Method for identification of proteins from intracellular bacteria
OwnerAllan Christian Shaw, Brian Berg Vandahlinventors
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Abstract
The present invention relates to a novel combination of methods that enables identification of proteins secreted from intracellular bacteria regardless of the secretion pathway. The invention further provides proteins that are identified by these methods. Secreted proteins are known to be suitable candidates for inclusion in immunogenic compositions and/or diagnostic purposes. The invention also provides peptide epitopes (T-cell epitopes) from the identified secreted proteins, as well as nucleic acid compounds that encode the proteins. The invention further comprises various applications of the proteins or fragments thereof, such as pharmaceutical and diagnostic applications.
Claims72
A method for identifying proteins secreted from an intracellular bacteria, comprising the following steps:
- 1) infecting host cells by the intracellular bacteria,
- 2) labelling the intracellular bacteria present in the infected cells,
- 3) preparing
- a) whole cell lysates of the infected cells
- b) purified and lysed bacteria from the infected cells,
- 4) comparing 2D-gel electrophoresis protein profiles of i) the whole cell lysates from step 3a) with ii) the purified and lysed bacteria from step 3b),
- 5) detecting protein spots from step 4) which are present in the whole cell lysates but absent or present in significantly reduced amount in the purified bacteria,
- 6) identifying the proteins in the spots selected in step 5).
A method for identifying proteins secreted from an intracellular bacteria, comprising the following steps:
- 1) infecting host cells by the intracellular bacteria,
- 2) pulse labelling of the intracellular bacteria present in the infected cells,
- 3) preparing whole cell lysates of the infected cells after different periods of chase following step 2),
- 4) comparing 2D-gel electrophoresis protein profiles of the whole cell lysates prepared after different periods of chase from step 3),
- 5) detecting protein spots from step 4) which are present in decreasing amount as chasing periods increase in step 3),
- 6) identifying the proteins in the spots selected in step 5).
A method for identifying proteins secreted from an intracellular bacteria, comprising the following steps:
- 1) infecting host cells by the intracellular bacteria,
- 2) cultivating the host cells in the presence and in the absence of a proteasome inhibitor, respectively,
- 3) labelling the intracellular bacteria present in the infected cells cultivated in the presence and in the absence of a proteasome inhibitor, respectively,
- 4) preparing whole cell lysates of the infected cells,
- 5) comparing 2D-gel electrophoresis protein profiles of the whole cell lysates of the infected cells cultivated in the presence and in the absence of a proteasome inhibitor, respectively,
- 6) detecting protein spots from step 5) which are present in the is whole cell lysates cultivated in the presence of a proteasome inhibitor, but absent or present in significantly reduced amount in the whole cell lysates cultivated in the absence of a proteasome inhibitor,
- 7) identifying the proteins in the spots selected in step 6).
A Chlamydia trachomatis polypeptide, characterized in that it is DT8 and comprises the following sequence (SEQ ID NO:
- 1):
- 12 MQHTIMLSLENDNDKLASMMDRVVAASSSILSASKDSESN RQFTISKAPDKEAPCRVSYVAASALSE
- or an immunogenic fragment thereof.
A peptide epitope comprising 7 to 10 consecutive amino acids of a Chlamydia trachomatis or Chlamydia p
A peptide epitope comprising 4 to 25 consecutive amino acids of a polypeptide comprising the sequence SEQ ID NO:1, preferably 6 to amino acids, and most preferably 7 to 10 amino acids.
Dependencies are inferred from the claim text.