| 研究実績の概要 |
The objectives for the fiscal year 2021-22 were to evaluate the cytotoxicity of recombinant cholix toxin (rChxA) on eukaryotic cells transformed with plasmid carrying candidate receptor cDNA. Further, cloning, expression and purification of candidate receptor protein (cRP) and binding assays were to be carried out.
The cytotoxicity assay was evaluated using wild-type and transformed cell lines (expressing cRP). The wild-type cells which did not express cRP did not show any cytotoxicity when treated with rChxA up to 20 µg/mL, although growth inhibition was observed at this concentration. On the other hand, transformed cells expressing cRP were susceptible to rChxA and demonstrated cytotoxicity with CD50 of 5 ug/mL rChxA. The results further support the idea that the cRP could be the target of ChxA.
In another objective, cloning and expression of cRP was attempted. Although, the E. coli could be transformed with plasmid carrying cDNA of candidate receptor but no expression was observed. Thus, the cDNA was cloned and the expression was carried out using Bac-to-Bac Baculovirus Expression System and Spodoptera frugiperda (Sf9) cells. The cRP could be expressed in Sf9 cells as confirmed by Western Blotting. As the cloning was carried out without any tag, purification could not be done. Further, cloning and expression of Histidine tagged cRP was carried out. The bacmid DNA carrying gene for His-tagged receptor candidate was prepared. The experiment to transfect the Sf9 cells and isolation of P1 viral stock is ongoing.
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| 今後の研究の推進方策 |
In vitro binding assays of rChxA with cRP will be carried out. The expressed and purified recombinant cRP 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. Finally, if the binding between the purified cRP and ChxA will be successful, they will be used for X-ray crystallography.
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