2.0，第8组为2.0；
(5)各组结肠紧密连接蛋白变化情况为：第2组与第1组相比：ZO-1(p<0.01)、ZO-2(p<0.0001)和occludin(p<0.0001)表达量显著下降；第5～6组与第2组相比：ZO-1(p<0.05)和occludin(p<0.05)表达量显著增加，ZO-2无显著差异；第7～8组与第2组相比：ZO-1(p<0.01)、ZO-2(p<0.01)和occludin(p<0.05)表达量显著增加；
(6)各组结肠炎症因子变化情况为：第2组与第1组相比：IL-6(p<0.0001)、IL-1β(p<0.0001)、TNF-α(p<0.0001)显著上升，IL-10(p<0.0001)显著下降；第5～8组与第2组相比：IL-6(p<0.05)、IL-1β(p<0.05)、TNF-α(p<0.05)显著下降，IL-10(p<0.05)显著上升；
(7)各组结肠关键转录因子蛋白水平变化为：第2组与第1组相比：p-ERK/ERK(p<0.0001)、p-p38/p38(p<0.0001)和p-NF-kB/NF-kB(p<0.0001)显著增加；第5组和第2组相比：p-ERK/ERK(p<0.05)显著降低，p-p38/p38(p>0.05和p-NF-kB/NF-kB(p>0.05)无显著差异性；第6组和第2组相比：p-ERK/ERK(p<0.01)、p-p38/p38(p<0.05)和p-NF-kB/NF-kB(p<0.05)显著降低；第7～8组和第2组相比：p-ERK/ERK(p<0.01)、p-p38/p38(p<0.05)和p-NF-kB/NF-kB(p<0.05)显著降低；
(8)各组结肠CRAMP蛋白表达情况：第2组和第1组相比CRAMP蛋白表达显著下降(p<0.0001)，第5～8组和第2组相比CRAMP蛋白表达显著增加(p<0.05)；
以上结果表明，口服重组菌L.plantarum FCQHC24L1/pMG36e-CRAMP全培养物的第5组，口服重组菌L.plantarum FCQHC24L1/pNZ8148-CRAMP全培养物的第6组，口服重组菌L.plantarum FCQHC24L1/pNZ8148-Usp45-Linker-CRAMP全培养物的第7组，口服重组菌L. plantarum FCQHC24L1/pMG36e-Usp45-Linker-CRAMP全培养物的第8组均具有很好的恢复结肠炎小鼠的体重，恢复结肠长度，减轻结肠炎症，抑制炎症细胞因子分泌，抑制炎症信号通路的激活，恢复肠道屏障，从而治疗结肠炎，且第7组L.plantarum FCQHC24L1/pNZ8148-Usp45-Linker-CRAMP组，第8组L.plantarum FCQHC24L1/pMG36e-Usp45-Linker-CRAMP组有更好的治疗效果。
发明人还尝试将植物乳杆菌与nisin联合使用，例如将植物乳杆菌L.plantarum FCQHC24L1/pNZ8148-CRAMP或L.plantarum FCQHC24L1/pNZ8148-Usp45-Linker-CRAMP对小鼠灌胃处理一段时间后，使植物乳杆菌L.plantarum FCQHC24L1/pNZ8148-CRAMP或L.plantarum FCQHC24L1/pNZ8148-Usp45-Linker-CRAMP在肠道内定植，再让处理对象摄入nisin，或内容物为nisin的产品，可达到CRAMP在肠道内定时、定向释放的效果。
对比例2 含CRAMP基因的分泌型重组植物乳杆菌的构建
对现有技术中含CRAMP基因的分泌型重组乳酸乳球菌作为对照，其表达的CRAMP基因未经过密码子优化(GGACTTCTCCGCAAAGGTGGGGAGAAGATTGGTGAAAAGCTTAAGAAAATTGGCCAGAAAATTAAGAATTTTTTTCAGAAACTTGTACCTCAGCCAGAG)，且无Usp45信号肽促进CRAMP在细胞外分泌，以及不能促进Usp45信号肽和CRAMP基因在胞内自剪切作用，上清中分泌的CRAMP蛋白含量低，表达产物约为1.5ng/μL。
将实施例11构建的重组菌与对比例1的重组菌表达CRAMP的能力进行比较，经ELISA检测发现(图18)，无Usp45诱导分泌的重组植物乳杆菌L.plantarum FCQHC24L1/pMG36e-CRAMP和L.plantarum FCQHC24L1/pNZ8148-CRAMP胞外CRAMP蛋白表达量约为10ng/μL，比对比例1的重组菌胞外蛋白分泌量高6～8倍；重组植物乳杆菌L.plantarum FCQHC24L1/pNZ8148-Usp45-Linker-CRAMP的CRAMP蛋白表达量约为20ng/μL，比对比例1的表达量(1.5ng/μL)高13倍；重组植物乳杆菌L.plantarum FCQHC24L1/pMG36e-Usp45-Linker-CRAMP的CRAMP蛋白胞外分泌量高于50ng/μL，比对比例1在相同条件下的表达量(1.5ng/μL)高30倍。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。

Claims (15)

  1. 一种编码CRAMP蛋白的基因，其特征在于，核苷酸序列如SEQ ID NO.1所示。
  2. 携带权利要求1所述基因的载体。
  3. 根据权利要求2所述的载体，其特征在于，所述载体为pMG36e或pNZ8148。
  4. 表达CRAMP蛋白的乳酸菌，其特征在于，表达SEQ ID NO.1所示的基因，或
    表达CRAMP蛋白；所述CRAMP蛋白为(a)或(b)：
    (a)如SEQ ID NO.2所示的CRAMP蛋白；
    (b)在(a)的基础上对一个或几个氨基酸进行缺失、取代或增减且具有抑菌特性的由(a)衍生的蛋白质。
  5. 根据权利要求4所述乳酸菌，其特征在于，以植物乳杆菌或乳酸乳球菌为宿主。
  6. 根据权利要求4或5所述乳酸菌，其特征在于，以Usp45信号肽促进CRAMP蛋白的表达。
  7. 根据权利要求6所述乳酸菌，其特征在于，所述Usp45信号肽与CRAMP蛋白之间通过linker连接。
  8. 根据权利要求7所述的乳酸菌，其特征在于，所述linker包含2个或更多个选自Gly和Ser的氨基酸残基。
  9. 根据权利要求5所述的乳酸菌，其特征在于，所述植物乳杆菌以植物乳杆菌FCQHC24L1为宿主。
  10. 根据权利要求5所述的乳酸菌，其特征在于，所述乳酸乳球菌以乳酸乳球菌NZ9000为宿主。
  11. 含有权利要求3～10任一所述乳酸菌的食用或药用组合物。
  12. 根据权利要求11所述的组合物，其特征在于，所述乳酸菌的含量≥1×10 5CFU/mL或1×10 5CFU/g。
  13. 一种疫苗，其特征在于，含有权利要求3～10任一所述的乳酸菌，或所述乳酸菌的纯培养物。
  14. 权利要求3～10任一所述的乳酸菌在制备可引入肠道的产品中的应用，其特征在于，所述产品具有如下至少一种功能：
    (a)抑制肠道炎症；
    (b)重塑肠黏膜屏障；
    (c)改善肠黏膜通透性；
    (d)预防或治疗肠道炎症以及肠道炎症引起的疾病；
    (e)改善或治疗因肠道稳态失衡引起的或相关的疾病；所述肠道稳态失衡引起的或相关的疾病包括但不限于糖尿病、胰腺炎或代谢综合症；
    (f)降低炎症因子IL-6、IL-1β、TNF-α水平；
    可选地，所述产品单独或与nisin联合使用。
  15. 权利要求3～10任一所述乳酸菌在制备发酵食品中的应用。
PCT/CN2021/083282 2020-03-27 2021-03-26 高表达抗菌肽cathelicidin基因的乳酸菌 WO2021190634A1 (zh)

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CN111411054A (zh) * 2020-03-27 2020-07-14 江南大学 一种表达小鼠抗菌肽基因的乳酸乳球菌

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CN105816854A (zh) * 2016-04-08 2016-08-03 苏州大学 抗菌肽cramp在防治病毒性心肌炎中的应用
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