Newcastle disease vaccine strain with high antibody escape capacity and transformation method thereof

Abstract

The invention discloses a Newcastle disease vaccine strain with high antibody escape ability and a modification method thereof, the cell strain is preserved in China General Microbiological Culture Collection Center on January 19, 2024, the address of the preservation unit is CGMCC No.45870, No.1 yard, Beichen West Road, Chaoyang District, Beijing, and the classification name of the strain is Newcastle disease virus strain. The recombinant NDV vaccine strain has the same antigen site as the current NDV gene VII type epidemic strain and has the efficient proliferation efficiency superior to that of a classical vaccine strain La Sota, the thermal stability of the NDV vaccine strain is improved by the constructed recombinant vaccine strain, and the cost of vaccine cold-chain transportation is reduced.

Description

Technical Field

[0001] The 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 transformation method thereof. Background Art

[0002] Newcastle disease virus (NDV) is one of the highly contagious and severe animal infectious pathogens that endanger 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 Class II animal disease.

[0003] NDV belongs to the family Paramyxoviridae, the genus Orthocheiritis virus, and the avian Orthocheiritis virus type I. It is a type of single-stranded negative-stranded non-segmented RNA virus with an envelope. Its genome encodes six structural proteins, NP-PMF-HN-L, in sequence. Among them, the HN protein is a membrane surface protein and the most important antigenic protein of NDV. Its genome length is 2002nt, but different NDV strains have different protein lengths due to the different positions of the termination codons of their HN open reading frames (ORFs). At present, at least 9 different lengths of HN proteins have been reported, with protein molecular weights ranging from 72 to 75KD. The main biological function of the HN protein is to mediate the adsorption of the virus to the host cell (HA activity), participate in the completion of membrane fusion by the auxiliary F protein, and at the same time, in the budding process of the progeny virus, play the NA activity to lyse the sialic acid receptors on the cell surface, so that the progeny virus can smoothly leave the host cell.

[0004] All NDVs belong to the same serotype, so cross-protection between NDVs of different genotypes is better. The widely used NDV vaccine is mainly derived from the La Sota strain of genotype II. Although this vaccine can effectively protect poultry from the attack of NDV virulent, it also faces a major problem, namely the interference of maternal antibodies. It is reported that NDV vaccine can provide complete immune protection in SPF chickens, while in the presence of maternal antibodies, the same dose of NDV-H5 vaccine has only 50% immune protection efficiency. In another study, researchers found that in the presence of NDV maternal antibodies, the immune dose of the vaccine needs to be increased to overcome the interference of maternal antibodies. Therefore, the development of a new ND vaccine that can antagonize maternal antibody interference and has high proliferation efficiency is of great significance for the prevention and control of NDV. Therefore, a Newcastle disease vaccine strain with high antibody escape ability and its modification method 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 by reverse genetics technology, wherein 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 improve 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 the biggest feature is:

[0015] (1) 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 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 neutralization by 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 a method for its transformation according to the present invention;

[0018] Figure 2 A schematic diagram of glycosylation identification of a Newcastle disease vaccine strain with high antibody escape ability and a recombinant virus of a modification method thereof according to 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 of a modification method thereof in more than 50 SPF chicken embryos of the present invention;

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

[0021] in Figure 4 A is the copy number of NP gene at different time 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 A schematic diagram of the effect of heat treatment on recombinant virus HA of a Newcastle disease vaccine strain with high antibody escape ability and a modification method thereof according to 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 after the virus was treated at 56°C for different time periods;

[0026] Figure 6 A schematic diagram of the change in serum antibody HI titer of SPF chickens inoculated with LaSota and recombinant virus inactivated vaccines according to a Newcastle disease vaccine strain with high antibody escape ability and a method for modifying the same of the present invention;

[0027] in Figure 6 A is the change of 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 A schematic diagram of the change in serum antibody HI titer of commercial chickens inoculated with LaSota and recombinant virus inactivated vaccines according to a Newcastle disease vaccine strain with high antibody escape ability and a method for modifying the same of the present invention;

[0030] in Figure 7 A is the change of 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 A schematic diagram of the change in serum antibody HI titer of commercial chickens inoculated with LaSota and recombinant virus live vaccine according to a Newcastle disease vaccine strain with high antibody escape ability and a method for modifying the same in the present invention;

[0033] in Figure 8 A is the change of 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 scheme of the present invention is further described 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] The current NDV vaccine strain La Sota is used as the skeleton, the HN head antigen region of the genotype VII NDV JSD0812 virulent strain is replaced, and the HN protein is subjected to double point mutations S379N and T394N to add an additional glycosylation site. In addition, in the prior art, the double point mutations R247K and S263R on the M protein can improve the budding efficiency of the virus by affecting the nuclear efficiency of the M protein, thereby promoting the replication of the virus. Therefore, the R247K and S263R double point mutations are 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 site is utilized, the present invention uses HN-specific polyclonal antibodies to detect the expression of HN protein in the allantoic fluid of each recombinant virus by Western blotting (WB). Figure 2 As shown, when not treated with PNGase F glycosidase (PNGase F glycosidase can hydrolyze the chemical bond connecting aspartic acid and oligosaccharide chain on N-glycoprotein or N-glycopeptide and release the complete N-oligosaccharide), the HN protein of S379N and T394N strains with one glycosylation site migrated slower in the gel compared with DM-JSDHN (M1 N, no additional glycosylation site), and the migration of HN protein was slower as the glycosylation site increased (M2N). After digestion with PNGase F glycosidase, the migration rate of HN protein of each recombinant virus was consistent, indicating that the change in the migration rate of HN protein after mutation was caused by N-glycosylation.

[0043] Implementation Case 2 Test Method

[0044] Reagent configuration

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

[0046]

[0047] Use 800mL ddH2O to dissolve and dilute the above solid reagents. After complete dissolution, add 200mL ddH2O to make up to 1L and sterilize under high pressure for storage.

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

[0049]

[0050] Use 800mL ddH2O to dissolve and dilute the above solid reagents. After complete dissolution, add 200mL ddH2O to make up to 1L. Autoclave and sterilize. When cooled to about 60°, add Amp icillin or Kana with a final concentration of 100μg / mL. Mix well with the culture medium and slowly pour into a disposable bacterial culture dish on a clean bench. After cooling and solidifying, seal and store at 4°.

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

[0057] (Plasmid description: ①pCMV-HA-huHN-JSD0812: The vector is pCMV, and the inserted fragment is the HN gene of the NDV JSD0812 strain. This plasmid is used to amplify the gene type VII HN gene for later plasmid construction. ②TVT-La SotaC5-ZP-B: The vector is TVT, and the inserted fragment 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, for the amplified product, DpnⅠ enzyme needs to be added to digest the methylated template plasmid in a 37℃ water bath for 2h or overnight before homologous recombination connection can be performed. The digestion system is as follows:

[0063]

[0064] After digesting the template plasmid, the digestion product was recovered using an agarose gel recovery kit, and the product concentration was measured using a NanoDrop 2000 micro-spectrophotometer. The recovered product was then connected using the full-gold homologous recombination kit. The steps are as follows: First, configure the system in the PCR tube according to the table below. It should be noted that the optimal ratio of the target fragment (shorter fragment) to the vector (longer fragment) is 2:1. Then flick the system to mix, centrifuge instantly to collect the tube wall liquid at the bottom of the tube, and place it in a 50°C water bath for reaction for 30-60 minutes. After the reaction is completed, place the recombinant product on ice for a few seconds, and then it can be used for transformation immediately or stored at -20°C.

[0065] Homologous recombination reaction system:

[0066]

[0067] The recombinant product was transformed into HB101 competent cells, and after overnight culture, a single clone was selected for PCR identification. The bacterial solution with positive PCR identification was sent for testing, and the bacterial solution with correct sequencing was selected for expansion culture and plasmid extraction.

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

[0069] First, the constructed intermediate plasmid ZP-B-JSDHN was used as a template, and the primers were La4571F and La10050R to amplify one of the fragments required for the full-length plasmid. Then, the full-length plasmid TVT-La SotaC5-DM constructed in the laboratory was used as a template, and the primers were La10033F and La4586R to amplify another part of the full-length plasmid. After the template was digested with DpnⅠ enzyme and purified by gel recovery, the two fragments were connected by homologous recombination, and the connection was immediately carried out. After that, the bacteria that were positive for PCR identification after transformation were cultured in small quantities and sent for sequencing. The bacteria that were sequenced correctly continued to be expanded for plasmid extraction. (ZP-B-JSDHN plasmid: except for the antigenic determining region of the HN gene, which is the HN of the genotype VII NDV, the rest of the parts are the same as TVT-LaSotaC5-ZP-B, and the purpose is to be used as an intermediate plasmid for the construction of the full-length plasmid)

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

[0071] Two pairs of primers containing two pairs of mutation points were designed for the two mutation positions of S379N and T394N, respectively, which were S379N-F, S379N-R and T394N-F, T394-R. First, the full-length plasmid of rLa-DM-JSDHN was used as a template, and a part of each full-length plasmid was amplified using primer AMP-F and primers S379N-R and T394N-R, and the other part was amplified by primer AMP-R and primer S379N-F, respectively. The above two fragments were homologously recombined and connected by homologous recombination to form the S379N single-point mutation full-length plasmid rLa-DM-JSDHN-S379N (the vector was TVT, containing a T7 promoter, and the inserted fragment was the full-length gene of NDV-M1N, in which the amino acid at position 379 of the HN protein was mutated to asparagine, and the plasmid was used as a template for the next mutation).

[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 a double-point mutation full-length plasmid rLa-DM-JSDHN-S379N / T394N (the vector is TVT, and the inserted fragment is the full-length gene of NDV-M2N, which is used to rescue the NDV-M2N recombinant virus).

[0073] The recombinant product was transformed into HB101 competent cells, and after overnight culture, a single clone was selected for PCR identification. The bacterial solution with positive PCR identification was sent for testing, and the bacterial solution with correct sequencing was selected for expansion culture and plasmid extraction. The extracted and 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 the rescue efficiency, BSR-T7 / 5 cells were revived two days before rescue and cultured in DMEM medium containing 1 mg / mL G418 without repeated passages. 12 h before transfection, cells were seeded in six-well plates and cultured in DMEM without antibiotics and containing 10% FBS. Cells were used for transfection when the cell density was about 60% to 80%;

[0076] Before transfection, dilute the frozen MVA 500 times with blood-free and antibody-free DMEM for later use. Take out the six-well plate, wash the cells three times with high-pressure sterile PBS, add 600 μL of the vaccinia virus MVA dilution to each well of the six-well plate, and place it in a 37°C, 5% CO2 incubator for adsorption for 30 minutes;

[0077] First, add 100 μL of Opt i-MEM to EP tube A, add 10 μL of FuGENE HD to tube A at a ratio of 1:3 for the total number of FuGENE HD transfection reagent and plasmids, mix well and let stand for 5 minutes. After 5 minutes, add the four point mutation M protein LaSota full-length plasmids and auxiliary plasmids to EP tube A at a ratio of pCI-NP: pCI-P: pCI-L: full-length plasmid = 2.0 μg: 1.0 μg: 0.5 μg: 5.0 μg, gently blow and mix, and incubate for 15 minutes to form a transfection complex; a negative control needs to be designed at the same time. During this period, the BSR cells incubated with vaccinia virus MVA were washed three times with autoclaved sterile PBS, and 1 mL of Opt i-MEM was added to each well of the six-well plate. After the incubation is completed, the transfection complex is evenly added to each well and cultured normally 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, and observe the cell status every 8-12 hours. If the cells show dead floating phenomenon, add 200 μL FBS to each well.

[0079] 60h after transfection, collect the cell supernatant in a sterile EP tube, gently digest the adherent cells with 200μL 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 taken to inoculate 9-day-old SPF chicken embryos, 200 μL / egg. The inoculated SPF chicken embryos were placed in a 37°C chicken embryo incubator for 96 hours.

[0081] After 96 hours, the chicken embryos were taken out and placed at 4°C overnight. The allantoic fluid was collected the next day and the hemagglutination titer of the chicken embryos 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 into cDNA, PCR was amplified to amplify the full-length sequence of the M protein point mutation specific fragment for testing and identification. Implementation Case 3 Determination of M2N Biological Characteristics

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

[0083] In order to find out whether the modified vaccine strain will affect the replication of the virus in chicken embryos, the present invention inoculated the recombinant virus M2N and La Sota in the allantoic cavity of more than 50 SPF chicken embryos, and collected the allantoic fluid of each chicken embryo 96 hours after inoculation to measure 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 recombinant viruses 50 and TCID 50 Determination

[0085] After the first generation of recombinant virus was rescued, the virus was continuously subcultured to the fifth generation, and each virus was diluted to 10 -4 Each recombinant virus was inoculated with 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 to measure the HA titer, mean embryo mortality time (MDT), and embryo infection dose (EID) of the chicken embryo. 50 ) and TCID 50 ). The results showed (as shown in Table 1) that the recombinant virus had a higher titer and reduced virulence compared to 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 / 36h. RNA was extracted from one part of the supernatants and reverse transcribed into cDNA for RT-PCR amplification of the La Sota NP gene. The other part was inoculated into BHK21 cells for TC ID 50 The results showed that the recombinant strain M1 N was 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 vaccines is of great significance for reducing the cost of cold chain transportation of vaccines. To this end, the present invention measured the HA activity of the recombinant virus after treatment at 37°C and 56°C. The results showed ( Figure 5 and Table 2), the HA activity of the recombinant virus was not greatly affected when stored at 37°C for 72h, but the HA activity and infectivity of La Sota were completely lost after heating at 56°C for 10 minutes, while the HA activity of the recombinant virus M2N slowly decreased after heating at 56°C, and the HA activity was completely lost after 30 minutes. These results indicate that the thermal stability of the recombinant virus is improved compared with the parent strain.

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

[0093]

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

[0095] In order to investigate the ability of the recombinant virus to escape neutralizing antibodies in vitro, the present invention conducted a chicken embryo neutralization test using the collected La Sota high immune serum. The results showed (as shown in Table 3) that the neutralization titer of the M2N virus was significantly reduced compared with La Sota, indicating that the S379N and T394N double point mutations increased the ability of the virus antigen to escape neutralizing antibodies.

[0096] Table 3 Neutralizing antibody titer of La Sota high immune serum against recombinant virus

[0097]

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

[0099] After the recombinant virus was inactivated with β-propiolactone at a final concentration of 0.05%, it was evenly mixed with ISA78 adjuvant in a ratio of 3:7, and SPF chickens were immunized as shown in Table 4. Serum was collected at 3, 7, 10, 14, 21 and 28 days after immunization, and the antibody titer was detected by hemagglutination inhibition (HI). The results showed that one week after SPF chickens were immunized with the inactivated vaccine, the antibody level in the body began to rise significantly, reaching a peak three weeks after immunization. It is worth noting that compared with La Sota, the antibody level stimulated by the recombinant virus M2N was significantly higher one week after immunization, especially the antibody titer against genotype VII JSD0812 HN, and the recombinant virus was 1 to 2 log2 higher than the parent strain. The results show 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 antibodies

[0103] In order to investigate the antibody stimulating ability of the inactivated recombinant virus vaccine in commercial chickens with high maternal antibodies, the present invention used the same inactivated vaccine as mentioned 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 antibodies against La Sota and genotype VII JSD0812 of the 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, the antibody titers stimulated by all inactivated vaccines were at a low level. After 14 days of immunization, the serum antibody levels stimulated by the recombinant virus M2N began to rise significantly, while the serum antibody titers of chickens immunized with the parental strain remained at a low level. These results show that LaSota is seriously interfered by maternal antibodies, while the recombinant virus M2N can overcome the interference of maternal antibodies and stimulate effective antibodies.

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

[0105]

[0106] 3.2.8 The ability of M2N to escape maternal antibodies is higher than that of 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 to detect antibody HI titers at 3, 7, 14, and 21 days after immunization. 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 the 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 antigenic determining region of the HN protein of the optimized and modified genotype VII strain JSD0812 was used to replace the original La Sota HN protein corresponding sequence, 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 characteristics of the recombinant virus M2N were measured, and the results showed that compared with the NDV vaccine strain LaSota, the recombinant strain M2N had improved proliferation efficiency in chicken embryos and significantly improved thermal stability. The results of the La Sota serum in vitro neutralization test showed that the virus's ability to escape neutralizing antibodies was improved after the double point mutations of S379N and T394N. In addition, the results of animal experiments showed that the antibody level stimulated by the recombinant virus M2N in SPF chickens was significantly higher than that of La Sota, and it could also stimulate effective antibody levels in commercial chickens with high maternal antibody levels. A series of complex modifications were carried out on the HN protein of the NDV vaccine strain La Sota, which successfully improved the proliferation efficiency, thermal stability, antigenicity and neutralizing antibody escape ability of the NDV vaccine strain.

[0113] In summary, the invention of the present invention carries out a series of complex modifications on the HN protein of the NDV vaccine strain La Sota, and successfully improves 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 implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which are all covered by the protection scope 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 transforming a Newcastle disease vaccine strain with high antibody escape ability, characterized in that: include: The recombinant virus M2N was rescued by double point mutations S379N and T394N on the HN protein of the M1N strain.

4. A Newcastle disease M2N strain according to claim 1, characterized in that: The construction method of 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 double point mutations of S379N and T394N. 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.

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

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