Recombinant chimeric antigen for poxvirus, subunit vaccine comprising same and use thereof

2:

• The recombinant chimeric antigen for poxvirus according to claim 1, wherein:
• (a) the antigenic fragment I or II of the A35 protein is an extracellular segment of the protein or a part thereof;
• or,
• the antigenic fragment I or II of the M1R protein is an extracellular segment of the protein or a part thereof.

3:

• The recombinant chimeric antigen of claim 1, wherein:
• (a) A1 represents the amino acid sequence as shown in SEQ ID NO:1, or an amino acid sequence having the same or substantially the same immunogenicity thereto obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence as shown in SEQ ID NO:
• 1;
• (b) A2 represents the amino acid sequence as shown in SEQ ID NO:1, or an amino acid sequence of the amino acid sequence as shown in SEQ ID NO:1 with a fragment of 1-30 amino acids extended to the N-terminus of the A35 protein, or an amino acid sequence having the same or substantially the same immunogenicity thereto obtained by substituting, deleting or adding one or more amino acids to the above amino acid sequences;
• (c) A2 represents the amino acid sequence as shown in SEQ ID NO:2, or an amino acid sequence having the same or substantially the same immunogenicity thereto obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence as shown in SEQ ID NO:
• 2;
• or,
• (d) M1 and M2 are the same and represent the amino acid sequence as shown in SEQ ID NO:3, or an amino acid sequence having the same or substantially the same immunogenicity thereto obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence as shown in SEQ ID NO:
• 3.

4:

• The recombinant chimeric antigen for poxvirus according to claim 3, wherein:
• (a) A1 represents the amino acid sequence as shown in SEQ ID NO:
• 1, A2 represents the amino acid sequence as shown in SEQ ID NO:
• 1 or SEQ ID NO:
• 2, and M1 and M2 are the same and represent the amino acid sequence as shown in SEQ ID NO:
• 3;
• (b) the C1, C2, C3 are all absent;
• (c) the recombinant chimeric antigen comprises the amino acid sequence as shown in SEQ ID NO:4;
• or
• (b) the recombinant chimeric antigen is a single-chain dimer structure.

5:

• The recombinant chimeric antigen for poxvirus according to any one of claims 1-4, wherein:
• (a) the N-terminus of the recombinant chimeric antigen further comprises a signal peptide sequence;
• (b) the signal peptide comprises a sequence as shown in SEQ ID NO:5;
• (c) the C-terminus of the recombinant chimeric antigen further comprises a tag sequence;
• or
• (d) the C-terminus of the recombinant chimeric antigen further comprises a tag sequence is selected from the group consisting of a Flag tag, a His tag, a MBP tag, a HA tag, a myc tag, a GST tag, and a SUMO tag.

6:

• A method for preparing the recombinant chimeric antigen of claim 1, comprising the following steps:
• (a) adding a Kozak sequence and a coding sequence of a signal peptide to the 5′ end, and adding a coding sequence of a histidine tag and a stop codon to the 3′ end of the nucleotide sequence encoding the recombinant chimeric antigen of claim 1,
• (b) performing cloning and expression,
• (d) screening for the correct recombinants,
• (d) transfecting them into expression system cells for expression,
• (e) collecting cell culture supernatant, and
• (f) isolating the recombinant chimeric antigen peptide therefrom.

7:

• The method of claim 6, wherein:
• (a) the expression system cell is a mammalian cell, an insect cell, a yeast cell or a bacterial cell;
• (b) the expression system cell is a HEK293T cell, a 293F series cell or a CHO cell;
• (c) the expression system cell is a HEK293F cell, a Freestyle293F cell or an Expi293F cell;
• (d) the insect cell is a sf9 cell, a Hi5 cell, a sf21 cell or a S2 cell;
• (e) the yeast cell is a Pichia cerevisiae cell or a yeast cell transformed therefrom;
• or
• (f) the bacterial cell is an Escherichia coli cell.

10:

• The vaccine or immunogenic composition according to claim 9, wherein:
• (a) the vaccine or immunogenic composition is in the form of a nasal spray, an oral preparation, a suppository or a parenteral preparation;
• (b) the nasal spray is selected from the group consisting of an aerosol, a spray and a powder aerosols;
• (c) the oral preparation is selected from the group consisting of a tablet, a powder, a pill, a granule, a soft/hard capsule, a film-coated preparation and a paste;
• (d) the tablet is a sublingual tablet;
• (e) the granule is a fine granule;
• (f) the powder is a bulk powder;
• (g) the pill is a minipellet;
• (h) the parenteral preparation is selected from the group consisting of a transdermal preparation, an ointment, a plaster, a topical liquid, and an injectable preparation;
• or
• (i) the injectable preparation is a bolus-injectable preparation.

11:

• The method of claim 8, wherein:
• (a) the poxvirus is selected from the group consisting of Monkeypox virus, Cowpox virus, Variola virus and Vaccinia virus;
• (b) the recombinant chimeric antigen is formulated as a vaccine;
• (c) the recombinant chimeric antigen is formulated as a recombinant protein vaccine;
• (d) the recombinant protein vaccine further comprises an adjuvant selected from the group consisting of an aluminum adjuvant, MF59 adjuvant and an MF59-like adjuvant;
• (e) the vaccine is in the form of a nasal spray, an oral preparation, a suppository or a parenteral preparation;
• (f) the nasal spray is selected from the group consisting of an aerosol, a spray and a powder aerosol;
• (g) the oral preparation is selected from the group consisting of a tablet, a powder, a pill, a granule, a soft/hard capsule, a film-coated preparation and a paste;
• (h) the oral preparation is formulated as a sublingual tablet;
• (i) the granule is a fine granule;
• (j) the powder is a bulk powder;
• (k) the oral preparation is formulated as a minipellet pill;
• (l) the parenteral preparation is selected from the group consisting of a transdermal preparation, an ointment, a plaster, a topical liquid, an injectable preparation;
• or
• (m) the injectable preparation is a bolus-injectable preparation.

12:

• The vaccine or immunogenic composition of claim 9, wherein:
• (a) the vaccine or immunogenic composition is a poxvirus recombinant protein vaccine, which comprises the recombinant chimeric antigen according of claim 1 and an adjuvant;
• or
• (b) the adjuvant is selected from the following adjuvants:
• an aluminum adjuvant, MF59 adjuvant and an MF59-like adjuvant.