人noggin基因克隆及真核表达载体构建

发布时间：2018-05-26 04:37

本文选题：基因 + noggin　；参考：《山东大学》2005年硕士论文

【摘要】：[目的]Noggin基因是1992年California大学的Harland, R. M.和Smith, W. C.从非洲爪蟾的胚胎中分离出来的,由于将其mRNA注入爪蟾的胚胎可使其头部明显增大,因此得名。此后发现该基因在胚胎发育中有重要神经诱导作用,尤其是诱导外胚层的背侧发育。Noggin作为胚胎发育的重要基因,在进化过程中相当保守,在不同的物种间具有高度同源性。它涉及许多的发育过程,如神经管的形成和关节软骨的形成。作为一种内源性神经诱导信号,它的作用不仅局限于胚胎期,在成年哺乳动物CNS与外周神经系统也可检测到noggin基因的表达。此外,noggin基因亦是目前在体内与体外均被证实有神经诱导作用的神经诱导剂。其神经诱导功能主要是通过拮抗骨形成蛋白(bone morphogenetic protein-4, BMP4)的作用实现的。对于神经细胞的体外诱导分化研究和实验国内外均有广泛报道,但通过构建人Noggin基因载体并用其诱导分化的研究尚属领先。 T alpha-1微管蛋白启动子是一种神经细胞特异性启动子,有选择的在神经干细胞和不成熟的神经细胞内表达。为了进一步研究noggin基因的神经诱导功能和在细胞分化过程中的表达情况,本研究利用基因工程的方法构建pCS2+[Tα1]-Noggin载体。使我们可以很便利的研究noggin在活体细胞和组织的生物学特性(基因表达、信号转导、代谢活动等),为下一步进行神经移植或基因治疗打下基础。 [方法]应用逆转录多聚酶链式反应(RT-PCR)技术,从正常人胎脑组织中扩增出noggin cDNA,并将其连接到T载体上,经酶切鉴定无误后,送公司进行
[Abstract]:[objective] Noggin gene was Harland, R. M. from California University in 1992. And Smith, W. C. Isolated from the embryo of Xenopus laevis, it is named after the embryo injected with mRNA to Xenopus. After that, it was found that this gene plays an important role in the embryonic development, especially the dorsal ectodermal development. Noggin, as an important gene in embryonic development, is quite conserved in the course of evolution and has high homology among different species. It involves many developmental processes, such as the formation of nerve tubes and articular cartilage. As an endogenous neuro-inducible signal, its function is not only limited to embryonic stage, but also the expression of noggin gene can be detected in CNS and peripheral nervous system of adult mammals. In addition, the noggin gene is also a neuroinducer which has been proved to have neuroinductive effects in vivo and in vitro. Its neural induction is mainly achieved by antagonizing the action of bone morphogenetic protein bone morphogenetic protein-4 (BMP4). The research and experiment of neural cell differentiation in vitro have been widely reported at home and abroad, but the research on the construction and differentiation of human Noggin gene vector is still leading. T alpha-1 tubulin promoter is a neuronal specific promoter that is selectively expressed in neural stem cells and immature nerve cells. In order to further study the neural induction function of noggin gene and its expression during cell differentiation, pCS2 [T 伪 1] -Noggin vector was constructed by genetic engineering. So that we can conveniently study the biological characteristics (gene expression, signal transduction, metabolic activity, etc.) of noggin in living cells and tissues, and lay the foundation for the next step of nerve transplantation or gene therapy. [methods] using reverse transcriptase polymerase chain reaction (RT PCR) technique, noggin cDNA was amplified from normal human fetal brain tissue and ligated to T vector.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R346

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