Prokaryotic Expression，Purification，Polyclonal Antibody Preparation and Identification of the Chlamydomonas reinhardtii BBSome Protein BBS2

Abstract:

BBS2 is a BBSome subunit and its loss causes Bardet-Biedl syndrome. Chlamydomonas reinhardtii is a model organism used for studying the Bardet-Biedl syndrome. To investigate the role of BBS2 in causing Bardet-Biedl syndrome，a prokaryotic expression vector，pET28a-bbs2 is constructed by inserting the 5' 399 bp fragment of the bbs2 gene into the pET28a plasmid. The recombinant vector is then transformed into Escherichia coli BL21（DE3） to induce the 6×His-BBS2 fusion protein expression. SDS- PAGE electrophoresis shows that this fusion protein is insoluble and expressed with a molecular weight of approximately 15 kDa as expected. In the presence of 8 mol/L urine，the fusion protein is purified by Ni-affinity purification and used to immunize New Zealand white rabbits. After the 5th immunization，the antisera are determined to have a titer of as high as 256 000. The antisera are then purified using protein A sepharose CL-4B. The polyclonal antibody prepared can specifically bind the C. reinhardtii BBS2 as shown in Western blotting assay and identified BBS2 in the basal bodies and in the flagellum as determined by immunofluorescent microscopy. The availability of this antibody will strengthen the research to investigate the role of BBS2 in causing Bardet-Biedl syndrome.