Purification,characterization And Cloning Of Cystatin Protease Inhibitor From Eggs Of Cyprinus Carpio

As a natural protease inhibitor,cystatin protease inhibitor are small molecular weight,can restrain activity of protease in cysteine and play an important function in defense mechanism of organism immune system.It is extensive distributed in animals,plants and microorganisms.CPIs have potential application value in pharmaceutical,food processing,plant resistance to pets and diseases, immune escape mechanism of parasitic by inhibiting enzyme active site.Carp belongs to Carp,Cyprinus and vertebrates.Therefore,the experimental subject are carp eggs in this paper,analysis the inhibitory activity of protease inhibitor,successfully extracted a cystatin protease inhibitor from carp eggs and further tested its partial characterization by biochemistry and molecular biology methods.The main contents and results are as follow:The protease inhibitor activity was measured based on the method of colorimetric,extracted four freshwater eggs(Cyprinus carpio,Hypophthalmichthys molitrix,Mylopharyngodon piceus,Hypophthalmichthys nobilis),analysis protease(thrombin,trypsin,papin,subtilisin,chymosin) inhibitory activity. The results displayed that thrombin,trypsin,subtilisin were highly inhibitory action,besides papin,chymosin. A protease inhibitor with a purification fold of 117 was purified from the egg of carp by Sephadex G50 and SephadexG75 gel filtration chromatography and reverse phase high performance liquid chromatography(RP-HPLC) steps.Assay the thermal and pH stability of carp eggs protease inhibitor. The results showed that the inhibitor was inactivation when at temperature above 80%, pH stability of inhibitor displayed a wide range. The antibacterial activity test results displayed that inhibitor could inhibited Aeromonas hydrophila and bacteria Bacillus subtilis.Cloned cystatin genes from carp eggs using RT-PCR and RACE methods, the length of cDNA was 546 bp. It includes a 390 bp open reading frame corresponding to a deduced protein of 129 amino acid, a 80 bp 3’UTR including Poly(A)tail, caudal recognition sequence(AATAAA),18 mature signal peptide. Analysis to the deduced amino acid sequences from the nucleic acid sequence showed that there exists cystatin domain in position 20-129. The BLAST search at NCBI showed that the protein encoded by the cDNA has a high degree of homology with ovarian cystatin.So we speculated that the protein belongs to a cystatin family.