Expression Product Of Lumbrukinase Activity In Vivo Of Animal Model

The thrombus disease,has become our country and the western nation population death with the main reason which cripples.According to the World Health Organization's statistics,approximately has 18 million people to die every year of all kinds of heart blood vessel of brain disease.Our country existing blood vessel of brain disease patient more than 5 million,newly arises every year the number approximately 1.5 million,for population death second reason.Thrombotic disease is caused loss of labor,reduced quality of life and increase the burden of disease mainly.Annual medical costs billions of dollars,a serious impediment to the country's economic development and people's living standards further improved.Fibrinolytic activity.It consists of plasmin and similar tissue-type plasminogen activator of various components.Dissolution with thrombosis and to prevent thrombosis dual action.Clinically,the extract of earthwore is widely used to treat angina pectoris,acute cerebral infarction is effective,Diabetic peripheral neuropathy effects Lumbrokinase right pulmonary heart disease,Meniere's disease, sudden deafness retinal vein occlusion,nephrotic syndrome, and have made significant effect. Lumbrokinase as a thrombolytic drug, which has been a more in-depth development. Current clinical application of Lumbrokinase are from earthworms the direct extraction of the body,its Preparation complicated, and the lower yield,thus through genetic engineering technology to produce high-purity Lumbrokinase has become a very effective way.With the mechanism of the enzyme and its application has deepened Some scholars use molecular biology methods in Escherichia coli,Pichia pastoris,COS-7 cells. BHK cells in the breast,such as goats,lumbrokinase gene expression.Lumbrokinase gene in both prokaryotic cells,or in which the eukaryotic expression it have.Although the expression is not high, to Lumbrokinase in molecular biology research has laid the foundation.Strain eukaryotic expression vector containing CMV promoter early, Synthesis of intron and the ribosome binding site and bovine growth hormone (BGH) polyadenylation signal sequence,In the same promoter could also both protein expression of genes, gene pairs were constructed expression vector,containing ampicillin resistance gene.Neomycin phosphotransferase gene can be inserted into the gene through G418 selection to be stable expression.In the aforementioned study,based on known kappa chain sequence, designed and synthesized in a long 25-30nt long primers,Expand the use of overlapping PCR method was kappa chain genes.Cloning of kappa chain of releasing the carrier,and the room has been the lumbrokinase gene connected to the kappa chain,as a signal peptide containing the gene.Then pass the signal peptide with kappa Lumbrokinase connected to the eukaryotic expression vector pIRES-neo.will be named:pIRES-kappa-LK.Expression of the recombinant plasmid liposome-mediated transfection of BHK-21 cells between,300μg/mlG418 week by the selection,pIRES-kappa-LK cell clone.On the cloning of cells,the protein used Pipe Dream plate tPA (DPP) on the expression product activity.The results showed that the recombinant plasmid pIRES-kappa-LK be in BHK expression Lumbrokinase expression of extracellular protein is secreted protein expression Lumbrokinase about the fragment encoding the 28 kDa molecular weight.Expression efficiency of 2.6%, protein expression with fibrinolytic activity,by gel chromatography activity 42000U/L up.Gel purified after Lumbrokinase product after protein,thrombosis in vivo animal model for activity detection.Activity in vivo experiments based on the commonly used clinical thrombolytic monitoring indicators (t-PA and PAI, the FDP, D-dimer).The results showed that the experimental group plasma therapy,increased fibrinolytic activity after thrombosis and degradation products values decrease.The efficacy of the experimental group were significantly lower than the control group.But before and after thrombolysis thrombosis and fibrinolysis degradation products of the change,statements of the Lumbrokinase thrombosis in vivo animal model active.