Analysis Of Complement And Coagulation Cascade System Regulatory Related Factors In Sipunculus Nudus

Thrombotic disease has become a serious health problem worldwide.However,there is still lack of ideal thrombolytic drugs without side effects.Coagulation and the complement cascade are ubiquitous in multicellular organisms,and they are one of the most basic cell signaling regulatory systems for homeostasis and physiological activities.Sipunculus nudus is an ancient kind of marine benthic worm that is delicious and of has a high therapeutic value,which is very popular for Fujian residents in the Taiwan Strait areas.A number of previous studies have shown that bodies of Sipunculus nudus contained a large amount of component which could improve the body's various metabolic pathways significantly,protecting people's cardiovascular and cerebrovascular systems in particular.Many types of cells and organs in the body fluid of Sipunculus nudus,are similar to mammalian blood vessels,with low evolutionary status and simple structure,and this character makes it becoming an excellent species for studying coagulation and complement cascade.In the present study,thrombus powder dissolved was subjected to stimulate the coelomic fluid system of Sipunculus nudus to activate the coagulation and complement system.The transcriptome sequencing and real-time quantitative PCR were used to investigate the coagulation and complement cascade in coelomic fluid system.The results of the present study revealed as below:1.In light of the database of transcriptome sequencing and annotation,the differential expressed genes of coagulation and complement cascades of coelomic fluid system were obtained.These genes were further verified by RT PCR.The results suggested that the intrinsic pathway,the fibrinolytic pathway and lectin pathway may be activated,whereas the classical and alternative pathways,the extrinsic pathway and the kinin system were not activated.2.Partial A2 M and CPAMD8 sequences from transcriptome database of S.nudus were acquired and their ORF sequences were cloned by RACE.Conserved Domains analysis showed that A2 M was of three conserved functional domains,while CPAMD8 had two conserved functional domains.The protein tertiary structure could be predicted.Both phylogenetic tree analysis and cluster analysis showed that the S.nudus had high homology with Lingula anatina.Real-time quantitative PCR was used to analyze the expression of A2 M in each tissue and it was found that its expression was of tissue specific.In conclusion,the gene excavation and functional studies on coagulation and complement cascades have for the first time been carried out on the S.nudus by transcriptome sequencing and gene cloning.This study preliminarily provides us better understanding of the evolution of coagulation and complement cascades in invertebrates.