CN115894636A - Seasonal influenza A universal Mosaic recombinant antigen, vaccine and application thereof - Google Patents
Seasonal influenza A universal Mosaic recombinant antigen, vaccine and application thereof Download PDFInfo
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- CN115894636A CN115894636A CN202210840150.8A CN202210840150A CN115894636A CN 115894636 A CN115894636 A CN 115894636A CN 202210840150 A CN202210840150 A CN 202210840150A CN 115894636 A CN115894636 A CN 115894636A
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- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/185—Escherichia
- C12R2001/19—Escherichia coli
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention relates to the technical field of biological pharmacy, in particular to a seasonal influenza A universal Mosaic recombinant antigen, a vaccine and an application thereof, wherein the influenza virus HA and NA Mosaic recombinant antigen sequences with the maximum diversity capable of covering potential T cell epitopes of natural sequences are obtained by performing data analysis on the existing known amino acid sequences of HA and NA of all human H1N1 and H3N2 in 2009-2021 and utilizing a Mosaic design strategy; the influenza virus HA and NA Mosaic recombinant antigen designed by the invention is formed by assembling short peptides of natural sequences, HAs high coverage rate with antigen through epitope coverage rate analysis, genetic evolution analysis and space conformation analysis, HAs close relationship with vaccine strains and good structural similarity with natural protein, and HAs the potential of being developed into vaccine antigens.
Description
Technical Field
The invention relates to the technical field of biological pharmacy, in particular to a seasonal influenza A universal Mosaic recombinant antigen, a vaccine and application thereof.
Background
Influenza virus (Influenza virus, IV), called Influenza virus for short, HAs many subtypes, HA and NA genes from different sources can be recombined to form hundreds of viruses with different subtypes, and many different strains exist in the same subtype and can be transmitted among different hosts. The HA antigen of the influenza A virus HAs high variation frequency, and the antigenic variation of the HA antigen is more frequent under the immunological pressure, so that the antigenic drift and the antigenic conversion are easy to occur, and the influenza virus can escape from the immunity, thereby causing seasonal influenza epidemics and global influenza pandemics.
Vaccination with influenza vaccines is the best intervention to prevent influenza, reduce influenza harm, and reduce various complications. Because of the variable nature of influenza viruses, the World Health Organization (WHO) predicts and recommends circulating strains in the southern and northern hemispheres each year, vaccine manufacturers produce influenza vaccines for vaccine strains, and the population also needs to be vaccinated with influenza vaccines each year to match strains that are predicted to circulate the year. If the strain used in the vaccine does not match the circulating strain, the protective efficacy is greatly reduced and the morbidity and mortality of influenza increases. Therefore, the development of a general vaccine with a relatively broad spectrum has become a key issue of attention.
Currently, a number of common influenza vaccine strategies are being studied internationally. Viral surface glycoproteins, hemagglutinin (HA) and Neuraminidase (NA) are the most common targets for vaccines, but their cross-protection is limited. Recent studies have focused on the HA stem region, which is more conserved than the HA head region. HA and NA targets rely on the induction of antibody responses to provide protection against IAV. However, the importance of the T cell immune response has often been overlooked during previous influenza vaccine development. The T cell immune response is a key factor for resisting virus infection and has an important function for controlling influenza virus and AIDS virus. There is increasing evidence that T cell immunity may be key to better vaccine cross-protection, playing an important role in preventing influenza.
Currently, influenza vaccines on the market in China are all traditional chick embryo culture vaccines, including inactivated and attenuated vaccines, usually trivalent (H1N 1a, H3N2 a and b) or tetravalent (H1N 1a, H3N2 a, yamagata b and Victoria b) vaccines. These vaccines on the one hand only elicit systemic humoral responses and on the other hand have limited protection against newly emerging strains. Based on the diversity of influenza viruses and frequent mutation of antigens, the development of a universal vaccine capable of resisting various subtypes of influenza viruses is a very ideal choice.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a seasonal influenza A universal Mosaic recombinant antigen, a vaccine and application thereof.
In order to achieve the purpose, the invention adopts the technical scheme that:
the invention provides a seasonal influenza A universal Mosaic recombinant antigen, which is prepared from the following components in parts by weight:
(a) An antigen consisting of an amino acid sequence shown in any one of SEQ ID Nos. 1 to 4; alternatively, the first and second electrodes may be,
(b) Antigen formed by substituting, deleting or adding one or more amino acids in the amino acid sequence in (a).
As a preferred embodiment of the seasonal influenza a universal Mosaic recombinant antigen of the present invention, the coverage rate of Th cell epitopes of the Mosaic recombinant antigen is greater than 81% compared with Th cell epitopes on a natural HA protein of influenza virus; compared with the Th cell epitope on the natural NA protein of the influenza virus, the Th cell epitope coverage rate of the Mosaic recombinant antigen is more than 84%.
The Mosaic recombinant antigen of the present application is one that produces a small number of "Mosaic" sequences on the native HA or NA protein sequence, such that it contains the greatest diversity of potential T cell epitopes from the native protein sequence. The optimized "mosaic" proteins have abundant T cell epitopes assembled from fragments of the native protein using genetic algorithms (computational optimization methods). Through simulating and predicting a three-dimensional structure model of the Mosaic recombinant antigen, the Mosaic recombinant antigen obtained by screening has higher structural similarity with natural protein and has the potential of being developed into a vaccine antigen.
As a preferred embodiment of the seasonal influenza a universal Mosaic recombinant antigen according to the invention, the HA protein comprises H1 protein or H3 protein and the NA protein comprises N1 protein or N2 protein.
As a preferred embodiment of the seasonal influenza a universal Mosaic recombinant antigen according to the invention, more than 81% of the 12 amino acids of the Th cell epitope of the Mosaic recombinant antigen are perfectly matched with 12 amino acids of the Th cell epitope on the native H1 protein or H3 protein of influenza virus.
As a preferred embodiment of the universal Mosaic recombinant antigen for seasonal influenza a according to the invention, more than 11 amino acids of the Th cell epitope of more than 96% of the Mosaic recombinant antigen completely match 12 amino acids of the Th cell epitope on the native H1 protein or H3 protein of influenza virus.
As a preferred embodiment of the seasonal influenza a universal Mosaic recombinant antigen according to the invention, more than 99% of the 10 amino acids of the Th cell epitopes of the Mosaic recombinant antigen are perfectly matched with 12 amino acids of Th cell epitopes of the native H1 protein or H3 protein of influenza virus.
As a preferred embodiment of the seasonal influenza a universal Mosaic recombinant antigen according to the invention, more than 84% of the 12 amino acids of the Th cell epitope of the Mosaic recombinant antigen are perfectly matched with 12 amino acids of the Th cell epitope on the native N1 protein or N2 protein of influenza virus.
As a preferred embodiment of the seasonal influenza a universal Mosaic recombinant antigen according to the invention, more than 11 amino acids of a 96% Th cell epitope of the Mosaic recombinant antigen are perfectly matched with 12 amino acids of a Th cell epitope on a native N1 protein or N2 protein of influenza virus.
As a preferred embodiment of the universal Mosaic recombinant antigen for seasonal influenza a according to the invention, more than 10 amino acids of the Th cell epitope of more than 98% of the Mosaic recombinant antigen completely match 12 amino acids of the Th cell epitope on the natural N1 protein or N2 protein of influenza virus.
Through the experimental verification of the protein biological function, the Mosaic recombinant antigen with the amino acid sequence shown as SEQ ID No. 1 can generate 2 6 The Mosaic recombinant antigen with the amino acid sequence shown as SEQ ID No. 2 can generate 2 7 The hemagglutination titer of (1).
The Mosaic recombinant antigen has binding capacity for both sialic acid a2, 3-galactose receptor and sialic acid a2, 6-galactose receptor, and has significant difference (P is less than or equal to 0.01) compared with PBS (phosphate buffer solution) group, wherein the binding capacity for the a2, 6-galactose receptor is stronger.
As a preferred embodiment of the seasonal influenza a universal Mosaic recombinant antigen, the Mosaic recombinant antigen is further added with gp67 signal peptide, thrombin cleavage site and 8 × His tag, and original signal peptide of HA or NA is removed; adding a GCN4pII sequence aiming at HA protein and a VASP sequence aiming at NA protein;
the amino acid sequence of the gp67 signal peptide is: MLLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAAD;
the GCN4pII sequence is MKQIEDKIEEILSKIYHIENEIARIKKLIGEV;
the VASP sequence is SSSDYSDLQRVKWELLEVKELQKVKEEIEAFVQELKRG.
The invention also provides a gene for coding the seasonal influenza A universal Mosaic recombinant antigen.
The invention optimizes the gene of the treated Mosaic recombinant antigen, and carries out gene modification according to the codon preference of insect cells to obtain the optimized gene.
As a preferred embodiment of the gene, the sequence of the gene is shown in any one of SEQ ID No. 5-8.
The invention also provides a vector containing the gene.
As a preferred embodiment of the vector of the present invention, the vector is obtained by ligating the gene to a plasmid. The plasmid is preferably pFastBac-Dual.
The invention also provides a cell containing the gene or the vector.
As a preferred embodiment of the cell of the present invention, the cell is obtained by transferring a gene or a vector into an Escherichia coli host cell.
In addition, the invention provides an application of the seasonal influenza A universal Mosaic recombinant antigen in preparation of a seasonal influenza A universal vaccine.
The invention provides a vaccine preparation, which comprises the seasonal influenza A universal Mosaic recombinant antigen, the gene and the vector.
As a preferred embodiment of the vaccine formulation according to the present invention, the vaccine formulation further comprises an immunologically and pharmaceutically acceptable carrier or adjuvant. The adjuvant comprises one or more of aluminum adjuvant, freund's adjuvant, aluminum phosphate, calcium phosphate, paraffin oil, lanolin, surfactant, calcium alginate, polynucleotide, muramyl peptide, saponin, RIBI adjuvant system, cholera toxin, polymer of acrylic acid or methacrylic acid, water-in-oil emulsion, and oil-in-water emulsion.
The invention also provides application of the vaccine preparation in preparation of a medicine for preventing and/or treating seasonal influenza A.
Compared with the prior art, the invention has the following beneficial effects:
(1) According to the invention, the amino acid sequences of HA and NA of all human H1N1 and H3N2 in 2009-2021 are subjected to data analysis, and a Mosaic design strategy is utilized to obtain the influenza virus HA and NA Mosaic recombinant antigen sequence which can cover the maximum diversity of potential T cell epitopes of a natural sequence;
(2) The influenza virus HA and NA Mosaic recombinant antigen designed by the invention is formed by assembling short peptides of natural sequences, HAs high coverage rate with antigen through epitope coverage rate analysis, genetic evolution analysis and space conformation analysis, HAs close relationship with vaccine strains and good structural similarity with natural protein, and HAs the potential of being developed into vaccine antigens.
(3) The influenza virus HA and the constructed Mosaic recombinant protein have potential application prospect and value in the aspect of developing influenza virus universal vaccines, and the T cell immune response of the antigen is expected to become an important consideration direction for developing influenza vaccines.
Drawings
FIG. 1 is a schematic diagram of the average value of the epitope coverage of HA amino acid sequence of H1m recombinant sequence for all human H1N1 in 2009-2021;
FIG. 2 is a schematic diagram of the average value of the HA amino acid sequence epitope coverage of the H3m recombinant sequence for all human H3N2 in 2009-2021 years;
FIG. 3 is a graph showing the average value of the coverage rate of the N1m recombinant sequence on the epitope of the NA amino acid sequence of all human H1N1 in 2009-2021 years;
FIG. 4 is a graph showing the average value of the epitope coverage of the N2m recombinant sequence on the NA amino acid sequence of all human H3N2 in 2009-2021;
FIG. 5 is a schematic representation of epitope coverage of each amino acid of the H1m recombination sequence;
FIG. 6 is a schematic representation of epitope coverage per amino acid of the H3m recombination sequence;
FIG. 7 is a schematic representation of epitope coverage of each amino acid of the N1m recombination sequence;
FIG. 8 is a schematic representation of epitope coverage per amino acid of the N2m recombination sequence;
FIG. 9 is a schematic diagram showing epitope deletion ratios of H1m recombinant sequences;
FIG. 10 is a diagram showing epitope deletion ratios of H3m recombinant sequences;
FIG. 11 is a schematic diagram showing epitope deletion ratios of N1m recombinant sequences;
FIG. 12 is a schematic diagram showing epitope deletion ratios of N2m recombinant sequences;
FIG. 13 is a schematic diagram of the genetic evolution analysis of the H1m recombination sequence;
FIG. 14 is a schematic diagram of the genetic evolution analysis of the H3m recombination sequence;
FIG. 15 is a schematic diagram of the genetic evolution analysis of the N1m recombination sequence;
FIG. 16 is a schematic diagram of the genetic evolution analysis of N2m recombination sequences;
FIG. 17 is a schematic diagram of a three-dimensional structure model simulation of a Mosaic recombinant antigen;
FIG. 18 is a Western Blot result chart of baculovirus system expressing Mosaic recombinant protein;
FIG. 19 is a chart showing the result of Coomassie blue staining of the eluate of Mosaic recombinant protein;
FIG. 20 is a graph showing the results of hemagglutination assay of the Mosaic recombinant protein;
FIG. 21 is a graph showing the results of a sugar receptor binding experiment of the recombinant Mosaic protein;
FIG. 22 is a graph showing the results of the neuraminidase activity assay for the Mosaic recombinant protein;
FIG. 23 is a graph showing the results of animal experiments on hemagglutination inhibition of the Mosaic recombinant protein;
FIG. 24 is a graph showing the results of animal experiments on neuraminidase inhibition by the Mosaic recombinant protein.
Detailed Description
To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to the accompanying drawings and specific embodiments.
In the following examples, the experimental methods used were all conventional methods unless otherwise specified, and the materials, reagents and the like used were commercially available without otherwise specified.
The HA and NA proteins of the influenza virus play an important role in virus invasion and release, have strong immunogenicity, and can induce strong specific antibody and T cell reaction. Mosaic vaccines are primarily designed against viruses with variable epitopes, with the goal of using the native sequence to generate a small number of "Mosaic" sequences that contain the greatest diversity of potential T cell epitopes from the native sequence. From fragments of native proteins, optimally generated "mosaic" proteins are assembled using genetic algorithms (computational optimization methods) similar to proteins from native viruses and have abundant T cell epitopes and can therefore be used as antigens for candidate vaccine design.
Example 1 construction of a Universal seasonal influenza A Mosaic recombinant antigen
1. Designing and optimizing a universal Mosaic recombinant antigen sequence:
1) Downloading the HA and NA amino acid sequences of all human H1N1 and H3N2 in 2009-2021 by using GISAID and NCBI databases, and removing repeated sequences and sequences with poor quality to obtain 7609H 1 amino acid sequences, 9262 amino acid H3 sequences, 8590N 1 amino acid sequences and 9942 amino acid N2 sequences.
2) The processed amino acid sequence was uploaded to the pharmaceutical Vaccine Designer program in FAS format with the following parameters set: cocktail Size is set to "1" to obtain 1 Mosaic sequence for further use; epitope length was set to "12" to obtain coverage of more CD4 + A Mosaic sequence of a Th cell epitope; the threshold was set to "3" to reduce the number of rare epitopes that are rare and occur a low number of times in the native epitope; after genetic algorithm operation, a series of Mosaic sequences assembled by short peptides consisting of 12 amino acids are finally obtained. Each population was then optimized in turn using a genetic algorithm, where new recombinants were generated and tested computationally for epitope coverage, and finally 4 Mosaic recombinant antigen sequences (shown in SEQ ID NOS: 1-4) were obtained.
2. Screening and identification of universal Mosaic recombinant antigen sequence
And performing epitope coverage analysis, genetic evolution analysis and spatial conformation analysis on the obtained Mosaic recombinant antigen sequence.
1) Epitope Coverage of mosaics was evaluated using an Epitope Coverage assay Tool (Epicover). The Mosaic recombinant antigen sequence was first added as an antigenic protein to the corresponding position. Meanwhile, the amino acid sequences of the complete strains which are subjected to the loading analysis alignment on GISAID and NCBI are set as test protein sets and are also added to corresponding positions. The epitope length was set to 12 and the maximum number of amino acid mismatches was set to 2, and the final results are expressed as the calculated average of the epitope coverage of the Mosaic recombinant antigen sequences over all background protein sets.
As shown in Table 1, more than 81% of the 12 amino acids of the Th cell epitopes on the Mosaic recombinant antigen sequence (designated H1m or H3 m) were perfectly matched (12/12 match) with 12 amino acids of the Th cell epitopes on the native H1 protein or H3 protein of influenza A, more than 96% of the 11 or more amino acids of the Th cell epitopes on the Mosaic recombinant antigen sequence were perfectly matched (11/12 match) with 12 amino acids of the Th cell epitopes on the native H1 protein or H3 protein of influenza A, and more than 99% of the 10 or more amino acids of the Th cell epitopes on the Mosaic recombinant antigen sequence were perfectly matched (10/12 match) with 12 amino acids of the Th epitopes of the native H1 protein or H3 protein of influenza A.
More than 84% of 12 amino acids of Th cell epitopes of the Mosaic recombinant antigen are completely matched with 12 amino acids of Th cell epitopes on natural N1 protein or N2 protein of influenza virus (12/12 matching) in the Mosaic recombinant antigen sequence (named N1m or N2 m), more than 11 amino acids of Th cell epitopes of more than 96% of the Mosaic recombinant antigen are completely matched with 12 amino acids of Th cell epitopes on natural N1 protein or N2 protein of influenza virus (11/12 matching), and more than 10 amino acids of Th cell epitopes of more than 98% of the Mosaic recombinant antigen are completely matched with 12 amino acids of Th cell epitopes on natural N1 protein or N2 protein of influenza virus (10/12 matching).
The average coverage of the Mosaic recombinant antigen sequences for the entire epitope is shown in FIGS. 1-4. The Mosaic recombinant antigen sequences are HAm and NAm.
TABLE 1
| Mosaic recombinant antigen | Number of natural sequences | Off-by-0 | Off-by-1 | Off-by-2 |
| H1m | 7609 | 88.27％ | 98.27％ | 99.61％ |
| H3m | 9262 | 81.82％ | 96.81％ | 99.26％ |
| N1m | 8590 | 84.47％ | 96.16％ | 98.48％ |
| N2m | 9942 | 84.84％ | 97.69％ | 99.73％ |
2) Epitope Coverage at each amino acid of the Mosaic recombinant antigen was analyzed using the Positional Epitope Coverage analysis Tool (Posicover). Firstly, the Mosaic recombinant antigen is taken as an antigen protein and added to a corresponding position, and simultaneously, the amino acid sequence of a strain which is subjected to downloading analysis and comparison on GISAID and NCBI before is set as a test protein set and also added to the corresponding position. The epitope length was set to 12 and the final results are expressed as mean epitope coverage. Schematic representation of epitope coverage of each amino acid of the Mosaic recombinant antigen sequence is shown in FIGS. 5-8; the schematic diagram of the epitope deletion rate of the Mosaic recombinant antigen sequence is shown in FIGS. 9-12, and the Mosaic recombinant antigen sequence has high epitope coverage rate and low overall deletion rate of the 12 mer.
3) Selecting HA and NA genes of the seasonal influenza A virus vaccine strain in 2009-2022, carrying out genetic evolution analysis on the HA and NA genes, HAm and NAm, carrying out statistical analysis by using a maximum likelihood method, and then drawing an evolutionary tree of the HA and NA genes. The schematic diagram of genetic evolution analysis of the Mosaic recombinant antigen sequence is shown in FIGS. 13-16, and the genetic relationship between the Mosaic recombinant antigen and various vaccine strains is relatively close, which indicates the potential of the designed Mosaic recombinant antigen as a vaccine antigen.
4) And (3) evaluating the natural immune function of the Mosaic recombinant antigen by simulating and predicting a three-dimensional structure model of the Mosaic recombinant antigen. As shown in fig. 17 (fig. 17-a is a schematic diagram of three-dimensional structure Model simulation of H1m recombinant antigen, fig. 17-B is a schematic diagram of three-dimensional structure Model simulation of H3m recombinant antigen, fig. 17-C is a schematic diagram of three-dimensional structure Model simulation of HA protein of a/Puerto Rico/8/1934 strain, fig. 17-D is a schematic diagram of three-dimensional structure Model simulation of N1m recombinant antigen, fig. 17-E is a schematic diagram of three-dimensional structure Model simulation of N2m recombinant antigen, fig. 17-F is a schematic diagram of three-dimensional structure Model simulation of NA protein of a/aici/2/1968 strain), scores of Global Model Quality (QMQE) of 4 Mosaic recombinant antigens and their natural proteins are respectively: 0.74 parts of H1m protein, 0.78 parts of H3m protein and 0.80 parts of HA protein; 0.81 portion of N1m protein, 0.78 portion of N2m protein and 0.79 portion of NA protein, which shows that the Mosaic recombinant antigen has higher structural similarity with natural protein.
3. Gene optimization and synthesis of universal Mosaic recombinant antigen sequence
For the amino acid sequences of the 4 designed Mosaic recombinant antigens H1m, H3m, N1m and N2m, original signal peptides of HA and NA are further removed, a gp67 signal peptide (MLLVNQSHQGFNKEHTSVSVAIVLYLLAAAAHSAFAAD) is added, and a GCN4pII sequence (MKQIEDKIEEILSKIYHNERIKKLIGEV) is added aiming at the HA protein sequence; the VASP sequence (SSSDYSDLQRVKWEEVKELQKVKEEEIIEAFVQELKRG) was added to the NA protein sequence, followed by the co-addition of the thrombin cleavage site (LVPRGS) and 8 × His tag (HHHHHHHH). And optimizing the encoding gene of the treated Mosaic recombinant antigen sequence, and performing gene modification according to the codon preference of insect cells to obtain an optimized gene sequence (shown as SEQ ID NO. 5-8).
Example 2 expression of Mosaic recombinant protein
This example used the Invitrogen company Bac-to-Bac baculovirus expression system to express the desired protein.
1. Construction of baculovirus recombinant plasmid:
performing multiple cloning site analysis on coding genes of Mosaic recombinant antigens H1m, H3m, N1m and N2m and genes of a pFastBac-Dual vector, selecting two restriction enzyme sites (EcoRI and HindIII) which are provided on the pFastBac-Dual vector and are not contained in a target fragment, performing amplification and digestion recovery on the target fragment, inserting the target fragment into a multiple cloning site behind a pH promoter of the pFastBac-Dual vector, and transforming escherichia coli DH5 alpha competent cells to obtain the recombinant plasmid containing the H1m, H3m, N1m and N2m antigens.
2. Extraction of baculovirus recombinant shuttle plasmid (bacmid):
and (2) transforming the recombinant plasmid obtained in the step (1) by an escherichia coli DH10Bac competent cell, screening by blue-white spots, culturing overnight in LB (lysogeny broth) containing kanamycin (50 mu g/mL), tetracycline (10 mu g/mL) and gentamicin (7 mu g/mL), and extracting bacmid (purchased from Biyun, goods number D0031) to obtain the recombinant baculovirus shuttle plasmid containing the H1m, H3m, N1m and N2m antigens.
3. Bacmid cell transfection and protein expression:
and (3) transfecting Bacmid and the empty rod (serving as blank control) described in the step 2 to sf9 insect cells respectively, and performing shaking table culture at the constant temperature of 27 ℃ for 72 hours to obtain the P0 generation recombinant baculovirus. After sf9 insect cells were inoculated according to MOI =3, P1 recombinant baculovirus generations were obtained, and the expression of the target protein in the cell supernatants was examined by Western blotting (Western Blot, WB), as shown in fig. 18 (fig. 18-a is a Western Blot result of H1M recombinant protein, fig. 18-B is a Western Blot result of H3M recombinant protein, fig. 18-C is a Western Blot result of N1M recombinant protein, fig. 18-D is a Western Blot result of N2M recombinant protein, 1 in fig. 18 represents the cell supernatant of P1 recombinant baculovirus generations, 2 represents the cell supernatant of P2 recombinant baculovirus generations, 3 represents the cell lysate of P2 recombinant baculovirus generations, 4 represents blank control, and M is protein ladder).
4. Continuous transmission and amplification culture:
after the expression and identification of the recombinant protein are successful, observing the cell death rate daily, inoculating sf9 insect cells for two consecutive generations according to MOI =3 when the cell death rate of the P1 culture is more than 90%, obtaining P3 generation recombinant baculovirus, centrifuging at 3000 Xg for 30min at 4 ℃ when the cell death rate of the P3 generation culture is about 50%, discarding the precipitate, and collecting the supernatant.
5. Protein concentration and protein purification:
and (3) concentrating the supernatant obtained in the step (4) by a vivaflow200 membrane package, performing three-time replacement by using PBS to obtain 100mL of concentrated solution, centrifuging at 4 ℃ at 10000rpm for 10min, collecting the supernatant, filtering by using a 0.22 mu M filter membrane to obtain liquid, placing the liquid at 4 ℃, and enabling the liquid to flow through a nickel affinity column by using a micro-peristaltic pump at the flow rate of 5mL/min until all the liquid passes through the column, wherein the protein is combined in the nickel affinity column. And (3) performing affinity chromatography by using an AKTA protein purification system, firstly removing the original column at the column position No. 1, flowing a column position valve into the pipeline 1A, plugging the two-line joint of which the outflow pipeline is 1B by using a communicating vessel, and then replacing the Buffer in the system. After the Buffer displacement is finished, the nickel affinity column is connected to the column position No. 1, the continuous concentration gradient elution is carried out by using phosphate equilibrium Buffer solution containing 5mM imidazole and phosphate elution Buffer solution containing 500mM imidazole, and the eluent is collected.
According to the eluted ultraviolet absorption peak images, the eluate containing the target protein and obtained after affinity chromatography is selected and subjected to SDS-PAGE electrophoresis, and then Coomassie brilliant blue staining is used, for example, FIG. 19 (FIG. 19-A is a Coomassie brilliant blue staining result image of H1m recombinant protein eluate, FIG. 19-B is a Coomassie brilliant blue staining result image of H3m recombinant protein eluate, FIG. 19-C is a Coomassie brilliant blue staining result image of N1m recombinant protein eluate, and FIG. 19-D is a Coomassie brilliant blue staining result image of N2m recombinant protein eluate (correct band for target protein expression in a square frame), according to the staining result, an ultrafiltration tube is used for centrifugal concentration until the volume is 0.5mL, the concentrated solution is centrifuged for 10min at 13000 Xg, supernatant is taken out and is packaged, and liquid nitrogen is frozen and stored at-80 ℃ for later use.
Example 3 verification experiment of biological function of Mosaic recombinant protein
1. And (3) hemagglutination activity verification:
hemagglutination assay of 1% guinea pig erythrocytes was performed on hemagglutination titers of purified Mosaic recombinant protein and cell culture supernatants of blank controls: adding 50 mu L of PBS into each well of 2-12 columns of the 96-well hemagglutination plate, sucking 50 mu L of purified Mosaic recombinant protein and a control sample, respectively adding into the 1 st column of the 96-well hemagglutination plate, sucking 50 mu L of purified Mosaic recombinant protein and the control sample, adding into the 2 nd column, sucking 50 mu L of purified Mosaic recombinant protein and the control sample, sucking 50 mu L of purified Mosaic recombinant protein, adding into the 3 rd column after uniformly blowing, sequentially carrying out multiple dilution until 50 mu L of purified Mosaic recombinant protein and the control sample are discarded in the 11 th column. And (3) replacing the gun head after blowing and mixing uniformly each time, adding 1% guinea pig red cells into each hole, shaking and mixing uniformly, standing at room temperature for 25min, and reading, wherein the hole with complete agglutination is used as the hemagglutination titer of the sample during reading.
As a result, as shown in FIG. 20, it was observed that 10. Mu.g of the H1m recombinant protein was able to produce 2 6 The hemagglutination titer of 10. Mu.g of the H3m recombinant protein was able to produce 2 7 The hemagglutination titer of (c), while no hemagglutination was observed in the blank control group (Mock).
2. Sugar receptor binding capacity verification:
add 50. Mu.L PBS to the 1 st column of the 96-well plate and 50. Mu.L PBS to the 2,3 th columnPBS diluted to 500ng/mL of 3 'SLN-PAA-biolt, 6' SLN-PAA-biolt (from GlycoNZ, cat # GNZ-0036-BP vs. GNZ-0997-BP), two duplicate wells per protein, incubated overnight at 4 ℃. Placing the plate into an ultraviolet crosslinking machine, performing action at a wavelength of 254nm for about 10min, discarding the liquid in the plate, washing the plate with PBS for 1 time, each time for 3min, adding 100 mu L/well of 1w/v% BSA in PBS for blocking, and incubating at 4 ℃ overnight. The solution in each well was discarded, washed 3 times with PBS for 3min each, 50. Mu.L of 2. Mu.g of HAm recombinant protein was added to each well, and incubated overnight at 4 ℃. Plates were washed 6 times for 3min using PBST, 50. Mu.L of 1:4000 diluted HA antibody against Influenza A virus H1N1 (purchased from GeneTex, cat. GTX 127357) was added to each well; HA antibody against infilunza a virus H3N2 purchased from GeneTex under the code GTX 127363) and incubated at room temperature for 2H. Plates were washed 6 times 3min using PBST, 100 μ L1: HRP-labeled goat anti-rabbit IgG (purchased from Fred, cat # FDR 007) at 8000 dilutions was incubated for 1h at room temperature. The plates were washed 6 times for 3min using PBST. Adding 100 mu of LTMB staining solution into each hole, and placing at room temperature for reaction for 30min; add 50. Mu.L of 2M H per well 2 SO 4 The reaction was stopped and absorbance values (OD 450nm and OD630 nm) at 450nm and 630nm were measured immediately using a microplate reader.
The results are shown in FIG. 21, where the H1m and H3m recombinant proteins have binding capacity for both sialic acid a2, 3-galactose receptor and sialic acid a2, 6-galactose receptor and a significant difference (P.ltoreq.0.01) compared to PBS group, with stronger binding capacity for a2, 6-galactose receptor.
3. Neuraminidase Activity assay
The experiment adopts a neuraminidase detection kit for detection (purchased from Biyun, with the product number of P0306):
1) Preparation of positive and negative control tests: a. 70. Mu.L of neuraminidase detection buffer was added to each well of a 96-well fluorescent plate. b. 10 or 0. Mu.L of neuraminidase were added to each well. c. mu.L of the solution dissolving the neuraminidase sample was added to each well. d. 0 or 10. Mu.L of LMilli-Q water was added to each well to make the total volume of 90. Mu.L per well.
2) Preparation of sample detection: a. 70. Mu.L of neuraminidase detection buffer was added to each well of a 96-well fluorescent plate. b. mu.L of neuraminidase sample was added to each well. c. Add 10. Mu.L of LMilli-Q water to make the total volume of each well 90. Mu.L.
3) And (3) detection: a. mix by shaking for about 1 minute. b. Add 10. Mu.L of neuraminidase fluorogenic substrate per well. c. Mix again by shaking for about 1 minute. Fluorescence measurements were performed after 30min incubation at 37 ℃. The excitation wavelength was 322nm and the emission wavelength was 450nm.
As shown in FIG. 22, the recombinant proteins N1m and N2m have relatively high neuraminidase activities and are significantly different from PBS group ((P.ltoreq.0.05 or P.ltoreq.0.01).
Example 4 evaluation of immune Effect of Mosaic recombinant protein
BALB/c mice were immunized with 4 kinds of recombinant proteins of Mosaic (H1 m, H3m, N1m, and N2m recombinant proteins) obtained by expression and purification in example 2 as immunogens, and the immune effect of the recombinant proteins of Mosaic was examined.
1. Immunizing a mouse:
the concentrations of 4 Mosaic recombinant proteins were each detected using a BCA assay kit (purchased from bi yun sky, cat # P0012), and the proteins were mixed uniformly with 7 ten thousand units/mL of IL-2 and 0.1% chitosan to obtain Mosaic recombinant protein vaccines (HAm protein vaccine and NAm protein vaccine).
Selecting 15 BALB/c female mice of 6-8 weeks old to randomly divide into 3 groups (blank control group: intramuscular injection of 100 muL PBS; immune HAm group: intramuscular injection of 100 muL of 2 HAm protein vaccines containing 60 mug; immune NAm group: intramuscular injection of 100 muL of 2 NAm protein vaccines containing 60 mug), immunizing the BALB/c female mice according to grouping conditions at 0 week and 2 week, performing orbital blood sampling on each group of mice at 0 week and 4 week (28 day) after immunization, standing at 4 ℃ overnight, centrifuging at 3000rpm for 10min to obtain serum, subpackaging and placing in a refrigerator at-80 ℃ for later use.
2. Hemagglutination inhibition (HAI) assay:
1) Preparation of RDE-treated mouse sera: the receptor-disrupting enzyme (RDE, purchased from dai ben institute, cat No. 340122) was mixed with the sera of each group of mice in a volume ratio of 3; taking out the test tube, and putting the test tube in a 56 ℃ water bath for 30min to inactivate the RDE; PBS was added to the tube to bring the serum dilution to 1; cooling to room temperature, adding chicken erythrocyte with 1/2 volume of original serum, mixing, storing at 4 deg.C for 1 hr, and mixing again every 15 min; centrifuging at 1200rpm for 1min, sucking the supernatant to obtain RDE treated mouse serum, and standing at 4 deg.C for use.
2) Preparation of four units of standard antigen: respectively detecting HA valence of A/Victoria/2570/2019 (H1N 1 subtype strain, presented by China center for disease prevention and control) and A/Cambodia/E0826360/2020 (H3N 2 subtype strain, presented by China center for disease prevention and control), diluting each antigen into 8 hemagglutination units by PBS, confirming HA valence again, and further diluting into 4 hemagglutination units to obtain four-unit standard antigen.
3) Hemagglutination inhibition assay: adding 25 mu LPBS into each hole of the 2 nd to 10 th rows and the 12 th row of the 96-well plate, and adding 50 mu LPBS into each hole of the 11 th row; adding 25 mu L of mouse serum treated by RDE into each hole of the 1 st row and the 2 nd row, and uniformly mixing; sucking 25 mu L of the mixed solution in the column 2, adding the solution into the column 3, and uniformly mixing; repeating the operation till the column 10, and discarding 25 μ L of the mixed solution in the column 10; adding 25 mu L of four unit standard antigens into the 1 st to 10 th columns and the 12 th column, wherein the 12 th column is used as a virus control column, and meanwhile, adding positive serum (mouse serum obtained at the early stage of a laboratory and having hemagglutination titer on corresponding strains through verification) into the 11 th column to be used as a standard positive control; after fully and uniformly mixing, placing the 96-well plate at room temperature and standing for 45min; 50 mul of 1% chicken red blood cell suspension is added into each hole, the mixture is kept stand for 25min at room temperature, a 96-hole plate is inclined at 45 ℃, and whether the red blood cells flow in a teardrop shape or not is observed.
The results are shown in FIG. 23, and the influenza virus specific hemagglutination-inhibiting antibody shows that at 4 weeks (D28) after the first immunization, the sera of mice in the immune HAm group have certain cross-protection effect against two vaccine strains of the seasonal influenza virus in 2021-2022 years, and the cross-protection effect is significantly higher than that of the blank control group (P is less than or equal to 0.05)
3. Neuraminidase Inhibition (NI) assay
1) Measurement of the amount of PNA-HPRO: adding 1% BSA in PBST as sample diluent in a 96-well plate,columns 1-11 were loaded with 216. Mu.L per well. The virus was thawed and mixed well and 24. Mu.L was added to each well in columns 1-11. The plates were washed 6 times for 3min each time with PBST in 96-well plates coated with fetoprotein. And transferring 50 mu L of the diluted virus to a 96-well plate coated with the fetal protein in parallel, supplementing 50 mu L of sample diluent to each well, supplementing 100 mu L of sample diluent to the 12 th column, shaking gently, mixing uniformly, and putting the mixture into an incubator at 37 ℃ for incubation for 16 hours. After incubation, the plate was aspirated and washed 6 times with PBST for 3min each. Columns 1-10 were added 100 μ L in sequence as per 1: 200. 1: 400. 1: 500. 1: 800. 1: 1000. 1: 1600. 1: 2000. 1: 3000. 1: 4000. 1: PNA-HRPO (1 mg/mL original concentration) diluted 5000 was incubated at room temperature for 2h. The plates were washed 6 times for 3min using PBST. Adding 100 mu of LTMB staining solution into each hole, and placing at room temperature for reaction for 30min; add 50. Mu.L of 2M H per well 2 SO 4 The reaction was stopped and the absorbance at 450nm (OD 450 nm) was immediately measured using a microplate reader. The amount of PNA-HPRO used was determined from the results.
2) And (3) determination of the NA dosage: adding 1% BSA in PBST as sample diluent to a 96-well plate, 120. Mu.L per well for columns 2-12 and 216. Mu.L per well for column 1. The virus was thawed and mixed well, and 24. Mu.L was added to column 1, and 2-fold dilution was performed continuously to column 11. The plates were washed 6 times for 3min each time with PBST in 96-well plates coated with fetoprotein. And transferring 50 mu L of the diluted virus to a 96-well plate coated with the fetal protein in parallel, supplementing 50 mu L of sample diluent into each well, shaking gently, mixing uniformly, and putting the mixture into an incubator at 37 ℃ for incubation for 16 hours. After incubation, the plate was aspirated, and washed 6 times for 3min with PBST. In each of columns 1-11, 100. Mu.L of PNA-HPRO in the amount determined in the above procedure was added and incubated at room temperature for 2h. The plates were washed 6 times for 3min using PBST. Adding 100 mu of LTMB staining solution into each hole, and placing at room temperature for reaction for 30min; add 50. Mu.L of 2M H per well 2 SO 4 The reaction was stopped and the absorbance at 450nm (OD 450 nm) was immediately measured using a microplate reader. And determining the amount of NA according to the result.
3) Preparation of RDE-treated mouse sera: the receptor-disrupting enzyme (RDE, purchased from the japanese national institute, cat No. 340122) was mixed with the sera of each group of mice in a volume ratio of 3; taking out the test tube, and putting the test tube in a 56 ℃ water bath for 30min to inactivate the RDE; PBS was added to the tube to bring the serum dilution to 1; cooling to room temperature, adding chicken erythrocyte with 1/2 volume of original serum, mixing, storing at 4 deg.C for 1 hr, and mixing again every 15 min; centrifuging at 1200rpm for 1min, sucking supernatant to obtain RDE treated mouse serum, and standing at 4 deg.C for use.
4) Neuraminidase inhibition assay: add 1% BSA in PBST as sample diluent to 96-well plates, 120. Mu.L per well in columns 3-11 and 216. Mu.L per well in column 2. The RDE-treated mouse sera were added in 24. Mu.L to column 1 and serially diluted 2-fold to column 11. The plates were washed 6 times for 3min each time with PBST in 96-well plates coated with fetoprotein. And transferring 50 mu L of diluted serum to a 96-well plate coated with the fetal protein in parallel, adding 50 mu L of the virus with the determined NA dosage in the steps into each well of the 1 st to 11 th columns, shaking gently, mixing uniformly, and then putting the mixture into an incubator at 37 ℃ for incubation for 16 hours. After incubation, the plate was aspirated and washed 6 times with PBST for 3min each. Add 100. Mu.L of PNA-HPRO in the well defined amount of the previous step to each of columns 1-11 and incubate for 2h at room temperature. Plates were washed 6 times for 3min using PBST. Adding 100 mu of LTMB staining solution into each hole, and placing at room temperature for reaction for 30min; add 50. Mu.L of 2M H per well 2 SO 4 The reaction was stopped and the absorbance at 450nm (OD 450 nm) was immediately measured using a microplate reader.
The results are shown in FIG. 24, at 4 weeks (28 days) after the first immunization, the sera of mice in NAm immunization group generated specific neuraminidase inhibitory antibodies against both A/Victoria/2570/2019 and A/Cambodia/E0826360/2020 vaccine strains, and were significantly higher than those in blank control group (P.ltoreq.0.05 or P.ltoreq.0.01), suggesting that the immunization with Mosaic recombinant protein of the present invention has a certain protective effect on mice.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
SEQ ID NO.1:
MKAILVVLLYTFATANADTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDKHNGKLCKLRGVAPLHLGKCNIAGWILGNPECESLSTARSWSYIVETSNSDNGTCYPGDFINYEELREQLSSVSSFERFEIFPKTSSWPNHDSNKGVTAACPHAGAKSFYKNLIWLVKKGNSYPKLNQTYINDKGKEVLVLWGIHHPSTTADQQSLYQNADAYVFVGTSRYSKKFKPEIATRPKVRDQEGRMNYYWTLVEPGDKITFEATGNLVVPRYAFTMERNAGSGIIISDTPVHDCNTTCQTPEGAINTSLPFQNVHPITIGKCPKYVKSTKLRLATGLRNVPSIQSRGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADLKSTQNAIDKITNKVNSVIEKMNTQFTAVGKEFNHLEKRIENLNKKVDDGFLDIWTYNAELLVLLENERTLDYHDSNVKNLYEKVRNQLKNNAKEIGNGCFEFYHKCDNTCMESVKNGTYDYPKYSEEAKLNREKIDGVKLESTRIYQILAIYSTVASSLVLVVSLGAISFWMCSNGSLQCRICI
SEQ ID NO.2:
MKTIIALSYILCLVFAQKIPGNDNSTATLCLGHHAVPNGTIVKTITNDRIEVTNATELVQNSSIGEICDSPHQILDGENCTLIDALLGDPQCDGFQNKKWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFKNESFNWTGVKQNGTSSACIRGSSSSFFSRLNWLTHLNYTYPALNVTMPNKEQFDKLYIWGVHHPGTDKDQIFLYAQSSGRITVSTKRSQQAVIPNIGSRPRIRDIPSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDAPIGKCKSECITPNGSIPNDKPFQNVNRITYGACPRYVKQSTLKLATGMRNVPEKQTRGIFGAIAGFIENGWEGMVDGWYGFRHQNSEGRGQAADLKSTQAAIDQINGKLNRLIGKTNEKFHQIEKEFSEVEGRVQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKTKKQLRENAEDMGNGCFKIYHKCDNACIGSIRNETYDHNVYRDEALNNRFQIKGVELKSGYKDWILWISFAISCFLLCVALLGFIMWACQKGNIRCNICI
SEQ ID NO.3:
MNPNQKIITIGSICMTIGMANLILQIGNIISIWVSHSIQIGNQSQIETCNQSVITYENNTWVNQTYVNISNTNFAAGQSVVSVKLAGNSSLCPVSGWAIYSKDNSVRIGSKGDVFVIREPFISCSPLECRTFFLTQGALLNDKHSNGTIKDRSPYRTLMSCPIGEVPSPYNSRFESVAWSASACHDGINWLTIGISGPDSGAVAVLKYNGIITDTIKSWRNNILRTQESECACVNGSCFTIMTDGPSDGQASYKIFRIEKGKIIKSVEMKAPNYHYEECSCYPDSSEITCVCRDNWHGSNRPWVSFNQNLEYQMGYICSGVFGDNPRPNDKTGSCGPVSSNGANGVKGFSFKYGNGVWIGRTKSISSRKGFEMIWDPNGWTGTDNKFSIKQDIVGINEWSGYSGSFVQHPELTGLDCIRPCFWVELIRGRPEENTIWTSGSSISFCGVNSDTVGWSWPDGAELPFTIDK
SEQ ID NO.4:
MNPNQKIITIGSVSLTISTICFFMQIAILITTVTLHFKQYEFNSPPNNQVMLCEPTIIERNITEIVYLTNTTIEKEICPKPAEYRNWSKPQCGITGFAPFSKDNSIRLSAGGDIWVTREPYVSCDPDKCYQFALGQGTTLNNVHSNNTVRDRTPYRTLLMNELGVPFHLGTKQVCIAWSSSSCHDGKAWLHVCITGDDKNATASFIYNGRLVDSVVSWSKDILRTQESECVCINGTCTVVMTDGNATGKADTKILFIEEGKIVHTSKLSGSAQHVEECSCYPRYPGVRCVCRDNWKGSNRPIVDINIKDHSIVSSYVCSGLVGDTPRKNDSSSSSHCLDPNNEEGGHGVKGWAFDDGNDVWMGRTINETSRLGYETFKVVEGWSNPKSKLQINRQVIVDRGDRSGYSGIFSVEGKSCINRCFYVELIRGRKEETEVLWTSNSIVVFCGTSGTYGTGSWPDGADLNLMHI
SEQ ID NO.5:
ATGCTCCTGGTTAACCAAAGTCACCAGGGCTTTAATAAGGAACACACATCGAAGATGGTCAGCGCCATAGTCCTTTACGTACTTCTCGCGGCGGCCGCACACTCCGCCTTTGCTGCAGATGATACTTTGTGCATCGGTTACCATGCTAACAATTCCACCGACACGGTTGACACGGTCCTGGAAAAGAATGTCACGGTCACCCACAGCGTCAACCTCCTCGAAGATAAACACAACGGCAAGCTGTGTAAGCTGCGCGGTGTGGCGCCCCTCCACCTGGGCAAGTGCAACATCGCTGGTTGGATCTTGGGCAACCCCGAGTGCGAGTCTCTGTCTACGGCTCGTTCCTGGTCCTACATAGTCGAGACATCGAACAGTGACAATGGTACCTGTTACCCGGGAGATTTCATCAACTACGAAGAGCTTCGTGAACAGCTGAGCTCCGTATCTTCTTTCGAGAGGTTCGAAATCTTTCCGAAGACAAGTTCCTGGCCTAACCACGACTCGAATAAGGGTGTCACCGCTGCCTGCCCGCATGCCGGTGCTAAATCTTTCTACAAGAATCTGATTTGGCTGGTGAAGAAGGGCAACTCCTACCCAAAACTGAACCAGACCTACATCAACGACAAGGGAAAGGAAGTACTGGTTCTGTGGGGCATCCATCATCCCTCTACTACCGCAGACCAGCAGTCACTGTACCAGAACGCAGACGCCTACGTGTTCGTGGGAACATCACGTTACTCGAAAAAATTTAAGCCAGAAATTGCGACAAGACCAAAGGTACGTGACCAGGAGGGTCGTATGAACTACTACTGGACTCTGGTGGAGCCCGGTGACAAAATTACGTTCGAAGCGACAGGAAACTTGGTGGTCCCGCGCTATGCATTCACTATGGAAAGGAACGCAGGTTCTGGTATCATCATCTCAGATACACCCGTTCACGACTGTAATACCACCTGCCAGACCCCCGAAGGCGCTATCAACACTTCACTGCCATTCCAGAACGTACACCCTATTACAATCGGTAAGTGCCCAAAATACGTTAAAAGTACCAAGTTGCGTTTGGCGACGGGACTTCGCAACGTACCGTCCATCCAAAGCCGTGGTTTGTTCGGTGCTATTGCAGGTTTCATCGAAGGTGGCTGGACTGGTATGGTGGATGGTTGGTACGGATACCACCACCAGAACGAGCAGGGCAGCGGCTACGCAGCCGACCTGAAGTCTACCCAGAACGCTATCGATAAGATCACCAATAAGGTCAACAGCGTCATTGAAAAGATGAACACCCAGTTTACGGCTGTGGGCAAGGAGTTCAATCACCTCGAGAAGAGGATCGAAAATCTCAACAAAAAGGTGGACGACGGCTTCCTGGATATCTGGACCTATAATGCAGAACTGCTCGTTCTCCTCGAGAACGAACGCACGCTGGACTACCACGACTCAAACGTCAAGAACCTGTACGAAAAGGTGCGTAACCAGCTCAAAAACAACGCTAAAGAAATAGGCAACGGCTGCTTCGAGTTCTACCACAAATGCGACAATACCTGCATGGAAAGCGTCAAAAATGGAACCTACGATTACCCGAAGTACTCCGAGGAAGCTAAGCTCAATAGGGAGAAAATCGACGGTGTGAAGCTCGAATCAACTCGCATCTATCAGATCCTTGCGTTGGTTCCCAGGGGATCCATGAAACAAATCGAGGATAAGATCGAAGAAATTTTGTCCAAAATCTACCATATAGAAAACGAGATTGCCCGCATCAAGAAGTTGATCGGAGAAGTCCACCACCACCACCACCACCATCACTGA
SEQ ID NO.6:
ATGCTCTTGGTCAATCAGAGTCACCAAGGCTTCAACAAGGAGCACACATCTAAGATGGTGAGTGCTATCGTACTGTACGTCCTTCTTGCTGCGGCTGCTCACTCCGCTTTCGCTGCTGACCAGAAAATCCCCGGAAATGATAACAGTACTGCCACCCTCTGTCTGGGTCACCATGCTGTCCCCAATGGTACCATCGTGAAAACCATTACAAATGACCGTATCGAGGTCACAAACGCCACCGAACTGGTGCAGAACAGCTCGATTGGTGAAATATGTGATAGCCCACACCAAATCCTGGACGGCGAAAACTGCACCTTGATCGACGCTCTTCTGGGCGACCCTCAGTGCGATGGCTTCCAAAATAAAAAGTGGGACCTCTTTGTGGAGAGAAGTAAGGCCTACAGTAACTGCTACCCATATGACGTGCCAGACTACGCATCACTGAGGAGCCTCGTGGCGAGCTCTGGTACCCTGGAGTTCAAAAACGAATCTTTCAACTGGACTGGTGTGAAGCAAAATGGAACTTCCAGCGCTTGCATCAGGGGTTCATCCTCCAGCTTCTTTAGCAGATTGAACTGGCTGACACATTTGAACTACACATACCCAGCTCTGAATGTCACTATGCCCAACAAGGAGCAGTTCGACAAGTTGTACATTTGGGGTGTACATCACCCCGGAACCGACAAGGACCAGATTTTCCTCTACGCCCAATCAAGCGGCCGTATCACTGTTTCAACTAAGAGGAGCCAACAGGCTGTCATCCCTAATATCGGTAGCAGGCCTAGGATCAGGGACATCCCTTCTAGGATCTCTATCTACTGGACAATTGTGAAGCCCGGCGATATTTTGCTCATCAATTCTACTGGAAACCTGATCGCGCCACGCGGATACTTCAAGATCAGAAGTGGCAAGAGCAGTATTATGCGCAGTGATGCGCCAATTGGAAAATGTAAGTCTGAGTGCATCACCCCTAACGGATCGATCCCCAACGACAAGCCGTTCCAGAATGTCAACAGAATTACCTACGGCGCCTGTCCCCGTTACGTAAAGCAGAGTACTCTCAAATTGGCTACTGGCATGCGTAACGTTCCGGAGAAGCAGACACGTGGCATTTTCGGAGCCATCGCCGGCTTCATTGAAAACGGATGGGAAGGTATGGTGGACGGATGGTACGGCTTCCGTCACCAGAACTCCGAAGGCCGCGGTCAAGCTGCCGACTTGAAGTCGACCCAGGCTGCGATAGATCAAATCAATGGAAAACTGAACCGCCTTATTGGCAAGACTAACGAGAAGTTCCACCAGATTGAGAAAGAGTTCAGCGAAGTAGAGGGAAGAGTCCAGGACTTGGAGAAATACGTTGAGGATACCAAGATCGACCTGTGGTCTTACAACGCGGAGCTCCTTGTCGCACTGGAGAATCAACACACAATTGATCTTACCGATAGTGAGATGAACAAGCTCTTTGAGAAAACAAAGAAGCAACTGAGAGAGAACGCAGAAGATATGGGAAACGGTTGCTTTAAAATTTACCATAAATGCGACAATGCTTGCATCGGATCGATCCGCAACGAGACGTACGATCACAACGTTTATCGTGATGAAGCTCTGAACAACCGTTTCCAGATCAAAGGAGTAGAGCTCAAAAGCGGCTACAAGGATTGGATCCTCGTGCCGAGAGGATCAATGAAGCAAATTGAAGATAAGATCGAAGAGATCCTCTCAAAAATTTACCACATCGAAAACGAAATCGCCAGAATCAAAAAATTGATCGGTGAGGTTCATCACCATCACCACCACCACCACTGA
SEQ ID NO.7:
ATGCTTTTGGTGAACCAGTCGCACCAGGGATTTAACAAGGAGCACACCTCAAAGATGGTATCTGCTATCGTCCTCTATGTACTTTTGGCAGCAGCTGCGCACTCTGCCTTTGCTGCTGATCATCACCACCATCATCACCACCATTCATCGTCCGACTACTCAGATCTCCAGCGTGTCAAACAGGAATTGCTGGAAGAAGTAAAGAAGGAGCTCCAAAAGGTGAAGGAGGAGATCATCGAGGCTTTCGTGCAGGAACTTCGCAAGAGAGGCTTGGTTCCTCGCGGCTCCCAGATCGGAAACATCATCTCCATATGGGTCAGTCATTCAATCCAAATCGGTAATCAGAGCCAGATTGAAACGTGCAACCAAAGCGTCATCACGTACGAGAATAACACTTGGGTGAATCAAACCTACGTGAACATCTCTAACACAAATTTCGCAGCTGGACAATCTGTCGTCTCGGTCAAGTTGGCAGGTAATTCCAGTCTCTGCCCAGTTAGTGGCTGGGCCATTTACTCTAAGGACAATTCTGTTCGTATTGGCTCCAAGGGCGACGTCTTCGTAATCCGCGAACCTTTTATATCCTGCTCTCCTCTCGAATGCCGCACTTTCTTCCTGACTCAGGGAGCACTTTTGAACGACAAGCATTCCAACGGAACTATTAAGGATCGTAGCCCTTATCGTACTCTGATGTCATGTCCAATCGGAGAGGTTCCGAGCCCATACAACTCTCGCTTTGAGTCAGTAGCCTGGTCAGCTTCCGCTTGCCACGACGGTATAAACTGGTTGACAATCGGAATTAGTGGACCAGACTCTGGCGCTGTCGCAGTTCTCAAGTACAACGGCATTATCACTGACACCATCAAGTCCTGGCGCAACAATATCTTGAGAACACAGGAAAGTGAATGCGCGTGTGTCAACGGCAGCTGCTTTACCATCATGACAGACGGACCAAGCGACGGACAGGCTAGTTACAAGATCTTCCGCATAGAAAAGGGCAAAATCATCAAGTCTGTAGAGATGAAAGCCCCTAACTACCACTACGAGGAGTGTTCGTGCTACCCTGATTCATCTGAGATCACCTGTGTTTGTAGGGACAATTGGCACGGATCCAACAGACCATGGGTTTCTTTCAACCAAAATCTCGAATACCAAATGGGTTACATCTGTAGCGGTGTCTTCGGAGATAACCCCCGTCCGAACGACAAAACTGGCTCTTGCGGTCCCGTGTCCTCCAACGGAGCTAACGGTGTGAAGGGTTTCTCTTTCAAGTACGGTAACGGTGTGTGGATTGGTAGGACAAAGTCAATCTCAAGTAGGAAGGGATTCGAGATGATTTGGGACCCCAACGGTTGGACTGGTACCGACAACAAGTTCTCGATAAAGCAAGATATTGTTGGAATCAACGAGTGGAGCGGTTACTCCGGCAGTTTTGTGCAGCACCCCGAACTGACTGGATTGGACTGCATACGCCCTTGCTTCTGGGTAGAGTTGATCCGTGGTCGTCCAGAGGAGAACACTATCTGGACGAGTGGAAGCAGCATCAGCTTTTGTGGCGTCAACTCCGACACAGTGGGATGGTCATGGCCTGACGGTGCTGAGTTGCCGTTCACTATCGACAAGTGA
SEQ ID NO.8:
ATGCTGCTTGTGAATCAGTCGCATCAGGGATTCAACAAGGAGCATACCTCTAAAATGGTTAGCGCTATCGTTTTGTACGTCTTGCTGGCTGCTGCCGCTCATAGTGCTTTCGCTGCAGATCATCACCACCATCACCACCACCACAGTAGCTCGGACTATTCCGACCTTCAGAGAGTCAAGCAGGAATTGTTGGAAGAAGTCAAGAAGGAATTGCAAAAGGTCAAGGAAGAGATTATCGAAGCCTTCGTACAGGAGCTTCGCAAACGCGGAACGCTCCACTTTAAACAATATGAGTTTAATTCTCCTCCTAACAACCAAGTGATGCTCTGCGAACCGACAATCATTGAGCGCAACATTACTGAAATCGTGTATTTGACAAATACTACCATCGAAAAGGAAATCTGCCCCAAGCCTGCTGAATACCGTAACTGGAGCAAACCCCAGTGCGGAATCACTGGTTTTGCTCCCTTCTCCAAGGACAACTCTATCCGCCTGTCCGCTGGAGGTGATATCTGGGTGACGAGGGAGCCCTACGTCTCGTGTGACCCAGATAAATGCTATCAGTTCGCTCTTGGCCAAGGTACTACGCTCAATAACGTGCACTCGAATAACACCGTACGCGACAGAACACCATACAGGACTCTGTTGATGAACGAATTGGGCGTCCCCTTCCACCTGGGTACCAAGCAAGTGTGTATAGCCTGGAGTAGCTCATCGTGTCACGACGGAAAGGCTTGGCTGCACGTCTGTATCACCGGTGATGATAAGAACGCTACCGCAAGTTTCATCTACAACGGTAGGTTGGTTGATAGCGTAGTCTCCTGGTCAAAGGACATCCTGAGAACGCAAGAGTCCGAATGCGTGTGCATTAACGGTACCTGCACTGTCGTGATGACGGACGGTAACGCCACTGGAAAAGCTGATACCAAAATCTTGTTTATCGAGGAAGGAAAGATCGTCCATACCAGTAAGTTGAGTGGCTCGGCGCAGCACGTCGAGGAATGTAGTTGCTACCCCAGGTACCCTGGTGTACGCTGCGTCTGCAGGGACAACTGGAAGGGAAGTAATAGGCCAATCGTGGATATCAACATCAAGGACCACTCCATCGTGTCTAGTTATGTGTGTTCTGGACTCGTGGGTGACACTCCACGCAAAAATGATTCCAGCTCATCTAGCCATTGTCTGGATCCTAACAACGAAGAGGGAGGTCATGGTGTTAAGGGCTGGGCCTTCGACGATGGCAACGATGTGTGGATGGGACGCACGATAAACGAAACGTCCAGACTCGGATACGAAACGTTCAAGGTCGTCGAGGGATGGTCGAATCCTAAGTCCAAACTGCAGATCAACAGGCAGGTCATCGTGGACCGTGGTGACAGGAGCGGCTACAGCGGTATTTTCTCGGTAGAGGGTAAGTCCTGTATCAACAGGTGTTTCTACGTAGAGCTGATCAGGGGCAGAAAAGAAGAAACAGAGGTCTTGTGGACATCAAACAGTATCGTGGTGTTTTGCGGTACCTCAGGTACTTACGGAACCGGTTCCTGGCCTGATGGTGCCGACCTTAACCTGATGCACATCTGA
Claims (20)
1. A universal Mosaic recombinant antigen for seasonal influenza A, which is characterized in that:
(a) An antigen consisting of the amino acid sequence shown in any one of SEQ ID Nos 1 to 4; alternatively, the first and second electrodes may be,
(b) Antigen formed by substituting, deleting or adding one or more amino acids in the amino acid sequence in (a).
2. The seasonal influenza a universal Mosaic recombinant antigen of claim 1, wherein the Th cell epitope coverage of the Mosaic recombinant antigen is greater than 81% compared to a Th cell epitope on a native HA protein of an influenza virus; compared with the Th cell epitope on the natural NA protein of the influenza virus, the Th cell epitope coverage rate of the Mosaic recombinant antigen is more than 84%.
3. The seasonal influenza a universal Mosaic recombinant antigen of claim 2, wherein the HA protein comprises an H1 protein or an H3 protein and the NA protein comprises an N1 protein or an N2 protein.
4. The seasonal influenza a universal Mosaic recombinant antigen of claim 3, wherein more than 81% of the 12 amino acids of the Th cell epitope of the Mosaic recombinant antigen are a perfect match to the 12 amino acids of the Th cell epitope on the native H1 protein or H3 protein of influenza virus.
5. The universal Mosaic recombinant antigen of claim 3, wherein said Mosaic recombinant antigen has more than 11 amino acids of a Th cell epitope of greater than 96% that are a perfect match to 12 amino acids of a Th cell epitope on a native H1 protein or H3 protein of influenza virus.
6. The seasonal influenza a universal Mosaic recombinant antigen of claim 3, wherein more than 99% of the 10 amino acids of the Th cell epitopes of the Mosaic recombinant antigen are a perfect match of 12 amino acids of Th cell epitopes of the native H1 protein or H3 protein of influenza virus.
7. The seasonal influenza a universal Mosaic recombinant antigen of claim 3, wherein more than 84% of the 12 amino acids of the Th cell epitope of the Mosaic recombinant antigen are a perfect match to the 12 amino acids of the Th cell epitope on the native N1 protein or N2 protein of influenza virus.
8. The seasonal influenza a universal Mosaic recombinant antigen of claim 3, wherein more than 11 amino acids of a 96% Th cell epitope of the Mosaic recombinant antigen completely matches 12 amino acids of a Th cell epitope on a native N1 protein or N2 protein of an influenza virus.
9. The universal Mosaic recombinant antigen of claim 3, wherein more than 10 amino acids of a Th cell epitope of said Mosaic recombinant antigen greater than 98% completely match 12 amino acids of a Th cell epitope on a native N1 or N2 protein of influenza virus.
10. The seasonal influenza a universal Mosaic recombinant antigen of claim 1, further added with a gp67 signal peptide, a thrombin cleavage site and an 8 xhis tag, removing the HA or NA native signal peptide; adding GCN4pII sequence aiming at HA protein and VASP sequence aiming at NA protein;
the amino acid sequence of the gp67 signal peptide is: MLLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAAD;
the GCN4pII sequence is MKQIEDKIEEILSKIYHIENEIARIKKLIGEV;
the VASP sequence is SSSDYSDLQRVQELLEVKELQKVKEEIEAFVQELKRG.
11. A gene encoding the universal seasonal influenza a Mosaic recombinant antigen of any one of claims 1 to 10.
12. The gene of claim 11, wherein the sequence of the gene is as shown in any one of SEQ ID Nos. 5-8.
13. A vector comprising the gene of claim 11 or 12.
14. The vector of claim 13, wherein the vector is obtained by ligating the gene of claim 11 or 12 into an expression plasmid.
15. A cell comprising the gene of claim 11 or 12 or the vector of claim 13 or 14.
16. The cell of claim 15, wherein the cell is obtained by transferring a gene or vector into an escherichia coli host cell.
17. Use of a seasonal influenza a universal Mosaic recombinant antigen as defined in any one of claims 1 to 10 in the preparation of a seasonal influenza a universal vaccine.
18. A vaccine preparation comprising the seasonal influenza a universal Mosaic recombinant antigen of any one of claims 1 to 10, the gene of claim 11 or 12, the vector of claim 13 or 14.
19. The vaccine formulation of claim 18, further comprising an immunologically and pharmaceutically acceptable carrier or adjuvant.
20. Use of a vaccine formulation according to claim 18 or 19 in the manufacture of a medicament for the prophylaxis and/or treatment of seasonal influenza a.
Priority Applications (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311374200.9A CN117534738A (en) | 2022-07-15 | 2022-07-15 | Seasonal influenza A universal Mosaic recombinant antigen, vaccine and application thereof |
| CN202311374197.0A CN117534737A (en) | 2022-07-15 | 2022-07-15 | Seasonal influenza A universal Mosaic recombinant antigen, vaccine and application thereof |
| CN202210840150.8A CN115894636A (en) | 2022-07-15 | 2022-07-15 | Seasonal influenza A universal Mosaic recombinant antigen, vaccine and application thereof |
| CN202311374201.3A CN117534739A (en) | 2022-07-15 | 2022-07-15 | Seasonal influenza A universal Mosaic recombinant antigen, vaccine and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210840150.8A CN115894636A (en) | 2022-07-15 | 2022-07-15 | Seasonal influenza A universal Mosaic recombinant antigen, vaccine and application thereof |
Related Child Applications (3)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311374197.0A Division CN117534737A (en) | 2022-07-15 | 2022-07-15 | Seasonal influenza A universal Mosaic recombinant antigen, vaccine and application thereof |
| CN202311374200.9A Division CN117534738A (en) | 2022-07-15 | 2022-07-15 | Seasonal influenza A universal Mosaic recombinant antigen, vaccine and application thereof |
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| US20110229518A1 (en) * | 2008-11-28 | 2011-09-22 | Statens Serum Institut | Optimized influenza vaccines |
| CN112996539A (en) * | 2018-09-12 | 2021-06-18 | Acm生物实验室私人有限公司 | Polymersomes comprising covalently bound antigens, methods of making and uses thereof |
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| KR101825407B1 (en) * | 2016-03-18 | 2018-02-06 | 한국생명공학연구원 | An antibody against a drug-resistant influenza virus |
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| US20110229518A1 (en) * | 2008-11-28 | 2011-09-22 | Statens Serum Institut | Optimized influenza vaccines |
| CN112996539A (en) * | 2018-09-12 | 2021-06-18 | Acm生物实验室私人有限公司 | Polymersomes comprising covalently bound antigens, methods of making and uses thereof |
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| GENBANK: AKE37534.1: "neuraminidase [Influenza A virus (A/India/Pun151245/2015(H1N1))]", GENBANK: AKE37534.1 * |
| GENBANK: ARW80332.1: "hemagglutinin [Influenza A virus (A/Washington/35/2017(H1N1))]", GENBANK: ARW80332.1, vol. 80332 * |
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