Prokaryotic Expression And Purification Of BBS5 From Chlamydomonas Reinhardtii And Preparation Of Its Polyclonal Antibody

Chlamydomonas reinhardtii is a unicellular algae model organism with an early start.The cell has two cilia of equal length on one side,and its growth conditions are simple and it is easy to culture.Cilia is a conserved structure on the surface of cell membrane,which is involved in regulating various life activities of cells.There is a two-way transport mechanism from the matrix to the top protein inside the cilia.and the intraflagellar transport(IFT)complex and the BBSome protein complex are important components of this transport mechanism.BBS5 protein is a key component of the BBSome protein complex that regulates signal transduction.Defects of BBS5 can lead to Bardet-Biedl syndrome.In this experiment,we extracted RNA from C.reinhardtii CC-125 by trizol and then reverse-transcribed it into cDNA as temlate to obtain the target gene with a length of 1059 bp.Then the target gene was amplified by PCR and ligated to the E.coli XL1 expression vector and the recombinant plasmids pET-28a(+)-bbs5 and pMAL-C2X-bbs5 with 6×His tag and MBP tag were successfully constructed and then transformed into E.coli BL21 competent cells.We add IPTG into concentration to induce BBS5 to express,then after purification and concentration,the antigen with a purity of 90%or more was obtained and immunized to New Zealand white rabbit.We used ELISA to detect the titer of polyclonal antibody after ench immunization.The 3rd immune serum was collected and purified by protein A purification and MBP-BBS5 antigen affinity purification.And we used Western blotting to test its specificity.The results showed the titer was 102400 and this polyclonal antibody had a higher specificity to bbs5 generated from C.reinhardtii CC-125.In this experiment,the prokaryotic expression of C.reinhardtii bbs5 gene was achieved,and a highly specific polyclonal Anti-BBS5 was prepared,which laid the foundation for the subsequent study of cilia diseases and human health.