Preparation and Application of a Polyclonal Antibody to Chlamydomonas reinhardtii RSP10

Abstract:

[Objective]This study aims to explore the cilium assembly of the radial spoke protein RSP10 and develop a highly specific polyclonal antibody to RSP10.[Method]Firstly, the prokaryotic expression vector pMal-C2X-rsp10 was constructed, and the expression of MBP-labeled RSP10 was induced in Escherichia coli BL21 (DE3). After the fusion protein was purified, and the MBP label was excised. The obtained protein was used to immunize New Zealand male white rabbits, and the antibody titer in the anti-serum was determined by indirect ELISA.[Result]The rabbit anti-Chlamydomonas reinhardtii RSP10 antibody with high specificity and high sensitivity was obtained after purification of Protein A and antigen antibody. The immunofluorence assay showed that RSP10 was mainly located in the kinetosome. The isolation of ciliary components showed that a large amount of RSP10 was detected in the axial filament and a small amount in the matrix of cilia.[Conclusion]This finding provides strong data support for the subsequent study of the assembly of RSP complex and the molecular mechanism underpinning its influence on cilium movement.