蚓激酶转基因苜蓿（Medicago sativa）及丹参（Salvia miltiorrhizae）的构建及检测
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摘要
蚯蚓纤溶酶(Earthworm Fibrinolytic Enzymes,EFE),又称蚓激酶,属于多组分的蛋白水解酶类,它们广泛分布于多种蚯蚓的消化道内腔及体液中,可能与摄取食物的吸收和利用有关。蚓激酶可以直接水解纤维蛋白,因此,它们既具有溶解现有血栓、也同时具有阻止血栓继续形成的功效;此外,有部分种类的蚓激酶还可以将纤溶酶原激活并转化为纤溶酶、或刺激人血管内皮细胞释放组织纤溶酶原激活剂(t-PA),从而能够间接溶解血栓。与其它溶栓剂所不同的是,蚓激酶的毒副作用比较小,而且还可以口服给药,因此,作为一种新型的溶栓药剂,无疑具有十分广阔的应用前景。
目前,在临床上应用的蚓激酶均源自人工饲喂的蚯蚓,然而,依靠人工从蚯蚓中提取蚓激酶仍然存在很多问题,比如蚯蚓饲养和繁殖周期长,生产工艺复杂和生产质量不稳定等,这些不利因素都在很大程度上制约了蚓激酶的规模化生产、利用及普及。
在转基因植物中表达蚓激酶单一组分,开辟了利用植物生物反应器生产蚓激酶的新领域,可为今后蚓激酶药物降低生产成本和在临床上的更广泛应用奠定坚实基础。本研究选用苜蓿和丹参这两种常见的植物作为表达蚓激酶单一组分的宿主植物,这是因为:1)苜蓿是世界上最重要的豆科牧草之一,具有品质优、产量高、适应性强、易于贮存以及加工等许多的优点,此外,其作为生物反应器生产的重组类蛋白药物可直接口服,而且无毒无害,便于今后的规模化生产和推广;2)丹参作为一味历史悠久的中药,其本身具有扩张冠状动脉、降低胆固醇和血脂、抑制凝血和激活纤溶系统等多种作用,在临床治疗心脑血管疾病方面应用广泛且疗效十分显著。若实现蚓激酶在丹参中的表达,可通过二者的协同作用,进一步增强丹参在防治心血管疾病方面的疗效,改善丹参的药用品质。
在苜蓿生物反应器构建过程中,利用本课题组先前建立的比较成熟的苜蓿组织培养再生体系,通过农杆菌介导方法,成功地将蚓激酶CST1和CST2-1基因分别导入到保定苜蓿中,先后获得转CST1基因的卡那霉素抗性再生苗130株和转CST2-1基因的卡那霉素抗性再生苗92株。对这些转基因植株进行PCR和PCR Southern检测,结果证实多数卡那霉素抗性植株分别含有上述蚓激酶编码组分。从PCR检测为阳性的转CST2-1基因再生植株中,任意选取两株进行基因组DNA Southern Blot检测,结果证实目的基因CST2-1确实已整合到苜蓿基因组中。通过ELISA,对外源蛋白表达情况进行了检测与分析,结果发现多数卡那霉素抗性植株在405nm处的吸光值都大于非转基因苗(阴性对照),但与蚓激酶肠溶胶囊溶剂相比,其数值偏低,这表明蚓激酶基因在转基因苜蓿细胞中是有表达的,但表达效率非常低。外源转基因遗传稳定检测结果发现,经多3～5次无性继代培养后的转基因苜蓿叶片在含有卡那霉素的培养基上仍能正常分化出健康的愈伤组织,这表明外源的蚓激酶基因在转基因苜蓿中通过无性繁殖可以稳定遗传给后代。
在丹参生物反应器构建过程中,首先通过对丹参的转化和培养条件进行了优化,使丹参再生幼苗的褐化和玻璃化现象大大减轻,更加完善了丹参的组织培养遗传转化再生体系。通过农杆菌介导方法,同样成功地将蚓激酶CST1和CST2-1基因分别导入到丹参中,分别获得卡那霉素抗性再生苗25株和33株。对这些再生植株进行PCR和PCR Southern检测和分析,结果证实多数卡那霉素抗性植株分别含有上述蚓激酶编码组分。从PCR检测为阳性的转CST2-1基因再生植株中,任意选取两株进行基因组DNA Southern Blot检测,结果证实目的基因CST2-1确实已整合到丹参基因组中。同样利用ELISA法对蚓激酶外源蛋白表达情况进行了检测与分析,结果发现外源蛋白表达普遍微弱,在一定程度上稍微高于转基因苜蓿的结果类似。外源转基因遗传稳定检测结果发现,经多3～5次无性继代培养后的转基因丹参叶片在含有卡那霉素的培养基上仍能正常分化出健康的愈伤组织,这表明外源的蚓激酶基因在转基因丹参中通过无性繁殖可以稳定遗传给后代。
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.
目前,在临床上应用的蚓激酶均源自人工饲喂的蚯蚓,然而,依靠人工从蚯蚓中提取蚓激酶仍然存在很多问题,比如蚯蚓饲养和繁殖周期长,生产工艺复杂和生产质量不稳定等,这些不利因素都在很大程度上制约了蚓激酶的规模化生产、利用及普及。
在转基因植物中表达蚓激酶单一组分,开辟了利用植物生物反应器生产蚓激酶的新领域,可为今后蚓激酶药物降低生产成本和在临床上的更广泛应用奠定坚实基础。本研究选用苜蓿和丹参这两种常见的植物作为表达蚓激酶单一组分的宿主植物,这是因为:1)苜蓿是世界上最重要的豆科牧草之一,具有品质优、产量高、适应性强、易于贮存以及加工等许多的优点,此外,其作为生物反应器生产的重组类蛋白药物可直接口服,而且无毒无害,便于今后的规模化生产和推广;2)丹参作为一味历史悠久的中药,其本身具有扩张冠状动脉、降低胆固醇和血脂、抑制凝血和激活纤溶系统等多种作用,在临床治疗心脑血管疾病方面应用广泛且疗效十分显著。若实现蚓激酶在丹参中的表达,可通过二者的协同作用,进一步增强丹参在防治心血管疾病方面的疗效,改善丹参的药用品质。
在苜蓿生物反应器构建过程中,利用本课题组先前建立的比较成熟的苜蓿组织培养再生体系,通过农杆菌介导方法,成功地将蚓激酶CST1和CST2-1基因分别导入到保定苜蓿中,先后获得转CST1基因的卡那霉素抗性再生苗130株和转CST2-1基因的卡那霉素抗性再生苗92株。对这些转基因植株进行PCR和PCR Southern检测,结果证实多数卡那霉素抗性植株分别含有上述蚓激酶编码组分。从PCR检测为阳性的转CST2-1基因再生植株中,任意选取两株进行基因组DNA Southern Blot检测,结果证实目的基因CST2-1确实已整合到苜蓿基因组中。通过ELISA,对外源蛋白表达情况进行了检测与分析,结果发现多数卡那霉素抗性植株在405nm处的吸光值都大于非转基因苗(阴性对照),但与蚓激酶肠溶胶囊溶剂相比,其数值偏低,这表明蚓激酶基因在转基因苜蓿细胞中是有表达的,但表达效率非常低。外源转基因遗传稳定检测结果发现,经多3～5次无性继代培养后的转基因苜蓿叶片在含有卡那霉素的培养基上仍能正常分化出健康的愈伤组织,这表明外源的蚓激酶基因在转基因苜蓿中通过无性繁殖可以稳定遗传给后代。
在丹参生物反应器构建过程中,首先通过对丹参的转化和培养条件进行了优化,使丹参再生幼苗的褐化和玻璃化现象大大减轻,更加完善了丹参的组织培养遗传转化再生体系。通过农杆菌介导方法,同样成功地将蚓激酶CST1和CST2-1基因分别导入到丹参中,分别获得卡那霉素抗性再生苗25株和33株。对这些再生植株进行PCR和PCR Southern检测和分析,结果证实多数卡那霉素抗性植株分别含有上述蚓激酶编码组分。从PCR检测为阳性的转CST2-1基因再生植株中,任意选取两株进行基因组DNA Southern Blot检测,结果证实目的基因CST2-1确实已整合到丹参基因组中。同样利用ELISA法对蚓激酶外源蛋白表达情况进行了检测与分析,结果发现外源蛋白表达普遍微弱,在一定程度上稍微高于转基因苜蓿的结果类似。外源转基因遗传稳定检测结果发现,经多3～5次无性继代培养后的转基因丹参叶片在含有卡那霉素的培养基上仍能正常分化出健康的愈伤组织,这表明外源的蚓激酶基因在转基因丹参中通过无性繁殖可以稳定遗传给后代。
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.
引文
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