A fusion protein HN-RBD, a coding gene, a recombinant NDV virus carrier, a recombinant NDV virus and application thereof

Abstract

The application belongs to the technical field of genetic engineering, and particularly relates to a fusion protein HN-RBD, a coding gene, a recombinant NDV virus carrier, a recombinant NDV virus and application thereof; the fusion protein HN-RBD comprises a partial domain of a Newcastle disease virus HN protein and a RBD of a S protein of a new coronavirus; the partial domain of the Newcastle disease virus HN protein comprises an intracellular domain and a transmembrane domain of the Newcastle disease virus HN protein. When a coding sequence of the fusion protein HN-RBD is combined with a coding sequence of the S protein of the new coronavirus or a variant of the S protein of the new coronavirus and is jointly inserted into the NDV virus carrier, more S proteins of the new coronavirus can be displayed on the surface of a recombinant NDV virus particle, and the ability of the recombinant NDV virus to produce mucosal immune antibody IgA is improved, which has great significance for developing an excellent recombinant NDV virus vaccine candidate strain.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of genetic engineering, and particularly relates to a fusion protein HN-RBD, a coding gene, a recombinant NDV virus carrier, a recombinant NDV virus and application thereof. BACKGROUND

[0002] The new crown (SARS-CoV-2) virus is a major respiratory virus, and the new crown virus infects the human body by interacting with the ACE2 protein on the host cell membrane through the S protein on the envelope surface. Therefore, producing efficient antibodies against the S protein of the new crown virus is the key to vaccine research and development.

[0003] The Newcastle disease virus (NDV) is a negative-strand RNA virus, and the genome includes NP, P, M, F, HN and L six genes, wherein the F protein and the HN protein belong to the viral envelope protein, the HN protein includes an intracellular domain, a transmembrane domain and an extracellular domain, and the extracellular domain contains Stalk and Head two parts. The NDV carrier can load an exogenous gene, and the recombinant NDV virus particle produced by the reverse genetics method with the NDV as the carrier has good safety, and in addition to producing humoral and cellular immunity as a vaccine strain, it can also produce mucosal immunity to prevent respiratory pathogens from infecting and colonizing the organism at the infection site.

[0004] There are two factors for the recombinant NDV virus to produce more antigen antibody IgG and IgA, one is that the antigen protein expressed by the antigen protein gene carried by the recombinant DNV virus particle has a good immunogenic conformation, and the other is that the recombinant NDV virus particle surface can display more antigen proteins. How to make the recombinant NDV virus particle surface display more antigen proteins has been a long-standing problem. SUMMARY

[0005] Therefore, the purpose of the present application is to provide a fusion protein HN-RBD, a coding gene, a recombinant NDV virus carrier, a recombinant NDV virus and application thereof, and the method of the present application can make the recombinant NDV virus particle surface display more new crown virus antigen S proteins.

[0006] The present application provides a fusion protein HN-RBD, comprising a partial domain of Newcastle disease virus HN protein and RBD of new crown virus S protein; the partial domain of Newcastle disease virus HN protein includes the intracellular domain and the transmembrane domain of the Newcastle disease virus HN protein.

[0007] Preferably, the partial domain of the Newcastle disease virus HN protein further includes the extracellular Stalk domain of the Newcastle disease virus HN protein.

[0008] Preferably, the amino acid sequence of the partial domain of the HN protein of the Newcastle disease virus is shown as SEQ ID NO. 1 or SEQ ID NO. 2.

[0009] The application also provides a gene encoding the fusion protein HN-RBD described in the above scheme.

[0010] The application also provides the application of the fusion protein HN-RBD described in the above scheme or the gene encoding in improving the display amount of the S protein on the surface of the Newcastle disease virus envelope particle and / or improving the ability of the recombinant Newcastle disease virus to produce mucosal immune antibody IgA.

[0011] The application also provides a recombinant NDV virus vector, taking the Newcastle disease virus as the original vector, and inserting the gene encoding described in the above scheme and the gene encoding the S protein of the new coronavirus or the S protein variant of the new coronavirus; the gene encoding the S protein variant of the new coronavirus refers to the gene encoding a protein with a homology of more than 90% with the S protein of the new coronavirus; the S protein variant of the new coronavirus includes S2P, S2P / GSAS or S6P; the S2P is a K986P and V987P mutation of the extracellular domain of the S protein of the new coronavirus; the S2P / GSAS is a mutation of the amino acids RRAR at positions 682-685 to GSAS based on S2P; the S6P is a F817P, A892P, A899P and A942P mutation based on S2P.

[0012] Preferably, the gene encoding the S protein of the new coronavirus or the S protein variant of the new coronavirus is inserted between the P gene and the M gene of the Newcastle disease virus vector; and the gene encoding the fusion protein HN-RBD is inserted between the M gene and the F gene of the Newcastle disease virus vector or between the P gene and the M gene of the Newcastle disease virus vector.

[0013] The application also provides a recombinant NDV virus obtained by virus reverse genetics rescue from the recombinant NDV virus vector described in the above scheme.

[0014] The application also provides the application of the fusion protein HN-RBD described in the above scheme or the recombinant NDV virus vector or the recombinant NDV virus described in the above scheme in preparing a vaccine for preventing the infection of the new coronavirus.

[0015] The application also provides a vaccine for preventing the infection of the new coronavirus, comprising the recombinant NDV virus described in the above scheme.

[0016] The application provides a fusion protein HN-RBD, which comprises a partial domain of a Newcastle disease virus HN protein and a RBD of a SARS-CoV-2 S protein; the partial domain of the Newcastle disease virus HN protein comprises an intracellular domain and a transmembrane domain of the Newcastle disease virus HN protein. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 Results of Western Blot detection of the content of the SARS-CoV-2 S protein in the envelope of the virus particles, wherein Lane 1: NDV-S2P; Lane 2: NDV-S2P / GSAS; Lane 3: NDV-S2P-minispike; Lane 4: NDV-S2P-M-HSA-RBD-Fc-F; Lane 5: NDV-S2P / HN-RBD; Lane 6: NDV-S2P-HN-RBD; Lane 7: NDV-S2P / GSAS-HN-RBD; Lane 8: NDV-S2P-HN-GFP; Lane 9: Marker; HN is an internal reference protein for measuring the content of the virus particles;

[0018] Figure 2 A schematic diagram of the constructed recombinant virus, wherein the recombinant virus envelope surface containing the combination of S and HN RBD has more S protein;

[0019] Figure 3 Results of ELISA detection of S1 IgA in the nasal wash and lung lavage of mice, *P<0.05;

[0020] Figure 4 Results of ELISA detection of S1 IgG antibodies in the serum of mice immunized with inactivated recombinant NDV vaccine, **P<0.05. DETAILED DESCRIPTION

[0021] The application provides a fusion protein HN-RBD, which comprises a partial domain of a Newcastle disease virus HN protein and a RBD of a SARS-CoV-2 S protein; the partial domain of the Newcastle disease virus HN protein comprises an intracellular domain and a transmembrane domain of the Newcastle disease virus HN protein.

[0022] In the application, the partial domain of the Newcastle disease virus HN protein preferably further comprises an extracellular stalk domain of the Newcastle disease virus HN protein.

[0023] In the present application, the RBD is derived from the S protein of the original strain or the mutant strain of the novel coronavirus.

[0024] In the present application, the amino acid sequence of the intracellular domain and the transmembrane domain of the HN protein of the Newcastle disease virus is shown in SEQ ID NO. 1, specifically: MDRAVSQVALENDEREAKNTWRLIFRIAILFLTVVTLAISVASLL.

[0025] In the present application, when the domain of the HN protein of the Newcastle disease virus is composed of the intracellular domain, the transmembrane domain and the extracellular stalk domain of the HN protein of the Newcastle disease virus, the amino acid sequence is shown in SEQ ID NO. 2, specifically:

[0026] MDRAVSQVALENDEREAKNTWRLIFRIAILFLTVVTLAISVASLLYSMGASTPSDLVGIPTRISRAEEKITSTLGSNQDVVDRIYKQVALESPLALLNTETTIMNAITSLSYQINGAANNSG.

[0027] In the present application, the C terminal of the domain of the HN protein of the Newcastle disease virus is connected with the N terminal of the RBD of the S protein of the novel coronavirus through a protein linker. The present application does not have special restrictions on the sequence of the protein linker, and any protein linker known in the art can be used; the amino acid sequence of the protein linker is preferably: GGSG (SEQ ID NO: 3).

[0028] The present application also provides a coding gene of the fusion protein HN-RBD described in the above-mentioned scheme. In the present application, the coding gene of the fusion protein HN-RBD is codon-optimized. In the present application, the nucleotide sequence of the coding gene is preferably shown in SEQ ID NO. 4, specifically:

[0029]

[0030] The application also provides the fusion protein HN-RBD or the coding gene described in the above scheme for use in increasing the display amount of the surface S protein of the Newcastle disease virus envelope particle and / or improving the ability of the recombinant Newcastle disease virus to produce mucosal immune antibody IgA.

[0031] When the coding sequence of the fusion protein HN-RBD of the application and the coding sequence of the novel coronavirus S protein or the novel coronavirus S protein variant are inserted into the NDV virus carrier together, more novel coronavirus antigen S proteins can be displayed on the surface of the recombinant NDV virus particles, and the ability of the recombinant NDV virus to produce mucosal immune antibody IgA is improved.

[0032] The application also provides a recombinant NDV virus carrier, taking the Newcastle disease virus as the original carrier, and inserting the coding gene described in the above scheme and the coding gene of the novel coronavirus S protein or the novel coronavirus S protein variant; the coding gene of the novel coronavirus S protein variant refers to the coding gene of a protein with a homology of more than 90% with the novel coronavirus S protein; the novel coronavirus S protein variant includes S2P / GSAS, S2P or S6P; the S2P is a K986P and V987P mutation of the extracellular domain of the novel coronavirus S protein; the S2P / GSAS is a mutation of the amino acids 682-685 RRAR to GSAS based on S2P; the S6P is a F817P, A892P, A899P and A942P mutation based on S2P.

[0033]

[0034] In the present application, the amino acid sequence of the S2P is shown as SEQ ID NO. 6 or SEQ ID NO. 7; the S2P shown as SEQ ID NO. 6 is from the original strain, in particular:

[0035]

[0036] S2P shown in SEQ ID NO. 7 is from the Delta variant, specifically:

[0037]

[0038] In the present application, the coding gene of the novel coronavirus S protein or the novel coronavirus S protein variant is preferably inserted between the P gene and the M gene of the Newcastle disease virus vector; the coding gene of the fusion protein HN-RBD is preferably inserted between the M gene and the F gene of the Newcastle disease virus vector or between the P gene and the M gene of the Newcastle disease virus vector; and the coding gene is preferably inserted in the form of an expression cassette. The method for constructing the recombinant NDV virus vector in the present application is not particularly limited, and is preferably cloned into a backbone vector by homologous recombination to form a recombinant virus vector.

[0039] The present application also provides a recombinant NDV virus obtained by virus reverse genetics rescue of the recombinant NDV virus vector described in the above scheme. The method for virus reverse genetics rescue in the present application is not particularly limited, and a conventional method in the art can be used.

[0040] The present application also provides the use of the fusion protein HN-RBD described in the above scheme or the recombinant NDV virus vector or the recombinant NDV virus described in the present application in the preparation of a vaccine for preventing novel coronavirus infection.

[0041] The technical solutions in the present application will be described clearly and completely in combination with the embodiments in the present application.

[0042] Example 1: Antigen gene design and synthesis

[0043] The novel coronavirus S protein is subjected to V986P and K987P amino acid mutations to form a S2P novel coronavirus S protein variant. On the basis of S2P, the novel coronavirus R682-R685 amino acids are mutated to GSAS to form a S2P / GSAS novel coronavirus S protein variant, and the transmembrane structure region and the intracellular structure region of the S2P protein are deleted, and the amino acid sequence containing the transmembrane structure region and the intracellular structure region of the NDV F protein is fused to form a S2P / F34 novel coronavirus S protein variant. The RBD sequence at the N-terminus of the novel coronavirus S protein is fused to the 1-122 amino acids of the NDV HN protein through a protein linker GGSG (SEQ ID NO: 3) to form a HN-RBD fusion protein.

[0044] The N-terminal of green fluorescent protein GFP was fused with the 1-122 amino acids of NDV HN gene through a protein linker GGSG to form an HN-GFP fusion protein. The N-terminal of the new crown RBD protein was added with a human IgG heavy chain secretion signal peptide MKHLWFFLLLVAAPRWVLS (SEQ ID NO. 29), and the C-terminal was connected with the rabies virus G protein containing the transmembrane region and intracellular region part through a linker GSG to form a minispike fusion protein. After codon optimization of the coding sequence of the above protein, it was sent to Jinweizhi for gene synthesis.

[0045] Example 2 Construction of recombinant NDV vector

[0046] In this example, the variant sequence of the original strain S protein and the variant sequence of the Delta strain S protein were used. The NDV_SARS-COV-2_S (published in patent CN112011521A) recombinant virus vector was used as a template to design primers to clone S2P, S2P / GSAS, S2P / F34 into the P gene and M gene of the NDV vector by PCR and homologous recombination, replacing the original S gene in the vector, respectively, to form NDV-S2P / F34, NDV-S2P and NDV-S2P / GSAS recombinant virus vectors. Using the NDV-S2P and NDV-S2P / GSAS recombinant virus vectors as templates, primers were designed to clone HN-RBD, HN-GFP, minispike into the M gene and F gene positions of the above recombinant virus vectors by PCR and homologous recombination, respectively, to form NDV-S2P-HN-RBD, NDV-S2P-HN-GFP, NDV-S2P / GSAS-HN-RBD and NDV-S2P-minispike recombinant virus vectors. Using the NDV-S2P virus vector as a template, primers were designed to insert HN-RBD into the NDV virus vector between the P gene and the M gene after the S2P gene in the form of an expression cassette by PCR and homologous recombination to form an NDV-S2P / HN-RBD recombinant virus vector. The amplification primers are shown in Table 1, wherein the PCR process is to amplify the template according to the requirements of the Primer STAR enzyme instruction, and the homologous recombination is carried out according to the method of the above example 1. The HiFi DNA Assembly kit instructions require recombination ligation of PCR products. Among them, the S protein sequence in NDV-S2P / GSAS-HN-RBD, NDV-S2P-minispike, NDV-S2P / HN-RBD virus vectors all come from the original strain S protein variant; the S protein sequence in S2P / F34, NDV-S2P-HN-RBD and NDV-S2P-HN-GFP virus vectors all come from the Delta strain S protein variant. The ligation product is transformed into E. coli competent cells, and after single colony verification to confirm that the construction is correct, it is shaken overnight at 37°C in LB medium and the plasmid is extracted.

[0047] The amino acid sequence of S2P / F34 is shown in SEQ ID NO. 8, specifically:

[0048]

[0049] Table 1

[0050]

[0051]

[0052] Rescue of recombinant NDV virus of Example 3

[0053] The recombinant Newcastle disease virus vector of Example 2, and the plasmids of virus rescue helper pCI-NP, pCI-P and pCI-L, respectively, were transfected into BHK21-T7 cells after measuring the concentration of the plasmids by Nano drop. The transfection method is as follows: BHK21-T7 cells were cultured in a 6-well plate, and when the cells grew to 80%-90%, they were washed with sterile PBS for 3 times; 500 μL of Opti-MEM was taken in a 1.5 mL centrifuge tube, and then the recombinant Newcastle disease virus vector containing the S gene of the new coronavirus, pCI-NP, pCI-P and pCI-L plasmids were added in a ratio of 1:1:1:1, with a total of 4 μg of plasmids per tube for one well; 3 μL of Plus reagent was added to the above solution, and reacted at room temperature for 5 min; 9 μL of Lipofectamine LTX was added and mixed gently; incubate at room temperature for 30 min; add the transfection complex and 2 mL of Opti-MEM, and incubate in a 37°C carbon dioxide incubator for 18-48 h.

[0054] After 24 h of transfection, TPCK trypsin was added at a final concentration of 0.5 μg / μL, and after 72 h of transfection, the cells were repeatedly frozen and thawed 3 times, and the mixture was inoculated into 9-day-old SPF chicken embryos at 0.5 mL / embryo. Discard the dead embryos within 24 h, collect the allantoic fluid of all dead and surviving chicken embryos within 24-120 h, and determine the HA titer one by one. The obtained embryo fluid with HA activity was sequenced to verify that the insertion position was correct and the inserted sequence had not been mutated; the obtained rescue virus was named NDV-S2P / GSAS, NDV-S2P, NDV-S2P / HN-RBD, NDV-S2P / GSAS-HN-RBD, NDV-S2P-HN-RBD, NDV-S2P-minispike and NDV-S2P-HN-GFP.

[0055] The virus HA titer is shown in Table 2:

[0056] Table 2

[0057]

[0058]

[0059] As shown in the results in Table 2, the rescued viruses containing S protein or S protein variants and fusion protein HN-RBD all have HA titers of 7.5 or more, and generally after passage adaptation, the virus titers can be significantly improved, and the titers can reach 8-11 Log2. It can be seen that the recombinant viruses produced by the method of the present application can meet the actual production requirements of vaccines.

[0060] Example 4 Purification of Virions

[0061] A 5X PEG8000+NaCl solution was configured, 8.766g of NaCl and 50g of PEG8000 were dissolved in 200ml of Milli-Q pure water, wet heat sterilized at 121℃ for 30min, and stored at 4℃ for standby. The NDV recombinant virus supernatant was filtered through a 0.45μm pore size filter, 7.5ml of 5X PEG-8000+NaCl stock solution was added to each 30ml of filtered virus initial liquid, and it was inverted and mixed every 20-30min, mixed for 3-5 times, and then left at 4℃ overnight. Then centrifuged at 4℃, 4000g for 20min, the supernatant was aspirated, the tube was left to stand for 1-2min, the residual liquid was aspirated, and an appropriate amount of Opti-MEM was added to dissolve the NDV virus precipitate, which was used later.

[0062] Example 5 Western Blot

[0063] The purified virus was added to the SDS loading buffer, boiled at 95℃ for 10min, and added to the assembled precast gel well, with a loading amount of 10μl per well, and 10μl of protein marker was loaded. Electrophoresis at 80V for 20min, and then electrophoresis at 120V for 50min, the electrophoresis gel plate was carefully pried open, and the whole gel was cut and placed in the transfer solution for 10min. A "sandwich" was formed by placing 1 layer of sponge, 2-3 filter papers, the separation gel, the PVDF membrane treated with methanol, 2-3 filter papers, and 1 layer of sponge from top to bottom. The wet transfer system was assembled, filled with pre-cooled transfer solution, and the "sandwich" was immersed. A constant current of 300mA was applied for wet transfer for 50-70min. The membrane was removed from the wet transfer system and blocked with blocking solution on a shaker at room temperature for 20min. The blocked membrane was rinsed with 0.1% PBST for 3 times, 5min each time, incubated with primary antibody at room temperature for 1h, rinsed with 0.1% PBST for 3 times, 5min each time, and then incubated with secondary antibody on a shaker at room temperature for 60min. After incubation, the membrane was rinsed with 0.1% PBST for 3 times, 10min each time. ECL luminescent solution A and B were mixed, and the membrane was exposed and photographed under a chemiluminescence instrument. The results are shown in Figure 1 .

[0064] From Figure 1The results show that in the NDV-S2P / HNRBD, NDV-S2P-HNRBD and NDV-S2P / GSAS-HNRBD recombinant viruses, more S protein or S1 cleavage product of S protein expression is detected (using HN protein as the internal reference of virus particle content) than the recombinant virus NDV-S2P / GSAS and NDV-S2P containing only S protein sequence, and the NDV-S2P-minispike and NDV-S2P-deltaRBD-Fc recombinant viruses containing one S protein and one non-HN-RBD fusion protein sequence. The two RBD fusion proteins minispike and deltaRBD-Fc represent two forms of RBD expression, membrane expression and secretory expression, among which minispike belongs to membrane protein expression together with HN-RBD. Therefore, when the S protein and RBD fusion protein coding sequences are inserted into the NDV virus together, only the recombinant virus containing the combination of S protein and HN-RBD fusion protein has a higher display amount of S protein on the surface of the viral envelope. It can be seen that HN-RBD can increase the display amount of S protein on the surface of the viral envelope Figure 2 )。And this effect is not affected by the insertion site of HN-RBD, that is, insertion between the P gene and the M gene of the NDV vector, or between the M gene and the F gene can have the same effect.

[0065] Example 6 Mouse immunization and mucosal antibody detection

[0066] Select the allantoic fluid of the strain with HA titer reaching 7Log2 or more, and immunize 4-5 week old Balb\c female mice through the nose. The immunization method is as follows: 5% chloral hydrate is injected intraperitoneally, with a dose of 0.1 mL per 20 g of mouse weight to anesthetize the animals. After anesthesia, the animals are immunized by nose drops using a pipette, 25 μl per nostril. A total of two immunizations are performed, with a second boost immunization 7 days after the first immunization. At 16 days after the first immunization, nasal wash and alveolar lavage of the anesthetized mice are collected. According to the requirements of the KIT007 kit of Yiqiao Shenzhou, the content of mouse mucosal new crown S1 IgA antibody is detected. All the obtained OD450 data are divided by the background OD450 value of the blank control mice, and the relative S1 IgA content is obtained after normalization of the data. The results are shown in Figure 3The results show that there is no significant difference in the content of new crown S1 IgA antibody in nasal wash, which may be related to the weak ability of nasal mucosa to secrete antibodies. In the bronchoalveolar lavage fluid, the ability of recombinant viruses containing S protein variants combined with fusion protein HN-RBD to stimulate mucosa to produce new crown S1 IgA antibody has been greatly improved. The production of new crown S1 IgA antibody by NDV-S2P / HNRBD and NDV-S2P-HNRBD recombinant viruses is significantly different from the control group NDV-S2P / GSAS, NDV-S2P-minispike and NDV-S2P-deltaRBD-Fc (*P<0.05). Therefore, recombinant NDV viruses containing S protein variants combined with fusion protein HN-RBD have the ability to produce higher mucosal immune antibodies.

[0067] Example 7 Preparation of inactivated recombinant NDV vaccine and immunization of mice

[0068] The above-mentioned recombinant viruses NDV-S2P / GSAS-HNRBD and NDV-S2P / GSAS harvest liquid were used to inoculate chicken embryos, and the allantoic fluid of the chicken embryos was harvested after 72 h of culture at 34°C. After inactivation treatment with 0.1% formaldehyde at 4°C, 10-day-old chicken embryos were inoculated, and security checks confirmed that the virus had been inactivated. The inactivated virus was mixed uniformly with CpG adjuvant, and 20 μl per mouse was injected intramuscularly into 4-5-week-old Balb / c female mice. Blood was collected 14 days after immunization to obtain serum. According to the requirements of the reagent instruction manual of the Yiqiao God China New Crown Virus SARS-CoV-2 Spike S1 Antibody Titer Assay kit, the serum samples were diluted 100 times and the S1 IgG content of the serum was detected. The results are shown in Figure 4 Compared with NDV-S2P / F34, the OD450 value of the antibody produced by NDV-S2P / GSAS-HN-RBD inactivated virus is nearly doubled, i.e. the recombinant virus containing S and fusion protein HN-RBD produces stronger blood immune S1 IgG antibody (**P<0.01). Since the virus has been inactivated, the inactivated recombinant virus can no longer infect host cells to express the corresponding antigen protein, so the immunogenicity of the inactivated virus is mainly from the proteins carried by the virus particles. Combined with the results of Example 6, it can be seen that the recombinant viruses containing S protein variants combined with fusion protein HN-RBD have the ability to produce higher mucosal immune antibodies and blood immune S1 IgG antibodies. Figure 1The results show that the ability of S1 IgG antibody production is mainly from the S protein or S protein variant on the surface of viral envelope particles and the trace amount of HN-RBD. Since the HN-RBD in this embodiment is inserted downstream of the S protein variant, its content is not higher than that of S2P / GSAS, and the S1 IgG antibody produced by the HN-RBD is not as good as that of the S2P / GSAS protein. Therefore, it can be inferred that the increase of nearly one time of S1 IgG antibody is mainly contributed by more S2P / GSAS protein on the surface of viral envelope. It is further illustrated that when the HN-RBD is inserted into the NDV vector with the S protein or its variant, the HN-RBD can increase the display amount of the S protein on the surface of the NDV viral envelope. At the same time, more S protein on the surface of the viral particles can greatly improve the immunogenicity of the inactivated vaccine.

[0069] Although the above embodiments have made a detailed description of the present application, it is only a part of the embodiments of the present application but not all the embodiments. Other embodiments can be obtained according to the above embodiments without creativity, and these embodiments all belong to the protection scope of the present application.

Claims

1. A fusion protein HN-RBD, composed of a partial domain of the Newcastle disease virus HN protein, the RBD of the SARS-CoV-2 S protein, and a protein linker; The amino acid sequence of a portion of the structural domain of the Newcastle disease virus HN protein is shown in SEQ ID NO.2; The C-terminus of the Newcastle disease virus HN protein is connected to the N-terminus of the RBD of the SARS-CoV-2 S protein via a protein linker; the amino acid sequence of the protein linker is: GGSG; The amino acid sequence of the RBD of the SARS-CoV-2 S protein is: RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGSKPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCS.

2. The gene encoding the fusion protein HN-RBD as described in claim 1.

3. The encoding gene according to claim 2, characterized in that, The nucleotide sequence of the encoding gene is shown in SEQ ID NO.

4.

4. The use of the fusion protein HN-RBD of claim 1 or the encoding gene of claim 2 or 3 in increasing the display of S protein on the surface of Newcastle disease virus envelope particles and / or increasing the ability of recombinant Newcastle disease virus to produce mucosal immune antibodies IgA.

5. A recombinant NDV viral vector, using Newcastle disease virus as the original vector, inserting the coding gene as described in claim 2 or 3, and the coding gene for the SARS-CoV-2 S protein or a variant of the SARS-CoV-2 S protein; The SARS-CoV-2 S protein variant is either S2P / GSAS or S2P; The S2P is a mutation of the extracellular domain of the SARS-CoV-2 S protein using K986P and V987P. The S2P / GSAS is based on S2P, with the amino acid RRAR mutation at positions 682-685 being replaced by GSAS. The amino acid sequence of the S2P / GSAS is shown in SEQ ID NO.5; The amino acid sequence of the S2P is shown in SEQ ID NO.6 or SEQ ID NO.7; the S2P shown in SEQ ID NO.6 is from the original strain; the S2P shown in SEQ ID NO.7 is from the Delta variant strain. The gene encoding the SARS-CoV-2 S protein or a variant of the SARS-CoV-2 S protein is inserted between the P gene and the M gene in the Newcastle disease virus vector, replacing the S gene in the original vector. The coding gene of claim 2 or 3 is inserted between the M gene and the F gene of the Newcastle disease virus vector or between the P gene and the M gene of the Newcastle disease virus vector.

6. A recombinant NDV virus, obtained by viral reverse genetics rescue from the recombinant NDV virus vector of claim 5.

7. The use of the recombinant NDV viral vector of claim 5 or the recombinant NDV virus of claim 6 in the preparation of a vaccine for the prevention of novel coronavirus infection.

8. A vaccine for preventing novel coronavirus infection, comprising the recombinant NDV virus as described in claim 6.

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