Preparation of Polyclonal Antibodies Against Chlamydomonas reinhardtii ChR2

Abstract:

Objective This study prepared the polyclonal antibodies against Chlamydomonas reinhardtii (C. reinhardtii) channelrhodopsin 2 (ChR2) to study the mechanism by which ChR2 in flagella guides cell phototaxis by sensing light signals.Method The constructed expression vector pET-28a(+)-chr2 was transformed into Escherichia coli BL21(DE3) competent cells to induce the expression of the 6×His-ChR2 fusion protein, which was then purified through a nickel column. The fusion protein was used as an antigen to immunize animals for the production of corresponding antibodies. The antiserum was treated with Protien A to enrich the antibodies, and thus high-purity polyclonal antibodies against C. reinhardtii ChR2 were obtained. Western blot was employed to assess the specificity of the prepared antibodies, and immunofluorescence was used to determine the localization of ChR2 in flagella.Result The purification results of the 6×His-ChR2 fusion protein demonstrated a purity exceeding 85% after purification. The indirect ELISA results showed that the titer of the polyclonal antibodies against C. reinhardtii ChR2 reached 1:25 600. The target band (relative molecular mass 93 000) appeared in electrophoresis of the whole protein extract of wild-type C. reinhardtii CC-125 cells but did not appear in that of the whole protein extract of mutant C. reinhardtii CC-5499 (a mutant with knockout of both chr1 and chr2) cells, indicating that the obtained polyclonal antibodies had high specificity. The localization of ChR2 was in the eyespots and flagella of C. reinhardtii cells.Conclusion The findings lay a foundation for subsequent studies on the mechanism by which ChR2 in flagella guides cell phototaxis by sensing light signals.