Purification And Molecular Cloning Of Fibrinolytic Factors From Eupolyphaga Sinensis Walker

Nowadays thrombi has became the most serious sick in the world.It is important for medicinals expert to invent some usefull medicine to cure the thrombi. Eupolyphaga sinensis Walker is a kind of tradional Chinese herbal medicine. It proves to be an useful medicine for deal with thrombi. But for a long time, people have known a little about it. According to this situaion, this experiment research the useful parts for cure thrombi in Eupolyphaga sinensis Walker by modern biology technology.At first, A kind of the plasminogen activator of Eupolyphaga sinensis Walker has been separated and purified by ammonium sulphate precipitation, DEAE-cellulose and SephadexG-75 Column chromatography. The activator which named EFE1 is appeared to be a single band on SDS-PAGE. It is showed that the MW of the activator is 41300. In addition, it includes ten point five percent sugar. Its specific activity is 547.9U/mg. Three kind of fibrinolytic factors of Eupolyphaga sinensis Walker which are named as EFF-1 EFF-2 and EFF-3 have been separated and purified by ammonium sulphate precipitation, DEAE-cellulose and preparative PAGE electropheresis. Three purified components were proved to be homogeneous in SDS-PAGE electropheresis with moleculare weights 41000,32900 and 30600 respectively. Their Activity as plasminogen activator were 171.3U/mg 234.0U/mg和 148.5U/mg. In addition, EFF-2 and EFF-3 showed fibrinolytic activities on fibrinous plate lacked of Plasminogen. So we could consider EFF-2 and EFF-3 as two new kind of fibrinolytic enzymes.A section of trypsin(ET-I)cDNA was amplified by RT-PCR from female Eupolyphaga sinensis Walker. Sequence analysis revealed that the cDNA was 434 base pair, and that encoded a protein of 144 amino acid residues. With the help of protein Blast, it was proved that the gene belongs to the serine protease trypsin family. In addition, it shows homology in protein sequence compared with Earthworm fribrinolytic enzyme, Scolopendra subspinipes mutilans scolonase and...