Molecular Cloning And Expression In Yeast Of CDNA Encoding Urechis Unicinctus Fibrinolytic EnzymeⅡ

Thrombosis has been a serious threat to human health. Acording to some relevant statistics, in the world, each year there are 17.5 million patients die of thrombotic disease. As to our country, the patients with thrombotic disease are more than 270 million, and 3 million of them ended with death each year. Nowadays China is facing the most severe condition of morbidity and mortality caused by cardiovascular and cerebrovascular disease. Thrombolytic therapy is the most important pathway in treatment of thrombotic disease. Currently thrombolytic agents used for clinical application has been developed into the third generation, but the healing efficacy of many kinds is far from satisfaction. They have poor specificity, serious side effects, short half-life or difficult to producing and too expensive for ordinary people. Therefore, evey patient and scientist is eager to the development of new thrombolytic which will overcome all these shortcomings.After years of research, a novel fibrinolysin is isolated and purified from Urechis unicinctus a marine invertebrate in our laboratory named Urechis unicinctus Fibrinolytic enzymes (UFE). It includs 4 different compounds, they are Urechis unicinctus Fibrinolytic enzymesⅠ(molecular weight 45KDa), Urechis unicinctus Fibrinolytic enzymesⅡ(molecular weight 26KDa), Urechis unicinctus Fibrinolytic enzymesⅢ(molecular weight 20KDa), and Urechis unicinctus Fibrinolytic enzymes IV(molecular weight 8-10KDa). Each of them not only improves the secondary plasmin activity of the organisms but also has great anticoagulant activity, plasmin activity and bio-security. In conclusion, it has great value of development. However, there have been many drawbacks when it is produced by means of separation and purification directly from living organisms, such as the complex production process, instability of production quality and low yield.Therefor we hope that through this study, the gene of Urechis unicinctus Fibrinolytic enzymesⅡwill be cloned, and the genetically engineered strains which can yield recombinant Urechis unicinctus Fibrinolytic enzymesⅡwill 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 UFEⅡ. According to this sequence, degenerate primers were designed to amplify part of the UFEⅡgene by using cDNA as a template through 3 '-RACE PCR. Then the complete cDNA (full length 906bp) sequence encoding the UFEⅡwas amplyfied on the basis of the result of 5'-RACE PCR, thereamong the open reading frame has 796bp. The comparison result of ufeⅡcDNA sequence analysised by BLASTx indicates that the protein sequence has high homology with the tryptase family members. The analysis results of ScanProsite software by PROSITE demonstrate that the protein contains a trypsin domain (No.32-264 bit) and according to the CDD database of NCBI, the protein is a trypsin-like serine protease.2. Secreted experssion plasmids pPICZaA- ufeⅡ-P1/P2 and pINA 1317-uefⅡ-Y1/Y2 have been successfully constructed, and respectively transformed in Pichia.pastoris include X-33,SMD1168 and Yarrowia lipolytica. After repeated screening on a large number of transformants still no strains with the ability of producing fibrinolysin was found. The fermentation supernatant after dialysis was concentrated by freeze dehydration. The samples were detected by SDS-PAGE and no interest protein band were found. When using the method of western blot we got the same result that no interest protein was produced. Experimental results show that this gene is failed to be expressed in these three kind of yeast.To sum up, In this study, the complete cDNA sequence of Urechis unicinctus Fibrinolytic enzymesⅡhas been cloned by general approach of molecular biology. The gene has been analysised by methods of bioinformatics, and come to the conclusion that the protein encoded by ufeⅡis a kind of trypsin-like serine protease. Its molecular weight is 2390.85 Daltons; Expression vectors have been successfully constructed and respectively transformed in three different kinds of yeast host strain. All these experiments laid the foundation for further research about this new gene and Urechis unicinctus Fibrinolytic enzymesⅡ.