Novel protein molecule useful for inhibition of anthrax toxin

• US 20040235136A1
• Filed: 02/17/2004
• Published: 11/25/2004
• Est. Priority Date: 03/29/2001
• Status: Active Grant

• Current Assignee: Hemant Khanna, Yogendra Singh
• Original Assignee: Hemant Khanna, Yogendra Singh
• Inventors: Singh, Yogendra, Khanna, Hemant
• Granted Patent: US 7,282,580 B2
• Time in Patent Office: Days
• US Class Current: 435/252.3
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: C07K 14/32 from Bacillus (G)
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The present invention relates to a novel molecule useful for anthrax toxin inhibition in vivo and also provides a method for in vivo inhibition of anthrax toxin action using the new molecule.

Claims

1. A recombinant protein designated as PA-I of SEQ:

ID NO;

1, useful for inhibiting anthrax toxin.

2. A recombinant protein as claimed in claim 1, wherein the said protein is non toxic to host cells.

3. A recombinant protein as claimed in claim 1, wherein the said protein inhibits native protein Protective Antigen (PA) mediated cellular intoxication.

4. A recombinant protein as claimed in claim 1, wherein the said protein inhibits the channel forming ability of PA protein.

5. A recombinant protein as claimed in claim 1, wherein the said protein when applied with PA in the ratio of about 1:

1, completely inhibits the anthrax lethal toxin.

6. A recombinant protein as claimed in claim 1, wherein the PA-I has oligopeptide of SEQ ID NO:

2 instead of oligopeptide of SEQ ID NO;

3 of native PA.

7. The gene encoding the recombinant protein PA-I, defined in claim 1, having sequence SEQ ID NO:

4.

9. A process for constructing a recombinant protein PA-I as defined in claim 1, said process comprising steps:

v) amplifying a region of PA gene encoding 2β

2-2 β

3 loop using the primers of SEQ ID NO;

5 and SEQ ID NO;

6;

vi) mutating the amplified PA gene by replacing SEQ ID NO;

3 of native PA with SEQ ID NO;

2,

vii) cloning the amplified mutated PA gene of step (ii) into a vector, and

viii) expressing the clone in a host to obtain the recombinant protein PA-I.

10. A method as claimed in claim 9, wherein the host used is selected from a group comprising E. coli and Bacillus anthracis.

11. A method as claimed in claim 9, wherein the vector for cloning the mutant gene is selected from a group of expression vector comprising plasmid pYS5 and pMS1.

12. A process as claimed in claim 9, wherein the said protein is non toxic to cells.

13. A process as claimed in claim 9, wherein the said protein inhibits native PA mediated cellular intoxication.

14. A process as claimed in claim 9, wherein the said protein inhibits the channel forming ability of PA toxin.

15. A process as claimed in claim 9, wherein the said recombinant protein PA-I completely inhibits the anthrax lethal toxins.

16. A method as claimed in claim 9, wherein the concentration of PA-I used for testing anthrax toxin inhibition is in the range of 0.01 μ

g/ml to 0.1 μ

g/ml.

8. A pair of oligonucleotide primers of SEQ ID NO:

5 and SEQ ID NO;

6, wherein the primers are reverse primer and forward primer respectively.

17. A composition useful in inhibiting anthrax toxin, said composition comprising a recombinant protein PA-I of SEQ ID NO:

1 and pharmacologically acceptable additive(s).

18. A composition as claimed in claim 17, wherein the additives are selected from a group comprising glucose and PBS.

19. A method of treating anthrax infection in a subject in need thereof, said method comprising step of administering pharmacologically effective amount of PA-I optionally along with pharmacologically acceptable additive(s).

20. A method of treatment as claimed in claim 19, wherein the additives are selected from a group comprising glucose and PBS.

21. A method as claimed in claim 19, wherein the PA-I is administered intravenously.

22. A method of treatment as claimed in claim 19, wherein the subject is a mammals, preferably human.

23. A method as claimed in claim 19, wherein the recombinant protein PA-I completely inhibits the toxicity of anthrax lethal toxin.

24. A method as claimed in claim 23, wherein recombinant protein PA-I results in 100% survival of rats even after 72 hours of injecting toxin.

25. A method as claimed in claim 23, wherein recombinant protein PA-I inhibits pore formation by native PA in cells.