Recombinant mycobacterium encoding a heparin-binding hemagglutinin (HBHA) fusion protein and uses thereof
Recombinant Mycobacteria (rMyc) which contain sequences encoding a heparin-binding hemagglutinin (HBHA) fusion protein are provided, as are methods of making and using the rMyc and the fusion protein. The fusion protein includes an amino terminal mycobacterial antigen Ag85B leader peptide and transcription of the fusion protein is driven by an Ag85B promoter sequence. The recombinant fusion protein is produced in abundance by the rMyc, is post-translationally methylated, and is highly antigenic.
1. A Mycobacterium comprising a nucleic acid fusion sequence encoding an Ag85B leader sequence attached to an amino terminus of a mycobacterial heparin-binding haemagglutinin (HBHA) protein or antigenic fragment thereof, wherein said fusion sequence is operably linked to a promoter, and wherein said nucleic acid fusion sequence comprises a nucleotide sequence at least 90% homologous to SEQ ID NO: 1.
2. The Mycobacterium of claim 1 , wherein said Mycobacterium is selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium bovis , and Mycobacterium smegmatis.
3. The Mycobacterium of claim 1 , wherein said Mycobacterium is Mycobacterium bovis ( Bacille Calmette-Guérin) (BCG).
4. The Mycobacterium of claim 3 , wherein said BCG is a BCG Danish Statens Serum Institut (SSI) strain.
5. The Mycobacterium of claim 4 , wherein said BCG SSI strain expresses a pfo gene.
6. The Mycobacterium of claim 5 , wherein said pfo gene is a pfo gene from Clostridium perfringens.
7. The Mycobacterium of claim 1 , wherein said Mycobacterium is an auxotroph.
8. The Mycobacterium of claim 7 , wherein said Mycobacterium is a pantothenic acid auxotroph.
9. A Mycobacterium comprising a nucleic acid fusion sequence encoding an Ag85B leader sequence attached to an amino terminus of a mycobacterial heparin-binding haemagglutinin (HBHA) protein or antigenic fragment thereof, wherein said fusion sequence is operably linked to a promoter, and wherein a polypeptide encoded by said nucleic acid fusion sequence comprises an amino acid sequence at least 90% identical to SEQ ID NO: 3.
10. The Mycobacterium of claim 1 , where said mycobacterial HBHA protein is Mycobacterium tuberculosis HBHA.
11. The Mycobacterium of claim 1 , wherein said promoter is a mycobacterial Ag85B promoter.
12. An isolated nucleic acid molecule comprising a nucleotide sequence at least 90% homologous to SEQ ID NO: 1.
13. A fusion protein, comprising an Ag85B leader sequence attached to an amino terminus of a mycobacterial heparin-binding haemagglutinin (HBHA) protein or antigenic fragment thereof, wherein the fusion protein comprises an amino acid sequence at least 90% identical to SEQ ID NO: 3.
14. A method of producing the fusion protein of claim 13 , comprising:
transfecting a Mycobacterium cell with a nucleic acid sequence encoding said fusion protein; and
growing said transfected Mycobacterium cell under conditions which allow said Mycobacterium cell to produce said fusion protein.
15. A method of producing recombinant heparin-binding haemagglutinin (rHBHA) protein, comprising the steps of:
growing a culture of Mycobacterium of claim 1 .
16. A seed lot of recombinant Mycobacterium , comprising:
recombinant Mycobacterium of claim 1 ; and
medium suitable for maintaining said recombinant Mycobacterium in a viable state during storage of said seed lot.
17. A seed lot of recombinant Mycobacterium , comprising:
recombinant Mycobacterium of claim 9 ; and
medium suitable for maintaining said recombinant Mycobacterium in a viable state during storage of said seed lot.