Extraction Tissue Factor By Yeast And Research Of Hemostasis Effect

Tissue factor (TF),a type of transmembrane glucoprotein with 47kD molecular weight,is a cellular receptor for plasma clotting factor VII, playing a very important role in physiological hemostasis. The protein of tissue factor contains 295 amino-acid residues, including 219 amino-acid residues consisting an extracellular domain,23 amino-acid residues consisting an hydrophobic transmembrane region and 21 amino-aeids residues consisting an intracellular tail. In normal physiological condition,TF is constitutively expressed in many cells,such as fibroblasts and keratinocytes,but is absent in vascular cells that come in contact with blood. However, tissue factor is induced in vascular cells in certain pathophysilogical conditions.Thus,vessel wall injury or a disease condition allows blood to come in contaet with TF,resulting in faetor VII binding to tissue factor. Once native factor VII complexed with tissue factor is converted to the enzyme factor VIIa,the complex triggers the clotting cascade that ultimately leads to fibrin formation.In addition to its established role in coagulation,molecular links between factor Vila/tissue factor and various biological processes.It has been reported that impaired angiogenesis, yeast mycelia contains tissue factor. Choose four kinds of yeast:alcohol yeast,active dry,saccharomyces cerevisiae and geotrichum candidum. Taking yeast as the engineering material, and isolating tissue factor through the fermentation.Tissue factor was isolated from the mycelium of Saccharomyces cerevisiae by the alcohol-deposit and aether-absorption procedure. Homogenicity of tissue factor was tested by SDS-PAGE, meanwhile its molecular weight was estimated too. Purify though gel filtration of Superdex G-200. Purified tissue factor, with each yeast extract tissue factor qualitative to concentration for 0.4mg/ml,0.6mg/mL,0.8mg/ml,lmg/ml, In mice respectively in vitro clotting (each blood drops plus 50μ1), Traumatic clotting test in vitro(each wound drops add 50μ1)and Injected into the body blood coagulation experiment(each injected mice with only 50μ1). Refer to the clotting qualitative standard product, screen the bacteria yeast extracts with best clotting effect.Screen the yeast fermentation, select fermentation conditions, deep fermentate, extract and purify tissue factor, and check their blood coagulation effect.A white crystalline substance extracted from yeast mycelia.Homogenicity of tissue factor was tested by SDS-PAGE and meanwhile its molecular weight was estimated too. The image of SDS-PAGE showed one band both innative and denaturing with molecularweight of 47kD.In clotting trials, the vitro time of clotting tissue factor is 13s, the clotting tissue factor extracted from alcohol yeast blood clots outside clotting time for geologic 18s; The outside clotting time of tissue factor extracted from active dry yeast is 21s; The outside clotting time of tissue factor extracted from the saccharomyces cerevisiae is 23s; The outside clotting time of tissue factor extracted from the geotrichum candidum is 14s. The vitro traumatic clotting time of clotting tissue factor for 44s, the plastids traumatic clotting time of clotting tissue factor extracted from alcohol yeast is for 48s; the plastids traumatic clotting time of clotting tissue factor extracted from active dry yeast is 58s; the plastids traumatic clotting time of clotting tissue factor extracted from saccharomyces cerevisiae is 58s; the plastids traumatic clotting time of clotting tissue factor extracted from the geotrichum candidum geological blood coagulation is 44s. From the comparision a conclusion is drawn that the blood clots extracted from geotrichum candidum has the best qualitative clotting effect. The best wave bottle cultivate conditions of geotrichum candidum is as below: revolving speed 200 r/min, temperature 30℃, outfit fluid volume 50im1/250ml, pH value 5.0. Tisse factor after deep fermentation extraction and purification still have tlle function of blood clotting.