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US 2004/0170972 A1Published application
Ehrlichia canis genes and vaccines
AssigneeCornell Research Foundation, Inc.
InventorYung-Fu Chang
Life of this patent
Estimating the term…
Abstract
This invention provides the sequence of 5,299 nucleotides from the E. canis genome. There are four proteins, ProA, ProB, MmpA, and a cytochrome oxidase homolog, as well as a partial lipoprotein signal peptidase homolog at the carboxy terminus, coded for in this cloned fragment. The antigenic properties of these proteins allow them to be used to create a vaccine. An embodiment of this invention includes the creation of a DNA vaccine, a recombinant vaccine, and a T cell epitope vaccine. Another embodiment of this invention includes the use of serological diagnosis techniques.
Claims65
A recombinant DNA comprising said DNA selected from the group consisting of:
- a) a recombinant DNA that encodes a protein having an amino acid sequence as shown in SEQ. ID. NO. 3;
- b) a recombinant DNA that encodes a protein having an amino acid sequence as shown in SEQ. ID. NO. 5;
- c) a recombinant DNA that encodes a protein having an amino acid sequence as shown in SEQ. ID. NO. 7;
- d) a recombinant DNA that encodes a protein having an amino acid sequence as shown in SEQ. ID. NO. 9;
- e) a recombinant DNA that encodes a protein having an amino acid sequence as shown in SEQ. ID. NO. 11;
- and
- f) any portion of said DNA above that encodes a protein that elicits an immune response against E. canis.
4 more elements
A recombinant protein comprising said protein selected from the group consisting of:
- a) a protein having an amino acid sequence as shown in SEQ. ID. NO. 3;
- b) a protein having an amino acid sequence as shown in SEQ. ID. NO. 5;
- c) a protein having an amino acid sequence as shown in SEQ. ID. NO. 7;
- d) a protein having an amino acid sequence as shown in SEQ. ID. NO. 9;
- e) a protein having an amino acid sequence as shown in SEQ. ID. NO. 11;
- and
- f) any portion of any of the above proteins that elicits an immune response against E. canis.
4 more elements
A vaccine wherein said vaccine protects dogs against E. canis infection.
A vaccine comprising:
- a) a vector capable of expressing a recombinant DNA inserted into said vector such that a recombinant protein is expressed when said vector is provided in an appropriate host;
- and
- b) the recombinant DNA inserted into said vector wherein said DNA is selected from the group consisting of:
- i) a recombinant DNA that encodes a protein having an amino acid sequence as shown in SEQ. ID. NO. 3;
- ii) a recombinant DNA that encodes a protein having an amino acid sequence as shown in SEQ. ID. NO. 5;
- iii) a recombinant DNA that encodes a protein having an amino acid sequence as shown in SEQ. ID. NO. 7;
- iv) a recombinant DNA that encodes a protein having an amino acid sequence as shown in SEQ. ID. NO. 9;
- v) a recombinant DNA that encodes a protein having an amino acid sequence as shown in SEQ. ID. NO. 11;
- and
- vi) any portion of said DNA above that encodes a protein fragment that is greater than 25 amino acids.
7 more elements
A method of identifying a T cell epitope against E. canis comprising:
- a) synthesizing overlapping peptide fragments over an entire length of a protein wherein said protein is selected from the group consisting of:
- i) a protein having an amino acid sequence as shown in SEQ. ID. NO. 3;
- ii) a protein having an amino acid sequence as shown in SEQ. ID. NO. 5;
- iii) a protein having an amino acid sequence as shown in SEQ. ID. NO. 7;
- iv) a protein having an amino acid sequence as shown in SEQ. ID. NO. 9;
- v) a protein having an amino acid sequence as shown in SEQ. ID. NO. 11;
- and
- vi) any portion of any of the proteins above that elicits an immune response against E. canis;
- b) testing said peptide fragment to determine if said peptide fragment elicits an immune response in a host animal;
- and
- c) identifying said peptide fragment as said T cell epitope of E. canis if said fragment elicits an immune response.
8 more elements
A method of creating a vaccine against Ehrlichia canis comprising:
- a) selecting a vector capable of expressing a recombinant DNA inserted into said vector;
- and
- b) inserting a recombinant DNA into said vector such that a recombinant protein is expressed when said vector is provided in an appropriate host wherein said DNA is selected from the group consisting of:
- i) a recombinant DNA that encodes a protein having an amino acid sequence as shown in SEQ. ID. NO. 3;
- ii) a recombinant DNA that encodes a protein having an amino acid sequence as shown in SEQ. ID. NO. 5;
- iii) a recombinant DNA that encodes a protein having an amino acid sequence as shown in SEQ. ID. NO. 7;
- iv) a recombinant DNA that encodes a protein having an amino acid sequence as shown in SEQ. ID. NO. 9;
- v) a recombinant DNA that encodes a protein having an amino acid sequence as shown in SEQ. ID. NO. 11;
- and
- vi) any portion of said DNA above that encodes a protein fragment that is greater than 25 amino acids.
7 more elements
A method of creating a vaccine against E. canis comprising:
- a) selecting a vector capable of expressing a recombinant protein inserted into said vector;
- b) insertion of a recombinant DNA into said vector such that said recombinant protein is expressed when said vector is transformed into a bacterial strain wherein said DNA is selected from the group consisting of:
- i) a recombinant DNA that encodes a protein having an amino acid sequence as shown in SEQ. ID. NO. 3;
- ii) a recombinant DNA that encodes a protein having an amino acid sequence as shown in SEQ. ID. NO. 5;
- iii) a recombinant DNA that encodes a protein having an amino acid sequence as shown in SEQ. ID. NO. 7;
- iv) a recombinant DNA that encodes a protein having an amino acid sequence as shown in SEQ. ID. NO. 9;
- v) a recombinant DNA that encodes a protein having an amino acid sequence as shown in SEQ. ID. NO. 11;
- and
- vi) any portion of said DNA above that encodes a protein that elicits an immune response against E. canis;
- and
- c) harvesting said recombinant protein from said bacterial strain.
8 more elements
A method of creating a T cell epitope vaccine comprising:
- a) selecting a recombinant protein that includes a T cell epitope wherein said T cell epitope comprises an amino acid peptide fragment of a protein selected from the group consisting of:
- i) a protein having an amino acid sequence as shown in SEQ. ID. NO. 3;
- ii) a protein having an amino acid sequence as shown in SEQ. ID. NO. 5;
- iii) a protein having an amino acid sequence as shown in SEQ. ID. NO. 7;
- iv) a protein having an amino acid sequence as shown in SEQ. ID. NO. 9;
- v) a protein having an amino acid sequence as shown in SEQ. ID. NO. 11;
- and
- vi) any portion of any of the above proteins that elicits an immune response against E. canis;
- b) identifying said T cell epitope from said protein;
- c) incorporating said T cell epitope into a construct capable of expressing said epitope as a protein;
- and
- d) harvesting said protein.
9 more elements
A recombinant DNA comprising said DNA selected from the group consisting of
- a) a recombinant DNA that encodes a protein having an amino acid sequence as shown in SEQ. ID. NO. 3;
- b) a recombinant DNA that encodes a protein having an amino acid sequence as shown in SEQ. ID. NO. 5;
- c) a recombinant DNA that encodes a protein having an amino acid sequence as shown in SEQ.ID. NO. 7;
- d) a recombinant DNA that encodes a protein having an amino acid sequence as shown in SEQ. ID. NO. 9;
- and
- e) a recombinant DNA that encodes a protein having an amino acid sequence as shown in SEQ. ID. NO. 11.
3 more elements
A vector capable of expressing a recombinant DNA comprising:
- a) a recombinant DNA inserted into said vector such that a recombinant protein is expressed when said vector is provided in an appropriate host wherein said DNA is selected from the group consisting of:
- i) a recombinant DNA sequence that encodes a protein having an amino acid sequence as shown in SEQ. ID. NO. 3;
- ii) a recombinant DNA sequence that encodes a protein having an amino acid sequence as shown in SEQ. ID. NO. 5;
- iii) a recombinant DNA sequence that encodes a protein having an amino acid sequence as shown in SEQ. ID. NO. 7;
- iv) a recombinant DNA sequence that encodes a protein having an amino acid sequence as shown in SEQ. ID. NO. 9;
- v) a recombinant DNA that encodes a protein having an amino acid sequence as shown in SEQ. ID. NO. 11;
- and
- vi) any portion of said DNA above that encodes a protein that elicits an immune response against E. canis.
5 more elements
A vector capable of expressing a recombinant DNA comprising:
- a) a recombinant DNA inserted into said vector such that a recombinant protein is expressed when said vector is provided in an appropriate host wherein said DNA is selected from the group consisting of:
- i) a recombinant DNA that encodes a protein having an amino acid sequence as shown in SEQ. ID. NO. 3;
- ii) a recombinant DNA that encodes a protein having an amino acid sequence as shown in SEQ. ID. NO. 5;
- iii) a recombinant DNA that encodes a protein having an amino acid sequence as shown in SEQ. ID. NO. 7;
- iv) a recombinant DNA that encodes a protein having an amino acid sequence as shown in SEQ. ID. NO. 9;
- and
- v) a recombinant DNA that encodes a protein having an amino acid sequence as shown in SEQ. ID. NO. 11.
4 more elements
Serological diagnosis techniques using:
- a) a recombinant DNA that encodes a protein having an amino acid sequence as shown in SEQ. ID. NO. 3;
- b) a recombinant DNA that encodes a protein having an amino acid sequence as shown in SEQ. ID. NO. 5;
- c) a recombinant DNA that encodes a protein having an amino acid sequence as shown in SEQ. ID. NO. 7;
- d) a recombinant DNA that encodes a protein having an amino acid sequence as shown in SEQ. ID. NO. 9;
- and
- e) a recombinant DNA that encodes a protein having an amino acid sequence as shown in SEQ. ID. NO. 11.
3 more elements
The method of kinetic enzyme-linked immunosorbent assay comprising the steps of:
- a) selecting an antigen to be added to microtiter plates that includes an immunogenic epitope comprising a recombinant protein selected from the group consisting of:
- i) a protein having an amino acid sequence as shown in SEQ. ID. NO. 3;
- ii) a protein having an amino acid sequence as shown in SEQ. ID. NO. 5;
- iii) a protein having an amino acid sequence as shown in SEQ. ID. NO. 7;
- iv) a protein having an amino acid sequence as shown in SEQ. ID. NO. 9;
- v) a protein having an amino acid sequence as shown in SEQ. ID. NO. 11;
- vi) any portion of said DNA above that encodes a protein that elicits an immune response against E. canis
- b) adding an antiserum of the species allowing it to complementarily bind to the antigen;
- c) adding the antibody to the microtiter plate, allowing the antibody to bind to the antigen;
- d) washing the microtiter plate to remove any unbound antibodies;
- e) adding an enzyme the microtiter plates allowing the enzyme to bind to the antibody;
- f) washing the microtiter plate to remove any unbound enzyme;
- and
- g) adding the enzyme's substrate, allowing it to bind to the enzyme, which produces a color change when bound.
11 more elements
The method of western blot analysis comprising the steps of:
- a) obtaining the species serum with antigens, where said antigen includes an immunogenic epitope comprising a recombinant protein selected from the group consisting of:;
- i) a protein having an amino acid sequence as shown in SEQ. ID. NO. 3;
- ii) a protein having an amino acid sequence as shown in SEQ. ID. NO. 5;
- iii) a protein having an amino acid sequence as shown in SEQ. ID. NO. 7;
- iv) a protein having an amino acid sequence as shown in SEQ. ID. NO. 9;
- v) a protein having an amino acid sequence as shown in SEQ. ID. NO. 11;
- vi) any portion of said DNA above that encodes a protein that elicits an immune response against E. canis
- b) running the serum through sodium dodecyl sulfate-polyacrylamide gel electrophoresis, allowing proteins to be fractionated into a series of bands arranged in order of molecular weight;
- c) transferring the proteins to a filter by blotting;
- d) adding antibodies tagged with a dye are washed over the filter, allowing the antibodies to bind to the fractionated proteins;
- and
- e) adding substrates to develop the bands on the filter.
9 more elements
The method of polymerase chain reaction comprising the steps of:
- a) selecting a target strand of DNA that will serve as a template for DNA synthesis comprising recombinant DNA selected from the group consisting of:
- i) a recombinant DNA that encodes a protein having an amino acid sequence as shown in SEQ. ID. NO. 3;
- ii) a recombinant DNA that encodes a protein having an amino acid sequence as shown in SEQ. ID. NO. 5;
- iii) a recombinant DNA that encodes a protein having an amino acid sequence as shown in SEQ. ID. NO. 7;
- iv) a recombinant DNA that encodes a protein having an amino acid sequence as shown in SEQ. ID. NO. 9;
- v) a recombinant DNA that encodes a protein having an amino acid sequence as shown in SEQ. ID. NO. 11;
- and
- vi) any portion of said DNA above that encodes a protein that elicits an immune response against E. canis;
- b) adding a mixture containing enzymes, nucleotides, DNA polymerase, and primers;
- c) subjecting above mixture to a number of cycles of amplification in an automated DNA cycler;
- and
- d) using products of said cycles of amplification and performing gel electrophoresis.
9 more elements
Dependencies are inferred from the claim text.