Streptococcus suis and escherichia coli bigeminy ghost as well as preparation method and application thereof

Abstract

The invention provides a streptococcus suis and escherichia coli bigeminy ghost and a preparation method and application thereof, and the preparation method of the bigeminy ghost comprises the following steps: constructing a recombinant plasmid containing a streptococcus suis multi-epitope fusion antigen FAS expression gene, and transferring the recombinant plasmid and a ghost induction plasmid into porcine parenteral pathogenic escherichia coli, the method comprises the following steps: firstly, inducing an FAS antigen to be expressed on the surface of a thallus, and then culturing and inducing generation of bacterial ghost to prepare the streptococcus suis and escherichia coli bigeminy bacterial ghost PGs; the amino acid sequence of the streptococcus suis multi-epitope fusion antigen FAS is as shown in SEQ ID NO. 1. The obtained PGs can be used for preparing bivalent bacterial ghost vaccines of streptococcus suis and escherichia coli, the preparation is simple, the yield is high, an organism can be stimulated to generate protective antibodies so as to resist infection of streptococcus suis of various serotypes and mixed infection with parenteral pathogenic escherichia coli, and the PGs have a good application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of veterinary biological products, and particularly relates to a Streptococcus suis and Escherichia coli double bacterial ghost, a preparation method thereof and an application thereof. Background Art

[0002] Bacterial ghost is a special cell structure mainly composed of the cell membrane and cell wall of bacteria. Through specific physical or chemical treatment, the internal contents of these cells (such as cytoplasm and DNA) are removed, and the cell morphology and external characteristics are retained. Since the outer membrane components (including lipopolysaccharide, peptidoglycan or flagella) are retained, it has the same envelope structure as natural bacteria and is an agonist of many innate immune stimulants. Therefore, bacterial ghosts can be effectively recognized and absorbed by immune and non-immune cells, and are effective activators for various cell types participating in innate immunity and adaptive immunity. In addition, bacterial ghosts do not contain nucleic acid substances, thus avoiding the harm of transgenes, are relatively stable in the environment, have no pollution risk, are convenient for storage and transportation, and are suitable for application in the development of medicine and biotechnology, and are a kind of biomaterial with broad application potential. However, the existing plasmid for inducing bacterial ghosts cannot achieve 100% inactivation, and chemical substances such as β-propiolactone still need to be added to assist in achieving complete inactivation, which limits the development of bacterial ghost vaccines. Although β-propiolactone can be used for vaccine inactivation, it has inhalation toxicity and may cause cancer. Cell-penetrating peptides are a new type of bacterial inactivation tool. The combination of lytic plasmids and cell-penetrating peptides can reduce environmental pollution and chemical hazards caused by organic reagents, and are expected to be used for developing bacterial ghost vaccines with intact surface antigens.

[0003] The bacterial ghosts of Gram-negative bacteria can be used as reliable carriers for inactivated vaccines. Research shows that they can induce strong immune responses and enhance the interaction between APCs and T cells, playing an important role in the treatment of bacterial infections. Currently, the most mature bacterial vaccine under research is the combination of the lysis gene E-mediated cell lysis and the degradation of internal DNA by staphylococcal nuclease A for developing Escherichia coli vaccines. In addition, some bacterial ghost vaccines have been verified in animal models to be able to successfully induce immune responses and resist pathogen attacks, and their safety has been evaluated, such as Actinobacillus pleuropneumoniae, Vibrio cholerae, etc.

[0004] Bacterial surface display is a method that relies on fusing target proteins with surface proteins of bacteria to carry exogenous proteins or peptide fragments to the surface of the carrier bacteria, enabling them to be directly exposed to antigen-presenting cells. The carrier bacteria commonly used in bacterial surface display technology itself are usually Escherichia coli and Salmonella. This technology is widely applied in vaccine development, especially for displaying antigenic epitopes of pathogens. By displaying pathogen antigens on the surface of bacteria, it can mimic the immune response during vaccination and screen out immunogenic antigens. Moreover, due to the fast proliferation rate of bacteria, directly displaying target molecules on the surface of bacteria can also avoid traditional protein purification steps, saving time and costs. Combining surface display technology with the preparation technology of bacterial ghosts can maximize the retention of the natural antigenic structure of the bacterial surface and exogenous proteins, ensuring the immunogenicity of the vaccine, which is a new direction in vaccine research. However, both the surface display of antigens and the induction of bacterial ghosts rely on plasmid systems to achieve. There is mutual exclusivity between plasmids, and plasmids with the same replication type cannot coexist in the same bacterium, restricting the development of the above technologies. Summary of the Invention

[0005] Object of the Invention: The present invention provides a two-component bacterial ghost of Streptococcus suis and Escherichia coli, its preparation method and application. In the present invention, by combining the preparation of bacterial ghosts and bacterial surface display technology, a co-transformable dual-plasmid system is constructed. Using extraintestinal pathogenic Escherichia coli as a carrier, relying on the lpp’OmpA protein to carry the screened fusion antigen (Fusion Antigen of S.suis, FAS) with multi-serum protection effect against Streptococcus suis to the surface of the bacterial cells, and forming bacterial ghosts (PGs) by E-SNA lysing Escherichia coli, achieving a large amount of expression and display of the fusion antigen FAS in a short time.

[0006] Technical Solution: To achieve the above object of the invention, the present invention adopts the following technical solutions:

[0007] In the first aspect, the present invention provides a preparation method of a two-component bacterial ghost of Streptococcus suis and Escherichia coli, comprising the following steps:

[0008] Construct a recombinant plasmid containing the expression gene of the multi-epitope fusion antigen FAS of Streptococcus suis, co-transfer it with the bacterial ghost induction plasmid into porcine extraintestinal pathogenic Escherichia coli, first induce the expression and surface display of the FAS antigen, and then culture to induce the generation of bacterial ghosts, thereby obtaining the two-component bacterial ghost PGs of Streptococcus suis and Escherichia coli; the amino acid sequence of the multi-epitope fusion antigen FAS of Streptococcus suis is as shown in SEQ ID NO.1.

[0009] As a specific embodiment, the recombinant plasmid containing the expression gene of the Streptococcus suis multi-epitope fusion antigen FAS has a vector selected from pBAD33; more specifically, the recombinant plasmid is pBAD33-lpp’OmpA FAS, and its nucleotide sequence is as shown in SEQ ID NO.2.

[0010] As a specific embodiment, the porcine extraintestinal pathogenic Escherichia coli is the PU-1 strain; for inducing the expression of the FAS antigen, L-arabinose is used for induction.

[0011] As a specific embodiment, the vector of the bacterial ghost induction plasmid is selected from pBV220; more specifically, the bacterial ghost induction plasmid is pBV220 E-SNA, and its nucleotide sequence is as shown in SEQ ID NO.3.

[0012] As a specific embodiment, the method for culturing and inducing the production of bacterial ghosts includes the following steps: in LB containing Cm + and Amp + , culture at 28±2 °C until OD = 0.6±0.05, raise the temperature to 42±2 °C to induce the lysis of the bacterial cells by E-SNA, and add WR 12 for inactivation.

[0013] In a second aspect, the present invention provides a Streptococcus suis and Escherichia coli double bacterial ghost, which is prepared by the above preparation method.

[0014] In a third aspect, the present invention provides a pharmaceutical composition, which contains the above-mentioned Streptococcus suis and Escherichia coli double bacterial ghost.

[0015] In a fourth aspect, the present invention provides a recombinant bacterium, which contains an expression plasmid and a lysis plasmid. The expression plasmid is a recombinant plasmid containing the expression gene of the Streptococcus suis multi-epitope fusion antigen FAS, and the lysis plasmid is a bacterial ghost induction plasmid; the amino acid sequence of the Streptococcus suis multi-epitope fusion antigen FAS is as shown in SEQ ID NO.1.

[0016] As a specific embodiment, the vector of the recombinant plasmid is selected from pBAD33; more specifically, the recombinant plasmid is pBAD33-lpp’OmpA FAS, and its nucleotide sequence is as shown in SEQ ID NO.2;

[0017] the vector of the bacterial ghost induction plasmid is selected from pBV220; more specifically, the bacterial ghost induction plasmid is pBV220 E-SNA, and its nucleotide sequence is as shown in SEQ ID NO.3.

[0018] Fifth aspect, the present invention provides the application of the Streptococcus suis and Escherichia coli double bacterial ghosts, the pharmaceutical composition, and the recombinant bacteria in the preparation of drugs for preventing Streptococcus suis and extraintestinal pathogenic Escherichia coli infections.

[0019] First, the present invention uses extraintestinal pathogenic Escherichia coli as a vector to surface-display the Streptococcus suis fusion antigen (by inserting the FAS expression sequence into the OmpA coding sequence, so that the expressed FAS antigen is assembled on the surface of the bacterial cells instead of accumulating inside the bacteria), and combines it with the Escherichia coli bacterial ghost preparation technology to produce a large number of bacterial ghosts (PGs) that highly express the Streptococcus suis protective antigen. The PGs provided by the present invention can be used to prepare a combined vaccine against Streptococcus suis and Escherichia coli. Subsequently, laboratory immune effect evaluation studies were carried out, and it was preliminarily determined that PGs can provide protection against multiple serotype strains of Streptococcus suis and extraintestinal pathogenic Escherichia coli for mice and piglets, significantly reducing the bacterial load of bacteria in various organs of the host. The serum after immunization has strong opsonophagocytic ability against multiple serotype Streptococcus suis and Escherichia coli. Therefore, PGs can be used to prepare a combined Streptococcus suis and Escherichia coli bacterial ghost vaccine, which is simple to prepare and has a high yield, and can stimulate the body to produce protective antibodies to resist infections of multiple serotype Streptococcus suis and mixed infections with extraintestinal pathogenic Escherichia coli, and has good application prospects.

[0020] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0021] (1) The pBAD33-lpp’OmpA FAS constructed by the present invention can efficiently display the fusion antigen FAS on the surface of PU-1 and can be co-transformed with the bacterial ghost induction plasmid pBV220 E-SNA into Escherichia coli. After immunizing mice with PGs, they can be recognized by the body and produce a strong immune response, and specific antibodies targeting Streptococcus suis can be produced by indirect ELISA.

[0022] (2) In the present invention, a challenge protection test of Streptococcus suis SS2 and SS9 virulent strains and Escherichia coli PU-1 was carried out on PGs-immunized mice. The immune protection rates of PGs against SS2 and SS9 were 70% and 90% respectively, and the protection rate against PU-1 infection was 80%, and it can significantly reduce the bacterial load of two serotype Streptococcus suis and PU-1 in different organs of mice.

[0023] (3) The antibodies produced after immunization with PGs generated by the present invention can have neutralizing ability and can have good antibacterial ability against at least five serotype Streptococcus suis and two extraintestinal pathogenic Escherichia coli.

[0024] (4) The antibodies produced after immunization with PGs generated by the present invention all show good opsonophagocytic ability in macrophages RAW264.7. Description of the Drawings

[0025] Figure 1 It is the plasmid pBAD33-lpp’OmpA FAS map for antigen surface display.

[0026] Figure 2 It is the identification diagram of pBAD33-lpp’OmpA FAS expression in PU-1 by Western Blot.

[0027] Figure 3 It is the identification diagram of FAS surface expression in PU-1 by indirect ELISA.

[0028] Figure 4 It is the identification diagram of FAS surface expression in PU-1 by indirect immunofluorescence.

[0029] Figure 5 It is the plasmid pBV220 E-SNA map for bacterial ghost induction.

[0030] Figure 6 It is the schematic diagram of the lysis kinetics detection of recombinant bacterium PU-1-pBAD33-lpp’OmpA FAS-pBV220 E-SNA induced at 42°C.

[0031] Figure 7 It is the schematic diagram of the structure of PU-1 under transmission electron microscopy under normal culture conditions, after WR 12 treatment and after E-SNA lysis.

[0032] Figure 8 It is the schematic diagram of the detection of the titers of specific antibodies against FAS and PU-1 in the sera of mice immunized with PGs.

[0033] Among them, Figure A is the schematic diagram of the detection of the titer of specific antibody against FAS in the sera of mice immunized with PGs; Figure B is the schematic diagram of the detection of the titer of specific antibody against PU-1 in the sera of mice immunized with PGs.

[0034] Figure 9 It is the schematic diagram of the detection of the titers of specific antibodies against FAS and PU-1 in the sera of piglets immunized with PGs.

[0035] Among them, Figure A is the schematic diagram of the detection of the titer of specific antibody against FAS in the sera of piglets immunized with PGs; Figure B is the schematic diagram of the detection of the titer of specific antibody against PU-1 in the sera of piglets immunized with PGs.

[0036] Figure 10Schematic diagram of the proliferation indices of splenic lymphocytes against FAS and PU-1 in PGs-immunized mice. Among them, Figure A is the schematic diagram of the proliferation index of splenic lymphocytes against FAS in PGs-immunized mice; Figure B is the schematic diagram of the proliferation index of splenic lymphocytes against PU-1 in PGs-immunized mice.

[0037] Figure 11 Survival curves of mice after immunization with PGs and infection with SS2-ZY05719, SS9-GZ0565, and PU-1. Among them, Figure A is the survival curve of mice infected with ZY05719; Figure B is the survival curve of mice infected with GZ0565; Figure C is the survival curve of mice infected with PU-1.

[0038] Figure 12 Record form of the onset and death of piglets.

[0039] Figure 13 Bacterial loads in various tissues and organs of mice after immunization with PGs and infection with SS2-ZY05719, SS9-GZ0565, and PU-1. Among them, Figure A is the bacterial load in various tissues and organs of mice infected with ZY05719; Figure B is the bacterial load in various tissues and organs of mice infected with GZ0565; Figure C is the bacterial load in various tissues and organs of mice infected with PU-1.

[0040] Figure 14 Changes in bacterial loads in the blood of piglets after immunization with PGs and infection with SS2-ZY05719, SS9-GZ0565, and PU-1. Among them, Figure A is the change in bacterial load in the blood of piglets infected with ZY05719; Figure B is the change in bacterial load in the blood of piglets infected with GZ0565; Figure C is the change in bacterial load in the blood of piglets infected with PU-1.

[0041] Figure 15 Temperature changes in piglets after immunization with PGs and infection with SS2-ZY05719, SS9-GZ0565, and PU-1. Among them, Figure A is the temperature change in piglets infected with ZY05719; Figure B is the temperature change in piglets infected with GZ0565; Figure C is the temperature change in piglets infected with PU-1.

[0042] Figure 16 Evaluation diagram of the antibacterial ability of sera from PGs-immunized rabbits against different serotypes of Streptococcus suis and Escherichia coli. Among them, Figure A is the evaluation diagram of the antibacterial ability of sera from PGs-immunized rabbits against representative strains of five representative serotypes of Streptococcus suis: SS2-ZY05719, SS5-HN105, SS9-GZ0565, SS31-WUSS147, SS chz-CZ130302; Figure B is the evaluation diagram of the antibacterial ability of sera from PGs-immunized rabbits against Escherichia coli PU-1 and PE164.

[0043] Figure 17 It is a graph for evaluating the antibacterial ability of the serum of PGs piglets after immunization against Streptococcus suis and Escherichia coli of different serotypes. Among them, Figure A is a graph for evaluating the antibacterial ability of the serum of PGs piglets after immunization against representative strains of five representative serotypes of Streptococcus suis: SS2-ZY05719, SS5-HN105, SS9-GZ0565, SS31-WUSS147, SS chz-CZ130302; Figure B is a graph for evaluating the antibacterial ability of the serum of PGs piglets after immunization against Escherichia coli PU-1 and PE164.

[0044] Figure 18 It is a graph for evaluating the opsonophagocytic ability of PGs hyperimmune serum against Streptococcus suis and Escherichia coli of different serotypes with macrophage RAW264.7 as the object. Among them, Figure A is a graph for evaluating the opsonophagocytic ability of PGs hyperimmune serum against representative strains of five representative serotypes of Streptococcus suis: SS2-ZY05719, SS5-HN105, SS9-GZ0565, SS31-WUSS147, SS chz-CZ130302; Figure B is a graph for evaluating the opsonophagocytic ability of PGs hyperimmune serum against Escherichia coli PU-1 and PE164.

[0045] Figure 19 It is a graph for evaluating the opsonophagocytic ability of the serum of PGs piglets after immunization against Streptococcus suis and Escherichia coli of different serotypes with macrophage RAW264.7 as the object. Among them, Figure A is a graph for evaluating the opsonophagocytic ability of the serum of PGs piglets after immunization against representative strains of five representative serotypes of Streptococcus suis: SS2-ZY05719, SS5-HN105, SS9-GZ0565, SS31-WUSS147, SS chz-CZ130302; Figure B is a graph for evaluating the opsonophagocytic ability of the serum of PGs piglets after immunization against Escherichia coli PU-1 and PE164. Detailed implementation mode

[0046] Example 1: Synthesis and expression identification of surface display vector

[0047] The multi-epitope fusion antigen FAS of Streptococcus suis used in the present invention has the amino acid sequence shown in SEQ ID NO.1. This antigen has been disclosed in Chinese Patent CN118255852 A. For the specific screening, synthesis, identification and other contents, please refer to the above patent.

[0048] Nanjing Qingke Biotechnology Company was entrusted to synthesize the plasmid pBAD33-lpp’OmpA FAS. The plasmid sequence is shown in SEQ ID NO.2. This plasmid contains the expression gene of the above multi-epitope fusion antigen FAS. The component composition, gene name, annotation information and restriction enzyme sites in its coding sequence are as Figure 1As shown. The plasmid was electrotransformed into extraintestinal pathogenic Escherichia coli (ordinary extraintestinal pathogenic Escherichia coli is fine. In this example, the Escherichia coli PU-1 strain disclosed in the prior art is taken as an example. The chromosome accession number of this strain is CP042246, and the plasmid accession number is CP0422451), and screened on an LB plate containing 25 μg / mL Cm + After culturing at 37 °C for 12 h, suspected colonies were picked and cultured in LB containing 25 μg / mL Cm + PCR amplification was performed using the pBAD33 universal primers, and the positively identified colonies were preserved (PU-1-pBAD33-lpp’OmpA FAS).

[0049] The positive strain was cultured in LB containing 25 μg / mL Cm + 20% arabinose (L-ara) was added to induce the expression of lpp’OmpA FAS. Samples without L-ara were set as controls. Bacterial cells were collected to prepare protein samples. After 12% SDS-PAGE, the proteins were transferred to the NC membrane by semi-dry transfer printing method, blocked with 5% skim milk powder, incubated with commercial His-tag monoclonal antibody at 4 °C overnight, washed 3 times with PBST, 10 min each time; HRP-labeled goat anti-mouse IgG was used as the secondary antibody and incubated at room temperature for 1 h; washed 3 times with PBST, 10 min each time; added chemiluminescent solution for development. The results showed that the induced protein could be recognized by the His-tag antibody, and the target protein appeared at about 55 kDa( Figure 2 ), and the size of the expressed protein was consistent with the theoretical value.

[0050] Example 2: Surface display evaluation

[0051] To identify whether FAS was successfully displayed on the surface of PU-1, the FAS on the surface of PU-1 was identified by indirect ELISA and indirect immunofluorescence.

[0052] 1. Indirect ELISA to detect the surface display level of FAS

[0053] 1) Coating: After washing the L-ara-induced PU-1-pBAD33 and PU-1-pBAD33-lpp’OmpA FAS three times with sterile PBS, they were diluted to 1×10 7CFU / mL, and add 100 μL per well into the ELISA plate, incubate overnight at 4°C; 2) Blocking: Discard the coating solution, wash the plate 3 times with PBST (200 μL per well) for 5 minutes each time. Add 200 μL per well of 0.5% BSA and block at 37°C for 2 hours; 3) Incubate with primary antibody: Dilute the mouse-derived his-tag antibody at a ratio of 1:200 respectively, discard the blocking solution, add the diluted antibody, 100 μL per well, and incubate at 37°C for 1 hour; 4) Incubate with secondary antibody: Discard the primary antibody, wash the plate 3 times with PBST (200 μL per well) for 5 minutes each time. Add 100 μL per well of HRP-goat anti-mouse IgG diluted at 1:100 and incubate in an incubator at 37°C for 1 hour; 5) Color development: Discard the secondary antibody, wash the plate 3 times with PBST (200 μL per well) for 5 minutes each time; Add 50 μL per well of TMB color development solution and incubate at 37°C in the dark for 15 minutes until the reaction is complete; Add the stop solution, 25 μL per well, to terminate the color development reaction; 6) Reading: Measure the OD value at 450 nm with an ELISA reader.

[0054] As Figure 3 shown, compared with the coating of PU-1-pBAD33 induced by L-ara, the coating of PU-1-pBAD33-lpp’OmpA FAS can be recognized by the his tag antibody, indicating that FAS can be displayed on the surface of PU-1.

[0055] 2. Indirect immunofluorescence assay for detecting the surface display level of FAS

[0056] 1) Fixation: Wash the PU-1-pBAD33 and PU-1-pBAD33-lpp’OmpA FAS induced by L-ara three times with sterile PBS and then resuspend with 4% formaldehyde for fixation at room temperature for 15 minutes; 2) Blocking: Centrifuge to remove the fixation solution, wash three times with PBS and then replace it with 1% BSA for resuspension, and block at room temperature for 30 minutes; 3) Incubate with primary antibody: Dilute the his-tag antibody with 1% BSA and incubate at room temperature for 1 hour; 4) Incubate with secondary antibody: Wash three times with PBST to remove the primary antibody, add the FITC-labeled fluorescent secondary antibody, and incubate at room temperature for 45 minutes; 5) Counterstain the nucleus: Wash three times with PBST to remove the secondary antibody, add DAPI to counterstain nucleic acids at room temperature for 15 minutes; 6) Mounting: Wash 3 times with PBST, take samples, air-dry and fix on a glass slide, add the anti-quenching agent, and cover with a coverslip; 7) Observation: Observe the fluorescence under a laser confocal microscope.

[0057] As Figure 4 shown, compared with the PU-1-pBAD33 induced by L-ara, green fluorescence signals can be observed at 488 nm for PU-1-pBAD33-lpp’OmpA FAS, indicating that FAS can be recognized by the His antibody, that is, FAS can be displayed on the surface of PU-1.

[0058] Example 3: Preparation of PGs

[0059] 1. Preparation of PU-1-pBAD33-lpp’OmpA FAS-pBV220 E-SNA

[0060] Entrust Nanjing Qingke Biotechnology Co., Ltd. to synthesize the plasmid pBV220 E-SNA, and the plasmid sequence is shown in SEQ ID NO.3. The component composition, gene name, annotation information and restriction enzyme sites in its coding sequence are as Figure 5 shown. Electroporate the plasmid into PU-1-pBAD33-lpp’OmpA FAS, and screen on the LB plate containing 25 μg / mL Cm + and 100 μg / mL Amp + . After culturing at 37 °C for 12 h, pick the suspected colonies into the LB containing 25 μg / mL Cm + and 100 μg / mL Amp + for culture, and perform PCR amplification with the pBV220 universal primers. Preserve the colonies identified as positive (PU-1-pBAD33-lpp’OmpA FAS-pBV220 E-SNA).

[0061] 2. Bacteriolysis kinetics of pBV220 E-SNA on PU-1

[0062] PU-1-pBAD33-lpp’OmpA FAS-pBV220 E-SNA is cultured in LB containing 25 μg / mL Cm + and 100 μg / mL Amp + at 28 °C until OD≈0.6, then raise the temperature to 42 °C to induce the lysis of bacteria by E-SNA. Measure the growth curve after raising the temperature and take samples for plate counting 2 h after induction.

[0063] The results of bacteriolysis kinetics show that pBV220 E-SNA has the ability to lyse PU-1, and the bacteriolysis efficiency can reach 99.06% 2 h after induction( Figure 6 ). Add 75 μM WR 12 to inactivate PGs by 100%.

[0064] 3. Observation of bacterial morphology

[0065] After culturing PU-1 to the logarithmic phase, wash it 3 times with PBS, centrifuge and resuspend it with 2.5% glutaraldehyde for fixation; take another logarithmic phase PU-1 and add 75 μM WR 12Incubate at room temperature for 2 hours, wash 3 times with PBS, centrifuge and resuspend with 2.5% glutaraldehyde; PU-1-pBAD33-lpp'OmpA FAS-pBV220 E-SNA was cultured at 28℃ until OD≈0.6, heated to 42℃ for induction for 2 hours, and the cells were collected, washed 3 times with PBS, centrifuged and resuspended with 2.5% glutaraldehyde. Incubate overnight at 4℃; After the subsequent sample preparation was completed by the College of Life Sciences of Nanjing Agricultural University, it was placed under a transmission electron microscope for observation.

[0066] like Figure 7 As shown, PU-1 is expressed by WR 12 After incubation, the internal structure was loose, but the bacterial structure was not damaged; after induction at 42 degrees Celsius, almost all the internal substances of PU-1-pBAD33-lpp'OmpA FAS-pBV220 E-SNA flowed out, leaving only the outer shell structure.

[0067] Example 4: PGs Immunity Assay

[0068] In order to enhance the immune protection effect of PGs, the PGs obtained in Example 3 were mixed with ISA 206VG at a volume ratio of 1:1 and fully vortexed to prepare the vaccine. 5-week-old SPF female Blab / c mice were randomly divided into 2 groups, 40 mice in each group. The first group was set as the control group (PBS) with PBS+adjuvant; the second group was set as the PGs protection group (PGs), and the immunization dose was 5×10 8 CFU / mouse. Mice in each experimental group were immunized by subcutaneous multiple injections. The immunization procedure was as follows: the second immunization was performed 14 days after the first immunization, and the third immunization was performed 14 days later, 0.2 mL / mouse. Blood was collected from each group of mice by orbital venous blood collection on the 11th day after each immunization. After incubation at 37°C for 1 hour, it was placed at 4°C overnight, centrifuged at 1000 rpm, 4°C for 10 minutes, and serum was collected and sterilely packaged and stored at -80°C for later use.

[0069] Four-week-old SPF piglets were randomly divided into two groups, each with 15 piglets. The piglets were immunized by neck injection. The PGs+Adjuvant group (Immune) was used as the control group, and the PBS+Adjuvant group (Control) was used as the control group. The immunization dose was 5×10 9 CFU / head. Immunization was performed once every two weeks, for a total of two immunizations. Blood was collected every 7 days after the first immunization, and blood collection continued until the 7th week. Blood was collected from piglets by ear vein blood collection, incubated at 37℃ for 1 hour, then placed at 4℃ overnight, centrifuged at 1000rpm, 4℃ for 10 minutes, serum was collected, and sterile aliquots were stored at -80℃ for later use.

[0070] Example 5: ELISA specific antibody titer detection

[0071] 1. Coating: The purified FAS and PU-1 proteins after fragmentation were diluted to 10 μg / mL with protein coating solution, and 100 μL per well was added to the enzyme-linked immunosorbent assay (ELISA) plate and incubated overnight at 4°C; 2. Blocking: The coating solution was discarded, and the plate was washed 3 times with PBST (200 μL per well) for 5 minutes each time. 200 μL per well of 0.5% BSA was added and incubated at 37°C for 2 hours for blocking; 3. Incubation with primary antibody: The sera collected after immunization were diluted at ratios of 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12800, 1:25600, 1:51200, and 1:102400 respectively. The blocking solution was discarded, and the diluted sera were added, 100 μL per well, and incubated at 37°C for 1 hour; 4. Incubation with secondary antibody: The primary antibody was discarded, and the plate was washed 3 times with PBST (200 μL per well) for 5 minutes each time. According to the source of the sera, 100 μL per well of HRP-goat anti-mouse or goat anti-pig IgG diluted at 1:100 was added and incubated in an incubator at 37°C for 1 hour; 5. Discard the secondary antibody, and wash the plate 3 times with PBST (200 μL per well) for 5 minutes each time; 6. Add 50 μL per well of TMB chromogenic solution and incubate at 37°C in the dark for 15 minutes until the reaction is complete; 7. Add the stop solution, 25 μL per well, to terminate the chromogenic reaction; 8. Measure the OD value at 450 nm with an ELISA reader and calculate the P / N value. The positive critical value is 2.1.

[0072] Figure 8 For the change in antibody titers against FAS ( Figure 8 A in Figure 8 ) and PU-1 ( B in

[0073] ) in mice after three immunizations, it shows that as the number of immunizations increases, the antibody content in the mouse sera increases accordingly. Figure 9 Figure 9 For the change in antibody titers against FAS ( Figure 9 A in ) and PU-1 (

[0074] B in ) in piglets after two immunizations. The antibody levels against FAS and PU-1 in the sera increase accordingly. After stopping immunization, the antibody titers in the sera can still remain at a high level.

[0075] To further evaluate whether PGs immunization in mice can induce cellular immune responses against FAS and PU-1, the detection of splenic lymphocyte proliferation levels was carried out.

[0076] 1. Isolation of splenic lymphocytes

[0077] 1) Sacrifice the BALB / c mice that have been immunized three times by cervical dislocation, aseptically remove the spleen, and wash it once with sterile PBS; 2) Gently grind it inside a syringe on a 70 μM sterile cell strainer, add 3 mL of RPMI 1640 medium, and after filtering the tissue residues, prepare a spleen cell suspension; 3) Isolate spleen lymphocytes according to the steps of the spleen lymphocyte separation kit (Solarbio); 4) Resuspend the spleen lymphocytes with RPMI 1640 medium containing 10% fetal bovine serum, count the lymphocyte suspension by trypan blue staining, and adjust the cell density to 5×105 cells / mL.

[0078] 2. Spleen lymphocyte proliferation assay

[0079] 1) Inoculate the above cell suspension into a 96-well plate at an inoculation density of 200 μL / well; 2) Add 10 μg of the purified FAS and PU-1 fragmented proteins, and an equal amount of PBS as a control; 3) Continue to culture the cells at 37 °C and 5% CO2 for 48 h; 4) After 48 h, add CCK8 reagent (Beyotime), 20 μL / well, and continue to culture the cells at 37 °C and 5% CO2 for 4 h; 5) After 4 h, measure the absorbance value of the 96-well plate at 450 nm with an enzyme-linked immunosorbent assay (ELISA) reader, and calculate the stimulation index (SI) of the spleen cells. The calculation formula is as follows: Stimulation index (SI) = (OD value of the stimulated group - OD value of the blank group) / (OD value of the unstimulated group - OD value of the blank group).

[0080] The results are as Figure 10 shown. When the spleen cells of the mice immunized with PGs were stimulated by either FAS or PU-1, the stimulation index (SI) was significantly higher than that of the control group, indicating that immunization could stimulate the mice to produce a higher level of spleen lymphocyte proliferation response.

[0081] Example 7: Challenge protection test

[0082] To better evaluate the protective efficacy of PGs, the immunized mice were inoculated with the representative strains ZY05719 and GZ0565 of the two main epidemic serotypes of Streptococcus suis, SS2 and SS9, by intraperitoneal infection, 200 μL / mouse, and the challenge bacterial dose was 3×10 8 CFU / mouse. Observe the mice daily after challenge, record the morbidity and mortality, and the survival curve is as Figure 11 shown. When the mice were challenged with ZY05719, PGs could provide a 70% protection rate for the mice, while all the mice in the immunized PBS group died ( Figure 11 in A); when challenged with GZ0565, the survival rate of the protected group of mice could reach 90% ( Figure 11In (B) of the figure, it shows that PGs can provide a high protection rate against infection with multiple serotypes of Streptococcus suis in mice. Similarly, PU-1 was inoculated by intraperitoneal challenge at a dose of 200 μL / mouse, and the challenge bacterial amounts were 1×10 7 CFU / mouse. The survival curve shows that PGs can provide 80% protection effect for mice ( Figure 11 in (C) of the figure). In summary, after immunizing mice with PGs, it can provide considerable protection against both Streptococcus suis and Escherichia coli.

[0083] In piglets, a challenge protection test was also carried out by intraperitoneal infection. The challenge doses were ZY05719 - 1.6×10 9 CFU / head; GZ0565 - 2×10 9 CFU / head, and PU-1 - 1.5×10 9 CFU / head. The records of the morbidity and mortality of piglets are shown in Figure 12 . After challenging the control group, the piglets showed fever symptoms and even died; after immunizing piglets with PGs, whether it was infection with Streptococcus suis or Escherichia coli, the piglets did not die.

[0084] Example 8: Bacterial load in organs

[0085] To more comprehensively evaluate the protective effect provided by PGs against Streptococcus suis infection after immunization, representative strains ZY05719 and GZ0565 of SS2 and SS9, as well as PU-1, were respectively used to infect mice at doses of 3×10 8 CFU / mouse, 3×10 8 CFU / mouse, and 1×10 7 CFU / mouse. At 12 h after challenge, the bacterial loads in different organs and tissues of the mice were measured.

[0086] The results are as Figure 13 shown. By counting the bacteria in different tissues, whether infected with Streptococcus suis or Escherichia coli, compared with the control group, the bacterial loads in the blood, brain tissue, liver, spleen, and kidney of the mice in the PGs-immunized group were all reduced.

[0087] In piglets, the change of bacterial load in the blood after challenge was carried out. Blood samples were taken at 3 h, 12 h, 24 h, 36 h, 48 h, 60 h, and 72 h after challenge to record the bacterial load in the blood. As Figure 14 shown, the bacterial load reached the peak at 36 h after infection with ZY05719, GZ0565, and PU-1, and the bacterial load in the blood of the PGs-protected group was significantly lower than that of the control group.

[0088] Example 9: Monitoring the body temperature of piglets after challenge

[0089] At 3 h, 12 h, 24 h, 36 h, 48 h, 60 h, and 72 h after challenging with the virulent agents, the body temperatures of the piglets were measured at the above time points using an infrared thermometer. The body temperature change curve is as Figure 15 shown. As the infection time prolonged after infection with the three strains of bacteria, the body temperature increased, reached the peak at 36 h after infection, and some piglets died. The body temperatures of the surviving piglets gradually decreased to the normal level.

[0090] Example 10: Detection of the antibacterial level mediated by serum after immunization

[0091] PBS and PGs were mixed with ISA 206VG at a volume ratio of 1:1 and vortexed thoroughly for emulsification for later use. Six New Zealand white rabbits were used as the immunization subjects and randomly divided into 2 groups with 3 rabbits in each group. The first group was immunized with the PBS + adjuvant group (PBS); the second group was immunized with the PGs group (PGs) at a dose of 1 mg / rabbit. The immunization route was subcutaneous multi-point injection at 1 mL / rabbit, and booster immunization was carried out every 14 days for three consecutive times. Blood was collected after each immunization, and the serum was separated and reserved after specific antibody titer detection.

[0092] To evaluate whether the serum after PGs immunization could produce antibodies against more serotypes of Streptococcus suis and Escherichia coli, a bacteriostatic test mediated by hyperimmune serum was carried out. Clinical isolates representative strains ZY05719, HN105, GZ0565, WUSS147, CZ130302 of five main serotypes SS2, SS5, SS9, SS31, and SS chz of Streptococcus suis; clinical isolates PU-1 and PE164 of Escherichia coli were cultured to the logarithmic phase and diluted to 1×10 6 CFU / mL with the medium. 100 μL of the bacterial solution was mixed with the serum after immunization of each group and co-incubated at 37 °C and 5% CO2 for 2 h. After incubation, the samples were serially diluted and the number of viable bacteria after incubation was recorded. The serum bacteriostatic percentage SBA = 1 - (treatment group / PBS treatment group).

[0093] As Figure 16 shown, after incubation with the PGs hyperimmune serum, the above 8 kinds of bacteria all showed good bacteriostatic effects compared with the control group, indicating that antibodies against multiple serotypes of Streptococcus suis and Escherichia coli could be produced after PGs immunization.

[0094] Similarly, the bacteriostatic effects of the serum after PGs immunization of piglets against Streptococcus suis and Escherichia coli were evaluated. Compared with the control group serum, although there was no significant difference in the inhibitory effect on GZ0565 and WUSS147, PGs could widely inhibit the growth of Streptococcus suis ( Figure 17 ); and the serum after PGs immunization could significantly inhibit the growth rate of three strains of Escherichia coli.

[0095] Example 11: Opsonophagocytosis mediated by hyperimmune serum

[0096] Using RAW264.7 cells as the object, the ability of opsonophagocytosis mediated by PGs hyperimmune serum was simulated.

[0097] 1) Adjust the culture media of the 7 strains mentioned in Example 9 to an OD600 of 0.6. Wash the bacterial suspension three times with sterile PBS and then dilute it with PBS to 1×10 8 CFU / mL. 2) Mix 100 μL of the post-immunization serum and the negative serum with an equal volume of the bacterial suspension and incubate at 37 °C for 30 min. 3) Centrifuge at 5000 rpm for 5 min, discard the supernatant, and resuspend with 100 μL of DMEM medium. 4) Add the above bacterial suspension to a 24-well plate filled with RAW264.7 cells and incubate in a 37 °C, CO2 incubator for 1.5 h. 5) After discarding the supernatant, wash three times with sterile PBS and then replace it with DMEM medium containing 5 μg / mL penicillin and 100 μg / mL gentamicin to kill extracellular bacteria. 6) Incubate in a 37 °C, CO2 incubator for 1.5 h. 7) After discarding the supernatant, wash seven times with sterile PBS, and take the last washing solution to drop on the plate to detect the amount of extracellular bacteria. 8) Add 500 μL of ddH2O to lyse the cells, perform serial dilution, and drop on the plate for counting, which is the total amount inside the cells at this time.

[0098] The results are as Figure 18 and Figure 19 shown. After incubating Streptococcus suis of five serotypes and two strains of Escherichia coli with the post-immunization serum and then stimulating RAW264.7, the phagocytosis rate of macrophages against them can be enhanced to varying degrees. The post-immunization serum of piglets immunized with PGs can also promote the phagocytosis of RAW264.7 against Streptococcus suis and Escherichia coli. It further shows that the specific antibodies contained in the serum can bind to Streptococcus suis of five serotypes and two strains of Escherichia coli, and activate macrophages, enhancing the phagocytosis ability of macrophages against Streptococcus suis and Escherichia coli.

Claims

1. A preparation method of a Streptococcus suis and Escherichia coli double bacterial ghosts, characterized in that, Comprising the following steps: Construct a recombinant plasmid containing the expression gene of the Streptococcus suis multi-epitope fusion antigen FAS, co-transfer it with the bacterial ghost induction plasmid into porcine extraintestinal pathogenic Escherichia coli, first induce the expression of the FAS antigen, and then culture to induce the production of bacterial ghosts, thus obtaining the Streptococcus suis and Escherichia coli dual bacterial ghosts; the amino acid sequence of the Streptococcus suis multi-epitope fusion antigen FAS is shown in SEQ ID NO.

1.

2. The preparation method of the Streptococcus suis and Escherichia coli double bacterial ghosts according to claim 1, characterized in that, The recombinant plasmid containing the expression gene of the Streptococcus suis multi-epitope fusion antigen FAS, the vector is selected from pBAD33; as a specific implementation, the recombinant plasmid is pBAD33-lpp’OmpA FAS, and its nucleotide sequence is shown in SEQ ID NO.

2.

3. The preparation method of the Streptococcus suis and Escherichia coli double bacterial ghosts according to claim 1, characterized in that, The porcine extraintestinal pathogenic Escherichia coli is the PU-1 strain; for inducing the expression of the FAS antigen, L-arabinose is used for induction.

4. The preparation method of the streptococcus suis and escherichia coli double bacterial ghosts according to claim 1, wherein, The vector of the bacterial ghost induction plasmid is selected from pBV220; as a specific implementation, the bacterial ghost induction plasmid is pBV220 E-SNA, and its nucleotide sequence is shown in SEQ ID NO.

3.

5. The preparation method of the streptococcus suis and escherichia coli double bacterial ghosts according to claim 1, characterized in that The method for culturing and inducing the production of bacterial ghosts includes the following: in LB containing Cm + and Amp + , culture at 28±2°C until OD = 0.6±0.05, raise the temperature to 42±2°C to induce the lysis of bacteria by E-SNA, and add WR 12 for inactivation.

6. A Streptococcus suis and Escherichia coli dual bacterial ghost, characterized in that, The Streptococcus suis and Escherichia coli dual bacterial ghosts are obtained by the preparation method according to any one of claims 1-5.

7. A pharmaceutical composition, characterized in that, The pharmaceutical composition contains the Streptococcus suis and Escherichia coli dual bacterial ghosts according to claim 6.

8. A recombinant bacterium, characterized in that, The recombinant bacterium contains an expression plasmid and a lysis plasmid, the expression plasmid is a recombinant plasmid containing the expression gene of the Streptococcus suis multi-epitope fusion antigen FAS, and the lysis plasmid is a bacterial ghost induction plasmid; the amino acid sequence of the Streptococcus suis multi-epitope fusion antigen FAS is shown in SEQ ID NO.

1.

9. The recombinant bacterium according to claim 8, wherein The vector of the recombinant plasmid is selected from pBAD33; as a specific implementation, the recombinant plasmid is pBAD33-lpp’OmpA FAS, and its nucleotide sequence is shown in SEQ ID NO.2; The vector of the bacterial ghost induction plasmid is selected from pBV220; as a specific implementation, the bacterial ghost induction plasmid is pBV220 E-SNA, and its nucleotide sequence is shown in SEQ ID NO.

3.

10. Use of the Streptococcus suis and Escherichia coli dual bacterial ghosts according to claim 6, the pharmaceutical composition according to claim 7, and the recombinant bacterium according to claim 8 in the preparation of drugs for preventing Streptococcus suis and extraintestinal pathogenic Escherichia coli infections.

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