Antigen for enhancing immune effect of newcastle disease virus and preparation method and application thereof

Abstract

This invention relates to the field of animal immunology, and more particularly to an antigen that enhances the immune response to Newcastle disease virus (NDV), its preparation method, and its application. The antigen is a fusion protein obtained by expressing the Ii-key-F-Fc recombinant vector using engineered bacteria, named the Ii-key-F-Fc fusion protein. The Ii-key-F-Fc recombinant vector is obtained by splicing three major antigenic epitope fragments from the NDV F protein and then tandemly connecting them with chicken IgY Fc and Ii-key. This invention constructs the pCold-TF-Ii-key-F-Fc prokaryotic expression plasmid by tandemly connecting the novel chicken vector Ii-key-Fc with the F antigenic epitope, and then expresses the fusion protein using an E. coli expression system to obtain a new antigen that enhances the immune response in poultry. This provides a new approach for developing a novel NDV vaccine and a new strategy for improving the immune response of subunit vaccines in poultry.

Description

Technical Field

[0001] This invention relates to the field of animal immunology, and more particularly to an antigen that enhances the immune response to Newcastle disease virus, its preparation method, and its application. Technical Background

[0002] Newcastle disease (ND) is a poultry disease syndrome caused by the Newcastle Disease Virus (NDV), characterized by high fever, respiratory distress, and digestive system lesions. To date, ND has caused four major epidemics (Alexander DJ, Aldous EW, Fuller C M. The long view: a selective review of 40 years of Newcastle disease research[J]. Avian Pathology, 2012, 41(4):329-335), and each epidemic has resulted in the emergence of new genotypes, rendering existing vaccines ineffective against viral invasion.

[0003] Current ND vaccines are mainly inactivated and live attenuated vaccines. While live attenuated vaccines offer good protection, they suffer from drawbacks such as susceptibility to maternal antibody interference, incomplete protection, and virulence reversion. Inactivated vaccines, although safe to use, require larger doses and are more expensive, posing significant challenges to ND control. Therefore, enhancing the immunizing effect of vaccines while ensuring safety is of great importance for ND control and eradication.

[0004] Mucosal immunity can induce local mucosal immune responses to resist the invasion of microorganisms such as bacteria and viruses, while also stimulating a systemic immune response. The close connection between mucosal epithelial cells and immune effector cells in the lamina propria suggests that delivering immunogens through the mucosal surface is an ideal pathway to achieve mucosal immunity and potential systemic immunity.

[0005] Newcastle disease (NDV) can infect the body through the respiratory mucosa, but there is still a lack of highly effective vaccines that can protect against this pathogen that invades the body through the mucosa. How to induce local mucosal immunity and systemic immune response in poultry through vaccines and reduce the incidence of Newcastle disease is a problem that needs to be solved by existing technologies. Summary of the Invention

[0006] In order to solve the problems existing in the prior art, one of the objectives of the present invention is to provide an antigen for enhancing the immune effect of Newcastle disease virus. The antigen is a fusion protein obtained by expressing the Ii-key-F-Fc recombinant vector in competent cells, named Ii-key-F-Fc fusion protein, and its amino acid sequence is shown in SEQ ID NO.1.

[0007] The Ii-key-F-Fc recombinant vector was obtained by splicing three major antigenic epitope fragments on the NDV F protein and then tandemly connecting them with chicken IgY Fc and Ii-key, respectively. The nucleotide sequence of the Ii-key-F-Fc recombinant vector is shown in SEQ ID NO.2. The NDV F protein is a surface glycoprotein on the cell membrane of Newcastle disease virus. The three major antigenic epitope fragments are located at positions 72 (66-91 aa), 161 (147-182 aa), and 343 (326-360 aa) of the NDV F protein, respectively, and their sequences are shown in SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5, respectively.

[0008] Preferably, the product obtained by splicing the three major antigenic epitope fragments on the NDV F protein is an antigenic epitope combinatorial peptide, the amino acid sequence of which is shown in SEQ ID NO.6.

[0009] Another object of the present invention is to provide a method for preparing the antigen for enhancing the immune effect of Newcastle disease virus, comprising the following steps:

[0010] S1. Chicken IgY Fc gene and three major antigenic epitope fragments of NDV F protein were obtained by enzyme digestion. The three major antigenic epitope fragments of NDV F protein were linked to form an antigenic epitope combination peptide, which was named F fragment. The F fragment was tandemly linked with IgY Fc and constant chain active fragment Ii-key to obtain the target gene fragment.

[0011] S2. After the target gene fragment is ligated into the pCold-TF prokaryotic expression vector, the Ii-key-F-Fc recombinant vector is transformed into competent cells BL21, and the competent cells are used to express the Ii-key-F-Fc fusion protein, which is an antigen that enhances the immune effect of Newcastle disease virus.

[0012] Preferably, in step S1, chicken macrophage RNA is extracted, and cDNA is obtained by reverse transcription. The IgY Fc gene is amplified using cFc-F / cFc-R as primers. Using pcDNA3-F plasmid as template, gene fragments containing the major NDV antigenic epitopes, namely the 72nd, 161st, and 343rd genes, are amplified using primer pairs NDV-F-F1 / NDV-F-R1, NDV-F-F2 / NDV-F-R2, and NDV-F-F3 / NDV-F-R3, respectively. Finally, using the first three PCR products as templates, the antigenic epitope combination peptide is amplified using primer pair NDV-FF / NDV-FR.

[0013] The cFc-F / cFc-R sequences are shown in SEQ ID NO.7 / SEQ ID NO.8, the NDV-F-F1 / NDV-F-R1 sequences are shown in SEQ ID NO.9 / SEQ ID NO.10, the NDV-F-F2 / NDV-F-R2 sequences are shown in SEQ ID NO.11 / SEQ ID NO.12, the NDV-F-F3 / NDV-F-R3 sequences are shown in SEQ ID NO.13 / SEQ ID NO.14, and the NDV-FF / NDV-FR sequences are shown in SEQ ID NO.15 / SEQ ID NO.16.

[0014] Preferably, in step S1, the F fragment, the constant chain active fragment Ii-key, and the IgY Fc fragment are ligated using the Overlap PCR method, wherein the upstream primer F-up of the F fragment uses the KpnI restriction site, and the downstream primer F-down inserts into the linker sequence; the upstream primer Fc-up of the Fc fragment inserts into the linker sequence, and the downstream primer Fc-down uses the HindIII restriction site.

[0015] The F-up sequence is shown in SEQ ID NO.17, the F-down sequence is shown in SEQ ID NO.18, the Fc-up sequence is shown in SEQ ID NO.19, the Fc-down sequence is shown in SEQ ID NO.20, and the Linker sequence is shown in SEQ ID NO.21.

[0016] Preferably, in step S2, the specific operation of expressing the Ii-key-F-Fc fusion protein using competent cells is as follows: Ii-key-F-Fc positive monoclonal antibodies are picked and inoculated into cells containing 50ug / mL Ampicillin. + Expression was induced at 15°C for 24 h in LB liquid medium at 37°C with a final IPTG concentration of 0.4 mmol / L.

[0017] Preferably, the Ii-key-F-Fc fusion protein obtained in step S2 can also undergo a purification step, specifically: the expressed fusion protein is sonicated and the supernatant is collected by centrifugation, and the supernatant is further collected by nickel affinity chromatography to obtain the purified Ii-key-F-Fc fusion protein, which is an antigen that enhances the immune effect of Newcastle disease virus.

[0018] Another object of the present invention is to provide the application of the above-mentioned Ii-key-F-Fc fusion protein in the preparation of Newcastle disease vaccines.

[0019] The present invention also provides an agent for inducing or enhancing immunity in poultry against Newcastle disease virus, the agent comprising an effective dose of the Ii-key-F-Fc fusion protein as described above, the amino acid sequence of which is shown in SEQ ID NO.1.

[0020] Preferably, the formulation is a liquid formulation or a spray, and is administered to the desired individuals via mucosal immunization.

[0021] The beneficial effects of this invention are as follows:

[0022] The F protein, as an important component of NDV virulence, is a key gene that enables viral invasion and transmission within the body. In this invention, a multi-epitope antigen gene was constructed by selecting three different epitopes of the F protein, which was then introduced into the pET-32a prokaryotic expression vector and Rosetta bacteria was selected as the host bacterium to obtain well-expressed F protein.

[0023] Chicken egg yolk immunoglobulin IgY Fc receptor (FcRY) can recognize and transport IgY. Using the IgY Fc fragment as a carrier, it fuses with viral protective antigens, thereby crossing the mucosal immune barrier and enhancing the body's immune response. Ii-key, a constant-chain functional fragment, has been extensively studied as an immune carrier; its tandem with antigenic peptides can significantly enhance the immune response.

[0024] This invention constructs a pCold-TF-Ii-key-F-Fc prokaryotic expression plasmid by tandemly linking a novel chicken vector Ii-key-Fc with an F antigenic epitope, and then expresses the fusion protein using an E. coli expression system to obtain a new antigen that enhances the immune response in poultry.

[0025] Experiments have demonstrated that the novel vector (Ii-key-F-Fc) fusion protein possesses a structure similar to IgY, enabling it to cross the chicken respiratory mucosal immune barrier via bidirectional transcellular translocation of FcRy, prolonging its half-life and maintaining an effective immune response. Simultaneously, it also possesses the antigen-presenting function of the Ii chain of MHC class II molecular chaperone proteins. Therefore, the novel vector (Ii-key-Fc) antigen can stimulate the body to produce effective local mucosal and systemic immune responses.

[0026] This invention provides new ideas for developing new Newcastle disease vaccines and offers a new strategy for improving the immunization effect of poultry subunit vaccines. Attached Figure Description

[0027] Figure 1 The figures show the PCR amplification results of the IgY Fc gene fragment and the NDV-F gene fragment. In the figure, a represents the IgY Fc gene, and b represents the NDV-F gene. Figure 1In both a and b, M is DNAMarker (DL2000); in a, 1 is the PCR amplification result of the IgY Fc gene, and in b, 1 is the PCR amplification result of the NDV-F gene.

[0028] Figure 2 The figures show the identification results of recombinant plasmids pET-32a-Fc and pET-32a-F. Figure a shows the identification results of recombinant plasmid pET-32a-Fc by PCR and double enzyme digestion in bacterial culture, and figure b shows the identification results of recombinant plasmid pET-32a-F by PCR and double enzyme digestion in bacterial culture. In the figures, M represents DNA Marker (DL2000). In figure a, 1 represents the identification results of pET-32a-Fc by PCR and double enzyme digestion in bacterial culture, 2 represents the identification results of pET-32a-Fc recombinant plasmid by double enzyme digestion, and 3 represents the identification results of pET-32a vector by double enzyme digestion. In figure b, 1 represents the identification results of pET-32a-F by PCR in bacterial culture, 2 represents the identification results of pET-32a-F recombinant plasmid by double enzyme digestion, and 3 represents the identification results of pET-32a vector by double enzyme digestion.

[0029] Figure 3 For the identification of recombinant plasmid pCold-TF-Ii-key-F-Fc by bacterial culture PCR and double enzyme digestion, M is DNA Marker (DL5000), 1 is PCR identification of pCold-TF-Ii-key-F-Fc bacterial culture, 2 is double enzyme digestion identification of pCold-TF-Ii-key-F-Fc recombinant plasmid, and 3 is double enzyme digestion identification of pCold-TF vector.

[0030] Figure 4 The expression levels of pCold-TF-Ii-key-F-Fc induced by different IPTG final concentrations are shown in the figure. M represents the Protein marker, and lanes 1-6 represent the expression levels induced by 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mmol / L IPTG, respectively.

[0031] Figure 5 The expression and solubility of the pCold-TF-Ii-key-F-Fc fusion protein are shown in the figure. M is the Protein marker, 1 is the result of uninduced pCold-TF-Ii-key-F-Fc fusion protein, 2 is the result of supernatant after induction and sonication, and 3 is the result of precipitation.

[0032] Figure 6 The results of SDS-PAGE analysis of pCold-TF-Ii-key-F-Fc protein after purification are shown in the figure. M represents the protein marker, 1 represents the flow buffer result, 2-3 represent the washing buffer result, and 4-7 represent the elution buffer result.

[0033] Figure 7 The serum antibody titer of mice immunized with His-F fusion protein was determined.

[0034] Figure 8 The figure shows the Western blot results of the F antibody. M is the pre-stained protein marker, 1 indicates that the F protein was not induced, and 2 indicates the F fusion protein.

[0035] Figure 9 In the figure, a, b, and c represent the Western blot identification results of the Ii-key-F-Fc fusion protein, the F-Fc fusion protein, and the F fusion protein, respectively; M in the figure represents the Pre-stained Protein marker.

[0036] Figure 10 The results show the detection of the fusion protein crossing the chicken respiratory mucosal barrier.

[0037] Figure 11 OD level of serum IgY antibody 450nm value.

[0038] Figure 12 The figure shows the antibody levels of sIgA in the lung lavage fluid of immunized chickens. Figure a shows the sIgA content in the lung lavage fluid, and figure b shows the sIgA content in the tracheal lavage fluid.

[0039] Figure 13 In the figures, a, b, and c show the serum levels of IL-2, IL-4, and IFN-γ, respectively.

[0040] Figure 14 Results of RT-qPCR detection of the expression levels of immune-related genes in chicken spleen. Detailed Implementation

[0041] To facilitate understanding, the technical solution of the present invention will be described in more detail below with reference to embodiments:

[0042] Example 1 Test Materials

[0043] 1. Source of laboratory animals

[0044] Seven-day-old Hy-Line Brown chickens were purchased from a hatchery in Lu'an, Anhui Province. Six- to eight-week-old female Balb / c mice were provided by Hangzhou Ziyuan Laboratory Animal Technology Co., Ltd.

[0045] 2. Strains and plasmids

[0046] The prokaryotic expression vector pET-32a and Escherichia coli DH5α, Rosetta, and BL21 strains were preserved in our laboratory; the pCold-TF plasmid was provided by Anhui Agricultural University.

[0047] 3. Mucosal adjuvants

[0048] Sangon Biotech (Shanghai) Co., Ltd. synthesized mouse mucosal adjuvant CPG ODN2007: 5'-TCGTCGTTGTCGTTTTGTCGTT-3', which was modified by full-chain thiophosphorylation and purified by HPLC.

[0049] 4. Preparation of culture medium and main antibiotics

[0050] LB liquid medium: NaCl 1%, tryptone 1%, yeast extract 0.5%, adjust pH to 7.4, autoclave at 121℃ for 20 min, store at 4℃ for later use.

[0051] LB solid medium: NaCl 1%, tryptone 1%, yeast extract 0.5%, agar powder 1.5%, adjust pH to 7.4, autoclave at 121℃ for 20 min, store at 4℃ for later use.

[0052] Preparation of ampicillin solution: Dissolve 2.0 g of ampicillin in 20 mL of ddH2O, filter through a 0.22 μm filter, dispense into aliquots, and store at -20 °C.

[0053] 5. Preparation of reagents for agarose gel electrophoresis

[0054] Preparation of reagents for agarose gel electrophoresis: For 50×TAE: Add 121g Tris and 14.6g EDTA to 400mL of deionized water and stir thoroughly to dissolve. Add 28.55mL of acetic acid and mix thoroughly. Make up to 500mL with deionized water and store at room temperature.

[0055] Preparation of 1.5% agarose gel: 0.45g agarose, 30mL 1×TAE buffer, mix and heat, then add 0.9μL nucleic acid dye and mix well.

[0056] 6. Preparation of reagents for protein induction expression and purification

[0057] Preparation of IPTG solution: Dissolve 0.24 g of IPTG in 10 mL of ddH2O, filter through a 0.22 μm filter, dispense into smaller containers, and store at -20 °C.

[0058] Binding buffer: 20 mM sodium phosphate, 0.5 M NaCl, 40 mM imidazole, pH 7.4, filtered using a 0.45 μm filter membrane.

[0059] Elution buffer: 20 mM sodium phosphate, 0.5 M NaCl, 500 mM imidazole, pH 7.4, filtered using a 0.45 μm filter membrane.

[0060] Chromatography packing material preservation solution: Dissolve 2 mL of ethanol in 8 mL of dH2O and store at 4 °C for later use.

[0061] 7. Preparation of purification reagents

[0062] Binding (washing) buffer: Na2HPO4 0.28g, NaCl 0.88g, dissolved in ddH2O, pH adjusted to 7.0, and volume brought to 100mL.

[0063] Eluent: 0.75g Glycine, mix well and bring to a final volume of 100mL, pH adjusted to 3.0.

[0064] Neutralization solution: Dissolve 12.1g of Tris in 80mL of ddH2O, adjust the pH to 8.5, and bring the volume to 100mL.

[0065] Filter the buffer solution through a 0.45 μm filter membrane before use.

[0066] 8. Main Instruments

[0067]

[0068]

[0069] Example 2: Construction of pET-32a-cFc and pET-32a-NDV-F recombinant plasmids

[0070] 2.1 Primer design for IgY Fc and F genes

[0071] Referring to the CDS region of the chicken IgG heavy chain gene sequence (accession number: X07174) in the NCBI-GenBank database, a pair of specific primers containing KpnI and SalI restriction enzyme sites and protective bases were designed using Primer Premier 5.0. Referring to the NDV-F genome sequence (accession number: AY508514) in the NCBI-GenBank database, 5 pairs of specific primers were designed, and EcoRI and XhoI restriction enzyme sites and protective bases were added to the 5' end of primers NDV-FF and NDV-FR. The primer sequences are shown in Table 1.

[0072] Table 1 Primers related to IgY Fc and F genes

[0073]

[0074] 2.2 Cloning of NDV-F gene and chicken IgY-Fc gene

[0075] RNA was extracted from chicken macrophages and cDNA was obtained by reverse transcription. Using cDNA as a template and cFc-F / cFc-R as primers, the IgY Fc gene was amplified. Using the laboratory-preserved pcDNA3-F plasmid as a template, gene fragments containing the major NDV antigenic epitopes, numbers 72, 161, and 343, were amplified using primer pairs NDV-F-F1 / NDV-F-R1, NDV-F-F2 / NDV-F-R2, and NDV-F-F3 / NDV-F-R3, respectively. Finally, using the first three PCR products as templates, the antigenic epitope combination peptide was amplified using primer pairs NDV-FF / NDV-FR. The high-fidelity enzyme reaction system is shown in Table 2-6. The reaction was pre-denatured at 98℃ for 5 min; denatured at 98℃ for 10 s, annealed at 60℃ for 15 s, extended at 68℃ for 1 min, for 30 cycles; and finally extended at 72℃ for 5 min to obtain the coding gene for constructing the prokaryotic expression plasmid.

[0076] Immediately after the PCR reaction, agarose gel electrophoresis was performed, and the target fragment was collected according to the instructions of the gel recovery kit.

[0077] like Figure 1 As shown in Figure a, using chicken macrophage mRNA extracted with Fc-F / Fc-R primers as a template, an Fc gene fragment of 960 bp was amplified by RT-PCR, which is consistent with the expected size of the chicken IgY-Fc gene fragment; Figure 1 As shown in Figure b, gene fragments containing antigenic epitopes were amplified using three pairs of NDV-F primers. Using the laboratory-preserved pcDNA3-F plasmid as a template, an NDV-F fragment of 294 bp was obtained by overlap PCR amplification, and the electrophoresis results were consistent with expectations.

[0078] 2.3 Double digestion of vector and target fragment

[0079] The gel-recovered product and plasmid pET-32a were double-digested with the corresponding restriction endonucleases. The digestion system is shown in Table 2. After incubation at 37°C for 30 min, the samples were detected by agarose gel electrophoresis and then recovered from the gel.

[0080] Table 2 Double enzyme digestion reaction system

[0081]

[0082] 2.4 Ligation and Transformation of Vector and Target Fragment

[0083] 2.4.1 The recovered target fragment was ligated into the pET-32a prokaryotic expression vector. The corresponding components (see Table 3) were added to the EP tube. After mixing, the mixture was reacted in a metal bath at 16°C for 1 h.

[0084] Table 3. Construction of recombinant plasmid ligation system

[0085]

[0086] 2.4.2 Preparation of competent cells

[0087] The steps are as follows:

[0088] S1. Inoculate DH5α or BL21 glycerol bacteria on a regular LB agar plate and incubate overnight at 37°C.

[0089] S2. Under aseptic conditions, pick a single colony and inoculate it into 4 mL of Amp. + / LB culture medium was activated overnight at 37℃ and 180r / min for 12-16h.

[0090] S3. Inoculate the bacterial suspension into fresh LB liquid medium at a ratio of 1:100 and incubate at 37°C and 200 rpm for 3-4 hours.

[0091] Dispense S4.1.3mL / tube, incubate on ice for 30min, then at 4℃ and 4000r / min for 5min, discarding the supernatant.

[0092] S5. Resuspend in 120 μL of pre-cooled CaCl2 and centrifuge at 4000 rpm for 5 min at 4℃, discarding the supernatant. Add another 120 μL of pre-cooled CaCl2 and resuspend in an ice bath for 30 min, then centrifuge at 4000 rpm for 3 min at 4℃, discarding the supernatant.

[0093] Pre-cool 6.50 μL of CaCl2 + 50 μL of 30% glycerol and resuspend. Store at -80°C.

[0094] Transform the ligation system into competent cells: Remove competent cells from the -80°C freezer and thaw on ice. Add the ligation product, gently tap to mix, incubate on ice for 30 min, heat shock at 42°C for 45 s, and incubate on ice for 2 min. Add 500 μL of antibiotic-free LB liquid medium, incubate at 37°C for 1 h at 180 rpm, centrifuge at 5000 rpm for 1 min, discard part of the supernatant, resuspend the pellet in approximately 100 μL of supernatant, and pipette the bacterial culture to a container with Amp... + / LB plates were used to spread the bacteria evenly with a spreader, and the plates were incubated upside down in a 37°C incubator for 12 hours. Single colonies were then picked for screening and identification.

[0095] 2.5 Identification of positive clones

[0096] From Amp + Pick several single colonies from an LB plate and place them on a plate containing Amp. + After shaking culture in LB liquid medium for 6 hours, the bacterial culture was aspirated for PCR identification.

[0097] Selected bacterial cultures that have been identified as positive for bacterial growth were expanded and the bacterial strain was preserved. Plasmids were extracted according to the instructions of the Axygen plasmid mini-DNA extraction kit. The specific steps are as follows:

[0098] S1. Take 4 mL of bacterial culture that has been cultured overnight in LB medium, centrifuge at 12,000 rpm for 1 min, and discard the supernatant;

[0099] S2. Add 250 μL of Buffer S1 to suspend the bacterial precipitate until a sterile block is present;

[0100] S3. Add 250 μL of Buffer S2, gently and thoroughly mix by turning the container up and down 4-6 times to ensure complete lysis of the bacteria until a clear solution is formed. The lysis process should not exceed 5 minutes.

[0101] S4. Add 350 μL of Buffer S3, gently and thoroughly mix by inverting the container 6-8 times, and centrifuge at 12,000 rpm for 10 min;

[0102] S5. Take the supernatant from step d and transfer it to the preparation tube (placed in a 2 mL centrifuge tube), centrifuge at 12,000 rpm for 1 min, and discard the filtrate;

[0103] S6. Place the preparation tube back into the centrifuge tube, add 500 μL Buffer W1, centrifuge at 12,000 rpm for 1 min, and discard the filtrate;

[0104] S7. Place the preparation tube back into the centrifuge tube, add 700 μL of Buffer W2, centrifuge at 12,000 rpm for 1 min, and discard the filtrate; then wash once with 700 μL of Buffer W2 and discard the filtrate.

[0105] S8. Place the preparation tube back into a 2mL centrifuge tube and centrifuge at 12,000rpm for 1min;

[0106] S9. Transfer the preparation tube into a new 1.5 mL centrifuge tube (provided in the kit), add 30 μL of LEluent or deionized water to the center of the membrane in the preparation tube, let stand at room temperature for 1 min, and centrifuge at 12,000 rpm for 1 min.

[0107] The extracted plasmids were identified by double enzyme digestion, and the recombinant plasmids that tested positive were sent to Chuzhou General Biotechnology Co., Ltd. for sequencing.

[0108] like Figure 2As shown in Figure a, the Fc target fragment obtained after double enzyme digestion and the pET-32a expression vector were recovered via gel extraction, ligation, and transformation to obtain single colonies. These were then subjected to sequential bacterial culture PCR identification, plasmid extraction, and double enzyme digestion identification. Electrophoresis results showed a fragment size of 960 bp, consistent with expectations. Sequencing results of the recombinant plasmid pET-32a-Fc showed 99.79% homology with the sequence uploaded to NCBI. Figure 2 As shown in Figure b, the digested NDV-F target fragment and pET-32a expression vector were recovered by gel, ligated and transformed to obtain single colonies, which were then subjected to bacterial PCR identification, plasmid extraction and double enzyme digestion identification. The electrophoresis results showed that the fragment size was 294 bp, which was consistent with the expectation. The sequencing results showed that the sequence was completely consistent with the expected target sequence.

[0109] Example 3 Construction of pCold-TF-Ii-key-F-Fc recombinant plasmid

[0110] A 36 bp linker [(G4S)3] was added between the NDV-F gene and the IgY Fc gene. The sequence of the linker [(G4S)3] is ACCGCCAGAGCCACCTCCGCCTGAACCGCCTCCACC. The F fragment, linker fragment, and Fc fragment were ligated using overlap PCR. The upstream primer (F-up) of the NDV-F fragment used a KpnI restriction site, and the downstream primer (F-down) was inserted into the linker sequence. The upstream primer (Fc-up) of the Fc fragment was inserted into the linker sequence, and the downstream primer (Fc-down) used a HindIII restriction site. The primer design is shown in Table 4.

[0111] Table 4 Primers related to the novel vector (Ii-key-Fc)

[0112]

[0113]

[0114] Since the Ii-key fragment is small, only 12bp in size, this sequence was directly designed on the upstream primer of the F gene. The Ii-key-FF / Fc-down primer and pCold-TF-F-Fc plasmid template were used to amplify the Ii-key-F-Fc spliced ​​gene.

[0115] The target gene, with a length of 1302 bp, was amplified and ligated into the pCold-TF vector, then transformed into BL21 competent cells. Identification was performed by bacterial culture PCR, plasmid extraction, and double enzyme digestion. Figure 3As shown, the electrophoresis results were consistent with the expected fragment size. The bacterial culture sequencing results, after alignment, were consistent with the expected designed sequence, indicating that the pCold-TF-Ii-key-F-Fc recombinant plasmid was successfully constructed.

[0116] Example 4: Induction and purification of fusion protein

[0117] 4.1 Optimal Inducer Concentration Optimization for Fusion Protein

[0118] A single colony of pCold-TF-Ii-key-F-Fc (sequencing) was picked and inoculated into a solution containing 50 μg / mL of Amp. + Incubate overnight in LB broth. The next day, inoculate 6 groups with fresh 1 mL Amp at a 1:100 ratio. + After incubation in LB liquid medium at 37°C with shaking for 4 hours, one group served as a blank control group without the inducer. The remaining 5 groups were incubated with IPTG solutions of different final concentrations (0.2, 0.4, 0.6, 0.8, and 1.0 mmol / L) and labeled. The cultures were then incubated at 37°C with shaking for 5 hours. The bacteria were harvested and detected by SDS-PAGE gel electrophoresis to determine the optimal concentration of the inducer.

[0119] like Figure 4 As shown, the SDS-PAGE electrophoresis results indicate that when the final IPTG concentration reaches 0.4 mmol / L, the protein expression level does not increase significantly. Therefore, the optimal induction concentration is 0.4 mmol / L.

[0120] 4.2 Identification of Fusion Protein Expression Form

[0121] The pCold-TF-Ii-key-F-Fc recombinant bacteria cultured overnight were inoculated into 100 mL LB (Amp) at a ratio of 1:100. + Liquid culture medium, incubated at 37℃ and 180 r / min until OD 600 =0.4-0.5, immediately bring the culture medium to 15℃ for 30 min. Then add the optimal IPTG concentration and induce expression at 15℃ for 24 h. After induction, the bacterial culture is sonicated and 20 μL of supernatant and precipitate resuspended are taken for detection. The expression form of the protein is detected by SDS-PAGE electrophoresis.

[0122] like Figure 5 As shown, the electrophoresis results indicate that the pCold-TF-Ii-key-F-Fc fusion protein is mainly present in the supernatant, expressed primarily in a soluble form, and the expression level of the fusion protein is relatively high.

[0123] 4.3 Large-scale expression and purification of fusion proteins

[0124] Recombinant bacterial expression was induced under established soluble expression conditions. The precipitate was collected by centrifugation, resuspended in PBS, and then sonicated in an ice-water bath. After sonication, the mixture was centrifuged at 8000 rpm for 10 min at 4°C, and the supernatant was collected. The His-tagged protein was purified and collected according to the following purification steps.

[0125] S1. Take 2mL His Ni 2+ The affinity chromatography packing material was loaded into a gravity column, and 20% ethanol in the nickel column was washed away with dH2O. The gravity column was then equilibrated with 8 column volumes of binding buffer.

[0126] S2. Add 3 mL of protein to the gravity column and mix by pipetting. Incubate at low temperature with shaking for 1 h. Collect the flow solution and wash with binding buffer. Collect the wash solution.

[0127] S3. Elute with 500mM imidazole elution buffer, collect 4-5mL of eluent, and identify all collected buffer fractions by SDS-PAGE gel electrophoresis.

[0128] S4. Collect the eluent in a dialysis bag and place it in the dialysis solution. Stir slowly and evenly with a magnetic stirrer during the process. Change the buffer every 6-8 hours. Perform gradient dialysis at 4°C to remove high concentrations of salt and imidazole. After complete dialysis, concentrate the solution with sucrose at 4°C and determine its concentration according to the BCA protein concentration kit.

[0129] like Figure 6 As shown, a band consistent with the target protein was detected at 99.5 kDa in the collected eluent, indicating the soluble expression of the pCold-TF-Ii-key-F-Fc fusion protein.

[0130] Example 5: Preparation and titer detection of polyclonal antibodies

[0131] pCold-TF-F-Fc and pCold-TF-F fusion proteins were obtained as controls, referring to the methods in Examples 3 and 4.

[0132] 5.1 Western blot identification of fusion proteins

[0133] The purified Ii-key-F-Fc, F-Fc, and F fusion proteins were used as antigens and Western blots were performed using anti-His tag and mouse anti-F polyclonal antibody primary antibody, and HRP goat anti-mouse secondary antibody, respectively.

[0134] like Figure 7As shown, after DAB staining, only the corresponding specific protein bands were observed in the PVDF membrane, consistent with the molecular weight of the protein bands detected by SDS-PAGE gel. Therefore, multiple results indicate that the Ii-key-F-Fc, F-Fc, and F fusion proteins possess good reactivity.

[0135] 5.2 Detection of fusion protein crossing the chicken respiratory mucosal immune barrier

[0136] Seven-day-old Hy-Line Brown chickens were immunized intranasally with biotin-labeled fusion protein. They were divided into four groups, with three chickens in each group receiving 200 μg per chicken. Eight hours later, blood was collected from the subwing vein of each group, and serum was separated. The content of the fusion protein was indirectly detected by measuring the OD value of biotin in the serum using an indirect ELISA.

[0137] Biotin labeling methods:

[0138] S1. Dissolve 2 mg of the protein to be labeled in 1 mL of phosphate buffer. Add the protein solution to an ultrafiltration tube and add labeling buffer not exceeding the maximum volume of the ultrafiltration tube. Centrifuge at 12,000 rpm for 10 min. This step can be repeated multiple times. After the last ultrafiltration is completed, add an appropriate amount of labeling buffer to adjust the antibody concentration to about 2 mg / mL.

[0139] S2. Remove the kit 20 minutes in advance and allow it to equilibrate to room temperature. Prepare a 10 mM solution of active super biotin using a solubilizer. Add 13.3 μL of the dissolved active biotin to the ultrafiltration tube and gently mix by pipetting. Incubate at 37°C in the dark for 30 minutes.

[0140] S3.1 Centrifuge at 12,000 rpm for 10 min, add an appropriate amount of labeling buffer to the ultrafiltration tube, and gently pipette to mix. Centrifuge at 12,000 rpm for 10 min. Repeat this step several times.

[0141] S4. Collect the solution (i.e., biotin-labeled protein) in the ultrafiltration tube, add an equal volume of preservation solution, and store at -20°C.

[0142] like Figure 8 As shown, the detection results showed that the content of Ii-key-F-Fc and F-Fc fusion proteins was not significantly different from that of the IgY positive control group (P>0.05), but was extremely significantly different from that of the F fusion protein (P<0.01), indicating that the Ii-key-F-Fc fusion protein can cross the respiratory mucosal immune barrier of chickens and enter the blood.

[0143] 5.3 Study on the effect of nasal immunization of chickens with novel carrier fusion protein

[0144] 5.3.1 Immunization Program

[0145] Eighty-eight 7-day-old Hy-Line Brown chickens were divided into four groups of 22 each. The grouping and immunization dosage are shown in Table 6. The chickens were immunized three times at 7, 21 and 35 days of age.

[0146] Table 6 Nasal Drop Immunization Grouping

[0147]

[0148] 5.3.2 Sample Preparation

[0149] 1) Serum preparation

[0150] At weeks 2, 3, 4, 5, and 6 post-immunization, each group was deprived of water and food for 8 hours in advance. Three chickens were randomly selected from each group, and blood was collected from the wing veins. The serum was separated and stored at -20°C.

[0151] 2) Preparation of tracheal and lung lavage fluid

[0152] At weeks 3, 4, 5, and 6 post-immunization, all groups were deprived of water and food for 8 hours prior to immunization. Three birds from each group were randomly selected, euthanized by cardiac blood sampling, and then disinfected in 75% alcohol for several minutes. The chickens were then fixed supine on a dissecting board. The neck skin was cut open using sterile ophthalmic forceps and scissors to expose and separate the trachea. The trachea was inserted below the larynx and flushed three times repeatedly with 1 mL of sterile PBS. The resulting flushing solution was centrifuged at 12,000 rpm for 5 min and stored at -20°C. The lungs were separated and placed in a 10 mL centrifuge tube. 2 mL of sterile PBS was added, and the lungs were cut into small pieces with scissors. The mixture was centrifuged at 12,000 rpm for 5 min, and the supernatant was used as the lung flushing solution, which was stored at -20°C.

[0153] 3) Separate the spleen

[0154] Remove the chicken spleen intact, divide it into appropriate sizes, place it in a general tissue fixation solution, and store it at -80℃ for later use.

[0155] 5.3.3 Detection of relevant immune indicators

[0156] Indicators include:

[0157] 1) Indirect ELISA detection of chicken serum-specific IgY antibody levels;

[0158] 2) ELISA was used to detect the level of specific sIgA secretion in tracheal and lung lavage fluid;

[0159] 3) Detection of immune factors IL-2, IL-4 and IFN-γ in serum.

[0160] All experimental data were processed by Excel, and the results were expressed as mean (S) ± standard deviation (SD, n=3). One-way ANOVA was performed using GraphPad Prism 9.0 software, and the significance of the data was marked. No common lowercase letters indicated a significant difference (P<0.05), *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0161] The results are as follows:

[0162] like Figure 11 As shown, after 14 days of initial immunization with three proteins fused with the F domain, specific IgY antibodies began to be produced in each experimental group. After booster immunization, the level of specific IgY antibodies induced in each experimental group showed an upward trend. Between 35 and 42 days post-immunization, the antibody level induced by the Ii-key-F-Fc intranasal immunization group reached the highest level, with a specific IgY antibody titer of 1:6400 as detected by ELISA. In contrast, the antibody titers of the F-Fc and F intranasal immunization groups were 1:1600 and 1:800, respectively. This indicates that the novel vector (Ii-key-F-Fc) group can stimulate chickens to produce higher levels of IgY antibodies. Statistical analysis revealed that the serum IgY antibody level against NDV-F in the novel vector (Ii-key-F-Fc) group was significantly higher than that in other groups (P<0.05). The IgY antibody level in the single vector (F-Fc) group was significantly different from that in the F antigen group (P<0.05). The IgY antibody levels in each fusion protein immunization group were extremely significantly different from those in the PBS control group (P<0.001).

[0163] like Figure 12 As shown in Figure a, sIgA was detected in the tracheal lavage fluid of each immunization group on day 21 post-immunization, and the difference was significant compared with the PBS control group (P < 0.05). After booster immunization, the sIgA antibody level in each immunization group gradually increased, reaching its highest point on day 35. The average sIgA level at the peak point in the tracheal lavage fluid of each group was: F group: 1811.09 ng / mL; F-Fc group: 1960.83 ng / mL; Ii-key-F-Fc group: 2400.39 ng / mL. Statistical analysis revealed that at any time point during testing, the sIgA level in the novel vector (Ii-key-F-Fc) group was higher than that in other groups (P<0.05). On days 21 and 35 post-immunization, the sIgA antibody level in the single vector (F-Fc) group was not statistically significant compared to that in the F antigen group. At other time points, the sIgA antibody level in the single vector (F-Fc) group was significantly different from that in the F antigen group (P<0.05), and the differences between each fusion protein immunization group and the PBS control group were extremely significant (P<0.001).

[0164] like Figure 12As shown in Figure b, the sIgA antibody level in the lung lavage fluid reached its highest point on day 35 post-immunization. The average sIgA content at the peak point for each group was as follows: F group: 1980.59 ng / mL; F-Fc group: 2102.05 ng / mL; Ii-key-F-Fc group: 2265.19 ng / mL. Statistical analysis revealed that from day 28 to day 35 post-immunization, the sIgA level in the lung lavage fluid of the novel vector group (Ii-key-F-Fc) was significantly higher than that of the single vector group (F-Fc) (P<0.05) and the F antigen group (P<0.01). The sIgA level in the single vector (F-Fc) group was significantly different from that in the F antigen group (P<0.01). The sIgA antibody levels in each fusion protein immunization group were significantly different from those in the PBS control group (P<0.05).

[0165] like Figure 13 As shown in Figure a, the average IFN-γ level reached its highest value between 28 and 35 days post-immunization. The highest IFN-γ level in serum of the novel vector group (Ii-key-F-Fc) was 875.34 pg / mL, while that in the single-vector group (F-Fc) was 802.47 pg / mL, with a highly significant difference between the two groups (P < 0.01). The highest IFN-γ level in the F antigen group was 692.58 pg / mL, with a highly significant difference between the novel vector group and the F antigen group (P < 0.01). At the same detection time point after immunization, the IFN-γ level in the novel vector group (Ii-key-F-Fc) was higher than that in other immunization groups (P < 0.05). The average IFN-γ level in the single-vector group (F-Fc) was significantly different from that in the F antigen group (P < 0.05). The IFN-γ level in each immunization group was significantly different from that in the PBS control group (P < 0.05).

[0166] like Figure 13 As shown in Figure b, the serum IL-2 levels in all immunization groups reached their highest levels between days 28 and 35 post-immunization. The highest IL-2 level in the novel vector group (Ii-key-F-Fc) was 266.37 pg / mL, the single-vector group (F-Fc) had a level of 246.53 pg / mL, and the F antigen group had a level of 229.87 pg / mL. Statistical analysis revealed that, except for day 21 post-immunization, the IL-2 level in the Ii-key-F-Fc group was not significantly different from that in the single-vector group, but was significantly higher than that in other immunization groups at all other time points (P<0.01). The single-vector group (F-Fc) was significantly different from the F antigen group (P<0.01), and the average IL-2 levels in all fusion protein immunization groups were higher than those in the control group (P<0.01).

[0167] like Figure 13As shown in Figure c, the average IL-4 level reached its highest value between 28 and 35 days post-immunization. The highest IL-4 level in the serum of the novel vector group (Ii-key-F-Fc) was 116.07 pg / mL, while that in the single-vector group (F-Fc) was 93.92 pg / mL, with a highly significant difference between the two groups (P < 0.05). The highest IL-4 level in the F antigen group was 78.04 pg / mL, which was extremely significantly different from that in the novel vector group (P < 0.0001). The average IL-4 level in all immunization groups was higher than that in the PBS control group (P < 0.001).

[0168] 5.3.4 Expression of immune-related genes

[0169] To further evaluate the immunomodulatory effect of the novel vector, the expression levels of immune-related genes in chicken spleen were detected using RT-qPCR after immunization. The relevant genes detected were major histocompatibility complex (MHCI) class α, MHCII class β, nuclear factor-κB (NF-κB), and tumor necrosis factor-α (TNF-α).

[0170] The results are as follows Figure 14 As shown: by Figure 14 As shown in Figure a, the transcriptional level of MHCIα reached its highest point on day 42, and the difference between the novel vector group (Ii-key-F-Fc) and the single vector group (F-Fc) was extremely significant (P<0.001). The differences between each immunization group and the control group were also extremely significant (P<0.0001). Figure 14 As shown in Figure b, the MHCIIβ transcription level reached its highest value on day 42, and the MHCIIβ transcription level of the novel vector group (Ii-key-F-Fc) was significantly different from that of other groups at any detection time point (P<0.01). Figure 14 As shown in Figure c, at any given time point, the NF-κB gene transcription level in the novel vector group (Ii-key-F-Fc) was significantly different from that in other groups (P<0.0001); Figure 14 As shown in Figure d, at any time point during the detection, the TNF-α transcription level of the novel vector group (Ii-key-F-Fc) was significantly different from that of other groups (P<0.001), and this was consistent with the results of the NF-κB gene transcription level detection.

[0171] The above analysis shows that the Ii-key-F-Fc group antigen has a stronger stimulating effect on the body than the F-Fc group and the F group, and the F-Fc group is stronger than the F group. Therefore, the novel vector (Fc-Ii-key) antigen group has a stronger stimulating effect on the body than the single vector (Fc), and the single vector (Fc) has a better immune effect on the body than the "naked" F antigen peptide.

[0172] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An antigen that enhances the immune response to Newcastle disease virus, characterized in that, This antigen is a fusion protein obtained by expressing the Ii-key-F-Fc recombinant vector with engineered bacteria, named Ii-key-F-Fc fusion protein, and its amino acid sequence is shown in SEQ ID NO.1; The Ii-key-F-Fc recombinant vector was obtained by splicing three major antigenic epitope fragments on the NDV F protein and then tandemly connecting them with chicken IgYFc and Ii-key, respectively. The nucleotide sequence of the Ii-key-F-Fc recombinant vector is shown in SEQ ID NO.

2. The NDV F protein is a surface glycoprotein on the cell membrane of Newcastle disease virus. The three major antigenic epitope fragments are located at positions 72, 161, and 343 of the NDV F protein, respectively, and their amino acid sequences are shown in SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5, respectively.

2. The antigen for enhancing Newcastle disease virus immunity as described in claim 1, characterized in that, The product obtained by splicing the three major antigenic epitope fragments on the NDV F protein is an antigenic epitope combinatorial peptide, the amino acid sequence of which is shown in SEQ ID NO.

6.

3. A method for preparing an antigen that enhances the immune response to Newcastle disease virus as described in claim 1, comprising the following steps: S1. Extract RNA from chicken macrophages, reverse transcribe to obtain cDNA, and amplify the IgY Fc gene using cFc-F / cFc-R as primers; using pcDNA3-F plasmid as template, amplify gene fragments containing the major NDV antigenic epitopes at levels 72, 161, and 343 using primer pairs NDV-F-F1 / NDV-F-R1, NDV-F-F2 / NDV-F-R2, and NDV-F-F3 / NDV-F-R3, respectively. Finally, using the first three PCR products as templates, amplify the antigenic epitope combination peptide using primer pair NDV-FF / NDV-FR, and name it fragment F; The target gene fragment was obtained by tandem concatenating the F fragment, the constant-strand active fragment Ii-key, and the IgY Fc fragment using the overlap PCR method, wherein the upstream primer F-up for the F fragment was Kpn. I For the restriction enzyme site, the downstream primer F-down is inserted into the linker sequence; for the Fc fragment, the upstream primer Fc-up is inserted into the linker sequence, and the downstream primer Fc-down uses Hind. III Enzyme cleavage sites; The cFc-F / cFc-R sequences are shown in SEQ ID NO.7 / SEQ ID NO.8, the NDV-F-F1 / NDV-F-R1 sequences are shown in SEQ ID NO.9 / SEQ ID NO.10, the NDV-F-F2 / NDV-F-R2 sequences are shown in SEQ ID NO.11 / SEQ ID NO.12, the NDV-F-F3 / NDV-F-R3 sequences are shown in SEQ ID NO.13 / SEQ ID NO.14, and the NDV-FF / NDV-FR sequences are shown in SEQ ID NO.15 / SEQ ID NO.

16. The F-up sequence is shown in SEQ ID NO.17, the F-down sequence is shown in SEQ ID NO.18, the Fc-up sequence is shown in SEQ ID NO.19, the Fc-down sequence is shown in SEQ ID NO.20, and the Linker sequence is shown in SEQ ID NO.21; S2. After the target gene fragment is ligated into the pCold-TF prokaryotic expression vector, the Ii-key-F-Fc recombinant vector is transformed into competent cells BL21, and the competent cells are used to express the Ii-key-F-Fc fusion protein, which is an antigen that enhances the immune effect of Newcastle disease virus.

4. The preparation method according to claim 3, characterized in that, In step S2, the specific operation of expressing the Ii-key-F-Fc fusion protein using competent cells is as follows: Ii-key-F-Fc positive monoclonal antibodies are picked and inoculated into cells containing 50ug / mL Ampicillin. + Expression was induced at 15°C for 24 h in LB liquid medium at 37°C with a final IPTG concentration of 0.4 mmol / L.

5. The preparation method according to claim 3 or 4, characterized in that, The Ii-key-F-Fc fusion protein obtained in step S2 includes a purification step, specifically: the expressed fusion protein is sonicated and the supernatant is collected by centrifugation. The supernatant is then further collected by nickel ion affinity chromatography to obtain the purified Ii-key-F-Fc fusion protein. This Ii-key-F-Fc fusion protein is an antigen that enhances the immune effect of Newcastle disease virus.

6. The use of an antigen that enhances the immune response to Newcastle disease virus in the preparation of a Newcastle disease vaccine, wherein the antigen that enhances the immune response to Newcastle disease virus is the Ii-key-F-Fc fusion protein as described in claim 1.

7. A preparation that provides or enhances the body's immunity to Newcastle disease virus, characterized in that, The formulation comprises an effective dose of the Ii-key-F-Fc fusion protein as described in claim 1, the amino acid sequence of which is shown in SEQ ID NO.

1.

8. The formulation as described in claim 7, characterized in that, The formulation is a liquid formulation or a spray.

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