Bacillus based delivery system and methods of use
AssigneeThe Curators of the University of Missouri
InventorsGeorge C. Stewart, Brian Matthew Thompson
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Abstract
Herein a Bacillus exosporium antigen delivery (BEAD) system that provides a means to introduce recombinant proteins or small molecules into the exosporium of members of the B. cereus family of bacteria, i.e. B. anthracis, B. cereus, and B. thuringiensis, is disclosed. The system results in the surface display of recombinant proteins or small molecules such that they can stimulate an immune response. In addition, methods of making and using the system are described.
Claims24
A Bacillus exosporium molecule delivery (BEMD) system comprising a recombinant Bacillus cereus family member expressing a fusion construct, wherein the fusion construct comprises at least one molecule of interest (MOI) and:
- (a) a targeting sequence consisting of a 16 amino acid sequence having at least 43% identity with amino acids 20-35 of SEQ ID NO.7, wherein the identity with amino acids 25-35is at least 54%;
- (b) a targeting sequence consisting of amino acids 1-35 of SEQ ID NO. 7;
- (c) a targeting sequence consisting of amino acids 20-35 of SEQ ID NO. 7;
- (d) a targeting sequence comprising SEQ ID NO. 13;
- (e) a targeting sequence consisting of SEQ ID NO.7;
- (f) a targeting sequence comprising amino acids 1-27 of SEQ ID NO. 8;
- (g) a targeting sequence comprising amino acids 12-27 of SEQ ID NO. 8;
- (h) a targeting sequence comprising SEQ ID NO. 8;
- (i) an amino acid sequence comprising the amino acid sequence encoded by the entire open reading frame of BAS3290, wherein BAS3290 comprises an N-terminal domain comprising SEQ ID NO:
- 8;
- (j) a targeting sequence comprising amino acids 1-38 of SEQ ID NO. 9;
- (k) a targeting sequence comprising amino acids 23-38 of SEQ ID NO. 9;
- (l) a targeting sequence comprising SEQ ID NO. 9;
- or
- (m) a targeting sequence comprising a methionine residue covalently bound to an amino acid sequence consisting of 16 amino acids having at least 43% identity with amino acids 20-35 of SEQ ID NO.7, wherein the identity with amino acids 25-35 is at least 54%.
A method of making a delivery system comprising:
- a) constructing a fusion construct, wherein the fusion construct comprises at least one MOI and:
- (i) a targeting sequence consisting of a 16 amino acid sequence having at least 43% identity with amino acids 20-35 of SEQ ID NO. 7, wherein the identity with amino acids 25-35 is at least 54%;
- (ii) a targeting sequence consisting of amino acids 1-35 of SEQ ID NO. 7;
- (iii) a targeting sequence consisting of amino acids 20-35 of SEQ ID NO. 7;
- (iv) a targeting sequence comprising SEQ ID NO. 13;
- (v) a targeting sequence consisting of SEQ ID NO. 7;
- (vi) a targeting sequence comprising amino acids 1-27 of SEQ ID NO. 8;
- (vii) a targeting sequence comprising amino acids 12-27 of SEQ ID NO. 8;
- (viii) a targeting sequence comprising SEQ ID NO. 8;
- (ix) an amino acid sequence comprising the amino acid sequence encoded by the entire open reading frame of BAS3290, wherein BAS3290 comprises an N-terminal domain comprising SEQ ID NO:
- 8;
- (x) a targeting sequence comprising amino acids 1-38 of SEQ ID NO. 9;
- (xi) a targeting sequence comprising amino acids 23-38 of SEQ ID NO. 9;
- (xii) a targeting sequence comprising SEQ ID NO. 9;or
- (xiii) a targeting sequence comprising a methionine residue covalently bound to an amino acid sequence consisting of 16 amino acids having at least 43% identity with amino acids 20-35 of SEQ ID NO.7, wherein the identity with amino acids 25-35 is at least 54%;
- b) cloning the fusion construct into a shuttle plasmid;
- and
- c) electroporating the shuttle plasmid containing the fusion construct into a Bacillus cereus family member such that the fusion construct is expressed on the Bacillus cereus family member's exosporium.
Dependencies are inferred from the claim text.