Recombinant IL-36Ra food-grade lactococcus lactis expression plasmid as well as construction method and application thereof

Abstract

The invention provides a recombinant IL-36Ra food-grade lactococcus lactis expression plasmid as well as a preparation method and application thereof. The expression plasmid comprises a base fragment for coding the interleukin-36 receptor antagonist; the expression plasmid can express IL-36Ra with multiplied activity, and the expression plasmid is a food-grade intracellular expression vector of lactococcus lactis and does not have antibiotic genes. Compared with traditional IL-36Ra protein injection and plasmid injection, the food-grade lactococcus lactis expression plasmid is simple to operate and relatively low in cost, and a new thought is provided for research on in-vivo gene therapy and IL-36Ra gene functions.

Description

Technical Field

[0001] The invention belongs to the field of molecular biology and immunology, and specifically belongs to food-grade lactic acid bacteria capable of expressing an interleukin-36 receptor antagonist and a preparation method thereof. Background Art

[0002] Interleukin-36 receptor antagonist (IL-36Ra) belongs to the IL-36 subfamily of the IL-1 family. IL-1 family members are associated with acute and chronic inflammation and play an important role in nonspecific immune responses to infection. The IL-36 subfamily consists of IL-36α, IL-36β, IL-36γ agonists, and IL-36Ra, all of which act through a specific IL-36 receptor (IL-36R). Although IL-36 and IL-1 signaling pathways have similar characteristics, the disease manifestations caused by abnormal IL-36 signaling are significantly different from those caused by abnormal IL-1 signaling.

[0003] Abnormal activation of IL-36 signaling promotes the occurrence of inflammatory diseases of the lungs, kidneys, and intestines, indicating that IL-36 has the potential to serve as an auxiliary disease diagnosis and therapeutic target. IL-36Ra and IL-36 agonists do not have caspase-1-specific cleavage sites, indicating that they utilize secretion pathways different from other IL-1 family members. Among them, IL-36 agonists induce inflammatory responses through intracellular signaling cascades to activate nuclear factor κB and mitogen-activated protein kinase, while IL-36Ra inhibits proinflammatory signals by competitively binding to IL-36R and acts as a negative regulator. Elastase can enzymatically cleave IL-36Ra in human skin cells, resulting in antagonistic activation of IL-36Ra. IL-36Ra is highly expressed in the skin, brain, tonsils, spleen, and various immune cells.

[0004] Studies have shown that IL-36Ra plays a certain anti-inflammatory role in inflammatory bowel inflammation, psoriasis, rheumatoid arthritis and tumorigenesis. In addition, IL-36Ra plays an important role in health and maintaining normal immune homeostasis, so it has certain development prospects in the treatment of inflammatory diseases.

[0005] Lactococcus lactis is a species of lactic acid bacteria, a representative species of lactic acid bacteria, and the most well-known probiotic in the intestines of humans and animals. More importantly, it is a generally recognized as safe (GRAS) food-grade microorganism. The characteristics of Lactococcus lactis include rapid growth and food-grade safety, which further proves that Lactococcus lactis can be an ideal strain for expressing foreign proteins and immune live carrier vaccines.

[0006] With the deepening of research on genetic engineering of Lactococcus lactis, many constitutive and inducible Lactococcus lactis expression systems, protein localization and antigen presentation systems have been developed. However, there are no reports on IL-36Ra mediated by Lactococcus lactis as a carrier. Summary of the invention

[0007] In view of the shortcomings of the prior art, the object of the present invention is to provide a food-grade lactic acid bacteria expression plasmid that can express IL-36Ra, an expression system comprising the plasmid, and a preparation method and application thereof.

[0008] To achieve this object, the present invention adopts the following technical scheme: In the first aspect, the present invention provides a recombinant IL-36Ra food-grade Lactococcus lactis expression plasmid, wherein the expression plasmid encodes a base fragment of the amino acid sequence shown in SEQ ID NO.1; the amino acid sequence shown in SEQ ID NO.1 is an interleukin-36 receptor antagonist.

[0009] In the present invention, the amino acid sequence of the interleukin-36 receptor antagonist in the expression plasmid is: VLSGALCFRMKDSALKVLYLHNNQLLAGGLHAEKVIKGEEISVVPNRALDASLSPVILGVQGGSQCLSCGTEKGPILKLEPVNIMELYLGAKESKSFTFYRRDMGLTSSFESAAYPGWFLCTSPEADQPVRLTQIPEDPAWDAPITDFYFQQC (SEQ ID NO.1) In the present invention, the IL-36Ra encoding gene sequence connected to the recombinant plasmid is a gene sequence whose expression activity is increased exponentially after the IL-36Ra gene fragment is excised and the N-terminal methionine is removed. Compared with the IL-36Ra gene sequence without excision of the N-terminal methionine, the protein expression amount and activity of the present application are significantly higher. The inventors believe that the N-terminal methionine may cause the target protein to be more arbitrarily recognized and degraded by the components in the expression system, and after the removal of methionine, the structure of the IL-36Ra protein is not significantly affected, and its expression amount and activity are significantly increased.

[0010] As a preferred technical solution of the present invention, the present invention also adopts molecular regulation means to add Usp45 signal peptide and LEISS nonapeptide amino acid sequence to the N-terminus of IL-36Ra to increase the secretory protein activity; therefore, the expression plasmid also contains a base fragment encoding Usp45 signal peptide and / or LEISS nonapeptide.

[0011] In the present invention, a variety of signal peptides can be selected in the expression plasmid, such as SP310, SPExp4, AL9 and Usp45 signal peptides. After comparison, it can be seen that the Usp45 signal peptide can significantly promote the expression of the target protein. In addition, when the LEISS nonapeptide is not added, the expression of the target protein is also significantly lower.

[0012] Preferably, the amino acid sequence of the Usp45 signal peptide is: MKKKIISAILMSTVILSAAAPLSGVYA (SEQ ID NO.2) Preferably, the amino acid sequence of the LEISS nonapeptide is: LEISSTCDA (SEQ ID NO.3) As a preferred technical solution of the present invention, the expression plasmid is the Lactococcus lactis food-grade intracellular expression vector pNZ8149.

[0013] Preferably, the expression plasmid further comprises a LacF selection gene.

[0014] In the present invention, the vector pNZ8149 is used, which may contain the LacF screening gene. If other vectors such as pNZ8148 are used, which do not contain the LacF gene, lactose cannot be used for positive colony screening; if antibiotics are used for screening, it does not meet the food-grade application purpose of the present invention.

[0015] Preferably, the expression plasmid further comprises a His tag.

[0016] As a preferred technical solution of the present invention, in the expression plasmid, the base sequence encoding the IL-36Ra amino acid sequence is optimized according to the codon preference of Lactococcus lactis, and the specific sequence is shown in SEQ ID NO.4: GTTCTTTCAGGTGCTCTTTGTTTCCGTATGAAAGATTCAGCTCTTAAAGTTCTTTACCTTCACAACAACCAACTTCTTGCTGGTGGTCTTCACGCTGAAAAAGTTATCAAAGGTGAAGAAATCTCAGTTGTTCCAAACCGTGCTCTTGATGCTTCACTTTCACCAGTTATCCTTGGTGTTCAAGGTGGTTCACAATGTCTTTCATGTGGTACTGAAAAAGGTCCAATCCTT AAACTTGAACCAGTTAACATCATGGAACTTTACCTTGGTGCTAAAGAATCAAAATCATTCACTTTCTACCGTCGTGATATGGGTCTTACTTCATCATTCGAATCAGCTGCTTACCCAGGTTGGTTCCTTTGTACTTCACCAGAAGCTGATCAACCAGTTCGTCTTACTCAAATCCCAGAAGATCCAGCTTGGGATGCTCCAATCACTGATTTCTACTTCCAACAATGTGAT The base sequence encoding the Usp45 signal peptide is shown in SEQ ID NO.5; GGCAAGAAGAAGATCATCAGCGCCATCCTGATGAGCACCGTGATCCTGAGCGCCGCCCGCCCTCTGAGCGGAGTTTACGCC The base sequence encoding the amino acid sequence of LEISS nonapeptide is shown in SEQ ID NO.6: CTGGAGATCAGCAGCACCTGCGACGCT As a preferred technical solution of the present invention, the expression plasmid comprises the base sequence shown in SEQ ID NO.7: CCATGGGCAAGAAGAAGATCATCAGCGCCATCCTGATGAGCACCGTGATCCTGAGCGCCGCCCGCCCCTCTGAGCGGAGTTTACGCCCTGGAGATCAGCAGCACCTGCGACGC TGTTCTTTCAGGTGCTCTTTGTTTCCGTATGAAAGATTCAGCTCTTAAAGTTCTTTACCTTCACAACAACCAACTTCTTGCTGGTGGTCTTCACGCTGAAAAAGTTATCAAAGGTGAAGAAATCTCAGTTGTTCCAAACCGTGCTCTTGATGCTTCACTTTCACCAGTTATCCTTGGTGTTCAAGGTGGTTCACAATGTCTTTCATGTGGTACTGAAAAAGGTCCAATCCTTAAACTTGAACCAG TTAACATCATGGAACTTTACCTTGGTGCTAAAGAATCAAAATCATTCACTTTCTACCGTCGTGATATGGGTCTTACTTCATCATTCGAATCAGCTGCTTACCCAGGTTGGTTCCTTTGTACTTCACCAGAAGCTGATCAACCAGTTCGTCTTACTCAAATCCCAGAAGATCCAGCTTGGGATGCTCCAATCACTGATTTCTACTTCCAACAATGTGATCATCATCATCATCACTAGTCTAGA In a second aspect, the present invention further provides a method for preparing the recombinant IL-36Ra food-grade Lactococcus lactis expression plasmid as described in the first aspect, the preparation method comprising: A gene fragment expressing IL-36Ra is constructed, and then the gene fragment is connected to a Lactococcus lactis food-grade intracellular expression vector and transformed into Lactococcus lactis.

[0017] As a preferred technical solution of the present invention, the step of constructing a gene fragment expressing IL-36Ra is: After the gene fragment encoding IL-36Ra was cut, the base sequence encoding the Usp45 signal peptide and the LEISS nonapeptide was added to the N-terminus, and a histidine tag was added to the C-terminus.

[0018] In the present invention, Usp45 signal peptide and LEISS nonapeptide amino acid sequence are added to the N-terminus of IL-36Ra to increase the secretory protein activity, 6 histidine tags are added to the C-terminus for subsequent affinity purification, and two restriction sites, Nco Ⅰ and Xba Ⅰ, are added at both ends for subsequent restriction digestion and linkage with the vector.

[0019] As a preferred technical solution of the present invention, the preparation method comprises the following steps: First, the IL-36Ra encoding gene is constructed on a cloning vector; the IL-36Ra encoding gene comprises the base sequence as described in SEQ ID NO.4, and the N-terminus of the sequence comprises the base sequence encoding the Usp45 signal peptide and the LEISS nonapeptide; Then, the recombinant plasmid produced by the cloning vector was cloned and extracted, and the obtained base sequence was connected to the expression vector pNZ8149 with the LacF screening gene after enzyme digestion to construct the recombinant IL-36Ra food-grade Lactococcus lactis expression plasmid.

[0020] In a third aspect, the present invention provides a recombinant IL-36Ra food-grade Lactococcus lactis expression system, the expression system comprising the expression vector as described in the first aspect; Preferably, the chassis cell of the expression system is Lactococcus lactis NZ3900.

[0021] The expression vector used in the present invention is Lactococcus lactis NZ3900. Compared with other expression strains, such as Lactobacillus casei, Lactobacillus plantarum and Lactobacillus helveticus, Lactococcus lactis does not use antibiotics as screening markers, and there is no potential risk of antibiotic contamination.

[0022] If Escherichia coli is used as a plasmid vector, the target plasmid needs to be extracted using a kit and then injected into the tail vein. Compared with the present invention, the operation is more complicated and the mortality rate of the animal model is higher.

[0023] In the chassis cells, the gene fragment of IL-36Ra can be secreted at a good level without any rejection or other inability to express, which enables the expression system established by the present invention to achieve good application prospects.

[0024] As a preferred technical solution of the present invention, the recombinant plasmid and the recombinant expression system can be prepared by the following method, which specifically includes the following steps: First, a gene fragment expressing IL-36Ra was constructed; Then, the Lactococcus lactis food-grade intracellular expression vector pNZ8149 with the LacF selection gene was used to construct a recombinant plasmid expressing IL-36Ra; Finally, Lactococcus lactis NZ3900 was transformed by electroporation and recombinant screening was performed using lactose as a carbon source to construct a food-grade genetically engineered bacterium expressing IL-36Ra.

[0025] Therefore, the recombinant plasmid and recombinant expression system of the present invention can be operated as follows: (1) Constructing the artificially synthesized IL-36Ra encoding gene on a cloning vector; (2) cloning and extracting the recombinant plasmid obtained in step (1), and obtaining the IL-36Ra DNA sequence after enzyme digestion; (3) Connecting the IL-36Ra DNA sequence obtained in step (2) to an expression vector with a LacF screening gene to construct an IL-36Ra expression plasmid; (4) The IL-36Ra expression plasmid obtained in step (3) is transformed into Lactococcus lactis NZ3900 competent cells, and Lactococcus lactis expressing IL-36Ra is screened using the LacF gene, and verified to obtain the food-grade lactic acid bacteria expression system that can express IL-36Ra.

[0026] Preferably, in step (1), the cloning vector is a pUC57 vector.

[0027] Preferably, in step (3), the expression vector is the Lactococcus lactis expression vector pNZ8149 with the LacF selection gene.

[0028] Preferably, in step (3), Hind III is used to perform restriction enzyme digestion verification on pNZ8149.

[0029] Preferably, in step (4), the positive recombinant clone strains are screened and determined by Elliker screening plates, and then positive colonies are selected and identified by bacterial liquid PCR, thereby verifying the successful construction of the food-grade lactic acid bacteria expression plasmid expressing IL-36Ra.

[0030] Preferably, in the step (4), in the PCR identification step, it is designed to prove that pNZ8149 and IL-36Ra are successfully connected and exist in NZ3900.

[0031] In a fourth aspect, the present invention further provides a use of the expression plasmid as described in the first aspect, the preparation method as described in the second aspect, or the expression system as described in the third aspect in the preparation of a drug comprising IL-36Ra.

[0032] Preferably, the medicine includes medicines for treating diseases such as inflammatory bowel disease, psoriasis, rheumatoid arthritis or tumors.

[0033] The numerical range described in the present invention not only includes the point values ​​listed above, but also includes any point values ​​between the above numerical ranges that are not listed. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values ​​included in the range.

[0034] Compared with the prior art, the advantages of the present invention are: (1) Compared with other expression vectors, especially the most commonly used Escherichia coli, the expression plasmid of recombinant IL-36Ra provided by the present invention is a Lactococcus lactis-specific vector, including pNZ8149, which does not contain antibiotic genes and is a food-grade expression vector. Therefore, the recombinant IL-36Ra food-grade Lactococcus lactis expression plasmid constructed by the present invention is a food-grade expression vector; (2) The present invention uses a food-grade expression system to solve the problem of IL-36Ra protein being degraded in the digestive tract after entering the body orally. The probiotic Lactococcus lactis briefly colonizes the body after entering the body, and regular supplementation allows IL-36Ra to be continuously produced in the body. Moreover, the modified IL-36Ra sequence does not change its structure or lose its activity, and is highly expressed and active in the chassis cells, thereby exerting its effects safely and persistently. Compared with other expression vectors, it can express proteins for a longer period of time and at a higher content. (3) The advantages of the vector in the present invention are, among which the most important one is that compared with the IL-36Ra protein injection and plasmid tail vein injection in traditional mouse experiments, the expression plasmid has the advantages of low price and simple operation. Therefore, the present invention provides a new idea for in vivo gene therapy and the study of IL-36Ra gene function. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is a schematic diagram of the structure of the recombinant IL-36Ra food-grade Lactococcus lactis expression plasmid; Figure 2 The electrophoresis diagram is for the verification of HindⅢ restriction enzyme digestion of pNZ8149; Figure 3 This is the positive colony picture of successful electroporation; Figure 4 This is the negative colony image of electrotransfer failure; Figure 5 This is the result of bacterial liquid PCR; Figure 6 The graph shows the protein levels at different induction times; Lane 1 is the protein marker; Lanes 2-8 are purified proteins at different expression times; Lane 9 is the blank plasmid; Figure 7 It is a comparison chart of protein expression in Comparative Example 2; wherein lane 1 is the purified protein corresponding to SEQ ID NO.11; lane 2 is the purified protein corresponding to SEQ ID NO.12; lane 3 is the purified protein corresponding to SEQ ID NO.1; and lane 4 is the protein Marker. DETAILED DESCRIPTION

[0036] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and through specific implementation methods. However, the following examples are only simple examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0037] In the following examples, unless otherwise specified, all reagents and consumables used were purchased from conventional reagent manufacturers in the field; unless otherwise specified, all experimental methods and technical means used were conventional methods and means in the field.

[0038] Example 1 Design and synthesis of IL-36Ra encoding gene sequence Usp45 signal peptide, LEISS nonapeptide and 6×His tag were added to both ends of the IL-36Ra target gene.

[0039] Specifically, the Usp45 signal peptide, an important secretory peptide of Lactococcus lactis, and the LEISS nine-peptide amino acid sequence were added to the N-terminus to improve the secretion efficiency, yield and activity of the secreted protein of the recombinant bacteria, so that the secreted protein exists in the cell in a soluble form, and a 6-histidine tag was added to the C-terminus for subsequent affinity purification.

[0040] (1) Usp45 Amino acid sequence: MKKKIISAILMSTVILSAAAPLSGVYA (SEQ ID NO. 2) Base sequence: AAAAAAAAGATTATCTCAGCTATTTTAATGTCTACAGTGATACTTTCTGCTGCAGCCCCGTTGTCAGGTGTTTACGCT (SEQ ID NO.5) (2) LEISSTCDA nonapeptide Amino acid sequence: LEISSTCDA (SEQ ID NO. 3) Base sequence: CTCGAAATATCGTCGACTTGTGATGCA (SEQ ID NO.6) (3) 6×His Tag base sequence: CATCATCATCATCATCAC (SEQ ID NO. 8) Two restriction sites, Nco Ⅰ and Xba Ⅰ, were added at both ends for subsequent restriction digestion and ligation with the pNZ8149 vector. The ligation product of pNZ8149 and the target gene (such as Figure 1 pNZ8149 was digested with Hind Ⅲ to verify the result. Figure 2 shown.

[0041] Example 2 Preparation of NZ3900 competent cells (1) Take 200 μL of bacterial solution from the NZ3900 culture tube stored at -80°C and inoculate it into 5 mL of liquid GSGM17 medium. Place it in a constant temperature incubator and culture it at 30°C overnight. (2) Add 5 mL of the overnight culture medium to 45 mL of GSGM17 culture medium and incubate at 30°C for 12 h. (3) Take 50 mL of the above bacterial solution and transfer it into 200 mL of GSGM17 medium. Incubate at 30°C until the OD600 is about 0.2. (4) Transfer 50 mL of the cultured bacterial solution into a clean centrifuge tube and centrifuge at 4000 rpm, 4°C, for 20 min to collect the bacteria; (5) Wash once with 50 mL of ice-cold washing solution I (0.5 mol / L sucrose, 100 mL / L glycerol) at 4000 rpm, 4°C, for 20 min to collect the cells; (6) Resuspend the cells in 25 ml of ice-cold washing solution II (0.5 mol / L sucrose, 100 ml / L glycerol, 50 mM EDTA), place on ice for 15 min, incubate at 4000 rpm, 4°C for 20 min, and collect the cells. (7) Resuspend the cells in 12.5 ml of ice-cold Wash Buffer I and centrifuge at 4000 rpm at 4°C for 20 min to collect the cells. (8) Resuspend the cells in 0.5 mL of ice-cold Wash Buffer I and divide the suspension into 1.5 mL EP tubes that have been placed on ice, 50 μL per tube, and store at -80°C.

[0042] Example 3 Transformation of the recombinant vector into the Lactococcus lactis expression strain NZ3900 (1) Add 20 μL of the ligation product of pNZ8149 and the target gene to the NZ3900 competent cells, transfer to a pre-cooled 0.22 mm electroporation cuvette, and place on ice for 5 min; (2) Electric shock conditions: voltage 2500 V, resistance 200 Ω, pulse 25 µF, and electric shock time 6 ms; (3) After the electric shock, add GM17MC recovery medium to the electrode cup; (4) Place on ice for 10 min and at 30°C for 2 h; (5) After standing, take 200 μL of the above bacterial solution and spread it on the Elliker screening plate.

[0043] Using the lacF screening gene, the successfully constructed Lactococcus lactis can use Elliker to screen the lactose in the plate and produce lactic acid, causing the purple culture plate containing the bromocresol purple indicator to turn yellow, thereby screening for positive colonies carrying the recombinant plasmid.

[0044] The results of positive and negative colonies are shown in Figure 3 , Figure 4 .

[0045] Example 4 Identification of positive colonies Select positive clones and perform colony PCR identification. The identification primer sequences are as follows: pNZ8149-36RaF: AGCATAATAAACGGCTCTG (SEQ ID NO.9) pNZ8149-36RaR: TGAACCACCTTGAACACC (SEQ ID NO.10) The positive colony PCR identification results are shown in Figure 5 .

[0046] Comparing the lengths of PCR products obtained from positive clone colonies, the results showed that they were all about 400 bp.

[0047] Example 5 In this example, the obtained positive colonies were cultured, induced and protein purified.

[0048] The specific steps are as follows: a single colony of successfully constructed recombinant lactic acid bacteria is selected, inoculated and cultured until OD600 is 0.6, the inducer Nisin is added to a predetermined final concentration, and the inducer is allowed to stand for induction. After the induction is completed, the product is centrifuged to collect the bacterial precipitate.

[0049] The cells were washed, the cell pellet was resuspended and frozen, and then ultrasonically disrupted after repeated freezing and thawing. The supernatant was purified by affinity chromatography, wherein the HIS tag was bound to the chromatography column, and then eluted with imidazole elution solution to obtain the purified protein, which was then analyzed by Western Blot.

[0050] The results of protein expression and purification are as follows Figure 6 As shown, lane 1 is the protein marker; lanes 2-8 are purified proteins at different expression times (2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 24 h); lane 9 is a blank plasmid; it can be seen from the protein gel image that the recombinant IL-36Ra is obviously expressed.

[0051] Comparative Example 1 During the design and synthesis of the IL-36Ra encoding gene sequence, the N-terminal methionine of the amino acid sequence was not removed (i.e., a methionine was added to the N-terminus of the SEQ ID NO.1 sequence). Experimental comparison showed that compared with the expression system constructed in the embodiment, its protein expression level and activity were significantly lower.

[0052] Comparative Example 2 In the present invention, two other IL-36Ra amino acid sequences described in NCBI (as shown in SEQ ID NO.11 and SEQ ID NO.12) were used to construct an expression system using the same method as SEQ ID NO.1 selected in the present invention.

[0053] SEQ ID NO.11: VLSGALCFRMKDSALKVLYLHNNQLLAGGLHAEKVIKGEEISVVPNRALDASLSPVILGVQGGSQCLSCGTEKGPILKLEPVNIMELYLGAKESKSFTFYRRDMGLTSSFESAAYPGWFLCTSPEADQPVSLTQIPEDPTCDAPITDFYFQQC (two methionine residues at the N-terminus have been removed, with a similarity of 98% to SEQ ID NO.1) SEQ ID NO.12: VLSGALCFRMKDSALKVLYLHDNQLLAGGLHAGKVIKGEEISVVPNRSLDASLSPVILGVQGGSQCLSCGTEKEPILKLEPVNIMELYLGTKESKSFTFYRRDMGLTSSFESAAYPGWFLCTAPEADQPVRLTQIPEDAAWDAPITDFYFQQC (N-terminal methionine has been removed, with a similarity of 95% to SEQ ID NO.1) Therefore, this comparative example proves that the protein expression level of other IL-36Ra amino acid sequences disclosed in NCBI is lower than that of the present application, such as Figure 7 As shown, although both of them expressed proteins at the location of the target protein, the expression levels were significantly lower.

[0054] The results show that the expression system constructed by the present invention is compatible with the selected amino acid sequence, and the two are adapted to each other. The possible reason for the significantly lower expression level when other protein sequences are used is that the removal of the methyl thio sequence causes the spatial structure of the protein to change or there is a mutual exclusion effect between the protein and the expression system, resulting in a lower expression level.

[0055] In summary, the present invention transforms the IL-36Ra amino acid sequence selected from NCBI to construct an expression vector that can highly express the IL-36Ra target protein with high activity. At the same time, the expression system is a food-grade Lactococcus lactis, which colonizes briefly after entering the body, and regular supplementation can enable IL-36Ra to be continuously produced in the body, providing a new idea for in vivo gene therapy and the study of IL-36Ra gene function.

[0056] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. A recombinant IL-36Ra food-grade Lactococcus lactis expression plasmid, characterized in that: The expression plasmid contains a base fragment encoding the amino acid sequence shown in SEQ ID NO.1; The amino acid sequence shown in SEQ ID NO.1 is an interleukin-36 receptor antagonist.

2. The expression plasmid according to claim 1, characterized in that The expression plasmid also comprises a base fragment encoding a Usp45 signal peptide and / or a LEISS nonapeptide.

3. The expression plasmid according to claim 1, characterized in that The expression plasmid is a food-grade intracellular expression vector pNZ8149 of Lactococcus lactis; The expression plasmid also includes a LacF screening gene; The expression plasmid also includes a His tag.

4. The expression plasmid according to claim 1, characterized in that In the expression plasmid, the base sequence encoding the IL-36Ra amino acid sequence is shown in SEQ ID NO.4; The base sequence encoding the Usp45 signal peptide is shown in SEQ ID NO.5; The base sequence encoding the amino acid sequence of LEISS nonapeptide is shown in SEQ ID NO.

6.

5. A method for preparing a recombinant IL-36Ra food-grade Lactococcus lactis expression plasmid according to any one of claims 1 to 4, characterized in that: The preparation method comprises: A gene fragment expressing IL-36Ra is constructed, and then the gene fragment is connected to a Lactococcus lactis food-grade intracellular expression vector and transformed into Lactococcus lactis.

6. The preparation method according to claim 5, characterized in that: The steps of constructing the gene fragment expressing IL-36Ra are: After the gene fragment encoding IL-36Ra was cut, the base sequence encoding the Usp45 signal peptide and the LEISS nonapeptide was added to the N-terminus, and a histidine tag was added to the C-terminus.

7. The preparation method according to claim 5 or 6, characterized in that: The steps include: First, the IL-36Ra encoding gene is constructed on a cloning vector; the IL-36Ra encoding gene comprises the base sequence as described in SEQ ID NO.4, and the N-terminus of the sequence comprises the base sequence encoding the Usp45 signal peptide and the LEISS nonapeptide; Then, the recombinant plasmid produced by the cloning vector was cloned and extracted, and the obtained base sequence was connected to the expression vector pNZ8149 with the LacF screening gene after enzyme digestion to construct the recombinant IL-36Ra food-grade Lactococcus lactis expression plasmid.

8. A food-grade Lactococcus lactis expression system for recombinant IL-36Ra, characterized in that: The expression system comprises the expression vector according to any one of claims 1 to 4; The chassis cell of the expression system is Lactococcus lactis NZ3900.

9. Use of the expression plasmid according to any one of claims 1 to 4, the preparation method according to any one of claims 5 to 7, or the expression system according to claim 8 in the preparation of a medicament comprising IL-36Ra.

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