ABSTRACT
Objective: To investigate the role of BBS protein in ciliary signal transduction by studying the pro- karyotic expression, purification and polyclonal antibody preparation of Chlamydomonas reinhardtii protein BBS4. Methods: Prokaryotic Expression Vector pET-28a(+)-bbs4 and pMAL-c2X-bbs4 were constructed by the cDNA sequence of bbs4 Gene from C. reinhardtii, and then transformed into Escherichia coli BL21(DE3)for protein expression. The fusion protein with maltose binding protein(MBP)and 6×His tag was obtained by inducing expression. The purified fusion protein 6×His-BBS4 were used to immunize New Zealand white rabbits and the antiserum was isolated from the blood collected from the ear vein. The titer of the antiserum was measured by indirect ELISA essay, the specificity of the antibody was tested by Western blotting method and immunofluorescence test. Results: Prokaryotic expression plasmids pET-28a(+)-bbs4 and pMAL-c2X-bbs4 were successfully constructed. The relative molecular weights of 6×His-BBS4 and MBP-BBS4 fusion proteins were 45 kDa and 85 kDa, respectively. The purity of the fusion proteins was more than 85%, and the concentration of the fusion proteins was more than 0.5 mg/ml. The proteins were used for immunization. The titer of the fusion proteins was 51 200. Western blotting showed a high specificity for the detection of C. reinhardtii CC-125. Prokaryotic expression of BBS4 protein of C. reinhardtii and preparation of polyclonal antibody were realized. Conclu- sion: The polyclonal antibody against BBS4 of C. reinhardtii was prepared successfully, which laid a foundation for further study on the role of BBS4 in ciliopathies.