人骨形成蛋白2和7成熟肽编码区基因克隆和真核表达载体的构建
发布时间:2018-06-17 20:49
本文选题:人骨形成蛋白 + 成熟肽 ; 参考:《广州医学院》2009年硕士论文
【摘要】: 骨的愈合有三个重要的因素:一为骨质再生细胞(骨间质干细胞,定向造骨细胞),二为骨质诱导蛋白(生长因子),三为愈合区域的环境(载体和来源组织)。在目前的整形外科手术中,组织工程学技术可以利用一些生物活性物质,如骨形成蛋白(bone morphogenetic proteins, BMPs),来改变骨移植过程的模式。BMPs是一类酸性糖蛋白,属于乙型转化生长因子(transforming growth factor-bate, TGF-βs)超家族,能够诱导促进软骨和骨的形成,在胚胎发育过程中发挥重要的作用,包括脑的发育和骨的形成。BMPs在体外与多种不同性质的载体连接,可以组成各种类型的有生物诱骨活性的骨修复材料,在矫形外科等领域有广泛的应用前景及重要的应用意义。目前最少有20种BMPs被确认,这些BMPs被广泛应用于骨的再生、牙质的生长和肾脏细胞的生长发育等研究中。 BMP-2是BMPs中生物学活性最高的蛋白之一,也是研究最为广泛的生长因子之一,其主要生物学作用是诱导未分化的间质细胞分化增殖,形成软骨和新生骨。直接局部应用BMP-2,因为药物的弥散和免疫反应,疗效较低。故应用转基因技术将编码BMP-2的基因导入恰当的靶细胞内,使之连续释放BMP-2,并以自分泌或旁分泌的形式作用于靶细胞,则可以大大弥补外源性细胞因子局部应用的缺陷。BMP-7最初从骨基质中分离纯化得到,其功能是调控骨和软骨的发生,它与早前从牛成骨蛋白提取物中分离的成骨蛋白1(osteogenetic protein,OP-1)是同一种蛋白。BMP-7具有细胞因子的特点,用量少而能发挥很大的作用,但它进入体内后易随体液流失降解,且半衰期短,不能局限在骨缺损局部发挥作用,因此必须寻找更有效的替代方法,使其稳定表达,基因治疗成为备选的方法之一。 BMP-2和-7基因开放读码框(open reading frame, ORF)均可编码由信号肽、前肽和成熟肽组成的多肽,由蛋白酶切去信号肽和前肽后可得到有功能的成熟肽。在本研究将利用基因重组技术,从BMP-2和-7基因表达量丰的骨肉瘤细胞中克隆两基因的成熟肽的基因序列,并构建它们的真核表达载体。实验中应用的真核表达载体为pcDNA3.1(+)。 结果显示:本研究已成功克隆和构建了BMP-2和-7成熟肽的真核表达载体,重组子序列与Genebank中登录的序列完全一致。实验结果可为在骨修复和细胞分化增殖等方面得到广泛应用,可为应用BMP-2和-7进行基因治疗等提供有力的实验支持。
[Abstract]:There are three important factors in bone healing: one is bone regeneration cell (bone interstitial stem cell), the other is bone inducible protein (growth factor), and the third is environment (carrier and source tissue) of healing area. In current orthopedic surgery, tissue engineering techniques can use some bioactive substances, such as bone morphogenetic protein morphogenetic, BMPsN, to change the pattern of bone transplantation. BMPs are a class of acidic glycoproteins. TGF- 尾, which belongs to the growth transforming factor-bate (TGF- 尾) superfamily, can induce the formation of cartilage and bone and play an important role in embryonic development, including brain development and bone formation. It can form various kinds of bone repair materials with biological osteoinductive activity, and has a wide application prospect and important application significance in orthopaedic surgery and other fields. At least 20 BMPs have been identified, which have been widely used in bone regeneration, odontoplasmic growth and renal cell growth, etc. BMP-2 is one of the most bioactive proteins in BMPs. It is also one of the most widely studied growth factors, whose main biological role is to induce the differentiation and proliferation of undifferentiated interstitial cells, forming cartilage and new bone. Direct local application of BMP-2, because of the drug dispersion and immune response, the efficacy is low. Therefore, the gene encoding BMP-2 was introduced into the appropriate target cells by transgenic technology, and the BMP-2 was continuously released and acted on the target cells in the form of autocrine or paracrine. BMP-7 was originally isolated and purified from bone matrix and its function was to regulate the occurrence of bone and cartilage. It is the same protein, BMP-7, which was isolated from bovine osteogenic protein extract (OP-1), has the characteristics of cytokine, and can play a great role in less dosage. However, it is easy to degrade with the loss of body fluid and has a short half-life after entering the body. It is important to find more effective alternative methods to stabilize the expression of bone defects. Gene therapy has become one of the alternative methods. BMP-2 and -7 genes can encode peptides composed of signal peptide, prepeptide and mature peptide by open reading frame (ORF). A functional mature peptide can be obtained by digesting signal peptide and propeptide from protease. In this study, gene sequences of mature peptides of BMP-2 and -7 genes were cloned from osteosarcoma cells with abundant expression of BMP-2 and -7 genes by gene recombination technique, and their eukaryotic expression vectors were constructed. The eukaryotic expression vector was pcDNA3.1. The results showed that the eukaryotic expression vectors of BMP-2 and -7 mature peptides were cloned and constructed successfully in this study. The experimental results can be widely used in bone repair and cell differentiation and proliferation, and can provide powerful experimental support for gene therapy with BMP-2 and -7.
【学位授予单位】:广州医学院
【学位级别】:硕士
【学位授予年份】:2009
【分类号】:R341
【参考文献】
相关期刊论文 前2条
1 郭勇,张西正,李娜,赵红斌,曾强成;人骨形成蛋白2基因克隆及真核表达载体的构建[J];生物医学工程研究;2004年03期
2 邓少林,郭强,黄昭明;骨形成蛋白-2表达载体的构建及其转录活性测定[J];生物技术通讯;2005年02期
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