A Newcastle disease vaccine strain with high antibody escape ability and its modification method

Abstract

The present invention discloses a Newcastle disease vaccine strain with high antibody escape ability and a method for its modification. The cell strain was deposited at the General Microbiology Center of the China Culture Collection Administration on January 19, 2024. The depository address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, CGMCC No. 45870. The strain is classified as Newcastle disease virus strain. The present invention has the same antigenic sites as the current NDV genotype VII epidemic strain and has a high proliferation efficiency that exceeds that of the classic vaccine strain La Sota. The constructed recombinant vaccine strain improves the thermal stability of the NDV vaccine strain, which helps reduce the cost of vaccine cold chain transportation.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering, and in particular relates to a Newcastle disease vaccine strain with high antibody escape ability and a modification method thereof. Background Art

[0002] Newcastle disease virus (NDV) is a highly contagious, severe zoonotic pathogen that threatens the global poultry industry. The World Organization for Animal Health (WOAH) lists Newcastle disease (ND) as a notifiable infectious disease, and my country also lists it as a Category II animal disease.

[0003] Newborn's disease virus (NDV) belongs to the Paramyxoviridae family, the genus Orthomyxovirus, and type I avian NDV. It is an enveloped, single-stranded, negative-sense, non-segmented RNA virus. Its genome encodes six structural proteins: NP, PMF, HN, and L. The HN protein is a membrane surface protein and the primary antigenic protein of NDV. Its genome is 2002 nt in length, but the length of the protein encoded by different NDV strains varies depending on the location of the stop codon in the HN open reading frame (ORF). At least nine different lengths of HN proteins have been reported, with molecular weights ranging from 72 to 75 kDa. The primary biological function of the HN protein is to mediate viral attachment to host cells (HA activity) and to assist the F protein in membrane fusion. Furthermore, during the budding process of progeny viruses, the NA activity cleaves cell surface sialic acid receptors, enabling the progeny viruses to successfully escape from the host cell.

[0004] All NDVs belong to the same serotype, resulting in good cross-protection between different genotypes. Currently, the most widely used NDV vaccine is derived from the La Sota strain of genotype II. While this vaccine effectively protects poultry against virulent NDV, it also faces a major challenge: interference from maternal antibodies (MDAs). NDV vaccines reportedly provide complete protection in SPF chickens, whereas the same dose of NDV-H5 vaccine offers only 50% protection in the presence of MDA. Another study found that in the presence of NDV MDA, a higher vaccine dose is required to overcome this interference. Therefore, developing a novel NDV vaccine that can antagonize MDA interference and exhibit high proliferation efficiency is crucial for NDV prevention and control. Therefore, a Newcastle disease vaccine strain with enhanced antibody escape potential and methods for its modification are needed. Summary of the Invention

[0005] The purpose of the present invention is to provide a Newcastle disease vaccine strain with high antibody escape ability and a modification method thereof.

[0006] The present invention is achieved through the following technical solutions:

[0007] The strain described in the present invention was deposited in the General Microbiology Center of the China Culture Collection Administration on January 19, 2024. The address of the depository unit is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, CGMCC No. 45870. The classification name of the strain is Newcastle disease virus strain.

[0008] Furthermore, the nucleotide sequence of the Newcastle disease M2N strain is shown in SEQ ID No.3.

[0009] A modification method for improving the neutralizing antibody escape ability of Newcastle disease vaccine strains comprises: performing double point mutations S379N and T394N on the HN protein of the M1N strain to rescue the recombinant virus M2N.

[0010] Furthermore, the method for constructing the M1N strain includes replacing the corresponding sequence of the HN protein of the LaSota vaccine strain with the optimized and modified HN protein, and rescuing the NDV-M1N vaccine strain matching the NDV genotype VII using reverse genetics technology. The nucleotide sequence of the optimized and modified HN protein is shown in SEQ ID No. 1, and the amino acid sequence of the HN protein is shown in SEQ ID No. 2.

[0011] Application of a Newcastle disease M2N strain in promoting the thermal stability of Newcastle disease virus.

[0012] A Newcastle disease M2N strain is used to promote the antibody escape ability of the Newcastle disease virus and enhance the antagonistic ability of maternal antibodies.

[0013] Application of a Newcastle disease M2N strain in promoting the thermal stability of Newcastle disease virus.

[0014] Compared with the prior art, the present invention adopts the above technical solution, and its biggest feature is:

[0015] (1) The present invention has the same antigenic sites as the current NDV genotype VII epidemic strain, and at the same time has a high proliferation efficiency that exceeds the classic vaccine strain La Sota. The constructed recombinant vaccine strain improves the thermal stability of the NDV vaccine strain, which is beneficial to reducing the cost of vaccine cold chain transportation.

[0016] (2) Compared with the traditional La Sota, the strain constructed by this patent has a stronger ability to escape the neutralization of maternal antibodies, the constructed recombinant vaccine strain inactivated vaccine has better antigenicity and stimulates higher antibody levels; and it can also stimulate effective antibody levels in chickens with high levels of maternal antibodies. The live strain vaccine has a better ability to stimulate antibodies in chicks with high levels of maternal antibodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 A schematic diagram of the construction of an M2N recombinant virus of a Newcastle disease vaccine strain with high antibody escape ability and its transformation method of the present invention;

[0018] Figure 2 This is a schematic diagram of glycosylation identification of a Newcastle disease vaccine strain with high antibody escape ability and a recombinant virus thereof according to a modification method of the present invention;

[0019] Figure 3 A schematic diagram of the HA titer of a Newcastle disease vaccine strain with high antibody escape ability and a recombinant virus thereof according to a modification method of the present invention in more than 50 SPF chicken embryos;

[0020] Figure 4 This is a schematic diagram of the growth curve determination of a Newcastle disease vaccine strain with high antibody escape ability and a recombinant virus thereof according to a modification method of the present invention;

[0021] in Figure 4 A is the copy number of NP gene at different times after virus infection of DF-1 cells;

[0022] Figure 4 B is the TCID at different time points after virus infection of DF-1 cells 50 ;

[0023] Figure 5 Schematic diagram of the effect of heat treatment on recombinant virus HA of a Newcastle disease vaccine strain with high antibody escape ability and its modification method of the present invention;

[0024] in Figure 5 A is the HA activity loss rate after the virus was treated at 37°C for different time periods;

[0025] Figure 5 B is the HA activity loss rate of the virus after treatment at 56°C for different time periods;

[0026] Figure 6 This is a schematic diagram of the changes in serum antibody HI titer of SPF chickens after inoculation with LaSota and recombinant virus inactivated vaccines, showing a Newcastle disease vaccine strain with high antibody escape ability and its transformation method according to the present invention;

[0027] in Figure 6 A is the change in serum antibody HI titer against La Sota antigen;

[0028] Figure 6 B shows the changes in serum antibody HI titer against genotype VII HN antigen;

[0029] Figure 7 This is a schematic diagram of the changes in serum antibody HI titer of commercial chickens after inoculation with LaSota and recombinant virus inactivated vaccines, according to a Newcastle disease vaccine strain with high antibody escape ability and its transformation method of the present invention;

[0030] in Figure 7 A is the change in serum antibody HI titer against La Sota antigen,

[0031] Figure 7 B is the change of serum antibody HI titer against genotype VII HN antigen;

[0032] Figure 8 This is a schematic diagram of the changes in serum antibody HI titer of commercial chickens after inoculation with LaSota and recombinant virus live vaccines, according to a Newcastle disease vaccine strain with high antibody escape ability and its transformation method of the present invention;

[0033] in Figure 8 A is the change in serum antibody HI titer against La Sota antigen;

[0034] Figure 8 B shows the changes in serum antibody HI titer against genotype VII HN antigen. DETAILED DESCRIPTION

[0035] The technical solution of the present invention is further illustrated below in conjunction with embodiments and comparative examples, but they should not be construed as limiting the present invention:

[0036] The Newcastle disease M2N strain was deposited in the General Microbiology Center of the China Culture Collection Administration on January 19, 2024. The address of the deposit unit is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, CGMCC No. 45870. The classification name of the strain is Newcastle disease virus strain, and the nucleotide sequence of the Newcastle disease M2N strain is shown in SEQ ID No. 3.

[0037] Case Study 1: Design and Rescue of Recombinant Viruses

[0038] 1.2.1 Sequence information of recombinant virus M2N

[0039] 1.2.2 Construction of M2N

[0040] Using the current NDV vaccine strain La Sota as the backbone, the HN head antigen region of the genotype VII NDV JSD0812 virulent strain was replaced, and the HN protein was subjected to double point mutations S379N and T394N to add an additional glycosylation site. In addition, in the prior art, the R247K and S263R double point mutations in the M protein can improve the budding efficiency of the virus by affecting the nuclear export efficiency of the M protein, thereby promoting the replication of the virus. Therefore, the R247K and S263R double point mutations were performed on the M protein of the recombinant virus to improve the proliferation efficiency of the virus. The structural diagram is shown in the figure. Figure 1 shown.

[0041] 1.2.3 Glycosylation identification of recombinant viruses

[0042] In order to determine whether the artificially introduced N-glycosylation sites are utilized, the present invention uses HN-specific polyclonal antibodies to detect the expression of HN proteins in the allantoic fluid of each recombinant virus by Western blotting (WB). Figure 2 As shown, when not treated with PNGase F glycosidase (which hydrolyzes the chemical bond between aspartic acid and oligosaccharide chains on N-glycoproteins or N-glycopeptides, releasing intact N-oligosaccharides), the HN proteins of the S379N and T394N strains, which had one additional glycosylation site, migrated slower in the gel compared to DM-JSDHN (M1 N, without additional glycosylation sites). Furthermore, the HN protein migrated even slower with the addition of additional glycosylation sites (M2N). After digestion with PNGase F glycosidase, the HN proteins of the recombinant viruses had consistent migration rates, indicating that the altered migration rate of the HN proteins after mutation is caused by N-glycosylation.

[0043] Implementation Case 2 Test Method

[0044] Reagent configuration

[0045] 1) Non-resistant liquid LB bacterial culture medium:

[0046]

[0047] Dissolve and dilute the above solid reagents with 800 mL of ddH2O. After complete dissolution, add 200 mL of ddH2O to make up to 1 L. Sterilize and store by autoclaving.

[0048] (2) Resistance solid LB bacterial culture medium:

[0049]

[0050] Dissolve and dilute the above solid reagents with 800 mL of ddH2O. After complete dissolution, add 200 mL of ddH2O to 1 L. Autoclave and sterilize. When cooled to about 60°C, add Ampicillin or Kana to a final concentration of 100 μg / mL. Mix well with the culture medium and slowly pour into a disposable bacterial culture dish in a clean bench. Let cool and solidify, then seal and store at 4°C.

[0051] Construction of full-length plasmid

[0052] (1) Homologous recombination primer design

[0053]

[0054] (2) Plasmid construction method

[0055] ① Construction of intermediate plasmid ZP-B-JSDHN

[0056] First, using the plasmid pCMV-HA-huHN-JSD0812 (constructed earlier in the laboratory) as a template, primers JSDHN-F and JSDHN-R were used to amplify the HN head coding sequence of NDV genotype VII strain JSD0812. Next, using the TVT-La SotaC5-ZP-B plasmid as a template, primers ZP-BF and ZP-BR were used to amplify the vector fragment of the intermediate plasmid. The amplification system and procedure are shown in the table below.

[0057] (Plasmid Description: ①pCMV-HA-huHN-JSD0812: The vector is pCMV, and the insert is the HN gene of the NDV JSD0812 strain. This plasmid is used to amplify the genotype VII HN gene for later plasmid construction. ②TVT-La SotaC5-ZP-B: The vector is TVT, and the insert contains part of the NP gene, M gene, F gene, HN gene, and part of the L gene of La Sota. This plasmid is used as an intermediate plasmid to facilitate genetic modification and later full-length plasmid construction)

[0058]

[0059] PCR reaction conditions:

[0060]

[0061]

[0062] Since the amplified product contains a methylated template plasmid, false positive colonies will be generated during subsequent transformation. Therefore, the amplified product needs to be digested with DpnⅠ enzyme in a 37°C water bath for 2 hours or overnight before homologous recombination ligation can be performed. The digestion system is as follows:

[0063]

[0064] After digesting the template plasmid, the digestion products were recovered using an agarose gel recovery kit, and the product concentration was measured using a NanoDrop 2000 microspectrophotometer. The recovered products were then ligated using the All-Gold Homologous Recombination Kit. The steps are as follows: First, prepare the system in a PCR tube according to the table below. Note that the optimal ratio of target fragment (shorter fragment) to vector (longer fragment) is 2:1. Then, gently flick the system to mix, centrifuge briefly to collect the liquid at the bottom of the tube, and incubate in a 50°C water bath for 30-60 minutes. After the reaction is complete, place the recombinant product on ice for a few seconds. It can then be used for transformation immediately or stored at -20°C.

[0065] Homologous recombination reaction system:

[0066]

[0067] Transform the recombinant product into HB101 competent cells. After overnight culture, single clones are selected for PCR identification. Strains that are positive for PCR identification are sent for testing. Strains that have been sequenced correctly are selected for expansion and plasmid extraction.

[0068] ② Construction of full-length plasmid rLa-DM-JSDHN

[0069] First, using the constructed intermediate plasmid ZP-B-JSDHN as a template and primers La4571F and La10050R, one fragment required for the full-length plasmid was amplified. Next, using the full-length TVT-La SotaC5-DM plasmid constructed earlier in the laboratory as a template and primers La10033F and La4586R, another fragment of the full-length plasmid was amplified. After digestion with Dpn I and gel purification, the two fragments were ligated by homologous recombination and immediately transformed. Transformed bacteria that tested positive for PCR were cultured in small quantities and sent for sequencing. Successfully sequenced bacteria were further expanded for plasmid extraction. (The ZP-B-JSDHN plasmid is identical to TVT-LaSotaC5-ZP-B, except that the HN gene antigenic determinant region is that of genotype VII NDV HN. It serves as an intermediate plasmid for full-length plasmid construction.)

[0070] ③Construction of full-length plasmid with double-point mutations of HN protein S379N and T394N

[0071] Two pairs of primers were designed for the two mutation sites, S379N and T394N, respectively. These primers, S379N-F and S379N-R, and T394N-F and T394-R, were used. First, using the full-length rLa-DM-JSDHN plasmid as a template, a portion of each full-length plasmid was amplified using primers AMP-F, S379N-R, and T394N-R. Another portion was amplified using primers AMP-R and S379N-F, respectively. These two fragments were then homologously ligated to construct the full-length plasmid rLa-DM-JSDHN-S379N with the S379N single-point mutation. (The vector was TVT, containing a T7 promoter, and the insert was the full-length NDV-M1N gene, in which amino acid 379 of the HN protein was mutated to asparagine. This plasmid served as a template for the next mutagenesis step.)

[0072] Next, using the full-length plasmid rLa-DM-JSDHN-S379N as a template, primers AMP-F and T394N-R were used to amplify one fragment of the full-length plasmid, and primers T394N-F and AMP-R were used to amplify the other fragment of the full-length plasmid. The two fragments were connected by homologous recombination to form the double-point mutation full-length plasmid rLa-DM-JSDHN-S379N / T394N (the vector was TVT, and the inserted fragment was the full-length gene of NDV-M2N, which was used to rescue the NDV-M2N recombinant virus).

[0073] The recombinant product was transformed into HB101 competent cells. After overnight culture, single colonies were selected and shaken for PCR identification. PCR-positive bacteria were sent for testing. After expansion and plasmid extraction, the correctly sequenced rLa-DM-JSDHN-S379N / T394N full-length plasmid was stored at -20°C for later virus rescue.

[0074] NDV La Sota was rescued using the NDV T7 RNA polymerase-dependent rescue system. The specific steps are as follows:

[0075] To maximize rescue efficiency, thaw BSR-T7 / 5 cells two days before rescue and culture them in DMEM medium containing 1 mg / mL G418 without repeated passage. 12 hours before transfection, seed the cells in a six-well plate and culture them in DMEM without antibiotics and containing 10% FBS. Transfection is performed when the cell density is approximately 60% to 80%.

[0076] Before transfection, dilute the frozen MVA 500-fold in blood- and antibiotic-free DMEM. Remove the six-well plate and wash the cells three times with autoclaved PBS. Add 600 μL of the vaccinia virus MVA dilution to each well of the six-well plate and place in a 37°C, 5% CO2 incubator for 30 minutes.

[0077] First, add 100 μL of Opt i-MEM to each tube in EP tube A. Then, add 10 μL of FuGENE HD transfection reagent to tube A at a ratio of 1:3 for the total number of plasmids. Mix well and let stand for 5 minutes. After 5 minutes, add the four point-mutated M protein LaSota full-length plasmids and the helper plasmids to tube A at a ratio of 2.0 μg: 1.0 μg: 0.5 μg: 5.0 μg. Gently pipette to mix and incubate for 15 minutes to form the transfection complex. A negative control should also be included. During this time, wash BSR cells incubated with vaccinia virus (MVA) three times with autoclaved PBS. Add 1 mL of Opt i-MEM to each well of a six-well plate. After incubation, evenly add the transfection complex to each well and incubate in a 37°C incubator for 50-60 hours.

[0078] 24 hours after transfection, add 1 μg / mL TPCK trypsin or 200 μL fresh SPF chicken embryo allantoic fluid to each well of the six-well plate. Observe the cell status every 8-12 hours. If the cells appear dead and floating, add 200 μL FBS to each well.

[0079] 60 hours after transfection, collect the cell supernatant in a sterile EP tube, gently digest the adherent cells with 200 μL of trypsin in each well of the six-well plate, discard the trypsin digestion solution, and gently blow off the cells with the collected cell supernatant and collect them together in the EP tube. This step requires caution to avoid contamination that affects the rescue efficiency.

[0080] Fresh supernatant was directly inoculated into 9-day-old SPF chicken embryos at 200 μL / egg. The inoculated SPF chicken embryos were placed in a 37°C chicken embryo incubator for 96 h.

[0081] After 96 hours, the chicken embryos were removed and placed at 4°C overnight. The next day, the allantoic fluid was collected and the hemagglutination titer was measured according to the OIE standard to determine whether the rescue was successful. If the rescue was successful, the virus was passaged to the third generation and the allantoic fluid RNA was extracted. After reverse transcription to cDNA, PCR was performed to amplify the full-length sequence of the M protein point mutation-specific fragment and send it for testing and identification.

[0082] 3.2.4.1 Determination of proliferation efficiency in SPF chicken embryos

[0083] In order to investigate whether the modified vaccine strain would affect the replication of the virus in chicken embryos, the present invention inoculated the recombinant viruses M2N and La Sota into the allantoic cavity of more than 50 SPF chicken embryos, and collected the allantoic fluid of each chicken embryo 96 hours after inoculation to determine the hemagglutination (HA) titer. The results showed that (see Figure 3 ), compared with La Sota, the proliferation efficiency of M2N was significantly higher.

[0084] 3.2.4.2 HA, MDT, and EID of the recombinant virus 50 and TCID 50 Determination

[0085] After the first generation of recombinant virus was rescued, the virus was continuously passaged to the fifth generation, and each virus was diluted to 10 -4 Each recombinant virus was inoculated into at least 10 embryos, and each embryo was inoculated with 100 μL of virus dilution. After 96 hours of inoculation and culture, the allantoic fluid containing the recombinant virus was collected and the HA titer, mean embryo mortality time (MDT), and embryo median infectious dose (EID) were determined. 50 ) and the TCID 50 The results showed (as shown in Table 1) that the recombinant virus had a higher titer and was less virulence than La Sota.

[0086] Table 1 HA, MDT, E ID of the fifth-generation recombinant virus 50 and TCID 50 Determination

[0087]

[0088] 3.2.4.3 Growth Curve Determination of M2N

[0089] The M2N recombinant virus was inoculated into DF-1 cells at an MOI of 5. The cell supernatants were collected at 6 / 12 / 18 / 24 / 30 / 36 h. RNA was extracted from one portion of the supernatants and reverse transcribed into cDNA for RT-PCR amplification of the La Sota NP gene. The other portion was inoculated into BHK21 cells for TC ID. 50 The results showed that the recombinant strain M1 N released faster on cells than La Sota (see Figure 4 ).

[0090] 3.2.4.4 Improved thermal stability of M2N

[0091] The thermal stability of the vaccine is of great significance for reducing the cost of cold chain transportation of the vaccine. Therefore, the present invention measured the HA activity of the recombinant virus after treatment at 37°C and 56°C. The results showed that ( Figure 5 (See Table 2 for details). The HA activity of the recombinant virus was not significantly affected when stored at 37°C for 72 hours. However, after heating at 56°C for 10 minutes, the HA activity and infectivity of La Sota were completely lost. The HA activity of the recombinant virus M2N slowly decreased after heating at 56°C, and was completely lost after 30 minutes. These results indicate that the recombinant virus has improved thermal stability compared to the parental strain.

[0092] Table 2 TCID of recombinant viruses after heat treatment 50 Detection

[0093]

[0094] 3.2.5 Double point mutations of S379N and T394N enhance the ability of viral antigens to escape neutralizing antibodies

[0095] To investigate the recombinant virus's ability to escape neutralizing antibodies in vitro, we conducted chicken embryo neutralization assays using La Sota high-immune serum. The results (Table 3) show that the neutralization titer of the M2N virus was significantly lower than that of the La Sota virus, indicating that the S379N and T394N double-point mutations enhance the virus's ability to escape neutralizing antibodies.

[0096] Table 3 Neutralizing antibody titers of La Sota high immune serum against recombinant viruses

[0097]

[0098] 3.2.6 Recombinant virus inactivated vaccine induces higher antibodies in SPF chickens

[0099] The recombinant virus was inactivated with β-propiolactone at a final concentration of 0.05% and then mixed with ISA78 adjuvant in a 3:7 ratio. SPF chickens were immunized as shown in Table 4. Serum was collected 3, 7, 10, 14, 21, and 28 days after immunization, and antibody titers were measured using hemagglutination inhibition (HI) assays. The results showed that antibody levels in SPF chickens began to rise significantly one week after immunization with the inactivated vaccine, reaching a peak three weeks after immunization. Notably, compared with La Sota, the recombinant virus M2N elicited significantly higher antibody levels one week after immunization, particularly against genotype VII JSD0812 HN, with titers 1 to 2 log2 higher than those of the parental strain. These results demonstrate that the recombinant virus has a stronger ability to stimulate antibody production.

[0100] Table 4 SPF chicken test grouping

[0101]

[0102] 3.2.7 M2N can effectively stimulate antibodies in chickens with high maternal antibody levels

[0103] In order to investigate the antibody stimulation ability of the recombinant virus inactivated vaccine in commercial chickens with high maternal antibodies, the present invention used the same inactivated vaccine as above to immunize three-day-old commercial chickens as shown in Table 5, and collected serum to measure HI titers before immunization and 3, 7, 10, 14, 21, and 28 days after immunization. The HI titers of the anti-La Sota and anti-genotype VII JSD0812 antibodies of commercial chickens before immunization were (7.2±0.75) log2 and (7.3±1.35) log2, respectively. The results showed (as shown in Table 5) Figure 7). Within 10 days after immunization, antibody titers elicited by all inactivated vaccines remained low. After 14 days, serum antibody levels elicited by the recombinant M2N virus began to rise significantly, while serum antibody titers in chickens immunized with the parental strain remained low. These results suggest that LaSota is severely affected by maternal antibody interference, while the recombinant M2N virus can overcome this interference and elicit effective antibodies.

[0104] Table 5 Grouping of commercial chickens in the experiment

[0105]

[0106] 3.2.8 M2N has a higher ability to escape maternal antibodies than La Sota

[0107] In order to evaluate the ability of the recombinant virus M2N to escape maternal antibodies, the present invention used M2N to immunize 3-day-old commercial chickens with high levels of maternal antibodies, and collected serum 3, 7, 14, and 21 days after immunization to detect antibody HI titers. The anti-La Sota and anti-genotype VII HN antibody HI titers of commercial chickens before immunization were (6.50±1.03)log2 and (6.25±0.96)log2, respectively. The results showed that the anti-La Sota serum antibody HI titer induced by M2N was not significantly different from that of La Sota, but the serum HI titer induced by it against genotype VII HN antigen was significantly higher than that of La Sota ( Figure 8 ). This indicates that the recombinant virus M2N has an improved ability to escape maternal antibodies.

[0108] Table 6 Grouping of commercial chickens in the experiment

[0109]

[0110]

[0111] Using the classic vaccine strain La Sota as the skeleton, the original La Sota HN protein corresponding sequence was replaced by the optimized and modified HN protein antigenic determining region of the genotype VII strain JSD0812, and the M1 N vaccine strain matching the NDV genotype VII was rescued using reverse genetics technology. Based on the M1 N strain, the HN protein was subjected to double point mutations S379N and T394N to rescue the recombinant virus M2N.

[0112] The biological properties of the recombinant virus M2N were evaluated. Results showed that compared to the NDV vaccine strain LaSota, the recombinant strain M2N exhibited increased proliferation efficiency and significantly improved thermal stability in chicken embryos. In vitro neutralization tests with LaSota serum demonstrated that the double point mutations, S379N and T394N, enhanced the virus's ability to escape neutralizing antibodies. Furthermore, animal studies demonstrated that the recombinant virus M2N elicited significantly higher antibody levels in SPF chickens than LaSota, and also elicited effective antibody levels in commercial chickens with high levels of maternal antibodies. A series of complex modifications to the HN protein of the NDV vaccine strain LaSota successfully improved the NDV vaccine strain's proliferation efficiency, thermal stability, antigenicity, and neutralization antibody escape ability.

[0113] In summary, the present invention conducts a series of complex modifications on the HN protein of the NDV vaccine strain La Sota, successfully improving the proliferation efficiency, thermal stability, antigenicity and neutralizing antibody escape ability of the NDV vaccine strain.

[0114] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes based on the technical solutions and inventive concepts of the present invention within the technical scope disclosed by the present invention, and they are all covered by the scope of protection of the present invention.

Claims

1. A Newcastle disease M2N strain, characterized in that The strain was deposited in the General Microbiology Center of China Culture Collection Administration on January 19, 2024. The address of the depository unit is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, CGMCC No. 45870. The classification name of the strain is Newcastle disease virus strain.

2. A Newcastle disease M2N strain according to claim 1, characterized in that The nucleotide sequence of the Newcastle disease M2N strain is shown in SEQ ID No.

3.

3. A method for modifying a Newcastle disease vaccine strain with high antibody escape ability, characterized in that: include: The recombinant virus M2N was rescued by performing double point mutations S379N and T394N on the HN protein of the M1N strain.

4. The method for transforming a Newcastle disease vaccine strain with high antibody escape ability according to claim 3, characterized in that: The method for constructing the M1N strain includes replacing the corresponding sequence of the HN protein of the LaSota vaccine strain with the optimized and modified HN protein, and using reverse genetics technology to rescue the NDV-M2N vaccine strain with the S379N and T394N double point mutations. The nucleotide sequence of the optimized and modified HN protein is shown in SEQ ID No. 1, and the amino acid sequence of the HN protein is shown in SEQ ID No.

2.

5. Use of the Newcastle disease M2N strain as claimed in claim 1 in promoting the thermal stability of Newcastle disease virus.

6. Use of the Newcastle disease M2N strain as claimed in claim 1 in improving the antibody escape ability of Newcastle disease virus and improving the antagonistic ability of maternal antibodies.

Images