A kind of avian pathogenic Escherichia coli antigen multi-epitope fusion protein and its preparation method and application

Abstract

The invention discloses a preparation method and application of a hexavalent multi-epitope fusion protein of avian pathogenic Escherichia coli. The invention preferably selects antigenic epitopes of the curli fimbriae subunit CsgA, lipoprotein SlyB, maltose porin LamB, outer membrane protein PhoE, proteolytic enzyme activator NlpD and lipoprotein Pal main structural subunit of avian pathogenic Escherichia coli, and preferably connects with the heat-labile enterotoxin LTb fusion protein in series to construct an antigenic multi-epitope fusion protein of six proteins of avian pathogenic Escherichia coli. The antigenic multi-epitope fusion protein has a wide serotype coverage, is non-allergenic, has strong immunogenicity, and produces strong specific humoral immunity and cellular immune response after immunizing animals.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to an avian pathogenic Escherichia coli antigen multi-epitope fusion protein and a preparation method and application thereof. Background Art

[0002] Avian pathogenic Escherichia coli (APEC) belongs to extraintestinal pathogenic Escherichia coli (ExPEC), which can cause Escherichia coli disease in poultry, including symptoms such as air sacculitis, pericarditis, ovum peritonitis, perihepatitis, omphalitis, and cellulitis. With the intensive and large-scale development of my country's poultry industry, avian Escherichia coli disease has become an important bacterial disease that endangers the poultry industry, causing serious economic losses. At present, the treatment of APEC infection is mainly based on drug therapy, but the widespread use of antibiotics has led to an increasing number of APEC resistant strains. With the formation of multidrug-resistant strains and the increase in drug residue problems, the quality and safety of poultry products have received more and more attention. At the same time, the country has promulgated the "National Action Plan for Containing Microbial Resistance" to reduce the irrational use of antibiotics and the development of antibiotic substitutes. Therefore, the use of antibiotic substitutes to prevent and control APEC will be an inevitable trend in the future. Vaccination is an important way to effectively prevent and control the occurrence and spread of avian Escherichia coli disease, reduce poultry product contamination, and ensure food safety.

[0003] In recent years, a new vaccine paradigm has emerged, namely multi-epitope vaccines composed of a series of epitopes. Multi-epitope vaccines are attractive alternatives to traditional live attenuated and inactivated vaccines, with good safety and the ability to induce targeted immune responses against specific epitopes. The identification of B cell and T cell epitopes is crucial for the development of multi-epitope vaccines. B cell epitopes play a key role in initiating humoral immune responses, while T cell epitopes recognized by T cells can induce cellular immune responses and promote overall immune activation. Thanks to advances in computational immunology and vaccine informatics, there are now a large number of powerful tools available for the precise design and development of epitope-based vaccines. Computational design has become a common approach for the development of multi-epitope vaccines against a variety of pathogens, including viruses, bacteria, parasites, and even cancer. In addition, work has been done on the development of multi-epitope vaccines for some bacterial diseases, such as Streptococcus agalactiae and Streptococcus pneumoniae. However, to date, no multi-epitope vaccine research has been conducted specifically for APEC. The present invention constructs a multi-epitope fusion protein of avian pathogenic Escherichia coli for the first time. By optimizing the multi-epitope fusion protein of avian pathogenic Escherichia coli antigen, the protein is more stable in structure and has a better binding effect with Toll-like receptors, while also being able to induce better immunogenicity and reactogenicity. Summary of the invention

[0004] In order to overcome the shortcomings of the prior art, the purpose of the present invention is to provide a multi-epitope vaccine for avian pathogenic Escherichia coli, that is, to screen out proteins with good immunogenicity in the genome sequences of different serotypes of avian pathogenic Escherichia coli through pan-genome analysis bioinformatics and analyze their epitopes, and to obtain an antigen protein by concatenating helper T cell epitopes, toxic T cell epitopes and B cell epitopes through Linker.

[0005] In order to solve the above-mentioned purpose, the present invention adopts the following technical scheme:

[0006] In a first aspect, the present invention provides an optimized avian pathogenic Escherichia coli antigen multi-epitope fusion protein, comprising the following steps:

[0007] The pan-genome was used to analyze the genome of avian pathogenic Escherichia coli and screen out the core genes of avian pathogenic Escherichia coli. The location and antigenicity of the core genes were further obtained through subcellular localization and antigenicity analysis, and the highly antigenic proteins CsgA, SlyB, LamB, PhoE, NlpD and Pal proteins located outside the cell / membrane were obtained. Anti-toxic T cell epitopes, helper T cell epitopes and B cell epitopes were further designed. The helper T cell epitopes, toxic T cell epitopes, B cell epitopes and heat-labile enterotoxin LTb were connected in series to form fusion proteins through Linker.

[0008] First, based on the amino acid sequences of the curli pilus subunit CsgA, lipoprotein SlyB, maltoporin LamB, outer membrane protein PhoE, proteolytic enzyme activator NlpD and lipoprotein Pal, NetCTL1.2 was used to predict toxic T cell epitopes online, IEDB was used to predict helper T cell epitopes online, and ABCpred was used to predict B cell epitopes online. According to the prediction results, toxic T cell epitopes of SEQ ID No.1-SEQ ID No.22, helper T cell epitopes of SEQ ID No.23-SEQ ID No.32, and B cell epitopes of SEQ ID No.33-SEQ ID No.44 were selected for the construction of APEC antigen multi-epitope fusion protein.

[0009] Based on the dominant antigenic epitopes of the curled pilus subunit CsgA, lipoprotein SlyB, maltose porin LamB, outer membrane protein PhoE, proteolytic enzyme activator NlpD and lipoprotein Pal, the CTL epitope is connected by Linker (AAY), the HTL epitope is connected by Linker (GPGPG), the B cell epitope is connected by Linker (KK), and the heat-labile enterotoxin LTb is connected to the N-terminus of the fusion protein by Linker (EAAAK). Secondary structure, tertiary structure, allergenicity, molecular docking and immune simulation analysis are performed. Its amino acid sequence is shown in SEQ ID No.45.

[0010] In combination with the first aspect, the present invention further provides an optimized coding gene sequence of avian pathogenic Escherichia coli antigen multi-epitope fusion protein, as shown in SEQ ID No.46.

[0011] On the other hand, the present invention also provides a method for preparing a multi-epitope fusion protein of avian pathogenic Escherichia coli antigen, comprising the following steps:

[0012] Gene synthesis was performed according to the optimized gene sequence, and the gene was inserted between the BmaH I and Hind III restriction sites of the expression vector pET28a (+), and the recombinant expression vector pET28a-MEFA was constructed, which was transferred into Escherichia coli BL21 (DE3) competent cells, and the expression was induced by IPTG, preferably at 0.05 μg IPTG and 28°C overnight, and the cells were collected by centrifugation, broken and purified to obtain the antigen multi-epitope fusion protein.

[0013] In another aspect, the present invention also provides a use of the multi-epitope fusion protein of avian pathogenic Escherichia coli antigen, which is used as an antigen in preparing vaccines.

[0014] Furthermore, the present invention provides a composition, wherein the active ingredient is the above-mentioned avian pathogenic Escherichia coli antigen multi-epitope fusion protein.

[0015] Preferably, the present invention provides an avian pathogenic Escherichia coli subunit vaccine, comprising the above-mentioned avian pathogenic Escherichia coli antigen multi-epitope fusion protein and an immunologically acceptable vaccine adjuvant mixed in a volume ratio of 1:1, wherein the vaccine adjuvant comprises Freund's adjuvant.

[0016] Beneficial Effects

[0017] Compared with the prior art: the antigen multi-epitope fusion protein of 6 proteins of APEC is preferably designed through pan-genome and reverse vaccinology analysis, and the serotype coverage is wide. The subunit vaccine prepared by using the protein has more capsid protein antigen epitopes, which stimulates the body to produce more types and more protective antibodies, and significantly improves the protection against avian pathogenic Escherichia coli. The selected multi-antigen epitopes and the codons of the encoding DNA of the multi-linked fusion recombinant protein are optimized according to the codon preference of Escherichia coli codons, which significantly improve the protein amount of the multi-linked fusion recombinant protein expressed by the Escherichia coli expression system, and ensure that there are sufficient multi-linked fusion recombinant proteins for the preparation of avian pathogenic Escherichia coli subunit vaccines. There is no allergy, it will not cause allergies in the immune animals, and it is highly safe. The antigen multi-epitope fusion protein has strong immunogenicity. As an antigen for preparing vaccines, the antibody titer is high after the immunized animals are immunized, and specific humoral immunity and cellular immune responses are generated. Moreover, the multi-linked fusion recombinant protein subunit vaccine prepared by the present invention only needs simple immunization to produce complete immune protection, which significantly reduces the cost expenditure of immune prevention. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 :Phylogenetic tree and matrix generated based on the presence or absence of genes in the genome of avian pathogenic Escherichia coli through pan-genome analysis.

[0019] Figure 2 : Detailed information on proteins of the core genome predicted using PSORTb.

[0020] Figure 3 :Tertiary structure prediction and quality assessment. (A) The tertiary structure of the developed APEC antigen multi-epitope fusion protein predicted by Phyre 2 server. (B) Ramachandran plot of the final structure of the APEC antigen multi-epitope fusion protein. (C) Z-score plot of the tertiary structure of the APEC antigen multi-epitope fusion protein.

[0021] Figure 4 :Molecular docking of APEC antigen multi-epitope fusion protein with Toll-like receptors. (A) Docking complex of MEFA-TLR2. (B) Residue interactions between MEFA and TLR2. (C) Docking complex of MEAF and TLR4. (D) Residue interactions between MEFA and TLR4.

[0022] Figure 5:Computer immune simulation. (A) Antigen and antibody levels after protein injection. (B) B lymphocyte population after three protein injections. (C) B lymphocyte population at each stage. (D) T lymphocyte population after two protein injections. (E) T lymphocyte number at each stage. (F) CD8 T cytotoxic lymphocyte number at each stage. (G) Dendritic cell number at each stage. (H) Macrophage number at each state. (I) Cytokine and interleukin concentrations.

[0023] Figure 6 : Cloning and purification of APEC antigen multi-epitope fusion protein. (A) APEC antigen multi-epitope fusion protein was cloned into pET28a(+) expression vector. The red part represents the gene coding. (B) Identification of recombinant plasmid pET-28a-MEFA. (C) Purification of recombinant APEC antigen multi-epitope fusion protein by nickel column. (D) Western Blot analysis of purified APEC antigen multi-epitope fusion protein.

[0024] Figure 7 :Immune response induced by APEC antigen multi-epitope fusion protein. (A) The IgY titer in serum was determined by ELISA method and expressed as dilution titer. (B) The proliferation of chicken spleen lymphocytes was detected by CCK-8 test. Spleen samples of 3 chickens were randomly selected from each group. Specific implementation plan

[0025] The present invention is further described below in conjunction with the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and cannot be used to limit the protection scope of the present invention.

[0026] Example 1: Screening of avian pathogenic Escherichia coli immune protective antigens

[0027] In this implementation, the core genes in the genomes of different serotypes of avian pathogenic Escherichia coli were obtained through pan-genome analysis, and then five proteins with good immunogenicity located in the bacterial outer membrane and extracellular space were obtained through reverse vaccinology screening. T cell and B cell epitopes were predicted through bioinformatics analysis, and the antigenic protein was obtained by further concatenating helper T cell epitopes, toxic T cell epitopes, B cell epitopes and heat-labile enterotoxin LTb through Linker.

[0028] The coding DNA sequences (CDS) of the complete genome of avian pathogenic Escherichia coli were annotated by using Prokka and a GFF3 format file of all genome sequences was generated for pan-genome analysis. The proteome was analyzed using Roary with the aim of determining the core proteome according to the default settings (genes present in 99% to 100% of the isolates were considered as core genes).

[0029] The core proteome was uploaded to the PSORTb server for subcellular localization prediction, and proteins predicted to be located in the outer membrane or predicted to be extracellular proteins were screened out for antigenicity prediction by VaxiJen. This prediction method based on the physicochemical properties of proteins does not require sequence alignment and has an accuracy rate of between 70% and 89%. In order to maximize the screening efficiency, proteins with a VaxiJen score greater than 0.7 were considered to be proteins for priority epitope extraction.

[0030] The core set of proteins identified by pan-genomic analysis was subjected to reverse vaccinology analysis to prioritize proteins from which epitopes could be extracted in subsequent analyses. Figure 1 and Figure 2 As shown, 2367 core genes were screened for subcellular localization using PSORTb, of which 1199 proteins were identified as cytoplasmic proteins, 597 proteins were identified as cytoplasmic membrane-bound proteins, 10 proteins were identified as extracellular proteins, 34 proteins were identified as outer membrane proteins, 95 proteins were identified as periplasmic proteins, and 432 proteins were of unknown localization ( Figure 2 ). Among them, proteins with extracellular secretion function and proteins located in the outer membrane were selected for further analysis.

[0031] The proteins initially identified were scored by VaxiJen. Of these proteins, 23 were identified as potential antigens, meeting the default critical value of 0.4. Further screening revealed that five proteins scored over 0.7. They were the curled pilus subunit CsgA, lipoprotein SlyB, maltose porin LamB, outer membrane protein PhoE, proteolytic enzyme activator NlpD, and lipoprotein Pal protein. These proteins were identified as excellent candidate proteins and were selected to extract epitopes to construct APEC antigen multi-epitope fusion proteins.

[0032] Example 2: Design of APEC antigen multi-epitope fusion protein

[0033] T cell and B cell epitopes were predicted through bioinformatics analysis, and the antigen protein was obtained by concatenating helper T cell epitopes, toxic T cell epitopes, B cell epitopes and heat-labile enterotoxin LTb through Linker.

[0034] Prediction of cytotoxic T lymphocyte (CTL) epitopes is crucial for designing optimal multi-epitope fusion proteins. The web server NetCTL1.2 was used to predict the CTL epitopes of the selected proteins. The CTL epitopes of the proteins were predicted using the NetCTL-1.2 server, and the screening was based on the comprehensive high score, i.e., low sensitivity and high specificity to the MHC-I supertype. A total of 47 epitopes were predicted and screened based on their binding affinity scores to the respective MHC I supertypes, of which SEQ ID No.1-SEQ IDNo.22 were selected for the construction of APEC antigen multi-epitope fusion proteins. Using the IEDB (MHC-II class molecule binding prediction) tool, IEDB recommended was selected to predict the proteins with percentile rank less than 0.2 and IC 50 The peptides with a value of less than 50 nM were selected to ensure strong binding to the HLA reference allele. A total of 38 epitopes were screened, and according to their positions in the amino acid sequence, these epitopes were integrated, of which SEQ ID No.23-SEQ ID No.32 were selected for the construction of the final APEC antigen multi-epitope fusion protein. The B cell epitopes of the selected proteins were predicted using the ABCpred server (http: / / crdd.osdd.net / raghava / abcpred / ), and the two epitopes with the highest scores in each protein were selected for the construction of the APEC antigen multi-epitope fusion protein, with sequences of SEQ ID No.33-SEQ ID No.44.

[0035] Based on the selected epitopes of the curled pilus subunit CsgA, lipoprotein SlyB, maltose porin LamB, outer membrane protein PhoE, proteolytic enzyme activator NlpD and lipoprotein Pal protein, the heat-labile enterotoxin LTb sequence was combined. The CTL epitope was connected by Linker (AAY), the HTL epitope was connected by Linker (GPGPG), and the B cell epitope was connected by Linker (KK). In addition, the heat-labile enterotoxin LTb was connected to the N-terminus of the fusion protein by Linker (EAAAK).

[0036] Example 3: Bioinformatics evaluation of APEC antigen multi-epitope fusion protein

[0037] First, we used two bioinformatics tools, AllergenFP and AllerTOP, to predict the allergenicity of the designed multi-epitope fusion protein. At the same time, we used VaxiJen to evaluate the antigenicity of the amino acid sequence of the multi-epitope fusion protein. Second, we used the web server ProtParam to evaluate the physicochemical properties of the fusion protein.

[0038] The allergenicity of the fusion protein was evaluated by analysis on the AllergenFP and AllerTOP servers. Both platforms indicated that the fusion protein sequence was non-allergenic. In addition, to reconfirm the antigenicity of the fusion protein, we re-evaluated it using the VaxiJen server, which resulted in a score of 0.8310.

[0039] The web server ProtPara was used to evaluate various physicochemical properties of the fusion protein we designed. The fusion protein consists of 767 amino acids, contains 57 negatively charged residues and 66 positively charged residues, and has a molecular weight of 80.7 kDa. The theoretical pI value of the fusion protein is 8.82, contains 11156 atoms, and has a chemical formula of C 3561 H 5464 N 984 O 1125 S 22 . It is estimated that its half-life is 30h in vitro, more than 20h in yeast (in vivo), and more than 10h in Escherichia coli (in vivo). The calculated instability index (II) is 32.23, so the fusion protein is a stable protein. It is worth noting that its fat index is 64.78, indicating that its thermal stability is relatively high. In addition, the fusion protein is also hydrophilic, with a hydration average value (GRAVY) of -0.402.

[0040] In order to improve the conformational structure of the predicted protein, the designed fusion protein tertiary structure was modeled using the Phyre 2 server, and then further improved using the GalaxyRefine server (http: / / galaxy.seoklab.org / cgi-bin / submit.cgi?type=REFINE). The tertiary structure of the PDB file of the improved structure was evaluated using the SWISS-MODEL server (https: / / swissmodel.expasy.org / assess). Subsequently, the protein structure was verified using the ProSA web server (https: / / prosa.services.came.sbg.ac.at / prosa.php).

[0041] The tertiary structure of the designed fusion protein was initially constructed by the Phyre 2 server and subsequently refined by the Galaxyrefine server. Among the five models generated, one showed the highest Lagrangian rational region score and was selected as the final structure for downstream analysis. The structure of this model was visualized using PyMOL ( Figure 3 A).

[0042] The ideal model is characterized by a high proportion of residues in the Lagrange reasonable region, and fewer residues in the abnormal region and helical region. The analysis of the Lagrange plot of the final structure of the fusion protein we developed showed that 80.13% of the residues were located in the Lagrange reasonable region, only 2.88% of the residues were located in the abnormal region, and 0.18% of the residues were located in the helical region ( Figure 3 B). The calculated MolProbity and Clash scores were 1.87 and 3.40, respectively. In addition, the overall quality and potential errors of the final structure were evaluated by ProSA, and the resulting Z score was -4.1. This score indicates that the fusion protein structure is within the experimental structure range ( Figure 3 C).

[0043] To evaluate the binding affinity of our designed fusion proteins with Toll-like receptors (TLR2 and TLR4), we performed docking studies separately. We obtained the crystal structures of TLR2 (PDB code: 2Z7X) and TLR4 (PDB code: 3FXI) from the Protein Data Bank. Molecular docking used the web-based ClusPro tool (https: / / cluspro.bu.edu / ). The three-dimensional structures of Toll-like receptors and fusion protein-TLR complexes generated by molecular docking were visualized using PyMOL software. In addition, PDBsum also helped to draw the amino acid residue map of the interaction between the designed fusion protein and TLR.

[0044] Molecular docking simulations were performed on the designed fusion proteins with TLR2 and TLR4 using the ClusPro server. Each docking simulation generated 30 clusters, and the cluster with the lowest energy score was considered the docking result. The lowest energy score of the fusion protein-TLR2 docking cluster was 21315.4, while the lowest energy score of the fusion protein-TLR4 docking cluster was 21264.1. The results of the molecular docking simulation were visualized using PyMOL ( Figure 4 A and C). To gain insight into the interactions between the fusion protein and these TLRs, we used the PDBsum tool to elucidate the residues involved in the receptor-ligand interaction and the binding forces. In the docking of the fusion protein with TLR2 ( Figure 5 B), the number of interface residues between the fusion protein and TLR2 is 59:41, and the interface areas are and A total of 6 salt bridges, 32 hydrogen bonds and 317 non-bonded contacts were detected. Figure 4 D), the number of interface residues between the fusion protein and TLR4 is 38:30, and the interface areas are and The complex exhibited 9 salt bridges, 23 hydrogen bonds, and 233 non-bonded contacts. These findings suggest that the designed fusion protein has high affinity to both TLR2 and TLR4.

[0045] Finally, we performed immune simulations using the C-ImmSim server (https: / / kraken.iac.rm.cnr.it / C-IMMSIM / ). C-ImmSim is an agent-based simulator that uses the Celada-Seiden model to simulate mammalian immune responses to designed fusion proteins. The simulator can generate humoral and cellular immune response curves.

[0046] Simulation analysis showed that after immunization, the antibody (Ab) titer increased significantly ( Figure 5 A), indicating a strong humoral immune response. Compared with the primary immune response, the secondary immune response was significantly enhanced, as shown by B cells ( Figure 5 B and C), helper T cells ( Figure 5 D and E) and cytotoxic T cells ( Figure 5 F) activity is enhanced. In addition, dendritic cells ( Figure 5 G) and macrophages ( Figure 5 H) concentrations, indicating that these antigen-presenting cells (APCs) can effectively present antigens. Of note, the levels of cytokines and interleukins ( Figure 5 I) was significantly increased after the second injection. In summary, these results indicate that our designed fusion protein has the potential to induce high levels of antibodies, activated immune cells, cytokines and antigen-presenting cells.

[0047] Example 4: Expression and purification of APEC antigen multi-epitope fusion protein

[0048] (1) Construction and identification of recombinant plasmid pET28a-MEFA

[0049] The nucleotide sequence was designed by selecting the corresponding epitope gene sequence based on the genome sequence of avian pathogenic Escherichia coli (SEQ ID No. 46). The optimized gene sequence was cloned into the pET28a (+) expression vector by using restriction enzymes Hind III and Bam HI at the N-terminal and C-terminal positions of the fusion protein nucleotide sequence to form a recombinant plasmid ( Figure 6 A). The gene was delivered to a biological company for synthesis of the gene sequence and cloned into the pET28a(+) expression plasmid, which was named pET28a-MEFA. The recombinant plasmid pET28a-MEFA was analyzed by PCR to obtain a gene fragment with the expected size, indicating that the recombinant plasmid pET28a-MEFA was successfully constructed ( Figure 6 B).

[0050] (2) Expression and purification of APEC antigen multi-epitope fusion protein

[0051] The correctly constructed recombinant expression plasmid pET28a-MEFA was transformed into competent E. coli BL21 (DE3), coated on LB / Kana plates, and a single colony was picked and inoculated into LB / Kana medium, and cultured overnight at 37°C in a shaker. The next day, fresh bacterial liquid was inoculated into LB / Kana medium at a ratio of 1:100, and cultured at 37°C, 180 rpm, until OD 600nm The concentration of IPTG was 0.6, and IPTG was added to a final concentration of 0.05 μg. Expression was induced overnight at 28°C. The next day, the cells were collected by centrifugation and crushed under high pressure in an ice-water bath. The APEC antigen multi-epitope fusion protein was purified using a nickel column. The washing solution containing different concentrations of imidazole was loaded onto the column, and the flow-through was collected for SDS-PAGE identification. The results showed that the APEC antigen multi-epitope fusion protein was successfully expressed, with a size of about 80.7 kDa ( Figure 6 C), which was purified and further verified by Western blot to verify the expression of APEC antigen multi-epitope fusion protein. It can be seen that the target band is clear and single ( Figure 6 D).

[0052] Example 5: Evaluation of the immune effect of APEC antigen multi-epitope fusion protein as a candidate vaccine antigen

[0053] (1) Experimental animal grouping and immunization

[0054] Freshly hatched SPF chickens were randomly divided into two groups, namely, APEC antigen multi-epitope fusion protein immunization group and PBS control group. The purified APEC antigen multi-epitope fusion protein was emulsified with Freund's adjuvant at a ratio of 1:1 and injected into the leg muscle for immunization. The immunization dose for each chicken was 100 μg of fusion protein, and the immunization was performed twice with an interval of 2 weeks. The APEC antigen multi-epitope fusion protein immunization group was Freund's complete adjuvant for the first immunization, and the second immunization adjuvant was Freund's incomplete adjuvant.

[0055] (2) Immunogenicity determination of APEC antigen multi-epitope fusion protein

[0056] 7 days after the second immunization, blood was collected from the chicken's wing vein to separate the serum. The APEC antigen multi-epitope fusion protein was used to coat the ELISA plate, and the indirect ELISA method was used to detect the titer and fluctuation of chicken serum IgY antibody.

[0057] The results showed that the specific antibody titer of the APEC antigen multi-epitope fusion protein immunization group was as high as 1:25600 7 days after the second immunization, while the PBS control group had no specific antibody titer ( Figure 7A). The above results indicate that the APEC antigen multi-epitope fusion protein has good immunogenicity and can stimulate the body to produce a high level of humoral immune response.

[0058] (3) Splenic lymphocyte proliferation assay

[0059] Seven days after the second immunization, the spleen of the chicken was taken and a single spleen lymphocyte suspension was prepared and diluted to 5×10 6 cells / mL; in a 96-well plate, add 100 μL of cell suspension to each well, then add 50 μL of 1640 medium containing 10% fetal bovine serum, 10 μL of PMA (100 ng / mL) + ionomycin (3 mM) stimulator) and 10 μL of APEC antigen multi-epitope fusion protein (10 μg / mL). Tests were performed in the unstimulated group, MEFA stimulation group, and positive stimulation group; cultured at 37°C for 72 hours, then added 10 μL of CCK8 to each well and continued to culture for 4 hours; OD was measured using an enzyme-labeled instrument 450nm , and calculated the stimulation index (SI), SI = (OD value of MEFA stimulation group - OD value of blank control group) / (OD value of unstimulated group - OD value of blank control group).

[0060] The results showed that 7 days after the second immunization, the SI value of the APEC antigen multi-epitope fusion protein stimulation group was significantly higher than that of the control group (P<0.01) ( Figure 7 B), indicating that APEC antigen multi-epitope fusion protein can well stimulate the proliferation of chicken spleen lymphocytes, thereby inducing a strong cellular immune response.

[0061] The above results fully confirm that the APEC antigen multi-epitope fusion protein prepared by the present invention has good immunogenicity. As an antigen for preparing vaccines, the antibody titer is high after immunizing animals, and specific humoral immunity and cellular immune responses are generated. It can be used as a candidate antigen for APEC genetic engineering vaccines.

[0062] The above description of the embodiments is to facilitate the understanding and use of the present invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments. Improvements and modifications made by those skilled in the art based on the principles of the present invention without departing from the scope of the present invention should be within the protection scope of the present invention.

Claims

1. A multi-epitope fusion protein of avian pathogenic Escherichia coli antigen, characterized in that: The amino acid sequence of the antigen multi-epitope fusion protein is shown in SEQ ID No.

45.

2. A nucleic acid encoding the avian pathogenic Escherichia coli antigen multi-epitope fusion protein according to claim 1.

3. A method for expressing the avian pathogenic Escherichia coli antigen multi-epitope fusion protein of claim 1, characterized in that the nucleic acid of claim 2 is synthesized, inserted between the HindⅢ and BamHI restriction sites of the expression vector pET28a(+), and a recombinant expression vector pET28a-MEFA is constructed, which is transferred into Escherichia coli BL21(DE3) competent cells, induced by IPTG, and purified to obtain the avian pathogenic Escherichia coli antigen multi-epitope fusion protein.

4. The expression method according to claim 3, characterized in that: The induction conditions were: 0.05 μg IPTG, 28° C. induction, centrifugation to collect the bacteria, and then breaking and purifying to obtain the antigen multi-epitope fusion protein.

5. Use of the fusion protein according to claim 1 and / or the nucleic acid according to claim 2 in the preparation of an avian pathogenic Escherichia coli vaccine.

6. A composition, wherein the active ingredient is the multi-epitope fusion protein of avian pathogenic Escherichia coli antigen according to claim 1.

7. An avian pathogenic Escherichia coli subunit vaccine, comprising the avian pathogenic Escherichia coli antigen multi-epitope fusion protein according to claim 1 and an immunologically acceptable vaccine adjuvant.

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