Genotype VII NDV recombinant virus expressing IBV S protein and its preparation method and application

Abstract

This invention provides a recombinant NDV genotype VII expressing the IBV S protein, its preparation method, and its application. The method includes inserting a modified S gene 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. This invention rescues the recombinant virus rDHN3mF-MH20S1-IBVS2 by replacing the S2 region of the adapted IBV-P65 cell line with the S2 region of QX-type IBV MH20; rescues the recombinant virus rDHN3mF-H120-S6P by mutating the hydrophobic amino acids at positions 692, 768, 772, 815, 862, and 863 of the H120-S protein to proline; and rescues the recombinant virus rDHN3mF-H120-S6P-GSAS by mutating six hydrophobic amino acids to proline and then mutating amino acids at positions 533-537 to glycine, serine, alanine, and serine respectively. The three rescued recombinant viruses were then used to infect BHK-21 cells, and Western blotting confirmed the high-efficiency expression of the S protein and the complete NDV protein.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology design, and in particular to a recombinant NDV genotype VII virus expressing IBV S protein, its preparation method, and its application. Background Technology

[0002] Infectious bronchitis (IB) is a highly contagious disease of chickens caused by the avian infectious bronchitis virus (IBV), affecting the respiratory, reproductive, and urogenital tracts. The disease typically causes respiratory symptoms such as increased nasal and ocular discharge, wheezing, coughing, and sneezing. It can also cause kidney dysfunction and decreased egg production through infection of the urogenital tract. Mortality rates are high in chicks, especially after IBV infection, which often leads to other secondary complications, severely impacting the production performance of both egg-laying and meat-producing poultry, resulting in significant economic losses for the poultry industry.

[0003] The S protein is the largest structural protein in IBV, with a molecular weight of approximately 128-160 kDa before glycosylation and reaching 150-200 kDa after glycosylation. The S protein contains viral neutralizing epitopes and is a major protective antigen for the host. The continuous evolution and mutation of the S protein pose a significant challenge to vaccine development. To date, nine genotypes have been identified worldwide based on the S1 gene, with the QX, Mass, and TW genotypes being the most prevalent. The QX genotype is currently the most prevalent strain in China. However, the IBV vaccine strain currently used in China is primarily a Mass-type attenuated vaccine, which cannot provide sufficient protection against the emerging IBV variants in recent years. Therefore, developing a novel IBV vaccine targeting different serotypes is urgently needed.

[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 transition from a metastable to a hyperstable conformation. Maintaining the pre-fusion metastable conformation is beneficial for improving the stability of the S protein and increasing its immunogenicity. Existing research indicates that mutating 2 or 6 specific amino acids in the SARS-CoV-2 S protein to proline or mutating the furin site helps to form a metastable conformation of the S protein.

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

[0006] The purpose of this invention is to provide a recombinant NDV genotype VII expressing the IBV S protein, its preparation method, and its application. The recombinant virus rDHN3mF-MH20S1-IBVS2 is rescued by replacing the S2 region of the adapted cell line IBV-P65 with the S2 region of 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. Furthermore, the recombinant virus rDHN3mF-H120-S6P-GSAS is rescued by mutating six hydrophobic amino acids to proline and then sequentially mutating amino acids at positions 533-537 to glycine, serine, alanine, and serine. The three rescued recombinant viruses were used to infect BHK-21 cells, and Western blot analysis confirmed the efficient expression of the S protein and the full-length NDV protein.

[0007] In a first aspect, the present invention provides a method for preparing a recombinant NDV genotype VII virus expressing the IBV S protein, the method comprising:

[0008] By inserting a modified S gene between the P and M genes in the pBR322-DHN3mF viral genome, any one of the recombinant viruses rDHN3mF-MH20S1-IBVS2, rDHN3mF-H120-S6P, and rDHN3mF-H120-S6P-GSAS can be obtained.

[0009] Further, the step of inserting the modified S gene between the P gene and the M gene in the pBR322-DHN3mF viral genome includes:

[0010] Three recombinant plasmids were obtained by homologous recombination of 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. The three recombinant plasmids were then transfected into cells to obtain three recombinant viruses.

[0011] Furthermore, the sequence of the F1 gene fragment is shown in SEQ ID NO: 1;

[0012] The sequence of the F2 fragment of the gene is shown in SEQ ID NO: 2;

[0013] The sequence of the MH20S1-IBVS2 gene fragment is shown in SEQ ID NO: 3;

[0014] The sequence of the H120-S6P fragment of the gene is shown in SEQ ID NO: 4;

[0015] The sequence of the gene H120-S6P-GSAS fragment is shown in SEQ ID NO: 5;

[0016] The sequence of the F3 fragment of the gene is shown in SEQ ID NO: 6.

[0017] Furthermore, the preparation process of the F1 fragment of the gene is as follows: using the pBR322-DHN3mF vector as a template, the F1 fragment is obtained by digestion with SmaI.

[0018] The preparation process of the F2 fragment of the gene is as follows: using pBR322-DHN3mF plasmid as a template, the F2 fragment is amplified using primers FDHN-SmaI-F1 and FDHN-R1; primers FDHN-SmaI-F1 and FDHN-R1 are shown in the sequence listing SEQ ID NO: 7-8;

[0019] The preparation process of the MH20S1-IBVS2 gene fragment is as follows: using MH20 cDNA as a template, the MH20-S1 fragment is amplified using primers MH20-S-F1 and MH20-S-R1; using the pXJ40-IBV-S vector as a template, the IBV-S2 fragment is amplified using primers IBV-S2-F1 and IBV-S2-R2; using the MH20-S1 and IBV-S2 fragments as templates, the MH20S1-IBVS2 fragment is amplified using primers MH20-S-F1 and IBV-S2-R2. Primers MH20-S-F1, MH20-S-R1, IBV-S2-F1, and IBV-S2-R2 are shown in SEQ ID NO: 9-12 of the sequence listing.

[0020] Further, the preparation process of the gene H120-S6P fragment is as follows: using pXJ40-H120-S2P plasmid as a template, the H120-1 fragment is amplified using primers H120-S-F1 and H120-6P-R1; the H120-2 fragment is amplified using primers H120-6P-F2 and H120-6P-R2; the H120-3 fragment is amplified using primers H120-6P-F3 and H120-6P-R3; the H120-4 fragment is amplified using primers H120-6P-F4 and H120-SR; and the H120-S6P fragment is amplified using fragments H120-1, H120-2, H120-3, and H120-4 as templates using primers H120-S-F1 and H120-SR. 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 SEQ ID NO: 13-20, respectively;

[0021] The preparation process of the H120-S6P-GSAS fragment is as follows: using the rDHN3mF-H120-S6P plasmid as a template, the GSAS-1 fragment is amplified using primers H120-S-F1 and GSAS-R1; the GSAS-2 fragment is amplified using primers GSAS-F1 and H120-SR; and the H120-S6P-GSAS fragment is amplified using the GSAS-1 and GSAS-2 fragments as templates using primers H120-S-F1 and H120-SR, respectively. Primers GSAS-R1 and GSAS-F1 are shown in SEQ ID NO: 21-22 of the sequence listing, respectively.

[0022] The preparation process of the F3 fragment of the gene is as follows: using pBR322-DHN3mF plasmid as a template, the F3 fragment is amplified with primers FDHN-F2 and FDHN-SmaI-R2; primers FDHN-F2 and FDHN-SmaI-R2 are shown in the sequence listing SEQ ID NO: 23-24, respectively.

[0023] Furthermore, the amounts of each substance used in the recombination process were as follows: 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, and ddH2O was added to 20 μL; the reaction conditions were 50℃ for 50 minutes.

[0024] Furthermore, during the transfection process, the transfected cells were the BHK-T7 cell line, and the components of the transfection reagent were as follows:

[0025] Solution A: Opti-MEM Medium 100μL, Lipomaster 2000 Transfection Reagent 18μL;

[0026] 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.

[0027] Secondly, the present invention also proposes a genotype VII NDV recombinant virus expressing the IBV S protein, which is obtained according to the above-described method for preparing the genotype VII NDV recombinant virus expressing the IBV S protein.

[0028] Thirdly, this invention also proposes the application of a type VII NDV recombinant virus expressing the IBV S protein in the preparation of vaccines.

[0029] Compared with the prior art, the present invention has the following advantages:

[0030] By modifying the IBV S protein of different genotypes, including replacing the S2 of the cell-adapted IBV-P65 with the S2 of the non-cell-adapted MH20, mutating six hydrophobic amino acids in the H120 S protein to proline, and further mutating the furin site, the rescued recombinant virus is more stable than the recombinant strain with the inserted unmodified S protein. These three recombinant viruses are expected to be further developed into bivalent vaccines against avian bronchitis virus and Newcastle disease virus, providing a new defense against avian bronchitis virus and Newcastle disease virus infection. Attached Figure Description

[0031] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0032] Figure 1 The image shows a gel electrophoresis diagram of the F1 gene fragment, where M represents the DNA Marker and 1 represents the F1 PCR fragment (13399 bp).

[0033] Figure 2This is a gel electrophoresis image of the F2 gene fragment, where M: DNA Marker; 1 is the F2 PCR fragment (1549bp).

[0034] Figure 3 The image shows a gel electrophoresis diagram of the MH20S1-IBVS2 gene fragment, where M represents the DNA Marker and 1 represents the MH20S1-IBVS2 PCR fragment (3498 bp).

[0035] Figure 4 The image shows a gel electrophoresis diagram of the H120-S6P gene fragment, where M represents the DNA Marker and 1 represents the H120-S6P overlap PCR fragment (3462 bp).

[0036] Figure 5 Gel electrophoresis image of H120-S6P-GSAS gene fragment; where M: DNA Marker; 1 is GSAS-1 PCR fragment (1619bp), and 2 is GSAS-2 PCR fragment (1871bp).

[0037] Figure 6 This is a gel electrophoresis image of the F3 gene fragment; where M: DNA Marker; 1 is the F3 PCR fragment (4704bp).

[0038] Figure 7 Schematic diagram of the rDHN3mF-MH20S1-IBVS2 plasmid;

[0039] Figure 8 Schematic diagram of rDHN3mF-H120-S6P plasmid;

[0040] Figure 9 Schematic diagram of rDHN3mF-H120-S6P-GSAS plasmid;

[0041] Figure 10 This is a diagram showing the enzyme digestion and identification of recombinant plasmids, where M: DNA Marker; 1: recombinant plasmid rDHN3mF-MH20S1-IBVS2; 2: recombinant plasmid rDHN3mF-H120-S6P; 3: recombinant plasmid rDHN3mF-H120-S6P-GSAS;

[0042] Figure 11 This is a schematic diagram of the sequencing results of the H120-S gene in the recombinant plasmid after mutation at the proline and furin sites. In the diagram, a is the sequencing result of the recombinant plasmid rDHN3mF-H120-S6P; b is the sequencing result of rDHN3mF-H120-S6P-GSAS.

[0043] Figure 12A schematic diagram illustrating the expression of the S protein of recombinant virus rDHN3mF-MH20S1-IBVS2 in BHK-21 cells after passage in chicken embryos at different dilutions. Here, 10⁻² represents passage in chicken embryos after a 100-fold virus dilution, 10⁻⁴ represents passage in chicken embryos after a 10,000-fold virus dilution, and 10⁻⁶ represents passage in chicken embryos after a 1,000,000-fold virus dilution; 1, 2, and 3 represent passage in different chicken embryos.

[0044] Figure 13 Enlarged image of BHK-21 cells infected with allantoic fluid from recombinant virus rDHN3mF-H120-S6P;

[0045] Figure 14 Enlarged image of BHK-21 cells infected with recombinant virus rDHN3mF-H120-S6P-GSAS allantoic fluid;

[0046] Figure 15 Enlarged view of BHK-21 cells infected with SPF chicken embryo allantoic fluid;

[0047] Figure 16 This is a schematic diagram showing the expression of the full-length NDV protein of the recombinant virus rDHN3mF-H120-S in BHK-21 cells.

[0048] Figure 17 A schematic diagram showing the expression of the S protein of the recombinant virus rDHN3mF-H120-S in BHK-21 cells;

[0049] Figure 18 This is a schematic diagram of the immune challenge procedure;

[0050] Figure 19 This is a schematic diagram of weight gain in chickens after immunization, where ns represents no significant difference;

[0051] Figure 20 This is a schematic diagram showing the detection results of NDV-specific antibodies in chicken serum after immunization. ** indicates a significant difference (p < 0.01); *** indicates an extremely significant difference (p < 0.001). Blood samples were collected from 5 chickens in each group, with 3 replicates for each serum sample.

[0052] Figure 21 This diagram illustrates the NDV shedding in the throat of SPF chickens after challenge, where n / m represents the number of positive samples / total number of samples.

[0053] Figure 22Figure 1 shows the IBV shedding status of SPF chicken swabs after challenge with M41. Figure 2 shows the viral shedding status of SPF chicken throat swabs after M41 challenge; Figure 3 shows the viral shedding status of chicken cloacal swabs after M41 challenge. * indicates a significant difference (p < 0.05); ** indicates a significant difference (p < 0.01); *** indicates an extremely significant difference (p < 0.001).

[0054] Figure 23 This diagram illustrates the IBV shedding in the tissues and organs of SPF chickens after challenge. Here, ns represents no significant difference; * represents a significant difference (p < 0.05); ** represents a significant difference (p < 0.01); and *** represents an extremely significant difference (p < 0.001). Detailed Implementation

[0055] The present invention will now be described in detail with reference to the accompanying drawings and embodiments. It should be noted that, unless otherwise specified, the embodiments and features described herein can be combined with each other.

[0056] The following detailed description is exemplary and intended to provide further detailed explanation of the invention. Unless otherwise specified, all technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used in this invention is for describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention.

[0057] Main reagents and consumables:

[0058] TransZol-up, DNA marker, nucleic acid dyes, and 6× Loading Buffer were purchased from Beijing TransGen Biotech Co., Ltd. 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 (7.5 x 10 cm), 1.0 M Tris-HCl pH 13 6.8, ST768-500 mL, and 10% sodium dodecyl sulfate solution were purchased from Shanghai Beyotime 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), and horseradish peroxidase-labeled goat anti-rabbit IgG (H+L) antibody were purchased from Beijing TransGen Biotech Co., Ltd. Primary antibody Anti-IBV-S Rabbit Polyclonal Antibody was a laboratory-preserved antibody. Lipomaster 2000 Transfection Reagent was purchased from Nanjing Novizan Biotechnology Co., Ltd. SV30010 penicillin-streptomycin bispecific antibody was purchased from Cytiva. 10cm², six-well plates, T25, etc., were all purchased from Guangzhou Jetech Co., Ltd. Plasmids pXJ40-IBV-S, pXJ40-H120-S2P, and pBR322-DHN3mF are laboratory-preserved plasmids.

[0059] Main instruments:

[0060] Electric thermostatic incubator HZ-100 (Yiheng Scientific Instruments Co., Ltd., Shanghai, China); Three-hole electric thermostatic water bath DK-8D (Yiheng Scientific Instruments Co., Ltd., Shanghai, China); Haier BCD-579WE refrigerator (Haier, Shanghai, China); CO2 thermostatic incubator Forma 371 (Thermo Scientific, USA); Clean bench SW-CJ-2FD (Suzhou Antai Air Technology Co., Ltd., Jiangsu, China); Biosafety cabinet 1300SERIES A2 (Thermo Scientific, USA); Inverted optical microscope (Nikon, Japan); High-speed centrifuge Centrifuge 5804R (Eppendorf, Germany); Pipettes Research plus (Eppendorf, Germany); PCR instrument C1000 Touch (Bio-Rad, USA); Electrophoresis apparatus PowerPac Basic (Bio-Rad, USA); Vertical electrophoresis tank MiniProtean Tetra (Bio-Rad, USA); Gel Imaging System 2500(R) (Tanon, Shanghai, China); Milli-Q Ultrapure Water System (Millipore, USA); Forma 994 Ultra-low Temperature Freezer (Thermo Scientific, USA); Nano Drop 2000 Nucleic Acid and Protein Analyzer (Thermo Scientific, USA); LRH-250 Biochemical Incubator (Yiheng Scientific Instruments Co., Ltd., Shanghai, China); BSA224S Analytical Balance (Sartorius, Germany); Vortex Oscillator (Thermo Scientific, USA); Azure Biosystems C600 Multifunctional Molecular Imaging System (Azure Biosystems, USA).

[0061] It should be noted that the primer synthesis and Sanger sequencing work in this application were both completed at Suzhou Genewiz Biotechnology Co., Ltd.

[0062] Example 1

[0063] This embodiment provides a method for preparing a recombinant NDV genotype VII virus expressing the IBV S protein, as detailed below:

[0064] (1) Obtaining IBV-S gene fragments of different genotypes

[0065] Strains MH20 (isolated from 12-day-old Ephedra broilers at a farm in Penglai, Shandong Province in 2017) and H120 were inoculated into 10-day-old chicken embryos and cultured at 37°C for 72 hours. The embryos were then observed. After the embryos with clearly visible blood vessels were placed in a 4°C refrigerator overnight, the allantoic fluid was collected, and IBV genomic RNA was extracted using the Trizol extraction method, followed by random primer reverse transcription.

[0066] Using the cDNA reverse transcription product of MH20 as a template, the 1620bp MH20-S1 fragment was amplified using primers MH20-S-F1 (SEQ ID NO: 9) and MH20-S-R1 (SEQ ID NO: 10). Using the pXJ40-IBV-S vector as a template, the 1878bp IBV-S2 fragment was amplified using primers IBV-S2-F1 (SEQ ID NO: 11) and IBV-S2-R2 (SEQ ID NO: 12). Using the MH20-S1 and IBV-S2 fragments as templates, the 3498bp MH20S1-IBVS2 fragment was amplified using primers MH20-S-F1 (SEQ ID NO: 9) and IBV-S2-R2 (SEQ ID NO: 12). The banding pattern of the MH20S1-IBVS2 gene fragment after electrophoresis is shown in the image. Figure 3 As shown.

[0067] Using the pXJ40-H120-S2P vector as a template, primers H120-S-F1 (SEQ ID NO: 13) and H120-6P-R1 (SEQ ID NO: 14) were used to amplify the 2086 bp H120-1 fragment; using the pXJ40-H120-S2P vector as a template, primers H120-6P-F2 (SEQ ID NO: 15) and H120-6P-R2 (SEQ ID NO: 16) were used to amplify the 255 bp H120-2 fragment; using the pXJ40-H120-S2P vector as a template, primers H120-6P-F3 (SEQ ID NO: 17) and H120-6P-R3 (SEQ ID NO: 18) were used to amplify the 2086 bp H120-1 fragment. NO: 18) Amplified the 165bp H120-3 fragment; using the pXJ40-H120-S2P vector as a template, amplified the 1028bp H120-4 fragment using primers H120-6P-F4 (SEQ ID NO: 19) and H120-SR (SEQ ID NO: 20); using fragments H120-1, H120-2, H120-3, and H120-4 as templates, amplified the 3462bp H120-S6P fragment using primers H120-S-F1 (SEQ ID NO: 13) and H120-SR (SEQ ID NO: 20). See the electrophoresis band diagram of the H120-S6P gene fragment for reference. Figure 4 As shown.

[0068] Using the rDHN3mF-H120-S6P plasmid as a template, primers H120-S-F1 (SEQ ID NO: 13) and GSAS-R1 (SEQ ID NO: 21) were used to amplify the 1619 bp GSAS-1 fragment; using the rDHN3mF-H120-S6P plasmid as a template, primers GSAS-F1 (SEQ ID NO: 22) and H120-SR (SEQ ID NO: 20) were used to amplify the 1871 bp GSAS-2 fragment; using the GSAS-1 and GSAS-2 fragments as templates, primers H120-S-F1 (SEQ ID NO: 13) and H120-SR (SEQ ID NO: 20) were used to amplify the 3462 bp H120-S6P-GSAS fragment. See the electrophoresis band diagrams of gene fragments GSAS-1 and GSAS-2 for reference. Figure 5 As shown.

[0069] (2) Obtaining F1, F2, and F3 gene fragments

[0070] The plasmid pBR322-DHN3mF was digested with SmaI enzyme to obtain the 13399bp F1 fragment. Using pBR322-DHN3mF as a template, the 1549bp F2 fragment was amplified using primers FDHN-SmaI-F1 (SEQ ID NO: 7) and FDHN-R1 (SEQ ID NO: 8). Using pBR322-DHN3mF as a template, the 4704bp F3 fragment was amplified using primers FDHN-F2 (SEQ ID NO: 23) and FDHN-SmaI-R2 (SEQ ID NO: 24). The banding patterns of gene fragments F1, F2, and F3 after electrophoresis are shown in the image below. Figure 1 , 2 As shown in Figure 6.

[0071] The PCR reaction conditions described above are as follows:

[0072] Reverse transcription was performed using the Tiangen FastKing one-step premixed kit for first-strand synthesis of genomic cDNA. The reaction system and PCR setup are shown in Table 1-3.

[0073] Table 1 Reverse transcription reaction system

[0074] Reagent Name volume 5×FastKing-RT superMix 4 μL Total RNA 2 μg RNase-Free ddH2O To bring the volume up to 20 μL

[0075] Table 2 Reverse Transcription PCR Program Settings

[0076] Reaction temperature reaction time 42℃ 15 min 95℃ 3 min 4℃ ∞

[0077] Table 3 High-fidelity PCR amplification reaction system

[0078] 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

[0079] The high-fidelity PCR amplification reaction program is as follows: 95℃ for 30 s; (denaturation, annealing, extension) × 31: 95℃ for 15 s, 55℃ (annealing temperature adjusted according to the primers shown) for 15 s, 72℃ for 1 min; 72℃ for 5 min; cool at 4℃ for several minutes.

[0080] The enzyme digestion system of the above plasmid pBR322-DHN3mF is shown in Table 4 below.

[0081] Table 4 Composition of pBR322-DHN3mF enzyme digestion system

[0082] Reagent Name mass / volume pBR322-DHN3mF 4 μg Sma I 4 μL Buffer 5 μL depcwater Make up to 50 μL

[0083] After purification of the enzyme digestion products and PCR products, homologous recombination was performed using ABclonal 2X MultiF Seamless Assembly Mix. The reagents used for homologous recombination are shown in Table 5-7 below.

[0084] Table 5. Reaction system of rDHN3mF-MH20S1-IBVS2

[0085] Components Dosage F1 870ng F2 201ng MH20S1-IBVS2 455ng F3 611ng Mix 10 μL DEPC water Make up to 20 μL

[0086] Table 6. Reaction system of rDHN3mF-H120-S6P

[0087] Components Dosage F1 870ng F2 201ng H120-S6P 450ng F3 611ng Mix 10 μL DEPC water Make up to 20 μL

[0088] Table 7. Reaction system of rDHN3mF-H120-S6P-GSAS

[0089] Components Dosage F1 870ng F2 201ng H120-S6P-GSAS 450ng F3 611ng Mix 10 μL DEPC water Make up to 20 μL

[0090] After preparing the above systems on ice, incubate at 50°C for 50 minutes to obtain recombinant products. Add 5 μL of the recombinant product to a 0.2 mL sterile PCR tube, add 100 μL of competent cells, gently tap the tube wall to mix, and incubate on ice for 30 min. Set the metal bath temperature to 42°C, and heat-shock the mixture after 30 min in the 42°C metal bath for 45 s. Immediately after 45 s, place it on ice for 2–3 min. Add 900 μL of antibiotic-free SOC medium to a 0.2 mL sterile PCR tube and incubate at 200 rpm and 37°C for 1 h. Remove the bacterial culture, centrifuge at 5000 rpm for 5 min at room temperature, discard 800 μL of bacterial culture, mix the bacterial culture with the remaining 100 μL of liquid by pipetting, and spread it onto a solid culture medium containing ampicillin antibiotic using a spreader. After standing at room temperature for 2-3 min, invert the solid culture medium and place it in a 37℃ constant temperature incubator for overnight incubation for 12-18 h.

[0091] Colony PCR was performed on the colonies grown on the plates. Positive colonies were selected for amplification and culture, and plasmid DNA was extracted. A schematic diagram of the constructed recombinant plasmid is shown in Figure 7-9. 500 ng of the plasmid to be validated was used for validation with Sma I. The reaction results are shown in Table 8 below.

[0092] Table 8 Recombinant plasmid enzyme digestion verification system

[0093] Components volume carrier 500 ng Sma I 1 μL Buffer 1 μL Deionized water Make up to 10 μL

[0094] Electrophoresis gel image after enzyme digestion is shown below. Figure 10As shown, the correctly digested plasmids were sent for sequencing. The sequencing results showed that the recombinant plasmids rDHN3mF-MH20S1-IBVS2, rDHN3mF-H120-S6P, and rDHN3mF-H120-S6P-GSAS were successfully constructed. Figure 11 As shown, both the proline and furin sites have been successfully mutated.

[0095] Example 2: Transfection into BHK-T7 cell line to rescue the virus.

[0096] Beforehand, seed the BHK-T7 cell line into 6-well plates. Once the cell density reaches approximately 80%-90%, transfect the cells using Novizan's Lipomaster 2000 Transfection Reagent according to the manufacturer's instructions. The transfection reagent is prepared as follows:

[0097] Solution A: Opti-MEM Medium 100μL, Lipomaster 2000 Transfection Reagent 18μL;

[0098] Solution B: Opti-MEM Medium 100μL, rDHN3mF-MH20S1-IBVS2 or rDHN3mF-H120-S6P or rDHN3mF-H120-S6P-GSAS 4μg, pXJ40-NP 2.5μg, pXJ40-P 1.25μg, pXJ40-L 1.25μg;

[0099] Add solution B dropwise to solution A, gently mix with a pipette, and let stand at room temperature for 5 minutes. Then, add the AB mixture dropwise to the culture medium, gently shaking the culture dish to disperse it evenly. Incubate at 37℃ and 5% CO2 for approximately 72–96 hours. Observe the cell state under an inverted microscope. If a large number of cells in the sample group show pathological changes, place the six-well plate in a -80℃ freezer. After three freeze-thaw cycles, collect the cell freeze-thaw solution into a 1.5 mL sterile EP tube and store it at -80℃ for later use. After passing the repeatedly freeze-thawed virus solution 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.

[0100] Example 3: Identification of the stability of the recombinant virus

[0101] (1) HA assay of recombinant virus

[0102] Prepare a clean 96-well blood coagulation plate and add 25 μL of PBS buffer to each well using a pipette. Then, add 25 μL of the sample to be tested to the first well on the left, repeating this process 3-4 times per sample. Mix the first well by pipetting up and down with a pipette. Then, transfer 25 μL from the first well to the second well, mixing again by pipetting up and down. Repeat this process until the eleventh well. Discard the liquid from the eleventh well. The last well is the red blood cell control group. Add 25 μL of 1% red blood cells to the 96-well plate using a pipette. After incubating at room temperature for 25-30 min, slightly tilt the 96-well blood coagulation plate to facilitate observation of blood coagulation. Repeat this process three times for each sample.

[0103] (2) RT-PCR verification of recombinant virus stability

[0104] 200 μL of chicken embryo allantoic fluid (detectable by hemagglutination in HA assay) was used to extract viral genomic RNA, which was then amplified by RT-PCR. The F gene in DHN3mF was amplified using primers FF (SEQ ID NO: 25) / FR (SEQ ID NO: 26). The inserted S gene was amplified using primers IBV-SF (SEQ ID NO: 27) / IBV-SR (SEQ ID NO: 28). 5 μL of the PCR reaction mixture was run on agarose gel electrophoresis. Bands with the correct size were sent to Genewiz for sequencing to check for mutations. Sequencing results showed that the inserted IBV-S gene was free of mutations, indicating that the recombinant virus was relatively stable.

[0105] (3) Western blot verification of recombinant viral protein expression

[0106] BHK-21 cells were infected with allantoic fluid from chicken embryos (where hemagglutination was detected in the HA assay) for 48 hours, and the lesions were observed as follows: Figure 13 CPE of BHK-21 cells 48 h after infection with recombinant virus rDHN3mF-H120-S6P. Figure 14 CPE of BHK-21 cells 48 h after infection with recombinant virus rDHN3mF-H120-S6P-GSAS. Figure 15 This serves as a blank control. Viral supernatant and cell samples were collected after 48 hours.

[0107] Western blot analysis of IBV-S protein expression:

[0108] Prepare an 8% SDS-PAGE gel. Pour the freshly prepared 1× electrophoresis buffer into the electrophoresis tank, clamp the protein gel, and spot 8-50 μL of sample. Set the electrophoresis conditions to 80 V for 120 min or 60 V for 180 min. After electrophoresis, perform membrane transfer at 80 V for 120 min. After transfer, rinse the NC membrane once with TBST. Block in 5% skim milk for 2 h, then wash the membrane six times with TBST for 5 min each time, for a total of 30 min. Discard the washing solution, add 1:2000 diluted Anti-IBV-S Rabbit Polyclonal Antibody, and pour in enough liquid to cover the NC membrane. Incubate overnight on a shaker at 4°C with gentle shaking. Wash the membrane six times with TBST for 5 min each time, for a total of 30 min. After washing the membrane, add goat anti-rabbit secondary antibody diluted 1:10000 and incubate on a shaker at room temperature for 1 h. After incubation, wash the membrane six times with TBST for 5 min each time. Then add an equal volume of ultrasensitive ECL chemiluminescence developing solution A / B, place the NC membrane in a membrane scanning instrument for scanning, and save the image.

[0109] Western blot analysis of DHN3mF full protein expression:

[0110] Prepare a 10% SDS-PAGE gel and a fresh 1× running buffer for electrophoresis. Pour the electrophoresis buffer into the electrophoresis tank, clamp the protein gel, and load 8–50 μL of sample. Set the electrophoresis conditions to 80 V for 120 min or 60 V for 180 min. After electrophoresis, transfer the NC membrane using an NC membrane at 80 V for 120 min. After transfer, wash the NC membrane once with TBST. Block the membrane in 5% skim milk for 2 h, then wash it six times with TBST for 5 min each time, for a total of 30 min. Discard the washing solution, add 1:1000 diluted NDV positive serum, and pour in enough liquid to cover the NC membrane. Incubate overnight at 4°C on a shaker with gentle shaking, then wash the membrane six times with TBST for 5 min each time, for a total of 30 min. After washing the membrane, rabbit anti-chicken secondary antibody diluted 1:10000 was added, and the membrane was incubated on a shaker at room temperature for 1 h. After incubation, the membrane was washed six times with TBST for 5 min each time. Then, an equal volume of ultrasensitive ECL chemiluminescence developing solution A / B was added, and the NC membrane was placed in a membrane scanning instrument for scanning. The image was then saved.

[0111] like Figure 16 , 17The image shows that after infecting BHK-21 cells with recombinant viruses rDHN3mF-H120-S6P and rDHN3mF-H120-S6P-GSAS, respectively, high-efficiency expression of S protein and NDV whole protein was detected in both cell samples and supernatant after 48 hours. Considering the stability and isolator quantity of the three recombinant viruses, recombinant virus rDHN3mF-H120-S6P was used for subsequent immune challenge experiments.

[0112] Example 4 Immunogenicity test of recombinant virus

[0113] (1) Grouping and Immunization Experiments of SPF Chickens

[0114] Twenty-eight 9-day-old SPF chickens were randomly divided into four groups of seven chickens each. After being cultured in isolators until 14 days of age, they underwent their first immunization, as shown in Table 9. Groups 1 and 2 were inoculated with 0.2 mL of PBS solution, while groups 3 and 4 were inoculated with 0.2 mL of 10⁶ EID⁵⁰ recombinant viral rDHN3mF-H120-S6P via eye drops and nasal drops. On day 14 after the first immunization, each group of chicken embryos underwent a second immunization via the same route and dose. After immunization, the chickens' mental state, appetite, and defecation were observed daily for any abnormalities. On days 7, 14, 21, 28, and 35 post-immunization, five chickens from each group were randomly selected to collect blood from the subwing venous vein. Serum was separated, and the production of specific antibodies was detected. The immunization challenge flowchart is as follows. Figure 18 As shown.

[0115] Table 9. SPF Chicken Immunization Groups and Immunization Schedules

[0116] Grouping label Immunoassay reagent name Immunization dose SPF chicken quantity / each 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

[0117] (2) Determination of body weight of SPF chickens after immunization

[0118] Chicken body weight was measured at 7, 14, 21, 28, and 35 days post-immunization. Five chickens were randomly selected from each group for each weighing, and the weighing time was kept consistent. The data were processed and analyzed using GraphPad Prism 9.

[0119] Depend on Figure 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 increased, the weight gain of each group was also basically the same, indicating that the recombinant virus had no significant effect on the growth of SPF chickens.

[0120] (3) Detection of NDV antibodies by hemagglutination inhibition test

[0121] The hemagglutination inhibition (HI) assay was used to detect the NDV antibody titer level in immunized chicken flocks. A four-unit antigen was prepared using DHN3 and validated before being used in the experiment. A clean 96-well hemagglutination plate was prepared, and 25 μL of PBS buffer was added to the plate. 25 μL of serum sample was added to the first well, with 3-4 replicates per sample. The first well was mixed by pipetting up and down using a pipette. Then, 25 μL of the first well was transferred to the second well, and the mixture was repeated until the tenth well. The liquid from the tenth well was discarded. 25 μL of the prepared four-unit antigen was added to wells 1 through 11, and the plate was incubated at room temperature for 20-30 min. Then, 25 μL of 1% chicken red blood cells were added dropwise to each well, and the plate was incubated at room temperature for 20-30 min. The plate was then held upright, and the wells from which the red blood cells could flow completely were considered positive. The data were recorded.

[0122] like Figure 20 As shown, NDV antibodies were produced in all experimental groups on day 7 post-immunization, with antibody titers ranging from 2.6 log2 to 3.4 log2. There was no significant difference between the two experimental groups (P > 0.05), but the antibody titers in both experimental groups were significantly higher than those in the PBS group. The NDV antibody level in the SPF chicken flock increased with immunization time, reaching a peak on day 21 post-immunization, with antibody titers ranging from 6.2 log2 to 7.2 log2. In conclusion, immunization with rDHN3mF-H120-S6P can produce high levels of NDV antibodies in chicken flocks, demonstrating good immunoprotective efficacy.

[0123] (4) SPF chicken challenge groups

[0124] Twenty-one days after the second immunization, groups 1 and 3 were designated as IB groups, and groups 2 and 4 as ND groups. Group IB was challenged with the virus 10 days after immunization via eye drops and nasal drops. 6 EID50 M41, attacking ND group 10 5 EID50 virulent strain DHN3. Specific challenge details are shown in Table 10.

[0125] Table 10 SPF Chicken Challenge Grouping

[0126] Group numbering Immunoassay reagent name Virus attack strain Infectious drug dosage SPF chicken quantity / each 1 PBS M41 106EID50 7 2 PBS DHN3 106EID50 7 3 rDHN3mF-H120-S6P M41 106EID50 7 4 rDHN3mF-H120-S6P DHN3 106EID50 7

[0127] (5) ND group tested for detoxification after DHN3 treatment.

[0128] Throat swabs were collected from chickens in the ND groups (groups 2 and 4) at 3, 5, and 7 days post-challenge to detect NDV shedding. The collected throat swabs were placed in 1 mL of PBS buffer solution containing 1000 U / mL penicillin antibody (pre-cooled to 4°C). After centrifugation at 8000 rpm for 5 minutes at 4°C, the supernatant was transferred to a new 1.5 mL centrifuge tube and stored at -80°C for later use. 9-11 day old healthy SPF chicken embryos were used, with 3 SPF embryos inoculated per sample, 100 μL per embryo. After incubation at 38°C for 48 h, the allantoic fluid from the embryos was collected for the hemagglutination assay (HA). If one of the three embryos showed a hemagglutination titer, the throat swab from that chicken was considered to contain NDV.

[0129] like Figure 21 As shown, NDV shedding was detectable in the larynx of the PBS group on days 3, 5, and 7 after challenge, while the experimental group (rDHN3mF-H120-S6P) effectively inhibited NDV shedding on day 3, and DHN3 shedding was undetectable in the experimental group over time. This preliminarily demonstrates that immunization with rDHN3mF-H120-S6P can, to some extent, resist DHN3 infection and provide good protection for the flock.

[0130] (6) Detection of viral shedding in swabs after M41 treatment in Group IB.

[0131] Throat swabs and cloacal swabs were collected from chickens in groups IB (groups 1 and 3) at 3, 7, and 10 days after challenge to detect M41 shedding. Quantitative real-time PCR was used to detect M41 shedding in the IB group swabs, with each sample tested in triplicate.

[0132] like Figure 22 It can be seen that the viral shedding in the PBS group and the cloacal swabs after challenge was significantly higher than that in the experimental group. This indicates that chickens immunized with rDHN3mF-H120-S6P can resist infection by the M41 strain to a certain extent.

[0133] (7) After attacking M41, the IB group tested the detoxification status of tissues and organs.

[0134] In Group IB (Groups 1 and 3), 2-3 SPF chickens were randomly selected for necropsy at 5, 10, and 14 days after viral challenge. Trachea, heart, kidney, spleen, liver, forestomach, lung, small intestine, cecal tonsils, and bursa of Fabricius were collected, ground, and RNA was extracted. After reverse transcription into cDNA, the viral load in the tissues and organs was detected by real-time quantitative PCR.

[0135] from Figure 23It can be seen that M41 viral RNA was detected in all 10 tissues and organs taken from both the experimental and control groups after challenge. On day 5 post-challenge, the viral load in the trachea, small intestine, and bursa of Fabricius of the experimental group (rDHN3mF-H120-S6P) was lower than that of the PBS group. Except for these three tissues and organs, the M41 viral load in the other tissues and organs on day 5 was either higher than or similar to that of the PBS group. On days 10 and 14 post-challenge, the viral load in the tissues and organs of the experimental group was lower than that of the control group. This indicates that immunization with rDHN3mF-H120-S6P can provide a certain degree of protection against M41 attack in chickens, playing a good protective role.

[0136] In summary, based on the above-mentioned method for preparing a recombinant NDV genotype VII virus expressing the IBV S protein, the S protein was precisely modified, including replacing the S2 of the IBV-P65 cell line with the S2 of the non-cell-adapted MH20 cell line; mutating six hydrophobic amino acids in the H120 S protein to proline; and further mutating the furin site to enhance the stability and immunogenicity of the pre-fusion S protein, thus obtaining a recombinant virus capable of efficiently expressing both the full NDV protein and the S protein. Immunization challenge experiments using the recombinant virus rDHN3mF-H120-S6P showed that chicken flocks immunized with the rDHN3mF-H120-S6P vaccine could generate sufficient immunity to resist genotype VII NDV and M41 infection, indicating that rDHN3mF-H120-S6P shows promise as a safe and effective bivalent vaccine candidate.

[0137] As is known from common technical knowledge, this invention can be implemented through other embodiments that do not depart from its spirit or essential characteristics. Therefore, the disclosed embodiments described above are merely illustrative in all respects and are not the only ones. All modifications within the scope of this invention or equivalent to the scope of this invention are included in this invention.

[0138] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the specific implementation of the present invention. Any modifications or equivalent substitutions that do 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 recombinant NDV genotype VII virus expressing the IBV S protein, characterized in that, The method includes: By inserting a modified S gene between the P and M genes in the pBR322-DHN3mF viral genome, either the recombinant virus rDHN3mF-H120-S6P or rDHN3mF-H120-S6P-GSAS can be obtained. Specifically, the F1 fragment, F2 fragment, H120-S6P fragment or H120-S6P-GSAS fragment, and F3 fragment were used to obtain two recombinant plasmids through homologous recombination. The two recombinant plasmids were then transfected into cells to obtain two recombinant viruses. The sequence of the F1 fragment of the gene is shown in SEQ ID NO: 1; The sequence of the F2 fragment of the gene is shown in SEQ ID NO: 2; The sequence of the H120-S6P fragment of the gene 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 F3 fragment of the gene is shown in SEQ ID NO:

6.

2. The method for preparing genotype VII NDV recombinant virus expressing IBV S protein according to claim 1, characterized in that, The preparation process of the F1 fragment of the gene is as follows: using the pBR322-DHN3mF vector as a template, the F1 fragment is obtained by digestion with SmaI. The preparation process of the F2 fragment of the gene is as follows: using pBR322-DHN3mF plasmid as a template, the F2 fragment is amplified with primers FDHN-SmaI-F1 and FDHN-R1; primers FDHN-SmaI-F1 and FDHN-R1 are shown in the sequence listing SEQ ID NO: 7-8.

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

4. The method for preparing genotype VII NDV recombinant virus expressing IBV S protein according to claim 1, characterized in that, The amounts of each substance used in the recombination process were as follows: 870 ng gene fragment F1, 201 ng gene fragment F2, 450 ng gene fragment H120-S6P or 450 ng gene fragment H120-S6P-GSAS, 611 ng gene fragment F3, 10 μL LABclonal 2X MultiFSeamlessAssembly Mix, and ddH2O was added to 20 μL; the reaction conditions were 50℃ for 50 minutes.

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

6. A recombinant NDV genotype VII expressing the IBV S protein, characterized in that, The method for preparing genotype VII NDV recombinant virus expressing IBV S protein according to any one of claims 1-5 is obtained.

7. The use of a genotype VII NDV recombinant virus expressing the IBV S protein as described in claim 6 in the preparation of a vaccine.

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