Purification, Identification And Cloning Of Serine Protease Inhibitors From Eggs Of Carassius Auratus

As a kind of inhibition substances, Serine protease inhibitors are distributed widelyin animals, plants and microorganisms. Based on the protease inhibitor primary andthree-dimensional structures, together with inhibition mechanisms, they are classifiedinto at least18families. They act as modulators, playing key roles in a variety ofphysiological functions in vivo, such as blood coagulation, fibrinolysis, apoptosis,development, inflammation and complement activation in vertebrate and Bloodlymphatic agglutinate, polyphenoloxidase activity, cytokines and antimicrobial peptidessynthesis in invertebrate. Due to exogenous protease is an important pathogenic factor,a major function of proteinase inhibitors is to avoid the hydrolysis of protease bypathogenic organisms.Studies show that there exist a variety of protease inhibitors in fish eggs. Inaddition to perform physical adjustment function, also play an important role in defenseof pathogens. However, only a few kinds of eggs have been studied so far, and themechanism of the defense itself is still doubtful. Therefore, carp eggs are used as themain object in this study, using biochemistry and molecular biology methods,successfully obtained a serine protease inhibitor from carp eggs and further studiedthe characterization of it. To further explore the physiological defense function, andprovides experimental basis on food, medicine and other applications in the future. Themain contents and the results of this experiment are as follows:1. Collected four common fish eggs (Odontobutis obscurus, Carassius auratus,Ctenopharyngodon idellus, Channa argus), and measured serine protease(trypsin,elastase, subtilisin,chymotrypsin) inhibitory activity by chromogenic substrate. Theresults showed that in addition to chymotrypsin, the other three kinds of serine proteaseswere strongly inhibitored.2. A inhibitor with an apparent molecular weight of43kDa was purified from theegg of carp by a combination of ConA Sepharose4B chromatography, gel filtrationchromatography and reverse phase high performance liquid chromatography (RP-HPLC) steps. It was named CaSPI and showed a trypsin inhibitor activity. The partialN-terminal amino acid sequence of43kDa protein was EAHPSHSGED. Glycoproteinstaining results showed that the CaSPI a glycoprotein. In the determination of jointactivity, CaSPI formed a stable complex with trypsin in SDS-PAGE electrophoresis.This result suggests that it may belong to a superfamily of Serpins.3. A1397bp length of cDNA was got by RT-PCR and RACE. It contains a1233bpopen reading frame corresponding to a deduced protein of410amino acid. Analysisto the deduced amino acid sequences from the nucleic acid sequence showed that thereexists SERPIN domain in position52-408. The BLAST search at NCBI showed that theprotein encoded by the cDNA has a high degree of homology with the Serpinssuperfamily of serine protease inhibitor such as CP9from the carp meningeal fluid. Andthe P1position was occupied by Arg. So we speculated that the protein belongs to aSerpins superfamily.4. The CaSPI/proteinase molar ratio and the inhibitory constant Kiwas determinedand calculated. When the molar ratio up to1.15:1, it could completely inhibit theprotease hydrolytic activity. And Kiwas0.46μM. This indicated that CaSPI is acompetitive inhibitor. The stability of CaSPI (time, temperature, pH, metal ions anddenaturing agents) was detected. The results showed that CaSPI is considerable stability.The antibacterial activity test results showed CaSPI could inhibited gram-negativebacteria Escherichia coli and gram-positive bacteria Bacillus subtilis.