Gene VII type NDV recombinant virus for expressing IBV S protein as well as preparation method and application of gene VII type NDV recombinant virus

Abstract

The invention provides a gene VII type NDV recombinant virus for expressing IBV S protein as well as a preparation method and application of the gene VII type NDV recombinant virus, and the method comprises the following steps: inserting a modified S gene between a P gene and an M gene of a pBR322-DHN3mF virus genome to obtain any one of a recombinant virus rDHN3mF-MH20S1-IBVS2, a recombinant virus rDHN3mF-H120-S6P and a recombinant virus rDHN3mF-H120-S6P-GSAS. According to the present invention, the S2 region of IBV-P65 adapted to a cell strain is replaced with the S2 region of QX type IBV MH20, such that the recombinant virus rDHN3mF-MH20S1-IBVS2 is rescued; the preparation method comprises the following steps: mutating hydrophobic amino acids at the 692nd site, the 768th site, the 772nd site, the 815th site, the 862nd site and the 863rd site on an H120-S protein into proline to rescue a recombinant virus rDHN3mF-H120-S6P; on the basis that six hydrophobic amino acids are mutated into proline, amino acids from the 533th site to the 537th site are mutated into glycine, serine, alanine and serine in sequence, and then the recombinant virus rDHN3mF-H120-S6P-GSAS is rescued. The three rescued recombinant viruses are respectively infected with BHK-21 cells, and high-efficiency expression of S protein and NDV holoprotein can be detected through WB verification.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering technology design, and in particular to a gene type VII NDV recombinant virus expressing IBV S protein, and a preparation method and application thereof. Background Art

[0002] Avian infectious bronchitis (IB) is a highly contagious disease of the respiratory, reproductive and urogenital tracts of chickens caused by avian infectious bronchitis virus (IBV). The disease usually causes respiratory symptoms such as increased eye and nasal discharge, wheezing, coughing and sneezing in chickens. It can also cause renal dysfunction and decreased egg production by infecting the urogenital tract. The mortality rate of chicks is very high, especially after infection with IBV, it will be accompanied by other secondary complications, which seriously affects the production performance of egg-laying and meat-producing poultry, causing huge economic losses to the poultry industry.

[0003] S protein is the structural protein with the largest molecular weight among IBV structural proteins. Before glycosylation, the molecular weight of S protein is about 128-160 KDa. After glycosylation modification, the molecular weight of S protein can reach 150-200 KDa. S protein contains virus neutralizing antigen epitopes and is the main protective antigen of the host. The continuous evolution and mutation of S protein has brought severe challenges to the development of vaccines. So far, 9 genotypes have been identified based on S1 gene worldwide. The more popular ones are QX type, Mass type, TW type, etc. QX type is the most popular strain in China. The IBV vaccine strain currently used in China is mainly Mass type attenuated vaccine, which cannot provide sufficient protection for the IBV variants that have emerged in recent years. Therefore, it is urgent to develop a new IBV vaccine that can target different serotypes.

[0004] The S protein is a type I membrane fusion protein. When S1 binds to the host cell receptor, it causes the S protein to transform from a metastable conformation to a hyperstable conformation. Maintaining the metastable conformation before fusion is beneficial to improving the stability of the S protein and increasing its immunogenicity. Existing studies have shown that mutating 2 or 6 specific amino acids in the S protein of SARS-CoV-2 to proline or mutating the furin site can help form a metastable conformation of the S protein.

[0005] Newcastle disease (ND) is an acute, highly contagious disease caused by a virulent strain of Newcastle disease virus (NDV). The disease can cause highly contagious respiratory and nervous system diseases in chickens, causing serious economic losses to the poultry industry. In recent years, with the continuous development of reverse genetics systems, Newcastle disease virus vectors have been widely used in reverse genetics systems due to their advantages such as convenient large-scale production, convenient immunization routes, good immune response induction, only infection of specific hosts, and safety and reliability. Currently, vaccines for preventing ND are mainly inactivated vaccines and attenuated vaccines. Inactivated vaccines have a large immunization dose and require multiple immunizations; while attenuated vaccines mainly target genotype II NDV, which is not compatible with the genotype VII NDV strain currently prevalent in my country, thus causing huge economic losses to my country's poultry industry. Summary of the invention

[0006] The object of the present invention is to provide a gene VII type NDV recombinant virus expressing IBV S protein, and a preparation method and application thereof, wherein the recombinant virus rDHN3mF-MH20S1-IBVS2 is rescued by replacing the S2 region of the IBV-P65 adapted to the cell line with the S2 region of the QX type IBV MH20; the recombinant virus rDHN3mF-H120-S6P is rescued by mutating the hydrophobic amino acids at positions 692, 768, 772, 815, 862 and 863 on the H120-S protein to proline; and the recombinant virus rDHN3mF-H120-S6P-GSAS is rescued after the amino acids at positions 533 to 537 are mutated to glycine, serine, alanine and serine in sequence on the basis of mutating the six hydrophobic amino acids to proline. The three rescued recombinant viruses were used to infect BHK-21 cells respectively, and WB verification revealed that efficient expression of S protein and complete NDV protein could be detected.

[0007] In a first aspect, the present invention provides a method for preparing a genotype VII NDV recombinant virus expressing IBV S protein, the method comprising: The modified S gene is inserted between the P gene and the M gene of the pBR322-DHN3mF viral genome to obtain any one of the recombinant viruses rDHN3mF-MH20S1-IBVS2, rDHN3mF-H120-S6P, and rDHN3mF-H120-S6P-GSAS.

[0008] Further, the step of inserting the modified S gene between the P gene and the M gene of the pBR322-DHN3mF viral genome comprises: The gene F1 fragment, gene F2 fragment, gene MH20S1-IBVS2 fragment or gene H120-S6P fragment or gene H120-S6P-GSAS fragment and gene F3 fragment are used for homologous recombination to obtain three recombinant plasmids, and the three recombinant plasmids are used for transfecting cells to obtain three recombinant viruses.

[0009] Furthermore, the sequence of the gene F1 fragment is shown in SEQ ID NO: 1; The sequence of the gene F2 fragment is shown in SEQ ID NO: 2; The sequence of the gene MH20S1-IBVS2 fragment is shown in SEQ ID NO: 3; The sequence of the gene H120-S6P fragment is shown in SEQ ID NO: 4; The sequence of the gene H120-S6P-GSAS fragment is shown in SEQ ID NO: 5; The sequence of the gene F3 fragment is shown in SEQ ID NO: 6.

[0010] Furthermore, the preparation process of the gene F1 fragment is as follows: using the pBR322-DHN3mF vector as a template and performing enzyme digestion with SmaI to obtain the F1 fragment; The preparation process of the gene F2 fragment is as follows: using the pBR322-DHN3mF plasmid as a template and using primers FDHN-SmaI-F1 and FDHN-R1 to amplify the F2 fragment; the primers FDHN-SmaI-F1 and FDHN-R1 are shown in the sequence table SEQ ID NO: 7-8; The preparation process of the gene MH20S1-IBVS2 fragment is as follows: using MH20 cDNA as a template and using primers MH20-S-F1 and MH20-S-R1 to amplify the MH20-S1 fragment; using pXJ40-IBV-S vector as a template and using primers IBV-S2-F1 and IBV-S2-R2 to amplify the IBV-S2 fragment; using MH20-S1 and IBV-S2 fragments as templates and using primers MH20-S-F1 and IBV-S2-R2 to amplify the MH20S1-IBVS2 fragment. Primers MH20-S-F1, MH20-S-R1, IBV-S2-F1, and IBV-S2-R2 are shown in the sequence table as SEQ ID NOs: 9 to 12.

[0011] Furthermore, the preparation process of the gene H120-S6P fragment is as follows: using the pXJ40-H120-S2P plasmid as a template, primers H120-S-F1 and H120-6P-R1 are used to amplify the H120-1 fragment; using primers H120-6P-F2 and H120-6P-R2 to amplify the H120-2 fragment; using primers H120-6P-F3 and H120-6P-R3 to amplify the H120-3 fragment; using primers H120-6P-F4 and H120-SR to amplify the H120-4 fragment; using fragments H120-1, H120-2, H120-3, and H120-4 as templates, primers H120-S-F1 and H120-SR are used to amplify the H120-S6P fragment. Primers H120-S-F1, H120-6P-R1, H120-6P-F2, H120-6P-R2, H120-6P-F3, H120-6P-R3, H120-6P-F4, and H120-SR are shown in the sequence listing as SEQ ID NOs: 13 to 20, respectively; The preparation process of the gene H120-S6P-GSAS fragment is as follows: using rDHN3mF-H120-S6P plasmid as a template, primers H120-S-F1 and GSAS-R1 are used to amplify the GSAS-1 fragment; using primers GSAS-F1 and H120-SR to amplify the GSAS-2 fragment; using the GSAS-1 fragment and the GSAS-2 fragment as templates, primers H120-S-F1 and H120-SR are used to amplify the H120-S6P-GSAS fragment. Primers GSAS-R1 and GSAS-F1 are shown in the sequence table as SEQ ID NOs: 21 to 22, respectively; The preparation process of the gene F3 fragment is as follows: using pBR322-DHN3mF plasmid as a template and primers FDHN-F2 and FDHN-SmaI-R2 to amplify the F3 fragment; the primers FDHN-F2 and FDHN-SmaI-R2 are shown in the sequence table SEQ ID NO: 23-24 respectively.

[0012] Furthermore, the amount of each substance used in the recombination process is: 870ng gene fragment F1, 201ng gene fragment F2, 455ng gene fragment MH20S1-IBVS2 or 450ng gene fragment H120-S6P or 450ng gene fragment H120-S6P-GSAS, 611ng gene fragment F3, 10μL ABclonal 2X MultiF Seamless Assembly Mix, ddH2O is added to 20μL; the reaction conditions are 50°C for 50 minutes.

[0013] Furthermore, during the transfection process, the transfected cells were BHK-T7 cell lines, and the components of the transfection reagent were as follows: Solution A: Opti-MEM Medium 100μL, Lipomaster 2000 Transfection Reagent 18μL; Solution B: Opti-MEM Medium 100μL, rDHN3mF-MH20S1-IBVS2 or rDHN3mF-H120-S6P; rDHN3mF-H120-S6P-GSAS 4μg, pXJ40-NP 2.5μg, pXJ40-P 1.25μg, pXJ40-L 1.25μg.

[0014] In a second aspect, the present invention further provides a genotype VII NDV recombinant virus expressing IBV S protein, which is obtained according to the above-mentioned method for preparing a genotype VII NDV recombinant virus expressing IBV S protein.

[0015] In a third aspect, the present invention further proposes the use of a gene type VII NDV recombinant virus expressing IBV S protein in the preparation of a vaccine.

[0016] Compared with the prior art, the present invention has the following advantages: By modifying IBV S proteins of different genotypes, including replacing S2 of the cell-adapted IBV-P65 with S2 of the non-cell-adapted strain MH20, mutating the six hydrophobic amino acids in the H120 S protein to proline and mutating the Flynn site on this basis, the rescued recombinant viruses are more stable than the recombinant strains inserted with unmodified S proteins. These three recombinant viruses are expected to be further developed into a bivalent vaccine against avian bronchitis virus and Newcastle disease virus, providing a new defense against avian bronchitis virus and Newcastle disease virus infections. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the accompanying drawings: Figure 1 It is the gel electrophoresis diagram of F1 gene fragment, where M: DNA Marker; 1 is F1 PCR fragment (13399 bp); Figure 2 It is the gel electrophoresis diagram of F2 gene fragment, where M: DNA Marker; 1 is F2 PCR fragment (1549 bp); Figure 3It is the gel electrophoresis diagram of MH20S1-IBVS2 gene fragment, where M: DNA Marker; 1 is the MH20S1-IBVS2 PCR fragment (3498 bp); Figure 4 It is the gel electrophoresis diagram of H120-S6P gene fragment, where M: DNA Marker; 1 is the H120-S6P overlapPCR fragment (3462bp); Figure 5 The figure is the gel electrophoresis of H120-S6P-GSAS gene fragment; M: DNA Marker; 1 is the GSAS-1 PCR fragment (1619 bp), 2 is the GSAS-2 PCR fragment (1871 bp); Figure 6 It is the gel electrophoresis diagram of F3 gene fragment; M: DNA Marker; 1 is F3 PCR fragment (4704bp); Figure 7 Schematic diagram of rDHN3mF-MH20S1-IBVS2 plasmid; Figure 8 Schematic diagram of rDHN3mF-H120-S6P plasmid; Fig. 9 Schematic diagram of rDHN3mF-H120-S6P-GSAS plasmid; Fig.10 It is the enzyme digestion identification diagram of the recombinant plasmid, where M: DNA Marker; 1: recombinant plasmid rDHN3mF-MH20S1-IBVS2; 2: recombinant plasmid rDHN3mF-H120-S6P; 3: recombinant plasmid rDHN3mF-H120-S6P-GSAS; Fig.11 Schematic diagram of sequencing results of H120-S gene in recombinant plasmid after proline and furin site mutation, wherein a is the sequencing result of recombinant plasmid rDHN3mF-H120-S6P; b is the sequencing result of rDHN3mF-H120-S6P-GSAS; Fig.12 Schematic diagram of the expression of S protein of the recombinant virus rDHN3mF-MH20S1-IBVS2 in BHK-21 cells after passage through chicken embryos at different dilutions, wherein 10-2 represents passage through chicken embryos after the virus is diluted 100 times, 10-4 represents passage through chicken embryos after the virus is diluted 10,000 times, and 10-6 represents passage through chicken embryos after the virus is diluted 1,000,000 times; 1, 2, and 3 represent passage through different chicken embryos; Fig.13 This is a magnified image of BHK-21 cells infected with the allantoic fluid of the recombinant virus rDHN3mF-H120-S6P; Fig.14 This is a magnified image of BHK-21 cells infected with the recombinant virus rDHN3mF-H120-S6P-GSAS allantoic fluid; Fig.15 This is a magnified picture of SPF chicken embryo allantoic fluid infected BHK-21 cells; Fig.16 Schematic diagram of the expression of the whole NDV protein of the recombinant virus rDHN3mF-H120-S in BHK-21 cells; Fig.17 Schematic diagram of the expression of S protein of the recombinant virus rDHN3mF-H120-S in BHK-21 cells; Fig.18 This is a schematic diagram of the immune attack procedure; Fig.19 This is a schematic diagram of the weight gain of chickens after immunization, where ns means no significant difference; Fig. 20 The figure is a schematic diagram of the detection results of NDV-specific antibodies in the serum of immunized chickens, where ** represents significant difference (p < 0.01); *** represents extremely significant difference (p < 0.001); 5 chickens were randomly selected from each group for blood drawing, and each serum was repeated 3 times; Fig.21 The figure is a schematic diagram of NDV excretion in the throat of SPF chickens after challenge, where n / m represents the number of positive samples / total number of samples; Fig. 22 Schematic diagram of IBV shedding in SPF chicken swabs after challenge, where Figure a shows virus shedding in laryngeal swabs of SPF chickens after M41 challenge; Figure b shows virus shedding in cloacal swabs of chickens after M41 challenge; * indicates significant difference (p < 0.05); ** indicates significant difference (p < 0.01); *** indicates extremely significant difference (p < 0.001); Fig.23 This is a schematic diagram of IBV shedding in tissues and organs of SPF chickens after infection, where ns represents no significant difference; * represents significant difference (p < 0.05); ** represents significant difference (p < 0.01); *** represents extremely significant difference (p < 0.001). DETAILED DESCRIPTION

[0018] The present invention will be described in detail below with reference to the accompanying drawings and in combination with embodiments. It should be noted that the embodiments and features in the embodiments of the present invention can be combined with each other without conflict.

[0019] The following detailed description is an exemplary description, which is intended to provide further detailed description of the present invention. Unless otherwise specified, all technical terms used in the present invention have the same meaning as those generally understood by those skilled in the art to which the present invention belongs. The terms used in the present invention are only for describing specific embodiments, and are not intended to limit the exemplary embodiments according to the present invention.

[0020] Main reagents and consumables: transZol up, DNA marker, nucleic acid dye, and 6× Loading Buffer were purchased from Beijing Quanshijin Company. 2×Phanta Flash Master Mix, 2× Taq Master Mix (Dye Plus), and ChamQ UnversalSYBR QPCR Master Mix were all purchased from Nanjing Vazyme Biotech Co., Ltd. Tetramethylethylenediamine (TEMED), 96% was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Ammonium persulfate APS powder, 30% Acr-Bis (29:1), and Tween-20 were purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd. 1.5 M Tris-HCL pH 8.8, ST789-500 mL, transfer filter paper (7.5x10 cm), 1.0 M Tris-HCL pH 13 6.8, ST768-500 mL, and 10% sodium dodecyl sulfate solution were purchased from Shanghai Biyuntian Biotechnology Co., Ltd. Nitrocellulose transfer membrane was purchased from Guangzhou Fangyuan Biotechnology Co., Ltd. Primary antibody ProteinFind® Anti-β-Actin Mouse Monoclonal Antibody, chemiluminescent secondary antibody ProteinFind® Goat Anti-Mouse IgG (H+L), horseradish peroxidase-labeled goat anti-mouse IgG (H+L) antibody, chemiluminescent secondary antibody ProteinFind® Goat Anti-Rabbit IgG (H+L), horseradish peroxidase-labeled goat anti-rabbit IgG (H+L) antibody were purchased from Beijing Quanshijin Biotechnology Co., Ltd. Primary antibody Anti-IBV-S Rabbit Polyclonal Antibody was stored in the laboratory. Lipomaster 2000 Transfection Reagent was purchased from Nanjing Novozyme Biotechnology Co., Ltd. SV30010 penicillin-streptomycin double antibody was purchased from Cytiva. 10cm2, six-well plates, T25, etc. were all purchased from Guangzhou Jiete Company. Plasmids pXJ40-IBV-S, pXJ40-H120-S2P, and pBR322-DHN3mF were stored in the laboratory.

[0021] Main instruments: Electric thermostatic incubator HZ-100 (Yiheng Scientific Instrument Co., Ltd., Shanghai, China); three-hole electric thermostatic water bath DK-8D (Yiheng Scientific Instrument Co., Ltd., Shanghai, China; Haier BCD-579WE refrigerator (Haier, Shanghai, China); CO2 thermostatic incubator Forma 371 (Thermo Corporation, USA); clean bench SW-CJ-2FD (Suzhou Antai Air Technology Co., Ltd., Jiangsu, China); biological safety cabinet 1300SERIES A2 (Thermo Corporation, USA); inverted optical microscope (Nikon Corporation, Japan); high-speed centrifuge Centrifuge 5804R (Eppendorf Corporation, Germany); pipette Research plus (Eppendorf Corporation, Germany); PCR instrument C1000 Touch (Bio-Rad Corporation, USA); electrophoresis instrument PowerPac Basic (Bio-Rad Corporation, USA); vertical electrophoresis tank MiniProtean Tetra (Bio-Rad, USA); gel imaging system 2500(R) (Tanon, Shanghai, China); ultrapure water instrument Milli-Q (Millipore, USA); ultra-low temperature refrigerator Forma 994 (Thermo, USA); nucleic acid and protein analyzer Nano Drop 2000 (Thermo, USA); biochemical incubator LRH-250 (Yiheng Scientific Instrument Co., Ltd., Shanghai, China); analytical balance BSA224S (Sartorius, Germany); vortex oscillator (Thermo, USA); Azure Biosystems C600 multifunctional molecular imaging system (Azure Biosystems, USA).

[0022] It should be noted that the primer synthesis and Sanger sequencing work in this application were completed at Suzhou Genewise Biotechnology Co., Ltd.

[0023] Example 1 This embodiment provides a method for preparing a genotype VII NDV recombinant virus expressing IBV S protein, as follows: (1) Obtaining IBV-S gene fragments of different genotypes The virus strains MH20 (isolated from 12-day-old Ephedra broilers in a farm in Penglai, Shandong in 2017) and H120 were inoculated into 10-day-old chicken embryos, and the embryos were cultured in a 37°C constant temperature incubator for 72 hours, and the embryos were observed. The chicken embryos with clearly visible blood vessels were placed in a 4°C refrigerator overnight, and the allantoic fluid was collected. The IBV genomic RNA was extracted using the Trizol extraction method, followed by random primer reverse transcription.

[0024] Using the cDNA of the reverse transcription product of MH20 as a template, primers MH20-S-F1 (SEQ ID NO: 9 in the sequence list) and MH20-S-R1 (SEQ ID NO: 10 in the sequence list) were used to amplify the MH20-S1 fragment with a length of 1620 bp; using the pXJ40-IBV-S vector as a template, primers IBV-S2-F1 (SEQ ID NO: 11 in the sequence list) and IBV-S2-R2 (SEQ ID NO: 12 in the sequence list) were used to amplify the IBV-S2 fragment with a length of 1878 bp; using the MH20-S1 and IBV-S2 fragments as templates, primers MH20-S-F1 (SEQ ID NO: 9 in the sequence list) and IBV-S2-R2 (SEQ ID NO: 12 in the sequence list) were used to amplify the MH20S1-IBVS2 fragment with a length of 3498 bp. The band diagram of the gene fragment MH20S1-IBVS2 after electrophoresis is shown in Figure 1. Figure 3 shown.

[0025] Using the pXJ40-H120-S2P vector as a template, primers H120-S-F1 (SEQ ID NO: 13 in the sequence list) and H120-6P-R1 (SEQ ID NO: 14 in the sequence list) were used to amplify the H120-1 fragment with a length of 2086 bp; using the pXJ40-H120-S2P vector as a template, primers H120-6P-F2 (SEQ ID NO: 15 in the sequence list) and H120-6P-R2 (SEQ ID NO: 16 in the sequence list) were used to amplify the H120-2 fragment with a length of 255 bp; using the pXJ40-H120-S2P vector as a template, primers H120-6P-F3 (SEQ ID NO: 17 in the sequence list) and H120-6P-R3 (SEQ ID NO: 18 in the sequence list) were used to amplify the H120-2 fragment with a length of 255 bp. NO: 18) to amplify the 165bp H120-3 fragment; using the pXJ40-H120-S2P vector as a template, primers H120-6P-F4 (SEQ ID NO: 19 in the sequence list) and H120-SR (SEQ ID NO: 20 in the sequence list) were used to amplify the 1028bp H120-4 fragment; using fragments H120-1, H120-2, H120-3, and H120-4 as templates, primers H120-S-F1 (SEQ ID NO: 13 in the sequence list) and H120-SR (SEQ ID NO: 20 in the sequence list) were used to amplify the 3462bp H120-S6P fragment. The band diagram of the gene fragment H120-S6P after electrophoresis is shown in Figure 1. Figure 4 shown.

[0026] The rDHN3mF-H120-S6P plasmid was used as a template and primers H120-S-F1 (SEQ ID NO: 13 in the sequence list) and GSAS-R1 (SEQ ID NO: 21 in the sequence list) were used to amplify a 1619 bp GSAS-1 fragment; the rDHN3mF-H120-S6P plasmid was used as a template and primers GSAS-F1 (SEQ ID NO: 22 in the sequence list) and H120-SR (SEQ ID NO: 20 in the sequence list) were used to amplify a 1871 bp GSAS-2 fragment; the GSAS-1 fragment and the GSAS-2 fragment were used as templates and primers H120-S-F1 (SEQ ID NO: 13 in the sequence list) and H120-SR (SEQ ID NO: 20 in the sequence list) were used to amplify a 3462 bp H120-S6P-GSAS fragment. The band diagram of the gene fragments GSAS-1 and GSAS-2 after electrophoresis is shown in Figure 2. Figure 5 shown.

[0027] (2) Obtaining F1, F2, and F3 gene fragments Plasmid pBR322-DHN3mF was digested with SmaI enzyme to obtain a 13399 bp F1 fragment; plasmid pBR322-DHN3mF was used as a template and primers FDHN-SmaI-F1 (SEQ ID NO: 7 in the sequence list) and FDHN-R1 (SEQ ID NO: 8 in the sequence list) were used to amplify a 1549 bp F2 fragment; plasmid pBR322-DHN3mF was used as a template and primers FDHN-F2 (SEQ ID NO: 23 in the sequence list) and FDHN-SmaI-R2 (SEQ ID NO: 24 in the sequence list) were used to amplify a 4704 bp F3 fragment; the band diagrams of gene fragments F1, F2, and F3 after electrophoresis were referenced to Figure 1 , 2 , as shown in Figure 6.

[0028] The above PCR reaction conditions are as follows: Reverse transcription: Tiangen FastKing One-step Genomic cDNA First-Strand Synthesis Premix Kit was used to reverse transcribe RNA. The reaction system and PCR setting procedures are shown in Table 1-3.

[0029] Table 1 Reverse transcription reaction system Reagent name volume 5×FastKing-RT superMix 4 μL Total RNA 2 μg RNase-Free ddH2O Make up to 20 μL Table 2 Reverse transcription PCR program settings Reaction temperature Reaction time 42℃ 15 min 95℃ 3 min 4℃ ∞ Table 3 High-fidelity PCR amplification reaction system Reagent name Volume / Mass cDNA / Plasmid / Genomic DNA 1 μL / 30ng / 400ng 2×Phanta Flash Master Mix 25 μL Upstream primer 2 μL Downstream primer 2 μL depc water 20 μL The high-fidelity PCR amplification reaction program was: 95°C 30 s; (denaturation, annealing, extension) × 31: 95°C 15 s, 55°C (annealing temperature was adjusted according to the primers shown) 15 s, 72°C 1 min; 72°C 5 min; cooling at 4°C for several minutes.

[0030] The restriction enzyme system of the above plasmid pBR322-DHN3mF is shown in Table 4 below Table 4 Composition of pBR322-DHN3mF restriction enzyme digestion system Reagent name Mass / Volume pBR322-DHN3mF 4 μg Sma I 4 μL Buffer 5 μL depcwater Make up to 50 μL After the enzyme digestion products and PCR products were purified, ABclonal 2X MultiF Seamless Assembly Mix was used for homologous recombination. The reagents for homologous recombination are shown in Table 5-7 below.

[0031] Table 5 rDHN3mF-MH20S1-IBVS2 reaction system Components Dosage F1 870ng F2 201ng MH20S1-IBVS2 455ng F3 611ng Mix 10 μL DEPC water Make up to 20 μL Table 6 rDHN3mF-H120-S6P reaction system Components Dosage F1 870ng F2 201ng H120-S6P 450ng F3 611ng Mix 10 μL DEPC water Make up to 20 μL Table 7 rDHN3mF-H120-S6P-GSAS reaction system Components Dosage F1 870ng F2 201ng H120-S6P-GSAS 450ng F3 611ng Mix 10 μL DEPC water Make up to 20 μL After the above systems were prepared on ice, they were reacted at 50°C for 50 minutes to obtain the recombinant product. Take 5 μL of the recombinant product and add it to a 0.2 mL sterile PCR tube. After adding 100 μL of competent cells, flick the tube wall to mix it evenly. After mixing, let it stand on ice for 30 min. The metal bath temperature was set to 42°C. After standing for 30 min, the mixture was placed in a 42°C metal bath for heat shock for 45 s. After 45 s, it was immediately placed on ice for 2~3 min. Add 900 μL of anti-SOC-free medium to a 0.2 mL sterile PCR tube and shake the bacteria at 200 rpm and 37°C for 1 h. Take out the bacterial solution, centrifuge at 5000 rpm at room temperature for 5 min, discard 800 μL of the bacterial solution, use the remaining 100 μL of liquid to blow and mix the bacterial solution, and then use a coating rod to apply it to the solid culture medium containing ampicillin antibiotics. After leaving it at room temperature for 2-3 min, turn the solid culture medium upside down and place it in a 37°C constant temperature incubator for overnight culture for 12-18 h.

[0032] The colonies grown on the plate were subjected to colony PCR, and the positive colonies were selected for amplification and culture, and plasmid DNA was extracted. The schematic diagram of the constructed recombinant plasmid is shown in Figure 7-9. 500 ng of the plasmid to be verified was verified with Sma I. The reaction is shown in Table 8 below.

[0033] Table 8 Recombinant plasmid restriction enzyme digestion verification system Components volume Carrier 500 ng Sma I 1 μL Buffer 1 μL Deionized water Make up to 10 μL The electrophoresis gel after enzyme digestion is shown in the figure below. Fig.10 As shown, the plasmids with the correct enzyme digestion were sent for sequencing, and the sequencing results showed that the recombinant plasmids rDHN3mF-MH20S1-IBVS2, rDHN3mF-H120-S6P, and rDHN3mF-H120-S6P-GSAS were successfully constructed. Fig.11 As shown, both the proline and furin sites have been mutated successfully.

[0034] Example 2 Transfection into BHK-T7 cell line and rescue of virus.

[0035] In advance, the BHK-T7 cell line was plated in a 6-well plate. When the cell density reached about 80%-90%, the transfection was performed using Lipomaster 2000 Transfection Reagent from Novozymes according to the instructions. The transfection reagent was prepared as follows: Solution A: Opti-MEM Medium 100μL, Lipomaster 2000 Transfection Reagent 18μL; Solution B: Opti-MEM Medium 100 μL, rDHN3mF-MH20S1-IBVS2 or rDHN3mF-H120-S6P or rDHN3mF-H120-S6P-GSAS 4 μg, pXJ40-NP 2.5 μg, pXJ40-P 1.25 μg, pXJ40-L 1.25 μg; Add liquid B to liquid A, mix gently with a pipette, and let stand at room temperature for 5 min. Then add the AB liquid mixture drop by drop into the culture medium, and gently shake the culture dish to make it evenly dispersed. Culture at 37℃ 5% CO2 for about 72~96 hours, observe the state of the cells under an inverted microscope, and if a large number of cells in the sample group are found to be lesions, put the six-well plate in a -80℃ refrigerator. After repeated freezing and thawing three times, collect the cell freeze-thaw solution into a 1.5 mL sterile ep tube and store it at -80℃ for use. After the virus solution after repeated freezing and thawing is passed through a 0.22 μM filter 1~2 times, take 200 μL of the virus solution and inoculate it into 9~11-day-old SPF chicken embryos for passage, and harvest the chicken embryo allantoic fluid.

[0036] Example 3 Identification of the stability of recombinant viruses (1) Determination of recombinant virus HA Prepare a clean 96-well hemagglutination plate and add 25 μL of PBS buffer to the 96-well plate with a dispenser. Then add 25 μL of the sample to be tested to the first column on the left, and repeat 3 to 4 wells for each sample. Use a dispenser to mix the first column by blowing it up and down, then aspirate 25 μL of the first column into the second column, and mix it by blowing it up and down, and repeat this operation until the eleventh column. Discard the liquid mixed by blowing it up and down from the eleventh column. The last column is the red blood cell control group. Use a dispenser to add 25 μL of 1% red blood cells to the 96-well plate, let it stand at room temperature for 25 to 30 minutes, and then slightly tilt the 96-well hemagglutination plate to facilitate the observation of hemagglutination. Repeat three times for each sample.

[0037] (2) RT-PCR verification of recombinant virus stability Take 200 μL of chicken embryo allantoic fluid in which hemagglutination can be detected in the HA experiment, extract the viral genomic RNA, and amplify the cDNA after RT-PCR. Use primers FF (SEQ ID NO: 25 in the sequence list) / FR (SEQ ID NO: 26 in the sequence list) to amplify the F gene in DHN3mF. Primers IBV-SF (SEQ ID NO: 27 in the sequence list) / IBV-SR (SEQ ID NO: 28 in the sequence list) amplify the inserted S gene. Take 5 μL of the reaction after the PCR reaction and run agarose nucleic acid electrophoresis. The bands with the correct size are sent to GENEWISE for sequencing to check whether there are mutations. The sequencing results show that the inserted IBV-S gene has no mutations and the recombinant virus is relatively stable.

[0038] (3) Western blot verification of recombinant viral protein expression Chicken embryo allantoic fluid that can detect hemagglutination in the HA test was used to infect BHK-21 cells and then the pathological changes were observed after 48 hours, as follows: Fig.13 This is the CPE of BHK-21 cells 48 hours after infection with the recombinant virus rDHN3mF-H120-S6P. Fig.14 This is the CPE of BHK-21 cells infected with the recombinant virus rDHN3mF-H120-S6P-GSAS for 48 hours. Fig.15 The blank control was used. Virus supernatant and cell samples were collected after 48 hours.

[0039] Western blot detection of IBV-S protein expression: Prepare 8% SDS-PAGE gel, pour the prepared fresh 1× electrophoresis solution into the electrophoresis tank, clamp the protein gel, take 8-50 μL sample for spotting, set the electrophoresis conditions to: 80 V, 120 min or 60 V, 180 min. After the electrophoresis, transfer the membrane, the transfer conditions are: 80 V, 120 min. After the transfer, rinse the NC membrane with TBST. After blocking in 5% skim milk for 2 h, wash the membrane with TBST six times, 5 min each time, for a total of 30 min. After pouring out the washing solution, add 1:2000 diluted Anti-IBV-S Rabbit Polyclonal Antibody, pour the liquid covering the NC membrane, then slowly shake overnight on a shaker in a 4℃ refrigerator, wash the membrane six times with TBST, 5 min each time, for a total of 30 min. After washing, add goat anti-rabbit secondary antibody diluted 1:10000, incubate on a shaker at room temperature for 1 h, and wash the membrane six times with TBST after incubation, 5 min each time. Then add equal volumes of ultrasensitive ECL chemiluminescent colorimetric solution A / B, put the NC membrane into a membrane scanner for scanning, and save the image.

[0040] Western blot detection of DHN3mF protein expression: Prepare 10% SDS-PAGE gel, prepare fresh 1× running buffer electrophoresis liquid, pour the electrophoresis liquid into the electrophoresis tank, clamp the protein gel, take 8~50 μL sample for spotting, set the electrophoresis conditions to: 80 V, 120 min or 60V, 180 min. After the electrophoresis, use NC membrane for transfer, the transfer conditions are: 80 V, 120 min. After the transfer, wash the NC membrane with TBST. After blocking in 5% skim milk for 2 h, wash the membrane with TBST six times, 5 min each time, for a total of 30 min. After pouring out the washing liquid, add 1:1000 diluted NDV positive serum, pour the liquid covering the NC membrane, then slowly shake and incubate overnight on a shaker in a 4℃ refrigerator, wash the membrane six times with TBST, 5 min each time, for a total of 30 min. After washing, add rabbit anti-chicken secondary antibody diluted 1:10000, incubate on a shaker at room temperature for 1 h, and wash the membrane six times with TBST after incubation, 5 min each time. Then add equal volumes of ultrasensitive ECL chemiluminescent colorimetric solution A / B, put the NC membrane into a membrane scanner for scanning, and save the image.

[0041] like Fig.16 , 17The figure shows that BHK-21 cells were infected with recombinant viruses rDHN3mF-H120-S6P and rDHN3mF-H120-S6P-GSAS, and the efficient expression of S protein and NDV whole protein was detected in the cell samples and supernatant after 48 hours. Considering the stability of the three recombinant viruses and the number of isolators, the recombinant virus rDHN3mF-H120-S6P was used for subsequent immune challenge experiments.

[0042] Example 4 Immunogenicity experiment of recombinant virus (1) Grouping and immunization experiments of SPF chickens Twenty-eight 9-day-old SPF chickens were randomly divided into 4 groups, with 7 chickens in each group. The first immunization was performed after they were cultured in an isolator for 14 days. As shown in Table 9 below, groups 1 and 2 were inoculated with 0.2 mL PBS solution, and groups 3 and 4 were inoculated with 0.2 mL 106EID50 of the recombinant virus rDHN3mF-H120-S6P. The immunization routes adopted were eye drops and nasal drops. On the 14th day after the first immunization, the chicken embryos of each group were immunized for the second time through the same route and the same dose. After the immunization, the mental state, appetite changes, and defecation of the chickens were observed daily for abnormalities. On the 7th, 14th, 21st, 28th, and 35th days after immunization, 5 chickens were randomly selected from each group to collect subwing venous blood, separate the serum, and detect the production of specific antibodies. The immune attack flow chart is as follows Fig.18 shown.

[0043] Table 9 Immunization grouping and immunization procedure of SPF chickens Group number Immunoassay reagent name Immunization dose Number of SPF chickens Immune pathway 1 PBS 0.2 mL 7 Eye drops, nose drops 2 PBS 0.2 mL 7 Eye drops, nose drops 3 rDHN3mF-H120-S6P 106EID50 7 Eye drops, nose drops 4 rDHN3mF-H120-S6P 106EID50 7 Eye drops, nose drops (2) Determination of body weight of SPF chickens after immunization The body weight of the chickens was measured on days 7, 14, 21, 28, and 35 after immunization. Five chickens were randomly selected from each group for weighing at a consistent time, and the data were processed and analyzed using GraphPad Prism 9.

[0044] Depend on Fig.19 It can be seen that the body weight of each group remained basically the same at the same time, with no significant difference (p>0.05). As the immunization time prolonged, the weight growth rate of each group was basically the same, indicating that the recombinant virus had no obvious effect on the growth of SPF chickens.

[0045] (3) Hemagglutination inhibition test to detect NDV antibodies The hemagglutination inhibition test (HI) was used to detect the titer of NDV antibodies produced by immunized chickens. Four-unit antigen was prepared with DHN3 and verified. It was used in the experiment after verification. Prepare a clean 96-well hemagglutination plate and add 25 μL PBS buffer solution to the plate. Add 25 μL of the sample serum to be tested in the first column, and repeat 3 to 4 wells for each sample. Use a spray gun to mix the first column up and down, then aspirate 25 μL of the first column into the second column, mix it up and down, and repeat this operation until the tenth column. Discard the liquid mixed by mixing up and down from the tenth column. Add 25 μL of the prepared four-unit antigen to the 1st to 11th columns and let it stand at room temperature for 20 to 30 min. Then, add 25 μL of 1% chicken red blood cells to the 96-well plate in mid-air. After standing at room temperature for 20 to 30 min, stand the hemagglutination plate upright, and take the wells where the red blood cells can flow down completely as positive, and record the data.

[0046] like Fig. 20 As shown in the figure, on the 7th day after immunization, NDV antibodies were produced in all experimental groups, and the antibody titers were between 2.6log2-3.4log2. There was no significant difference between the two experimental groups (P>0.05), and the antibody titers of the two experimental groups were significantly higher than those of the PBS group. The NDV antibody level of the SPF chicken group in the experimental group increased with the increase of immunization time and reached a peak on the 21st day after immunization, with an antibody titer between 6.2log2-7.2log2. In summary, it can be shown that after immunization with rDHN3mF-H120-S6P, the chickens can produce a higher level of NDV antibodies, which has a better immune protection effect.

[0047] (4) SPF chicken virus attack grouping 21 days after the second immunization, groups 1 and 3 were named IB group, and groups 2 and 4 were named ND group. The IB group was challenged with the virus by eye drops and nasal drops for 10 6 EID50 M41, attack 10 on ND group 5 EID50 strong strain DHN3. Specific attack conditions are shown in Table 10.

[0048] Table 10 SPF chicken challenge grouping Group No. Immunoassay reagent name Attack strain Challenge dose Number of SPF chickens 1 PBS M41 106EID50 7 2 PBS DHN3 106EID50 7 3 rDHN3mF-H120-S6P M41 106EID50 7 4 rDHN3mF-H120-S6P DHN3 106EID50 7 (5) Detection of detoxification after attacking DHN3 in the ND group On days 3, 5, and 7 after the challenge, throat swabs were collected from chickens in the ND group (groups 2 and 4) to detect NDV excretion. The collected throat swabs were placed in 1 mL of PBS buffer solution containing 1000 U / mL double antibody (precooled at 4°C). After centrifugation at 4°C, 8000 rpm for 5 minutes, the supernatant was taken into a new 1.5 mL centrifuge tube and stored at -80°C for later use. 9-11-day-old Jiankang SPF chicken embryos were taken, and 3 SPF chicken embryos were inoculated for each sample, with 100 μL inoculated for each embryo. After being placed in a 38°C constant temperature incubator for 48 hours, the chicken embryo allantoic fluid was collected for HA experiment. If one of the three chicken embryos has a hemagglutination titer, it is determined that the chicken throat swab contains NDV.

[0049] like Fig.21 As shown, the PBS group could detect NDV excretion in the larynx on days 3, 5, and 7 after the challenge, while the experimental group rDHN3mF-H120-S6P could effectively inhibit NDV excretion on the third day, and no DHN3 excretion was detected in the experimental group as time went on. This preliminarily proves that after the chickens are immunized with rDHN3mF-H120-S6P, they can resist the infection of DHN3 strain to a certain extent, which plays a good protective role for the chickens.

[0050] (6) IB group tested the virus release in the swab after attacking M41 On days 3, 7, and 10 after the challenge, throat swabs and cloacal swabs were collected from chickens in groups IB (groups 1 and 3) to detect the shedding of M41. Fluorescence quantitative PCR was used to detect the shedding of swabs in the IB group, with three replicates for each sample.

[0051] like Fig. 22 It can be seen that the amount of virus excreted from the laryngeal swab and cloacal swab of the PBS group after the virus attack was significantly higher than that of the experimental group, indicating that the chickens can resist the infection of the M41 strain to a certain extent after being immunized with rDHN3mF-H120-S6P.

[0052] (7) Detection of tissue and organ detoxification after IB group attacked M41 In Group IB (Groups 1 and 3), 2-3 SPF chickens were randomly selected for autopsy on days 5, 10, and 14 after infection. The trachea, heart, kidney, spleen, liver, forestomach, lung, small intestine, cecal tonsils, and bursa of Fabricius were ground and then RNA was extracted. After reverse transcription into cDNA, the viral load in tissues and organs was detected by fluorescent quantitative PCR.

[0053] from Fig.23It can be seen that the viral RNA of M41 can be detected in the 10 tissue organs taken from the experimental group and the control group after the virus attack. On the 5th day after the virus attack, the viral load in the trachea, small intestine and bursa of the experimental group rDHN3mF-H120-S6P was lower than that in the PBS group. Except for these three tissue organs, the M41 viral load of the remaining tissue organs on the fifth day was higher than that of the PBS group or not much different from that of the PBS group. On the 10th and 14th days after the virus attack, the viral load in the tissue organs of the experimental group was lower than that of the control group. This shows that after immunization with rDHN3mF-H120-S6P, the chickens can resist the attack of M41 to a certain extent and play a good protective role.

[0054] In summary, according to the above-mentioned method for preparing a genotype VII NDV recombinant virus expressing IBV S protein, the S protein was precisely modified, including replacing S2 of the cell-adapted IBV-P65 with S2 of the non-cell-adapted strain MH20; the six hydrophobic amino acids in the H120 S protein were mutated to proline and the Flynn site was mutated on this basis to enhance the stability and immunogenicity of the pre-fusion S protein, and obtain a recombinant virus that can efficiently express the NDV full protein and S protein. The immune challenge experiment using the recombinant virus rDHN3mF-H120-S6P found that the chickens immunized with the rDHN3mF-H120-S6P vaccine were able to produce sufficient immune efficacy in vivo to resist infection with genotype VII NDV and M41, indicating that rDHN3mF-H120-S6P is expected to become a safe and effective two-in-one candidate vaccine.

[0055] It is known from common technical knowledge that the present invention can be implemented by other embodiments that do not deviate from its spirit or essential features. Therefore, the above disclosed embodiments are only illustrative in all respects and are not exclusive. All changes within the scope of the present invention or within the scope equivalent to the present invention are included in the present invention.

[0056] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, ordinary technicians in the relevant field should understand that the specific implementation methods of the present invention can still be modified or replaced by equivalents. Any modification or equivalent replacement that does not depart from the spirit and scope of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A method for preparing a genotype VII NDV recombinant virus expressing IBV S protein, characterized in that: The method comprises: The modified S gene is inserted between the P gene and the M gene of the pBR322-DHN3mF viral genome to obtain any one of the recombinant viruses rDHN3mF-MH20S1-IBVS2, rDHN3mF-H120-S6P, and rDHN3mF-H120-S6P-GSAS.

2. The method for preparing a genotype VII NDV recombinant virus expressing IBV S protein according to claim 1, characterized in that: The step of inserting a modified S gene between the P gene and the M gene of the pBR322-DHN3mF viral genome comprises: The gene F1 fragment, gene F2 fragment, gene MH20S1-IBVS2 fragment or gene H120-S6P fragment or gene H120-S6P-GSAS fragment and gene F3 fragment are used for homologous recombination to obtain three recombinant plasmids, and the three recombinant plasmids are used for transfecting cells to obtain three recombinant viruses.

3. The method for preparing a genotype VII NDV recombinant virus expressing IBV S protein according to claim 2, characterized in that: The sequence of the gene F1 fragment is shown in SEQ ID NO: 1; The sequence of the gene F2 fragment is shown in SEQ ID NO: 2; The sequence of the gene MH20S1-IBVS2 fragment is shown in SEQ ID NO: 3; The sequence of the gene H120-S6P fragment is shown in SEQ ID NO: 4; The sequence of the gene H120-S6P-GSAS fragment is shown in SEQ ID NO: 5; The sequence of the gene F3 fragment is shown in SEQ ID NO:

6.

4. The method for preparing a genotype VII NDV recombinant virus expressing IBV S protein according to claim 2, characterized in that: The preparation process of the gene F1 fragment is as follows: using pBR322-DHN3mF vector as a template and performing enzyme digestion with SmaI to obtain the F1 fragment; The preparation process of the gene F2 fragment is as follows: using the pBR322-DHN3mF plasmid as a template and using primers FDHN-SmaI-F1 and FDHN-R1 to amplify the F2 fragment; the primers FDHN-SmaI-F1 and FDHN-R1 are shown in the sequence table SEQ ID NO: 7-8; The preparation process of the gene MH20S1-IBVS2 fragment is as follows: using MH20 cDNA as a template and using primers MH20-S-F1 and MH20-S-R1 to amplify the MH20-S1 fragment; using pXJ40-IBV-S vector as a template and using primers IBV-S2-F1 and IBV-S2-R2 to amplify the IBV-S2 fragment; using MH20-S1 and IBV-S2 fragments as templates and using primers MH20-S-F1 and IBV-S2-R2 to amplify the MH20S1-IBVS2 fragment. Primers MH20-S-F1, MH20-S-R1, IBV-S2-F1, and IBV-S2-R2 are shown in the sequence table as SEQ ID NOs: 9 to 12.

5. The method for preparing a genotype VII NDV recombinant virus expressing IBV S protein according to claim 2, characterized in that: The preparation process of the gene H120-S6P fragment is as follows: using pXJ40-H120-S2P plasmid as a template and primers H120-S-F1 and H120-6P-R1 to amplify the H120-1 fragment; using primers H120-6P-F2 and H120-6P-R2 to amplify the H120-2 fragment; using primers H120-6P-F3 and H120-6P-R3 to amplify the H120-3 fragment; using primers H120-6P-F4 and H120-SR to amplify the H120-4 fragment; using fragments H120-1, H120-2, H120-3, and H120-4 as templates and primers H120-S-F1 and H120-SR to amplify the H120-S6P fragment. Primers H120-S-F1, H120-6P-R1, H120-6P-F2, H120-6P-R2, H120-6P-F3, H120-6P-R3, H120-6P-F4, and H120-SR are shown in the sequence listing as SEQ ID NOs: 13 to 20, respectively; The preparation process of the gene H120-S6P-GSAS fragment is as follows: using rDHN3mF-H120-S6P plasmid as a template, using primers H120-S-F1 and GSAS-R1 to amplify the GSAS-1 fragment; using primers GSAS-F1 and H120-SR to amplify the GSAS-2 fragment; The H120-S6P-GSAS fragment was obtained by using the GSAS-1 fragment and the GSAS-2 fragment as templates and primers H120-S-F1 and H120-SR respectively. Primers GSAS-R1 and GSAS-F1 are shown in the sequence listing as SEQ ID NOs: 21 to 22, respectively; The preparation process of the gene F3 fragment is as follows: using pBR322-DHN3mF plasmid as a template and primers FDHN-F2 and FDHN-SmaI-R2 to amplify the F3 fragment; the primers FDHN-F2 and FDHN-SmaI-R2 are shown in the sequence table SEQ ID NO: 23-24 respectively.

6. The method for preparing a genotype VII NDV recombinant virus expressing IBV S protein according to claim 2, characterized in that: The amounts of various substances used in the recombination process were: 870 ng gene fragment F1, 201 ng gene fragment F2, 455 ng gene fragment MH20S1-IBVS2 or 450 ng gene fragment H120-S6P or 450 ng gene fragment H120-S6P-GSAS, 611 ng gene fragment F3, 10 μL ABclonal 2X MultiF Seamless Assembly Mix, ddH2O was added to 20 μL; the reaction conditions were 50°C for 50 minutes.

7. The method for preparing a genotype VII NDV recombinant virus expressing IBV S protein according to claim 1, characterized in that: During the transfection process, the transfected cells were BHK-T7 cell lines, and the components of the transfection reagent were as follows: Solution A: Opti-MEM Medium 100μL, Lipomaster 2000 Transfection Reagent 18μL; Solution B: Opti-MEM Medium 100μL, rDHN3mF-MH20S1-IBVS2 or rDHN3mF-H120-S6P; rDHN3mF-H120-S6P-GSAS 4μg, pXJ40-NP 2.5μg, pXJ40-P 1.25μg, pXJ40-L 1.25μg.

8. A genotype VII NDV recombinant virus expressing IBV S protein, characterized in that: Obtained according to the method for preparing a genotype VII NDV recombinant virus expressing the IBV S protein according to any one of claims 1 to 7.

9. Use of the genotype VII NDV recombinant virus expressing IBV S protein as claimed in claim 8 in the preparation of a vaccine.

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