CN119331108A

CN119331108A - Preparation method and application of Escherichia coli biomimetic vesicles presenting H9 subtype avian influenza virus HA1 protein

Info

Publication number: CN119331108A
Application number: CN202411499731.5A
Authority: CN
Inventors: 彭大新; 李悦; 陈素娟; 秦涛; 印云聪
Original assignee: Yangzhou University
Current assignee: Yangzhou University
Priority date: 2024-10-25
Filing date: 2024-10-25
Publication date: 2025-01-21

Abstract

The present invention belongs to the field of biotechnology, and relates to a preparation method and application of an Escherichia coli bionic vesicle presenting H9 subtype avian influenza virus HA1 protein. The present invention improves the yield of bionic vesicles by constructing a defective bacterium ΔtolA-ΔaraD of O78 serotype Escherichia coli, and realizes the surface presentation of HA1 protein on bionic vesicles through L-arabinose induction and high-pressure homogenization technology. The present invention presents HA1 protein by using bionic vesicles derived from O78 serotype Escherichia coli for the first time, which not only improves the yield of vesicles, but also increases the soluble expression of HA1 protein, with low cost and high feasibility.

Description

Preparation method and application of escherichia coli bionic vesicle presenting H9 subtype avian influenza virus HA1 protein

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a preparation method of high-yield biomimetic vesicle stable presentation H9N2 subtype Avian Influenza Virus (AIV) HA1 protein derived from O78 serotype escherichia coli and application thereof in resisting H9 subtype avian influenza and avian colibacillosis.

Background

In intensive farms for poultry, mixed infection with multiple pathogens is common, and economic losses are enormous. H9N2 subtype avian influenza virus (Avian influenza virus, AIV) is one of the most prevalent pathogens in domestic poultry, and is less pathogenic to poultry when infected alone, usually does not lead to direct death, but in actual production many production losses are directly related to it. Avian pathogenic escherichia coli (Avian pathogenic e. Coll, APEC), one of the most common bacterial pathogens in poultry, often co-infects with AIV subtype H9N2 to produce a synergistic effect, leading to increased mortality in poultry, decreased productivity and risk of human food borne transmission.

Vaccination is currently one of the main strategies for controlling avian influenza and avian colibacillosis. Subunit vaccine is used as a novel vaccine, and specific antigen components of pathogens are presented, so that immune response is stimulated, and the subunit vaccine has the advantages of higher safety, simpler production and the like compared with the traditional vaccine. However, subunit vaccines are often less immunogenic and there is a need for effective carrier systems to enhance their immune effects. In recent years, outer membrane vesicles (Outer membrane vesicles, OMVs) have been increasingly attracting attention as subunit vaccine vectors. The OMVs of the escherichia coli become one of research hotspots because of easy preparation, low cost and strong controllability. Several recent studies have shown that OMVs can be targeted by producing heterologous proteins in the outer membrane or periplasm. In addition, the targeting process can be realized by fusion expression of the heterologous protein and the vesicle related protein, so that the method opens up possibility for displaying the heterologous protein by taking OMVs as a carrier. ClyA of escherichia coli acts as a membrane anchor protein, transported to OMVs by the bacterial own regulatory mechanism. However, the inability of high levels of ClyA to localize to the outer membrane results in OMVs with limited ability to present foreign proteins, which greatly limits the efficiency of ClyA anchoring to the outer membrane for antigen delivery.

Traditional vaccines have limitations in dealing with pathogen diversity and variability, and antibiotic abuse has raised bacterial resistance problems. APEC serotypes are numerous, with O78 being one of the common serotypes in china. However, existing engineered E.coli OMVs express foreign proteins that can only generate immune responses against foreign antigens, due to the non-optimal cross-protective effect between the different serotypes of APEC. Therefore, developing a bivalent vaccine against H9N2 AIV and O78 serotype APEC simultaneously becomes a scientific research problem and actual demand which need to be solved urgently.

Disclosure of Invention

Aiming at the problems, the invention improves the yield of the bionic vesicle by constructing O78 serotype escherichia coli defect bacteria delta tolA-delta araD, realizes the surface presentation of HA1 protein on the bionic vesicle by an L-arabinose induction and high-pressure homogenization technology, and evaluates whether the HA1 protein can be used as an immunogen to effectively protect chickens from being infected by H9N2 subtype avian influenza virus and O78 serotype escherichia coli.

The technical scheme provided by the invention is as follows:

The fusion protein is obtained by connecting ClyA protein and HA1 protein in series through a Linker, and the sequences of the ClyA protein, the Linker and the HA1 protein are shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO. 3.

The invention also provides a preparation method of the H9 subtype avian influenza virus HA1 protein presented by the escherichia coli bionic vesicle, clyA and H9N2 AIV HA1 proteins are connected in series through a Linker, fusion proteins are expressed in vesicle donors, the fusion proteins ClyA-HA1 are presented on the surface of the bionic vesicle by utilizing a high-pressure homogenization technology, and the recombinant bionic vesicle (BBV) is obtained, and the nucleotide sequences of the ClyA protein, the Linker and the HA1 proteins are subjected to codon optimization aiming at the escherichia coli, as shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO. 3.

SEQ ID NO.1:

ATGACAGAAATAGTAGCTGACAAGACCGTTGAGGTGGTGAAAAACGCCATCGAAACCGCAGATGGTGCACTCGACCTGTATAACAAGTATCTGGACCAGGTGATTCCGTGGCAGACCTTTGATGAAACCATCAAGGAGCTGAGTCGTTTTAAACAAGAATACAGCCAAGCGGCGTCTGTTCTGGTTGGTGACATCAAGACTTTGCTTATGGATTCGCAAGATAAGTACTTCGAAGCGACGCAAACCGTTTATGAGTGGTGTGGTGTCGCTACCCAGCTGTTGGCGGCATATATTCTGTTATTCGACGAGTACAACGAAAAGAAAGCGAGCGCGCAGAAAGACATTCTGATCAAGGTGCTGGACGACGGCATTACCAAATTGAATGAGGCTCAAAAGTCTTTGTTGGTTTCATCCCAGAGCTTCAACAACGCGTCTGGAAAGTTGCTGGCGCTGGACAGCCAGCTGACCAATGATTTCAGCGAAAAGTCCAGCTATTTCCAGAGCCAAGTGGACAAAATCCGTAAAGAAGCCTACGCAGGCGCGGCCGCGGGTGTTGTAGCCGGTCCGTTTGGTCTGATCATCAGCTACAGCATTGCCGCAGGTGTCGTGGAAGGCAAGCTCATCCCGGAACTGAAGAACAAGCTGAAATCAGTCCAGAATTTCTTTACCACCCTGAGCAATACCGTGAAGCAGGCAAATAAAGATATTGATGCAGCGAAATTAAAGCTCACCACAGAGATCGCGGCGATTGGCGAAATTAAAACCGAGACGGAAACGACCCGTTTTTATGTTGACTACGATGACCTGATGCTGAGCCTGCTTAAGGAGGCGGCTAAAAAAATGATTAACACGTGCAACGAGTACCAGAAGCGCCATGGTAAAAAAACTCTGTTCGAGGTTCCGGAGGTG;

SEQ ID NO.2:

GGCTCCGGCTCTGGTGAAGGTTCCGGC;

SEQ ID NO.3:

GACAAAATCTGCATTGGTTATCAAAGCACGAACAGCACCGAAACCGTTGATACGCTGACTGAGAACAACGTGCCGGTTACACATGCGAAAGAACTGCTTCACACCGAGCACAACGGCATGCTGTGTGCGACCGGTTTGGGCCACCCGCTCATCCTGGATACGTGCACCATTGAAGGTCTGATCTACGGCAATCCGAGCTGCGACCTGTTGTTGGGTGGACGTGAATGGAGCTATATCGTGGAAAGACCATCCGCGGTTAACGGTCTGTGCTACCCGGGTAACGTGGAGAACCTGGAAGAGCTGCGCAGCCTTTTCAGTTCTGCGTCCTCCTACCAACGTATCCAAATCTTTCCGGATACCATCTGGAATGTGACGTACAGCGGCACCAGCAAGGCGTGTTCGGACAGCTTCTATCGTAGCATGCGTTGGCTGACCCAGAAAAATAACGCCTACCCGATTCAGGATGCTCAATATACCAACAACCAAGAAAAAAACATTCTGTTCATGTGGGGTATTAACCATCCGCCTACTGACACTGCCCAAACGAACCTGTACACCCGTACCGACACCACAACCTCCGTTGCGACCGAGGAGATCAACCGTACCTTTAAACCGTTGATTGGTCCGCGTCCGTTGGTTAATGGTTTACAGGGTCGCATCGATTATTACTGGTCAGTCCTGAAGCCGGGTCAGACCTTGCGCATTCGTTCTAATGGCAATTTAATTGCACCGTGGTATGGTCACATCCTGTCCGGCGAATCCCATGGTCGCATCCTGAAGACCGACCTGAAACGTGGTAGCTGCACGGTACAGTGTCAGACCGAGAAGGGCGGACTCAATACCACCCTGCCATTTCAGAATGTCAGCAAGTACGCGTTCGGCAACTGCTCTAAGTACATCGGGATTAAAAGCCTGAAGCTGGCAGTGGGTCTGCGTAATGTTCCGAGTCGCTCTAGCCGC.

Further, clyA and H9N2 AIV HA1 proteins are connected in series through a Linker, a ribosome binding site is fused at the N end of the ClyA sequence to promote the expression of the fusion protein, a 6 Xhis tag is fused at the C end of the HA1 sequence, the ClyA-HA1 sequence is inserted between EcoRI and HindIII cleavage sites in a pBAD18-Cm vector by using T4 ligase, and a pBAD18-ClyA-HA1 recombinant plasmid is constructed, wherein the ribosome binding site sequence is shown as SEQ ID NO. 4.

Further, the primers for constructing the pBAD18-ClyA-HA1 recombinant plasmid are shown in SEQ ID NO. 11-14.

Furthermore, the vesicle donor is O78 serotype escherichia coli defect bacteria delta tolA-delta araD, and the vesicle donor knocks out araD genes through a lambda-Red homologous recombination technology.

Further, the primers of the targeting fragment for the araD gene knockout are shown in SEQ ID NO.5 and 6.

Furthermore, the identification primer of the O78 serotype escherichia coli defect bacteria delta tolA-delta araD is shown as SEQ ID No. 7-10.

The invention also provides a recombinant bionic vesicle, which is prepared by the method.

The invention also provides a recombinant expression vector, and the RBS-optimized pBAD18-Cm improves the expression quantity of fusion protein ClyA-HA 1.

Further, the RBS sequence is GAAGGAGATATACAT (SEQ ID NO. 4).

The invention also provides a bionic vesicle donor, which contains the recombinant expression vector.

Furthermore, the bionic vesicle donor knocks out araD gene through lambda-Red homologous recombination technology, eliminates metabolic decomposition of wild escherichia coli on an L-arabinose inducer, and increases the stability of expression of fusion protein ClyA-HA 1.

The invention also provides a method for presenting recombinant protein by using the bionic vesicle, which uses a high-pressure homogenization technology to quickly destroy bacterial membranes, forces intracellular fusion proteins to be released and carry out plasma membrane recombination, and effectively drives the bacterial membranes to generate recombinant bionic vesicles.

Further, the high pressure homogenizer pressure was set at 1000 bar,4 ℃.

The invention also provides application of the fusion protein or the method or the recombinant bionic vesicle in preparing reagents for preventing and treating H9 subtype avian influenza virus and O78 serotype escherichia coli infection.

Furthermore, the recombinant bionic vesicle is used for inhibiting the H9 subtype avian influenza virus from expelling toxin in chicken larynx and cloaca, reducing the bacterial load in chicken infected by O78 serotype escherichia coli, and reducing the inflammation degree of air sac and heart.

Advantageous effects

The invention firstly utilizes the bionic vesicle derived from O78 serotype escherichia coli to present the HA1 protein, thus not only improving the vesicle yield, but also increasing the soluble expression of the HA1 protein, and having lower cost and high feasibility. BBVs (BBV-HA 1) of the displayed HA1 protein can cause the specific sIgA level aiming at H9N2 AIV and outer membrane protein in nasal cavity and trachea to be obviously increased after the SPF chicken is immunized by nasal drip, igY level aiming at H9N2 AIV and outer membrane protein in immune serum is obviously increased, HI antibody titer reaches 5.2+/-0.84 log2, and in-vitro bactericidal activity of immune serum is obviously enhanced. CD4 ⁺ T cells in peripheral blood lymphocytes of immunized chickens were enhanced in immune activity. The lymphocytes were stimulated with HA1 and outer membrane proteins, respectively, and BBV-HA1 proliferated efficiently compared to PBS group. The immune protection result shows that BBV-HA1 can effectively reduce bacterial load in chicken, reduce inflammation degree of air sac and heart, promote weight recovery of chicken, and provide protection effect against H9 subtype strain attack. The recombinant bionic vesicle can provide effective protection for H9N2 AIV and O78 serotype APEC, and provides support for the development of bivalent vaccines.

Drawings

FIG. 1 shows PCR verification of the aroD gene knockout of J11 strain, A. With the result of PCR amplification of homology arm primer F1/R1, M: DL 2000,1-3 represents the non-mutant successful strain, 4 represents the mutant successful strain, B. With the result of PCR amplification of aroD gene specific primer F2/R2, M: DL 2000,1-3 represents the non-mutant successful strain, 4 represents the mutant successful strain;

FIG. 2 is a schematic diagram constructed for recombinant biomimetic vesicles;

FIG. 3 is a representation of recombinant biomimetic vesicles, A. Electron microscopy of recombinant biomimetic vesicles with scale of 100nm, B. Particle size of recombinant biomimetic vesicles;

FIG. 4 shows the successful construction result of the biomimetic vesicle presenting HA1 protein, wherein A. The biomimetic vesicle presenting ClyA-HA1 recombinant protein SDS-PAGE analysis, B. The biomimetic vesicle presenting ClyA-HA1 recombinant protein Western Blot identification, C. The biomimetic vesicle presenting ClyA-HA1 recombinant protein localization analysis, 1 represents the uninduced lysis supernatant, 2 represents the induced lysis supernatant, and 3 represents BBV-ClyA-HA;

FIG. 5 shows the amount of biomimetic vesicle presenting protein, A. BCA method for total vesicle determination, B. BSA gradient concentration for recombinant protein determination;

FIG. 6 shows sIgA antibody levels against TX, A. Anti-TX specific sIgA antibody levels in nasal cavity, B. Anti-TX specific sIgA antibody levels in trachea, expressed as OD ₄₅₀ values;

FIG. 7 shows IgY antibody levels against TX, A. Specific IgY antibody levels against TX in serum, expressed as OD ₄₅₀ values, B. HI antibody titers;

FIG. 8 shows sIgA antibody levels against Omps, A. Anti-TX specific sIgA antibody levels in nasal cavity, B. Anti-TX specific sIgA antibody levels in trachea, expressed as OD ₄₅₀ values;

FIG. 9 shows IgY level and bactericidal activity against Omps in serum, A. Specific IgY antibody level against Omps in serum expressed as OD ₄₅₀ value, B. Bactericidal activity of immune serum;

FIG. 10 shows lymphocyte proliferation results A. Proliferation results for HA1 protein B. Proliferation results for Omps;

FIG. 11 shows the immunoprotection of recombinant biomimetic vesicles against chicken infection O78 serotype APEC, A. Body weight changes, B. Pathological changes of heart and air sac, C. Lesion scoring, D. Bacterial load in blood, E. Bacterial load in various tissue organs.

Detailed Description

Example 1

Construction of the Δ tolA Δarad J11 Strain

The araD gene was knocked out using lambda-Red homologous recombination techniques. The primer of the targeting fragment (araD-DF/R) was designed according to the araD gene sequence of E.coli published in GenBank, and the sequence is shown in Table 1.

The chloramphenicol resistance gene containing the homology arm was amplified using the pKD3 plasmid as a template and the araD-DF and araD-DR primers in Table 1, and recovered after 1% gel electrophoresis. Subsequently, the Δ tolA J11 strain carrying the pKD46 plasmid was cultured in LB medium supplemented with ampicillin and 0.2% L-arabinose until OD ₆₀₀ =0.5 at 30 ℃. Bacterial cells were then harvested and washed three times with ice-cold 10% glycerol to prepare electrotransformed competent cells. Then the targeting DNA fragment is electrically transferred into competent cells, cmR positive single colonies are screened at 37 ℃, positive strains are prepared into electrically transferred competent cells, the electrically transferred competent cells are transferred into pCP20 plasmids, the electrically transferred competent cells are cultured at a constant temperature of 30 ℃ for 12-16 h, amR positive single colonies are selected and screened, the positive strains are cultured at a constant temperature of 42 ℃, pCP20 plasmids are eliminated, and then the electrically transferred competent cells are cultured at a constant temperature of 37 ℃ to obtain a non-resistant knockout strain delta tolA delta araD J11. PCR was performed with two homology arm primers (F2/R2), the size of the band of the strain Delta tolA J.DELTA.3911 was 797 bp, and the strain of the double gene Delta tolA DeltaaraD J11 mutant was 1122 bp. After the resistant gene knockout, the Δ tolA J11 strain band size was 696 bp and the Δ tolA Δaradj11 gene mutant strain did not amplify araD gene using araD gene specific primers (F1/R1) (fig. 1), indicating successful construction of the gene mutant strain.

Example 2

Delta tolA DeltaaraD J11 biomimetic vesicle presenting H9 subtype AIV HA1 recombinant protein

The recombinant biomimetic vesicle construction strategy is shown in figure 2. ClyA (GenBank: AF 240780.1) and HA1 (GenBank: JN 653620.1) were both codon optimized and synthesized for escherichia coli by nanjing gold sry. First, clyA and HA1 were connected in series by Linker using Overlap PCR, and ribosome binding site (Ribosomebinding site, RBS) was fused to the N-terminal of the ClyA sequence to promote expression of the fusion protein, and 6 Xhis tag was fused to the C-terminal of the HA1 sequence to verify protein expression, and the ClyA-HA1 sequence was inserted between EcoRI and HindIII cleavage sites in pBAD18-Cm vector using T4 ligase to construct pBAD18-ClyA-HA1 recombinant plasmid, the primers are shown in Table 2.

Note that the underlined indicates the cleavage site, bold is the ribosome binding site, italics is the 6 Xhis tag.

The recombinant plasmid pBAD18-ClyA-HA1 is electrotransferred into delta tolA delta araD J11 competent cells, then single clone is selected randomly in LB culture medium of 5 mL CmR, and is shake cultured at 37 ℃ and 220 rpm for 12h, then bacterial liquid is subjected to PCR identification, and the recombinant plasmid with correct PCR identification is sent to the biological company for sequencing.

The Delta tolA DeltaaraD J11 strain carrying pBAD18-ClyA-HA1 is inoculated into a chloramphenicol-resistant liquid LB at a ratio of 1:100, shake culture is carried out at 37 ℃ and 220 rpm, when the OD ₆₀₀ value is about 0.5, L-arabinose with a final concentration of 0.2% is added, induction is carried out for 12 hours at 25 ℃ and 220 rpm, bacterial cells are collected, and PBS is added to resuspend the bacterial cells. Delta tolA DeltaaraD J11 cells expressing ClyA-HA1 recombinant protein were resuspended in PBS buffer (pH 7.4). The separation BBVs was carried out by forcing the bacterial wall break under high pressure (1000 bar,4 ℃) using a high pressure homogenizer (AH-E). The high pressure homogeneous sample was collected, centrifuged at 6,000Xg for 30 min at 4℃to precipitate impurities, and the supernatant was collected. The supernatant was filtered through a 0.45 μm filter and concentrated using a 100 kDa ultrafiltration tube. Ultracentrifugation (150000 g, 1h, 4 ℃) was then performed and the pellet was resuspended in sterile PBS (pH 7.4) to give the enriched biomimetic vesicles. By carrying out electron microscope observation and particle size analysis on BBVs and BBV-ClyA-HA1 obtained by separation, and detecting the self-assembly condition of BBVs through a transmission electron microscope, it is found that the recombinant BBVs can keep a nano-scale spherical structure, which shows that the expression of the ClyA-HA1 recombinant protein does not influence the form of BBVs. The particle size was analyzed by a Markov particle analyzer (ES 90 Nano) and found to be 205.77.+ -. 1.76: 1.76 nm for BBV, while 233.70.+ -. 3.99: 3.99 nm for BBV-ClyA-HA1 (FIG. 3).

Subsequently, it was subjected to SDS-PAGE electrophoresis. The results showed that there was one over-expressed band of about 70 kDa in both the post-induction supernatant and BBVs compared to the uninduced bacterial solution (FIG. 4A). Western Blot verification was performed using H9N2 AIV TX polyclonal antibody as primary antibody, and the results showed that specific bands of about 70 kDa appeared in supernatant and BBVs after induction (FIG. 4B), consistent with the theoretical molecular weight of ClyA-HA1, demonstrating that recombinant proteins were able to be expressed correctly in supernatant and BBVs. Furthermore, we attempted to detect the localization of recombinant proteins at BBVs. BBVs expressing the ClyA-HA1 recombinant protein was incubated with proteinase K at 37℃in the presence or absence of 1% SDS. The samples were then subjected to Western Blot analysis using a 6 xhis monoclonal antibody as primary antibody. The data indicate that proteinase K is able to directly degrade the ClyA-HA1 protein, regardless of whether BBVs is disrupted by SDS, indicating that the HA1 protein is localized to the surface of BBVs (FIG. 4C).

The total amount of outer membrane vesicles and biomimetic vesicles were measured by BCA method, and found that per 1L of culture naturally produced outer membrane vesicles were 2.24 mg and biomimetic vesicles were 9.66 mg, in addition we used coomassie brilliant blue method to preliminarily estimate recombinant protein amount, and the biomimetic vesicles were subjected to SDS-PAGE analysis with Bovine Serum Albumin (BSA) standard of known concentration and stained with coomassie brilliant blue, and the loading condition of recombinant protein in the biomimetic vesicles was preliminarily analyzed according to the gray value of BSA protein bands, and found that each 9.66 mg biomimetic vesicles contained 3.26 mg recombinant protein, and the recombinant protein amount was 33.74% of the biomimetic vesicles (fig. 5).

Example 3

Immunoprotection assay of recombinant biomimetic vesicles

SPF chickens of 1 week old are randomly divided into 9 groups, H9N2 AIV TX is inactivated at a final concentration of 2 permillage formaldehyde at 4 ℃ for 24H, and the escherichia coli J11 strain is heat-inactivated for later use. The immunization groups are shown in table 3. The TX inactivated vaccine and the J11 inactivated vaccine are uniformly mixed with an adjuvant according to the proportion of 1:1, and are inoculated by subcutaneous multipoint injection, and the vaccine is immunized once every two weeks for 2 times. The BBV, HA1, BBV+HA1 and BBV-HA1 groups were immunized by nasal drip route, once every other week for 3 total immunizations.

The level of specific sIgA antibodies in serum against Omps and TX specific IgY and nasal and tracheal lavages was determined using an indirect ELISA method. Briefly, the elisa plate was incubated with 50 ng purified Omps or TX whole virus overnight at 4 ℃ in 100 μl of coating buffer (15.9 mM Na ₂CO₃、29.7 mM NaHCO₃, pH 9.6). Plates were then washed 3 times with Phosphate Buffer (PBST) containing 0.05% Tween-20, 10 min each. 2h was blocked with PBST containing 5% skim milk at 37 ℃. Serum samples are diluted according to 1:6400 times, nasal cavity and trachea lavage fluid is diluted according to 1:5 times, 100 mu L of diluted sample to be tested is added into each hole, and the mixture is placed at 37 ℃ for incubation of 1 h. Subsequently, 100. Mu.L of rabbit anti-chicken IgY-HRP (H+L) or rabbit anti-chicken sIgA-HRP (H+L) antibody (1:8000 dilution) was added and incubated at 37℃for 1H. 100. Mu.L of TMB was added to react 15min in the dark, and then 50. Mu.L of 2M H ₂ SO4 was added to terminate the reaction. Finally, the optical density value at OD ₄₅₀ nm is measured in an enzyme-labeled instrument.

Immune serum HI antibody level determination 4 units of antigen were prepared according to the TX strain HA test results. Using a 96-well V-shaped microplate, 25. Mu.L of PBS was added to each of wells 1 to 11, and 50. Mu.L of PBS was added to well 12. 25 mu L of serum to be detected is added into the 1 st hole, 25 mu L of serum to be detected is removed from the 2 nd hole after fully and uniformly mixing, the 25 mu L of serum to be detected is sequentially diluted to the 10 th hole by a multiple ratio, the 25 mu L of serum to be detected is discarded from the 10 th hole, the 11 th hole is used as a virus control, and the 12 th hole is used as a red blood cell control. 25 μL of 4 units of antigen was added to each of wells 1 through 11, the reaction plate was tapped and left to stand at 37℃for 15 min. Subsequently 25 μl of 1% chicken erythrocyte suspension was added to each well in order from right to left. The reaction plate was placed on a micro-oscillator with gentle shaking, and after standing at 37 ℃ for 15 min, the results were observed and judged when the erythrocytes in the control wells of erythrocytes showed significant sedimentation.

To determine the bactericidal capacity of antibodies in serum samples in vitro, we used an improved method of determining the bactericidal activity of serum. The APEC O78 strain was cultured to the logarithmic growth phase and then suspended in PBS to a concentration of 10 ⁶ CFU/mL for subsequent determination. The immune serum was diluted 1:10 in LB medium. 90. Mu.L of diluted serum was mixed with 10. Mu.L of conditioned bacteria in a round bottom 96 well plate and then incubated with shaking at 37℃under 100 rpm for 1 h. The reaction mixture was serially diluted 10-fold and incubated overnight at 37 ℃ to count viable colonies. To calculate the survival rate of O78 in the different serum samples, the number of serum treated bacteria/the number of original bacteria was calculated. All serum samples were tested independently in triplicate.

3.1 Evaluation of mucosal immunity against H9N2 AIV after vaccine immunization

One week after each immunization, the nasal and tracheal lavages of the chickens were collected, and the vaccine-induced chicken flocks were assayed for production of specific sIgA antibody levels against TX by coating with purified H9N2 AIV TX using an indirect ELISA method. The results showed that none of the groups immunized the trachea and nasal cavity of the chickens showed higher sIgA antibody levels after the first and second immunization. After the third boost, the antibody levels in nasal and tracheal of the BBV-HA1 immunized group were significantly increased compared to the PBS group, while the antibody levels of the inactivated vaccine group (TX-inactivated) and other immunized groups were not significantly different from the PBS group (fig. 6). It is demonstrated that only the use of BBV as a carrier to present HA1 protein stimulates the mucosal immune response in the body, inducing the body to produce specific mucosal immunity against H9N2 AIV TX.

3.2 Humoral immunity evaluation against H9N2 AIV after vaccine immunization

One week after each immunization, chicken blood was collected and serum was isolated. Humoral immune responses of each immune group against H9N2 AIV TX were determined by an indirect ELISA assay and an HI assay, respectively. The results of the indirect ELISA experiments showed that the IgY antibody levels in the sera of the immunized chickens of each group were not increased one week after the first immunization. One week after the second boost, only TX-inactive immune group IgY antibody levels were significantly elevated. One week after the third immunization, serum IgY levels were significantly increased in both TX-inactive and BBV-HA1 immunized groups. The HI test result shows that the HI antibody level of the chicken of the TX-inactive immune group is improved by one week after the immunization, the average HI antibody level can reach 2.4+/-1.52 log2, the HI antibody titer of the BBV-HA1 immune group is slightly improved by one week after the second boost, the HI antibody titer of the chicken of the TX-inactive immune group is obviously improved, and the HI antibody titer of the chicken of the TX-inactive immune group is 8.6+/-0.86 log2. One week after the third immunization, the HI antibody titers for TX-reactive, BBV+HA1 and BBV-HA1 were all significantly increased, with BBV+HA1 reaching 3+ -0.71 log2, BBV-HA1 reaching 5.2+ -0.84 log2, and TX-reactive reaching 10.8+ -0.45 log2 (FIG. 7). In addition to the highest antibody levels induced by the TX-reactive immune group, the BBV-HA1 immune group is also effective in stimulating the body to produce higher antibody levels, whereas the antibody levels produced by immunization of the HA1 protein alone or by immunization of the immune group with BBV as an HA1 protein adjuvant are significantly lower than those produced by BBV-HA1. It was demonstrated that the HA1 protein presented by BBV increased immunogenicity and was better recognized by the body resulting in a more specific humoral immune response.

3.3 Evaluation of mucosal immunity against APEC after vaccine immunization

One week after each immunization, the nasal and tracheal lavages of the chickens were collected, and the vaccine-induced chicken flocks were assayed for specific sIgA antibody levels against APEC by coating with purified Omps protein using an indirect ELISA method. The results showed that none of the air and nasal passages of the immunized chickens showed higher sIgA antibody levels after the first immunization. After the second boost, the antibody levels of BBV, BBV+HA1 and BBV-HA1 were increased. After the third boost, the antibody levels of BBV, bbv+ha1 and BBV-HA1 were further elevated and significantly different from the PBS group antibody levels. However, the antibody levels of the HA1 and J11-inactive groups were not significantly different from those of the PBS group (fig. 8). BBV was shown to induce specific mucosal immunity against APEC Omps in the body.

3.4 Humoral immunity evaluation against APEC after vaccine immunization

One week after each immunization, chicken blood was collected and serum was isolated. The humoral immune response of each immune group against APEC was determined by an indirect ELISA assay and a serum bactericidal assay, respectively. The results showed that the level of IgY antibodies in the serum of each group of immunized chickens was not elevated after the first immunization. After the second and third booster immunizations, anti-Omps IgY antibody levels were significantly elevated in the J11-inactive, BBV, BBV +H2 and BBV-HA1 immunized groups, with significant differences from the PBS and HA1 immunized groups. Among them, the J11-inactive group had the highest antibody level, and there was no significant difference between BBV, BBV+HA1 and BBV-HA1 immune groups (FIG. 9A). In addition, we examined the bactericidal activity of each group of immune sera. Freshly isolated immune serum was incubated with APEC in vitro, shake incubated at 37 ℃ for 1h and plated diluted to count viable colonies. The results showed that after the third boost, the BBV, BBV+HA1, BBV-HA1 and J11-inactive immune serum bacteria effectively inhibited bacterial survival, while neither PBS nor HA1 immune serum inhibited bacterial survival (FIG. 9B). BBVs was shown to be effective in stimulating body-specific humoral immune responses, producing specific antibodies against APEC.

3.5 PBMC cell proliferation

After three immunizations, we isolated chicken PMBC, stimulated lymphocytes with HA1 protein and Omps protein, respectively, and evaluated cell proliferation using CCK-8. Specifically, after PMBC isolation and counting, 96-well plates were seeded with 10 ⁶ cells per well in RPMI 1640 medium containing 10% FBS and stimulated with the addition of Omps (20 μg/well) and 48 h were incubated in an incubator at 37℃and 5% CO ₂. mu.L of CCK-8 solution was added to each well and incubated at 37℃in the absence of light for 1h and then the optical density (OD ₄₅₀) was measured at 450 nm using a microplate reader. The Stimulation Index (SI) was calculated as Omps OD ₄₅₀ value for stimulated cells/OD ₄₅₀ value for unstimulated cells. The results show that after stimulation with HA1 protein, the HA1, BBV+HA1, BBV-HA1 and TX inactivated vaccine groups proliferated very significantly (p.ltoreq.0.01) compared with the PBS group, wherein the BBV-HA1 group had the highest lymphocyte proliferation index (FIG. 10A), and that under Omps protein stimulation, the BBV, BBV+HA1, BBV-HA1 and J11 inactivated vaccine groups proliferated very significantly (p.ltoreq.0.01) compared with the PBS group (FIG. 10B).

Example 4

Toxicity attack protection test results

4.1 Toxicity attack protection against H9N2

After immunization of SPF chickens according to the immunization program described above, the H9N2 AIV TX was used to combat toxins via the nasal drip route, and the throat and cloacae swabs were collected at 3, 5 and 7d, respectively, after the challenge, and the protection of the SPF from H9N2 AIV by the vaccine was determined by real-time fluorescent quantitative RT-PCR according to the M gene-based H9N2 AIV probe method previously established in the laboratory, using the PCR Primer design software Primer 5.0 and Lasergene 7.1 software to analyze and design primers and probes, and synthesized by the Probiotics (Shanghai) Co., ltd. The primers are shown in Table 4.

To accurately determine the detoxification status of chickens, the collected throat and cloaca swabs were extracted to extract cDNA, and absolute fluorescent quantitative PCR detection was performed according to a system in which 10. Mu.L of 2X PERFECTSTART II Probe qPCR SuperMix UDG, 0.4. Mu.L of 10. Mu.M upstream and downstream primers, 0.4. Mu.L of probe, and 2. Mu.L of reverse transcription product were added to a total volume system of 20. Mu.L, and finally a ddH ₂ O filling system of 6.8. Mu.L was used.

The reaction conditions were set to 94℃for 5min, 94℃for 5 seconds, 60℃for 30 seconds, and 40 cycles were performed with fluorescence signal acquisition at the end of annealing.

The results showed that no cloacal detoxification was detected for each group of SPF chickens at 3, 5 and 7 d after challenge, but the throat detoxification was different. The TX inactivated vaccine group can completely inhibit the toxin expelling of the larynx (0/8), and other immune groups can not completely inhibit the toxin expelling of the larynx. Among them, BBV-HA1 is the best protective effect, and only 2 chicken throats in the No. 7 d are detoxified. The pure immune HA1 protein still HAs 5 chicken larynx toxin expelling, and the BBV and BBV+HA1 groups have 5 chicken larynx toxin expelling. These results demonstrate that HA1 protein provides better immunoprotection when presented to BBV (table 5).

4.2 Toxicity counteracting protection against O78 serotypes APEC

SPF chickens were immunized according to the immunization program described above, but challenged with APEC of the O78 serotype (10 ⁷ CFU/only) via the balloon injection route, with continuous observation of 7 d. To evaluate the effect of immunization on the weight gain of post-challenge SPF chickens, each group of chickens was weighed daily. The results showed that the body weight of each group of SPF chickens continued to decrease within 4 d after challenge, and then began to gradually increase after challenge at 5 th d. Compared to the Blank healthy control group, the body weight differences between SPF chickens of the BBV, BBV+HA1, BBV-HA1 and J11 inactivated vaccine groups disappeared (p > 0.05) at 7 d after challenge, while the body weights of SPF chickens of the PBS and HA1 groups remained significantly different from those of the Blank group (p < 0.001) (FIG. 11A).

To evaluate the effect of immunization on infection in post-challenge SPF chickens, chickens were killed at 7 d post-challenge, lesions of the heart and air sac were recorded and analyzed, and lesions were scored. The results showed that the PBS and HA1 group chickens had cardiac dysplasia, thickened pericardium, and a large amount of cellulose exudation, thickened air sacs and turbidity. While the hearts and air sacs of the BBV, BBV+HA1, BBV-HA1 and J11 inactivated vaccine group chickens were not significantly different from the Blank group (FIGS. 11B, C).

Furthermore, we assessed the dynamic distribution and content of bacteria in SPF chickens. After 24 h bacterial challenge, 3 were randomly dissected and liver, spleen, lung and kidney tissues were taken and colony counted after milling with sterile PBS. A blood sample of 24 h and 96 h after challenge was also collected for determination of bacterial load. The colonies were counted by serial 10-fold dilution in sterile PBS and final plating on MAIKAI medium. By colony counting we found that the bacterial load in blood of the immunized chickens of the BBV, bbv+ha1, BBV-HA1 and J11 inactivated vaccine group was significantly reduced (p < 0.05) 72h after challenge compared to the PBS group. Except for the trachea, bacterial loads of liver, spleen, lung and kidney were significantly reduced (p < 0.05) APEC in BBV, bbv+ha1, BBV-HA1 and J11 inactivated vaccine immune groups compared to PBS group (fig. 11d, e).

Claims

1. A fusion protein, wherein the fusion protein is obtained by connecting a ClyA protein and a HA1 protein in series via a linker; the sequences of the ClyA protein, the linker and the HA1 protein are shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3.

2. A method for preparing an Escherichia coli bionic vesicle presenting H9 subtype avian influenza virus HA1 protein, characterized in that ClyA and H9N2 AIV HA1 protein are connected in series through a Linker, the fusion protein is expressed in a vesicle donor, and the fusion protein ClyA-HA1 is presented on the surface of the bionic vesicle by high-pressure homogenization technology to obtain a recombinant bionic vesicle; the sequences of the ClyA protein, Linker and HA1 protein are shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3.

3. The method for preparing the H9 subtype avian influenza virus HA1 protein presented by Escherichia coli biomimetic vesicles according to claim 2 is characterized in that ClyA and H9N2 AIV HA1 proteins are connected in series through a linker, the ribosome binding site is fused to the N-terminus of the ClyA sequence to promote the expression of the fusion protein, the 6×his tag is fused to the C-terminus of the HA1 sequence, and the ClyA-HA1 sequence is inserted between the EcoRⅠ and HindⅢ restriction sites in the pBAD18-Cm vector using T4 ligase to construct the pBAD18-ClyA-HA1 recombinant plasmid; the ribosome binding site sequence is shown in SEQ ID NO.4.

4. The method for preparing the Escherichia coli biomimetic vesicle presenting H9 subtype avian influenza virus HA1 protein according to claim 3, characterized in that the primers for constructing the pBAD18-ClyA-HA1 recombinant plasmid are shown in SEQ ID NOs.11 to 14.

5. The method for preparing the Escherichia coli biomimetic vesicle presenting H9 subtype avian influenza virus HA1 protein according to claim 2, characterized in that the vesicle donor is a defective bacterium ΔtolA-ΔaraD of Escherichia coli of serotype O78; and the araD gene of the vesicle donor is knocked out by λ-Red homologous recombination technology.

6. The method for preparing the Escherichia coli biomimetic vesicle presenting H9 subtype avian influenza virus HA1 protein according to claim 5, characterized in that the primers for the targeting fragment of the araD gene knockout are as shown in SEQ ID NOs. 5 and 6.

7. The method for preparing the Escherichia coli biomimetic vesicle presenting the H9 subtype avian influenza virus HA1 protein according to claim 5, characterized in that the identification primers of the O78 serotype Escherichia coli defective bacteria ΔtolA-ΔaraD are shown in SEQ ID NOs. 7 to 10.

8. A recombinant bionic vesicle, characterized in that it is prepared by the method described in claims 2 to 7.

9. Use of the fusion protein according to claim 1, the methods according to claims 2 to 7, or the recombinant biomimetic vesicle according to claim 8 in preparing an agent for preventing and treating H9 subtype avian influenza virus and O78 serotype Escherichia coli infection.

10. The use according to claim 9, characterized in that the recombinant bionic vesicles are used to inhibit the detoxification of H9 subtype avian influenza virus in the chicken larynx and cloaca, reduce the bacterial load in chickens infected with O78 serotype Escherichia coli, and reduce the degree of inflammation of the air sacs and heart.