Signal peptide for promoting OMP25 expression, fusion protein containing signal peptide and application

Abstract

The invention provides a signal peptide for promoting OMP25 expression, a fusion protein containing the signal peptide and application, and belongs to the technical field of gene engineering. The signal peptide comprises a tPAa signal peptide or a tPAs signal peptide; the amino acid sequence of the tPAa signal peptide is as shown in SEQ ID NO.2, and the amino acid sequence of the tPAs signal peptide is as shown in SEQ ID NO.3. According to the invention, the secretion efficiency of the Brucella outer membrane protein OMP25 in a eukaryotic expression system is obviously improved by utilizing the signal peptide, and the fusion protein containing the signal peptide also enhances the antigen presentation capability of macrophages, induces Th1 type immune response and drives the macrophages to be polarized to M1 type phenotype, so that the immunogenicity of the OMP25 is obviously improved. The invention provides an efficient antigen preparation method for the development of brucellosis vaccines, and has important application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and in particular relates to a signal peptide for promoting OMP25 expression, a fusion protein containing the signal peptide, and applications thereof. Background Art

[0002] Brucellosis (abbreviated as "brucellosis") is a zoonotic infectious disease prevalent worldwide caused by infection with the Gram-negative bacterium Brucella. Brucellosis in animals is mainly transmitted vertically and can cause miscarriage, premature birth and stillbirth in livestock, resulting in high economic losses each year. Human brucellosis is mostly caused by direct contact with infected livestock and accidental ingestion of contaminated meat and dairy products. The clinical manifestations of human brucellosis are non-specific and complex and varied, including emaciation, fatigue, undulant fever, loss of appetite, impotence, hepatosplenomegaly, etc., which can be easily confused with diseases such as influenza. In addition, Brucella has potential properties as a biological warfare agent and also poses major risks in terms of biosafety. Therefore, it is urgent to develop an effective, safe vaccine that can prevent and control brucellosis.

[0003] Vaccination is the most effective means of preventing and controlling brucellosis. Currently, there is no clinically effective human vaccine with long-term efficacy. Skin-scratch vaccines used on humans cause a painful experience for recipients, significantly reducing their acceptance and compliance, and hindering the promotion and popularization of this vaccine. Currently available live attenuated vaccines include B. melitensis Rev.1, B. abortus RB51, B. suis S2, B. melitensis M5, and B. abortus A19. However, these vaccines have certain drawbacks, such as residual virulence, interference with serological test results after vaccination, inability to identify vaccine strains and wild-type strains, and the RB51 live attenuated vaccine carrying a resistance gene to rifampicin, the first-line drug for treating brucellosis, which affects the effectiveness of antibiotic treatment. OMP25 is a key outer membrane protein of Brucella. Among the many Brucella antigens, its native form has relatively low secretion efficiency and immunogenicity in eukaryotic systems, which greatly restricts its application in vaccine research and development.

[0004] In the research scope of molecular biology and biotechnology, signal peptides are key elements that guide protein secretion and expression. Their structural and functional characteristics have a decisive influence on the final fate of proteins. Most secretory proteins in the biological world (eukaryotes and prokaryotes) carry a short chain of 16 to 30 amino acid residues at their amino termini (N termini), which is called a signal peptide. The signal peptide is mainly composed of three regions, namely the N region, the H region, and the C region. The three regions together construct the classic structure of the signal peptide. The signal peptide can not only guide the newly formed peptide chain into the endoplasmic reticulum or other organelles, but its structural domain also has high immunogenicity and is rich in CD8. +T cell epitopes can significantly enhance antigen presentation efficiency and immune recognition efficacy. Numerous previous studies have demonstrated that structural modification of signal peptides using modern molecular biology techniques can significantly improve antigen expression levels and functional activity. However, the question remains: what signal peptide sequence should be used to effectively overcome the difficulties in expressing the membrane protein OMP25 in eukaryotic expression systems? Summary of the Invention

[0005] In view of this, the object of the present invention is to provide a signal peptide for promoting the expression of OMP25, a fusion protein containing the signal peptide, and its application, wherein the signal peptide can significantly promote the expression level of OMP25.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a signal peptide for promoting OMP25 expression, wherein the signal peptide comprises a tPAa signal peptide or a tPAs signal peptide; the amino acid sequence of the tPAa signal peptide is shown in SEQ ID NO.2, and the amino acid sequence of the tPAs signal peptide is shown in SEQ ID NO.3.

[0008] The present invention provides a nucleotide encoding the signal peptide for promoting the expression of OMP25.

[0009] Preferably, the nucleotide sequence encoding the tPAa signal peptide is shown as SEQ ID NO.4; the nucleotide sequence encoding the tPAs signal peptide is shown as SEQ ID NO.5.

[0010] The present invention provides an OMP25 fusion protein containing the above-mentioned signal peptide, wherein the fusion protein comprises tPAa-OMP25 or tPAs-OMP25; the amino acid sequence of the OMP25 is shown in SEQ ID NO.8.

[0011] Preferably, the nucleotide sequence of the tPAa-OMP25 is shown as SEQ ID NO.13; the nucleotide sequence of the tPAs-OMP25 is shown as SEQ ID NO.14.

[0012] The present invention provides a biomaterial expressing OMP25, wherein the biomaterial is any one of the following:

[0013] (1) an expression cassette containing the above-mentioned fusion protein;

[0014] (2) a recombinant plasmid containing the expression cassette described in (1);

[0015] (3) recombinant cells containing the above-mentioned fusion protein;

[0016] (4) a recombinant cell containing the expression cassette described in (1);

[0017] (5) A recombinant cell containing the recombinant plasmid described in (2).

[0018] The present invention provides the use of the above-mentioned signal peptide, nucleotide or biological material in the preparation of OMP25.

[0019] The present invention provides an application of the above-mentioned biomaterial in preparing an immunity-enhancing product.

[0020] Preferably, the biomaterial significantly enhances the antigen presenting ability of macrophages, induces Th1 immune response, and drives macrophage polarization toward M1 phenotype, thereby enhancing their anti-infection ability.

[0021] The present invention provides a method for preparing OMP25, comprising the following steps: transfecting the recombinant plasmid into host cells, culturing the obtained recombinant cells, and collecting the supernatant of the recombinant cells to obtain OMP25.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention provides a signal peptide that promotes OMP25 expression, a fusion protein containing the signal peptide, and its applications. The present invention utilizes the signal peptide to significantly improve the secretion efficiency of the Brucella outer membrane protein OMP25 in a eukaryotic expression system. The signal peptide-containing fusion protein also enhances macrophage antigen presentation, induces a Th1 immune response, and drives macrophage polarization toward an M1 phenotype, thereby significantly improving the immunogenicity of OMP25. This invention provides a highly efficient antigen preparation method for the development of brucellosis vaccines and has important application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The 2% agarose gel electrophoresis diagram of the sequences of the recombinant plasmids pIRES2-EGFP-tPAa-OMP25 (tPAa), pIRES2-EGFP-tPAs-OMP25 (tPAs), pIRES2-EGFP-OMP25-1 (sP25), and pIRES2-EGFP-OMP25-2 (nsP25) after double digestion with EcoRI and XhoI, as shown in SEQ ID NOs. 13-14 and SEQ ID NOs. 9-10, M: Marker (GeneRuler DNA Ladder Mix, 2894959, Thermo Fisher Scientific (China) Co., Ltd.); 1: sP25; 2: nsP25; 3: tPAa; 4: tPAs;

[0025] Figure 2Images of GFP (green fluorescent protein) expression in RAW264.7 cells after transfection of recombinant plasmids pIRES2-EGFP-tPAa-OMP25 (tPAa), pIRES2-EGFP-tPAs-OMP25 (tPAs), pIRES2-EGFP-OMP25-1 (sP25), and pIRES2-EGFP-OMP25-2 (nsP25);

[0026] Figure 3 RAW264.7 cells were transfected with recombinant plasmids pIRES2-EGFP-tPAa-OMP25 (tPAa), pIRES2-EGFP-tPAs-OMP25 (tPAs), pIRES2-EGFP-OMP25-1 (sP25), and pIRES2-EGFP-OMP25-2 (nsP25) for 48 hours, and the expression levels of OMP25 proteins in the cell culture supernatant were detected. A is the electrophoresis diagram of OMP25 protein expression in different groups, and B is the relative expression levels of OMP25 proteins in different groups.

[0027] Figure 4 The results are compared by flow cytometry on the expression levels of immune-related indicators TNF-α and IL-6 in RAW264.7 cells after transfection of different recombinant plasmids.

[0028] Figure 5 The results of flow cytometry detection of antigen presenting molecules MHC I and MHC II expression levels in different groups are compared. A is a scatter plot of cells expressing antigen presenting molecules MHC I and MHC II in different groups by flow cytometry detection; B is a bar graph comparing the expression levels of MHC I and MHC II in different groups;

[0029] Figure 6 The results of flow cytometry detection of the expression levels of immune activation-related factors and polarization markers CD86, CD80, C206, iNOS, and Arg-1 in different groups are compared. A is a scatter plot of the expression of immune activation-related factors and polarization markers CD86, CD80, C206, iNOS, and Arg-1 in different groups by flow cytometry detection; B is a bar graph comparison of the expression of immune activation-related factors and polarization markers CD86, CD80, C206, iNOS, and Arg-1 in different groups;

[0030] Figure 7The comparison results of the percentage of positive cells of proinflammatory cytokines IL-1β and IL-12 detected by flow cytometry in different groups. A is the cell scatter plot of the expression of proinflammatory cytokines IL-1β and IL-12 detected by flow cytometry in different groups; B is the bar chart comparison results of the proinflammatory cytokines IL-1β and IL-12 in different groups. DETAILED DESCRIPTION

[0031] The present invention provides a signal peptide for promoting OMP25 expression, wherein the signal peptide comprises a tPAa signal peptide or a tPAs signal peptide; the amino acid sequence of the tPAa signal peptide is shown in SEQ ID NO.2, and the amino acid sequence of the tPAs signal peptide is shown in SEQ ID NO.3.

[0032] The tPAa signal peptide or tPAs signal peptide described herein is a tPA signal peptide mutant. The tPAa signal peptide or tPAs signal peptide is obtained by mutating the proline (P) at position 17 of the wild-type tissue plasminogen activator (tPA) signal peptide to alanine (A) or serine (S), respectively. The amino acid sequence of the tPA wild-type signal peptide is shown in SEQ ID NO. 1. The present invention has found that, compared with the tPA wild-type signal peptide, the use of the tPAa signal peptide or tPAs signal peptide significantly improves the secretion efficiency of OMP25 in a eukaryotic expression system.

[0033] The present invention provides a nucleotide encoding the signal peptide for promoting the expression of OMP25.

[0034] In the present invention, the nucleotide sequence encoding the tPAa signal peptide is shown as SEQ ID NO.4; the nucleotide sequence encoding the tPAs signal peptide is shown as SEQ ID NO.5.

[0035] The present invention provides an OMP25 fusion protein containing the above-mentioned signal peptide, wherein the fusion protein comprises tPAa-OMP25 or tPAs-OMP25; the amino acid sequence of the OMP25 is shown in SEQ ID NO.8.

[0036] In the present invention, the tPAa-OMP25 is formed by directly linking the tPAa amino acid sequence shown in SEQ ID NO. 2 to the amino terminus (N-terminus) of the OMP25 amino acid sequence shown in SEQ ID NO. 8, and the tPAs-OMP25 is formed by directly linking the tPAs amino acid sequence shown in SEQ ID NO. 3 to the N-terminus of the OMP25 amino acid sequence shown in SEQ ID NO. 8, i.e., the tPA signal peptide sequence is located upstream of the OMP25 gene sequence. As a preferred embodiment, the nucleotide sequence of the tPAa-OMP25 is shown in SEQ ID NO. 13; the nucleotide sequence of the tPAs-OMP25 is shown in SEQ ID NO. 14.

[0037] The present invention provides a biomaterial expressing OMP25, wherein the biomaterial is any one of the following:

[0038] (1) an expression cassette containing the above-mentioned fusion protein;

[0039] (2) a recombinant plasmid containing the expression cassette described in (1);

[0040] (3) recombinant cells containing the above-mentioned fusion protein;

[0041] (4) a recombinant cell containing the expression cassette described in (1);

[0042] (5) A recombinant cell containing the recombinant plasmid described in (2).

[0043] In the present invention, the backbone plasmid of the recombinant plasmid is a eukaryotic expression vector, such as pIRES2-EGFP. The present invention does not specifically limit the source of pIRES2-EGFP, and commercially available products in the art can be used. The preparation of the recombinant plasmid comprises the following steps: tPAa-OMP25 as shown in SEQ ID NO.13 or tPAs-OMP25 as shown in SEQ ID NO.14 is connected between the XhoI and EcoRI restriction sites of pIRES2-EGFP. The recombinant plasmid is named pIRES2-EGFP-tPAa-OMP25 or pIRES2-EGFP-tPAs-OMP25. The recombinant cell is preferably a eukaryotic cell, such as macrophage RAW264.7, and the recombinant cell is prepared by transfecting the above-mentioned recombinant plasmid into a eukaryotic cell. The recombinant plasmid drives the expression of tPAa-OMP25 or tPAs-OMP25 in eukaryotic cells (such as RAW264.7) via a CMV promoter, and the transfection efficiency is visualized using the pIRES-EGFP system. After transfection of eukaryotic cells (such as RAW264.7), the recombinant plasmid can efficiently secrete the Omp25 protein extracellularly and induce a strong Th1 immune response.

[0044] The present invention provides the use of the above-mentioned signal peptide, nucleotide or biological material in the preparation of OMP25.

[0045] The present invention provides an application of the above-mentioned biomaterial in preparing an immunity-enhancing product.

[0046] In the present invention, the biomaterial significantly enhances the antigen presenting ability of macrophages, induces Th1 immune response, and drives macrophages to polarize toward M1 phenotype, thereby enhancing their anti-infection ability.

[0047] The present invention provides a method for preparing OMP25, comprising the steps of transfecting the recombinant plasmid into a host cell, obtaining the recombinant cell, culturing the cell, and collecting the supernatant of the recombinant cell to obtain OMP25.

[0048] In the above-mentioned method for preparing OMP25, the host cells secrete and express OMP25 protein in the culture supernatant, and the protein expression level is increased by about 2 to 3.8 times compared with the expression vector of sp25 (amino acid sequence as shown in SEQ ID NO.7). The polarization state of macrophages is judged by detecting the expression levels of iNOS and Arg-1. In the group transfected with the recombinant plasmid (such as pIRES2-EGFP-tPAa-OMP25 or pIRES2-EGFP-tPAs-OMP25), iNOS expression is upregulated while Arg-1 expression is downregulated, indicating a trend towards M1 polarization; in the group transfected with the recombinant plasmid (such as pIRES2-EGFP-tPAa-OMP25 or pIRES2-EGFP-tPAs-OMP25), MHCI and MHC expression are upregulated. The expression of OMP25 molecules was upregulated, indicating that the fusion protein (tPAa-OMP25 or tPAs-OMP25 fusion protein) enhanced the antigen presentation ability of RAW264.7 cells. The expression of IL-12 and IL-1β was significantly increased, indicating that the fusion protein induced a Th1 immune response. The expression of CD80 and CD86 co-stimulatory molecules was upregulated, indicating that the fusion protein enhanced the ability of macrophages to activate T cells. CD206 expression was downregulated, further supporting the trend of macrophages toward an M1 phenotype. In addition, multiple signal peptides were screened in preliminary experiments, including those derived from CD80, C3, and CCL3, but none of these signal peptides significantly increased OMP25 expression or immune activation ability and were therefore not used in the formal experiments.

[0049] In the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art.

[0050] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0051] In the following examples, the complete culture medium without antibiotics was prepared by uniformly mixing 10 mL of pretreated fetal bovine serum with high-glucose DMEM.

[0052] Example 1

[0053] Construction of recombinant expression vector of signal peptide OMP25

[0054] The present invention designed tPA mutant signal peptides SEQ ID NO. 2 and SEQ ID NO. 3, and compared them with the wild-type signal peptide SEQ ID NO. 1.

[0055] tPA wild-type signal peptide SEQ ID NO. 1: MKRELLCVLLLCGLAFP (GenBank: AAH11256.1);

[0056] tPAa signal peptide SEQ ID NO. 2: MKRELLCVLLLCGLAFA;

[0057] tPAs signal peptide SEQ ID NO.3: MKRELLCVLLLCGLAFS;

[0058] tPA signal peptide nucleotide sequence SEQ ID NO.4:

[0059] 5'-ATGAAGAGAGAGCTGCTGTGTGTACTGCTGCTTTGTGGACTGGCTTTCCCA-3';

[0060] tPAa signal peptide nucleotide sequence SEQ ID NO.5:

[0061] 5'-ATGAAGAGAGAGCTGCTGTGTGTACTGCTGCTTTGTGGACTGGCTTTCGCC-3';

[0062] tPAs signal peptide nucleotide sequence SEQ ID NO.6:

[0063] 5'-ATGAAGAGAGAGCTGCTGTGTGTACTGCTGCTTTGTGGACTGGCCTTCAGC-3';

[0064] OMP25 amino acid sequence SEQ ID NO.7:

[0065] MRTLKSLVIVSAALLPFSATAFAADAIQEQPPVPAPVEVAPQYSWAGGYTGLYLGYGWNKAKTSTVGSIKPDDWKAGAFAGWNFQQDQIVYGVEGDAGYSWAKKSKD GLEVKQGFEGSLRARVGYDLNPVMPYLTAGIAGSQIKLNNGLDDESKFRVGWTAGAGLEAKLTDNILGRVEYRYTQYGNKNYDLAGTTVRNKLDTQDFRVGIGYKF;

[0066] The amino acid sequence of OMP25 without the wild-type signal peptide is SEQ ID NO.8:

[0067] ADAIQEQPPVPAPVEVAPQYSWAGGYTGLYLGYGWNKAKTSTVGSIKPDDWKAGAFAGWNFQ QDQIVYGVEGDAGYSWAKKSKDGLEVKQGFEGSLRARVGYDLNPVMPYLTAGIAGSQIKLNNGLDD ESKFRVGWTAGAGLEAKLTDNILGRVEYRYTQYGNKNYDLAGTTVRNKLDTQDFRVGIGYKF;

[0068] Wild-type OMP25 nucleotide sequence SEQ ID NO.9:

[0069] ATGCGCACTCTTAAGTCTCTCGTAATCGTCTCGGCTGCGTTGCTGCCGTTCTCTGCGACCGCTTTTGCTGCCGACGCCATCCAGGAACAGCCTCCGGTTCCGGCTCCGGTTGAAGTAGCTCCCCAGTATAGCTGGGCTGGTGGCTATACCGGTCTTTACCTTGGCTACGGCTGGAACAAGGCCAAGACCAGCACCGTTGGCAGCATCAAGCCTGACGATTGGAAGGCTGGCGCCTTTGCTGGCTGGAACTTCCAGCAGGACCAGATCGTATACGGCGTTGAAGGTGATGCAGGTTATTCCTGGGCCAAGAAGTCCAAGGACGGCCTGGAAGTCAAGCAGGGCTTTGAAGGCTCGCTGCGTGCCCGCGTTGGCTACGACCTGAACCCGGTTATGCCGTACCTCACGGCTGGTATTGCCGGTTCGCAGATCAAGCTTAACAACGGCTTGGACGACGAAAGCAAGTTCCGCGTGGGTTGGACGGCTGGTGCCGGTCTCGAAGCCAAGCTGACGGACAACATCCTCGGCCGCGTTGAGTACCGTTACACCCAGTACGGCAACAAGAACTATGATCTGGCCGGTACGACTGTTCGCAACAAGCTGGACACGCAGGATTTCCGCGTCGGCATCGGCTACAAGTTCTAA;

[0070] OMP25 nucleotide sequence without the wild-type signal peptide SEQ ID NO.10:

[0071] GCCGACGCCATCCAGGAACAGCCTCCGGTTCCGGCTCCGGTTGAAGTAGCTCCCCAGTATAGCTGGGCTGGTGGCTATACCGGTCTTTACCTTGGCTACGGCTGGAACAAGGCCAAGACCAGCACCGTTGGCAGCATCAAGCCTGACGATTGGAAGGCTGGCGCCTTTGCTGGCTGGAACTTCCAGCAGGACCAGATCGTATACGGCGTTGAAGGTGATGCAGGTTATTCCTGGGCCAAGAAGTCCAAGGACGGCCTGGAAGTCAAGCAGGGCTTTGAAGGCTCGCTGCGTGCCCGCGTTGGCTACGACCTGAACCCGGTTATGCCGTACCTCACGGCTGGTATTGCCGGTTCGCAGATCAAGCTTAACAACGGCTTGGACGACGAAAGCAAGTTCCGCGTGGGTTGGACGGCTGGTGCCGGTCTCGAAGCCAAGCTGACGGACAACATCCTCGGCCGCGTTGAGTACCGTTACACCCAGTACGGCAACAAGAACTATGATCTGGCCGGTACGACTGTTCGCAACAAGCTGGACACGCAGGATTTCCGCGTCGGCATCGGCTACAAGTTCTAA;

[0072] The amino acid sequence of the wild-type signal peptide of OMP25, SEQ ID NO.11: MRTLKSLVIVSAALLPFSATAFA;

[0073] The nucleotide sequence of the wild-type signal peptide of OMP25, SEQ ID NO.12:

[0074] 5’-ATGCGCACTCTTAAGTCTCTCGTAATCGTCTCGGCTGCGTTGCTGCCGTTCTCTGCGACCGCTTTTGCT-3’

[0075] The codon-optimized nucleotide sequence of the tPAa-OMP25 fragment, SEQ ID NO.13:

[0076] ATGAAGAGAGAGCTGCTGTGTGTGCTGCTGCTGTGCGGCCTGGCCTTCGCCGCTGATGCCATCCAGGAGCAGCCTCCTGTGCCCGCCCCTGTGGAGGTGGCTCCACAGTACTCCTGGGCCGGCGGCTACACAGGCCTGTACCTGGGCTACGGCTGGAACAAGGCCAAGACATCCACAGTGGGCTCCATCAAGCCCGATGACTGGAAGGCCGGCGCCTTCGCCGGCTGGAACTTCCAGCAGGACCAGATCGTGTACGGCGTGGAGGGCGACGCCGGCTACTCCTGGGCTAAGAAGAGCAAGGACGGCCTGGAGGTGAAGCAGGGCTTCGAGGGCTCCCTGAGGGCCAGAGTGGGCTACGACCTGAACCCCGTGATGCCCTACCTGACAGCCGGCATCGCCGGCTCCCAGATCAAGCTGAACAACGGCCTGGATGATGAGAGCAAGTTCAGGGTGGGCTGGACCGCCGGCGCCGGACTGGAGGCTAAGCTGACCGACAACATCCTGGGCAGGGTGGAGTACAGGTACACACAGTACGGCAACAAGAACTACGACCTGGCCGGCACCACAGTGAGAAACAAGCTGGACACCCAGGATTTCAGAGTGGGCATCGGCTACAAGTTCCACCACCACCACCATCACTAA;

[0077] The codon-optimized nucleotide sequence of the tPAs-OMP25 fragment SEQ ID NO.14:

[0078] ATGAAGAGGGAGCTGCTGTGCGTGCTGCTGCTGTGTGGCCTGGCCTTCAGCGCCGACGCCATCCAGGAGCAGCCTCCTGTGCCCGCCCCTGTGGAGGTGGCTCCTCAGTACAGCTGGGCCGGCGGCTACACCGGCCTGTACCTGGGATACGGCTGGAACAAGGCCAAGACCAGCACAGTGGGCTCCATCAAGCCCGATGACTGGAAGGCCGGCGCCTTCGCCGGCTGGAACTTCCAGCAGGATCAGATCGTGTACGGCGTGGAGGGCGATGCCGGCTACAGCTGGGCTAAGAAGAGCAAGGACGGCCTGGAGGTGAAGCAGGGCTTCGAGGGCTCCCTGAGAGCCAGAGTGGGCTACGATCTGAACCCTGTGATGCCCTACCTGACCGCCGGCATCGCCGGCTCTCAGATCAAGCTGAACAACGGCCTGGATGATGAGTCCAAGTTCAGAGTGGGCTGGACCGCCGGCGCCGGACTGGAGGCTAAGCTGACAGATAACATCCTGGGCAGAGTGGAGTACAGGTACACACAGTACGGCAACAAGAACTACGACCTGGCCGGCACCACAGTGAGAAACAAGCTGGACACACAGGACTTCAGAGTGGGAATCGGCTACAAGTTCCACCACCACCACCATCACTAA;

[0079] Primers covering the full length of the target sequences SEQ ID NOs. 9-10 and SEQ ID NOs. 13-14 were designed using small fragment primers. Each primer was 60-80 bp long, with a 20 bp overlapping region (homologous arm). Through gel recovery and overlapping PCR amplification, the target fragments were cloned into the eukaryotic expression vector pIRES2-EGFP between the Xhol and EcoRI restriction sites by seamless cloning and homologous recombination. Four sets of plasmids were ultimately obtained: pIRES2-EGFP-tPAa-OMP25 (tPAa), pIRES2-EGFP-tPAs-OMP25 (tPAs), pIRES2-EGFP-OMP25-1 (wild type, sP25), and pIRES2-EGFP-OMP25-2 (signal peptide removed, nsP25). The pIRES2-EGFP-tPAa-OMP25 and pIRES2-EGFP-tPAs-OMP25 plasmids were synthesized by General Biotechnology (Anhui) Co., Ltd. (order number G0349964), and the pIRES2-EGFP-OMP25-1 and pIRES2-EGFP-OMP25-2 were synthesized by Sangon Biotechnology (Shanghai) Co., Ltd. (order numbers CK20036FABX37203-10; CK20037F ABX37203-11).

[0080] The target fragments were verified by sequencing using an endotoxin-free plasmid extraction kit (DP120-01, Tiangen Biochemical Technology (Beijing) Co., Ltd.), and the target fragments were double-digested with EcoRI and XhoI to verify the target fragments of tPAa-OMP25, tPAs-OMP25, wild-type OMP25, and OMP25 with the wild-type signal peptide removed, as well as the endotoxin content. The enzyme digestion diagram is shown in Figure 2. Figure 1

[0081] Each target fragment was sequenced, and the sequences of wild-type OMP25, OMP25 without the wild-type signal peptide, tPAa-OMP25, and tPAs-OMP25 were consistent with the sequences described in SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.13, and SEQ ID NO.14, respectively.

[0082] Figure 1 The results showed that the target band size was consistent with the expected value, indicating that the target fragment had been successfully cloned into the vector.

[0083] In summary, it was proved that the recombinant vectors of various purposes were successfully constructed.

[0084] Example 2 Liposome transfection experiment

[0085] In this example, the constructed recombinant plasmid was transfected into mouse RAW264.7 cells using liposome transfection. The specific steps are as follows:

[0086] Fetal bovine serum (FBS) pretreatment: The fetal bovine serum used needs to be heat-inactivated at 56°C for 30 minutes to remove complement and obtain pretreated fetal bovine serum to reduce its nonspecific effects on the entire system.

[0087] Cell seeding: One day before transfection, RAW264.7 cells were seeded at 1.5×10 5 The cells were seeded into 12-well plates at a density of 100 cells / well, and complete culture medium without antibiotics was added. The cells were cultured at 37°C and 5% CO2.

[0088] Medium Replacement: On the day of transfection, aspirate and replace the original medium with serum-free, high-glucose DMEM pre-warmed at 37°C 2 hours in advance to minimize the effects of serum on transfection efficiency. Initiate transfection after the cells reach 70%-80% confluence and normal morphology.

[0089] Liposome transfection: Liposomes were prepared according to the ratio recommended in the reagent manual (liposome: plasmid = 2.25 μL: 1 μg) and transfected with pIRES2-EGFP-tPAa-OMP25, pIRES2-EGFP-tPAs-OMP25, pIRES2-EGFP-OMP25-1 (sP25) and pIRES2-EGFP-OMP25-2 (nsP25) prepared in Example 1 for 48 hours to obtain transfected RAW264.7 cells. 2 hours before adding the transfectant plasmid, replace it with serum-free high-glucose DMEM, and then replace it with complete culture medium after 6 hours. Confirm by conventional methods that the cells are in good condition, without obvious toxic reactions, and suitable for subsequent expression and analysis experiments. The transfection effect is shown in Figure 2 .

[0090] Figure 2 The results showed that 48 hours after the recombinant plasmids pIRES2-EGFP-tPAa-OMP25, pIRES2-EGFP-tPAs-OMP25, sP25 and nsP25 were transfected into RAW264.7 cells, the GFP (green fluorescent protein) expression images observed under a fluorescence microscope indicated that the plasmids were successfully transfected and expressed, the GFP fluorescence showed well, and the transfection efficiency was strong, which were 56.2%, 57.4%, 57.8% and 56.5% respectively.

[0091] Example 3 Western blot detection

[0092] In this example, the expression of the target protein in RAW264.7 cells transfected with the vectors described in Example 2 was detected by Western blot to evaluate the effects of different constructed vectors on the expression of the OMP25 protein.

[0093] The recombinant plasmids were transfected into RAW264.7 cells, and the cell culture supernatant (S) was collected 48 hours after transfection for analysis of the expression of secreted OMP25 protein.

[0094] The expression levels of OMP25 protein in the cell culture supernatant were detected using conventional Western blot methods, including BCA quantification, electrophoresis transfer (wet transfer method), antibody incubation and color development.

[0095] Figure 3 The results showed that compared with the sP25 group, both pIRES2-EGFP-tPAa-OMP25 and pIRES2-EGFP-tPAs-OMP25 could significantly enhance the expression and secretion capacity of OMP25 protein, and the expression level of OMP25 protein increased by 2 to 3.8 times.

[0096] Example 4 Flow cytometry detection of immune indicators

[0097] The pIRES2-EGFP-tPAa-OMP25 (tPAa), pIRES2-EGFP-tPAs-OMP25 (tPAs), pIRES2-EGFP-OMP25-1 (sP25), and pIRES2-EGFP-OMP25-2 (nsP25) recombinant plasmids of Example 2 were transfected into RAW264.7 cells. 48 hours after transfection, the supernatants of the transfected RAW264.7 cells (respectively named tPAa, tPAs, sP25, and nsP25 groups) were collected and used for flow cytometry detection. Two strategies were used in the experiment:

[0098] First, using the CBA mouse cytokine detection kit (Cat. No. ABI560485, BD Biosciences (USA), cell culture supernatant was collected 48 hours after transfection, and the cytokine levels in the supernatant were dynamically detected to detect the secretion levels of pro-inflammatory cytokines (IL-6, TNF-α).

[0099] Figure 4 The results showed that compared with the control group sP25, the expression of proinflammatory cytokines (IL-6 and TNF-α) showed different degrees of upregulation.

[0100] Second, 48 hours after transfection, the collected transfected RAW264.7 cells were stained using conventional flow cytometry to detect surface and intracellular immune molecule expression, including indicators of antigen presenting molecules (MHC I, MHC II), immune activation-related factors and macrophage polarization markers (CD86, CD80, C206, iNOS, Arg-1), and proinflammatory cytokines (IL-1β, IL-12). Vector refers to the empty vector group (RAW264.7 cells were transfected with the empty vector pIRES2-EGFP according to the method described in Example 2 for 48 hours); Blank refers to RAW264.7 cells cultured normally in DMEM medium with 10% fetal bovine serum, which served as a blank control.

[0101] Figure 5 The results showed that compared with the control group sP25, both tPAa and tPAs could increase the expression of MHC I and MHC II, thereby improving the antigen presentation ability.

[0102] Within 48 hours after each recombinant plasmid was transfected into RAW264.7 cells, the differences in the expression levels of related immune factors (CD86, CD80, C206, iNOS, Arg-1) in different treatment groups were shown in Figure 6 .

[0103] Figure 6 Results showed that the tPAa group (tPAa-OMP25 signal peptide fusion group) and the tPAs group (tPAs-OMP25 signal peptide fusion group) showed significantly higher percentages of cells expressing immune activation markers (such as CD86 and CD80) and M1 polarization markers (such as iNOS) than the sP25, Vector, and Blank control groups, while the expression of M2 polarization markers Arg-1 and CD206 was significantly decreased. Data are expressed as percentages of positive cells, suggesting that the mutant signal peptides (tPAa and tPAs) enhance the immune regulatory ability of OMP25.

[0104] After 48 hours of transfection of RAW264.7 cells with different recombinant plasmids, the expression of intracellular proinflammatory cytokines IL-1β and IL-12 is shown in Figure 7 .

[0105] Figure 7The results showed that the expression levels of the above-mentioned proinflammatory factors in the tPAa group (tPAa-OMP25 signal peptide fusion group) and the tPAs group (tPAs-OMP25 signal peptide fusion group) were significantly higher than those in the control groups without fusion of mutant signal peptides (wild-type signal peptide group sP25, signal peptide-free group nsP25, and Blank group). This suggests that the OMP25 recombinant protein fused with the mutant signal peptide can effectively enhance the expression of cellular proinflammatory factors, activate the proinflammatory response of macrophages, and has the potential to enhance innate immune activation.

[0106] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A signal peptide that promotes OMP25 expression, characterized in that: The signal peptide includes a tPAa signal peptide or a tPAs signal peptide; the amino acid sequence of the tPAa signal peptide is shown in SEQ ID NO.2, and the amino acid sequence of the tPAs signal peptide is shown in SEQ ID NO.

3.

2. A nucleotide encoding the signal peptide for promoting OMP25 expression according to claim 1.

3. The nucleotide according to claim 2, characterized in that The nucleotide sequence encoding the tPAa signal peptide is shown in SEQ ID NO.4; the nucleotide sequence encoding the tPAs signal peptide is shown in SEQ ID NO.

5.

4. An OMP25 fusion protein containing the signal peptide according to claim 1, characterized in that: The fusion protein includes tPAa-OMP25 or tPAs-OMP25; the amino acid sequence of the OMP25 is shown in SEQ ID NO.

8.

5. The fusion protein according to claim 4, characterized in that The nucleotide sequence of the tPAa-OMP25 is shown in SEQ ID NO.13; the nucleotide sequence of the tPAs-OMP25 is shown in SEQ ID NO.

14.

6. A biomaterial expressing OMP25, characterized in that The biological material is any one of the following: (1) An expression cassette containing the fusion protein according to claim 4 or 5; (2) a recombinant plasmid containing the expression cassette described in (1); (3) A recombinant cell containing the fusion protein according to claim 4 or 5; (4) a recombinant cell containing the expression cassette described in (1); (5) A recombinant cell containing the recombinant plasmid described in (2).

7. Use of the signal peptide according to claim 1, the nucleotide according to claim 2 or 3, or the biomaterial according to claim 6 in the preparation of OMP25.

8. Use of the biomaterial according to claim 6 in the preparation of immunity-enhancing products.

9. The use according to claim 8, characterized in that The biomaterial significantly enhances the antigen presenting ability of macrophages, induces Th1 immune response, and drives macrophages to polarize toward M1 phenotype, thereby enhancing their anti-infection ability.

10. A method for preparing OMP25, characterized in that: The method comprises the following steps: transfecting the recombinant plasmid according to claim 6 into host cells, culturing the obtained recombinant cells, collecting the supernatant of the recombinant cells, and obtaining OMP25.

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