Method for displaying foreign protein by utilizing salmonella and application thereof

Abstract

The invention discloses a method for displaying foreign protein by utilizing salmonella and application of the method, and belongs to the technical field of biological medicine. According to the invention, the SpyCatcher and an outer membrane protein Lpp-OmpA gene are fused and are expressed in salmonella, a recombinant strain is constructed, and the SpyCatcher is displayed on the surface of bacteria; the foreign protein gene and the SpyTag gene are fused and subjected to prokaryotic expression purification, and recombinant foreign protein is obtained; after the recombinant foreign protein and the recombinant salmonella are incubated, the foreign protein can be anchored on the surface of the salmonella through a SpyCatcher-SpyTag covalent bond. When the foreign protein is an antigen protein, the foreign protein is displayed on the surface of the attenuated bacterial vector, and a live vector vaccine can be prepared. The invention provides a novel universal antigen display method, overcomes the limitation of the traditional antigen display method on the protein size and conformation, and has a good application prospect in live vector vaccine construction.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine and relates to a method for displaying foreign proteins using Salmonella and application of the method in constructing live vector vaccines. Background Art

[0002] Live vector vaccines are a type of vaccine that uses non-toxic or attenuated bacteria or viruses as live vectors to express or display foreign proteins, and present the foreign proteins to the host to trigger a corresponding immune protection response. Salmonella can activate the immune response of dendritic cells and macrophages in the host's intestinal mucosa, and can induce good humoral and cellular immune responses. At the same time, because the surface of Salmonella contains components such as lipopolysaccharide, it can activate the host's natural immunity and play the role of an immune adjuvant. Therefore, using Salmonella as a vector to display and present antigenic proteins has good application prospects.

[0003] The method of using bacteria as a carrier to display or present foreign proteins is mainly to fuse foreign proteins with known bacterial surface proteins, or to use bacterial secretion systems to display antigenic proteins on the surface or secrete them outside the cell. For example, foreign proteins can be displayed on the surface of bacteria by fusing them with Lpp-OmpA. However, the problem with this method is that it is not suitable for the display and presentation of all proteins. This method has certain requirements on the size and other characteristics of the target protein, and cannot display proteins that are too large or have special conformations.

[0004] The SpyCatcher-SpyTag system is a convenient protein covalent coupling system. It is derived from the fibronectin binding protein of Streptococcus pyogenes. By splitting the domain and rationally modifying the fragments, a peptide (SpyTag) was obtained, which can form an amide bond with its protein partner SpyCatcher in a short time and under mild conditions. The system can be used for protein coupling and display, and has good versatility and convenience.

[0005] Glaesserella parasuis (GPS), also known as Haemophilus parasuis, is a pathogen that seriously harms the pig farming industry. It can cause diseases such as polyfibrinous serositis and arthritis in pigs, causing serious economic losses. At present, vaccine immunization is an important means of preventing and controlling Glaesser's disease in pigs. It is of great significance to develop new vaccines with good immune protection effects. At present, there is no relevant commercial live vector vaccine. Summary of the invention

[0006] The purpose of the present invention is to provide a method and application of displaying foreign proteins by using Salmonella (Salmonella choleraesuis). The present invention uses the Lpp-OmpA bacterial surface display system and the SpyCatcher-SpyTag system to anchor and display foreign proteins (such as antigenic proteins) carrying SpyTag on the bacterial surface by covalent bonds ( Figure 1 ). When the foreign protein is an antigen protein, it is displayed on the surface of the attenuated bacterial carrier, and a live carrier vaccine can be prepared, which can activate the host's cellular immunity and humoral immunity and provide a good immune protection effect. The present invention has a good application prospect in the construction of live carrier vaccines.

[0007] In order to achieve the above object, the technical solution of the present invention is as follows:

[0008] A method for displaying foreign proteins using Salmonella, comprising the following steps:

[0009] (1) Inserting a DNA fragment encoding the Lpp-OmpA-SpyCatcher fusion protein into an expression plasmid to construct a recombinant plasmid capable of expressing the Lpp-OmpA-SpyCatcher fusion protein.

[0010] (2) The recombinant plasmid constructed in step (1) is transferred into Salmonella to obtain recombinant Salmonella capable of displaying SpyCatcher on its surface.

[0011] (3) The recombinant Salmonella obtained in step (2) is cultured or cultured and induced to express so that SpyCatcher is displayed on the surface of the recombinant Salmonella, and the recombinant exogenous protein carrying SpyTag is incubated with the recombinant Salmonella in a buffer solution, and the exogenous protein is displayed on the surface of the Salmonella through the covalent binding of SpyCather-SpyTag.

[0012] In step (1), the amino acid sequence of Lpp-OmpA is shown in SEQ ID NO.1, and the nucleotide sequence encoding Lpp-OmpA is preferably shown in SEQ ID NO.2. The amino acid sequence of Spycather is shown in SEQ ID NO.3, and the nucleotide sequence encoding Spycather is preferably shown in SEQ ID NO.4. Furthermore, Lpp-OmpA and Spycather are connected by a flexible linker, and the flexible linker is preferably a flexible tether, whose amino acid sequence is shown in SEQ ID NO.5, and the encoding nucleotide sequence is preferably shown in SEQ ID NO.6.

[0013] In step (1), the expression plasmid is preferably pBAD33.

[0014] In step (2), the Salmonella is preferably Salmonella C500.

[0015] In step (3), the amino acid sequence of the SpyTag is shown as SEQ ID NO.7, and its encoding nucleotide sequence is preferably shown as SEQ ID NO.8.

[0016] In step (3), the recombinant exogenous protein carrying SpyTag is preferably obtained by prokaryotic expression and purification, such as constructing a DNA fragment encoding the exogenous protein-SpyTag fusion protein into a prokaryotic expression plasmid, expressing and purifying it using a prokaryotic expression system to obtain a recombinant exogenous protein carrying SpyTag.

[0017] The buffer described in step (3) is preferably PBS buffer.

[0018] In the method of using Salmonella to display foreign proteins, the Salmonella can be replaced by other non-toxic or weakly toxic bacteria to display foreign proteins on the surface of other bacteria.

[0019] The method of using Salmonella to display foreign proteins is used in the construction of live vector vaccines. When the foreign protein is an antigen protein, a live vector vaccine can be obtained by the method.

[0020] A pig Glaser's disease vaccine with Salmonella as a carrier, which is obtained by the above method, wherein the exogenous protein is one or more of AfuA, CdtB, PalA and HPS06257 proteins of Glaseria parasuis. Among them, the amino acid sequence of AfuA protein is shown in SEQ ID NO.9, and its encoding nucleotide sequence is preferably shown in SEQ ID NO.10; the amino acid sequence of CdtB protein is shown in SEQ ID NO.11, and its encoding nucleotide sequence is preferably shown in SEQ ID NO.12; the amino acid sequence of PalA protein is shown in SEQ ID NO.13, and its encoding nucleotide sequence is preferably shown in SEQ ID NO.14; the amino acid sequence of HPS06257 protein is shown in SEQ ID NO.15, and its encoding nucleotide sequence is preferably shown in SEQ ID NO.16.

[0021] The present invention uses attenuated Salmonella as a carrier, uses the Lpp-OmpA bacterial surface display system to display SpyCatcher on the surface of Salmonella, constructs a recombinant strain, fuses the exogenous protein with SpyTag and performs expression purification, and after incubating the exogenous protein fused with SpyTag and the recombinant Salmonella displaying SpyCatcher on the surface, the exogenous protein can be covalently anchored to the surface of Salmonella through the SpyCatcher-SpyTag system. The present invention uses this method to display the four antigenic proteins AfuA, CdtB, PalA and HPS06257 of swine Grasseria on the surface of Salmonella, and constructs a live vector vaccine for swine Grasseria disease. Animal experimental results show that the vaccine can activate the host's cellular immunity and humoral immunity, and provide good immune protection. The method in the present invention can well display any exogenous protein on the surface of Salmonella, has versatility, and has a good application prospect in the construction of live vector vaccines.

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

[0023] (1) The present invention uses bacterial surface display technology to display the antigen protein of Glaseria suis on the surface of Salmonella to obtain a new recombinant Salmonella live vector vaccine.

[0024] (2) The recombinant Salmonella live vaccine can be used as an anti-swine Glaserostomiasis vaccine; the live vector vaccine can effectively stimulate the body to produce cytokines after intraperitoneal injection of Balb / c mice, proving that the recombinant live bacterial vaccine prepared by the present invention can effectively stimulate the body to produce cellular immunity; immune protection evaluation experiments have confirmed that the live bacterial vaccine can increase the immune protection rate of mice to more than 83.3%.

[0025] (3) The Salmonella live vector vaccine prepared by the present invention can also act as an adjuvant, stimulating a humoral immunity level equivalent to that of subunit vaccines and commercial vaccines.

[0026] (4) The present invention provides a new universal antigen display method, which overcomes the limitations of traditional antigen display methods on protein size and conformation. The live vaccine vector constructed using the present invention provides good immune protection effects and has a good application prospect in the construction of live vector vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Schematic diagram of using Lpp-OmpA bacterial surface display system and SpyTag-SpyCatcher system to display target antigen protein.

[0028] Figure 2 Fluorescence microscopy image showing the verification of the C500-SpyCatcher vector surface display system using GFP protein.

[0029] Figure 3 This is a Western Blot analysis showing the validation of the C500-SpyCatcher vector surface display system using GFP protein.

[0030] Figure 4 This is an identification diagram of the GPS antigen protein AfuA, CdtB, PalA and HPS 06257 expression plasmid.

[0031] M: 2000marker; negative control; 2: AfuA-His-Flag-SpyTag / pQE-80L / DH5α; 3: CdtB-His-HA-SpyTag / pQE-80 L / DH5α; 4: PalA-His-Flag-SpyTag / pQE-80L / DH5α; 5: HPS06257-His-HA-SpyTag / pQE-80L / DH5α.

[0032] Figure 5 This is the SDS-PAGE identification diagram of the expression and purification of GPS recombinant protein. M: protein marker; 1: rAfuA / BL21(DE3); 2: rCdtB / BL21(DE3); 3: rPalA / BL21(DE3); 4: rHPS 06257 / BL21(DE3).

[0033] Figure 6 This is a Western Blot analysis of the expression and purification of GPS recombinant protein. M: protein marker; 1: rAfuA / BL21(DE3); 2: rCdtB / BL21(DE3); 3: rPalA / BL21(DE3); 4: rHPS 06257 / BL21(DE3).

[0034] Figure 7 This is a Western Blot identification diagram of the combination of GPS antigen proteins rAfuA, rCdtB, rPalA, and rHPS 06257 with the C500-SpyCatcher vector surface display system.

[0035] Figure 8 These are the results of the protective efficacy evaluation after immunization with the recombinant Salmonella Glaseria live vector vaccine using the challenge strain GPS H0165.

[0036] Fig. 9 This is a picture of tissue lesions after immunization with the recombinant Salmonella live vector vaccine and challenged with the virus strain GPS H0165. In the figure, a, b, c, and d are the mouse heart, lung, liver, and mouse spleen respectively; 1, 2, 3, 4, and 5 are the NC group, C500 group, C500+Ag group, Ag group, and CV group respectively.

[0037] Fig.10 These are the detection results of specific antibody IgG in the serum after immunization with the recombinant Salmonella live vector vaccine using the challenge strain GPS H0165.

[0038] Fig.11 These are the test results of specific antibodies IgG1 and IgG2a in the serum after immunization with the recombinant Salmonella live vector vaccine and challenged with the challenge strain GPS H0165.

[0039] Fig.12 These are the results of the bactericidal rate test of the serum after immunization with the recombinant Salmonella live vector vaccine and challenged with the challenge strain GPS H0165. DETAILED DESCRIPTION

[0040] The technical scheme of the present invention is described clearly and completely below in conjunction with the embodiments and drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the protection scope of the present invention.

[0041] The strains used in the following examples are all commercial strains, and the competent E. coli cells DH5α and BL21 (DE3) were purchased from Takara Company.

[0042] Example 1: Display of foreign proteins on bacterial surfaces via covalent bonds

[0043] S1. Construction of Salmonella C500-SpyCatcher surface display system

[0044] The lpp-ompA-linker-spycatcher fragment (composed of SEQ ID NO.2-SEQ ID NO.6-SEQ ID NO.4, the sequence is shown in Table 1 below) and plasmid pBAD33 were PCR amplified using the primers in Table 1, and then the amplified Lpp-OmpA-linker-Spycather with homology arms was connected to the linearized plasmid pBAD33 by the DNA homologous recombination method to construct the recombinant plasmid pBAD33-Lpp-OmpA-Spycather-his, and then the Salmonella C500 strain was made into an electroporation competent state, and the plasmid pBAD33-Lpp-OmpA-Spycather-his was electroporated into C500 to obtain the recombinant Salmonella C500-Spycatcher.

[0045] Table 1 Target genes and their amplification primers with linearized vector

[0046]

[0047] S2. Purification of recombinant protein GFP-SpyTag

[0048] The Gfp-spytag fragment (sequence shown in Table 2 below) and plasmid pQE80L were PCR amplified using the primers in Table 2, and the amplified Gfp-spytag gene with homology arms was connected with the linearized plasmid pQE80L by DNA homologous recombination to construct the recombinant expression plasmid pQE80L-GFP-SpyTag. The ligation product was transferred into E. coli DH5α competent medium and then cultured overnight to obtain E. coli carrying the recombinant expression plasmid; the plasmid was extracted and transformed into E. coli BL21 (DE3) competent medium, and the bacteria were cultured at 37°C and 180 r / min until the bacterial solution OD 600 When the value reaches 0.6-0.8, IPTG with a final concentration of 0.5 mmol / L is added, and the expression is induced overnight at 16°C and 180 r / min. The recombinant protein GFP-SpyTag can be expressed in the supernatant after bacterial disruption. The supernatant is collected and purified to obtain the recombinant protein GFP-SpyTag.

[0049] Table 2 Target genes and their amplification primers with linearized vector

[0050]

[0051]

[0052] S3. Recombinant protein GFP-SpyTag is anchored on the surface of recombinant bacteria through covalent binding of Spycather-SpyTag

[0053] A single colony of C500-SpyCatcher was picked up in LB liquid culture medium, and arabinose with a final concentration of 0.1% was added to induce expression at 37°C for 4.5 hours. The bacteria were collected, washed 3 times with PBS, resuspended, and a sufficient amount of recombinant protein GFP-SpyTag was added. The cells were gently mixed and incubated at room temperature for 1 hour. During the period, the cells were gently flipped several times. The GFP fluorescent protein was loaded onto the surface of C500 using the specificity of the combination of SpyTag and SpyCatcher for presentation of GFP protein. The cells were then observed under a fluorescence microscope ( Figure 2 ) and Western Blot( Figure 3 ) were used to verify the surface display system, and the results showed that GFP fluorescent protein was displayed on the surface of Salmonella.

[0054] Example 2: Construction and application of a swine Glaser's disease vaccine using Salmonella as a carrier

[0055] The present invention provides a recombinant Salmonella live vector vaccine for swine Grasser's disease (Salmonella vector vaccine displaying GPS antigen on the surface). The prepared vaccine displays Grasser's AfuA, CdtB, PalA and HPS 06257 subunit proteins on the surface, which can induce an immune response in the body and protect the body from GPS infection. The vaccine has the advantages of not requiring additional adjuvants and having a simple preparation process.

[0056] S1. Preparation of antigenic protein of Glaseria suis

[0057] Using the genome of Glaseria suis as a template and the primers in Table 3, PCR amplification was performed to obtain Flag-afuA, HA-cdtB, Flag-palA, and HA-HPS 06257. Using the plasmid pQE80L-GFP-SpyTag constructed in Example 1 as a template and the primers in Table 3, PCR amplification was performed to obtain linearized pQE80L-SpyTag.

[0058] The amplified Flag-afuA, HA-cdtB, Flag-palA or HA-HPS 06257 was connected with pQE80L-SpyTag to construct recombinant expression plasmids pQE-80L-AfuA-His-Flag-SpyTag, pQE-80L-CdtB-His-HA-SpyTag, pQE-80L-PalA-His-Flag-SpyTag and pQE-80L-HPS 06257-His-HA-SpyTag. The constructed plasmids all carried a purification tag 6×his tag and different detection tags Flag or tag HA respectively; the ligation product was transferred into E.coli DH5α competent cell and then cultured overnight to obtain E. coli carrying the recombinant expression plasmid; the plasmid was extracted and verified by the plasmid universal primer pQE80L-F / R. The primers used are shown in Table 3.

[0059] Table 3 Amplification primers for target gene and linearized vector

[0060]

[0061]

[0062] The recombinant expression plasmids pQE-80L-AfuA-His-Flag-SpyTag, pQE-80L-CdtB-His-HA-SpyTag, pQE-80L-PalA-His-Flag-SpyTag and pQE-80L-HPS 06257-His-HA-SpyTag constructed and verified correctly by sequencing were transformed into E. coli BL21 (DE3) competent cells, spread on LB solid culture medium of corresponding resistance, placed in a 37°C incubator, and inverted for culture for 12 to 16 hours.

[0063] Pick a single recombinant expression colony and place it in 5 mL of LB liquid medium with corresponding resistance, and culture it at 37°C and 180 r / min until it becomes turbid. At the same time, pick E. coli BL21 (DE3) -pQE-80L empty bacteria as a negative control; transfer it to 800 mL of LB liquid medium with corresponding resistance at a ratio of 1:100, and culture it at 37°C and 180 r / min until the bacterial solution OD 600 When the value reaches 0.6-0.8, add IPTG with a final concentration of 0.5mmol / L and induce overnight at 16℃180r / min; centrifuge at 10000r / min for 10min, collect 800mL of recombinant expression bacteria, discard the supernatant, and resuspend with 40mL resuspension buffer.

[0064] Use a high-pressure crusher to lyse the bacterial resuspension 5-6 times until the bacterial suspension becomes clear and transparent. Centrifuge at 12000r / min for 60min at 4℃. Filter the supernatant with a 0.22μm filter to prevent the sample solution from clogging the nickel column. Collect the supernatant and place it on ice for later use.

[0065] Take 1.5-2 mL Ni 2+ Add the resin to the nickel column and let it stand for 30 minutes. Connect the nickel column to a peristaltic pump, wash the nickel column with 5 column volumes of Buffer B, and then balance the nickel column with 5 column volumes of Buffer A. Then, the protein can be loaded onto the column. Add the filtered supernatant after bacterial crushing and control the flow rate to be about 1 mL / min. After the supernatant of bacterial crushing has flowed out, add Buffer A to the column and wash for 30 minutes. Connect the nickel column with protein loading to the protein purifier, turn on the UV lamp and gradient purification program. The elution rate is 1 mL / min. First, elute 30 mL with 5% Buffer B (i.e., 25 mmol / L imidazole), then use a Buffer B gradient elution of 10%-50% (i.e., 50-250 mmol / L imidazole) for 60 minutes. Finally, elute 30 mL of the target protein with 100% Buffer B (i.e., 500 mmol / L imidazole) and collect the eluate.

[0066] The concentration and purity of the protein eluate were tested by SDS-PAGE, and the eluate with higher concentration and purity was selected for concentration and ultrafiltration; according to the size of the protein, a 5KD or 10KD ultrafiltration tube was selected for protein concentration. The protein solution to be concentrated was added, and the solution was centrifuged at 3000 rpm at 4°C and concentrated to 1-1.5 mL.

[0067] Use PBS as the replacement fluid, centrifuge at 3000 rpm at 4°C, and replace three times for more than 1000 times to remove high concentrations of imidazole. Determine the protein concentration according to the instructions of the BCA protein concentration determination kit (product of Kangwei Century Company); add glycerol with a final concentration of 25% to the purified protein solution, filter and sterilize with a 0.22μm filter, divide and store at -80°C after quick freezing in liquid nitrogen.

[0068] The results of SDS-PAGE and Western Blot identification of purified antigen protein are as follows Figure 5 , 6 As shown, four antigenic proteins were expressed in the supernatant of the disrupted cells.

[0069] S2. Preparation of Salmonella vector vaccine displaying GPS antigen on the surface

[0070] The single colony of the surface display strain C500-SpyCatcher of Salmonella constructed in Example 1 was picked up in LB liquid culture medium, and arabinose with a final concentration of 0.1% was added to induce expression at 37°C for 4.5 hours. After washing with PBS three times, the mixture was resuspended and sufficient amounts of the subunit AfuA-His-Flag-SpyTag, CdtB-His-HA-SpyTag, PalA-His-Flag-SpyTag and HPS 06257-His-HA-SpyTag antigen proteins of Glaseria suis were added respectively, and the mixture was gently mixed and incubated at room temperature for 1 hour, during which the mixture was gently flipped several times, and the specificity of the combination of SpyTag and SpyCatcher (such as Figure 1 ) GPS antigen protein was loaded onto the surface of Salmonella C500 for presentation of antigen protein. Western Blot results (such as Figure 7 ) showed that the molecular weight of the proteins increased significantly after binding.

[0071] In the above step S2, the SpyTag-Spycatcher system is used to display the antigen protein on the surface of Salmonella. The whole process is fast and simple.

[0072] S3. Animal experiments for evaluating the immune protection effect of GPS

[0073] The mice were divided into 5 groups, with a total of 150 4-week-old female SPF BALB / c mice, as shown in Table 4. Immunization was performed by intraperitoneal injection of 0.2 mL of the vaccine, and a booster immunization was performed on the 14th day after the first immunization. The dose of recombinant protein was 50 μg / mouse each time. The challenge dose was 4 LD 50 .

[0074] Table 4 Grouping of GPS recombinant Salmonella intraperitoneal injection immunization

[0075]

[0076]

[0077] Note: The commercial subunit vaccine is a product of Wuhan Keqian Animal Biological Products Co., Ltd.

[0078] (I) Evaluation of the protective efficacy of Salmonella vector vaccine displaying GPS antigens after immunization with the challenge strain GPS H0165

[0079] On the 24th day after the first vaccination, mice were given 4LD 50 The mice were challenged with the GPS strain SH 0165 of 1:1 dose by intraperitoneal inoculation. The mice were observed for one week and the mortality was recorded.

[0080] Results (such as Figure 8 ) showed that 48 hours after infection, facing the lethal dose of GPS SH 0165, the C500 group, C500+Ag group, Ag group and CV group provided 58%, 83%, 100% and 92% protection rates, respectively (12 mice in each group in the protection evaluation experiment). In the absence of adjuvant, the C500+Ag group was able to provide an immune protection rate equivalent to that of commercial vaccines and traditional subunit vaccines.

[0081] (II) Tissue lesions after immunization with Salmonella vector vaccine displaying GPS antigen and challenged with the challenge strain GPS H0165

[0082] The mice that just died during the challenge and the mice 48 hours after the challenge (death by cervical dislocation) were autopsied to observe the lesions of tissues and organs. The mice that survived one week after the challenge were decapitated and autopsied to observe the lesions of tissues and organs.

[0083] Results (such as Fig. 9) showed that the hearts of the five groups of mice had varying degrees of fibrinous exudation. The lungs and livers were severely congested, and the spleens were enlarged and severely congested. In addition, most mice in the Ag and CV groups had white spots or white patches on their livers, and white patches protruding from the spleen (which could not be peeled off), while the C500+Ag group provided comparable immune protection to the Ag and CV groups, and the livers and spleens of the mice in this group did not show abnormalities, indicating that the Salmonella vector vaccine displaying GPS antigens on the surface has fewer side effects than subunit vaccines.

[0084] (III) Detection of specific antibodies IgG, IgG1, and IgG2a in serum after immunization with Salmonella vector vaccine displaying GPS antigen and challenge with the challenge strain GPS H0165

[0085] Blood was collected from the retro-orbital venous plexus of mice on the 12th day and the 21st day after the first immunization (the 7th day after the second immunization). After 1 hour, the blood was placed at 4°C overnight and centrifuged at 3000r / min for 15min. The upper serum was collected and packaged and stored at -20°C.

[0086] The serum was separated and the IgG, IgG1 and IgG2a antibody levels of each antigen were detected by ELISA. The recombinant protein and Salmonella antigen were used as coating antigens. The recombinant protein was diluted to 2 μg / 100 μL with coating solution, and the inactivated Salmonella was diluted to 8×10 8 CFU / 100μL, 200μL per well was added to the ELISA plate A 1 to A 12, and 100μL of coating solution was used to dilute from top to bottom, and finally 100μL of residual liquid was discarded, and the plate was placed at 4°C overnight after shaking for 3min. After washing and blocking the next day, the negative and positive sera were diluted 80-1280 times in the EP tube with sample diluent (PBS containing 5% (m / v) skimmed milk powder). 100μL of positive serum diluted 80 times was added to A 1-F1, and 100μL of negative serum diluted 160 times was added to A2-F 2. Serum with different dilution multiples was added in turn, and each sample was repeated three times. After incubation at 37°C for 30min, subsequent experiments were carried out and OD was read. 630 The values ​​were averaged to calculate the corresponding P / N value (i.e., the ratio of the positive OD value to the corresponding negative OD value), thereby determining the optimal working concentrations of antigen and serum.

[0087] The immune serum was tested by the established indirect ELISA method for AfuA, CdtB, PalA, HPS 06257 and Salmonella somatic antigen. The serum samples were diluted to 10-40960 times with sample diluent, and two replicates were set. After color development, the OD was measured. 630 , take the average value to calculate the serum dilution titer. Fig.10) showed that Salmonella vector vaccines, homemade subunit vaccines and commercial subunit vaccines displaying GPS antigens on the surface can induce mice to produce high titers of specific IgG antibody levels. 12 days after the first immunization, the antibody levels stimulated by AfuA-SpyTag, CdtB-SpyTag, PalA-SpyTag, HPS06257-SpyTag and C500-SpyCatcher were significantly different from those of the negative control group, and 7 days after the second immunization, the antibody levels were further significantly increased.

[0088] The levels of specific IgG subclass antibodies IgG1 and IgG2a in immune serum were detected by the established indirect ELISA methods for AfuA, CdtB, PalA, HPS 06257 and Salmonella somatic antigens, respectively. Each serum sample was diluted to 80 times with sample diluent, and two replicates were set. After color development, OD was measured. 630 ΔIgG1 and ΔIgG2a were obtained by subtracting IgG1 and IgG2a of the negative control group (NC group) from IgG1 and IgG2a of the experimental group, respectively. R represents the ratio of ΔIgG1 to ΔIgG2a. Fig.11 ) showed that the immune mode stimulated by Salmonella vector and Salmonella vector vaccine displaying GPS antigen on the surface was mainly Th1 type, that is, cellular immunity; the immune mode stimulated by homemade subunit vaccine and commercial subunit vaccine was mainly Th2, that is, humoral immunity.

[0089] (IV) Serum bactericidal rate detection after immunization with Salmonella vector vaccine displaying GPS antigen and challenged with the challenge strain GPS H0165

[0090] The GPS SH 0165 strain was cultured in TSB, washed twice with sterile PBS, and diluted to 1.0 × 10 9 CFU / mL. The serum on the 21st day after the first immunization was inactivated in a water bath at 56°C for 30 minutes. The inactivated serum was diluted to 50% with sterile PBS. 10 μL of the diluted bacterial solution and 190 μL of the diluted serum were mixed in a 1.5 mL centrifuge tube and incubated at 37°C for 30 minutes. Then 100 μL of non-immunized and heparinized mouse whole blood was added to the centrifuge tube and incubated at 37°C for 1 hour. After incubation, all the liquid in the centrifuge tube was diluted 10 7 Times and 10 8 The dilution was multiplied by 100 μL and 100 μL of the diluted solution was applied to TSA plates respectively. The test was repeated three times in parallel. After culturing at 37°C for 12 h, the colonies were counted.

[0091] Bactericidal rate = (CFU of the control group after 1 hour of culture - CFU of the immune group after 1 hour of culture) / CFU of the control group after 1 hour of culture × 100%.

[0092] Results (such as Fig.12 ) showed that according to the calculation of the bactericidal rate, the GPS in the negative control group and C500 group grew well, with bactericidal rates of only 2.04% and 2.19%. However, the growth of GPS in the other three groups was inhibited, with bactericidal rates of 28.65%, 40.00% and 40.88%, respectively. Compared with the negative control group, the bactericidal rate was significantly different, and compared with the traditional subunit vaccine, the C500+Ag group also showed a comparable bactericidal effect, indicating that the immune response induced by the Salmonella vector vaccine displaying GPS antigens on the surface can exert a bactericidal effect through antibody mediation.

[0093] The above embodiments are only used to help illustrate the present invention. The implementation methods of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for displaying foreign proteins using Salmonella, characterized in that: The following steps are involved: (1) inserting a DNA fragment encoding the Lpp-OmpA-SpyCatcher fusion protein into an expression plasmid to construct a recombinant plasmid capable of expressing the Lpp-OmpA-SpyCatcher fusion protein; (2) transferring the recombinant plasmid constructed in step (1) into Salmonella to obtain recombinant Salmonella capable of displaying SpyCatcher on its surface; (3) The recombinant Salmonella obtained in step (2) is cultured or cultured and induced to express so that SpyCatcher is displayed on the surface of the recombinant Salmonella, and the recombinant exogenous protein carrying SpyTag is incubated with the recombinant Salmonella in a buffer solution, and the exogenous protein is displayed on the surface of the Salmonella through the covalent binding of SpyCather-SpyTag.

2. The method for displaying foreign proteins using Salmonella according to claim 1, characterized in that: In step (1), the amino acid sequence of Lpp-OmpA is shown in SEQ ID NO.1; the amino acid sequence of Spycather is shown in SEQ ID NO.3; In step (3), the amino acid sequence of the SpyTag is shown in SEQ ID NO.

7.

3. The method for displaying foreign proteins using Salmonella according to claim 1, characterized in that: In step (1), Lpp-OmpA and Spycather are connected via a flexible linker.

4. The method for displaying foreign proteins using Salmonella according to claim 3, characterized in that: The flexible linker is a flexible tether, and its amino acid sequence is shown in SEQ ID NO.

5.

5. The method for displaying foreign proteins using Salmonella according to claim 1, characterized in that: In step (1), the expression plasmid is pBAD33.

6. The method for displaying foreign proteins using Salmonella according to claim 1, characterized in that: In step (2), the Salmonella is Salmonella C500.

7. The method for displaying foreign proteins using Salmonella according to claim 1, characterized in that: In step (3), the recombinant exogenous protein carrying SpyTag is obtained by a method comprising the following steps: constructing a DNA fragment encoding the exogenous protein-SpyTag fusion protein into a prokaryotic expression plasmid, expressing and purifying using a prokaryotic expression system, and obtaining the recombinant exogenous protein carrying SpyTag.

8. The method for displaying foreign proteins using Salmonella according to claim 1, characterized in that: Replace Salmonella with other non-toxic or weakly toxic bacteria so that foreign proteins can be displayed on the surface of other bacteria.

9. Use of the method according to any one of claims 1 to 8 in the construction of live vector vaccines, characterized in that: When the foreign protein is an antigen protein, a live vector vaccine is obtained by the method.

10. A porcine Glaser's disease vaccine, characterized in that: It is obtained by the method according to any one of claims 1 to 8, wherein the exogenous protein is one or more of AfuA, CdtB, PalA and HPS06257 proteins of Glaseria parasuis.

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