The instant application contains a Sequence Listing in ASCII format as a file named seq.txt, created on Aug. 11, 2022, of 4 kB in size, which is hereby incorporated by reference in its entirety.

TECHNICAL FIELD

The disclosure relates to cathelicidin-expressing lactic acid bacteria, and belongs to the technical field of genetic engineering.

BACKGROUND

Antimicrobial peptides are the main components of the innate immunity and defense of many hosts such as plants, invertebrates and vertebrates (including humans). Cathelicidins are a major class of antimicrobial peptides, and are characterized by a conservative N-terminal precursor sequence called cathelin. The conservation of the cathelin sequence indicates that various members of this family evolved from duplication and modification of common ancestor genes. The CRAMP (Cathehcidin-Related AntiMicrobial Peptide) contains 34 amino acids (GLLRKGGEKIGEKLKKIGQKIKNFFQKLVPQPEQ, SEQ ID NO: 2) and has strong antimicrobial activities against gram-positive and gram-negative bacteria, but has no hemolytic activity on human red blood cells. CRAMP at 1 mM can directly cause immediate permeabilization of the inner membrane ofE. coli. Antibodies against CRAMP shows abundant CRAMP expression in bone marrow progenitors and neutrophils. In addition, studies have found that CRAMP has strong antimicrobial activities against some pathogenic fungi (Candia alicans andAspergillus fumigatus). In vitro studies have confirmed that CRAMP can significantly inhibit proliferation ofHelicobacter pylori. The lack of CRAMP can lead to aggravation of colitis in mice, while treatment of CRAMP knockout mice with colitis by CRAMP-expressingLactobacillushas a significant alleviation effect.

CRAMP level in the intestinal tract is significantly decreased due to destruction of the intestinal barrier, resulting in the inability to exert an immune-modulatory effect and regulate the balance of intestinal flora. Therefore, choosing a safe and non-toxic carrier system that can survive in the intestinal tract and express CRAMP, so that CRAMP can play a role in the intestinal tract, is of great significance for regulating the balance of intestinal flora.

Due to high adhesion of surface molecules,Lactococcus. lactisandLactobacillus. plantarumcan successfully colonize the intestinal tract of animal bodies and become the dominant flora in the intestinal tract to play a variety of functions such as improving the body's immunity, promoting absorption of nutrients, and maintaining the balance of the intestinal flora. The formed biologically stable barrier is an important guarantee for maintaining the balance of intestinal microbes. In terms of expressing foreign genes, aL. lactisexpression system and aL. plantarumexpression system as prokaryotic expression systems have the following advantages: (1) as food-grade bacteria, the expression systems are safer as live carrier vaccines; (2) a foreign gene can be expressed in the cell, and can also be expressed and displayed on the cell surface or secreted out of the cell; (3) the expression systems are safe and free of endotoxin, have no need to purify and express foreign protein, and can be taken directly with the bacteria; and (4) the expression systems can colonize on the surface of a body's mucosa (belonging to a common mucosal immune system), and inoculation to a certain site on the mucosa can induce a systemic mucosal immune response; last but not least, this kind of immunity can help the body gain longer-term immunological memory, so as to resist the invasion of pathogens for a long time.

SUMMARY

The disclosure aims at solving the technical problem of overcoming the shortcomings of the prior art that oral CRAMP is easily degraded by digestive tract enzymes, and intestinal targeted delivery of CRAMP and maximization of a local immunoregulatory effect cannot be achieved, and provides a recombinantLactobacillusthat secretes and expresses CRAMP protein and an application thereof.

The disclosure provides a gene encoding CRAMP protein, and the gene includes a nucleotide sequence as shown in SEQ ID NO: 1.

In an embodiment, the gene is also fused with a nucleotide sequence encoding a Usp45 signal peptide, the nucleotide sequence encoding the Usp45 signal peptide is shown in SEQ ID NO: 3, and a gene sequence linked to the signal peptide is shown in SEQ ID NO: 4.

In an embodiment, a linker sequence is 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 disclosure further provides a vector carrying the gene.

In an embodiment, the vector is pMG36e or pNZ8148.

In an embodiment, the vector is pMG36e-CRAMP, pNZ8148-CRAMP, pNZ8148-Usp45-CRAMP, pMG36e-Usp45-CRAMP, pMG36e-Usp45-Linker-CRAMP or pNZ8148-Usp45-Linker-CRAMP.

The disclosure further provides aLactobacillusexpressing the CRAMP protein and the gene as shown in SEQ ID NO. 1, or the CRAMP protein which is (a) or (b):

(a) a CRAMP protein as shown in SEQ ID NO. 2; and

(b) a protein derived from (a), with one or several amino acids deleted, substituted, or increase and decrease on the basis of (a), and has antimicrobial properties.

In an embodiment, theLactobacillusisL. plantarumorL. lactis.

In an embodiment,L. plantarumFCQHC24L1 is used as a host.

In an embodiment, theL. plantarumFCQHC24L1 was disclosed in a 2019 paper entitled “Study on the Differences in the Genome and Main Physiological Characteristics ofL. plantarumin Different Ecological Niches”. The applicant promises to distribute the strain to the public who implement the disclosure through legal channels within 20 years from the date of application.

In an embodiment, theL. plantarumexpresses the CRAMP protein-encoding gene with pMG36e or pNZ8148 as a vector.

In an embodiment, a Usp45 signal peptide is also introduced into theL. plantarumto promote expression of the CRAMP protein.

In an embodiment, the Usp45 is linked to the CRAMP gene through a linker, and the linker includes 2 or more amino acid residues selected from Gly and Ser.

In an embodiment, the amino acid sequence of the linker is GGGGS (SEQ ID NO: 13), and the nucleotide sequence encoding the linker is shown in SEQ ID NO. 9.

In an embodiment,L. lactisNZ9000 is used as a host.

In an embodiment, theL. lactisuses pMG36e or pNZ8148 as a vector.

In an embodiment, the Usp45 signal peptide is also introduced into theL. lactisto promote expression of the CRAMP protein.

The disclosure further provides a method for constructing the CRAMP-encodingL. plantarum, including: linking the CRAMP protein-encoding gene as shown in SEQ ID NO. 1 to a vector, and then transforming the gene into aL. plantarumcell; and the vector is pMG36e or pNZ8148.

In an embodiment, a Usp45 signal peptide is linked to the pMG36e or pNZ8148.

In an embodiment, the Usp45 is linked to the CRAMP gene through a linker, and the nucleotide sequence of the linker is GGCGGTGGCGGCAGC (SEQ ID NO:9).

In an embodiment, the method includes the following steps:

(1) synthesizing the CRAMP protein-encoding gene as shown in SEQ ID NO.1; and

(2) linking the gene synthesized in step (1) to pMG36e to obtain a recombinant plasmid pMG36e-CRAMP; and transforming the pMG36e-CRAMP recombinant plasmid intoL. plantarumFCQHC24L1 by an electrotransformation method to obtain recombinantL. plantarumFCQHC24L1/pMG36e-CRAMP.

In an embodiment, the method includes the following steps:

(1) synthesizing the CRAMP protein-encoding gene as shown in SEQ ID NO.10; and

(2) linking the gene synthesized in step (1) to pMG36e to obtain a recombinant plasmid pMG36e-Usp45-Linker-CRAMP; and transforming the pMG36e-Usp45-Linker-CRAMP recombinant plasmid intoL. plantarumFCQHC24L1 by an electrotransformation method to obtain recombinantL. plantarumFCQHC24L1/pMG36e-Usp45-Linker-CRAMP.

In an embodiment, specific operations of the electrotransformation method include: preparing competent cells ofL. plantarumFCQHC24L1, transforming the recombinant plasmid, mixing and transferring the cells to an electroporation cuvette, adding a recovery MRS medium after electric shock, and culturing the cells in a static state after treatment in an ice bath, and screening high-copy transformants on a plate.

The disclosure further provides a method for constructing any one of the mentioned above recombinantL. lactis, including: linking the CRAMP protein-encoding gene as shown in SEQ ID NO. 1 to a vector, and then transforming the gene into aL. lactiscell; and the vector is pMG36e or pNZ8148.

In an embodiment, a Usp45 signal peptide is linked to the pMG36e or pNZ8148.

In an embodiment, the method includes the following steps:

(1) synthesizing the CRAMP protein-encoding gene as shown in SEQ ID NO.1; and

(2) linking the gene synthesized in step (1) to pMG36e to obtain a recombinant plasmid pMG36e-Usp45-CRAMP; and transforming the pMG36e-Usp45-CRAMP recombinant plasmid intoL. lactisNZ9000 by an electrotransformation method to obtain recombinantL. lactisNZ9000/pMG36e-Usp45-CRAMP.

In an embodiment, the method includes the following steps:

(1) synthesizing the CRAMP protein-encoding gene as shown in SEQ ID NO. 4; and

(2) linking the gene synthesized in step (1) to pNZ8148 to obtain a recombinant plasmid pNZ8148-Usp45-CRAMP; and transforming the pNZ8148-Usp45-CRAMP recombinant plasmid intoL. lactisNZ9000 by an electrotransformation method to obtain recombinantL. lactisNZ9000/pNZ8148-Usp45-CRAMP.

In an embodiment, the electrotransformation method includes: preparing competent cells ofL. lactisNZ9000, transforming the recombinant plasmid, mixing and transferring the cells to an electroporation cuvette, adding a recovery MRS medium after electric shock, and culturing the cells in a static state after treatment in an ice bath, and screening high-copy transformants on a plate.

The disclosure further provides an edible or medicinal composition, and the composition contains theL. lactisand/orL. plantarum.

In an embodiment, the concentration ofL. lactisand/orL. plantarumin the composition is greater than or equal to 1×105CFU/mL or 1×105CFU/g.

In an embodiment, the composition is a medicine and contains a pharmaceutically acceptable carrier.

The disclosure further provides an application of theL. lactisand/orL. plantarumin preparing a vaccine.

In an embodiment, the vaccine is an oral vaccine for preventing acute colitis.

In an embodiment, the application is to culture theL. lactis, and then use the anaculture ofL. lactisas an oral vaccine.

In an embodiment, the application includes the following steps: inoculatingLactococcus/pMG36e-Usp45-CRAMP in a GM17 liquid medium, culturing the bacteria in a static state overnight, transferring the bacteria to a GM17 liquid medium at a certain ratio, continuing culturing until the bacteria reach a logarithmic growth phase, and directly using the anaculture as an oral vaccine.

In an embodiment, the static culture is performed at a temperature of 28-30° C.

In an embodiment, the transfer is to inoculateL. lactisNZ9000/pMG36e-Usp45-CRAMP in a GM17 medium at a volume ratio of 1:100.

In an embodiment, the OD value of a bacterial culture solution in the logarithmic growth phase is 0.4-0.6.

In an embodiment, the application includes the following steps: inoculatingL. lactisNZ9000/pMG36e-Usp45-CRAMP recombinant expression bacteria in a medium containing GM17 at a ratio of 1:100, continuing culturing for 2-3 h until the bacteria reach the logarithmic growth phase (OD600=0.4-0.6); culturing the bacteria until the concentration of the recombinant bacteria reach 1012CFU/mL, and collecting the induced anaculture as an oral vaccine.

In an embodiment, the application is to culture theL. plantarum, and then use the anaculture of theL. plantarumas an oral vaccine.

In an embodiment, the application is to culture the recombinantL. plantarum, and then use the anaculture of theL. plantarumas an oral vaccine or the main component of an oral vaccine.

In an embodiment, the application includes the following steps: inoculating recombinantL. plantarum/pMG36e-Usp45-Linker-CRAMP in an MRS liquid medium, culturing the bacteria in a static state overnight, transferring the bacteria to an MRS liquid medium at a certain ratio, continuing culturing until the bacteria reach a logarithmic growth phase, and directly using the anaculture as an oral vaccine.

In an embodiment, the static culture is performed at a temperature of 28-30° C.

In an embodiment, the transfer is to inoculateL. plantarumFCQHC24L1/pMG36e-Usp45-Linker-CRAMP in an MRS medium at a volume ratio of (1-10):100.

In an embodiment, the OD value of a bacterial culture solution in the logarithmic growth phase is 0.4-0.6.

In an embodiment, the application includes the following steps: inoculatingL. plantarumFCQHC24L1/pMG36e-Usp45-Linker-CRAMP recombinant bacteria in an MRS medium at a volume ratio of (1-10):100, continuing culturing for 2-3 h until the bacteria reach the logarithmic growth phase (OD600=0.4-0.6), culturing the bacteria until the concentration of the recombinant bacteria reach 1012CFU/mL, and collecting the induced anaculture as an oral vaccine.

The disclosure further claims to protect an application of theL. plantarumin preparation of a medicine for preventing or treating acute colitis.

In an embodiment, the medicine is used in combination with nisin.

In an embodiment, after the medicine is taken into the intestinal tract, nisin or a nisin-containing medicine is then taken to achieve the effect of directional release of CRAMP and increase of the amount of CRAMP secreted in the intestinal tract.

The disclosure further claims to protect an application of theL. lactisand/orL. plantarumin preparation of a product that can be introduced into the intestinal tract; and the product has at least one of the following functions:

(a) inhibiting intestinal inflammation;

(b) reconstructing intestinal mucosal barrier;

(c) improving intestinal mucosal permeability; and

(d) preventing and treating intestinal inflammation and diseases caused by intestinal inflammation.

The disclosure further claims to protect an application of theL. lactisand/orL. plantarumin preparation of a medicine for preventing or treating inflammatory bowel disease, diarrhea or diseases caused by or related to imbalance of intestinal homeostasis, and the diseases caused by or related to the imbalance of intestinal homeostasis include, but are not limited to, liver diseases, metabolic endocrine diseases, cardiovascular diseases, etc., such as diabetes, pancreatitis, or metabolic syndrome.

The disclosure further claims to protect an application of theL. lactisand/orL. plantarumin preparation of a medicine for preventing or treating acute colitis.

Beneficial effects: (1) The disclosure provides the optimized gene encoding the CRAMP protein, and introduces the Usp45 signal peptide to promote the secretion and expression of CRAMP. The expression level of the CRAMP protein inL. lactiscan reach 40 ng/μL, and inL. plantarumcan reach 20 ng/μL or even higher. Owing to probiotic properties ofL. lactisandL. plantarum, theLactobacillusexpression system becomes a food-grade expression system that can be taken together with bacteria.

(2) The disclosure uses the constructed CRAMP protein-expressingL. lactisandL. plantarumto prepare a vaccine for regulation of intestinal flora disorder, and theL. lactisandL. plantarumhave advantages in regulation of intestinal flora and intestinal immune response and maintenance. An anaculture can be directly taken as an oral vaccine to stimulate mice and cause a strong cellular immune response. The recombinantL. lactiscan be used as a new oral vaccine product with good industrial prospects, plays a positive role in reducing intestinal inflammation, and has important practical significance for promoting health development of the intestinal tract.

(3) The vaccine containing recombinantL. plantarumand/orL. lactisprepared by the disclosure can specifically regulate intestinal flora disorder, and help regulation of the intestinal flora and intestinal immune response and maintenance. Animal experiments have confirmed that the oral vaccine prepared by the disclosure can stimulate mice and cause a strong cellular immune response. The oral vaccine can be used as a new oral vaccine product with good industrial prospects, plays a positive role in alleviating intestinal inflammation, and has important practical significance in promoting health development of the intestinal tract.

