Vaccines comprising heat-sensitive transgenes
The present disclosure provides temperature sensitive essential nucleic acid molecules from a psychrophilic bacterium, proteins encoded by the nucleic acid molecules, as well as recombinant cells into which have been introduced such nucleic acid molecules. The disclosed recombinant cells containing one or more essential nucleic acid molecules from a psychrophilic bacterium are thereby made temperature sensitive, and can be administered to a mammal to induce an immune response in the mammal.
1. A method of making a recombinant temperature sensitive (TS) bacteria comprising an essential peptide, comprising:
screening a psychrophilic microbial genome for detection therein of an essential polynucleotide that confers temperature sensitivity to a bacterium upon expression of a protein from the essential polynucleotide that is inactivated at a temperature greater than 30° C.;
isolating said essential polynucleotide;
constructing a nucleic acid construct comprising the essential polynucleotide and one or more control sequences operably linked to the essential polynucleotide;
inserting the nucleic acid construct into the genome of a selected mesophilic bacterial host cell thereby functionally replacing the selected mesophilic bacterial host cell's homologue of the essential polynucleotide whereby the essential polynucleotide encodes a protein that is operable at a temperature less than 30° C. and inoperable at a temperature greater than 30° C.;
culturing the mesophilic bacterial host cell comprising the essential polynucleotide at a temperature less than 30° C. to confirm the viability of the mesophilic bacterial host cell comprising the essential polynucleotide;
further culturing the mesophilic bacterial host cell comprising the essential polynucleotide at a temperature greater than 30° C. to determine if the mesophilic bacterial host cell comprising the essential polynucleotide is killed; and
if the mesophilic bacterial host cell comprising the essential polynucleotide is killed, inserting the nucleic acid construct into the genome of a selected destination mesophilic bacterial host cell, thereby functionally replacing the selected destination mesophilic bacterial host cell's homologue of the essential polynucleotide, thereby generating a recombinant TS bacteria.
2. The method of claim 1 , wherein the selected mesophilic bacterial host cell is a Francisella novicida cell.
3. The method of claim 1 , wherein the wherein the psychrophilic microbial genome is from Colwellia sp., Pseudoalteromonas sp., or Shewanella sp.
4. The method of claim 1 , wherein the psychrophilic microbial genome is from Pseudoalteromonas haloplanktis.
5. The method of claim 1 , wherein the psychrophilic microbial genome is from C. psychrerhraea.
6. The method of claim 1 , wherein the selected destination mesophilic bacterial host cell is Salmonella sp. or Mycobacterium sp.
7. The method of claim 1 , wherein the selected destination mesophilic bacterial host cell is Haemophilus sp., Vibrio sp., Escherichia sp., Streptococcus sp., Burkholderia sp., Campylobacter sp., Neisseria sp., Helicobacter sp. or Francisella sp.
8. The method of claim 1 , wherein screening the psychrophilic microbial genome detects a polynucleotide sequence comprising the nucleotide sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25 or 27.
9. The method of claim 1 , wherein screening the psychrophilic microbial genome detects a polynucleotide sequence encoding a protein comprising the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, or 28.
10. The method of claim 1 , wherein the protein encoded by the essential polynucleotide is operable at a temperature of 4° C. to 30° C.
11. The method of claim 1 , wherein the nucleic acid construct comprising the essential polynucleotide comprises a vector.
12. The method of claim 1 , wherein screening the psychrophilic microbial genome detects a polynucleotide sequence selected from the group consisting of ligA, pyrG, hemC, ftsZ, cmk, murG, fmt, tyrS, or dnaK.
13. The method of claim 1 , wherein screening the psychrophilic microbial genome detects a polynucleotide sequence comprising the ligA polynucleotide sequence.