Recombinant protein, biological material, avian influenza and Newcastle disease chimeric virus-like particle, vaccine as well as preparation method and application of avian influenza and Newcastle disease chimeric virus-like particle

Abstract

The invention provides a recombinant protein, a biological material, an avian influenza and Newcastle disease chimeric virus-like particle, a vaccine and a preparation method and application thereof, the recombinant protein comprises an SOC protein, a Newcastle disease virus HN protein and an avian influenza virus HA protein, the amino acid sequence of the recombinant protein is shown as SEQ ID NO.1, the amino acid sequence of the SOC protein is shown as SEQ ID NO.2, the amino acid sequence of the Newcastle disease virus HN protein is shown as SEQ ID NO.1, and the amino acid sequence of the Newcastle disease virus HN protein is shown as SEQ ID NO.2. The amino acid sequence of the Newcastle disease virus HN protein is as shown in SEQ ID NO.3, and the amino acid sequence of the avian influenza virus HA protein is as shown in SEQ ID NO.4. The avian influenza and Newcastle disease chimeric virus-like particle is formed by assembling the recombinant protein in vitro. Compared with active avian influenza virus and Newcastle disease virus, the virus-like particle prepared by the invention can induce effective cell and humoral immune response under the condition of no adjuvant, has strong immunogenicity, does not have the risk of incomplete inactivation or virulence recovery, and can be prepared into a safe, stable, multivalent and efficient bivalent subunit vaccine.

Description

Technical Field

[0001] The present invention relates to the technical field of bioengineering agent virus vaccines, and in particular to a recombinant protein, a biomaterial, an avian influenza and Newcastle disease chimeric virus-like particle, a vaccine, and a preparation method and application thereof. Background Art

[0002] Avian influenza viruses, with their numerous subtypes, can infect humans and other mammals, posing a significant threat to both the livestock industry and public health. Vaccination is an effective measure for preventing and controlling major avian influenza outbreaks. However, current vaccines, primarily based on subtype-specific and protective antigens, induce antibodies that lack cross-protection, making avian influenza prevention and control extremely challenging. Failure to effectively and effectively prevent and control a pandemic could result in significant economic losses. Furthermore, the primary antigenic sites of influenza viruses are susceptible to antigenic drift and even mutation under long-term selective pressure. The search for a universal vaccine that protects against multiple, or even all, influenza viruses has become a hot topic in avian influenza vaccine research in recent years.

[0003] Newcastle disease (ND) is a viral infectious disease caused by Newcastle disease virus (NDV) of the Paramyxoviridae family. The World Organization for Animal Health lists it as a notifiable animal disease. Due to its high mortality rate and strong infectivity, it causes huge economic losses to the poultry industry.

[0004] Since George Smith reported in 1985 the fusion of a foreign peptide to the minor capsid gene product (gp) pIII on the filamentous phage f1, numerous phages have been developed as antigen delivery vectors. Over 5,000 phages have been studied, providing a vast selection of phages for vaccine delivery. Recent studies have demonstrated that phage T4 VLPs are highly immunogenic, require no adjuvant, and provide complete protection against bacterial and viral pathogens. Therefore, T4 phage could be designed as a novel multivalent, multi-component avian vaccine vector for complex and emerging pathogens. While there are reports on vaccine design using T4 as a vector, most suffer from low assembly efficiency, and there are no patents related to the development of a combined influenza and Newcastle disease vaccine vector. Summary of the Invention

[0005] In view of this, the present invention proposes a recombinant protein, a biomaterial, an avian influenza and Newcastle disease chimeric virus-like particle, a vaccine, and a preparation method and application thereof. The present invention overcomes the low in vivo assembly efficiency of phage by expressing the antigen protein in vitro and then recombining it with a bacteriophage in vitro. By providing chimeric virus-like particles of avian influenza virus and Newcastle disease virus for vaccine preparation, the safety problems of existing vaccines are effectively solved. The prepared double vaccine has high antibody titer and obvious effect. It has a wide range of applications and can be designed to prevent multiple avian influenza and Newcastle disease viruses at the same time. It has good practical application value and also provides a reference for the future design of more broad-spectrum and universal vaccines.

[0006] The technical solution of the present invention is achieved as follows:

[0007] In a first aspect, the present invention provides a recombinant protein comprising SOC protein, Newcastle disease virus HN protein and avian influenza virus HA protein,

[0008] Among them, the amino acid sequence of the recombinant protein is shown in SEQ ID NO.1, the amino acid sequence of the SOC protein is shown in SEQ ID NO.2, the amino acid sequence of the Newcastle disease virus HN protein is shown in SEQ ID NO.3, and the amino acid sequence of the avian influenza virus HA protein is shown in SEQ ID NO.4.

[0009] On the basis of the above technical solution, further, the avian influenza virus is an H3 subtype avian influenza virus.

[0010] The amino acid sequence of the recombinant protein (SEQ ID NO.1):MGGYVNIKTFTHPAGEGKEVKGMEVSVPFEIYSNEHRIADAHYQTFPSEKAAYTTVVTDAADWRTKNAAMFTPTPVSGGGGGSGGGGSKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNA ELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFGGGGSGGGGSDDEQARLNPVSAVFDNISRSRVTRVSSSSTKAAYTTSTCFKVVKTNKAYCLSIAEISNTLFGEFRIVPLLVEILKDDRV.

[0011] The amino acid sequence of the SOC protein (SEQ ID NO.2):

[0012] MGGYVNIKTFTHPAGEGKEVKGMEVSVPFEIYSNEHRIADAHYQTFPSEKAAYTTVVTDAADWRTKNAAMFTPTPVSG.

[0013] The amino acid sequence of the Newcastle disease virus HN protein (SEQ ID NO. 3) is: DDEQARLNPVSAVFDNISRSRVTRVSSSSTKAAYTTSTCFKVVKTNKAYCLSIAEISNTLFGEFRIVPLLVEILKDDRV.

[0014] The amino acid sequence of the avian influenza virus HA protein (SEQ ID NO. 4) is: KMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTL DFHDSNVKNLYDKVRLQLRDNAKELGNGCFEF.

[0015] On the basis of the above technical solution, further, the nucleotide sequence of the recombinant protein is shown as SEQ ID NO.5, the nucleotide sequence of the SOC protein is shown as SEQ ID NO.6, the nucleotide sequence of the Newcastle disease virus HN protein is shown as SEQ ID NO.7, and the nucleotide sequence of the avian influenza virus HA protein is shown as SEQ ID NO.8.

[0016] The nucleotide sequence of the recombinant protein (SEQ ID NO.5): ATGGGTGGTTATGTAAACATCAAAACCTTTACGCATCCTGCTGGTGAAGGTAAAGAAGTTAAAGGTATGGAAGTTTCTGTACCGTTTGAGATTTATTCAAACGAACATCGGATTGCTGATGCTCATTATCAGACTTTCCCATCTGAAAAAGCTGCTTACACTACTGTGGTGACTGACGCAGCAGATTGGCGTACTAAGAACGCTGCAATGTTTACCCCTACACCAGTAAGTGGTGGTGGCGGTGGTAGCAAAATGAACACCCAGTTTGAAGCAGTCGGCCGTGAATTCAACAATCTGGAGCGTCGTATCGAGAACCTGAACAAAAAAATGGAGGATGGCTTCCTGGACGTTTGGACCTACAATGCAGAACTGCTGGTTCTGATGGAGAACGAACGCACCCTGGATTTTCACGACTCTAACGTTAAAAACCTGTACGACAAAGTTCGTCTGCAGCTGCGTGATAACGCAAAAGAACTGGGTAACGGCTGCTTCGAATTTGGTGGCGGTGGTAGCGACGACGAGCAGGCGCGTTTGAACCCGGTTAGCGCAGTTTTCGACAACATAAGTCGTAGCCGCGTGACTCGCGTCTCCAGCTCGTCAACCAAAGCCGCATACACCACCAGCACCTGCTTTAAAGTCGTGAAAACCAATAAGGCCTACTGCCTGTCTATTGCAGAGATTTCCAATACCTTGTTCGGCGAATTCCGTATCGTGCCGCTGTTGGTGGAAATCCTTAAGGACGACCGCGTG。

[0017] As shown in the nucleotide sequence of the SOC protein (SEQ ID NO.6):

[0018] ATGGGTGGTTATGTAAACATCAAAACCTTTACGCATCCTGCTGGTGAAGGTAAAGAAGTTAAAGGTATGGAAGTTTCGTACCGTTTGAGATTTATTCAAACGAACATCGGATTGCTGATGCTCATTATCAGACTTTCCCATCTGAAAAAGCTGCTTACACTACTGTGGTGACTGACGCAGCAGATTGGCGTACTAAGAACGCTGCAATGTTTACCCCTACACCAGTAAGTGGT.

[0019] The nucleotide sequence of the Newcastle disease virus HN protein (SEQ ID NO.7) is: GACGACGAGCAGGCGCGTTTGAACCCGGTTAGCGCAGTTTTCGACAACATAAGTCGTAGCCGCGTGACTCGCGTCTCCAGCTCGTCAACCAAAGCCGCATACACCACCAGCACCTGCTTTAAAGTCGTGAAAACCAATAAGGCCTACTGCCTGTCTATTGCAGAGATTTCCAATACCTTGTTCGGCGAATTCCGTATCGTGCCGCTGTTGGTGGAAATCCTTAAGGACGACCGCGTG.

[0020] The nucleotide sequence of the avian influenza virus HA protein (SEQ ID NO.8): AAAATGAACACCCAGTTTGAAGCAGTCGGCCGTGAATTCAACAATCTGGAGCGTCGTATCGAGAACCTGAACAAAAAAATGGAGGATGGCTTCCTGGACGTTTGGACCTACAATGCAGAACTGCTGGTTCTGATGGAGAACGAACGCACCCTGGATTTTCACGACTCTAACGTTAAAAACCTGTACGACAAAGTTCGTCTGCAGCTGCGTGATAACGCAAAAGAACTGGGTAACGGCTGCTTCGAATTT.

[0021] On the basis of the above technical solution, further, the Newcastle disease virus HN protein and the avian influenza virus HA protein constitute an HA-HN concatemer, and the amino acid sequence of the HA-HN concatemer is shown in SEQ ID NO.9.

[0022] The amino acid sequence of the HA-HN concatemer (SEQ ID NO. 9): KMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFGGGGSGGGGSDDEQARLNPVSAVFDNISRSRVTRVSSSSTKAAYTTSTCFKVVKTNKAYCLSIAEISNTLFGEFRIVPLLVEILKDDRV.

[0023] On the basis of the above technical solution, further, the nucleotide sequence of the HA-HN concatemer is shown as SEQ ID NO.10.

[0024] The nucleotide sequence of the HA-HN concatemer (SEQ ID NO.10): AAAATGAACACCCAGTTTGAAGCAGTCGGCCGTGAATTCAACAATCTGGAGCGTCGTATCGAGAACCTGAACAAAAAAATGGAGGATGGCTTCCTGGACGTTTGGACCTACAATGCAGAA CTGCTGGTTCTGATGGAGAACGAACGCACCCTGGATTTTCACGACTCTAACGTTAAAAACCTGTACGACAAAGTTCGTCTGCAGCTGCGTGATAACGCAAAAGAACTGGGTAACGGCTGCTTCGAAT TTGGTGGCGGTGGTAGCGACGACGAGCAGGCGCGTTTGAACCCGGTTAGCGCAGTTTTCGACAACATAAGTCGTAGCCGCGTGACTCGCGTCTCCAGCTCGTCAACCAAAGCCGCATACACCACCAG CACCTGCTTTAAAGTCGTGAAAACCAATAAGGCCTACTGCCTGTCTATTGCAGAGATTTCCAATACCTTGTTCGGCGAATTCCGTATCGTGCCGCTGTTGGTGGAAATCCTTAAGGACGACCGCGTG.

[0025] In a second aspect, the present invention provides a biomaterial comprising the above-mentioned recombinant protein, wherein the biomaterial further comprises any one of a recombinant expression vector, a recombinant microorganism, an expression cassette, a cell strain, and a cell line.

[0026] In a third aspect, the present invention provides an avian influenza and Newcastle disease chimeric virus-like particle, which is assembled in vitro from the above-mentioned recombinant protein.

[0027] Compared to live avian influenza virus and Newcastle disease virus, the virus-like particles produced by this invention can induce effective cellular and humoral immune responses without an adjuvant, exhibit strong immunogenicity, and eliminate the risk of incomplete inactivation or reversion to virulence. They can be formulated into safe, stable, multivalent, and highly effective bivalent subunit vaccines. Compared to currently available attenuated or inactivated vaccines, virus-like particles offer a safer alternative.

[0028] On the basis of the above technical solution, further, the phage assembled in vitro is T4 phage.

[0029] On the basis of the above technical solution, further, the T4 phage is a Hoc-Soc-T4 phage.

[0030] On the basis of the above technical solution, the dosage ratio of the recombinant protein to the Hoc-Soc-T4 phage is 1 mg: 5×10 10 Hoc-Soc-T4 phage particles.

[0031] On the basis of the above technical solution, further, the buffer of the in vitro assembly buffer comprises 20 mM Tris, 150 mM NaCl, 0.1 mM CaCl2, 0.05 mM MgCl2, 0.5 mM sucrose, and 0.2% glycerol.

[0032] In a fourth aspect, the present invention provides a method for preparing the above-mentioned avian influenza and Newcastle disease chimeric virus-like particles, comprising the following steps:

[0033] S1. constructing an expression vector for the recombinant protein;

[0034] S2, expressing and purifying the recombinant protein;

[0035] S3. Assembling the purified recombinant protein with T4 phage in vitro to obtain the avian influenza and Newcastle disease chimeric virus-like particles.

[0036] In a fifth aspect, the present invention provides an avian influenza and Newcastle disease chimeric virus-like particle vaccine, comprising the above-mentioned avian influenza and Newcastle disease chimeric virus-like particles.

[0037] In a sixth aspect, the present invention provides a use of the above-mentioned recombinant protein, or biomaterial, or avian influenza and Newcastle disease chimeric virus-like particles in the preparation of vaccines or drugs for treating or preventing diseases caused by avian influenza and Newcastle disease viruses.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] (1) The present invention provides chimeric virus-like particles of avian influenza virus and Newcastle disease virus for use in vaccine preparation, thereby effectively solving the safety problems of existing vaccines. The prepared vaccine has high antibody titer and obvious effect. It has a wide range of applications and can be designed to prevent multiple avian influenza and Newcastle disease viruses at the same time. It has good practical application value and also provides a reference for the future design of more broad-spectrum and universal vaccines.

[0040] (2) The present invention can obtain a broad-spectrum avian influenza vaccine, a Newcastle disease virus and an avian influenza double vaccine.

[0041] (3) The vaccine obtained by the present invention is more economical, safer, more efficient and convenient to transport and use. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0043] Figure 1 is the PCR result of the avian influenza virus HA sequence in the present invention, and M is the molecular mass standard of 2000 bp;

[0044] Figure 2 is the PCR result of the HN sequence of Newcastle disease virus in the present invention, and M is the molecular mass standard of 2000 bp;

[0045] Figure 3 The PCR result of the tandem combination of the HA sequence of the avian influenza virus and the HN sequence of the Newcastle disease virus in the present invention, M is a 2000 bp molecular weight standard;

[0046] Figure 4 is the PCR result of HA-HN fusion expression in the present invention, M is the molecular weight standard of 2000 bp;

[0047] Figure 5 is the PCR result of the pER-28a vector fragment in the present invention, and M is the molecular mass standard of 5000 bp;

[0048] Figure 6 The colony PCR results of the fusion expression of the HA sequence of the H3 subtype avian influenza virus and the HN sequence of the Newcastle disease virus with the viral protein P in the present invention, M is the molecular weight standard of 2000 bp;

[0049] Figure 7The bands of P-HA-HN protein induced expression after fusion with viral protein P are shown in Figure 2. M is the 180KDa protein molecular weight standard. 1 is the P-HA-HN protein supernatant. 2 is the P-HA-HN protein precipitate.

[0050] Figure 8 is the standard curve of P-HA-HN;

[0051] Figure 9 is the purified band of P-HA-HN protein after fusion expression with viral protein P, M is the 180KDa protein molecular weight standard, and 1 is the pure P-HA-HN protein;

[0052] Figure 10 WB images of P-HA-HN and SDS-PAGE images of the assembled P-HA-HN protein and virus-like particles, M is a 180KDa protein molecular weight standard, 1 is WT phage, and 2 is P-HA-HN-T4-VLP;

[0053] Figure 11 This is the electron microscopic observation of P-HA-HN-T4 chimeric phage virus particles;

[0054] Figure 12 This is an Elisa diagram of avian influenza virus and Newcastle disease virus positive serum and P-HA-HN protein. The columns represent different dilution times of the positive serum. DETAILED DESCRIPTION

[0055] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0056] The method for preparing the avian influenza and Newcastle disease chimeric virus-like particles of the present invention comprises the following steps:

[0057] (1) Antigen design

[0058] Using the avian influenza virus antigen protein HA and the Newcastle disease virus antigen protein HN as antigens, a bioinformatics approach was applied to select conserved HA sequences from avian influenza viruses and combine them with highly antigenic conserved HN fragments to generate HA-HN concatemers. A prokaryotic expression vector for this concatemer was constructed, providing a theoretical basis for further studying the immunogenicity of the HA+HN proteins and developing broad-spectrum or universal avian influenza and Newcastle disease virus vaccines with cross-protection.

[0059] (2) Expression and purification of recombinant antigens

[0060] The recombinant protein including the above-mentioned recombinant antigen and SOC protein was constructed into the pET-28a vector for induced expression and purified using a nickel column. The purity of the recombinant protein was then detected by SDS-PAGE and the concentration was detected by a BCA kit.

[0061] (3) Production and purification of virus-like particles

[0062] The knockout phage obtained in the early stage is purified, and the purified recombinant protein and phage are assembled in vitro to obtain virus-like particles.

[0063] In the following specific embodiments, the Hoc-Soc-T4 phage is derived from and preserved by the research group.

[0064] Example 1: Construction and identification of Newcastle disease virus HN protein and H3N2 subtype avian influenza virus HA protein

[0065] The genome of H3N2 subtype avian influenza virus was reverse transcribed to obtain cDNA, and the cDNA was used as a template to amplify the HA fragment by PCR. The target gene size was 249 bp ( Figure 1 ), the nucleotide sequence is shown in SEQ ID NO.8, the primer sequences are shown in Table 1 below, namely SEQ ID NO.11 and SEQ ID NO.12, and the PCR program system is shown in Table 2.

[0066] The HN protein gene was synthesized and primers were designed for PCR. The target gene was 237 bp in size and the nucleotide sequence was shown in SEQ ID NO.7. The primer sequences were shown in Table 1, namely SEQ ID NO.13 and SEQ ID NO.14. The PCR program system was shown in Table 2.

[0067] The two ends of the gene were connected by overlap extension PCR, and the target gene size was 501 bp ( Figure 3 ), the nucleotide sequence is shown in SEQ ID NO.10.

[0068] Table 1 Primers for HA and HN

[0069]

[0070] Table 2 PCR program

[0071]

[0072] Example 2: Construction of a fusion fragment of SOC protein and HA-HN fragment

[0073] The relevant homology arm primers were designed, and the primer sequences were shown in Table 3. PCR was performed on the HA-HN fragment, which was approximately 547 bp ( Figure 4 ).

[0074] The vector fragment connected to the viral protein P (SOC protein) was subjected to PCR and the size was 5491 bp ( Figure 5 ) The results were confirmed by 1% agarose gel electrophoresis and were consistent with the expected bands. The nucleotide sequence of viral protein P (SOC protein) is shown in SEQ ID NO.6.

[0075] Table 3 Primers for viral protein P and HA-HN fragments

[0076]

[0077] Example 3: Construction and identification of expression vector pET28a-P-HA-HN

[0078] After the PCR product was confirmed to be correct by 1% agarose gel electrophoresis, the target gene was recovered and homologous recombination was performed.

[0079] The recombinant product was transformed into E. coli DH5α and plated on LB culture plates containing kanamycin. After culturing overnight at 37°C, colonies were picked for PCR identification ( Figure 6 ), after correct identification, pick the correct colony and place it in LB medium containing kanamycin and shake at 37℃ overnight.

[0080] Subsequently, a plasmid extraction kit (Tiangen) was used to extract the plasmid according to the instructions, and sequencing was performed to obtain the correct pET28a-P-HA-HN expression plasmid.

[0081] Example 4: Solubility analysis and purification of P-HA-HN recombinant protein

[0082] 1. Induce P-HA-HN recombinant protein expression

[0083] (1) The pET28a-P-HA-HN expression plasmid was transformed into Escherichia coli BL21 (DE3) competent cells by electroporation, and coated on LB solid plates containing 50 μg / mL kanamycin. The cells were cultured at 37°C overnight. A single colony with good growth was picked and placed in LB liquid medium containing 50 μg / mL kanamycin and cultured overnight.

[0084] (2) Inoculate 10 mL of the overnight culture into 1 L of fresh LB liquid medium containing 50 μg / mL kanamycin until the culture OD600 reaches 0.8.

[0085] (3) Induce with 1 mM isopropyl-b-dithiogalactoside (IPTG) at 30°C for 4 h.

[0086] (4) Centrifuge at 4300 g for 15 min to collect the E. coli cells and resuspend the bacteria in Binding buffer.

[0087] (5) Cells were lysed using a high-pressure cell disruptor at 4°C and cell debris was removed by centrifugation at 35,000 g for 20 min at 4°C.

[0088] After the expression product was crushed by high pressure, the supernatant and precipitate were analyzed by SDS-PAGE. The results showed that the P-HA-HN recombinant protein was mainly located in the precipitate and existed in the form of inclusion bodies ( Figure 7 ). After inclusion body purification, a relatively pure P-HA-HN recombinant protein (28Kda) was obtained ( Figure 7 ).

[0089] 2. Purification of P-HA-HN recombinant protein

[0090] (1) Rinse the HisTrap column with approximately 10 mL of ultrapure water.

[0091] (2) Use approximately 5 mL of lysis buffer (4×lysis buffer: 2 M NaCl, 80 mM Tris base, 400 mM EDTA plus distilled water to 100 mM, pH 7.9) to equilibrate the HisTrap column.

[0092] (3) The supernatant containing the P-HA-HN recombinant protein was passed through a 0.22 μm filter and loaded onto a HisTrap column using a peristaltic pump.

[0093] (4) Use approximately 30 mL of wash buffer to remove non-specifically bound proteins from the column.

[0094] (5) Elution was performed using 4× Elution Buffer (4× Elution Buffer: NaCl 11.668 g, 2 M Tris base 0.9691280 mM, imidazole 27.232 g, 4 M, distilled water to 100 ml, pH 7.9). Approximately 10 mL of protein-containing solution was collected.

[0095] (6) Use the Broadford protein concentration detection kit to detect the concentration of the protein collected in the EP tube.

[0096] (7) Load a Hi-load 16 / 60 Superdex 200 column onto a protein purifier.

[0097] (8) A Hi-load 16 / 60 Superdex 200 column was washed with 150 mL of gel filtration buffer using an AKTA system.

[0098] (9) Collect 5 mL of high-concentration protein from (6) and pass it through a chromatography column using an AKTA system.

[0099] (10) The peak protein in the gel filtration elution curve was collected and concentrated using a 10KD protein ultrafiltration centrifuge tube.

[0100] The protein concentration was determined by BCA assay and the cells were stored at -80°C for later use.

[0101] The BCA protein concentration was determined as follows:

[0102] (1) Prepare BSA protein standard solutions at concentrations of 5 mg / mL and 1 mg / mL.

[0103] (2) Prepare an appropriate amount of BCA working solution by adding 50 volumes of BCA reagent A to 1 volume of BCA reagent B (50:1) and mix thoroughly.

[0104] (3) Add 0 μL, 0.5 μL, 2.5 μL, 5 μL, 10 μL, 15 μL, and 20 μL of 1 mg / mL BSA protein standard solution and 6 μL and 8 μL of 5 mg / mL BSA protein standard solution to the standard wells of a 96-well plate, and dilute to 20 μL with appropriate amount of PBS.

[0105] (4) Add the appropriate protein sample to be tested to the sample wells of a 96-well plate and dilute to 20 μL with PBS.

[0106] (5) Add 200 μL of BCA working solution to each well, mix well, and incubate at 37°C for 30 min.

[0107] (6) Measure the absorbance at A562 nm using an enzyme-labeled instrument. Calculate the protein concentration of the sample by drawing a standard curve, such as Figure 8 shown.

[0108] Example 5: In vitro assembly of P-HA-HN-T4

[0109] 1. Phage proliferation and purification

[0110] 1. Phage proliferation:

[0111] (1) Transfer the overnight cultured E. coli P301 into LB / M9CA medium at a volume ratio of 1:100.

[0112] (2) When the bacterial concentration reaches 1.5-2.0×10 8When the concentration of cells / mL was 0.5, the Hoc-Soc-T4 phage stored by the previous research group was used to infect the bacteria at a multiplicity of infection (MOI) of 0.1. The cells were incubated at 37°C for 3 hours.

[0113] (3) The mixed culture of bacteria and phages was harvested by centrifugation at 30,000 g for 30 min.

[0114] (4) Resuspend the phage culture in Pi-Mg buffer containing chloroform and DNase I and incubate at 37°C for 20 min.

[0115] (5) Remove bacterial debris by centrifugation at 4,300 g for 20 min and obtain the supernatant.

[0116] (6) Pour the supernatant into a new 30 mL centrifuge tube and then centrifuge at 30,000 g for 30 min to harvest the phage.

[0117] (7) Resuspend the obtained phage with an appropriate amount of Pi-Mg buffer and store at 4°C.

[0118] 2. CsCl purification of Hoc-Soc-T4 phage:

[0119] (1) Prepare a CsCl gradient, the specific gradient is as follows:

[0120] Table 4 CsCl gradient

[0121] NO. Stock CsCl (mL) <![CDATA[H2O(mL)]]> Total Volume (mL) 1 1 4 5 2 1.5 3.5 5 3 2 3 5 4 2.5 2.5 5 5 3 2 5 6 3.5 1.5 5

[0122] (2) Then take a centrifuge tube and slowly stack equal volumes of CsCl separation solutions of different gradients, No. 6, No. 5, No. 4, No. 3, No. 2, and No. 1, from bottom to top along the tube wall.

[0123] (3) The obtained phage suspension was placed on the top layer of the gradient and centrifuged at 180,000 g for 1 h at 4°C in a Beckman ultrahigh-speed refrigerated centrifuge using rotor SW41 Ti.

[0124] (4) After centrifugation, a clear phage layer can be observed. Use a long needle to insert into the phage collection interface, aspirate the phage layer and transfer it to a new centrifuge tube.

[0125] (5) The phage sample was placed in a dialysis bag and dialyzed first in dialysis buffer I at 4°C for 5 h, and then in dialysis buffer II at 4°C overnight.

[0126] (6) Collect the dialyzed phage samples and store them at 4°C.

[0127] 2. In vitro fusion of P-HA-HN-T4

[0128] 1. Determination of Hoc-Soc-T4 phage concentration

[0129] (1) Prepare SDS-PAGE gel according to the recipe of SDS-PAGE polyacrylamide gel.

[0130] (2) Add the diluted BSA standard and phage to the SDS-PAGE polyacrylamide gel sample wells in sequence.

[0131] (3) Under 80 V voltage conditions, when the band migrates to the position of the separation gel, increase the voltage to 120 V. Continue electrophoresis until the minimum indicator band of the protein marker reaches the bottom of the separation gel.

[0132] (4) After electrophoresis, move the gel to a staining box, add Coomassie Brilliant Blue staining solution, and stain at room temperature for 2 hours.

[0133] (5) Pour out the staining solution and add Coomassie Brilliant Blue destaining solution until the staining is complete.

[0134] (6) Use Image Lab software to capture images of polyacrylamide gel.

[0135] (7) Image Pro Plus 6.0 was used to measure the grayscale values of the standard protein BSA and phage capsid protein gp23 bands at different concentrations, and the Hoc-Soc-T4 phage concentration was calculated.

[0136] 2. In vitro fusion of P-HA-HN protein and Hoc-Soc-T4 phage

[0137] (1) Take approximately 5×10 10 Hoc-Soc-T4 phage particles were centrifuged at 21,130 g and 4°C for 45 min.

[0138] (2) Wash twice with 1 mL of PBS and centrifuge at 21,130 g for 45 min at 4°C.

[0139] (3) According to the different molar ratios of P-HA-HN protein and Soc binding sites on Hoc-Soc-T4 phage (1:1-1:30), preferably 1:20, 1 mg of P-HA-HN protein was added to a 5×10 10 In a test tube containing Hoc-Soc-T4 phage particles, adjust the volume to 1 mL with in vitro assembly buffer (20 mM Tris, 150 mM NaCl, 0.1 mM CaCl2, 0.05 mM MgCl2, 0.5 mM sucrose, 0.2% glycerol) and incubate at 4°C for 45 min.

[0140] (4) Centrifuge at 21,130 g for 30 min at 4°C to remove unbound P-HA-HN protein. Wash the phage particles displaying P-HA-HN protein twice with 1 mL of assembly buffer and centrifuge to precipitate the phage.

[0141] (5) Resuspend the phage loaded with P-HA-HN protein in an appropriate amount of assembly buffer and transfer to a new 1.5 mL centrifuge tube.

[0142] (6) The copy number of P-HA-HN protein on the T4 phage capsid was estimated by SDS-PAGE analysis and relative gray value.

[0143] like Figure 9 Each T4 phage contains approximately 200 P-HA-HN proteins. Compared with the total amount of wild-type T4 assembled proteins, its packaging rate exceeds 20%, which can better meet industrial needs.

[0144] 3. Western Blot Identification

[0145] (1) To identify the expression of the target protein, 50 μL of the collected recombinant virus-like particles were mixed with 10 μL of 6× Protein loading buffer, and the mixture was centrifuged at 12000 rpm for 10 min. After that, 20 μL of the sample was loaded and the electrophoresis parameters were set to 120 V for 70 min.

[0146] After running SDS-PAGE as described above, the proteins in the gel were transferred to a PVDF membrane.

[0147] (2) Blocking: The blocking solution is PBST containing 5% skim milk powder. Place the PVDF membrane in the blocking solution and block at room temperature for 2 h.

[0148] (3) Primary Antibody Incubation: Dilute the primary antibody 3000-fold using blocking buffer as the antibody diluent. Transfer the PVDF membrane from the blocking buffer to the primary antibody diluent and incubate at room temperature for 2 h. The primary antibody used was NDV / AIV Spike Antibody, Rabbit PAb (purchased from Beijing Sino Biological Biotechnology Co., Ltd., Cat. No. 40589-T62).

[0149] (4) Washing the membrane: Wash the membrane three times with PBST buffer, shaking gently for 5 minutes each time.

[0150] (5) Secondary antibody incubation: Use blocking solution as antibody diluent, dilute HRP-labeled goat anti-rabbit secondary antibody 5000 times, and shake gently at room temperature for 1 hour.

[0151] (6) Washing the membrane: Wash the membrane three times with PBST buffer, shaking gently for 5 minutes each time.

[0152] (7) Color development: Use TMB, a special HRP color development solution, to develop the color until the bands are clear, and then rinse with ddH2O to stop the color development.

[0153] (8) Take a photo and save it.

[0154] Western Blot identification results Figure 10 shown.

[0155] 3. Electron microscopic observation of P-HA-HN-T4 chimeric phage virus particles

[0156] like Figure 11 As shown, the protein is smaller than the phage and cannot be clearly seen.

[0157] Example 6: Elisa analysis of P-HA-HN recombinant protein

[0158] To verify the antigenicity of the recombinant protein, the indirect ELISA method was performed as follows:

[0159] The antigen was coated on a 12-well enzyme-labeled strip at a concentration of 2 μg, 100 μL / well, and mixed by microshaker at 4°C overnight.

[0160] The next day, the plates were washed three times with PBST, each time with shaking for 3-5 minutes, and then dried. 100 μL of primary antibody (NDV positive serum diluted 1:2000) was added to the first well, and the dilution was serially diluted to 11 wells. The last well was used as a negative serum control. After incubation at 37°C for 45 minutes, the plates were washed three times with shaking for 5 minutes each time, and then dried.

[0161] Add 100 μL / well of secondary antibody (1:5000 diluted enzyme-labeled antibody HRP-goat anti-mouse IgG), incubate at 37°C for 30 min, wash three times, shaking for 5 min each time, and spin dry;

[0162] Add 100 μL of freshly prepared OPD substrate solution to each well and place it in the dark at room temperature for 15 min; add 100 μL of stop solution (2 mol / L H2SO4) to each well to stop the reaction and measure the OD450 value on a microplate reader.

[0163] The purified P-HA-HN recombinant protein was subjected to ELISA analysis, and the results showed that the recombinant protein had specific reactions with chicken H9N2 subtype avian influenza virus positive serum and Newcastle disease positive serum (clinical inactivated live vaccine immune serum), while the control PBS group had no specific reaction ( Figure 12 ), indicating that the prepared recombinant protein has good immunogenicity and the highest titer can reach 64,000.

[0164] Example 7: Chicken immunization results and HI titer detection of chimeric virus-like particle vaccine

[0165] Purchase SPF chicken embryos and breed them in the incubator for 20 days until chicks hatch one after another. Collect blood from the chicks at 7 days after hatching, give them the first immunization at 7 days, and give them a booster immunization at 21 days.

[0166] The 4HA antigen F48E9 virulent strain (type IX) strain / A / duck / Shandong / 12 / 2019(H3N2) (donated by Hubei Academy of Agricultural Sciences) and the 4HA antigen NDV commercial attenuated vaccine (donated by the Institute of Animal Husbandry and Veterinary Medicine, Jiangsu Academy of Agricultural Sciences) were used for testing.

[0167] Starting from the first immunization, blood was collected every other week until the fourth week after immunization. The supernatant was collected by centrifugation and tested for HI titer. The results are shown in Table 5.

[0168] The specific steps are as follows:

[0169] The serum to be tested was serially diluted 2-fold with PBS and an equal amount of hemagglutinin working solution containing 4HAU was added.

[0170] Set up PBS and hemagglutinin controls, shake thoroughly, and place in a 25°C constant temperature and humidity chamber for 20 minutes.

[0171] Add 1% chicken red blood cell suspension and place in a 25°C constant temperature and humidity chamber for 20 minutes. Determine the result when the red blood cells in the control wells become noticeably button-shaped. The highest serum dilution that completely inhibits red blood cell aggregation (red blood cells completely slide down) is used as the endpoint.

[0172] Table 5 HI titer test results of avian influenza and Newcastle disease

[0173]

[0174] According to the test results, the chimeric T4 virus-like particles prepared by the present invention can induce high HI titers through secondary immunization without the addition of an adjuvant. Therefore, the broad-spectrum avian influenza and Newcastle disease chimeric virus-like particles provided by the present invention overcome the shortcomings of existing vaccines and are expected to be economically and conveniently used in clinical prevention and control of multiple avian influenza and Newcastle disease subtypes.

[0175] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A recombinant protein, characterized in that Including SOC protein, Newcastle disease virus HN protein and avian influenza virus HA protein, Among them, the amino acid sequence of the recombinant protein is shown in SEQ ID NO.1, the amino acid sequence of the SOC protein is shown in SEQ ID NO.2, the amino acid sequence of the Newcastle disease virus HN protein is shown in SEQ ID NO.3, and the amino acid sequence of the avian influenza virus HA protein is shown in SEQ ID NO.

4.

2. The recombinant protein according to claim 1, wherein The nucleotide sequence of the recombinant protein is shown in SEQ ID NO.5, the nucleotide sequence of the SOC protein is shown in SEQ ID NO.6, the nucleotide sequence of the Newcastle disease virus HN protein is shown in SEQ ID NO.7, and the nucleotide sequence of the avian influenza HA protein is shown in SEQ ID NO.

8.

3. The recombinant protein according to claim 1, wherein The Newcastle disease virus HN protein and the avian influenza HA protein form an HA-HN concatemer, and the amino acid sequence of the HA-HN concatemer is shown in SEQ ID NO.

9.

4. The recombinant protein according to claim 3, characterized in that The nucleotide sequence of the HA-HN concatemer is shown in SEQ ID NO.

10.

5. A biomaterial, characterized in that The biomaterial comprises the recombinant protein according to any one of claims 1 to 4, and the biomaterial further comprises any one of a recombinant expression vector, a recombinant microorganism, an expression cassette, a cell strain, and a cell line.

6. A chimeric virus-like particle of avian influenza and Newcastle disease, characterized in that: The recombinant protein according to any one of claims 1 to 4 is assembled in vitro.

7. The chimeric virus-like particle of avian influenza and Newcastle disease according to claim 6, characterized in that: The phage assembled in vitro is T4 phage.

8. The method for preparing a chimeric virus-like particle of avian influenza and Newcastle disease according to claim 7, characterized in that: The following steps are involved: S1, constructing an expression vector for the recombinant protein; S2, expressing and purifying the recombinant protein; S3. Assembling the purified recombinant protein with T4 phage in vitro to obtain the avian influenza and Newcastle disease chimeric virus-like particles.

9. A chimeric virus-like particle vaccine for avian influenza and Newcastle disease, characterized in that: The invention comprises the avian influenza and Newcastle disease chimeric virus-like particles according to claim 6.

10. Use of the recombinant protein according to any one of claims 1 to 4, or the biomaterial according to claim 5, or the chimeric virus-like particle of avian influenza and Newcastle disease according to claim 6 in the preparation of vaccines or drugs for treating or preventing diseases caused by avian influenza and Newcastle disease viruses.

Images