Recombinant attenuated
The present invention discloses attenuated Clostridium perfringens organisms that express a substantially nontoxic alpha-toxin. The expressed alpha-toxin is a deletion mutein that relative to the alpha-toxin of the mature alpha-toxin of Clostridium perfringens strain 13, is missing at least nine consecutive amino acid residues including His 68 . The present invention also discloses attenuated organisms that encode the muteins, as well as the use of such attenuated organisms as vaccines.
1. A recombinant nucleic acid molecule that encodes a substantially nontoxic mutein of Clostridium perfringens alpha-toxin; wherein the mutein alpha-toxin comprises the amino acid sequence of SEQ ID NO: 3 minus at least 9 consecutive amino acid residues, with no more than 48 consecutive amino acid residues deleted; and wherein one of the deleted amino acid residues is His 68 .
2. The recombinant nucleic acid molecule of claim 1 wherein the mutein alpha-toxin comprises the amino acid sequence of SEQ ID NO: 3 minus at least 12 consecutive amino acid residues; and wherein one of the deleted amino acid residues is His 68 .
3. The recombinant nucleic acid molecule of claim 2 wherein the mutein alpha-toxin comprises the amino acid sequence of SEQ ID NO: 3 minus at least 18 consecutive amino acid residues; and wherein one of the deleted amino acid residues is His 68 .
4. The recombinant nucleic acid molecule of claim 1 wherein the mutein alpha-toxin comprises SEQ ID NO: 3 minus 9 consecutive amino acid residues ranging from Tyr 62 through Trp 70 .
5. The recombinant nucleic acid molecule of claim 1 comprising the nucleic acid sequence of SEQ ID NO: 2; wherein nucleotides 268-294 of the nucleic acid molecule are deleted.
6. The recombinant nucleic acid molecule of claim 5 wherein the deleted nucleotides are replaced by a nucleotide sequence encoding a single Leu residue.
7. A recombinant nucleic acid molecule that encodes a substantially nontoxic mutein of Clostridium perfringens alpha-toxin;
wherein the substantially nontoxic mutein alpha-toxin comprises the amino acid sequence of SEQ ID NO: 3 in which His 68 is deleted together with an additional 4 to 60 flanking amino acid residues; and
wherein the attenuated Clostridium perfringens organism is substantially nontoxic due to a lack of a functional wild-type plc gene.
8. The recombinant nucleic acid molecule of claim 1 wherein the substantially nontoxic mutein alpha-toxin comprises the amino acid sequence of SEQ ID NO: 3 with no more than 36 consecutive amino acid residues deleted.
9. The recombinant nucleic acid molecule of claim 8 wherein the substantially nontoxic mutein alpha-toxin comprises the amino acid sequence of SEQ ID NO: 3 with no more than 24 consecutive amino acid residues deleted.
10. The recombinant nucleic acid molecule of claim 9 wherein the substantially nontoxic mutein alpha-toxin comprises the amino acid sequence of SEQ ID NO: 3 with no more than 18 consecutive amino acid residues deleted.
11. A vector that expresses a recombinant nucleic acid molecule that encodes a substantially nontoxic mutein of Clostridium perfringens alpha-toxin; wherein the mutein alpha-toxin comprises the amino acid sequence of SEQ ID NO: 3 minus at least 9 consecutive amino acid residues, with no more than 48 consecutive amino acid residues deleted; and wherein one of the deleted amino acid residues is His 68 .
12. The vector of claim 11 wherein the mutein alpha-toxin comprises the amino acid sequence of SEQ ID NO: 3 minus at least 12 consecutive amino acid residues; and wherein one of the deleted amino acid residues is His 68 .
13. The vector of claim 12 wherein the mutein alpha-toxin comprises the amino acid sequence of SEQ ID NO: 3 minus at least 18 consecutive amino acid residues; and wherein one of the deleted amino acid residues is His 68 .
14. The vector of claim 11 wherein the mutein alpha-toxin comprises SEQ ID NO: 3 minus 9 consecutive amino acid residues ranging from Tyr 62 through Trp 70 .
15. The vector of claim 11 comprising the nucleic acid sequence of SEQ ID NO: 2; wherein nucleotides 268-294 of the nucleic acid molecule are deleted.
16. The vector of claim 15 wherein the deleted nucleotides are replaced by a nucleotide sequence encoding a single Leu residue.
17. A vector that expresses a recombinant nucleic acid molecule that encodes a substantially nontoxic mutein of Clostridium perfringens alpha-toxin;
wherein the substantially nontoxic mutein alpha-toxin comprises the amino acid sequence of SEQ ID NO: 3 in which His 68 is deleted together with an additional 4 to 60 flanking amino acid residues; and
wherein the attenuated Clostridium perfringens organism is substantially nontoxic due to a lack of a functional wild-type plc gene.
18. The vector of claim 11 wherein the substantially nontoxic mutein alpha-toxin comprises the amino acid sequence of SEQ ID NO: 3 with no more than 36 consecutive amino acid residues deleted.
19. The vector of claim 18 wherein the substantially nontoxic mutein alpha-toxin comprises the amino acid sequence of SEQ ID NO: 3 with no more than 24 consecutive amino acid residues deleted.
20. The vector of claim 19 wherein the substantially nontoxic mutein alpha-toxin comprises the amino acid sequence of SEQ ID NO: 3 with no more than 18 consecutive amino acid residues deleted.