Purification And Immunogenicity Analysis And Of A Noval Fibrinolytic Enzyme From The Echiurid (Urechis Unicinctus) And Trial Cloning Of Its Encoding Gene

Nowadays,thrombus has become the most severe disease in the world.It is urgent for medicinal expert to search for more useful and effective medicines to cure the patients.Urechis unicinctus is an animal living in the sediment of North China Sea. For a long time,people have known little about it.It is found that the coelomic fluid of U.unicinctus doesn't congeal after being separated for a long time.This suggests that it may contain anticoagulants or fibrinolytic substances.Objective:In this study,we try to purify the fibrinolytic enzyme from the coelomic fluid of U.unicinctus,estimate its relative molecular weight,study its immunogenicity(antibody titer),clone the cDNA of its encoding gene from digestive tract tissue of U.unicinctus.Methods:Ultrafiltration,sephacryl gel filtration chromatography and ion exchange chromatography were used to separate and purify Urechis unicinctus fibrinolytic enzyme(UFE).Folin's phenol reagent method was used to detect the in vitro fibrinolytic activity of UFE.Ultraviolet absorption and Bradford method were applied for definiting the content of protein.Dialysis was used to desalt the extract. The purity of UFE was identified by SDS-PAGE and high performance liquid chromatography(HPLC).Relative molecular weight of UFE was determined by SDS-PAGE.The sequences of oligopepetide fragments of UFE was determined with N- terminal sequencing and mass spectrum(de novo).Rabbit was used to test the immunogenicity of UFE.The animal was immunned with hypodermic injection and intramuscular injection.Polyclonal antibody was isolated from rabbit blood serum,with antibody titer and antigenicity determined with agar gel precipitation test.Total RNA was extracted from the digestive tract of the echiurid using RNA Extract Kit.UFE cDNA was amplified by RT-PCR using primer designed according to the N-terminal amino acid sequence and 3 oligopeptide fragments of UFE.The PCR product was inserted into pMD-18T vector and sequenced commercially.Results:A novel serine protease,named as Urechis unicinctus fibrinolytic enzyme(UFE) was purified from the coelomic fluid of the echiurid.The recovery of UFE reached 13.9%and the specific activity is 421.9U/mg,increased:by 10.2 folds. SDS-PAGE showed a single band and HPLC analysis revealed a single peak, indicating the purity of the enzyme was about 95%.Purified UFE showed an apparent molecular weight of 26.4kD as was determined by SDS-PAGE.The amino acid sequence of the 5 oligopeptide fragments was as follows:ICGGSPADIT;DMKR;RNRLPHACMPZR;ACVWYFVH;and BNEGGYLDACM.Homologous searching demonstrated that the UFE isolated was a noval protease.Three cDNA fragments were cloned with the amino acid sequence deduced, respectively.Online searching against the protein deposited in database showed that two of them were similar to beta-1,4-endo-glucanase which degrades polysaccharide possessing beta-1,4-glucan backbones,and the other was similar to septin 11,a member of septin family known to play diverse cellular roles,such as cytokinesis, polarity determination,vesicle trafficking and membrane dynamics.As the primers were designed according to the amino acid sequence of oligopeptides gained using mass-spectrogram de novo sequencing,we are sceptical about the correctness of these oligopeptides.Although database searching found the homologues;however,the similarity was low.Currently,we can not determine if the sequences obtained were transcripted from the gene encoding the fibrinolytic enzyme.