| This work was funded by National "863"High-tech Project (No.2002AA206653)- Goat Mammary Gland Bioreactor of Lumbrokinase. The purpose of the work is to establishment goat mammary gland bioreactor of lumbrokinase by genetic engineering way, and lumbrokinase can be expressed in milk. To drink milk and treat thrombus with the purpose of health. Now, three works have been done: Lumbrokinase has been expressed in the milk and it has fibrinolytic activity; Lumbrokinas's expression in mammal cells and retroviral-mediated transfection of lumbrokinase into goat mammary gland.The outbreak rate and death rate of thrombus are all high, and it has a up-trend. Up to the present, we have getten some kinds of thrombolytic enzyme from creature tissue and which have been maken into anti-thrombosis agents. For example, urokinase.streptokinase etc. But the raw material is very deficient, so those agents are very expensive. For example, one liter urine contains 5/10000 gram urokinase. And those agents have a lot of problem in the clinical application. They lose activity easily, stay in body in a short time and easily lead to bleed. Lots of studies and pharmacodynamics tests showed lumbrokinase abstracted from earthworm has the affects of preventing blood condensation and the form of thrombus. It can dissolve thrombus. The oral agent of lumbrokinase crude extract has been in the clinical application and the result is wonderful. For example, Baiao lumbrokinase capsule, Boluoke and so on. Now, to express lumbrokinase by genetic engineering way is in the stage of entry-level. Sun Zhao-jun etc. did such exprement. Lumbrokinas PI239 gene was inserted into eukaryotic expression vector pcDNA3, then the recomponent vector was used to infect BHK cell. The infected cell was tested by PCR and the same fragment as the target gene was amplified. But the lumbrokinase was not found by the fibrin agarose plate assay and SDS-PAGE. They constructed recombinant expression vector contains lumbrokinase matur peptide and green fluorescent protein to infect BHK cell. The result showed that PI239 matur peptide has expressed in BHK cell. Shen Yue etc. applied genetic recombinant technique to express lumbrokinase. Lumbrokinase PI239 gene was inserted into baculovirus expression vector by Bac-to-Bac expression system. The recombinant virus was used to infect insect sf9 cell. The SDS-PAGE analysis revealed that the molecular weight of the lumbrokinase was 30 000. Western blot showed that PI239 protein could be recognized by specific antiPI239 antibody.(1) Construction of mammary gland specific expression vectors of lumbrokinase and its expression in goat mammary gland. Lumbrokinase cDNA mutant was used to construct expression vectors, pIbCP-LK-neo and pIbCP-LK-LK, of which the gene regulatory element was a caprine beta-casein promoter. The two expression vectors were then injected into goatmammary glands for expression. The milk was collected and tested for fibrinolytic activity by the modified fibrin agarose plate assay. It was indicated that both plasmids were expressed in milk. The expression quantity of pIbCP-LK-LK was higher than that of pIbCP-LK-neo. SDS-PAGE showed that the molecular weight of lumbrokinase is 26kDa. This is the first report on lumbrokinase expression by genetic engineering and it has provided solid foundation for lumbrokinase production.(2) The expression of lumbrokinase gene in Vero cell and DK cell. LK gene in the vector pMD18T was cloned to the expression vector pIRES1neo, the CMV promoter is the upstream regulatory element, and the eukaryotic expression vector pILK was established. Liposome-mediated transfection of pILK was carried out on Vero-dhfr" cell and DK-dhfr" cell. After being screened with G418, the tolerance limits of Vero-dhfr" cell and DK-dhfr to G418 were 600ug/ml and 1100ug/ml. But lumbrokinase did not express in those two cells and all the cells died under G418, no cloned cells. Recently, people found lumbrokinase cannot only hydrolyze fibrin protein and fibrinolytic but also can hydrolyze immu...
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