Preparation And Application Of Polyclonal Antibody Of Chlamydomonas Reinhardtii Phospholipase D

Chlamydomonas reinhardtii is a unicellular eukaryotic organism with a relatively simple structure.It has the advantages of rapid growth,clear genetic background,easy acquisition of mutants,and easy separation of cilia.Animal cilia are highly conserved and can combine genetics,biochemistry and cell biology to study the structure and function of cilia,making them an ideal model organism for the study of ciliopathies.The generation of cilia or the homeostasis of signal molecules in cilia depend on “intraciliary transport(IFT)”,and there is also a stable protein composed of eight BBS proteins(BBS1/2/4/5/7/8/ 9/18)composed of BBSome multi-protein complexes,which act as cargo adapters to recognize signaling proteins and connect them to the IFT system for bidirectional transport within the cilia by molecular motors of axofilament microtubules.Absence or dysfunction of BBSome in the cilia can also lead to Bardet Biedlsyndrome(BBS),a disease with the main features of ciliopathies,and its clinical symptoms include polydactyly,bone deformities,and infertility.As a signal molecule on the inner membrane of cilia,PLD mainly enters the cilia by diffusion and relies on the BBSome to output the cilia.Therefore,PLD has an important indication for studying the homeostasis of cilia and the molecular mechanism of how BBSome maintains the homeostasis of signaling molecules effect.In this project,the target gene of Chlamydomonas reinhardtii pld(1-522)was obtained by PCR amplification,and the recombinant expression vector p MAL-c2x-pld(1-522)containing maltose binding protein(MBP)tag at the N-terminus was successfully constructed.Bacillus BL21(DE3)expresses the N-terminal MBP tag-labeled PLD(1-173)fusion protein,which is induced by IPTG to obtain the fusion protein MBP::PLD(1-173).The purity of the fusion protein MBP::PLD(1-173)after affinity purification met the requirements of immunity.It was used as an antigen to immunize New Zealand white rabbits.The serum was separated and the titer was determined by indirect ELISA method to be 1:102400.The obtained antiserum was enriched for Ig G subtype antibodies using protein A purification beads,followed by membrane purification of Ig G subtype antibodies using6×His::PLD(1-173)protein.Finally,the whole-cell protein extract of Chlamydomonas reinhardtii CC-125 was subjected to immunoblot analysis using the purified PLD polyclonal antibody,and the results showed that the obtained PLD polyclonal antibody had high specificity.And the antibody was used to successfully detect the localization of PLD protein in the cilia of Chlamydomonas reinhardtii,the content of PLD in the cilia of the BBSome subunit bbs1 mutant,and the accumulation site of PLD protein in the cilia of the bbs1 mutant.Molecular mechanisms of molecular homeostasis provide the basis.At the same time,a pld mutant of Chlamydomonas reinhardtii was identified by PLD antibody,which can be used for subsequent PLD function research.