【作者】 颜景斌；

【导师】 曾溢滔； 黄淑帧； 张敬之； 马晴雯；

【作者基本信息】 上海交通大学 ， 生物化学与分子生物学， 2007， 博士

【摘要】 转铁蛋白(Transferrin, TF)是一种与铁结合的糖蛋白,在细胞生长、分化、增殖过程中起着十分重要的功能。因此,利用生物反应器生产转铁蛋白在生物制药上具有重要的意义和前景。本文首先分析不同调控元件对人转铁蛋白在动物各脏器中表达的调节能力,分别克隆了兔转铁蛋白启动子、增强子,并将其与人转铁蛋白小基因(包括TF cDNA及内含子I)相连构建表达载体,同时构建了以山羊β乳球蛋白启动子启动的乳腺表达载体。在此基础上将上述表达单元装入慢病毒载体构建了转铁蛋白慢病毒表达载体。随后将质粒表达载体分别转染小鼠乳腺上皮细胞、大鼠肝脏细胞以及小鼠和山羊乳腺。结果显示所有表达载体均可指导人转铁蛋白在细胞以及小鼠和山羊乳腺中表达,兔转铁蛋白增强子与启动子组合可显著性增加人转铁蛋白的表达水平,且其指导表达的效率明显高于山羊β乳球蛋白启动子。然后通过原核显微注射和慢病毒感染等不同方式来制备转基因小鼠。实验结果也证明将一个兔转铁蛋白增强子与启动子串联组成的调控元件指导人转铁蛋白基因在血浆中的表达效率最高,转基因小鼠血浆和乳汁中人转铁蛋白最高的表达水平分别达到913.6μg/mL和697.3μg/mL。另外,初步的结果显示慢病毒介导的转基因小鼠制备方法不仅整合效率比原核显微注射高近3倍,而且人转铁蛋白的平均表达水平明显高于显微注射获得的小鼠。但外源基因在小鼠各脏器中的表达模式两者之间没有明显的差异,均主要在肝脏和乳腺中表达,心脏、肾脏等其它脏器仅有较低的表达。与此同时,本文还对利用细胞体外转染和体细胞核移植技术制备的转基因克隆胎牛中人转铁蛋白基因整合、表达情况进行了分析。结果证实外源基因以单拷贝的形式整合在牛基因组中,整合位点离最近的一个牛功能基因约220Kb,而人转铁蛋白主要在肝脏中检出了表达。另外,本文分别设计了两条针对小鼠β酪蛋白基因和三条针对乳清酸蛋白基因的干扰载体,将这些载体分别注射哺乳期的人转铁蛋白转基因小鼠乳腺。结果显示大部分干扰载体不仅能有效降低小鼠内源性乳蛋白的表达水平,而且在一定程度上提高了外源人转铁蛋白的表达,最高的提高了近一倍。同时通过显微注射的方法制备了整合干扰载体的转基因小鼠,对1只已进入泌乳期的小鼠分析显示内源性β酪蛋白相对浓度仅为正常小鼠的约70%。本文系统地分析了不同调控元件对人转铁蛋白在动物各脏器中表达的调节作用,为这些调控元件的功能研究提供了科学的数据,并获得了一批可在乳汁和血浆中高效表达人转铁蛋白的转基因动物。同时采用干扰内源性乳蛋白的方法来提高外源基因表达水平,为乳腺生物反应器的研究提供了新的思路和方法。更多还原

【Abstract】 Transferrin (TF) is an iron-binding, monomeric glycoprotein. It is an important factor involved in various cellular processes and plays complex physiological roles related to cell function, differentiation and proliferation. Conceivably, production of TF through transgenic approaches holds great potentials in bio-pharmaceutical industry.In this study, We have analyzed the capabilities of several regulatory elements in regulating the expression of human TF in diverse tissues of animal models. First, the enhancer and promotor of rabbit transferrin were cloned by PCR and ligated with human transferrin minigene (including TF cDNA and intron I). Meanwhile, human transferrin minigene was ligated with goat beta lactoglobulin promotor as control. Then, these expression elements were cloned into lentivirus vector. To investigate the expression of human transferrin, these plasmids were transfected into HC-11, BRL-3A cell lines and mouse or goat mammary gland, respectively. The results showed that human transferrin could be expressed in these cell lines and mouse or goat mammary gland following transfection, and its expression level could be improved dramatically when it was driven by rabbit enhancer and promotor as compared with goat beta lactoglobulin promotor. Transgenic mice were generated by either microinjection or lentivirus infection. The expression level of human transferrin was analyzed and the efficiencies of two different preparation methods were studied. The experimental data confirmed that human transferrin could be expressed at the highest level in the serum when it was regulated by the element including one rabbit enhancer and promotor. The expression level reached as high as 913.6μg/mL and 697.3μg/mL in serum and milk of the transgenic mice, respectively. The results also suggested that the transgenes could be not only integrated into the host chromosomes more efficiently (the integration rate was improved three times), but also expressed at higher level when the transgenic mice were prepared with lentivirus infection other than microinjection. However, the expression profiles of human transferrin in different organs of transgenic mice were not changed when the mice were prepared with different methods. Human transferrin was mainly expressed in liver and mammary gland of transgenic mice, and the expression levels in other organs such as heart, kidney and so forth, were very low.Meanwhile, the integration and expression of human transferrin in transgenic fetal bovine, which was prepared with in vitro transfection and somatic nuclear transfer, were analyzed. The data indicated that foreign gene was integrated into bovine chromosome with only one copy, and the integration site was located in the downstream of one funtional gene. And the expression of human transferrin was detected in liver of fetal bovine.In addition, two RNAi fragments targeting mouse beta casein gene and three RNAi fragments targeting whey acidic protein gene were synthesized and cloned into expression vectors. These vectors were injected into the mammary gland of transgenic mice integrated with human transferrin gene, and the relative quantity of beta casein and concentration of human transferrin were analyzed. The results showed that the concentration of endogenous milk protein was decreased and the expression levels of human transferrin were improved in most of the transgenic mice. Then, transgenic mice integrated with RNAi fragments were prepared and the data indicated that the relative abundance of beta casein in the milk of one transgenic mouse reached only 70% of normal mice.In this paper, the expression levels of human transferrin in different organs of transgenic animals were analyzed, and the data revealed that human transferrin could be expressed effectively in the serum and milk of transgenic mice when proper regulatory elements were utilized. Meanwhile, it was found that the expression of transgenes could be improved when the endogenous milk proteins were inhibited. These data should provide valuable information and offer a new idea to promote the study of transgenic mammary gland bioreactor.更多还原