CDNA Cloning And Expression Patterns Of Coagulation Factor In Eriocheir Sinensis

Objective: Coagulation factor as biological activity protein plays an important role in thrombin cascades of anticoagulation and coagulation.The main function of coagulation factor was to catalytic fibrinogen into fibrin and as a defense signal factor to activate the immune system. Studies have shown that human coagulation factor preparations were the effective drug for treatment of hemophilia A; Limulus blood preparation of Limulus reagent(Limulus amoebocyte lysate, LAL) for trace amounts of the virus and pollutants has strong sensitivity; The snake coagulation factor played an important role in the treatment of thrombosis and inflammation.Crab(Eriocheir sinensis)was a kind of important economic crab in our country. In recent years, due to the expansion of farming and degradation of water quality, more and more crabs were death by diseases. In addition to the use of antibiotics and other drugs, people attached great attention to enhance innate immune system of crab. How to stimulate the immune potential best were the main topics which were worth exploring in aquatic animal genetics and breeding areas.In this study, we cloned the cDNA sequence of coagulation factor gene and analyzed gene structure. Through prokaryotic expression system to obtian recombinant protein which used to preparate polyclonal antibodies,Using Realtime RT-PCR 、 Western blot to explore the characteristics of mRNA and protein expression in the condition of physiological and pathological, to reveal the gene function in coagulation and immune which providing important theoretical basis for breeding of disease resistance.Methods: By adopting RACE(rapid-amplification of cDNA ends) from hepatopancreas to clone the full-length cDNA of Eriocheir sinensis coagulation factor,Through the pET32a(+) prokaryotic expression system to express coagulation factor fusion protein, purified the fusion protein by Ni-ida. Respectively, we use Real-timeRT-PCR to detect :(1) the expression level of coagulation factor in health crab tissues;(2) relative expression of coagulation factor in heart, hepatopancreas and blood by injecting A.hydrophila 0 h, 1.5 h, 3 h, 6 h, 12 h, 24 h and 48 h to reflect the expression change with time;(3)relative expression of coagulation factor in heart,hepatopancreas and blood by breaking clamp claw 0.5 h, 1 h,1.5 h, 3 h and 6 h to reflect the expression change in autotomy. Using purified coagulation factor fusion protein as an immunogen injecting rabbit to prepare the polyclonal antibody of coagulation factor.Through AGAR double diffusion method and indirect enzyme-linked immunosorbent method to detect antibody titer. Also, we use western bolt to validate antibody specificity and explore the expression characteristics of natural protein in heart,hepatopancreas and blood after infecting A.hydrophila and artificial fracture.Result: The full-length cDNA of Eriocheir sinensis coagulation factor were2279 bp, which contained a 936 bp open reading frame to code for the peptide with 311 amino acids. Predicted the molecular weight was 33.75 kDa, theory of isoelectric point was 9.38. Phylogenetic tree analysis results show that the coagulation factor protein primary structure similar to most species prothrombin, ties of consanguinity was the most close with termite(Zootermopsis nevadersis). The research of coagulation factor mRNA relatively expression level in different tissues show that the expression in heart,hepatopancreas and blood were much higher than other tissues.Infecting A.hydrophila,the coagulation factor mRNA expression level in heart, hepatopancreas and blood were significantly higher and reached the highest at 24 h and 3 h; By artificial fracturing crab claws, the coagulation factor mRNA expression level in heart, hepatopancreas and blood reached the highest at 0.5 h,1 h,1.5 h respectively. According to the results of the western bolt, prepared polyclonal antibody could identify coagulation factor specificly and show that the expression of coagulation factor was a little in health crab,by the infection of bacterium and artificial fracture, the expression of coagulation factor was a lot in heart, hepatopancreas and blood.Conclusion: We have cloned and identified the coagulation factor of Eriocheir sinensis successfully, which proved that coagulation factor also existed in invertebrates.Coagulation factor gene mainly expressed in heart, hepatopancreas and blood, and expressed very few in the rest of the organization. By infecting A.hydrophila and artificial fracture, the relative expression of coagulation factor increased significantly,with the infection waned, the expression also reduced gradually. Western blot results show that coagulation factor protein expressed a little in health crab, but expressed a lot in heart, hepatopancreas and blood by infection and stimulation.In this study we detected coagulation factor characteristic of expression by infection and stimulation,speculating that coagulation factor might play an important role in immunity of Eriocheir sinensis.