2020 Fiscal Year Research-status Report
Analysis to identify the receptor for cholix toxin from Vibrio cholerae
| Project/Area Number |
20K16246
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| Research Institution | Osaka Prefecture University |
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Principal Investigator |
アワスティ シャルダ 大阪府立大学, 生命環境科学研究科, 客員研究員 (30751888)
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| Project Period (FY) |
2020-04-01 – 2023-03-31
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| Keywords | Cholix toxin |
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| Outline of Annual Research Achievements |
The objective for the year 2020 was to evaluate the cytotoxicity of cholix toxin (ChxA) towards the eukaryotic cells which either carry or not carry prospective receptor candidate of ChxA. The sensitivity of the cells to rChxA was evaluated using carcinoma cell line which express the potential receptor protein. The results were compared with the cytotoxicity data of non-sensitive cell lines which lacks the potential receptor candidate. The expression of the candidate receptor protein was confirmed by Western blotting using commercially available monoclonal antibodies. The cells expressing potential receptor were susceptible to rChxA with CD50 of 1.25 μg/mL. On the other hand, cells which did not carry potential receptor only showed growth inhibition effect when treated with ≧2.5 μg/mL of rChxA but no cytotoxicity. Further, another cell line, which do not carry potential receptor protein were also analyzed for ChxA cytotoxicity. As expected, this cell line was not susceptible to rChxA and no cytotoxic activity was observed up to 80 μg/mL rChxA. The results obtained in these experiments support the in-silico observation that the candidate receptor protein might be the target receptor for the ChxA.
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| Current Status of Research Progress |
Current Status of Research Progress
4: Progress in research has been delayed.
Reason
The research plan is delayed due to corona pandemic. Some of the efforts were diverted to other works and experiments.
As expected for year 2020, the cytotoxicity assay of ChxA with different cell lines expressing and non-expressing candidate receptor protein is completed. The experiment which could not be completed in 2020 was to evaluate the cytotoxicity of ChxA in transformed cells expressing candidate receptor protein. I expect to progress the work in the current fiscal year.
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| Strategy for Future Research Activity |
The non-susceptible cells which have been transformed with plasmid (pTJ-Neo) carrying cDNA encoding the candidate receptor protein will be evaluated for cytotoxicity with rChxA. Further, other non-susceptible cells will be transformed with plasmid (pTJ-Neo~cDNA-receptor), to see cell tropism between transformed and non-transformed cells. The CD50 values will be calculated to evaluate the fold changes in sensitivities of the transformed cells towards ChxA.
In vitro binding assays of rChxA with candidate receptor protein will be carried out. For this purpose, the candidate receptor protein will be expressed either in E. coli using pET vector system or Sf9 cells using baculovirus expression system. The expression will be confirmed by Western blotting and the protein will be purified. The purified recombinant receptor protein will be used for toxin overlay assay with rChxA. The binding assay will be also carried out by pull-down assay using receptor protein as ‘bait’ and rChxA as ‘prey’ protein.
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| Causes of Carryover |
Some of the experimental objectives of the year 2020 could not be completed due to corona pandemic. Those experiments will be carried out along with other objectives in the current fiscal year. The funds will be required to accomplish the incomplete objectives of the research.
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