Molecular Cloning And Expression Of Urechis Unicinctus Fibrinolytic Enzyme Ⅲ

Thrombosis is a serious threat to the world, especially the developed countries, also including the elderly of developing countries. Thrombosis has characteristics of high incidence, high mortality and high morbidity, especially the suddenness. Thrombolytic therapy is the most important pathway in treatment of thrombotic disease. After years of efforts to explore, thrombotic drugs has made great progress from the discovery of urokinase to the develodment of rPA and the usage of the Lumbrokinase. However, there still exist inadequacies such as short half-period and poor specificity.After years of research, a group of novel fibrinolysin are isolated and purified from Urechis unicinctus in our laboratory named Urechis unicinctus Fibrinolytic Enzyme(UFE). It includes four different compounds, they are UFEⅠ, UFEⅡ, UFEⅢ and UFEIV. With the same enzyme concenteation,the thrombolytic effect of Urechis unicinctus Fibrinolytic Enzyme is more apparent than that of lumbrokinase and has great bio-security. Urechis unicinctus Fibrinolytic Enzyme Ⅲ takes on potential applied values in the future due to its lower molecular weight and higer fibrinolytic activity. Conventional means of separation and purification has some drawbacks such as poor reproduciblity, low yield, therefore we hope that through this study, the gene of Urechis unicinctus Fibrinolytic EnzymeⅢ will be cloned, and the genetically engineered strains which can yield recombinant Fibrinolytic EnzymeⅢ will be constructed. The main work are as follows:1. Through N-terminal sequencing of protein we got the N-terminal amino acid sequence of the UFEⅢ. According to this sequence, degenerate primers were designed to amplify part of the UFEⅢ gene by using cDNA as a template through3’RACE PCR. Then the complete cDNA(full length863bp) sequence encoding the UFEⅢ was amplied on the basis of the result of5’-RACE PCR, the open reading frame has786bp.The comparison result of UFEⅢ cDNA sequence analysised by BLASTx indicates that the protein sequence has high homology with tryptase family members. The analysis result of ScanProsite software by PROSITE demonstrated that the protein contains a trypsin domain. According to the CDD database of NCBI, the protein is a trypsin-like serine protease.2.Expression plasmids pET32a-UFEⅢ、pGEX-6P-1-UFEⅢ及pET28a-UFEⅢ have been successfully constructed, and respective transformed in Rosetta2(DE3), expressed under the IPTG induced. Fermentation supernatant was purified by affinity chromatography and inclusion body was refolded. We obtained soluble target protein, but no enzyme activity was detected.3.Secreted expression plasid pINA1317-UFEⅢ has been successfully constructed, and transformed in Y. lipolytica Polh. After repeated sceening on a large number of transformants, two strains with higher plasmin activity was obtained. SDS-PAGE electrophresis showed it is30KD, larger than expected molecular weight which reason was that the protein was modified of glycosylation. The positive strain supernatant was diluted with pINA1317supernatant, the lower concentration of target protein, the lower activity of the enzyme, which further evidented that the target protein was expressed.We obtained the recombinant protein for the first time, which laid the foundation for the further protein structure research and drug development, but still needs further exploration for the prokaryotic expression.