Construction And Identification Of Medicago Sativa And Salvia Miltiorrhizae Expressing The Earthworm Fibrinolytic Enzymes

Earthworm Fibrinolytic Enzymes (EFE), are also called lumbrokinase, are a group of proteolytic enzymes, and they are distributed throughout the lumen of alimentary tract and Body fluid in many kinds of earthworms, and are probably responsible for digesting the food. EFE degrade fibrin directly, therefore it can degrade the blood clots and prevent them from growing up; besides that, some kinds of EFE activate plasminogen and turn them into fibrinolytic enzymes, or activate human vascular endothelial cells to release the t-PA and degrade the fibrin indirectly. Other than many other kinds of thrombolytics, EFE have weak side effect and are edible. Therefore, as a noval kind of thrombolytics, EFE have a very broad prospective.At present, the EFE which are used clinically are come from the earthworm by artificial feeding, however, extracting EFE from earthworm encounters some problems, such as the breeding cycle which lasts very long, complicated processing technique, and the unstable quality of the product, which entirely constrain the large-scaled production, application and popularity of the EFE.Expression of a single component of EFE in transgenic plants opens up a new research area of EFE produced by plant as bioreactor, and increases possibility of the novel thrombolytics in the clinic use. In this thesis, Alfalfa and Salvia are used as the host plants of expressing a single component of EFE, the reasons are as follows: 1) Alfalfa, one of the most important pasture grasses around the world, has characteristics of high yields, good quality, strong adaptability and easy storage. As a bioreactor for production of medical proteins in the plant, its product could be for oral use and accepted easily by common popularity; 2) Salvia miltiorrhizae, being used as a traditional Chinese medicine for a long history, has many medical functions, such as decreasing cholesterin and blood lipiods, inhibiting clotting and activating plasminogen and so on. If the EFE are expressed in the Salvia, it will be possible to improve the effect of prevention on angiocardiopathy by Salvia and medical quality of Salvia through the synergy of EFE and Salvia.In the process of establishing alfalfa as bioreactor, we used the alfalfa regeneration system which was firmly established formerly in our laboratory in order to introduce the EFE genes, such as CST1 and CST2-1 into the plants through the transformation mediated by agrobacterium tumefaciens, and obtained 130 kanamycin-resistant regenerated plantlets of Baoding alfalfa which was introduced the CST1 and 92 which was introduced the CST2-1. The PCR and PCR Southern assay on these regenerating plants with kanamycin resistance showed that the target genes has been introduced into these plants. And the Southern Blot results showed that CST2-1 was in the genome of the two plants selected randomly from the positive results of PCR Southern. According to the result of ELISA test, most of the regenerating alfalfa plants have higher OD405 than the negatives, however, in contrast with the results of positives made by Lumbrokinase Enteric-coated Capsules, they are still very low, which indicated that EFE had been expressed in the transgenic alfalfa cells, though the expected protein was in very low amount in the plants. In the assay of the stability of the foreign genes, transgenic plants were cultivated successively by 3～5 generations, and the leaves could also differentiate healthy callus on the culture medium contained kanamycin, which showed these plants were steady and hereditary transgenic plants.In the process of establishing alfalfa as bioreactor, we firstly alleviated enormously the problems of browning and vitrification in the regenerating plantlets and complete the Salvia regeneration system through optimize the conditions of the conformation and culture. We also successfully introduced the EFE genes, such as CST1 and CST2-1 into the plants through the transformation mediated by agrobacterium tumefaciens, and obtained 25 kanamycin-resistant regenerated plantlets of Salvia miltiorrhizae which was introduced the CST1 and 33 which was introduced the CST2-1. According to the PCR and PCR Southern assay on these regenerating plants with kanamycin resistance, it was shown that the target genes has been introduced into these plants. And the Southern Blot results showed that CST2-1 was in the genome of the two plants selected randomly from the positive results of PCR Southern. The results of ELISA test were slightly higher than those of alfalfa and also showed EFE had been expressed in the transgenic Salvia cells, though the expected protein was in very low amount in the plants. In the assay of the stability of the foreign genes, transgenic plants were cultivated successively by 3～5 generations, and the leaves could also differentiate healthy callus on the culture medium contained kanamycin, which showed these plants were steady and hereditary transgenic plants.